AFAB-Vol.1-Issue 2 by cielo shabatura

Volume 1, Issue 2 November 2011

EDITORIAL BOARD Sooyoun Ahn

W.K. Kim

Arkansas State University, USA

University of Manitoba, Canada

Walid Q. Alali

M.B. Kirkham

University of Georgia, USA

Kansas State University, USA

Kenneth M. Bischoff

Todd Kostman

NCAUR, USDA-ARS, USA

University of Wisconsin, Oshkosh, USA

Claudia S. Dunkley

Y.M. Kwon

University of Georgia, USA

University of Arkansas, USA

Lawrence Goodridge

Maria Luz Sanz

Colorado State University, USA

MuriasInstituto de Quimica Organic General, Spain

Leluo Guan

Melanie R. Mormile

University of Alberta, Canada

Missouri University of Science and Tech., USA

Joshua Gurtler

Rama Nannapaneni

ERRC, USDA-ARS, USA

Mississippi State University, USA

Yong D. Hang

Jack A. Neal, Jr.

Cornell University, USA

University of Houston, USA

Divya Jaroni

Benedict Okeke

Southern University, USA

Auburn University at Montgomery, USA

Weihong Jiang Shanghai

John Patterson

Institute for Biol. Sciences, P.R. China

Purdue University, USA

Michael Johnson

Toni Poole

University of Arkansas, USA

FFSRU, USDA-ARS, USA

Timothy Kelly

Marcos Rostagno

East Carolina University, USA

LBRU, USDA-ARS, USA

William R. Kenealy

Roni Shapira

Mascoma Corporation, USA

Hebrew University of Jerusalem, Israel

Hae-Yeong Kim

Kalidas Shetty

Kyung Hee University, South Korea

University of Massachusetts, USA

EDITORIAL STAFF EDITOR-IN-CHIEF Steven C. Ricke University of Arkansas, USA

EDITORS Todd R. Callaway FFSRU, USADA-ARS, USA

MANAGING EDITOR Ellen J. Van Loo Ghent, Belgium

LAYOUT EDITOR Melody Rust Eureka Springs Arkansas, USA

Cesar Compadre University of Arkansas for Medical Sciences, USA

TECHNICAL EDITOR

Philip G. Crandall University of Arkansas, USA

ONLINE EDITION EDITOR

Jessica C. Shabatura Fayetteville Arkansas, USA

C.S. Shabatura Fayetteville Arkansas, USA

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TABLE OF CONTENTS ARTICLES 105

Production of Cyclosporin A by Static Fermentation Using Tolypocladium inflatum MTCC 557 S. A. Survase, U. S. Annapure and R. S. Singhal

123

A Transcriptomic Expression Array, PCR and Disk Diffusion Analysis of Antimicrobial Resistance Genes in Multidrug-Resistant Bacteria S. A. Khan, K. Sung and M. Nawaz

140

Antibiotic Resistance and Plasmid Profiles in Bacteria Isolated from Market-Fresh Vegetables S. Akter, Rafiq-Un-Nabi, F. Ahmed Rupa, Md. L. Bari and M. A. Hossain

175

Survival of Salmonella in Organic and Conventional Broiler Feed as Affected by Temperature and Water Activity A. Petkar, W. Q. Alali, M. A. Harrison and L. R. Beuchat

BRIEF COMMUNICATIONS 98

Influence of Winter and Summer Hutch Coverings on Fecal Shedding of Pathogenic Bacteria in Dairy Calves R. L. Farrow, T. S. Edrington, B. Carter, T. H. Friend, T. R. Callaway, R. C. Anderson and D. J. Nisbet

116

Comparative Studies on the Survival of Verocytotoxigenic Escherichia coli and Salmonella in Different Farm Environments C. J. Oâ&#x20AC;&#x2122;Neill, D. J. Bolton and S. Fanning

150

Impact of Calcium Chloride Dip and Temperature on Microbial Quality of Organically and Conventionally Grown Melons H. T. Aldrich, L. Goodridge, M. Bunning, C. Stushnoff and P. Kendall

186

Isolation and Initial Characterization of Plasmids in an Acetogenic Ruminal Isolate R. S. Pinder and J. A. Patterson

REVIEWS 159

Minimizing the Risk of Listeria monocytogenes in Retail Delis by Developing Employee Focused, Cost Effective Training P. G. Crandall, J. A. Neal Jr., C. A. Oâ&#x20AC;&#x2122;Bryan, C. A. Murphy, B. P. Marks and S. C. Ricke

EXTRAS 193

Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

BRIEF COMMUNICATION Influence of Winter and Summer Hutch Coverings on Fecal Shedding of Pathogenic Bacteria in Dairy Calves R. L. Farrow1, T. S. Edrington1, B. Carter2, T. H. Friend2, T. R. Callaway1, R. C. Anderson1, and D. J. Nisbet1 Food and Feed Safety Research Unit, ARS, USDA, College Station, TX 77845 Texas A&M University, Department of Animal Science, College Station, TX 77843 1

‡Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.

ABSTRACT The effects of hutch coverings utilized during the summer and winter months to moderate extreme temperatures were examined on fecal prevalence of E. coli O157:H7 and Salmonella in newborn dairy calves. In the initial study, the effects of shade using screens in three treatment groups: no shade, partial shade, and full shade were examined. Two additional studies were designed where individual calf hutches were modified with a hutch blanket (treatment) or no hutch blanket (control) in the winter study and a ventilated hutch design added as a third treatment in the summer study. During the summer experiment, prevalence of E. coli O157:H7 and Salmonella was low; however, Salmonella was increased (P < 0.05) in the ventilated hutch versus the control treatment. In the winter study, quantifiable results for both E. coli O157:H7 and Salmonella were largely negative. Salmonella positive samples were numerically higher, however no treatment differences were observed. In the shade cloth study all fecal samples were E. coli O157:H7 negative. Salmonella was cultured from all treatment groups, however no differences were observed between treatments. Summarily, there is no evidence that hutch treatments decreased the period prevalence of fecal shedding of Salmonella, E. coli O157:H7 or Enterococcus. Keywords: Dairy calves, Salmonella, E. coli, Hutch covering Agric. Food Anal. Bacteriol. 1: 98-104, 2011

INTRODUCTION Dairy producers have long realized the need for individualized care of newborn calves from both management and health perspectives. Dairy calves are born without circulating antibodies that are critical Correspondence: T. S. Edrington, tom.edrington@ars.usda.gov Tel: +1 -979-260-3757 Fax: +1-979-260-9332

for immune function and current management practices strive to insure that newborns receive adequate amounts of colostrum to develop immunity to a host of potentially pathogenic organisms; however, these calves have an increased susceptibility to disease (Roy, 1970). In 2006 the U. S. Department of Agriculture reported a mortality rate of 11 percent for pre-weaned dairy calves with the majority of those deaths resulting from enteric and respiratory pathogens (USDA, 2006).

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Calf hutches have been used as a means to reduce direct contact among newborn calves in an effort to reduce transmission of disease. Dairy calves that were comingled had an increased prevalence of E. coli O157:H7 compared to calves that were housed individually (Garber et al., 1995). Poos and Sordillo (1982) reported that calves housed in hutches had improved growth and performance as well as reduced mortality when compared to other methods. In extreme cold weather environments, calves reared in hutches had an overall better performance, ate more starter feed and required fewer medical treatments (McKnight, 1978).

over hutches on pathogen shedding. The second (winter) and third (summer) studies evaluated the effects of reflective insulation applied to calf hutches; during the summer study an additional treatment designed to increase hutch ventilation was incorporated. Holstein heifer calves were placed alternately in treatments immediately after birth and managed as typical for dairy calves in this region. Heifers were fed approximately 2.0 L of pasteurized waste milk twice daily until they would drink from a bucket (1 to 3d), after which they were fed approximately 7.5L pasteurized waste milk twice daily. As per protocol of dairies typical of this region calves were provided approximately 7.5L water upon consumption of milk,

In warm climates, supplemental shade used during the summer months decreased severity of the heat stress experienced by calves when compared to those in hutches alone (Spain and Spears, 1996). Scott et al. (1976) reported that calves exposed to chronic heat loads had lower IgG than those that were housed at thermo-neutrality. Given the aforementioned research, it is hypothesized that reducing temperature variations in periods of extreme heat and cold will reduce the level of stress in calves and thereby increase their resistance to pathogenic organisms. The objectives of this research are to determine if the application of shade to dairy hutches decreases fecal prevalence of E. coli O157:H7 and Salmonella in newborn dairy calves.

and in addition calves were gradually introduced to solid feeds between 10 to 14 d of age. All hutches were commercially available polyethylene hutches (Calf-Tel Pro®, Hampel Corp., Germantown, WI) affixed with a 1 x 2 m outdoor pen made of welded wire panels.

MATERIALS AND METHODS This research was conducted on a single, large commercial Holstein dairy (> 2000 head) located in the Texas Panhandle and managed as typical for dairies in Eastern New Mexico and the Texas Panhandle. During the periods at which the observations took place for the summer and winter studies temperatures averaged approximately 24°C (18.1 - 31.4°C) and 4.9°C (-2.9 - 12.7°C), respectively. At the time of these studies this dairy was a closed herd that reared its own replacement females. Three studies were designed to evaluate the effects of hutch modifications on pathogen shedding. The first study (shade cloth) evaluated the effects of a suspended shade cloth 99

Shade Cloth Study This study utilized 80% shade cloth (Sunblocker Premium, Farmtek., Dyersville IA) to provide partial (n = 4), full (n = 6) or no shade (n = 14). The cloth was suspended 3 m above the hutches so that a portion of the pen in front of the hutches was also shaded; partially shaded hutches were considered as those that received shade in the morning or evening because they were located near the edge of the shade structure. Heifers were assigned to treatment as described above. However, during the first collection calves had only been placed in half of the hutches, resulting in reduced sample sizes for partial (n = 2), full (n = 2) and no shade (n = 9). Fecal samples were collected via rectal palpation or from freshly voided, uncontaminated fecal pats within the hutch area on 20JUL2006 (day 1), 25AUG2006 (day 37), and 23SEP2006 (day 66).

Summer Study As described below, hutch coverings were placed on hutches upon placement of the calf in the hutch, shortly following birth starting June 10, 2007 (day 1),

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and were removed on September 1, 2007 (day 84). An additional 10 hutches were modified by creating a 14 x 18 inch hole, 18 inches off the ground, in the back wall of the hutch (ventilation treatment) to increase air flow through the hutch. Heifers were assigned to treatment as described above, calves were fed using this opening on the inside the hutch, versus normal feeding which occurred outside of the hutch in the wire pen, thus allowing them to remain shaded while eating. One calf in this type of hutch died early in the experimental period and was not replaced resulting in 9 hutches of this type at each collection. Fecal samples were collected as above on days 6, 19, 41, 65 and 84 of the experimental period as described

Edrington and co-workers (2009). Unless noted otherwise, all reagents and antibiotics were obtained from Sigma Chemical Co. (St. Louis, MO).

above.

Data were analyzed using SAS Version 9.2 (SAS Inst. Inc., Cary, NC, USA). Quantitative enumeration was infrequent and not sufficient to detect differences among treatments, therefore data is presented as prevalence (% positive) and not actual counts. The incidence of fecal pathogen shedding was subjected to a Chi-square analysis using the PROC FREQ procedure. Additionally, the PROC MIXED procedure was used to examine the main effects of treatment and day, and treatment x day interaction. Differences in means were considered significant at a 5% level of significance.

Winter Study During the winter and summer studies, treated hutches were covered with a 2.2 x 2.5 m sheet of Tempshield™ reflective insulation (Innovative Insulation Inc., Arlington, TX). Grommets were place on the edges of the “hutch blanket” to facilitate attachment to the hutch in three places on each side of the hutches long sides by elastic cord. Treated hutches (n = 20) alternated with control hutches (n = 19) and were all placed in a single row, contained within multiple rows of calf hutches. Heifers were assigned to treatments as above. Hutch blankets were applied on December 18, 2007 (day 1) and removed February 23, 2008 (day 68). Fecal samples were collected as described above on days 29, 42, 56 and 68 of the experimental period. Fecal samples were collected using sterile palpation sleeves, placed on ice and transported to our laboratory in College Station, Texas for bacterial culture described in the following section.

Antimicrobial susceptibility was determined on isolates using the Sensititre automated antimicrobial susceptibility system and the National Antibiotic Resistance Monitoring System’s (NARMS) testing panels (Trek Diagnostics Systems Inc., Cleveland, OH) for isolates as described previously (Edrington et al., 2009).

Statistical Analysis

RESULTS Shade Cloth Study

All fecal samples were processed the day follow-

When combined across sampling dates, partially shaded calves shed Salmonella more frequently (70%) when compared to no shade and full shade (54 and 43%, respectively), however no statistical differences between treatments were observed (Table 1). Salmonella isolates (n = 29) from the first two collection times were analyzed for antimicrobial susceptibility. Two isolates per positive sample from the first collection period and a single isolate from collec-

ing collection for qualitative analysis of Salmonella (Edrington et al., 2009) and E. coli O157:H7 as described previously (Robinson et al., 2004) and modified (Brichta-Harhay et al., 2007). Enterococcus was qualitatively cultured as previously described by

tion two were subjected to antimicrobial screening. Most isolates (86%) were pan susceptible; resistance to sulphathiazole was observed in two isolates and resistance to tetracycline was observed in a single isolate. One isolate that displayed resistance to

Bacterial Culture and Isolation

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Table 1. Prevalence (number and % positive) of Salmonella in fecal samples collected from dairy calves housed in control hutches or hutches with partial shade or full shade. Fecal samples were enriched for qualitative analysis prior to plating (ENR). Control

Partial Shade

Full Shade

Day

Method

no.

ENR

9/9

100

2/2

100

2/2

100

ENR

8/14

4/4

100

4/6

ENR

3/14

1/4

0/6

0.0

all days

ENR

20/37

7/10

6/14

No significant differences between treatments were observed

chlortetracycline, oxytetracycline, and tetracycline

ment, with a higher (P < 0.05) percentage of positive

belonged to serogroup C2. No differences were observed between treatments with respect to antimicrobial resistance. All fecal samples were E. coli O157:H7 negative throughout the study.

samples in the hutch blanket treatment compared to control and ventilated treatments. There were no treatment x day interactions observed. Five different Salmonella serogroups were identified (C1, C2, E1, K and poly A-I, vi) with C2 accounting for 55% of the isolates. Twenty-one Salmonella isolates were examined for antimicrobial susceptibility (7, 8 and 6 each from the control, blanket and ventilated hutch treatments, respectively) and all were susceptible to all of the antibiotics examined (data not shown). Enterococcus isolates (n = 14) cultured from hutch calves on this farm during the shade study were examined for antimicrobial susceptibility. All 14 isolates were resistant to quinupristin/dalfopristin and four of these isolates were additionally resistant to erythromycin, lincomycin, streptomycin, tetracycline, and tylosin. No treatment differences were observed and all Enterococcus isolates were resistant to vancomycin (data not shown). Based on this data and similar data generated in the Winter Study, Enterococcus isolates were not examined in the summer hutch covering study.

Summer Study Fecal prevalence of E. coli O157:H7 was low throughout the study, with 4 of 205 samples positive following enrichment and IMS (Table 2). Three of those positive samples were cultured on d 84 in the hutch covering treatment; however no significant differences were observed between control and ventilated hutch treatments (P = 0.06). No samples were culture positive for E. coli O157:H7 using quantitative methodology. Similarly, Salmonella was cultured infrequently following direct plating with positive samples detected only on d 84 in the control and ventilated treatments (22% positive in each treatment). Following enrichment however, Salmonella positive fecal samples were detected on all collection days except d 41. The highest incidence was observed on days 19 and 84, although only on d 19 were significant treatment differences observed. Salmonella prevalence was increased (P < 0.05) in the ventilated hutch compared to the control treatment, but was similar for control and hutch blanket treatments (17.2, 31.6 and 66.7% for control, hutch blanket and ventilated treatments, respectively). When averaged across collection days, only E. coli O157:H7 prevalence (as detected by IMS) was affected by treat101

Winter Study Results of the quantitative culture of E. coli O157:H7 and Salmonella over the course of the winter study were largely negative (Table 3). None of the samples contained quantifiable populations of Salmonella (0/136) while only two animals had quantifiable concentrations of E. coli O157:H7 (d 56 and

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Table 2. Prevalence (number and % positive) of E. coli O157:H7 (EC) and Salmonella (Salm) in fecal samples collected during the summer study, by day of collection (when at least on sample was culture positive) and across all collection days, from dairy calves housed in control hutches or hutches modified with a blanket or ventilated to improve calf comfort. Fecal samples were either plated directly to quantify bacterial populations (DIR) or enriched for qualitative analysis prior to plating (ENR). Control

Ventilated hutch

Bacteria

Day

Method

no.

ENR

0/4

1/4

ENR

0/18

3/15

0/9

All days

ENR

0/100

4/73

5.48

0/32

ENR

2/4

nsa

ENR

5/29

17.2b

6/19

31.6b

6/9

66.7c

ENR

1/22

4.55

0/16

0/5

DIR

4/18

22.2

0/15

2/9

22.2

ENR

3/18

16.7

1/15

6.67

1/9

11.1

All days

ENR

11/100

9/73

12.3

7/32

21.9

Salm

Hutch blanket

no samples collected. Treatment means within a row with different superscripts differ (P < 0.05).

68, control treatment). Enrichment of the samples followed by IMS identified the same two positive animals in the control treatment and an additional six positive animals in the hutch blanket treatment (2 on d 42, 4 on d 56). The number of Salmonella positive samples increased slightly with enrichment, although no treatment differences were observed. The majority of serogrouped Salmonella belonged to group K. Antimicrobial susceptibility screening was conducted on five E. coli O157:H7 isolates (data not shown). All isolates were resistant to sulphisoxazole and two of the five were also resistant to streptomycin. Enterococcus isolates from d 29 (n = 32) and 42 (n = 11) collections were also examined (data not shown). Most isolates however were susceptible to all of the antibiotics examined with the exception of quinupristin/dalfopristin, to which all but three were resistant. Isolates (in both treatments) displayed resistance to seven different antibiotics (chloramphenicol, erythromycin, lincomycin, quinupristin/dalfopristin, streptomycin, tetracycline and tylosin) including

three isolates in the control treatment and two in the hutch blanket treatment, accounting for most of the observed resistance. Of the 11 isolates examined 13 d later on d 42, only one isolate in the hutch blanket treatment was multi-resistant (6 antibiotics, exhibiting the same pattern as the previous isolates). As discussed above, the majority of the isolates were susceptible to all antibiotics with the exception of quinupristin/dalfopristin (data not shown). All Enterococcus isolates were susceptible to vancomycin. Due to the limited number of Salmonella positive samples, isolates were not retained for antimicrobial susceptibility screening.

DISCUSSION Results of the current research highlight the sporadic nature of pathogen shedding in naturally-colonized animals. Prevalence of E. coli O157:H7 and Salmonella was relatively low in this study compared to previous research conducted by our laboratory examining pathogen prevalence in dairy cattle during the summer months (Edrington et al., 2004). Even so,

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Table 3. Prevalence (number and % positive) of E. coli O157:H7 (EC) and Salmonella (Salm) in fecal samples collected during the winter study, by day of collection (when at least one sample was culture positive) and across all collection days, from dairy calves housed in control hutches or hutches modified with a reflective blanket to improve calf comfort. Fecal samples were either plated directly to quantify bacterial populations (DIR) or enriched for qualitative analysis prior to plating (ENR). Control

Hutch blanket

Bacteria

Day

Method

no.

ENR

0/17

2/17

11.8

DIR

1/18

5.6

2/15

13.3

ENR

1/18

5.6

4/15

26.7

DIR

1/18

5.6

0/16

ENR

1/18

5.6

0/16

All days

DIR

2/71

2.8

2/65

3.1

All days

ENR

2/71

2.8

6/65

9.2

ENR

1/18

5.6

0/17

ENR

0/17

1/17

5.9

ENR

2/18

11.1

1/15

6.7

All days

ENR

3/71

4.2

2/65

3.1

Salm

some significant treatment differences and trends were noted in fecal shedding of E. coli O157:H7 and Salmonella on various collection days, suggesting hutch coverings increased pathogen prevalence. Various scenarios including environmental changes within the hutch due to hutch coverings may have contributed to this outcome, however identifying a single cause is not possible. It is unclear to the authors what contributed to a greater proportion of Salmonella positive calves to have originated from the partially shaded treatment relative to both no shade and full shade within the shade study. It should be noted that for a study of this type to achieve an α = 0.95 and a ß = 0.20 with 20% prevalence expected in the control versus 10% in the treated groups it would require a sample of 219 animals per treatment group. Given the resources available to the researchers sample sizes of this magnitude were unobtainable. However, due to the low number of sampling units used in this study readers are cautioned not to over interpret these data. Serogrouping of the Salmonella isolates identified the majority as belonging to groups C2 (summer study), K (winter study) and C1 and C2 (shade cloth 103

study). This is not surprising as we have reported seasonal differences in serogroup prevalence in dairy cattle previously (Edrington et al., 2004). Salmonella Newport, a serotype frequently MultidrugRresistant (MDR), belongs to serogroup C2. However, antimicrobial susceptibility testing revealed no MDR isolates; therefore it is likely that these isolates belonged to another common dairy serotype within the C2 group, likely Kentucky. Serotyped isolates from previous dairy research belonging to serogroup K, were frequently identified as Cerro (Edrington et al., 2004). Antimicrobial susceptibility screening of the various isolates yielded few MDR isolates (mostly Enterococcus) and these were resistant to antimicrobials frequently utilized in veterinary medicine and susceptible to antibiotics used in human medicine. In summary, further research is necessary to provide additional evidence to support the use of hutch coverings to moderate temperatures and consequently increase calf comfort. These data indicate that in an effort to increase animal level comfort

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 2 - November 2011

dairy producers may inadvertently increase pathogen shedding. Additional studies should be focused on locating herds that have high enough levels of pathogen shedding and sufficient quantity of subjects to discern measures of effect should they exist.

CONCLUSION Efforts to mitigate extreme temperatures within calf hutches via hutch coverings have been shown to increase the pathogen burden of dairy calves within this study. However, due to the small number of observations further work is warranted to better understand the effects of temperature modification within hutches with various forms of shades and reflective material. Therefore, the authors recommend that each dairy assess their needs and situation individually to best determine how increase calf comfort while ensuring that an increased pathogen burden has not been imposed.

ACKNOWLEDGEMENT The authors wish to thank the Food Animal Concerns Trust for partial funding of this research and Shannon Garey for assistance during sample collection.

REFERENCES Brichta-Harhay D. M., T. M. Arthur, J. M. Bolsilevac, M. N. Guerini, N. Kalchayanand, M. Koohmaraie. 2007. Enumeration if Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods. J. Appl. Microbiol. 103:1657-1668. Edrington, T. S., B. H. Carter, T. H. Friend, G. R. Hagevoort, T. L. Poole, T. R. Callaway, R. C. Anderson, D. J. Nisbet. 2009. Influence of sprinklers, used to alleviate heat stress, on fecal shedding of E. coli

vese, K. M. Bischoff, J. L. McReynolds, R. C. Anderson, D. J. Nisbet. 2004. Variation in the faecal shedding of Salmonella and E. coli O157:H7 in lactating dairy cattle and examination of Salmonella genotypes using pulsed-field gel electrophoresis. Lett. Appl. Microbiol. 38:366-372. Garber, L. P., S. J. Wells, D. D. Hancock, M. P. Doyle, J. Tuttle, J. A. Shere, T. H. Zhao. 1995. Risk factors for shedding Escherichia coli O157:H7 in dairy calves. J. Am. Vet. Med. Assoc. 207:46-49. McKnight, D. R. 1978. Performance of newborn dairy calves in hutch housing. Can. J. Anim. Sci. 58:517520. Poos, M. I. and L. Sordillo. 1982. The effect of type of housing and supplementation on performance of dairy calves from birth to weaning. J. Dairy Sci. 65:121. Robinson, S. E, E. J. Wright, N. J. Williams, C. A. Hart, and N. P. French. 2004. Development and application of a spiral plating method for the enumeration of Escherichia coli O157:H7 in bovine feces. J. Appl. Microbiol. 97:581-589. Roy, J. H. B. 1970. The calf: management and feeding. Butterworths, London, UK. 224 p. Scott, G. H., F. Wiersma, B. E. Menefee, F. R. Radwanski. 1976. Influence of environment on passive immunity in calves. J. Dairy Sci. 59:1306-1311. Spain, J. N. and D. E. Spears. 1996. Effects of supplemental shade on thermoregulatory response of calves to heat challenge in a hutch environment. J. Dairy Sci. 79:639-646. USDA. 2006. Characterization and enhancement of immune responses of calves. Annual Report FY 2006. United States Department of Agriculture, Agricultural Research Services, National Animal Disease Center, Ames, IA. p 1-5.

O157:H7 and Salmonella and antimicrobial susceptibility of Salmonella and Enterococcus in lactating dairy cattle. Lett. Appl. Microbiol. 48:738-743. Edrington, T. S , M. E. Hume, M. L. Looper, C. L. Schultz, A. C. Fitzgerald, T. R. Callaway, K. J. GenoAgric. Food Anal. Bacteriol. â&#x20AC;˘ AFABjournal.com â&#x20AC;˘ Vol. 1, Issue 2 - November 2011

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Production of Cyclosporin A by Static Fermentation Using Tolypocladium inflatum MTCC 557 S. A. Survase1, U. S. Annapure1 and R. S. Singhal1 Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India

ABSTRACT The effect of different nutrients (carbon and nitrogen sources) and fermentation factors (such as inoculum size and volume of production media) was evaluated on the production of Cyclosporin A (CyA) in stationary culture of Tolypocladium inflatum MTCC 557 over a 21-day period at 25ºC. Glycerol was found to be the best carbon source and gave a maximum CyA production of 410 mg/L. Further, response surface methodology was used to optimize the concentrations of medium components which took the CyA production to 452 mg/L. The exogenous supplementation of various amino acids individually and in combination was also studied. The time of addition of the optimized combination of amino acids was also evaluated. A maximum CyA production of 1241 mg/L was obtained when L-valine and L-leucine were added after 4th day of the fermentation. The optimum concentrations of media components were (in g/L) glycerol 98.8, casein peptone 28.5, malt extract 20, peptone 10 and α-amino butyric acid 5.7.

Keywords: cyclosporin A, Tolypocladium inflatum, stationary culture, response surface method, fermentation, static, amino acids, production Agric. Food Anal. Bacteriol. 1: 105-115, 2011

INTRODUCTION Cyclosporin A (CyA), a cyclic undecapeptide, is one of the most commonly prescribed immunosuppressive drugs for the treatment of patients with organ transplantation, autoimmune diseases, including AIDS, owing to its superior T-cell specificity and low levels of myelotoxicity (Kahan, 1984; Schindler, 1985). The organisms reported to produce CyA in-

Correspondence: Shrikant A. Survase, shrikantraje1@rediffmail.com Tel: 91-022-24145616 Fax: +91-022-24145614

105

clude Tolypocladium inflatum (Agathos et al., 1986), Fusarium solani (Sawai et al., 1981), Neocosmospora varinfecta (Nakajima et al., 1988) and Aspergillus terreus (Sallam et al., 2003). CyA is reported to be produced by submerged culture fermentation (Agathos et al., 1986), static fermentation (Balaraman and Mathew, 2006), solid state fermentation (Survase et al., 2009a), and also synthesized enzymatically (Billich and Zocher, 1987). The production of CyA in submerged fermentation has been reported to vary with respect to the production strain, fermentation conditions, and the nutrient composition of the culture medium. The ef-

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fect of media components such as carbon and nitrogen sources (Abdel-fattah et al., 2007; Agathos and Parekh, 1990) and environmental factors such as pH and inoculum density (Issac et al., 1990) on CyA production have been studied. Static culture conditions were used successfully for production of glucansucrase using Leuconostoc dextranicum NRRL B-1146 (Majumdar and Goyal, 2008), cordycepin by Cordyceps militaris CCRC 32219 (Shih et al., 2007) and exo-polysaccharide of Agaricus brasiliensis (Fan et al., 2007). Balaraman and Mathew, (2006) reported higher production of CyA in static fermentation as compared to submerged and solid state fermentation using Tolypocladium

agar were procured from Himedia Ltd, Mumbai, India. Sodium chloride and calcium chloride; amino acids L-valine, L-leucine, glycine, DL-amino butyric acid, DL-methionine and solvents acetonitrile, n-butyl acetate, sodium hydroxide, concentrated hydrochloric acid and sulphuric acid were all purchased from S. D. Fine Chemicals Ltd. Mumbai, India. All the solvents used were of AR grade except acetonitrile, which was of HPLC grade. Standard CyA (authentic sample) was a gift sample through the kind courtesy of RPG Life Sciences Ltd., Mumbai, India.

sp (VCRC F21 NRRL No.18950). They studied medium optimization with respect to composition and reported maximum CyA production of 2.22 g/L medium or 5.85 g/kg biomass. With this background we have examined production of CyA using T. inflatum MTCC 557. Response surface methodology (RSM) is used to evaluate the relative significance of variables in the presence of complex interactions with limited number of experiments. It has been successfully employed for optimization of medium constituents for the production of metabolites such as cholesterol oxidase (Chauhan et al., 2009) and cephamycin C (Bussari et al., 2008). To the best of our knowledge, there is just one single report on optimization of the medium constituents for the production of CyA by static fermentation. In the present study, the effects of various carbon sources, fermentation time, inoculum size and production medium volume were investigated. Thereafter, the concentration of media components was optimized by using response surface methodology. The effect of various amino acids was also studied for the first time in static fermentation.

Strains of T. inflatum MTCC 989, T. inflatum MTCC 557 (indicated as Beauveria nivea in the MTCC catalog), T. inflatum NCIM 1283, were procured from MTCC, Chandigarh and NCIM, Pune, India. T. inflatum NRRL 18950 was a gift sample from the ARS Culture Collection (NRRL), Peoria, Illinois, USA. The cultures were maintained on agar slants containing malt extract 2% and yeast extract 0.4% (MYA), pH 5.4 at 4°C after growing it for 12 days at 24°C.

Microorganisms

Preparation of the seed inoculum and fermentation The organism was subcultured onto a fresh MYA slant and incubated at 25 ± 2°C for 12 days to a fully grown slant. To this slant, 10 ml of sterile saline containing 0.1% Tween 20 was added and well mixed. One milliliter of this saline containing approximately 108 to 109 spores was added to 50 ml of medium composed of malt extract 2%, yeast extract 0.4%, pH 5.4 taken in a 250 ml flask and incubated at 180 rpm for 72 h at 25 ± 2°C. This was used as the seed for static fermentation. Seed inoculum (10 % v/v) was used to inoculate sterile production medium. The fermentation was carried out at 25 ± 2 ˚C, pH of 5.7 ± 0.2 for 21 days.

MATERIALS AND METHODS Screening of microorganisms and fermentation media

Materials Glucose, maltose, sucrose, glycerol, yeast extract, casein peptone, bactopeptone, malt extract and

Four strains as indicated in the previous section were screened for the maximum production of CyA

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using medium reported by Balaraman and Mathew (2006), and the strain which gave maximum production was used for further optimization studies.

Table 1. The central composite rotatable design (CCRD) matrix of independent variables in coded as well as actual form with their corresponding response

Three different production media were tested for maximum production of CyA using T. inflatum MTCC 557. Various fermentation media screened were as follows; medium I containing (g/L) glucose 80, casein peptone 30, malt extract 20, bacto-peptone 10 and DL-amino butyric acid 5 (Balaraman and Mathew, 2006); medium II containing (g/L) glucose 58.46, casein peptone 8.66, KH2PO4 4.48 and KCl 3.23 (Survase et al., 2009b); and medium III developed in our laboratory containing (gL) glucose 50, ammonium sulphate

Run

15, K2HPO4 1.25. The production medium which supported maximum production of CyA was used as the basal medium for optimization of CyA production.

Effect of fermentation parameters on production of CyA using T. inflatum MTCC 557 T. inflatum MTCC 557 was tested for the production of CyA at different time intervals between 1 to 21 days using medium I as the production medium. Fermentation medium was inoculated with different inoculum size (5- to 20 % v/v), and its effect was observed on production of CyA. Glucose in the basal medium was replaced with various carbon sources such as sucrose, soluble starch, maltodextrin and glycerol as carbon source for the production of CyA using T. inflatum MTCC 557. All carbon sources were evaluated at 8 % w/v in the culture medium. Three nitrogen sources viz., casein peptone, malt extract and bacto-peptone at different concentration combinations (1:2:3; 1:3:2;2:3:1;2:1:3;3:2:1 and 3:1:2) were evaluated for maximum production of CyA. The effect of volume of production medium was investigated by putting different volumes (25 to 150 ml) in 250 ml Erlenmeyer flasks.

Response surface methodology A central composite rotatable design (CCRD) for three independent variables was used to obtain the combination of values that optimizes the response 107

Casein Amino Glycerol Peptone butyric CyAa (mg/l) (%) (%) acid (%)

6 (-1)b

2 (-1) b

0.25 (-1) b

201± 14

10 (1)

2 (-1)

0.25 (-1)

221 ± 24

6 (-1)

4 (1)

0.25 (-1)

186 ± 15

10 (1)

4 (1)

0.25 (-1)

315 ± 17

6 (-1)

2 (-1)

0.75 (1)

187 ± 22

10 (1)

2 (-1)

0.75 (1)

274 ± 16

6 (-1)

4 (1)

0.75 (1)

145 ± 24

10 (1)

4 (1)

0.75 (1)

405 ± 25

4.63 (-1.68)

3 (0)

0.5 (0)

213 ± 12

11.36 (1.68)

3 (0)

0.5 (0)

431 ± 14

8 (0)

1.31 (-1.68)

0.5 (0)

155 ± 21

8 (0)

4.68 (1.68)

0.5 (0)

247 ± 12

8 (0)

3 (0)

0.07 (-1.68)

213 ± 33

8 (0)

3 (0)

0.92 (1.68)

274 ± 12

8 (0)

3 (0)

0.5 (0)

410 ± 27

8 (0)

3 (0)

0.5 (0)

412 ± 20

8 (0)

3 (0)

0.5 (0)

408 ± 25

8 (0)

3 (0)

0.5 (0)

411 ± 24

8 (0)

3 (0)

0.5 (0)

413 ± 26

8 (0)

3 (0)

0.5 (0)

411 ± 22

values are mean ± SD of three determinations Values in the parenthesis are coded values of the independent variables CyA is cyclosporin A a

within the region of three dimensional observation spaces. The experiments were designed using the software, Design Expert Version 6.0.10 trial version (State Ease, Minneapolis, MN). The medium components (independent variables) selected for the optimization were glycerol, casein peptone, and amino butyric acid. The experimental design showing the coded as well as actual values of

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independent variables is shown in Table 1. Regression analysis was performed on the data obtained from the designed experiments. The second order polynomial coefficients were calculated to estimate the responses of the dependent variable. Response surface plots were also obtained using Design Expert Version 6.0.10.

umn was maintained at 70°C and the HPLC profile was monitored at 210 nm. Estimation of biomass A 10 ml sample of homogenized broth was centrifuged at 10,000 rpm for 20 min, washed twice with distilled water and taken on a preweighed Whatman filter paper. This was dried to a constant weight at 80°C.

Effect of amino acids RESULTS AND DISCUSSION The effect of different amino acid members of CyA molecule on drug production was evaluated by replacing α-amino butyric acid in the fermentation media with different amino acids such as L-valine, L-leucine, DL-valine, L-methionine and glycine at 6 g/L. They were screened individually as well as in combination with others. The time of addition of combination of amino acids (0 to 144 h) was also optimized to further increase the yield.

Screening of the available strains showed T. inflatum MTCC 557 to produce maximum CyA (256 mg/L) under static conditions followed by T. inflatum NRRL 18950

CyA extraction and estimation The CyA extraction from the culture broth was carried out according to the method of Agathos et al. (1986). The fermentation broth was homogenized in a blender. Ten milliliters of homogenized culture broth was extracted with equal volume of n-butyl acetate. Before extracting the sample, a concentrated solution of NaOH was added to reach the concentration of 1N and heated at 60°C for 30 min. The mixed sample was kept on rotary shaker (180 rpm) for 24h. After centrifuging, the extract was filtered using Whatman filter paper (No.1) and then through Pall 0.2 μm membrane filter (Ultipor® N66® Nylon 6, 6 membranes) to give clear extract. One milliliter of the extract was evaporated under vacuum to dryness. The dried extract was dissolved in equal volume (1 ml) of HPLC grade acetonitrile. Twenty microliters of sample was analyzed for CyA content using HPLC (Jasko system) fitted with

which gave 196 mg/L of CyA after 21 days. T. inflatum MTCC 989 and T. inflatum NCIM 1283 gave lower titers of CyA. Balaraman and Mathew (2006) reported use of Tolypocladium sp. (VCRC F21 NRRL No.18950) for production of CyA under static conditions. The reported maximum CyA production was 2.22 g/L medium or 5.85 g/kg biomass. The difference in the yield could be explained as a strain difference. They have used a modified strain of T. inflatum NRRL 18950 whereas we used a wild type strain for our studies. Among the three media screened by using T. inflatum MTCC 557 for maximum production of CyA, medium reported by Balaraman and Mathew (2006) which contained glucose as carbon source and combination of three different nitrogen sources was found to be promising. Medium I produced 254 mg/L of CyA followed by medium II which produced 145 mg/L. Biomass production was also higher 20.4 g/L (measured as dry cell weight (DCW)) in medium I as compared to other two media. Medium III produced the lowest amount (97 mg/L) of CyA and biomass 10 g/L. Medium II reported by Survase et al. (2009b) produced maximum CyA yield of 134.5 mg/L from shake flask cultures. Three flasks after every two days from day 1 to 23 days were evaluated to study the effect of fermenta-

a reverse phase column Waters Sperisorb® ODS (C18 octadecyl silane, 250 X 4.6 mm ID) by the method described by Survase et al. (2009a). The mobile phase consisted of 70:30 ratio of acetonitrile and water with a flow rate of 1 ml/min. The temperature of the col-

tion time on CyA production. It was observed that production of CyA started after 3rd day of fermentation which reached maximum of 254 mg/L after 21 days. The CyA production was found to be decreased to 238 mg/L on 23rd day which could be due to the

Analytical determinations

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Figure 1. Effect of fermentation time on production of CyA using T. inflatum MTCC 557 (DCW is dry cell weight and CyA is cyclosporin A)

autolysis of fungal cells (Fig. 1). The biomass was also found to be decreased after the 21st day. It was observed that 15 % v/v of 72 h old seed culture resulted in maximum CyA production of 312 mg/L as compared to 255 mg/l using 10 % v/v inoculum (data not shown). An increase in the seed culture age and inoculum size did not increase the yields significantly. Isaac et al. (1990) reported that a higher spore density gave higher production of CyA in submerged fermentation using T. inflatum UAMH 2472. In our previous study (Survase et al., 2009b), 10 % v/v inoculum was found to be optimum under shaking conditions (180 rpm). It was observed that variation in concentrations of nitrogen sources viz. casein peptone, malt extract and bacto-peptone in the basal medium changed the production of CyA (between 123 mg/L to 252 mg/L) where DCW values ranging from 16.2 to 20.6 g/L were observed (Table 2). The optimum concentrations included (in g/L) casein peptone 30, malt extract 20 and bacto-peptone 10. The effect of different carbon sources on production of CyA using T. inflatum MTCC 557 is shown in Fig. 2. It was observed that, glycerol supported 109

Table 2. Effect of various concentrations of nitrogen sources on CyA production in the basal medium where, glucose was used as a carbon source at 80 g/l Nitrogen Source (%)

CyAa DCWa (mg/l) (g/l)

Casein Peptone

Malt extract

Bactopeptone

197.4 ± 8.6

17.9 ± 0.58

225.2 ± 7.5

19.7 ± 0.45

252.5 ± 7.4

20.6 ± 0.64

146.2 ± 8.2

16.2 ± 0.52

188.2 ± 4.5

17.5 ± 0.55

123.4 ± 6.8

17.4 ± 0.41

values are mean ± SD of three determinations DCW is dry cell weight and CyA is cyclosporin A a

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Figure 2. Effect of carbon source on CyA production using T. inflatum MTCC 557. All carbon sources were added at 8 % w/v (DCW is dry cell weight and CyA is cyclosporin A)

maximum production of 410 mg/L with biomass production of 16.5 g/L DCW. This was followed by sucrose which supported maximum production of 287 mg/L. Glucose produced biomass of 19.65 g/L and CyA production was yielded 254 mg/L. Survase et al. (2009b) reported glycerol as a carbon source in agitated culture system supported biomass growth, but with a lower CyA production. Survase et al. (2009a) also reported use of glycerol as the best carbon source for supplementation in solid state fermentation system. Abdel-fattah et al. (2007) used three carbon sources as glucose, sucrose and starch in combination yielded maximum CyA production using T. inflatum DSMZ 915 in agitated culture system. Agathos et al. (1986) used sorbose as carbon source for production of CyA using T. inflatum ATCC 34921.

flask produced maximum amount of CyA (249 mg/L) and biomass (19.25 g/L). With increasing production medium volume, there was a decrease in CyA production as well as biomass production.

The volume of production medium was varied from 25 ml to 150 ml in 250 ml Erlenmeyer flasks and its effect was observed on production of CyA and biomass (data not shown). It was observed that 50 ml of the production medium in 250 mL

cated the model to be significant (Table 3). The Model F-value of 76.9 and Model P-value (Prob > F) of < 0.0001 implies the model to be significant. The P values were used as a tool to check the significance of each of the coefficients, which, in turn are

Response Surface Methodology The combined effect of three independent variables A: glycerol (g/L); B: casein peptone (g/L) and C: α-aminobutyric acid (g/L) on production of CyA was examined using CCRD with 20 experimental runs. The experimental values of yields of CyA are given in Table 1. The quadratic model was suggested for the given set of experimental results. The results were analyzed by using ANOVA i.e. analysis of variance suitable for the experimental design used. The ANOVA of the quadratic model indi-

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Table 3. Analysis of variance (ANOVA) for the experimental resultsv of the central-composite design (Quadratic Model) Source

Coefficient Sum of F estimate Squares Value 76.9

Prob >F

Model

267.09

96322.82

< 0.0001

63.16

54487.8

23.63

7626.31

54.8

< 0.0001

13.96

2659.79

19.11

0.0014

16.8

4069.69

29.24

0.0003

-25.98

9723.54

69.87

< 0.0001

-10.95

1727.73

12.41

0.0055

35.25

9940.5

71.43

< 0.0001

24.75

4900.5

35.21

0.0001

1.25

2.5

0.09

0.7705

391.51 < 0.0001

necessary to understand the pattern of the mutual interactions between the test variables. The smaller the magnitude of the P, the more significant is the corresponding coefficient. Values of P less than 0.050 indicate the model terms to be significant. The coefficient estimates and the corresponding P values suggested that, the variables A, B, C, A2, B2 and C2, are significant model terms whereas the interactions AB and AC were significant. The second order response model found after analysis for the regression was: CyA (mg/L) = 535.42 - 113.26 (glycerol) + 35.98 (casein peptone) - 179.98 (amino butyric acid) + 4.20 (glycerol2) - 25.97 (casein peptone2) -175.18 (amino butyric acid2) + 17.62 (glycerol x casein peptone) + 49.50 (glycerol x amino butyric acid) + 5.0 (casein peptone x amino butyric acid) (1) Eq. (1) represents the mathematical model relating the production of CyA with the independent process variables, and the second order polynomial coefficient for each term of the equation determined through multiple regression analysis using the Design Expert 6.0.10. The fit of the model was also expressed by the coefficient of regression (R2), which was found to be 111

Figure 3. 3D-surface plot for cyclosporin A production; A Effect of glycerol and casein peptone when other variables are held at zero level; B Effect of glycerol and amino butyric acid when other variables are held at zero level

0.99, indicating that 99 % of the confidence level of the model to predict the response (CyA yield). The “Pred R-Squared” of 0.98 is in reasonable agreement with the “Adj R-Squared” of 0.99. “Adeq Precision” measures the signal to noise ratio. A ratio greater than 4 is desirable. Here, the ratio of 32 indicates an adequate signal. Accordingly, three-dimensional graphs (Fig. 3) were

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Figure 4. Effect of different amino acids on production of CyA using T. inflatum MTCC 557 (DCW is dry cell weight and CyA is cyclosporin A)

generated for the pair-wise combination of the three variables, while keeping the other two at their center point levels. The optimum value of the combination of the three media constitutes was determined using the special features of the RSM tool such as “contour plot generation” and “point prediction” for the maximum production of CyA. The optimal combination of medium components obtained from the study included (in g/L) glycerol 98.8, casein peptone 28.5, malt extract 20, peptone 10 and α-amino butyric acid 5.7. The optimal composition was verified experimentally and compared with the data calculated from the model. The experimentally obtained CyA yield was 452.32 mg/L, whereas the predicted value from the polynomial model was 455.05 mg/L, thereby confirming the high accuracy of the model under the investigated conditions.

Effect of amino acids CyA consists of 11 amino acids in its structure and its production is affected by the addition of exogenous amino acids which are members of the CyA ring (Bal-

akrishnan and Pandey, 1996; Lee and Agathos, 1989; Survase et al., 2009b). Hoppert et al. (2001) reported that the supplemented amino acids modify the endogenous amino acid pool of the fungus and directed the biosynthesis of CyA as precursors. The effect of exogenous amino acid was studied in both submerged as well as solid state fermentation (Balakrishnan and Pandey, 1996; Lee and Agathos, 1989; Survase et al., 2009b), but the effect of amino acids in submerged fermentation under static conditions has not yet been reported. In the present study, α-amino butyric acid from the basal medium was replaced with constituent amino acids (Fig. 4) and their effect on CyA production and biomass production was observed. Of all the amino acids tested, L-valine produced the maximum CyA of 595 mg/L followed by L-leucine 556 mg/L. DL-valine, on the other hand, did not increase the product titer as that of L-valine. Addition of L-methionine in the production medium reduced the CyA production. Zocher et al. (1984) reported that methionine could not take part in the biosynthesis, as methylated amino acids in-

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Figure 5. Effect of combination of amino acids on production of CyA using T. inflatum MTCC 557 (DCW is dry cell weight and CyA is cyclosporin A)

terfere with the biosynthesis of CyA in vivo. When added together, L-valine and L-leucine increased drug production dramatically (897 mg/L) (Fig. 5). These two amino acids seem to act independently and their mode of action is different. This effect was not observed with other amino acid combinations. Supplementation of L-methionine with L-valine resulted in loss of the stimulatory effect of L-valine. Similar results were encountered by Balakrishnan and Pandey (1996), Lee and Agathos (1989) and Nisha et al. (2008) in CyA biosynthesis. The optimal amount and time of addition of L-valine was also investigated. It was observed that CyA production increased with an increase in concentration of L-valine up to 9 g/L, Maximum CyA production of 884 mg/L was observed at initial L-valine concentration of 9 g/L (data not shown). Balakrishnan and Pandey (1996) and Lee and Agathos (1989) reported that CyA production reached the saturation level at L-valine 4 g/L whereas, Survase et al. (2009b) reported the saturation level to be 6 g/L after which there was no further increase in CyA production. The optimum time for L-va113

line (9 g/L) and L-leucine (6 g/L) addition for maximum product titer was found to be 4 days (Data not shown). When added after 4 days, CyA production of 1241 mg/L was obtained. Balakrishnan and Pandey (1996) and Lee and Agathos (1989) reported that addition of L-valine in the fermentation medium after 18 h and 20 h respectively, gave the maximum production of CyA under shake flask conditions.

CONCLUSION Static fermentation could successfully be used for production of CyA. The exogenous supply of amino acids along with time of addition played an important role in maximizing the production of CyA.

ACKNOWLEDGEMENT We are thankful to Department of Biotechnology, Government of India for funding this project. The gift of CyA standard from RPG Life Sciences Ltd, Mumbai is gratefully acknowledged.

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Survase, S. A., N. S. Shaligram, R. C. Pansuriya, U. S. Annapure, and R. S. Singhal. 2009a. A novel medium for the enhanced production of cyclosporin A by Tolypocladium inflatum MTCC 557 using solid state fermentation. J. Microbiol. Biotechnol. 19:462-467. Survase, S. A., U. S. Annapure, and R. S. Singhal. 2009b. Statistical optimization of cyclosporin: A production on a semi-synthetic medium using Tolypocladium inflatum MTCC 557, Global J. Biotechnol. Biochem. 4:184-192 Zocher, R., N. Madry, H. Peeters, and H. Kleinkauf. 1984. Biosynthesis of cyclosporin A. Phytochemistry 23:549-551.

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BRIEF COMMUNICATION Comparative Studies on the Survival of Verocytotoxigenic Escherichia coli and Salmonella in Different Farm Environments C. J. O’Neill1, 2, D. J. Bolton1 and S. Fanning2

Teagasc-Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland Centre for Food Safety, School of Public Health, Physiotherapy and Population Science, University College Dublin, Belfield, Dublin 4, Ireland 1

ABSTRACT The aim of this study was to investigate the survival characteristics of verocytotoxigenic Escherichia coli (VTEC) and Salmonella in soil, slurry, farm water and bovine feces. Samples of each of the aforementioned media were inoculated with separate cocktails of VTEC and Salmonella and stored at 4 and 14 ºC, representing average Winter and Summer temperatures respectively. Samples were withdrawn periodically and surviving cells enumerated by directly plating onto selective media. Decimal reduction times (D-values) were calculated from the inverse of the slope obtained by linear regression of a plot of time versus the log of surviving cells. In the latter stages of the experiment, presence or absence was determined by enrichment and selective plating. VTEC and Salmonella D-values ranged from 3.59 to 23.84 days. Temperature significantly affected VTEC survival in water (P<0.05) and Salmonella survival in bovine feces (P<0.01) but not in any of the other farm media tested. In general there was no significant difference (P>0.001) between VTEC and Salmonella survival in a given medium under similar storage temperatures. However, Salmonella D-values were significantly higher in slurry (4°C) and bovine feces (4°C and 14°C). This study provides critical comparative data on VTEC and Salmonella death rates in a range of environments commonly encountered on farms to support the development of quantitative microbial risk assessment (QMRA) and provide the scientific basis for an effective good agricultural practice (GAP) food safety program.

Keywords: Salmonella Typhimurium, Salmonella Dublin, Verocytotoxigenic E. coli, persistence, slurry, soil, water, feces Agric. Food Anal. Bacteriol. 1: 116-122, 2011

Correspondence: D. J. Bolton, declan.bolton@teagasc.ie Tel: +353 (1) 805 9539 Fax: +353 (1) 805 9550

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INTRODUCTION Cattle serve as a source of both Salmonella and verocytotoxigenic E. coli especially E. coli O157:H7, which are shed in the feces without any clinical symptoms (Losinger et al. 1995; Alice, 1997; Rasmussen et al. 1993). Few studies have addressed the survival of E. coli O157:H7 in soil, feces, slurry and water and none has integrated survival in the different media into the same study nor have provided comparative data for Salmonella spp. Furthermore, data on Salmonella survival rates in the farming environment is absent. Under current EC regulations all food processors have a legal responsibility to provide safe food. Hazard analysis and critical control point (HACCP) is generally regarded to be the most effective approach to food safety, but is not a legal requirement during primary production in Europe. Effective food safety is therefore dependent on good agricultural practice (GAP) (Horchner et al., 2006). Risk assessment, which provides a quantifiable metric on the effectiveness or otherwise of specific farming activities, is a prerequisite to an effective science based GAP covering such issues as farm waste management and biosecurity. However, there is limited information available for on-farm risk assessments (McGee et al., 2002; Semenov et al., 2008; Williams et al., 2008,) and insufficient data to fully characterize verocytotoxigenic Escherichia coli (VTEC) and Salmonella enterica survival in the farming environment. Specific data on the persistence of these two pathogens in slurry, water, feces and in soil is therefore required. Mismanagement of animal waste, in particular, will lead to direct or indirect infection of humans (Vernozy-Rozand et al., 2002; Guan and Holley, 2003). Survival data for VTEC and Salmonella in the main environments encountered on the farm will aid in the development of proper science-based management practices which will in turn decrease farm to fork transmission. The objective of this study was therefore to provide comparative data on VTEC and Salmonella survival in slurry, soil, water and bovine feces.

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MATERIALS AND METHODS Organisms A VTEC cocktail and a Salmonella cocktail composed of isolates of farm origin were chosen for this study. The former included bovine and ovine E. coli O157:H7 strains and an untypable canine strain. The canine strain initially serotyped as O157:H7 (hence its addition in this study) but was later discovered to be untypable. The Salmonella organisms included bovine Salmonella ser. Dublin and Salmonella Typhimurium DT104 strains, along with a Salmonella Typhimurium DT193 isolated from farm trough water. All were obtained from the culture collection at Teagasc Food Research Centre (Ashtown), Dublin 15, Ireland.

Collection of samples Composite samples of farm water, slurry, cattle feces and soil were collected from a local farm in County Meath, Ireland. Approximately 10 kg each of slurry, feces and soil, in addition to 10 l of farmyard water were collected and transferred to sterile holding containers. Samples were transported in a coolbox at approximately 2°C to the laboratory.

Preparation of inoculum A cryogenic bead of each isolate of Salmonella and VTEC were aseptically transferred to 30 ml Brain Heart Infusion broth (BHI, Oxoid, Basingstoke, UK) and incubated for 18 h at 37ºC. One ml was then transferred to a 30 ml fresh BHI and incubated at 37ºC for 18 h to obtain a stationary phase culture. The cultures were then centrifuged at 3000 g (Eppendorf, Davidson and Hardy, Ireland). Cells were washed three times in sterile Maximum Recovery Diluent (MRD, Oxoid). The resulting pellets from the three VTEC isolates were then mixed in 100 ml MRD to form a VTEC cocktail. The same methods were used to prepare the Salmonella cocktail. A serial dilution was performed on both cocktails, followed by plating onto Plate Count Agar (Oxoid) to determine the initial inoculum.

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Inoculation of soil, fecal and slurry samples with VTEC strains Soil, fecal and slurry samples (400 g) were inoculated with 1 ml of approximately log10 8.0 cfu ml-1 of the VTEC cocktail to yield a final concentration of approximately log10 6.0 colony forming units (cfu) g-1. Samples were mixed and stored in 500 g sterile plastic containers, in triplicate, at both 4ºC and 14ºC representing mean Winter and Summer temperatures, respectively. Uninoculated slurry, fecal and soil samples were used as controls. Sampling took place on days 0, 3, 9, 13, 21, 34, 42, 64, 76, 88 and 102. Surviving cells were enumerated as described below.

Inoculation of Water Samples with VTEC strains Water samples (400 ml) were inoculated with 1 ml of the VTEC cocktail to yield a final concentration of approximately log10 6.0 cfu ml-1. Storage and sampling was as previously described.

Inoculation of soil, slurry and fecal samples with Salmonella species Soil, slurry and fecal samples (400 g) were inoculated with log10 8.0 cfu ml-1 of the Salmonella cocktail to yield a final concentration of approximately log10 6.0 cfu g-1. Storage was as per the VTEC above. Sampling took on days 0, 3, 7, 12, 19, 32, 40, 60, 69, 81 and 102.

Inoculation of water samples with Salmonella species Water samples (400 ml) were inoculated with the Salmonella cocktail to yield a final concentration of approximately log10 6.0 cfu ml-1. Uninoculated water samples were used as controls. Samples were stored in 500 ml sterile Duran bottles. Storage and sampling was as previously described.

Enumeration of VTEC

first mixed using a sterile spatula. Ten g/ ml samples were then obtained to which 90 ml MRD was added before stomaching for 90 seconds. Following serial dilution, samples were plated directly onto CT-SMAC (Oxoid) in 100 μl volumes in duplicate. Plates were then incubated at 37ºC for 24 h. Enrichment cultures were used to detect the presence of low numbers of bacteria in the latter stages of the experiment. Ten g/ ml of samples were enriched 1:10 in buffered peptone water (BPW) (Oxoid) and incubated at 37ºC for 24 h. Samples were then plated onto CT-SMAC in duplicate and incubated as before. The enrichment procedure was carried out until the organism was not detected for two consecutive sampling days.

Enumeration of Salmonella species Samples were diluted and stomached as previously described. Following serial dilution, samples were plated in 100 μl volumes onto XLD Agar (Oxoid) in duplicate. Plates were then incubated at 37ºC for 24 h. Enrichment cultures were used to detect the presence of low numbers of bacteria in the latter stages of the experiment. Ten g/ ml of samples were enriched 1:10 in BPW and incubated at 37ºC for 24 h. Samples were then plated onto XLD in duplicate and incubated as before. The enrichment procedure was carried out until the organism was not detected for two consecutive sampling days.

Statistical analysis of the results Each experiment was performed in triplicate. The rate of decline over time was obtained by plotting surviving cells against time. The line of best fit for each set of points was found using linear regression analysis (Genstat 5, Statistics Department, Rothamsted Experimental Station, Hertfordshire, UK). The decimal reduction values reported were calculated using the average slope (D = -1/slope). The decimal reduction values were then statistically analyzed using the t-test (Genstat 5, Statistics Department, Rothamsted Experimental Station, Hertfordshire, UK). Differences are reported at the 5%. 1% and 0.1% levels.

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Storage temperature (°C)

Medium

D-value1(days)

S.E.

4°C2 versus 14°C

Storage temperature (°C)

Medium

D-value1(days)

S.E.

4°C versus 14°C

VTEC3 versus Salmonella

VTEC

Soil

8.79 a

0.0343

Salmonella

Soil

6.78ab

0.0236

VTEC

Feces

7.56 a

0.0169

Salmonella

Feces

15.32 c

0.0155

P<0.001 P<0.05

VTEC

Slurry

3.90 a

0.070

Salmonella

Slurry

11.97bc 0.0215

VTEC

Water

8.63 a

0.0383

P<0.05

Salmonella

Water 4.89 a

VTEC

Soil

8.16 b

0.0132

Salmonella

14 Soil

VTEC

Feces

9.79 b

0.0288

Salmonella

VTEC

Slurry

6.50 ab 0.0311

VTEC

Water

3.59 a

P<0.05

0.0564

Bacteria

Table 1. VTEC and Salmonella D-values in soil, feces, slurry and water

P<0.05

0.0189

9.43 b

0.0079

14 Feces

23.84 c

0.0090

P<0.001 P<0.05

Salmonella

14 Slurry

10.51 b 0.0094

Salmonella

14 Water 5.25 a

0.0302

D-values with the same superscript letter are not significantly different (P>0.001). Comparisons were made within a given storage condition (4°C or 14°C) 1

NS=non significant; for significant differences the degree of significance is indicated. Comparisons were between storage conditions (4°C and 14°C) 2

NS=non significant; for significant differences the degree of significance is indicated. Comparisons were made within a given storage condition (4°C or 14°C) 3

S.E= Standard error

RESULTS Neither Salmonella nor VTEC were detected in the uninoculated control samples at any stage throughout the experiment. At 4°C there was no sig-

faster to that observed in soil (D14-value: 8.16 days) and feces (D14-value: 9.79 days). A comparison of the effect of storage temperature found that the differences observed between the D-values obtained at 4 and 14°C were only significant (P<0.05)

nificant difference (P> 0.001) in the VTEC D-values which ranged from 3.9 days (slurry) to 8.79 days (soil) (Table 1). At 14°C the rate of decline in water (D14-value: 3.59 days) was similar to that in slurry (D14-value: 6.5 days) but significantly (P<0.001)

in water. At 4°C there was no significant difference (P> 0.001) in the Salmonella D-values in water (4.89 days) and soil (6.78 days) but the rate of decline in water was significantly (P<0.001) faster than in feces (D4-

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value: 15.32 days) or slurry (D4-value: 11.97 days), which were statistically similar (Table 1). At 14°C the D-values in soil (9.43 days) and slurry (10.51 days) were statistically the same but significantly (P<0.001) slower than in water (D14-value 5.25 days) and significantly faster (P<0.001) than in feces (D14value: 23.84 days) A comparison of the effect of storage temperature found that the differences observed between the D-values obtained at 4 and 14°C were only significant (P<0.001) in feces. In general, the D-values obtained for VTEC and Salmonella were similar with significant differences only observed between the two bacterial cocktails

to end up in the slurry tank. The D4-value and D14value for VTEC in slurry of 3.9 days and 6.5 days were considerably lower than the 38.76 days reported by Himathongkham et al. (1999) for E. coli O157:H7 in slurry stored at 4°C and the 15.27 days reported by McGee et al. (2001) for the same organism in the same medium at 10 °C. This may be due to differences in slurry composition, in addition to bacterial strain variation. At 14°C, survival of VTEC in water was significantly reduced. This was probably due to increased microbiological competition at the higher temperature (Avery et al,. 2008). The D4 and D14-values of 8.63 and 3.59 days demonstrates a longevity that cor-

in feces at 4°C (P<0.05), slurry at 4°C (P<0.05) and feces at 14°C (P<0.05). In each case the Salmonella demonstrated better survival.

roborates the findings of other similar studies on the survival of these organisms in aquatic environments (Wang and Doyle, 1998; McGee et al., 2002). Farm water contaminated with this organism would remain a potential reservoir for VTEC, facilitating dissemination and livestock re-infection (Bolton et al., 1999; McGee et al., 2002). The Salmonella D4 and D14-values in soil, slurry, feces and water ranged from 4.89 to 23.84 days. In general, the survival rates for the VTEC and Salmonella cocktails were statistically similar with the exception of feces and slurry at 4 °C and feces at 14 °C, where the Salmonella survival rates were significantly higher. These mixed findings are comparable to those of Himathongkham et al., (1999) who reported similar D-values for E. coli O157:H7 and Salmonella Typhimurium, with the predominant organism varying depending on the temperature and location within the manure pile. Furthermore, in a study of the survival of enteric bacteria in inoculated feces spread on grass pasture land, Hutchinson et al,. (2005) also reported that there was no significant difference between the reduction times for the different bacterial pathogens including E. coli O157:H7 and Salmonella spp.

DISCUSSION The D-values for VTEC in feces at 4 and 14°C were 7.56 and 9.79 days, respectively. The former compares with the D4-value of 9.04 days reported by Himathongkham et al. (1999) for E. coli O157:H7 in the upper layers of manure piles. It has been reported that colonised cattle may shed up to 108 VTEC (Besser et al., 2001; Fukushima and Seki, 2004) cells per gram of feces, suggesting prolonged persistence of these bacteria in feces for up to 60 days (Winter) and 78 days (Summer) based on this study. Feces deposited onto grassland is washed by rainfall into the soil (Bolton et al., 1999). During the warmer months, when animals are grazing outdoors, cross-contamination of soil is therefore inevitable. The VTEC D-values in soil at 4°C (8.79 days) and 14°C (8.16 days) are similar to those previously reported (Bolton et al., 1999, Besser et al., 2001; Islam et al,. 2004; Fremaux et al., 2008) and provide further evidence that once contaminated, soil will remain a source of these pathogenic organisms for extended periods, contaminating plants (Islam et al., 2004; Islam et al., 2005), livestock (McGee et al., 2001) and posing a risk for recreational use (Moore et al., 1993; Chapman et al., 1997; Jackson et al., 1998). During the Winter months, VTEC are more likely

CONCLUSIONS In conclusion, the D-values provided in this study may be used in risk assessments to estimate survival in soil, feces, slurry and water under different envi-

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ronmental conditions and to predict the duration of slurry storage prior to grazing land application or duration of the soil resting period prior to crop sowing. If the target is a minimal 105-fold reduction of VTEC or Salmonella spp., then slurry should be stored at 60 days at 4°C or 53 days at 14°C before spreading. Fields that may be contaminated with these organisms, either as a result of grazing or fecal waste spreading should be left for a minimum of 44 days during Winter or 47 days during Summer before crops are grown.

ACKNOWLEDGEMENT This research was funded by the US-Ireland Cooperative Program in Agricultural Science and Technology administered by the Department of Agriculture, Fisheries and Food, Ireland.

Fremaux, B., C. Prigent-Combaret, and C. VernozyRozand. 2008. Long-term survival of Shiga toxinproducing Escherichia coli in cattle effluents and environment: An updated review. Vet. Microbiol. 132:1-18. Guan, T. Y. and R. A. Holley. 2003. Pathogen survival in swine manure environments and transmission of human enteric illness- A review. J. Environ. Qual. 32:383-392. Himathongkham, S., S. Bahari, H. Riemann, and D. Cliver. 1999. Survival of Escherichia coli O157:H7 and Salmonella Typhimurium in cow manure and cow manure slurry. FEMS Microbial. Lett. 178:251257.

Alice, N. P. 1997. Public and microbes: public and animal health problem. J. Dairy Sci. 80:2673-2681. Avery, L. M., A. P. Williams, K. Killham, and D. L. Jones. 2008. Survival of Escherichia coli O157:H7 in waters from lakes, rivers, puddles and animaldrinking troughs. Sci. Total Environ. 389:378-385. Besser, T. E., B. L. Richards, D. H. Rice, and D. D. Hancock. 2001. Escherichia coli O157:H7 infection of calves: infectious dose and direct contact transmission. Epidemiol. Infect. 127: 555-560. Bolton D. J., C. M. Byrne, J. J. Sheridan, D. A. McDowell and I. S. Blair. 1999. The survival characteristics of a non-toxigenic strain of Escherichia coli O157:H7. J. Appl. Microbiol. 86:407-11. Chapman, P. A., C. A. Siddons, J. Manning, and C. Cheetham. 1997. An outbreak of infection due to verocytotoxin-producing Escherichia coli O157 in four families: the influence of laboratory methods on the outcome of the investigation. Epidemiol.

Horchner, P. M., D. Brett, B. Gormley, I. Jenson, and A. M. Pointon. 2006. HACCP- based approach to the derivation of an on-farm food safety program for the Australian red meat industry. Food Control 17:497-510. Hutchison, M. L., L. D. Walters, T. Moore, D. J. Thomas, and S. M. Avery. 2005. Fate of pathogens present in livestock wastes spread onto farm plots. Appl. Environ. Microbiol. 71:691-696. Islam, M., M. P. Doyle, S. C. Phatak, P. Millner, and X. Jiang. 2004. Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water. J. Food Prot. 67:1365-1370. Islam, M., M. P. Doyle, S. C. Phatak, P. Millner, and X. Jiang. 2005. Survival of Escherichia coli O157:H7 in soil and on carrots and onions grown in fields treated with contaminated manure composts or irrigation water. Food Microbiol. 22:63-70. Jackson, S. G., R. B. Goodbrand, R. P. Johnson, V. G. Odorico, D. Alves, K. Rahn, J. B. Wilson, M. K. Welch, and R. Khakhria. 1998. Escherichia coli O157:H7 diarrhoea associated with well water and infected cattle on an Ontario farm. Epidemiol. Infect. 120:17-20.

Infect. 119:245-250. Fukushima, H. and R. Seki. 2004. High numbers of Shiga-toxin producing Escherichia coli found in bovine faeces collected at slaughter in Japan. FEMS Microbial. Lett. 238:189-197.

Losinger W. C., S. J. Wells, L. P. Garber, H. S. Hurd, and L. A. Thomas. 1995. Management factors related to Salmonella shedding by dairy heifers. J. Dairy Sci. 78:2464-72. McGee, P., D. J. Bolton, J. J. Sheridan, B. Earley and

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N. Leonard. 2001. The survival of Escherichia coli O157:H7 in slurry from cattle fed different diets. Lett. Appl. Microbiol. 32:152-155. McGee, P., D. J. Bolton, J. J. Sheridan, B. Earley, G. Kelly and N. Leonard. 2002. Survival of Escherichia coli O157:H7 in farm water: its role as a vector in the transmission of the organism within herds. J. Appl. Microbiol. 93:706-713. Moore, A. C., B. L. Herwaldt, G. F. Craun, R. L. Calderon, A. K. Highsmith and D. D. Jaunek. 1993. Surveillance for waterborne disease outbreaks in the United States 1991-1992. M M W R. 42:1-22. Rasmussen, M. A., W. C. J. Cray, T. A. Casey and S. C. Whipp. 1993. Rumen contents as a reservoir of Enterohemorrhagic Escherichia coli. FEMS Microbiol. Lett. 114:79-84. Semenov, A.V., E. Franz, E., L. van Overbeek, A. J. Termorshuizen and A. H. C. van Bruggen. 2008. Estimating the stability of Escherichia coli O157:H7 survival in manure-amended soils with different management histories. Environ. Microbiol. 10:1450-1459. Vernozy-Rozand, C., M. P. Montet, F. Lequerrec, E. Serillon, B. Tilly, C. Bavai, S. Ray-Gueniot, J. Bouvet, C. Mazuy-Cruchauet and Y. Richard. 2002. Prevalence of verotoxin-producing Escherichia coli in slurry, farmyard manure and sewage sludge in France. J Appl. Microbiol. 93:473-478. Wang, G. and M. P. Doyle. 1998. Survival of enterohemorrhagic Escherichia coli O157:H7 in water. J. Food. Prot. 61: 662–667. Williams, A. P., K. A. McGregor, K. Killham and D. L. Jones. 2008. Persistence and metabolic activity of Escherichia coli O157:H7 in farm animal faeces. FEMS Microbiol. Lett. 287:168.

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A Transcriptomic Expression Array, PCR and Disk Diffusion Analysis of Antimicrobial Resistance Genes in Multidrug-Resistant Bacteria S. A. Khan1, K. Sung1, and M. Nawaz1 U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Microbiology, 3900 NCTR Road, Jefferson, AR 72079, USA.

ABSTRACT We report the development of a microarray-based transcriptomic expression analysis method for the detection of 131 antimicrobial resistance genes in multidrug-resistant clinical (Enterococcus faecium, E. faecalis), aquaculture (Aeromonas veronii), poultry (Campylobacter jejuni, Staphylococcus aureus), and outbreak (Salmonella enterica serovar Typhimurium) strains. Unmodified oligonucleotide probes and 131 pair of primers for the genes conferring resistance to 22 different antimicrobials were used for transcriptomic array and PCR analysis. Detection of resistance genes by transcriptomic array and PCR methods correlated well with the susceptibility profiles of the isolates used in the study. However, some of the genes conferring resistance to ampicillin (amp), bleomycin (ble), lincomycin (linAn2, lmr, lmrA, lmrB, mgt), neomycin (neo, nptII, himaR), oleandomycin (oleB, oleC-orf4, oleC-orf5), penicillin (mecA), and rifampin (arr2) could not be detected by above methods. Co- or cross-resistance to these drugs and their extracellular transport due to the presence of 2 to 5 efflux pump genes (oleB, marA, tetA, tetB, vraD) is believed to confer resistance to the above antimicrobials. Moreover, the presence of resistance genes for bacitracin, chloramphenicol, erythromycin, kanamycin, streptomycin, and tetracycline in phenotypically sensitive isolates indicated either inactive versions of these genes or modulation of gene expression. Overall, the transcriptomic array method provided a valuable insight into the mechanism(s) of resistance, status of gene expression at transcription level, and detection of all the antimicrobial resistance genes among bacteria from different ecological sources. Keywords: Antimicrobial, resistance genes, microarray, hybridization, PCR, disk diffusion, oligonucleotide Agric. Food Anal. Bacteriol. 1: 123-139, 2011

Correspondence: S. A. Khan, saeed.khan@fda.hhs.gov Tel: +1 -870 543-7197 Fax: +1-870 543-7307

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INTRODUCTION The most common techniques used to detect susceptibility of bacteria to different antimicrobials are the disk diffusion (Clinical and Laboratory Standards Institute, 2009a) and broth dilution assays (Clinical and Laboratory Standards Institute, 2009b). While these assays show susceptibility of the organisms to different antimicrobial agents and their level of resistance in most cases, they cannot tell which of the multiple resistance genes for a given antimicrobial are present. Polymerase chain reaction and other genetic analysis tools that are used to detect them are time-consuming, expensive, and labor-intensive

the probes. Detection of the genes with labeled genomic DNA can pinpoint the presence of resistance genes but it cannot tell whether the gene is transcribed or expressed without using other genetic testing methods (Cockerill III, 1999). The transcriptomic array described in this study uses 40 nucleotide long unmodified oligonucleotide probes that were printed on epoxy and polylysine-coated glass slides and used to detect transcriptional expression of antimicrobial resistance genes from multidrug-resistant bacteria. A comparison of the transcriptomic array, PCR and disk diffusion data exhibited a very good correlation and offers a powerful combination for comprehensive analysis of antimicrobial resis-

for detecting hundreds of genes in a single experiment (Khan et al., 2000). The success of PCR technology, among various other factors, depends upon published genomic DNA sequences. Any variation of the PCR primer sequence, either due to a discrepancy in the published DNA sequences or because of point mutations, insertion and deletion events could lead to false or unsuccessful amplifications requiring other methods of verification. Lately, microarray technology has been employed to identify and type food-borne pathogens (Al-Khaldi et al., 2002; Garaizer et al., 2006; Majtan et al., 2007; Rasooly et al., 2008). It has also been used successfully to detect the presence of multiple antimicrobial resistance genes among multidrug-resistant bacteria (Antwerpen et al., 2007; Brazas and Hancock, 2005; Call et al., 2003; Cassone et al., 2008; Chen et al., 2005; Frye et al., 2006; Perreten et al., 2005; Van Hoek et al., 2005; Zhu et al., 2007) with enhanced sensitivity and specificity. The cost of detection still remains prohibitive for routine monitoring of resistance genes in a high-throughput manner. One of the factors that can substantially increase the cost of custom arrays is the use of long and modified oligonucleotide probes (Lyons, 2003). Modification of oligonucleotide probes requires the addition of amino, thiol, and biotin func-

tance genes and their transcriptomic expression in multidrug-resistant bacteria. Our results also indicated that expensive modification of oligonucleotide probes was not necessary for the detection of antimicrobial resistance genes when printed on epoxycoated glass slides.

tional groups to improve their binding efficiency on the chip surface and has been shown to be unnecessary (Schüler et al., 2009). The microarray-based methods described earlier used labeled genomic DNA for hybridization with

multiple antimicrobials, they were grown in the presence of antibiotics used for their initial selection. For example, E. faecium and E. faecalis were grown in the presence of vancomycin (30 µg ml-1), A. veronii in the presence of tetracycline (30 µg ml-1), C. jejuni and

MATERIALS AND METHODS Bacterial strains used in the study An in-house collection of multidrug-resistant bacteria from different ecological sources, including Enterococcus faecium ATCC51559 and E. faecalis ATCC 51299 (human clinical isolates), Aeromonas veronii isolates Avt101 and Aet2002 (catfish isolates), Campylobacter jejuni isolates TH200 and TH205, Staphylococcus aureus isolates P20 and P34 (poultry isolates) and S. enterica serovar Typhimurium phage types DT16 and DT21 (outbreak isolates) were used for the detection of antimicrobial resistance genes in them. The number of genes targeted for detection by microarray and PCR was 131. Most were related to antibiotics used either in clinical practice or the National Antibiotic Resistance Monitoring System (NARMS) panel. Although bacteria were resistant to

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S. enterica serovar Typhimurium phage types DT16 and DT21 in the presence of ampicillin (10 µg ml-1), and S. aureus in the presence of erythromycin (15 µg ml-1) in Luria Bertani (LB) broth.

Microarray supplies Cy3 and/or Cy5-labeled antisense (complementary to the probes) oligonucleotides (40-mer), 2 x hybridization buffer, epoxy- and polylysine-coated slides were purchased from Eurofins MWG Operon Biotech (Huntsville, AL, USA). An RT-PCR kit was purchased from Invitrogen (Carlsbad, CA, USA) and CyScribe first-strand cDNA labeling kit was purchased from Amersham Biosciences (Piscataway, NJ, USA). RNA isolation kit was purchased from Qiagen (Valencia, CA, USA). For the printing of oligonucleotide probes on the slides, a microarray core facility at the University of Arkansas for Medical Sciences was utilized. The slide scanner was purchased from PerkinElmer (Boston, MA, USA). Hybridization chambers were from Corning Life Sciences (Acton, MA, USA).

Antimicrobial susceptibility testing by disk diffusion All the isolates were tested for antimicrobial susceptibility (Clinical and Laboratory Standards Institute, 2009a, b) by the criteria of the Clinical and Laboratory Standards Institute (CLSI). Twenty two different antibiotics, with their concentrations noted after their names in µg ml-1, and three salts (tylosin, tellurium, and benzalkonium chloride) that are used in postharvest processing were tested to determine the sensitivity of bacteria to these compounds: ampicillin-10, apramycin-15, bacitracin-10, benzalkonium chloride-100, bleomycin-512, chloramphenicol-30, erythromycin-15, gentamicin-120, hygromycin-512, kanamycin-30, lincomycin-2, methicillin-5, neomycin-30, oleandomycin-512, penicillin-10, rifampin-5, spectinomycin-100, streptomycin-300, sulfamethoxazole-trimethoprim-1.25, tellurium-512, tetracycline-30, tylosin-128, tobramycin-10, vancomycin-30, and virginiamycin-512 (AB BIODISK, Piscataway, NJ, 125

USA). CLSI interpretive criteria for apramycin, bleomycin, oleandomycin, QUADs, sulphonamide and virginiamycin were not available for some of the bacterial species tested. In these cases, when there was growth around the disks, the isolates were marked as resistant but when a zone of clearance was present, they were marked as question marks (Tables 1 and 2).

Probe designing and printing The entire sequences for 131 genes, representing 22 different antimicrobials and three salts (tylosin, tellurium and QUADs), were copied from the nucleotide sequence database and BLAST searched for homologous sequences. The regions of a gene that had the least homology to unrelated sequences in the database were used to create a library of 40mer oligonucleotide probes and each of the probes was BLAST searched again. Three probes for each gene were selected based upon the minimum homology against the whole database. These were then aligned against each other and screened for the similarity index; finally, one probe per gene was selected. The probes were suspended in water at a concentration of 100 pmoles/µl. These were then diluted with 2 x printing buffer A (MWG Operon). Using a Gene Machine printer (San Carlos, CA, USA), the probes were printed on polylysine- and epoxycoated slides in triplicate and each experiment was done at least twice. The probes were printed in twelve rows and twelve columns and numbered as 1 to 131. Row number 1 had the probes from 1 to 12, row 2 had the probes from 13 to 24, and row 3 had the probes from 25 to 36 and so on. Antisense probes that were complementary to the 40-mer oligonucleotide probes were labeled with either Cy3 or Cy5 and obtained from MWG Operon.

Isolation of DNA and RNA Chromosomal DNA was isolated from bacterial cells grown overnight in MH broth at 35ºC. The cells (1 ml) were centrifuged at 10,000 X g and the pellet was resuspended in 100 μl of sterile Milli Q (MQ) water. The DNA was isolated by using QIAmp DNA

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Table 1. Disk Diffusion, PCR and Microarray Analysis of Multiple Antibiotic Resistance Markers

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Melting temperature of primers is in parentheses in the primer’s column. The primers within bold lines indicate 30-100% homology among the genes within same class of antibiotics a

Presence of a PCR product is indicated by “P” and the strength of the hybridization signal for various genes

in different bacteria is indicated by color in “S/N” column. ■ indicates S/N ≥ 3, less darker shade indicates S/N ≤ 3 but more than 2, and the least darker shade indicates S/N between 1 and 2. Antimicrobial susceptibility data is not available for apramycin, bleomycin, oleandomycin, QACs, and virginiamicin. Therefore, the resistance profile of bacteria for these antibiotics is indicated by “?” mark; S = sensitive, R = resistant; I = intermediate resistant Numbers within parenthesis in front of the gene names indicate their position on microarray slide.

Mini kit (Qiagen, Valencia, CA). RNA from a 1 ml bacterial culture was isolated using Qiagen’s RNeasy Mini kit as per manufacturer’s instructions. DNA and RNA concentrations were determined by measuring the absorbance at 260 nm in a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and using a conversion ratio, A260 of 1 = 50 µg/ml of DNA and 40 µg/ml of RNA. The integrity of an RNA sample was tested by measuring the A260/280 ratio and analysis on a 1.2% agarose gel containing 0.66 M formaldehyde. An RNA preparation with an A260/280 ratio of 1.8 to 2.0 and the absence of visible degradation on agarose gel was considered good. The gels were run in MOPS electrophoresis buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0) for 3 h at 90 mV (Antwerpen et al., 2007), stained with ethidium bromide (1 µg ml-1 in electrophoresis buffer) and photographed using a gel documentation system, GDS 8000 (UVP, Inc. Upland, CA, USA). All the procedures starting from RNA isolation to cDNA labeling, hybridization and PCR analysis were replicated at least twice unless indicated otherwise.

cDNA labeling by reverse transcription To minimize the loss of RNA by degradation and the loss of sensitivity, RNA isolated by the Qiagen method was immediately used to make Cy3-labeled cDNA by using CyScribe first-strand cDNA labeling kit (Amersham BioSciences). Total RNA (10 µg) in a volume of 10 µl double-distilled water (ddH2O) was mixed with 1 µl of random hexamer primers (Amersham Biosciences, USA) and incubated for 5 min at 70°C followed by incubation for 10 min at room temperature. After the incubation, 4 µl of 5 X CyScript buffer, 2 µl of 0.1 M dithiothreitol, 1 µl of 2 mM dNTP mix (1 mM dCTP + 1 µl of 1 mM Cy3-labeled dCTP), and 1 µl of CyScript reverse transcriptase were added to it. The synthesis of cDNA was carried out in the dark at 42°C for 1.5 h. RNA template was degraded by alkaline hydrolysis with 2 µl of 2.5 M NaOH at 37°C for 15 min. The reaction was then neutralized by adding 10 µl of 2 M HEPES free acid. Labeled cDNA was purified with CyScribe GFX purification kit (Amersham Biosciences) and dried prior to hybridization using DNA 120 SpeedVac (Thermo Savant, San

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Jose, CA, USA). It was stored at -20°C until use and dissolved in 10 µl of nuclease-free water before hybridization.

Hybridization and specificity A mock hybridization and washing procedure of the printed slides followed by staining with SYBR Green I (Molecular Probes, Inc., Eugene, OR, USA) was carried out to determine whether the probes remained immobilized or were washed off from the slides. To determine the specificity of the probes, Cy3- and/or Cy5-labeled antisense oligonucleotides were hybridized with probes on the

using a primer selection module of the Lasergene program (DNASTAR, Inc., Madison, WI, USA) and synthesized by MWG Biotech. PCR amplification was carried out in a reaction volume of 25 µl by using a PCR Kit (Applied Biosystems, Foster City, CA, USA). Each reaction tube contained 5 µl of bacterial DNA (0.1-0.5 µg), 5 µl of a 10 µM mixture of the forward and reverse primers (Table 1), and 15 µl of PCR mix (200 µl of PCR mix contains: 33.3 µl of 10 X XL buffer II, 27 µl of 25 mM magnesium acetate, 66 µl of 10 mM dNTP mix and 7 µl of Taq DNA polymerase and 66.7 µl of water). A total of 35 cycles of amplification were carried out. Each cycle consisted of 94°C denaturation for 1 min, annealing for 1 min

slide. Hybridization was carried out by first using a mixture of Cy3- and Cy5-labeled antisense oligonucleotides to find out if they hybridized specifically, inhibited hybridization or cross-hybridized with other probes. Antisense oligonucleotides or Cy3-labeled cDNA (10 µl) were heated at 95°C for 2 min followed by a quick cooling in ice bath for 30 sec. A 2 X hybridization buffer (MWG) was preheated to 55°C in a water bath and then combined with of Cy3-labeled antisense oligonucleotides or cDNA. The mixture was applied to the microarray slide and the hybridization solution was spread evenly. The slides were then covered with a cover slip and sealed in humid hybridization chambers that were then put in a 42°C water bath overnight. The hybridized slides were washed with 1X SSC and 0.2% (w/v) sodium dodecyl sulfate (SDS) at 55°C for 10 min. Subsequent two washes were carried out with 0.1X SSC, 0.2% (w/v) SDS at 55°C for 10 min followed by rinsing in distilled water for 10 sec. The slides were then placed in a 50 ml capacity plastic tube and centrifuged at 4,000 x g for 3 min to dry the slide surface. Hybridization with Cy3-labeled cDNA was also carried out using the above procedure.

at 1°C below the lowest Tm of a given primer pair, and 72°C extension for 5 min. The first denaturation and the last extension steps were extended for 2 and 15 min, respectively. The PCR amplicons were analyzed on 1.5% agarose gels.

PCR detection of antimicrobial resistance genes

and above are usually considered as positive for the expression of the genes and a S/N ratio of less than 2 was considered as negative (Al-Khaldi et al., 2002; Cassone et al., 2008). Genes with S/N ratios of ≥ 3 are represented by dark shade squares and those with

Primers for the partial amplification of different antimicrobial resistance genes were designed by 129

Signal detection, quantification and validation Two independent hybridization experiments were performed for each bacterial strain using Cy3-labeled cDNA with three replicates of an array on each slide. Thus, the microarray hybridization results were from six subarrays for each bacterium tested. After hybridization, the slides were immediately scanned using PerkinElmer’s ScanArray Express Scanner (Perkin Elmer, Boston, MA, USA) and ScanArray Express 1.1 software at 5 µ resolution. Quantification of the hybridized signal was carried out using QuantArray 3.0 software (Packard BioSciences, Billerica, MA, USA). Signal intensities were measured by the adaptive circle method followed by local background subtraction. The mean of intensities from six hybridization panels were calculated and signal-to-noise ratios (S/N) were determined for all the genes. Arbitrary signal intensity units of 2000 or a S/N ratio of 3

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S/N ratios of ≥ 2 but less than 3 are indicated by light shade squares in Table 1. In cases where bacteria exhibited resistance to an antimicrobial and the corresponding gene(s) indicated the S/N ratio between 2 and 3, PCR and sequence analysis was carried out to verify the presence of those genes. Genes were used as positive controls when detected by PCR and microarray and their presence corresponded with the antimicrobial resistance profile. Genes that were not detected by PCR and microarray and correlated well with the susceptibility profiles of bacteria were used as negative controls. Figure 1. Testing slide chemistry: Slides were stained with SYBR Green I before and after mock hybridization and photographed. A and B. SYBR Green I stained polylysine (A) and epoxy-coated (B) slides before mock hybridization, C and D. SYBR Green I stained polylysine (C) and epoxy-coated (D) slides after mock hybridization

Sequence Analysis PCR products were purified by a QIAquick gel extraction kit (Qiagen, Valencia, CA, USA), eluted in nuclease-free water and directly sequenced with PCR primers (Table 1) using Sanger’s sequencing method (Sanger et al., 1977). Nucleotide sequences of the PCR products were BLAST searched against the existing GenBank database to confirm their identity.

RESULTS Slide chemistry and specificity of the probe

Figure 2. Testing probe specificity: After hybridization with a mixture of Cy3- and Cy5-labeled oligonucleotidess, the slides were scanned under their respective channels. A. 1-11, 23-33, Cy3; and 12-22, 34-44, Cy5-labeled antisense oligonucleotide hybridization profile. B. 1-11, 23-33, 45-60, 75-90, Cy3 and 12-22, 34-44, 61-74, Cy5-labeled antisense oligonucleotide hybridization profile. C. 45-60, 75-90, Cy3 and 61-74 Cy5-labeled antisense oligonucleotide hybridization profile. D. 91-100, 112-122, Cy3 and 101-111, 123131, Cy5-labeled antisense oligonucleotide hybridization profile. All the figures are representatives of the replicate experiments.

Staining of slides with SYBR Green I after postprint processing indicated that the probes were present on both the polylysine- and epoxy-coated slides (Figs. 1A, B). However, after mock hybridization at 55°°C, the probes were washed off of polylysine (Fig. 1C) slides but not epoxy-coated slides (Fig. 1D). Repetition of the experiment yielded similar results. Hybridization with a mixture of Cy3- and Cy5-labeled antisense oligonucleotides indicated that they hybridized specifically with corresponding probes on the slide. Cross hybridization with other probes was also observed (Figs. 2A, B, C, D) but since the S/N ratio of cross-hybridized probes was 1 or less, it was considered insignificant. These genes that cross-hybridized were tetB and murX (Fig. 1A), tetB, murX, and vanD (Fig. 1B), cat, bcrB, tetA, lmrA, mgt, bla-TEM-1D, bla-VEB, and aacA4 (Fig.

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Figure 3. Hybridization of Cy3-labeled cDNA with antibiotic resistance gene probes. A. RNA analysis on a 1.2% agarose gel. Lanes 1 and 4, RNA ladder; lane 2, RNA from E. faecium ATCC 51559; lane 3, RNA from E. faecalis ATCC 51229. B. Hybridization profile of E. faecium ATCC 51559. C. Hybridization profile of S. aureus strain P34. D. Hybridization profile of A. veronii strain Aet2002. E. Hybridization profile of S. Typhimurium phage type DT16. All the figures are representatives of the replicate experiments.

1C), vraG, lmrA, bla-OXA10, bla-TLA1, aacA1b, aacC, aadA1, aadB, kamA and llm (Fig. 1D). Aminoglycoside resistance genes aadA1 and aadA2 had 98.8%, hptII and hyg had 99.2%, and aph4 and nos had 100% sequence homology. It did not pose a major problem because only 1 to 3 of them were present at a given time in a single bacterium and were considered present after verification by PCR and sequence analysis.

Electrophoretic analysis of RNA samples on agarose gel prior to using in microarray hybridization experiments indicated that it was intact (Fig. 3A). Lanes 2 and 3 in figure 3A show the RNA pro-

tion with the probes, hybridization signals with varying signal strengths were obtained (Figs. 3B, C, D, E) for different bacteria tested. The data shown are for E. faecium, ATCC 51559 (Fig. 3B), S. aureus isolate P34 (Fig. 3C), A. veronii isolate Aet2002 (Fig. 3D), and S. Typhimurium phage type DT16 (Fig. 3E). Most of the bacteria that exhibited resistance to multiple antimicrobials had strong hybridization signals (S/N = 3 or more) are shown by dark shades in Table 1. Those, exhibiting S/N ratios between 2 and 3 are shown by slightly lighter shading in Table 1. These included kamC, hmrB, aad9, tlrB, tet34, tetA(E), vanB2, and vanD in E. faecalis ATCC 51229; bacA, cmlA5, ermF, nos, lmrB, hmrB, oleB, oleC-orf5, spa, tlpD, tlrB, tetA, tetO, ddl2, murX, and vanB in E. faecium ATCC 51559; kamB, kamC, ermF, and tlrB in A. veronii isolate Avt101; amp, aac(3)IV, bacA, ermA,

files of E. faecium ATCC 51559 and E. faecalis ATCC 51229, respectively. Similar RNA profiles were obtained from other bacterial strains used in the study (data not shown). When total RNA was reverse transcribed to prepare Cy3-labeled cDNA for hybridiza-

aadD, kanR, oleB, oleC-orf5, mecA, aad9, spa, tlrB, tet, ddl2, and vanB2 in A. veronii Aet2002; mecA, sulI, tlrB, otrA, tet34, vanB2, and vgb in C. jejuni isolate TH200; aac(3)IV, hyg, kanR, oleB, mecA, aad9, and tet34 in C. jejuni isolate TH205; kamC, himaR,

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Table 2. Correlation of microarray and PCR data in bacteria exhibiting resistant, sensitive, and/or inconclusive susceptibility patterns.

Sensitivity Antibiotics and their corresponding resistance Profile markers used for detection by microarray and PCR

Organism Enterococcus faecalis ATCC 51299

Enterococcus faecium ATCC 51559

Aeromonas veronii Avt101

Aeromonas veronii Aet2002

Campylobacter jejuni TH200

R S

NEO [neo, himaR, nptII] TET [otrA, tetO]

SUL [spa, sulI]

AMP [amp]; BLE [ble]; NEO [neo, himaR, nptII]; PEN [mecA]; RIF [arr2]

TET [otrA, tetO]

OLE [oleB, oleC-orf5]

PEN [mecA]; RIF [arr2]

CHL [cmlA5]; STR/SPE [aad9]

APR [aac(3)IV, kamB, kamC]; SUL [spa, sulI]; QAC [qacH]

RIF [arr2]

S ?

CHL [cmlA5] SUL [spa, sulI]; QAC [qacH]

AMP [amp]; RIF [arr2]

CHL [cmlA5]; ERY [ermA, msrSA]; STR/SPE [aad9]

APR [aac(3)IV]; OLE [oleB]; QAC [qacH]; SUL [spa]; TEL [tlrB]; VIR [vgb] SUL [spa]; TEL [tlrB]; VIR [vgb]

Campylobacter jejuni TH205

Staphylococcus aureus P20

Staphylococcus aureus P34

Salmonella Typhimurium DT16

Salmonella Typhimurium DT21

AMP [amp]

CHL [cmlA5]; ERY [ermA, msrSA]; STR/SPE [aad9]

APR [aac(3)IV]; OLE [oleB]; QAC [qacH]; SUL [spa]

LIN [linAn2, lmr, lmrA, lmrB, mgt]; OLE [oleB, oleC-orf4, oleC-orf5]

BAC [bacA, bcrA]; KAN [aadB, kanR]]

SUL [spa]; VIR [vgb]

AMP [amp]

BAC [bacA, bcrA, bcrB];

APR [kamC]; SUL [spa]; VIR [vgb]

RIF [arr2]

CHL [cmlA5]]; STR/SPE [aad9]; TET [tetA(E), tetO]

APR [aac(3)IV, kamB]; QAC [qacH]

AMP [amp]; BLE [ble]; NEO [neo, himaR, nptII]; RIF [arr2]

APR [aac(3)IV, kamB]; QAC [qacE∂1, qacH]

Names of the antimicrobial agents are indicated by three capital letter codes in bold and their corresponding resistance determinant genes within parentheses. The gene names that are in italics were not detected by either microarray or PCR but those in bold and italics were detected by microarray and PCR. AMP = Ampicillin; APR = Apramycin; BAC = Bacitracin; BLE = Bleomycin; CHL = Chloramphenicol; ERY = Erythromycin; HYG = Hygromycin; KAN = Kanamycin; LIN = Lincomycin; NEO = Neomycin; OLE = Oleandomycin; PEN = Penicillin; QAC = Quaternary ammonium compounds; RIF = Rifampin; SUL = Sulphonamide; STR = Streptomycin; SPE = Spectinomycin; TEL = Tellurium; TET = Tetracycline; VAN = Vancomycin; VIR = Virginiamyciåån; S = Sensitive; ? = Antimicrobial agents for which either sensitivity criteria was not available or the results of the disk diffusion assay were inconclusive but their corresponding resistance genes were detected by microarray and PCR. Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 2 - November 2011

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tetM, tet34, ddl2, sfiX, vanB, vanC2-c3, vanE and vanG in S. aureus isolates P20; nos, lmrB, hmrB, neo, oleB, oleC-orf5, mecA, sulI, tet, ddl2, ddl, and vanB in S. aureus P34; ermF, lmr, femB, mecA, spa, tetA(E), dacB, and vanB in S. Typhimurium phage type DT16; lmr, femA, femB, oleC-orf5, spa, sulI, tlrB, dacB, sfiX, vanA, vanB2, and vanE in S. Typhimurium phage type DT21 (Table 1, Fig. 4A). Apart from the specific antimicrobial resistance genes, 1 to 6 additional aminoglycoside resistance genes were also present in all the bacteria except in C. jejuni isolate TH205 (Table1). The gene satG was present in A. veronii isolates Avt101 and Aet2002, and S. Typhimurium phage types DT16 and DT21.

ATCC 51559 and E. faecium ATCC 51229 (Table1, Fig. 4B). In the absence of CLSI interpretive criteria for apramycin, bleomycin, oleandomycin, QUADs, sulphonamide and virginiamycin, susceptibility patterns of bacteria could not be established for these antimicrobials. Using the criterion of S/N ratio >2 and PCR detection, sulphonamide resistance genes spa, and sulI were found in E. faecium ATCC 51559. Oleandomycin resistance gene oleB, and oleC-orf5 were detected in E. faecium ATCC 51559. Apramycin resistance genes aac(3)IV, kamB, and kamC, sulphonamide resistance genes spa, and sulI and QUADs resistance gene qacH were detected in A. veronii

E. faecalis ATCC 51299, S. aureus isolates P20 and P34 contained the gene aacA1b. The genes aacA4 and aacC were present in E. faecalis ATCC 51299, A. veronii isolate Avt101, C. jejuni isolate TH200, and S. Typhimurium phage types DT16 and DT21. The gene aadA was present in S. aureus isolate P20, aadA1 in A. veronii isolate Avt101 and S. Typhimurium phage type DT16, and aadA2 in E. faecalis ATCC 51299, A. veronii isolate Avt101, and S. Typhimurium phage type DT21. The gene speC was present in E. faecalis ATCC 51299 and E. faecium ATCC 51559. E. faecalis ATCC 51299, and S. aureus isolates P20 and P34 possessed the gene ant(3’’)-Ih-aac(6’)-Iid. Moreover, all the bacteria used in the study also contained 2 to 5 efflux pump genes (Table 1). In the case of bacteria that were sensitive to a given antimicrobial agents, some of them were still found to contain the corresponding antimicrobial resistance genes. These included bacitracin resistance genes bacA and bcrA in S. aureus isolate P20, and bacA, bcrA, and bcrB in S. aureus isolate P34; chloramphenicol resistance gene cmlA5 in A. veronii isolates, Avt101 and Aet2002, C. jejuni isolates TH200 and TH205, and S. Typhimurium phage types DT16; erythromycin resistance genes ermA and msrSA in C. jejuni isolates TH200 and TH205; kanamycin re-

isolate Avt101. A. veronii isolate Aet2002 contained sulphonamide resistance genes spa and sulI, and QUADs resistance gene qacH. C. jejuni isolates TH200 and TH205 possessed apramycin resistance genes aac(3)IV, hygromycin resistance genes hyg and nos, oleandomycin resistance gene oleB, QUADs resistance gene qacH, and sulphonamide resistance genes spa and sulI. C. jejuni isolate TH200 had additional tellurium and virginiamycin resistance genes tlrB and vgb, respectively (Table 1). S. aureus isolates P20 and P34 contained sulphonamide resistance gene spa and virginiamycin resistance gene vgb. S. enterica serovar Typhimurium phage types DT16 and DT21 contained QUADs resistance gene qacH.

sistance genes aadB and kanR in S. aureus isolate P20; streptomycin resistance gene aad9 in C. jejuni isolates TH200 and TH205, A. veronii isolate Avt101, and S. Typhimurium phage types DT16; and tetracycline resistance genes otrA and tetO in E. faecium

ATCC 51559 and S. Typhimurium phage type DT21. Ampicillin resistance gene amp was missing in E. faecium ATCC 51559, C. jejuni TH200, and TH205, S. aureus P34 and S. Typhimurium phage type DT21. Penicillin resistance gene mecA was absent in E.

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PCR detection of antimicrobial resistance genes Among resistant bacteria, most of the genes with S/N ratios of 3 and above could be detected by PCR. Some with S/N ratios > 2 but < 3 were also detected (Table 1, Fig. 4A). Some genes associated with the corresponding antimicrobial agents could not be amplified from resistant isolates (Table 1, 2). These included neomycin resistance genes neo, himaR, and nptII in E. faecalis ATCC 51299, E. faecium

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Figure 4. PCR amplification of antimicrobial resistance genes from bacteria that exhibited resistance to different antimicrobials and had S/N ratio of ≤ 3. A. Lanes 1, 13, and 24, 100 bp ladder; lane 2, kamC; lane 3, aad9; lane 4, bacA; lane 5, ermF; lane 6, hmrB; lane 7, tlpD; lane 8, oleC-orf5; lane 9, ermA; lane 10, amp; lane 11, aadD; lane 12, tlrB; lane 14, mecA; lane 15, bcrB; lane 16, kanR; lane 17, mecA; lane 18, nos; lane 19, neo; lane 20, mecA; lane 21, femB; lane 22, sulI; lane 23, femB. B. Resistance genes from bacteria that exhibited sensitivity to different antimicrobials and those with inconclusive susceptibility patterns. Lanes 1, 13, and 24, 100 bp ladder; lane 2, tetO; lane 3, sulI; lane 4, otrA; lane 5, oleB; lane 6, cmlA5; lane 7, spa; lane 8, cmlA5; lane 9, ermA; lane 10, tlrB; lane 11, aad9; lane 12, mgt; lane 14, aadB; lane 15, spa; lane 16, bacA; lane 17, bcrA; lane 18, vgb; lane 19, tetA(E); lane 20, tetO; lane 21, qacH; lane 22, aac(3)IV; lane 23, kamB. The names of bacteria from which these genes were amplified are shown on top of the lanes. All the figures are representatives of the replicate experiments. faecium ATCC 51559 and A. veronii Avt101. Bacterial strains E. faecium ATCC 51559, A. veronii Avt101, Aet2002, C. jejuni TH200 and S. Typhimurium phage types DT16 and DT21 were missing rifampin resistance gene arr2. E. faecium ATCC 51559 and S. Typhimurium phage type DT21 did not have bleomycin resistance gene ble. S. aureus P20 was missing lincomycin resistance genes linAn2, lmr, lmrA, lmrB and mgt, and oleandomycin resistance genes oleB, oleC-orf4 and oleC-orf5 (Table 1, 2). Most of the antimicrobial resistance genes did not amplify from sensitive isolates but some yielded am-

plicons corresponding to the antimicrobial agents which they were sensitive to (Table 1, 2, Fig. 4B). The antimicrobial agents for which no CLSI interpretive criteria were available, the corresponding resistance genes were also detected by PCR analysis (Table 1, 2, Fig. 4B).

DISCUSSION This study highlights the development of an inexpensive transcriptomic array method for the detection of multiple antimicrobial resistance genes in

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a variety of multidrug-resistant bacteria. Unlike the previous methods that utilized expensive modified oligonucleotide probes and the genomic DNA labeling for detection of multiple antimicrobial resistance genes (Call et al., 2003; Frye et al., 2006; Perreten et al., 2005; Relógio et al., 2002; Van Hoek et al., 2005; Volokhov et al., 2003), this method uses inexpensive and unmodified oligonucleotide probes. The data generated by this method was compared and correlated with the results obtained by PCR and disk diffusion assays. During the course of this study, we used 40 nucleotide long unmodified oligonucleotide probes to detect 131 genes that confer resistance to multiple antimicrobial agents in a lim-

deemed as good enough to confer resistance to the above antimicrobials. Similar observations were made earlier (Bernstein et al., 2002; Frye et al., 2006; Wain and Kidgell, 2004, Woodford, 2001) and corresponded with our findings. In most of the bacteria that exhibited resistance to an antimicrobial agent, the corresponding resistance genes were detected in them but the presence of resistance genes corresponding to ampicillin, bleomycin, erythromycin, lincomycin, neomycin, oleandomycin, penicillin, and rifampin could not be verified by the above techniques in some of the isolates. The absence of these genes suggested that the observed resistance in these isolates was either due to cross

ited set of bacteria. Preliminary tests revealed that the proper slide chemistry played an important role when unmodified oligonucleotide probes were used in a microarray experiment. Use of polylysine-coated slides resulted in the loss of hybridization signals due to the washing off of the probes during a mock hybridization experiment. Epoxy-coated slides performed better with unmodified probes as they remained bound to the surface of the slides. Further, a test of specificity of probes indicated that hybridizations with commercially synthesized and Cy3/Cy5-labeled antisense oligonucleotide sequences yielded specific hybridization signals. An insignificant level of cross-hybridization (S/N = 1 or less) was seen with other genes. In actual tests, hybridization with Cy3labeled cDNA, prepared by the reverse transcription of total RNA from ten different multidrug-resistant bacteria yielded high intensity (IU 2000 and above) and low intensity (IU < 2000) hybridization signals for different antimicrobial resistance genes. Most of the genes conferring resistance to multiple antimicrobial agents in bacteria could be detected by PCR and microarray (S/N > 3) but in some cases where bacteria exhibited resistance to antimicrobial agents, the observed S/N ratios for the corresponding genes were between 2 and 3. Using the S/N cut off limit of

resistance or the involvement of efflux pump genes. All the bacteria used in the study contained 2 to 5 efflux pump genes (oleB, marA, tetA, tetB, vraD). The presence of efflux pump genes causes the bacteria to become resistant to multiple unrelated antimicrobial agents and was probably responsible for the observed resistance in these organisms. For example, the gene marA itself has been shown to produce cross resistance to tetracycline, chloramphenicol, ampicillin, nalidixic acid, and ciprofloxacin, as well as to other toxic chemicals (QUADs) that are present in household, industrial, and hospital disinfectants (Alekshun and Levy, 1999). Cross resistance of kanamycin-resistant mutants of Escherichia coli Q13 has been shown to confer various degrees of crossresistance to streptomycin, gentamicin, neomycin, and dibekacin in vivo (Choi et al., 1980). Absence of neomycin resistance genes (neo, himaR and nptII) in E. faecalis ATCC51299, E. faecium ATCC 51559, and S. Typhimurium phage type DT21 could be due to the presence of kanamycin resistance observed in these isolates. Extended spectrum ß-lactamases such as bla-TEM1D and bla-OXA10 genes that have been shown to confer resistance to ampicillin and penicillin (Paterson and Bonomo, 2005) may be responsible for the observed resistance to these an-

3 (Al-Khaldi et al. 2002; Cassone et al. 2008), these genes would be considered as being absent but the fact that the susceptibility pattern of the bacteria matched with PCR and sequence analysis data, a lower level of gene expression in these cases was

tibiotics in the absence of ampicillin and/or penicillin resistance genes amp and mecA, respectively, in E. faecium ATCC 51559, A. veronii Avt101, C. jejuni TH200 and TH205, S. aureus P34 and S. Typhimurium phage type DT21. The absence of rifampin re-

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sistance gene arr2, an ADP-ribosylating transferase, in A. veronii Avt101, Aet2002, C. jejuni TH200, and S. Typhimurium phage types DT16 and DT21, could mean the involvement of multidrug resistance proteins. Cloning and expression of a chromosomallylocated multidrug resistance gene cmr from Corynebacterium glutamicum has been shown to confer increased resistance to rifampin, bleomycin, tetracycline and many other antibiotics in E. coli (Jäger et al., 1997). Similar proteins may have been involved in conferring resistance to rifampin in above isolates and bleomycin resistance observed in E. faecium ATCC 51559 and S. Typhimurium phage type DT21. Absence of lincomycin and oleandomycin-specific re-

approximately 20% of the C. jejuni genes (Stintzi, 2003). Change in DNA supercoiling induced by a wide range of environmental stresses in coli has been shown to affect the gene transcription (Cheung et al., 2003). Environmental factors such as heat, acid, and osmotic stress have been demonstrated to cause differential expression of 13 to 18% of the genes (Xie, 2004). Earlier, we have also shown that susceptibility profiles of the organisms could change from resistant to sensitive in a disk diffusion assay depending upon the media used for testing (Nayak et al., 2002). In the light of current observations and the earlier reports, it is possible for a resistance gene to be present without exerting its phenotypic effect for the reasons

sistance genes in S. aureus isolate P20 is probably due to the presence of ermB gene which is present in this isolate and known to confer cross resistance to all macrolides, lincosamides and stretogramins B (Malbruny et al., 2011). For most of the bacteria that exhibited sensitivity to an antimicrobial, disk diffusion data corroborated with microarray and PCR data but some of the bacteria that exhibited sensitivity to an antimicrobial agent possessed the corresponding antimicrobial resistance genes as demonstrated by transcriptomic array, PCR and partial sequence analysis. The question, therefore, arises that why did the bacteria show sensitivity to antibiotics when the corresponding resistance genes were present? They were detected by PCR for the simple fact that genomic DNA harboring these genes was used as a template. Their detection by transcriptomic array analysis indicated that the genes were transcribed. After transcription of the resistance genes, they have to be translated to exert their phenotypic effect but if the translation product is either inhibited or silenced by any of the components of the growth media and/or growth conditions, the organisms will exhibit a sensitive phenotype for a given antimicrobial agent. In E. coli, silencing of the resistance genes blaOXA-2, aadA1, sul1, and tetA has been re-

mentioned above. While susceptibility assays by disk diffusion methods are sensitive to various experimental conditions, transcriptomic array and PCR analyses can still provide information regarding the presence or absence of a resistance gene. The latter, however, can not differentiate between a transcribed or untranscribed gene unless cDNA derived from the RNA transcripts is used as a template in PCR analysis. Therefore, gene expression analysis by transcriptomic or proteomic analysis methods would provide more information compared to the genomic DNA-based PCR or microarray technologies. The transcriptomic array technique described in this study proved more useful in the sense that it was able to detect the presence of antimicrobial resistance genes and also provided information about the status of their transcriptomic expression. PCR and genomic DNA labeling and detection techniques, while able to detect the genes, do not offer an explanation about the presence of resistance genes in sensitive isolates or absence thereof in resistant isolates and whether they expressed or remained unexpressed. In genomic DNA labeling and detection method, the entire genomic DNA is labeled and used for the detection of antimicrobial resistance genes whether they are transcribed

ported to be responsible for the observed sensitive phenotype (Enne et al., 2006) and the results presented in this study also point to a similar phenomenon. On another note, a growth temperature increase from 37 to 42°C has been shown to up- or downregulate

or not. Since transcriptomic expression method involves the labeling of cDNA during reverse transcription of RNA, only the genes transcribed under experimental conditions are detected. While each of the methods described above have certain mer-

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its, the transcriptomic array method, apart from being able to detect the presence and absence of the antimicrobial resistance genes, appeared to provide more insight into the mechanism of antimicrobial resistance, status of gene expression, coresistance, and cross resistance compared to the genomic DNA labeling and PCR detection methods. Furthermore, the technique could also be used for research in food safety, probiotic product development, epidemiological screening, and a thorough and comprehensive screening of multiple antimicrobial resistance genes among bacteria from different ecological sources.

at NCTR for their help in printing the array slides. The work was supported by intramural funding by the US Food and Drug Administration. Views presented in this paper do not necessarily reflect those of the FDA.

CONCLUSIONS

JAOAC Int. 85:906-910. Antwerpen, M. H, M. Schellhase, E. EhrentreichFörster, F. Bier, W. Witte, U. Nübel. 2007. DNA microarray for detection of antibiotic resistance determinants in Bacillus anthracis and closely related Bacillus cereus. Mol. Cell. Probes. 21:152-160. Bernstein, J. A., A. B. Khodursky, P. H. Lin, S. LinChao, S. Cohen. 2002. Global analysis of mRNA decay and abundance in E. coli at a single-gene resolution using two-color fluorescent DNA microarrays. Proc. Nat. Acad. Sci. USA. 99:9697-9702. Brazas, M.D, R.E. Hancock. 2005. Using microarray gene signatures to elucidate mechanisms of antibiotic action and resistance. Drug Discov. Today 10:1245-1252. Call, D. R, M. K Bakko, M. J. Krug, M. C. Roberts. 2003. Identifying antimicrobial resistance genes with DNA microarrays. Antimicrob. Agents Chemother. 47:3290-3295. Cassone, M., M. Del Grosso, A. Pantosti, A. Giordano, G. Pozzi. 2008. Detection of genetic elements carrying glycopeptides resistance clusters in Enterococcus by DNA microarrays. Mol. Cell. Probes. 22:162-167. Chen, S., S. Zhao, P. F. McDermott, C. M. Schroeder, D. G. White, J. A. Meng. 2005. DNA microarray for

The transcriptomic array method utilizing unmodified oligonucleotide probes was successfully used to detect the presence of 131 antimicrobial resistance genes conferring resistance to 22 different antimicrobials described in this paper. It provides a valuable insight into the mechanism(s) of resistance, status of gene expression at transcription level, and detection of the antimicrobial resistance genes among bacteria from different ecological sources. For most of the genes, the transcriptomic array and PCR data correlated well with the susceptibility profiles of the isolates used in the study. However, some of the genes conferring resistance to certain antibiotics could not be detected by above methods. Co- or cross-resistance to these drugs and their extracellular transport due to the presence of 2 to 5 efflux pump genes is believed to confer resistance to the above antimicrobials. Moreover, the detection of resistance genes for few antibiotics in phenotypically sensitive isolates indicated either inactive versions of these genes or modulation of gene expression.

ACKNOWLEDGEMENT The authors thank Dr. Doug Wagner and Dr. John Sutherland of the Division of Microbiology, NCTR, for their critical reading and evaluation of the manuscript. We also thank Mr. William Branham and Dr. James Fuscoe of the Division of Systems Toxicology 137

REFERENCES Alekshun, M. and S. Levy. 1999. The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Trends Microbiol. 7:410-413. Al-Khaldi, S. F., S. A. Martin, A. Rasooly, J. D. Evans. 2002. DNA microarray technology used for studying food-borne pathogens and microbial habitats.

identification of virulence and antimicrobial resistance genes in Salmonella serovars and Escherichia coli. Mol. Cell. Probes. 19:195-201. Cheung, K. J., V. Badrinarayana, D. W. Selinger, D. Janse, G. M. Church. 2003. A microarray-based

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antibiotic screen identifies regulatory role for supercoiling in the osmotic stress response of Escherichia coli. Genome Res. 13:206-15. Choi, E. C., T. Nishimura, Y. Tanaka, N. Tanaka. 1980. In vivo and in vitro cross-resistance of kanamycinresistant mutants of E. coli to other aminoglycoside antibiotics. J. Antibiot. 33:1527-1531. Clinical and Laboratory Standards Institute. 2009a. Performance standards for antimicrobial disk susceptibility tests; 10th ed. M02-A10. Wayne, PA: CLSI. Clinical and Laboratory Standards Institute. 2009b. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; eighth ed.

Antimicrob. Chemother. 60:937-946. Malbruny, B., A. M. Werno, D. R. Murdoch, R. Leclercq, V. Cattoir. 2011. Cross-resistance to lincosamides, streptogramins A, and pleuromutilins due to the lsa(C) gene in Streptococcus agalactiae UCN70. Antimicrob. Agents Chemother. 55:1470-1474. Nayak, R., S. A. Khan, R. H. Watson, C. E. Cerniglia. 2002. Influence of growth media on vancomycin resistance of Enterococcus isolates and correlation with resistance gene determinants. FEMS Microbiol. Lett. 214:159-163. Paterson, D. L., R. A. Bonomo. 2005. Extended-spectrum ß-lactamases: a clinical update. Clin. Microbiol. Rev. 18:657-686.

M07-A8. CLSI , Wayne, PA. Cockerill III, F.R. 1999. Genetic methods for assessing antimicrobial resistance. Antimicrob. Agents Chemother. 43:199-212. Enne, V. I., A. A. Delsol, J. M. Roe, P. M. Bennett. 2006. Evidence of antibiotic resistance gene silencing in Escherichia coli. Antimicrob. Agents Chemother. 50:3003-3010. Frye, J. G, T. Jesse, F. Long, G. Rondeau, S. Porwollik, M. McClelland, C. R. Jackson, M. Englen, P. J. Fedorka-Cray. 2006. DNA microarray detection of antimicrobial resistance genes in diverse bacteria. Int. J. Antimicrob. Agents. 27:138-151. Garaizer, J., A. Rementeria, S. Porwollik. 2006. DNA microarray technology: a new tool for the epidemiological typing of bacterial pathogens? FEMS Immunol. Med. Microbiol. 47:178-189. Jäger, W., J. Kalinowski, A. Pühler. 1997. A Corynebacterium glutamicum gene conferring multidrug resistance in the heterologous host Escherichia coli. J. Bacteriol. 179:2449-2451. Khan, S. A, M. S. Nawaz, A. A. Khan, C. E. Cerniglia. 2000. Transfer of erythromycin resistance from poultry to human clinical strain of Staphylococcus aureus. J. Clin. Microbiol. 38:1832-1838. Lyons, P. 2003. Advances in spotted microarray re-

Perreten, V., L. Vorlet-Fawer, P. Slickers, R. Ehricht, P. Kuhnert, J. Frey. 2005. Microarray-based detection of 90 antibiotic resistance genes of Gram-positive bacteria. J. Clin. Microbiol. 43:2291-2302. Rasooly, A., K. E. Herold. 2008. Food microbial pathogen detection and analysis using DNA microarray technologies. Foodborne Pathog. Dis. 5:531-550. Relógio, A., C. Schwager, A. Richter, W. Ansorge, J. Valcárcel. 2002. Optimization of oligonucleotidebased DNA microarrays. Nucleic Acids Res. 30:1-30. Sanger, F., S. Nicklen, A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467. Schüler, T., A. Nykytenko, A. Csaki, R. Möller, W. Fritzsche, J. Popp. 2009. UV cross-linking of unmodified DNA on glass surfaces. Anal. Bioanal. Chem. 395:1097-1105. Stintzi, A. 2003. Gene expression profile of Campylobacter jejuni in response to growth temperature variation. J. Bacteriol. 185:2009-2016. Van Hoek, A. H., I. M. Scholtens, A. Cloeckaert, H. J. Aarts. 2005. Detection of antibiotic resistance genes in different Salmonella serovars by oligonucleotide microarray analysis. J. Microbiol. Methods. 62:13-23. Volokhov, D., V. Chizhikov, K. Chumakov, A. Rasooly. 2003. Microarray analysis of erythromycin resistance

sources for expression profiling. Brief Funct. Genomics Proteomics. 2:21-30. Majtan, T., L. Majtanova, J. Timko, V. Majtan. 2007. Oligonucleotide microarray for molecular characterization and genotyping of Salmonella spp. Strains. J.

determinants. J. Appl. Microbiol. 95:787-798. Wain, J., C. Kidgell. 2004. The emergence of multidrug resistance to antimicrobial agents for the treatment of typhoid fever. Trans. R. Soc. Trop. Med. Hyg. 98:423-430.

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Woodford, N. 2001. Epidemiology of the genetic elements responsible for acquired glycopeptides resistance in enterococci. Microb. Drug Resist. 7:229236. Xie, Y., L. S. Chou, A. Cutler, B. Weimer. 2004. DNA microarray profiling of Lactococcus lactis subsp. lactis IL1403 gene expression during environmental stresses. Appl. Environ. Microbiol. 70:6738-6747. Zhu, L. X., Z. W. Zhang, C. Wang, H. W. Yang, D. Jiang, Q. Zhang, K. Mitchelson, J. Cheng. 2007. Use of a DNA microarray for simultaneous detection of antibiotic resistance genes among staphylococcal clinical isolates. J. Clin. Microbiol. 45:3514-3521.

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Antibiotic Resistance and Plasmid Profiles in Bacteria Isolated from Market-Fresh Vegetables S. Akter1, Rafiq-Un-Nabi2, F. Ahmed Rupa1, Md. L. Bari3 and M. A. Hossain1 Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh 2 Virology Lab, LSD, ICDDR, B, Mohakhali, Dhaka-1212, Bangladesh 3 Food Analysis and Research Laboratory, Center for Advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh 1

ABSTRACT This study was carried out to evaluate the total bacterial load and to isolate and identify antibiotic resistant bacteria in fresh vegetables sold in the local markets of Dhaka city and also to determine its resistance pattern to antibiotics. The highest Aerobic Plate Count (APC) found in tomato and carrot samples of local markets were 5.17 log10 colony forming units (CFU/g) (Annanda Bazar Market) and 5.78 log10 CFU/g (Khilgaon Market), respectively. However, the lowest APC found in tomato and carrot samples of local markets were 4.90 log10 CFU/g (Malibagh Bazar) and 5.50 log10 CFU/g (Malibagh Market), respectively. Antibiotic sensitivity patterns of the isolates were determined and nearly all of them were resistant to commonly used antibiotics. The percentage of resistant bacteria to the total load was also high. Amoxicillin resistant bacteria counts were 3.4%, followed by cefixime 2.15%, and ciprofloxacin count at 1.61%. There were 0.26% of the bacteria found to be multi drug resistant. Therefore, the fresh vegetables samples collected from local markets were heavily contaminated with resistant bacteria and are of special concern for human consumption. Plasmid profile, curing and transformation study results demonstrated that resistance to amoxicillin is plasmid mediated but for cefixime and ciprofloxacin were not. These study results demonstrated that plasmids are one of the important ways to spread resistance but chromosomal mutation by environmental selection might also responsible for resistance. Keywords: Isolation, antibiotic resistant bacteria, fresh vegetables and plasmid profile Agric. Food Anal. Bacteriol. 1: 140-149, 2011

INTRODUCTION Fresh vegetables are essential parts of the diet of humans. An increased number of microbial infections associated with consumption of fresh vegetables have been reported in recent years. DocuCorrespondence: A. Hossain, hossaina@du.ac.bd Tel: +8802-9661920-59 Ext 7735 Fax: + 8802-8615583

mented illnesses have been caused by bacteria, parasites, and viruses and are transmitted via many types of fruits and vegetables (Beuchat, 1996; NACMCF, 1999). Since antimicrobials are used in livestock and crop production to control pathogens, there is concern about antibiotic resistance development in these pathogens and subsequent transfer to humans through contaminated food. Antibiotic resistance among bacteria associated with food animals has

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been well documented; research regarding resistance profiles of bacteria isolated from raw produce is very limited. New research shows that antibiotic resistant bacteria also may be ingested with vegetables (Kilonzo Nthenge, A., 2009). For half a century, meat producers have fed antibiotics to farm animals to increase their growth and stave off infections. Now scientists have discovered that those drugs are appearing in unexpected places. Vegetables such as corn, green onion and cabbage absorb antibiotics when grown in soil fertilized with livestock antibiotics contaminated manure (Kumar et al., 2005). Antibiotic resistance is a food safety problem for several reasons (CDC, 2005). First, antibiotic re-

antibiotic for another reason. The antibiotic can kill normal bacteria in the gut, allowing a few Salmonella that ordinarily would be unlikely to cause illness, take over and cause illness (CDC, 2005). A third possible reason for the resistance-related problem is that the food supply may be a source of antibiotic-resistant genes. Harmless bacteria present in food-producing animals could be resistant, and humans could acquire these bacteria when they eat meat products from these animals. Once ingested, resistant genes from these bacteria could be transferred to bacteria that cause disease (Woodford and Ellington, 2007). According to the Centers for Disease Control and

sistance is increasing to some antibiotics, such as fluoroquinolones and third-generation cephalosporins. These antibiotics are commonly used to treat serious infections caused by bacterial pathogens frequently found in food, such as Salmonella and Campylobacter. Each year, several million people in the United States are infected with Salmonella and Campylobacter, which usually cause diarrhea that lasts about a week (CDC, 2006). Antibiotics are not recommended for treatment of most of these diarrheal illnesses, but are used to prevent complications in infants, persons with weakened immune systems, and older persons. Antibiotics may be life-saving for several thousand people each year who have serious invasive infections, such as bacteremia (infection in the bloodstream) and meningitis (infection of the lining of the brain and spinal cord). Salmonella infections are treated with ampicillin, trimethoprim-sulfamethoxazole, fluoroquinolones or third-generation cephalosporins, but some Salmonella and Campylobacter infections have become resistant to these antibiotics (CDC, 2005). A second reason for antibiotic resistance as a food safety problem is that more people may become ill. Ordinarily, healthy persons who consume few Salmonella may carry them for a few weeks without hav-

Prevention (CDC, 2005), more than 70% of the bacteria that cause infections acquired in hospitals are resistant to at least one of the drugs most commonly used to treat them. Out of the total antibiotics consumed, about 25% are used in hospitals. In Germany, estimated use of antibiotics in 1998 was approximately 412 tons (Kümmerer and Henninger, 2004) and taking the excretion rate into consideration, the amount of antibiotics released into municipal wastewater was 305 tons. Furthermore it is predicted that most of the resistant bacteria originate from hospitals (Klaus Kümmerer, 2009) and which in turn end up with municipality sewage wastewater. This wastewater pollution is more alarming in developing countries such as Bangladesh, because of poor wastewater management system. Food like vegetables can be contaminated by resistant bacteria through this water and resistant bacteria can subsequently spread through the entire population very easily. Therefore, the aims of this study were: 1) to isolate and identify the resistant bacteria; 2) to examine their resistant pattern; and 3) to examine their plasmid profile and their effect on resistance.

ing any symptoms, because those few Salmonella are held in check by the indigenous bacteria in their intestines. However, even a few antibiotic-resistant Salmonella in food can cause illness if the person who consumes the contaminated food then takes an 141

MATERIALS AND METHODS Sample collection A total of four kilograms of carrot and tomato samples were collected from four different local markets (Khailgaon market, Anada market, Polashi mar-

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ket and Malibag market) of Dhaka city. One kilogram of each carrot and tomato samples were purchased from each local market and brought aseptically to the Laboratory of Microbiology, University of Dhaka for microbial analysis. Special sterile Ziploc bags were used to carrying all the samples to avoid further contamination. Samples were processed and used within 24 h of collection.

Processing of samples and microbial analyses The samples were washed with sterile distilled water and cut into small pieces in a biosafety cabinet using sterile knives. Twenty-five grams of each tomato and carrot samples were measured carefully and accurately by using a weighing machine and dissolved in 225 mL of sterile physiological saline (0.85% NaCl solution) in a stomacher bag, separately. Each sample was then stomached for 90 seconds and 0.1 mL of the diluted and undiluted stomacher treated samples was subsequently spread plated onto Plate Count Agar (PCA; Oxoid Ltd., Hampshire, England) for the determination of total bacterial load and onto DifcoTM Muller Hinton Agar (MHA; Becton, Dickinson Company Ltd, Sparks, MD, USA) containing antibiotics for the isolation of antibiotic resistant bacteria. The MHA plates were supplemented with 0.16 µg/mL ciprofloxacin, 5 µg/mL cefixime, 10 µg/ mL amoxicillin separately. For isolating multi drug resistance (MDR) bacteria, MHA agar plates supplemented with all three antibiotics simultaneously at the same concentrations mentioned were used. The plates were then incubated at 37°C for 24 h and viable bacteria were enumerated.

Biochemical tests Biochemical tests were done according to the manual for general bacteriology of the American Society of Microbiology (1981). Biochemical tests conducted in this study were as follows: Oxidase Test, Catalase Test, Carbohydrate fermentation/ Utilization test, Kligler’s Iron Agar (KIA) test, Hydrogen sulfide production (H2S), Methyl Red (MR) test,

Voges-Proscauer (VP) test, Citrate Utilization test, Nitrate reduction test, Indole production, Urea (MIU) tests, and Motility tests were performed to identify the bacteria of interest (Cappuccino and Sherman, 1990).

Antibiotic susceptibility tests Susceptibility of isolated bacteria to different antimicrobial agents was measured in vitro according to Bauer-Kirby methods (1996). Briefly, commercially available antimicrobial discs (Oxoid Ltd, Basingstoke, Hants, UK) of Streptomycin (S = 10 μg/mL), Ciprofloxacin (Cip = 5 μg/mL), Ampicillin (A = 25 μg/mL), Rifampicin (R® = 5 μg/mL), Oxytetracyclin (OT =30 μg/mL), Cephalosporin (Ce/Cef = 30 μg/ mL), Cefixime (CFM = 30 μg/mL) and Chloramphenicol (C© = 30 μg/mL) were placed on the inoculated agar plates and incubated in an upright position overnight at 37ºC. The results were expressed as the zone of inhibition around the antibiotic disc.

Minimum inhibitory and bactericidal concentrations The minimum inhibitory concentrations (MICs) of all antimicrobials were determined by microdilution techniques in Mueller-Hinton broth according to Sanches et al. (2005). The inocula were prepared at a density adjusted to a 0.5 McFarland turbidity standard 108 colony-forming units (CFU/mL) and diluted 1:10 for the broth microdilution procedure. Microtiter plates were incubated at 37°C and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolates. The MIC was defined as the lowest concentration of antimicrobials at which the microorganism tested did not demonstrate visible growth. Minimum bactericidal concentration (MBC) was defined as the lowest concentration yielding negative subcultures or only one colony.

Statistical analysis All trials were replicated three times. Reported

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Table 1. Total viable bacterial count & resistant bacterial count found in tomato & carrot samples from four different markets in Dhaka citya Sample Resistance

Tomato Total count

AmoxR

CefixR

Carrot CiproR

MDR

Total count

AmoxR

CefixR

CiproR

MDR

Sampling log log log log log log log log log log site (CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) Anondo Market

5.17A

4.17 A

3.71B

3.41 B

2.77B

5.71 A

3.85 B

3.74 A

2.00 B

Malibagh Market

4.98 B

4.04 B

3.90 A

3.64 A

2.60 B

5.56 B

3.94 A

3.75C

2.69 C

2.95 A

Khilgaon Market

5.11 A

4.00 B

3.93 A

3.49 B

2.84 A

5.78 A

3.97 A

3.86 A

3.77 A

3.04 C

Palashi Market

5.07 B

3.99 B

3.96 A

3.71 A

3.04 C

5.57B

3.95 A

3.89 A

3.81 A

3.11 C

Data represent the mean values (n=6) obtained from three individual trials, with each of these values being obtained from duplicated samples and are expressed in logarithmic colony forming unit per gram (CFU/g). Significant differences in plate count data were established by the least-significant difference at the 5% level of significance. Mean values with the same letter in the same column are not significantly different (P<0.05). a

plate count data represent the mean values obtained from three individual trials, with each of these values being obtained from duplicated samples. Data were subjected to analysis of variance using the Microsoft Excel program (Redmond, Washington DC, USA). Significant differences in plate count data were established by the least-significant difference at the 5% level of significance.

Isolation of Plasmid DNA

Doly, 1979). A portion (15 μL) of plasmid DNA was loaded on to a 1.0% agarose gel containing 0.5 μg m L-1 ethidium bromide and electrophoresed in TBE (Tris -Boric acid- EDTA) buffer. The plasmid DNA were visualized by placing the gel on a UV (300 nm) transilluminator and recorded using the digital documentation imaging system (model universal Hood II; BIO-RAD Laboratories, Hercules, CA , USA).

Plasmid curing

A single bacterial colony was transferred into a conical flask containing Luria Broth (LB) medium amended with the appropriate antibiotics and incubated at 37°C overnight with mild shaking (180 x g). After incubation, 1 mL of the culture was taken and centrifuged (16,000 x g; 30 s) at 4°C. The supernatant was removed and the pellet was resuspended in 150 μL of Tris- EDTA buffer 10 mM Tris chloride (pH 8), 1mM EDTA (pH 8)] solution by vigorous vortexing. Two hundred microliter of NaOH-SDS (0.2 M

Curing of R-plasmid was done according to the method of Tomeda et al. (1968). Test organisms were selected from which plasmids had been isolated previously. An overnight culture of each test organism in LB containing amoxicillin was diluted to 104 cfu/ mL using freshly prepared sterile LB by serial dilution technique. From this diluted culture, 0.5 mL was added with 4.5 mL LB containing different concentrations of curing agents Ethidium Bromide: 75 μg/ mL, 100 μg/mL and 125 μg/mL. Thus the resulting

NaOH, 1% SDS) solution and 150 μL of 3M potassium acetate (pH 4.8) were then added and vortexed for 10 s. The content was centrifuged (16,000 x g; 5 min) again at 4°C and the supernatant was precipitated with 600 μL of ice cold ethanol (Birnboim and

concentration was 103 cfu/mL and the cultures were then incubated at 37ºC in an orbital shaker at 180 x g for 48 hours. After incubation, the broth culture was again diluted to 103 cfu/mL with sterile physiological saline. A 10 μL aliquot was subsequently spread

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Table 2. Percentage of resistant bacteria to total viable bacterial count Total Bacteria

% amoxicillin resistant bacteria

% cefixime resistant bacteria

% ciprofloxacin resistant bacteria

% MDR bacteria

2.37×106

3.4

2.45

1.61

0.26

on Luria Agar medium. After 24 hour incubation at 37°C, plates were examined for growth. From this plate culture, individual colonies were selected and picked with a sterile toothpick on one Luria agar medium without antibiotic and one Luria agar containing amoxicillin (25 μg/mL), with a numbered grid line attached on the bottom of each plate. After 24-hour incubation at 37°C, plates were observed for the cured cells. The cured plasmid cells were detected

transferred into each of 3 chilled Eppendorf tubes and labeled the tubes. A 1 ng amount of known plasmid (DH5α) to one tube and 10 ng was added to the other and the tubes placed on ice for 30 min, then held at 42ºC for 120 seconds and the cells were returned to ice for 1 to 2 minutes. One hundred μL of the diluted and undiluted transformation mixtures were spread plated on to amoxicillin containing Luria agar plates and incubated at 37ºC overnight. To

comparing the development of bacterial colonies on antibiotic containing plate with that of the normal (without antibiotic) plate. The samples that showed colonies on normal LB agar but failed to grow on LB agar supplemented with amoxicillin were the possible cured isolates.

calculate the competencies of cells, the number of colonies on the plate were divided by the amount of DNA (in ng) added to the transformation (Sambrook et al. 1989).

Plasmid transformation

RESULTS AND DISCUSSION

Producing Competent Cells Plasmid DNA from different strains was extracted by the method of Birniboirn & Doly (1979). A single colony from a freshly grown plate was picked and dispersed in 100 mL of LB media in a 1 L flask. The culture was incubated at 37ºC with vigorous shaking for approximately 3 hours. Cell density was monitored by determining optical density (OD)600 and was kept at less than 108 cfu/mL (log phase of growth). A 50 mL aliquot of this culture was transferred to a 50 mL conical tube and centrifuge at 4,000 x g for 10 min at 4°C. The supernatant was decanted and the pellet was resuspended in 10 mL of ice cold 0.1 M CaCl2. After resuspension, cells were centrifuged at 4,000 x g for 10 min. The supernatant was decanted and the pellet was resuspended in 1.0 mL of ice cold 0.1 M CaCl2. Competent cells were stored by add-

Antibiotic resistance is a worldwide public health problem that continues to grow. It occurs when strains of bacteria harbored in the human body become resistant to antibiotics due to improper use and abuse of antibiotics. For this study, we collected samples from four different local markets in Dhaka city. The total bacterial load in tomato sample was between 4.98 to 5.10 log CFU/g in 4 different local markets in Dhaka city. However, the bacterial loads in the carrot samples were 5.50 to 5.70 log CFU/g, which is significantly (P<0.05) higher than that of tomato samples. (Table 1). The percentage of resistant bacteria to the total viable bacterial load was also high. Amoxicillin resistant bacterial count was 3.4%, followed by cefixime 2.15%, and ciprofloxacin count at 1.61%. There were 0.26% of bacteria that exhibited multi drug resistance (MDR) (Table 2). A total of 2607 resistant bacteria were found and of them, amoxicil-

ing ice-cold sterile glycerol to a final concentration of 10% (v/v). Cells were mixed and left on ice for 30 min, then stored at -70ºC. Plasmid Transformation To transform, 0.1 mL of competent cells were

lin resistant bacteria were 42%, cefixime resistant bacteria were 37%, ciprofloxacin resistant bacteria were 18% and multi drug resistant bacteria were 3% (Figure 1). The percentage of amoxicillin resistant bacteria to all resistant bacteria was found to

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be at the highest frequency. Based on morphology, colony characteristics, single colonies were picked up randomly by sterile loop and studied further. The colonies were then plated on several differential and selective media such as McConkey Agar media, Eosine Methylene Blue (EMB) agar, Cetrimide, XLD (Xylose-lysineDeoxycholate) agar and TCBS media. Morphological studies and biochemical tests of the isolates revealed that both Gram-negative bacteria including Escherichia coli, Shigella dysenteriae, Klebsilla pneumonia, Salmonella Typhimurium and Grampositive bacteria including Pseudomonas vulgaris, Micrococcus luteus, Corynebacterium xerosis, Streptococcus lactis, Staphyloccus aureus were present in the samples. The most common resistant bacteria were found to be E. coli. Based on the morphology, colony characteristics, gram staining and biochemical test, 22 colonies were picked for further study (data not shown). Minimum bactericidal concentration (MBC) and MIC tests were performed using a microdilution method. The MIC for amoxicillin ranged from 32 to 64 µg/mL and MBC ranged from 64 to 128 µg/mL. MIC for cefixime ranged from 16 to 32 µg/mL and MBC ranged from 32 to 64 µg/mL. MIC for ciprofloxacin ranged from 1 to 2 µg/mL and MBC ranged from 4 to 8 µg/mL (Table 3). These results are in agreement with the results of other researchers (Schwartz et al., 2003; Willis 2000; Nemi et al., 1983; Klech and Lee, 1978). These results also suggest that the bacteria can become resistant to higher concentrations of antibiotics. An antibiogram study of 22 isolates showed that the isolates were also resistant to streptomycin, ampicillin, rifampicin, oxytetracyclin, cephalosporin and chloramphenicol (Table 4). Most of isolates were resistant to ampicillin (18), followed by oxytetracyclin (14), and very low resistance to ciprofloxacin (4) and cephalosporin (4). These experimental results also suggested that multidrug resistance to environmental bacteria was increasing and almost every bacterium was resistant to more than one antibiotic (Table 4). Plasmid profile of the 22 isolates showed that 145

Figure 1. Total resistant bacteria and the percentages of resistant bacteria to particular antibiotics

five isolates (4, 7, 11, 14 and 21) contained plasmid. Isolate 4 had almost 8 kb plasmid and isolates 7, 11 and 21 had greater than 5 to 6 kb plasmid. Isolate 14 contained two plasmids and they were 8 and greater than 4 to 5 kb plasmid (Figure 2). Kalantar et al. (2011) studied the presence of plasmids of molecular sizes ranging from 1.4 kb to 4.5 kb among the acute diarrhea causing E. coli isolates showing resistance to ampicillin (A), chloramphenicol (C) and tetracycline (T), and stated that these resistances are plasmid mediated. However, the authors did not carry out conjugation or plasmid curing experiments in order to establish the involvement of the plasmid in carrying ACT-resistance among the isolates studied. Curing tests were performed to determine the role of plasmid on resistance. Curing of amoxicillin isolate 7, cefixime isolate 14 and ciprofloxacin isolate 21 were done. After curing they were subjected to an antibiotic susceptibility assay and it was found that only isolate 14 showed sensitive to amoxicillin after curing. However the other two isolates (7 and 21) were not sensitive to their respective resistant antibiotics (data not shown). For the confirmation of curing, the plasmid of cured bacteria was again isolated and after agarose

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Figure 2. Agarose gel electrophoresis of plasmid DNA of resistant isolates. E. coli PDK-9 is used as a marker; CHR indicates the banding position of the chromosomal DNA.

Table 3: The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the isolates Antibiotics

Number of isolates

Suspected microorganisms

MIC(µg/ml)

MBC(µg/ml)

Amoxicillin

1 2 14 15 16

E.coli S. dysenteriae E.coli K. pneumonia M. lucteus

32 64 32 32 64

64 128 64 64 128

Cefixime

3 4 5 6 7 17 18

K. pneumonia K. pneumonia S. Typhimurium K. pneumonia E.coli M. lucteus M. lucteus

16 16 16 16 32 16 16

64 32 32 64 64 32 64

Ciprofloxacin

8 9 10 11 19 20

Not identified E.coli P. vulgaris E. coli S. aureus S. lactis

2 2 1 2 2 1

4 8 4 4 8 4

gel electrophoresis, the three cured isolate showed that they had lost their plasmid. However, the growth of isolates 7 in cefixime and isolates 21 in ciprofloxacin indicate that their resistance is not dependent on the presence of plasmid (data not shown). Therefore, a transformation study was done to confirm that the antibiotic resistance is plasmid mediated. Transformation by plasmid on DH5α from isolate

14 was performed to confirm that plasmid was responsible for the resistance. After transformation, the bacteria was placed on media containing amoxicillin antibiotics and incubated at 37°C and after 48 h of incubation small colonies were observed. The colonies were then analyzed for amoxicillin susceptibility, and it was found that the transformed isolates were fully resistant to amoxicillin (data not shown).

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Table 4. Antibiotic sensitivity pattern of the 22 isolates against 8 commonly used antibiotics. Isolate No.

Suspected Microorganisms

Resistant

Intermediate Sensitive

E.coli

Amp, R, OT

Cip, Cef, S,C

S. dysenteriae

Amp, OT

Cip, Cef, C,S,OT,R

K. pneumonia

Amp, C, Cef, R, S,OT

CFM

Cip

K. pneumonia

Amp, C, Cef, R,OT

CFM

Cip, S

S. Typhimurium

Amp, Cef, CFM

Cip,R, S,OT

K. pneumonia

Amp, R, CFM

C, Cef, S, OT, Cip

E .coli

Amp, Cip, Cef, OT, CFM

C,R

Not identified

Cef

Amp, S, C, Cip, R, OT

E. coli

Amp, C, Cef, Cip, R,OT

P. vulgaris

Amp, C, Cef, Cip, R,OT

E.coli

Amp, S

C, Cef, Cip, R, OT,

M. lucteus

Amp, Cef, OT

C, Cip, R

C. xerosis

C, Cip, S

Amp

Cef, R,OT

E. coli

Amp, Cip, Cef, OT

C,S,R

K. pneumonia

Amp, C, Cef, R,S

Cip, OT

M. lucteus

Amp, Cef, OT

S, Cip, C

M. lucteus

Amp, C, Cef, Cip, S,OT, CFM R

M. lucteus

CFM

Amp, C, Cef, Cip, S, R, OT

S. aureus

R,OT

Cef

Amp,C,Cip,S

S. lactis

S,R

Amp, Cef, C, Cip

S. Typhimurium

Amp, Cef, C,Cip, OT

C,S

S. aureus

Cef, R,OT

Amp, C, Cip, S

Abbreviations: S= Streptomycin, Cip= Ciprofloxacin, A = Ampicillin, R = Rifampicin, OT= Oxytetracyclin, Cef= Cephalosporin, CFM= Cefixime and C= Chloramphenicol .

From plasmid profile, curing and transformation study, it can be concluded that resistance to amoxicillin is plasmid mediated but for cefixime and ciprofloxacin resistance was not plasmid mediated. The presence of plasmid mediated resistance for amoxicillin has also been reported by Cattoir et al. (2008). The presence of a similar plasmid in cefixime resistant bacteria has been reported by Kalantar et al. (2011). The ciprofloxacin resistant isolates plasmid profiles shows similarity to the data reported by Ferrero et al. (1995). These study results demon-

or occurs due to chromosomal mutation(s) or by acquiring extra-chromosomal DNA plasmid (Mandal et al., 2004).

strated that the plasmid is one of the important ways to spread resistance but chromosomal mutation by environmental selection might also responsible for resistance. Acquisition of antibiotic resistant properties of a bacterium could be its inherent properties

mation study results demonstrated that resistance to amoxicillin is plasmid mediated but for cefixime and ciprofloxacin were not. These study results demonstrated that the plasmid is one of the important ways to spread resistance but chromosomal mutation by

147

CONCLUSIONS In conclusion, the result of this study demonstrated that the fresh vegetable samples collected from local markets were heavily contaminated with resistant bacteria and is of special concern for human consumption. Plasmid profile, curing and transfor-

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environmental selection might also responsible for resistance. Further research is needed to determine all major sources of antibiotic resistant food borne pathogens in fresh produce. Data on the prevalence and types of antibiotic resistance in microorganisms isolated from fresh produce may help explain the role of foods in the transmission of antibiotic-resistant strains to human populations.

American Society of Microbiology. 1981. Manual for general bacteriology. Ourfood.com. http://our-

ics, science shows. Environmental Health News. Environmentalhealthnews.org. Available at http:// www.environmentalhealthnews.org/ehs/news/antibiotics-in-crops. Accessed January, 2011. Ferrero, L., B. Cameron, J. Crouzet. 1995. Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutant of Staphylococcus aureus. Antimicrob. Agents Chemother. 39:1554– 1558. Kalantar, E., F. Soheili, H, Salimi, D. M. Soltan. 2011. Frequency, antimicrobial susceptibility and plasmid profiles of Escherichia coli pathotypes obtained from children with acute diarrhea. Jundishapur J. Microbiol. 4:23-28.

food.com/general_bacteriology.html. Accessed July, 2010. Bauer, A. W., W. M. Kirby, J. C. Sheris, M. Turck. 1996. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45:149-158. Beuchat, L. R. 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59:204– 216. Birnboim, H. C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513–1523. Cappuccino, J. G., and N. Sherman. 1990. Microbiology: A laboratory manual. 4th Edition. The Benjamin-Cumming Publishing Company, Inc., Menlopark, CA. 186 p. Cattoir, V., L. Poirel, P. Nordmann. 2008. Plasmid-mediated quinolone resistance pump QepA2 in an Escherichia coli isolate from France. Antimicrob. Agents Chemother. 52:3801–3804. CDC. 2005. National Antimicrobial Resistance Monitoring System (NARMS) Frequently Asked Questions (FAQ) About Antibiotic Resistance - Why is antibiotic resistance a food safety problem? Cdc. gov. http://www.cdc.gov/narms/faq_pages/5.htm. Accessed January, 2011. CDC. 2006. Progress report: Implementation of a

Kilonzo Nthenge, A., E. Rotich, C. Thompson. 2009. Antibiotic-resistant bacteria isolated from organic and convectional fresh produce. National Institute of Food Technology p. 49. http://www.reeis.usda. gov/web/crisprojectpages/213697.html Accessed on May 2011. Kumar, K., S. C. Gupta, S. K. Baidoo, Y. Chander, C. J. Rosen. 2005. Antibiotic uptake by plants from soil fertilized with animal manure. J. Environ. Qual. 34: 2082-2085. Kümmerer, K. and A. Henninger. 2004. Promoting resistance by the emission of antibiotics from hospitals and households into effluents. Clin Microbiol Infect. 9:1203–1214. Kümmerer, K. 2009. Antibiotics in the aquatic environment – A review – Part II. Chemosphere 75: 435-44. Klech, W. J. and L. S. Lee. 1978. Antibiotic resistance patterns of Gram-negative bacteria isolated from environmental sources. Appl. Environ. Microbiol. 36:450-456. Mandal, S., M. D. Mandal, N. K. Pal 2004. Plasmidencoded multidrug resistance of Salmonella typhi and some enteric bacteria in and around Kolkata, India: a preliminary study. Online J. Health Allied Scs. 4:1-7.

public health action plan to combat antimicrobial resistance. Cdc.gov. http://www.cdc.gov/drugresistance/actionplan/2005report/CDC_6-21-06.pdf, Accessed January, 2011. Cimitile, M. 2009. Crops absorb livestock antibiot-

NACMCF (National Advisory Committee on Microbiological Criteria for Foods). 1999. Microbiological safety evaluation and recommendations on fresh produce. Food Control 10:117–143. Nemi, M., M. Sibakov, S. Niemela. 1983. Antibiotic

REFERENCES

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resistance among different species of fecal coliforms isolated from water samples. Appl. Environ. Microbiol. 45:79-83. Sambrook, J., E. F. Fritsch, T. Maniatis 1989. Molecular cloning: A laboratory manual. Second edition, Cold Spring Harbor Laboratory Press. 1:74-135. Sanches, N. R., D. A. Garcia, M.S. Schiavini, C. V. Nakamura, B. P. D. Filho. 2005. An evaluation of antibacterial activities of Psidium guajava. Brazillian Arch. Bio and Technol. 48:429-436. Schwartz, T., T. Kohnen, B. Jansen. 2003. Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiol. Ecol. 43:325–35. Tomeda, M., M. Inuzuka, N. Kubo, S. Nakamura. 1968. Effective elimination of drug resistance in sex factors in Escherichia coli by sodium dodecyl sulfate. J. Bacteriol. 95:1078-1089. Turnidge, J. D., M. J. Ferraro, J. H. Jorgensen. 2003. Susceptibility test methods: General considerations. In: P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, R. H. Yolken. Manual of clinical microbiology. 8th Ed. American Society of Clinical Microbiology, ASM Press, Washington. p 1102-1107. Willis, C. 2000. Antibiotics in the food chain: their impact on the consumer. Rev. Med. Microbiol. 11:153-160. Woodford, N. and M. J. Ellington. 2007. The emergence of antibiotic resistance by mutation. Clin. Microbiol. Infect. 13:5–18.

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BRIEF COMMUNICATIONS Impact of Calcium Chloride Dip and Temperature on Microbial Quality of Organically and Conventionally Grown Melons H. T. Aldrich1, L. Goodridge2, M. Bunning1, C. Stushnoff3 and P. Kendall1* Department of Food Science and Human Nutrition 2 Department of Animal Sciences 3 Department of Horticulture and Landscape Architecture Colorado State University, Fort Collins, CO 80523 1

ABSTRACT Melons (Cucumis melo L.) have been associated with many foodborne illness outbreaks. Current melon handling recommendations do not require cool temperature storage to maintain the safety of whole melons, yet storage temperature often influences microbial growth. While calcium chloride (CaCl2) dips have been shown to reduce post-harvest decay and increase shelf-life of many different fruits, the anti-microbial effect is unknown. It is also unclear whether production method (organic or conventional) impacts microbial growth on fresh produce. This study evaluated the impact of a 20 min 0.08 M CaCl2 post-harvest dip treatment and two storage temperatures (10˚C and 21˚C) on total aerobic and Enterobacteriaceae bacterial counts present on the surface of organically and conventionally grown melons (cultivar ‘Arava’) stored for 10 d. Storage temperature significantly impacted microbial growth, as higher levels of aerobic and Enterobacteriaceae bacteria grew overall on melons stored at 21˚C vs. 10˚C (p<0.05). Storage time did not impact bacterial counts within the 10 d. Organic melons had overall more aerobic growth than conventional melons (p<0.05), with non-dipped organic melons stored at 21˚C having the highest aerobic bacterial counts. Organic melons treated with CaCl2 and stored at 21˚C had lower (p<0.05) Enterobacteriaceae levels than non-dipped organic melons stored at 21˚C. Based on the results of this study, storing whole ‘Arava’ melons at cooler temperatures is best to slow microbial growth, and the use of a CaCl2 dip treatment may be a useful method to minimize growth on organic melons. Keywords: post-harvest storage, calcium chloride, food safety, aerobic bacteria, Enterobacteriaceae, organic production, melons, storage temperature Agric. Food Anal. Bacteriol. 1: 150-158, 2011

INTRODUCTION

ing the last thirty years (Sivapalasingam et al., 2004; Doyle and Erickson, 2008). Fresh fruits and vegetables

Foodborne illness outbreaks associated with fresh produce have increased in the United States dur-

are especially vulnerable to causing foodborne outbreaks due to the fact that they are often consumed raw or minimally processed and each step, from planting through post-harvest handling, may contribute to the microbial load (Johnston et al., 2005).

Correspondence: Patricia Kendall, Patricia.Kendall@colostate.edu, Tel: +1 -970-491-0799

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Melons (Cucumis melo L.) are a popular fruit in the United States that have been associated with numerous foodborne illness outbreaks. There were at least 28 documented outbreaks associated with cantaloupes and muskmelons between 1984 and 2003 (Bowen et al., 2006), indicating a strong need to increase the safety of fresh melons. The foodborne agents implicated in the contamination events included Salmonella enterica, Campylobacter jejuni, Escherichia coli O157:H7, and norovirus, with both domestic and imported melons implicated in past outbreaks. There is some concern among public health experts that organic production methods increase the

et al., 2007). One such calcium treatment showing positive results on fruits such as strawberries, lemons, peaches, and melons has been calcium chloride (CaCl2) dips (Garcia et al., 1996; Lester and Grusak, 2001; Tsantili et al., 2002; Manganaris et al., 2007; Martin-Diana et al., 2007; Lysiak et al., 2008). Concentrations of CaCl2 used in the above studies have ranged from 0.045-0.27 M, with the recommended concentrations falling within the 0.06-0.09 M range, depending on the fruit being studied and whether the fruit was treated whole or fresh-cut (Garcia et al., 1996; Lester and Grusak, 2001; Tsantili et al., 2002; Manganaris et al., 2007; Martin-Diana et al., 2007; Lysiak et al., 2008). The optimal CaCl2 concentration

risk for microbial contamination due to increased manure use, compared to using chemical fertilizers in conventional farming (Stephenson, 1997a,b). Because organic foods are one of the fastest growing food categories with sales increasing nearly 20% each year since 1990 (Winter and Davis, 2006), the impact of organic production practices on food safety remains an important issue to evaluate. Studies assessing consumer perceptions of organic produce have often found that consumers think organic produce is safer, more nutritious, and better tasting than conventionally grown produce (Torjusen et al., 2001; Magnusson et al., 2003; Shepherd et al., 2005; Yiridoe et al., 2005). However, research comparing such attributes has produced inconsistent or inconclusive results, most likely due to unparallel growing conditions, cultivar choices, and other uncontrolled variables (Harker, 2004; Lester, 2006). Well-controlled studies are needed to better understand the impact of organic and conventional growing methods on microbial growth on fresh produce. Calcium plays an important role in maintaining cell wall structure in fruit by interacting with pectic acid to form calcium pectate, which has a firming effect on cell walls; thus, calcium deficiency during the growing process has been shown to cause a vari-

found to slow senescence without any negative side effects in a study using whole honeydew melons was 0.08 M (Lester and Grusak, 2001). To our knowledge, the microbial effects of using CaCl2 as a post-harvest treatment have not been studied. Research by Chikthimmah et al. (2005) found that using CaCl2 in mushroom irrigation water during crop production resulted in lower levels of microbial growth during storage compared to mushrooms grown without CaCl2 added to the irrigation water, indicating the potential of such a treatment to impact microbial levels following harvest. This study was designed to assess selected postharvest effects on aerobic and Enterobacteriaceae microflora commonly found on fresh produce. Testing for aerobic bacteria provided an indicator of the impact of the post-harvest treatments on all bacteria that grow in oxygenated conditions, including spoilage as well as pathogenic organisms. Evaluating Enterobacteriaceae bacterial levels provided information on how the post-harvest treatments affected bacteria in this Gram-negative family, which includes common food pathogens, such as Salmonella spp., Shigella spp., and enterohemorrhagic Escherichia coli (Varnam and Evans, 1991). According to current melon handling recommen-

ety of physiological disorders in produce (Poovaiah, 1986). Because of the role calcium has in maintaining cell walls, researchers have investigated the use of post-harvest calcium treatments to increase the quality and shelf-life of fruit after harvest (Martin-Diana

dations (Fleming and Pool, 2005), cool temperature storage is not required to maintain the safety of whole melons. However, a study evaluating the effect of temperature on inoculated whole cantaloupe melons found significantly more microbial growth

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occurred at 19˚C than 4˚C (Annous et al., 2004). Most whole melon storage temperature recommendations are based on preventing chilling injury among commonly grown cultivars (Salunkhe and Kadam, 1998; Thompson, 2003). There is a lack of research available on optimal storage conditions for specialty melon cultivars such as ‘Arava.’ This project evaluated the impact of storage temperature and a CaCl2 dip treatment on total aerobic and Enterobacteriaceae bacterial concentrations present on organically and conventionally grown ‘Arava’ melons stored for up to 10 d.

MATERIALS AND METHODS Plant Material Galia melons (cultivar ‘Arava’) (Johnny’s Selected Seeds, Winslow, ME) were grown at the Colorado State University Horticulture Field Research Center in Fort Collins, CO during the summer of 2007. ‘Arava’ melons have a netted rind that is a grey-green color while growing and turns light yellow when ripe. Fruit flesh of ‘Arava’ melons is light green and the melons require approximately 80-90 d to reach maturity. Melons were grown simultaneously on organic and conventional plots spaced 50 m apart. Soil at the Horticulture Research Center is classified as Nunn clay with a pH of 7.8 and the organic plots have been USDA certified organic since 2001. Prior to planting, soil tests were conducted on both plots. Compared to the conventional plots, the certified organic plots contained 2.0-2.4% higher levels of organic matter derived from green manure plough-down of legume and cereal cover crops and from thoroughly composted chicken manure. The nitrogen, phosphorus, and potassium contents of the two plots were made approximately equivalent using organic or conventional fertilizers prior to field planting. For the organic plot, ‘Evergreen’ poultry compost (A1 Organics, Eaton, CO) was applied with a Millcreek spreader (Millcreek Manufacturing Co, Lancaster County, PA) and rototilled into the soil. To match nutrient levels in the organic fertilizer, urea (45-0-0) and triple superphosphate (0-20.1-0) were

applied to the conventional plot using a broadcast spreader. Melon plants were started in the Colorado State University Plant Environmental Research Center’s greenhouses in 3-in. peat pots using Sunshine Organic Basic planting media (Sun Gro Horticulture, Bellvue, WA) with 20% vermicompost (local source). After four weeks, the melons were transplanted to the field, spaced evenly in black plastic mulched beds (rows 24 in. apart and beds 50 in. apart). Crops were irrigated using drip irrigation with municipal water. Irrigation levels were determined using ‘Watermark’ granular matrix sensors (Irrometer Company, Riverside, CA). Irrigation levels were monitored to ensure the melons were watered adequately to prevent plant moisture stress throughout the growing season. During the growing season, pest management practices were used to minimize cucumber beetle (Acalymma vittatum) pressure on the melons. Synthetic insecticide Permethrin (Loveland Products Inc., Greeley, CO) was applied to the conventional plots and naturally derived pyrethrum (MGK Co., Golden Valley, MN) was used on the organic plots. Once the ‘Arava’ melons reached peak maturity (as indicated by light yellow rind and nearly full slip off the vine), they were harvested manually early in the morning, then transported at ambient temperature to the laboratory for processing within 30 min.

Treatments Organically and conventionally grown melons (17 ± 1-cm diam) were randomly assigned into CaCl2 dip and non-dip (control) groups (total n=144). Any visible soil was brushed off melons using paper towels. Half of all organically and conventionally grown melons were dipped in a 0.08 M CaCl2 solution (8.8 g CaCl2 per L of water) and half were left untreated. The CaCl2 concentration was chosen based on favorable results to increase shelf-life of fruit in other studies (Garcia et al., 1996; Lester and Grusak, 2001; Tsantili et al., 2002; Manganaris et al., 2007) as well as preliminary research conducted in our lab. For the dip, food grade CaCl2 (DOW Chemical

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Company, Midland, MI) was mixed with tap water (21 ± 1˚C) in 68 L plastic tubs (Sterile, Townsend, MA) until dissolved. Each growing method/storage time treatment set of melons (n=12) was completely immersed in the CaCl2 solution for 20 min, then removed, allowed to air dry on paper towels for 1 h and randomly assigned to room or refrigerated storage. Fresh dip solution was made for each set of dipped melons. All melons and dipping solutions were kept at ambient temperature (21± 1˚C) throughout the dipping and drying steps. Melons were then individually wrapped loosely in tissue paper labeled with sample ID information and placed into new 30.5×38.1×25.4-cm cardboard

samples were plated on aerobic and Enterobacteriaceae Petrifilm™ (3M, St. Paul, MN) according to manufacturer’s instructions. All Petrifilm™ plates were incubated overnight (37˚C). Colonies for each sample were counted on plates that contained 25250 colonies and expressed as CFU/mL.

boxes (Weyerhaeuser, Federal Way, WA), keeping treatment groups separate. Melons were stored at 21± 1˚C (relative humidity 30 ± 5%) or 10 ± 1˚C (relative humidity 70 ± 5%). At d 1, 5, and 10, total aerobic and Enterobacteriaceae counts were determined, with six melons evaluated individually per treatment group as test replications.

for multiple comparisons. Fixed effects included time, temperature, dip, and growing method; replication was included as a random effect.

Microbial Analysis

Data Analysis Results were transformed into log scale and analyzed using SAS Proc Mixed (Version 9.1, Cary, NC). A factorial analysis of variance was performed with differences between means assessed using a significance of p<0.05 with the Tukey-Kramer adjustment

RESULTS Aerobic Counts

Microbial testing was based on methods used by the United States Department of Agriculture’s (USDA) Microbiological Data Program (MDP, 20022006; MDP, 2003-2007) and modified for our lab (K. McCallum (Colorado Department of Agriculture, Denver, CO, personal communication)). Each whole melon was placed in a 38.1×50.8cm sterile bag (VWR, West Chester, PA) to which 300 mL of a solution of Universal Pre-Enrichment Broth (UPEB) (Difco, Sparks, MD) and 0.1% Tween 80 (Fisher Scientific, Fair Lawn, NJ) were added. The bags were sealed with a twist tie. The bagged melons were shaken for 20 up and down strokes and 20 side to side strokes to assure the UPEB solution adequately “washed” all surfaces of the melon, then stored with the melon remaining in the bag at 5˚C for

The mean aerobic bacterial counts recovered from the melon rinds ranged from 4.56 to 8.28 log CFU/ mL. Storage time did not impact aerobic growth (p>0.05), but storage temperature, dip treatment, and growing method did have significant overall affects on aerobic bacterial levels (p<0.05). Since overall storage time was not significant, results from d 1, 5, and 10 were combined (Figure 1). Overall, aerobic counts were lower (p<0.01) on CaCl2 dipped melons compared to non-dipped control melons (6.16 vs. 6.54 log CFU/mL, respectively). Also, melons stored at 10˚C had overall lower bacterial growth (p<0.0001) than those stored at 21˚C (5.98 vs. 6.72 log CFU/mL, respectively). Within each temperature/growing method combination, treatment with CaCl2 did not significantly impact aerobic counts, but CaCl2 dipped melons stored at 10˚C had the lowest bacterial counts (p<0.05), regardless

18-24 h. A 5 mL sample was taken from the bagged UPEB solution and transferred to a sterile Falcon tube (BD Falcon™, Franklin Lakes, NJ). Six, 10-fold serial dilutions were made using buffered peptone water (Difco, Sparks, MD). From each dilution, 1 mL

of growing method. Growing method impacted the presence of aerobic bacteria (p<0.05), with organically grown melons having higher overall mean aerobic counts than conventional melons (6.49 vs. 6.21 log CFU/mL, respectively). Much of this difference

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Log CFU/mL

Dip 7 No Dip 6 5 4 Figure 31. Aerobic and Enterobacteriaceae bacterial counts (log CFU/mL) of organically and conAerobic 2 ˚C 10 ˚C 21 in ˚Ca 0.08 M CaCl 21 ˚Csolution for 20 minutes ventionally grown10 ‘Arava’ melons. Melons were immersed 2 1 Organic Conventional Organic Conventional or not treated then stored at 10 or 21˚C. Each bar represents the mean bacterial counts of organic 0 CaCl2 Dip 5.67 5.64 6.69 6.65 Organic Conventional Organic Conventional or Dip conventional melons by storage treatment (n=18 per bar). Error bars No 6.45 6.16 7.15 6.41indicate standard error.

Within each graph, lowercase letters are not significantly 10 ˚Cbars with similar 10 ˚C 21 ˚C 21 ˚C different (p<0.05).

Log CFU/mL

SE=0.19

8 7 6 5 4 3 2 1 0

Aerobic Results

abc

Organic Conventional

Organic Enterobac

10 ˚C 10 ˚C Organic

Log CFU/mL

CaCl2 Dip No Dip

10 ˚C

ab 10 ˚C No Dip 6.45 6.16

Organic

10 ˚C CaCl2 Dip cd 5.67 cd 5.64

10 ˚C

bcd

aNo Dip 6.45 6.16

CaCl2 Dip No Dip 21 ˚C 21 ˚C CaCl2 Dip No Dip 6.69 7.15 6.65 6.41

abc

Conventional

10 ˚C 21 ˚C 10 ˚C 21 ˚C Conventional Organic 4.74 Aerobic Results 5.07 5.87 5.11

Organic abc Conventional

10 ˚C Organic Organic

10 ˚C CaCl2 Dip 5.67 5.64

Conventional

CaCl2 Dip No Dip 8 7 6 5 4 3 2 1 Enterobac 0

21 ˚C 21 ˚C Conventional 5.56 6.18 6.50 6.00 21 ˚C

21 ˚C

6.69 6.65

7.15 6.41

ab CaCl2 ab Dip No Dip CaCl2 Dip No Dip

10 ˚C Conventional Conventional 4.74 10 ˚C 5.87

21 ˚C 21 ˚C Organic Conventional Conventional Organic 5.07 5.56 6.18 21 ˚C 21 ˚C 5.11 6.50 6.00

Log CFU/mL

Enterobacteriaceae Results 8 7 6 10 ˚C 10 ˚C 21 ˚C 21 ˚C 5 to organic control (no dip) melons CaCl2 stored Dip No Dipstored at CaCl2 Dip vs. No6.06 Dip CaCl2 was due than melons 21˚C (5.20 log CFU/ 4 Organic 4.74 5.87 5.56 6.5 Dip at 21˚C3 having the highest aerobic counts (Figure 1). mL, respectively). Also, melons dippedNo in CaCl Dip 2 had Conventional 5.07 5.11 6.18 6 lower (p<0.001) overall Enterobacteriaceae counts 2 1 Enterobacteriaceae Counts compared to non-dipped control melons (5.38 vs. 0 5.87 log CFU/mL, respectively). The combination Organic Conventional Organic Conventional

The Enterobacteriaceae bacterial counts ranged 10 ˚C 10 ˚C from 4.19 to 7.15 log CFU/mL, and were significantly affected overall by temperature and dip treatment

of the CaCl2 dip and 10˚C storage temperature also 21 ˚C produced the lowest Enterbacteriaceae counts (Figure 1).

21 ˚C

Enterobacteriaceae Results

(p<0.05). Again, storage time was not significant (p>0.05), so results from d 1, 5, and 10 were com10 ˚C bined (Figure 1). CaCl2 Dip 4.74 at 10˚C Like the aerobicOrganic counts, melons stored Conventional 5.07 had lower (p<0.0001) Enterobacteriaceae counts

Growing method did not impact Enterobacteriaceae counts overall (p>0.05). When looking at the 10 ˚C 21 ˚C 21 ˚C results dip treatment and storage temperature Noby Dip CaCl2 Dip No Dip 5.871), organic and 5.56conventional 6.5 (Figure melons did 5.11 6.18 6 not follow a similar pattern. Organic CaCl2-dipped

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melons had significantly lower (p<0.05) Enterobacteriaceae counts than non-dipped control melons at a given temperature, whereas CaCl2 treatment did not impact conventionally grown melons. In contrast, conventionally-grown melons were significantly impacted (p<0.05) by storage temperature, with lower Enterobacteriaceae counts on melons stored at 10˚C than at 21˚C. Enterobacteriaceae counts on the organic melons were not impacted by storage temperature (Figure 1).

Using CaCl2 dips on fresh whole fruit has been

at room temperature, which is a common practice among consumers, or at cooler postharvest storage temperatures. Since the Enterobacteriaceae family contains many common foodborne pathogens such as Shigella spp., Salmonella spp. and Enterohemorrhagic E. coli (Varnam and Evans, 1991), CaCl2 may potentially increase the safety of organic melons during storage. CaCl2 is approved as a processing aid for organic food production (Code of Federal Regulations, 2008), is relatively inexpensive, and easily accessible (Dow-Chemical, 2007), making it a feasible solution for farmers. The USDA has monitored for the presence of several foodborne pathogens on cantaloupe as well as

shown to be an effective treatment for prolonging the shelf-life as well as improving quality characteristics during storage compared to fruit dipped in a water control (Garcia et al., 1996; Lester and Grusak, 2001; Tsantili et al., 2002; Manganaris et al., 2007) or left dry (Garcia et al., 1996; Lysiak et al., 2008). CaCl2 must be dissolved in water before being applied to fresh fruit; therefore, it is not possible to use a CaCl2 application without submerging fruit in a water-based dip. For the control in this study, we chose to use non-dipped melons, since that is how small-scale farmers in the Rocky Mountain region currently handle their fresh specialty melons (F. Stonaker (Specialty Crops Program Coordinator, Colorado State University Department of Horticulture and Landscape Architecture, personal communication)). CaCl2 dip treatments have been found to have many benefits compared to fruit left untreated or dipped in a water control (Lester and Grusak, 2001; Manganaris et al., 2007; Martin-Diana et al., 2007; Lysiak et al., 2008), so we wanted to compare the use of such a dip to the existing post-harvest handling methods used by small-scale farmers. The results of this study warrant further research with additional produce, with additional controls, and with inoculated samples to establish the role CaCl2 dips may have in

alfalfa sprouts, pre-cut bagged lettuce, spinach, and tomatoes since 2002 as part of the Microbiological Data Program (MDP). Among the thousands of samples tested for this program between 2002-2006, only 0.17% of cantaloupe samples tested were positive for Salmonella spp. and 0.24% for pathogenic E. coli (K. McCallum (Colorado Department of Agriculture, Denver, CO, personal communication) (MDP, 2002-2006). Though this shows pathogens can be found on cantaloupe, due to the very low rates of contamination, it would be difficult to determine post-harvest treatment effects on specific foodborne pathogens without inoculating the produce. The effect of storage temperature on microbial growth seen in this study is consistent with the results from other studies showing lower bacterial growth at lower temperatures for inoculated whole melons (Annous et al., 2004), as well as for fresh-cut produce (Zagory, 1999; Francis and O’Beirne, 2001). Storing melons at cooler temperatures may be an effective solution for limiting post-harvest microbial growth. Yet, many melons are susceptible to chilling injury when storage temperatures are decreased, and the specific temperature causing chilling injury symptoms varies greatly by cultivar (Miccolis and Saltveit, 1995). Most storage recommendations have been

minimizing microbial growth. The CaCl2 dip in this study appears to be a promising option for decreasing the bacterial counts on the surface of whole ‘Arava’ melons, especially when the melons are grown organically and stored either

based on commonly-grown commercial cantaloupe or honeydew cultivars (Salunkhe and Kadam, 1998; Thompson, 2003). We did not test for chilling injury in this study, but it would be useful to determine the effect of lower storage temperatures on quality and

DISCUSSION

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sensory characteristics of specialty cultivars such as ‘Arava.’ In this study, organic melons were observed to have higher levels of aerobic bacteria compared to conventionally grown melons, primarily due to significantly higher counts on non-dipped control organic melons stored at 21˚C. This indicates organic ‘Arava’ melons may be more conducive than conventionally grown melons to higher levels of microbial growth after storage at room temperature for several days. This is a potential concern because growers as well as consumers may store melons at room temperature before the fruit is consumed. Enterobacteriaceae counts were not significantly different between

melons the results may not be the same for whole melons, as the physiology of cut melons has been shown to be distinctly different than that of whole fruit (Lamikanra et al., 2003). Further assessment of the value of dipping whole melons in CaCl2 as well as sensory effects of CaCl2 on different melon cultivars is important to assure consumer acceptance.

the two growing methods, yet the growing method affected how the melons responded to dip treatment and storage temperature in this study. Other studies comparing organic and conventional produce safety have used produce from different farms (Magkos et al., 2006), which would greatly confound the results. Based on our research, it appears that growing method may impact the general microflora of ‘Arava’ melons when stored at room temperature as well as impacting possible methods to minimize Enterobacteriaceae bacteria. Additional well-controlled research is needed to determine the growing method effect on other produce as well as on specific microorganisms. Overall, time (up to 10 d) was not a significant factor impacting microbial growth in this study. This is encouraging as melons are often stored by growers or consumers for several days before consumption. However, other variables, such as temperature and growing method may eventually allow microbial levels to significantly increase over time. The sensory impact of using a CaCl2 treatment should also be addressed. Research on whole melons dipped in a 0.08 M CaCl2 solution indicate there may be no negative sensory effects (Lester and Grusak, 2001), yet another study found higher bitterness

ods. This risk may be minimized through the use of a CaCl2 dip at harvest. Regardless of growing method, storing whole ‘Arava’ melons at cooler temperatures is an effective method for slowing bacteria growth. Additional research should be conducted to explore the potential for a CaCl2 dip to decrease specific foodborne pathogens on melons and other produce as well as treatment and storage effects on sensory and quality characteristics.

and lower melon flavor scores for fresh cut melons dipped in 1% and 2.5% CaCl2 solutions (approximately 0.09 and 0.23 M, respectively) compared to the control and other treatments (Luna-Guzman and Barrett, 2000). Since the latter study used fresh cut

Bowen, A., A. Fry, G. Richards, and L. Beuchat. 2006. Infections associated with cantaloupe consumption: a public health concern. Epidemiol. Infect. 134:675-685. Chikthimmah N., L. F. LaBorde, and R. B. Beelman.

CONCLUSIONS Based on the results of this study, the common practice of storing melons at room temperature may pose a greater microbial risk for melons grown using organic compared to conventional production meth-

ACKNOWLEDGEMENTS This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2005-55618-15634. The assistance of Frank Stonaker and the horticultural crew at the CSU Horticulture Field Research Center in growing the melons is gratefully acknowledged.

REFERENCES Annous, B. A., A. Burke, and J. E. Sites. 2004. Surface pasteurization of whole fresh cantaloupes inoculated with Salmonella poona or Escherichia coli. J. Food Prot. 67:1876-1885.

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2005. Hydrogen peroxide and calcium chloride added to irrigation water as a strategy to reduce bacterial populations and improve quality of fresh mushrooms. J. Food Sci. 70:M273-M278. Code of Federal Regulations. 2008. Available from: http://ecfr.gpoaccess.gov/cgi/t/text/text-idx?c=e cfr&sid=4410780cb6a0c74813c0c51973cca3ff&rgn =div5&view=text&node=7:3.1.1.9.31&idno=7. Accessed May, 2008. Dow-Chemical. 2007. Calcium Chloride Product information. Available from: http://www.dow.com/ calcium/. Accessed June, 2007. Doyle, M .P., and M. C. Erickson. 2008. Summer meeting 2007 - the problems with fresh produce:

lines for comparison studies. HortScience. 41:296300. Lester, G. E., and M. A. Grusak. 2001. Postharvest application of chelated and nonchelated calcium dip treatments to commercially grown honeydew melons: Effects on peel attributes, tissue calcium concentration, quality, and consumer preference following storage. HortTechnology. 11:561-566. Luna-Guzman, I., and D. M. Barrett. 2000. Comparison of calcium chloride and calcium lactate effectiveness in maintaining shelf stability and quality of fresh-cut cantaloupes. Postharvest Biol. Tech. 19:61-72. Lysiak, G., W. J. Florkowski, and S. E. Prussia. 2008.

an overview. J. Appl. Microbiol. 105:317-330. Fleming ,P., and B. Pool. 2005. Commodity Specific Food Safety Guidelines for the Melon Supply Chain. Available from: http://www.fda.gov/ downloads/Food/FoodSafety/Product-SpecificInformation/FruitsVegetablesJuices/GuidanceComplianceRegulatoryInformation/UCM168625.pdf. Accessed April, 2011. Francis, G. A., and D. O’Beirne. 2001. Effects of vegetable type, package atmosphere and storage temperature on growth and survival of Escherichia coli O157:H7 and Listeria monocytogenes. J. Ind. Microbiol. Biot. 27:111-116. Garcia, J. M., S. Herrera, and A. Morilla. 1996. Effects of postharvest dips in calcium chloride on strawberry. J. Agr. Food Chem. 44:30-33. Harker, F. R. 2004. Organic food claims cannot be substantiated through testing of samples intercepted in the marketplace: a horticulturalist’s opinion. Food Qual. Prefer. 15:91-95. Johnston, L. M., L. A. Jaykus, D. Moll, M. C. Martinez, J. Anciso, B. Mora, and C. L. Moe. 2005. A field study of the microbiological quality of fresh produce. J. Food Prot. 68:1840-1847. Lamikanra, O., B. Juaraez, M. A. Watson, and O .A. Richard. 2003. Effect of cutting and storage on

Postharvest calcium chloride application and moisture barrier influence on peach fruit quality. HortTechnology. 18:100-105. Magkos, F., F. Arvaniti, and A. Zampelas. 2006. Organic food: Buying more safety or just peace of mind? A critical review of the literature. Crit. Rev. Food Sci. Nutr. 46:23-56. Magnusson, M. K., A. Arvola, U. K. K. Hursti, L. Aberg, and P. O. Sjoden. 2003. Choice of organic foods is related to perceived consequences for human health and to environmentally friendly behaviour. Appetite. 40:109-117. Manganaris, G. A., M. Vasilakakis, G. Diamantidis, and I. Mignani. 2007. The effect of postharvest calcium application on tissue calcium concentration, quality attributes, incidence of flesh browning and cell wall physicochemical aspects of peach fruits. Food Chem. 100:1385-1392. Martin-Diana, A. B., D. Rico, J. M. Frias, J. M. Barat, G. T. M. Henehan, and C. Barry-Ryan. 2007. Calcium for extending the shelf life of fresh whole and minimally processed fruits and vegetables: a review. Trends Food Sci. Tech. 18:210-218. MDP. 2002-2006. Microbiological Data Program, Program Reports. Available from: http://www.ams. usda.gov/science/MPO/Download.htm. Accessed

sensory traits of cantaloupe melon cultivars with extended postharvest shelf life. J. Sci. Food Agric. 83:702-708. Lester, G. E. 2006. Organic versus conventionally grown produce: Quality differences, and guide-

November, 2007. MDP. 2003-2007. Microbiological Data Program Standard Operating Procedures. Available from: http://www.ams.usda.gov/science/MPO/SOPs. htm. Accessed November, 2007.

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Miccolis, V., and M. E. Saltveit. 1995. Influence of storage period and temperature on the postharvest characteristics of 6 melon (Cucumis melo L., Inodorus Group) cultivars. Postharvest Biol. Tech. 5:211-219. Poovaiah, B.W. 1986. Role of calcium in prolonging storage life of fruits and vegetables. Food Tech. 40:86-89. Salunkhe, D. K., and S. S. Kadam. 1998. Handbook of Vegetable Science and Technology: Production, Composition, Storage, and Processing. Marcel Dekker, New York, NY. Shepherd, R., M. Magnusson, and P. O. Sjoden. 2005. Determinants of consumer behavior related

2005. Comparison of consumer perceptions and preference toward organic versus conventionally produced foods: A review and update of the literature. Renew. Agr. Food Syst. 20:193-205. Zagory, D. 1999. Effects of post-processing handling and packaging on microbial populations. Postharvest Biol. Tech. 15:313-321.

to organic foods. Ambio. 34:352-359. Sivapalasingam, S., C. R. Friedman, L. Cohen, and R. V. Tauxe. 2004. Fresh produce: A growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J. Food Prot. 67:23422353. Stephenson, J. 1997a. Public health experts take aim at a moving target: Foodborne infections. JAMA. 277:97-98. Stephenson, J. 1997b. New approaches for detecting and curtailing foodborne microbial infections. JAMA. 277:1337-1339. Thompson, A. K. 2003. Fruit and Vegetables: Harvesting, Handling, and Storage. Blackwell Pub. Co., Ames, IA. Torjusen, H., G. Lieblein, M. Wandel, and C. A. Francis. 2001. Food system orientation and quality perception among consumers and producers of organic food in Hedmark County, Norway. Food Qual. Prefer. 12:207-216. Tsantili, E., K. Konstantinidis, P. E. Athanasopoulos, and C. Pontikis. 2002. Effects of postharvest calcium treatments on respiration and quality attributes in lemon fruit during storage. J. Hortic. Sci. Biotech. 77:479-484. Varnam, A. H., and M. G. Evans. 1991. Foodborne Pathogens: An Illustrated Text. Wolfe Pub., London. Winter, C. K., and S. F. Davis. 2006. Organic foods. J. Food Sci. 71:R117-R124. Yiridoe, E .K., S. Bonti-Ankomah, and R. C. Martin. Agric. Food Anal. Bacteriol. â&#x20AC;˘ AFABjournal.com â&#x20AC;˘ Vol. 1, Issue 2 - November 2011

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REVIEW Minimizing the Risk of Listeria monocytogenes in Retail Delis by Developing Employee Focused, Cost Effective Training P. G. Crandall1*, J. A. Neal Jr.2, C. A. O’Bryan1, C. A. Murphy3, B. P. Marks4 and S. C. Ricke1 Address: Department of Food Science and Center for Food Safety, University of Arkansas, Fayetteville, AR 72704 2 College of Hotel and Restaurant Management, University of Houston, Houston, TX 77204 3 Department of Curriculum and Instruction, University of Arkansas, Fayetteville, AR 72704 4 Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, MI 48824

ABSTRACT Listeria monocytogenes is the one of most lethal of all food pathogens. It has a high fatality rate among immune compromised individuals and has been shown to contaminate ready-to-eat (RTE) deli meats, which have been linked to several outbreaks of listeriosis. Unfortunately, the incidence of listeriosis has not decreased significantly since 2001 and the 2010 Healthy People goal of 2.4 cases of listeriosis per million consumers has not been met. More than 8 out of 10 of the listeriosis cases linked to delis were attributed to RTE luncheon meats sliced in retail stores, which has led risk assessors to suggest that cross-contamination from the retail deli environment is likely responsible for the stubborn resistance in reducing listeriosis. Research has also shown that most food borne illnesses result from food handler error, which may be minimized when employees are properly trained and transfer their training to their jobs. There is a need for training that is specifically focused on the deli employee which could have a measurable impact in decreasing the risk of L. monocytogenes cross-contamination. Proper motivation and training of employees and managers is vital to keep consumers safe. Thus, there is a crucial need to fill gaps in the knowledge base for designing effective training for newly hired and hard-to-reach employees in a retail food service environment. Keywords: Worker training, delicatessen, Listeria monocytogenes Agric. Food Anal. Bacteriol. 1: 159-174, 2011

INTRODUCTION Every year, food borne illnesses devastate the lives of millions of Americans and take a significant economic toll, considering the costs of medical treatment and lost wages in addition to the costs of food Correspondence: Philip G. Crandall, crandal@uark.edu Tel: +1 -479-575-7686 Fax: +1-479-575-6936

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recalls much of which is paid for by the retailer and manufacturer. Scharff (2010) estimated the health care burden for the U.S. economy to be $152 billion for just the costs of the acute illness and a few of the long-term health-related costs, certainly not the “bottom-line” for the total cost to the U.S. economy. Scharff (2010) continued separating out these costs for individual pathogens. Listeria monocytogenes has the highest cost to the long-term “quality of life”

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and is second only to Vibrio for the highest cost to treat an individual case. L. monocytogenes is 3rd behind Campylobacter and Salmonella, both of which rarely result in mortality, in the total costs for treatments from a single food borne pathogen. One reason that L. monocytogenes accounts for such a disproportionately large portion of this cost burden is because when invasive listeriosis invades the brain or spinal cord, the fatality rates can approach 30%. Survivors may be left with neurological impairments requiring lifelong, constant care. Pregnant women account for approximately 30% of the cases of listeriosis, resulting in miscarriages, still- or premature births, and 20% of the surviving

ress in reducing L. monocytogenes cross-contamination in the retail deli. First, all currently available educational approaches need to be critically evaluated, including: 1) determining the strengths, weaknesses, knowledge content and usability of currently available Computer Based Trainings (CBT) for deli managers and hourly employees and 2) adapting the traditional face-to-face deli employee trainings to include newly developed techniques to minimize L. monocytogenes cross-contamination. Employers are required by he latest edition of the FDA Model Food Code (FDA, 2009) that serves as the basis of most health regulations for delis, to train and keep written documentation of training for employees

infants require lifelong care (Roberts and Pinner, 1990). Despite a wholesale commitment on the part of the food industry and Federal regulators to minimize this problem, the incidence of listeriosis has not decreased significantly since 2001. The current estimate of listeriosis in the U.S. is 2.7 cases/million persons, which is considerably higher than the 2010 Healthy People goal of 2.4 cases/million (CDC, 2009). Kause (2009) has stated that although FSIS in-plant testing programs have reported a dramatic decline in L. monocytogenes from deli ready-to-eat (RTE) meat and poultry products from 1990 to 2008, a corresponding decline in cases of listeriosis has not occurred. Furthermore, 83% of the listeriosis cases attributed to delis were attributed to RTE luncheon meats sliced at retail deli stores, which led Kause (2009) to suggest that cross-contamination from the retail environment is likely responsible for the plateau in reducing listeriosis in the U.S. Therefore, training that is specifically designed for deli managers and employees could have a measurable impact in decreasing the risk of L. monocytogenes cross-contamination. Cost-effective training can be accomplished through cooperative training efforts involving the industry, regulators and university researchers and educators. Cleaning a cutting

handling food. Secondly, there is a need to improve L. monocytogenes sanitation procedures by 1) filling knowledge gaps in current quantitative risk assessment for RTE foods by developing transfer coefficients for L. monocytogenes cross-contamination that can result from the of behavior of employees working improperly in potentially L. monocytogenes contaminated deli environments, 2) using a visual indicator for meat and fat residues to verify the cleaning of slicers and food contact surfaces prior to sanitation, 3) introducing a lethal kill step capable of reducing L. monocytogenes contamination by 5 logs and 4) modeling the risk reduction by verifying comprehension and permanent changes in employee behavior. Thirdly, new sanitation techniques need to be incorporated into the training and documentation must be made for changes in behavior of new and hard-to-reach employees, who may not have been effectively trained using previous interventions proposed to reduce L. monocytogenes cross contamination. Achieving the twin goals of cost-effective education of deli employees and improved sanitation procedures will require a coordinated research and education effort with the prospect of a verifiable decrease in the risk from L. monocytogenes cross-contamination in retail delis.

board or sanitizing a floor drain may seem to be mundane tasks, but they are critically important. Proper motivation and training of employees and managers is vital to keep consumers safe (Bricher, 2007). However, two issues must be addressed to make prog-

Regulations Retail delis are unique in that they may have to be knowledgeable of and compliant with regulatory

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Figure 1. Factors influencing Listeria monocytogenes Contamination of Deli Products Source of Listeria Listeria monocytogenes from a variety of sources: cutting boards, shoes, gloves, floors, drains, aerosols, coolers, mats, etc.

Cross-contamination potential factors

Contamination

Effectiveness of sanitation Transfer coefficients for L. monocytogenes

L. monocytogenes levels in final deli product.

Worker effect

statutes from multiple and sometimes confusing ju-

ability to the risk transfer model. Finally, new online

risdictions including: three levels of health departments--county, municipal, and state health departments, and requirements set out by the FDA Model Food Code. New regulations targeting reduced risk from L. monocytogenes in RTE foods must be based on quantitative risk assessment models. Currently, a significant number of assumptions have had to be incorporated into these risk models, because there is a lack of research data on L. monocytogenes transfer rates from contaminated surfaces to foods and attachment and cross-contamination from food contact surfaces. Additionally, the impact of practices and behaviors of retail deli employees must also be targeted to collect actual data to replace risk assessors’ assumptions. This could be termed the “worker effect” for risk assessment models. This review will assess the characteristics, strengths, weaknesses, completeness of information and the level of knowledge content, style of presentation and usability of current retail food safety training platforms for deli managers and hourly employees. As part of employees’ “hands-on” training, the use of fluorescent compounds has been shown to be an effective indicator of potential behaviors that can lead to cross-contamination. The use of these fluorescent compounds can be extended to esti-

training modules on sanitation for deli employees and current research that focuses on best practices to reduce or eliminate L. monocytogenes will be discussed, along with the impact of deli specific training targeting new employees and the potential for long-term changes in behavior.

mate the rates of L. monocytogenes cross-contamination, document the effectiveness of new sanitization procedures and identify potential opportunities for modeling supplemental risk assessment data by adding the “worker effect” component to the vari-

products from the same retail store. While these pulse-types were not recovered from patients with listeriosis, it is compelling evidence for the need to improve worker hygiene and cleaning/disinfection programs in conjunction with more effective clean-

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L. MONOCYTOGENES AND THE DELI First, we will discuss a systematic approach to leverage newly published information on the locations of persistent strains of L. monocytogenes isolated from retail delis (Chen et al., 2001; Sauders et al., 2004; Sauders et al., 2009). We will then discuss the requirements for training retail deli managers and employees. In a recent survey of the prevalence of L. monocytogenes in RTE brands of sliced luncheon meats in Europe, significant differences were found between deli meat that was sliced and packaged in a retail deli (8.5% positive) compared to commercially manufactured, sliced and packaged product that had only about 1/3 the incidence, 2.7% positive (Garrido et al., 2009). These researchers went on to publish pulsed-field gel electrophoresis (PFGE) that revealed the same L. monocytogenes pulse-type was repeatedly recovered from the same sliced RTE

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ing practices and constant food worker education for retail delis. L. monocytogenes cross-contamination sources are quite complex and can originate from a variety of sources (Figure 1). Cross-contamination can occur from deli meats to commercial cutting boards, including wood or high density polyethylene (HDPE), to 304 grade stainless steel and to latex, vinyl, or polyethylene in gloves. Consequently there is a need to quantify transfer coefficients to or from soles of shoes, gloves and all types of equipment and environmental surfaces, including stainless steel, cement, ceramic tile and deli flooring material. As an example of factors influencing sequential-contact

where N0 is the number of bacteria on the donor surface prior to a contact event, Nc is the number of bacteria on the donor surface after the contact event, and ki and n are model parameters. Those parameters can be estimated by using non-linear regression to minimize the sum of squared errors between the experimental data (for a given transfer scenario) and the model-predicted counts. Although all three candidate models are essentially empirical, the third is based on prior evidence of the impact of multiple, sequential contact events on the transfer rate, due to concurrent transfer of food product residue on contact. Given that each of the candidate models has a different number of parameters, the best mod-

transfer rates, Campos et al. (2009) demonstrated that the accumulation of food product residue on a slicer blade influenced L. monocytogenes transfer rates to and from deli meat products. In general, the data consistently exhibited a biphasic tendency, with a distinctive log-linear phase (less than 10 contacts), followed by a long tailing phase. The duration of first log-linear phase was product-specific, but corresponded roughly to the point where accumulated product residue exceeded approximately 5 microns in thickness on the blade. This would be consistent with a hypothesis that the mechanisms of bacterial transfer between a food product and equipment surface are significantly different when there is a food barrier thicker than the cells being transferred. This is an example of why any model forms generated for L. monocytogenes should be based on a phenomenological framework for the transfer process being modeled. Phenomenological evidence is based on observations or the results of experiments rather than derived theory basis and the boundaries may be referred to as “fuzzy.” Candidate transfer models to quantify the number of bacteria transferred include: (a) log-linear, (b) Weibull-type, and (c) a biphasic-like model, illustrated here with the example of a two-part, first-order

el should be selected utilizing both qualitative and quantitative criteria, such as Akaike’s Information Criterion (AIC) (Motulsky and Christopoulos, 2004). However, before models can be verified, biological data must be generated to provide input on transfer coefficients. The following section addresses the use of indicators for incorporation into food systems.

kinetic analogy:

the fluorescence indicator even in a nonlinear relationship, as long as the functional form is consistent across the tested cross-contamination transfer scenarios, in order to ensure robustness. The fluorescent indicators can be quantified fluorometrically using the

(a)

(b)

(c)

Fluorescent indicators for estimating cross-contamination The current state-of-the-art in modeling of surface-to-surface bacterial transfer in food processing environments is still essentially empirical. Because of commercial concerns of contaminating food processing plants with a non-pathogenic surrogate, such as Listeria innocua, it would be ideal to use a model fluorescent indicator as a surrogate for L. monocytogenes when assessing delicatessen-type transfer of L. monocytogenes and cross-contamination. In order to test the suitability of a fluorescent indicator surrogate for any system such as a simulated deli, it is not essential that the model parameters be identical between the Listeria and fluorescent indicator results. Rather, it is sufficient if a replicable, functional relationship can be identified between the L. monocytogenes and

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Table 1. Risk factors for foodborne illness at foodservice establishments

maximum excitation wavelength, then measuring the responding fluorescent wavelength compared to a standard curve for this fluorescent compound. Ideally, these measurements would yield a series of transfer coefficients relating the amount of fluorescence to the number of L. monocytogenes cells that would be transferred under identical conditions. A cocktail of L. monocytogenes strains isolated from retail delis together with a fluorescent indicator would allow for simultaneous assessment of the quantitative transfer of both L. monocytogenes and fluorescent indicator from deli meats (turkey, ham, salami) to and from a deli slicer. For example, a retail chub of delicatessen turkey, ham and salami can simulate

Modeling the “worker-effect” on the variability in bacterial transfer events

surface contamination following thermal processing by the manufacturer by directly dip-inoculating in the L. monocytogenes cocktail containing the fluorescent indicator and allowing it to air-dry. Using the fluorescent indicator Glo-Germ®, it has been possible to identify 5 product contact surfaces on deli slicers that were cross-contaminated following the slicing of fluorescent indicator -contaminated cooked, RTE turkey chubs (Vorst et al., 2006). This work served as the basis for the ability to quantify surface-to-product and product-to-surface transfer of L. monocytogenes during slicing of various deli meats (Keskinen et al., 2008; Vorst et al., 2006). More recently, this same research group examined quantitative transfer of E. coli O157:H7 during processing of leafy greens, using Glo-Germ-inoculated head lettuce to identify 23 key product contact surfaces on a commercial-scale flume tank, shaker table, and dewatering centrifuge (Buchholz et al., 2008) for subsequent sampling (Buchholz et al. 2009a,b). Significantly more data must be collected before practical working applications can be created, encompassing a wider range of contact scenarios to build the foundation for phenomenological models, which then can be applied across multiple pathogens and multiple foods. New Standard Sanitary Oper-

The recent draft FSIS risk assessment for L. monocytogenes in deli meats pointed to the fact that there is need for better assessment of how contamination of deli meats at retail occurs (FSIS, 2009). In addition, FDA has assessed foodborne illness risk factors in foodservice operations and the main risk factors determined are listed in Table 1 (FDA, 2010). Deli employee data generated, in a sense of experimental simulations, would provide extremely novel evidence of the interaction among transfer events in this environment. Those data could subsequently be utilized to model the “worker effect” on the overall transfer outcome and the variability in that outcome. This type of analysis typically follows the overall approach (Chen et al., 2001; Schaffner, 2004) to quantify variability in cross-contamination rates among the components of the system (i.e., the food product, the environmental surfaces, and the worker) and in quantifying the impact of the worker (and the impact of the proposed training interventions on that factor). Frequency distributions of aggregate transfer can be generated for a given contaminated food contact surface and non-contaminated surfaces in the deli environment, and the distributions for each test case (pre- and post-transfer) can be statistically

ating Procedures (SSOPs) will need to take into account the recent FSIS 2009 Listeria Risk Assessment and findings, which identify critical cleaning points to be targeted in a retail deli for deli employee specific training (Chen et al., 2001; Sauders et al., 2004, 2009).

compared to test for impact. There have been various studies on worker compliance with practices that reduce foodborne illness risk. Lynch et al. (2005), observed gloves were used by 46% of workers in fast food restaurants. They also

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Risk factors Contaminated equipment Food from unsafe source Improper holding time/temperature Inadequate cooking Poor personal hygiene Hand washing Glove use

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observed that 93% of workers in states that prohibited bare hand contact of food used gloves, while just less than 5% used gloves in states that did not prohibit bare hand contact (Lynch et al., 2005). Lubran et al (2010) in an observational study did not observe bare-hand contact with RTE foods at any time during their study in Maryland, another state that forbids bare hand contact of food. Observance of workers by customers was also cited as an incentive to wearing gloves (Lubran et al., 2010). The FDA (2010) reported that food employees in the deli departments of retail stores were compliant with proper, adequate hand washing procedures 48% of the time. In contrast, Lubran et al. (2010) found that employees at

by fluorescence (Crandall et al., 2009). This process would also allow assessment of cross-contamination of the sink area.

chain stores washed their hands 17% of the recommended times and food employees at independent stores washed their hands 2% of recommended times. Lubran et al. (2010) also found that employees wiped surfaces of equipment with cloths dipped in a sanitizer solution without washing and rinsing the surface first. Of course, sanitizer effectiveness is reduced when it is used on unclean surfaces such as slicer blades, which often contain greasy food residues from slicing luncheon meats and cheeses with high fat content (Vorst et al., 2006).

ture and relative humidity to achieve a 5 log reduction of L. innocua, a more heat stable surrogate for L. monocytogenes (Crandall et al., 2010). Crandall et al. (2010) determined on dissembled slicer food contact surfaces that 77º C for 3 hours under saturated moisture conditions could produce a 5 log reduction of the more heat resistant L. innocua. However, two questions remain: it must be determined whether repeated moist heat treatment of an entire slicer, including the electrical components, immediately followed by an extensive hot drying cycle is practical in a retail deli setting. Secondly, what are the heat transfer rates from a commercial convection oven or moist-heating bread proofing cabinet into the potential L. monocytogenes harborages on a commercial deli slicer?

Modeling transfer coefficients for Listeria monocytogenes Under SSOPs for deli operations, it is vital that any piece of equipment be clean prior to sanitizing (FDA, 2009). However, to systematically assess 1) the vast array of cleaning compounds and 2) cleaning protocols, in addition to variations in equipment and food contact surfaces, known concentrations, mixtures of blended deli meat, and fluorescent indicator would have to be used to purposefully contaminate a number of deli slicers from various manufacturers followed by cleaning according to accepted protocols. After proper disassembly and cleaning the slicer, we found in our lab that testing could be followed by staining any meat residue with a dilute water solution of food grade dye, FD&C 3 and 40, followed by re-cleaning any meat residues prior to quantifying the amount of remaining meat residues

Thermal inactivation of Listeria monocytogenes on food equipment In addition to the FDA mandate to clean all room temperature food contact surfaces “to sight and feel” every 4 hours, additional treatments are required to sanitize potential niches, Listeria harborages, that are difficult to reach with traditional cleaning and sanitizing treatments. Research conducted in our laboratory established threshold time, tempera-

DELI EMPLOYEE TRAINING AND ROLE IN SANITATION Many of the most important issues on sanitation, employee hygiene and training have been recently summarized for deli workers and will only briefly discussed here (Clayton and Griffith, 2008; Endrikat et al., 2010; Gapud, 2009). There are several factors to consider. First, while isolated incidences of food borne illness outbreaks (which involve more than a single individual) garner national publicity, the vast majority of food-borne illnesses are sporadic, where an individual person contracts a life-threatening illness that is not part of a recognized, multiple-per-

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son outbreak (Jones et al., 2004). Secondly, almost half (44%) of American adults eat out daily and more than 40% of the food borne illness outbreaks where a source had been identified were linked to retail food service between 1993 and 1997. Improving the safety of retail environments is an area where a significant impact can be achieved by improving the overall safety of RTE deli foods. Third, there is an ever-increasing, world-wide population who are at increased risk from listeriosis: the elderly, immunocompromised, the very young and pregnant women. In the seven years from 1993 to 2000, the elderly portion of the U.S. population (those over 65) grew 20%, and the elderly currently represent about 1 person

A key reason to focus on deli employee training is that in the majority of sporadic cases of listeriosis it is not possible to identify the specific contaminated food due to long L. monocytogenes incubation times, loss of samples and most consumers’ inability to accurately recall what they have eaten and where they ate during the past several days. For these reasons, many experts believe that improving employee behavior in retail establishments, many of which may also contain environmental L. monocytogenes, could be the best strategy for reducing the incidence of sporadic cases of listeriosis (Varma et al., 2007). However, any form of non-targeted, generalized training could be problematic for broad

in 8 living in the U.S. (U.S. Census Bureau, 2005). In addition, persons 60 and over have been shown to have little awareness of the preventative measures to take to minimize their risk of listeriosis (Cates et al., 2006), suggesting a dangerous combination of higher vulnerability and lack of knowledge. In addition, Dan Engeljohn, Deputy Assistant Administrator for the Food Safety and Inspection Service (FSIS), has been quoted as saying that his agency has “found that deli departments generally have insanitary conditions, which raises the risk that an outbreak of Listeria monocytogenes would occur.” The FSIS’s report estimates that “a person is 7 times more likely to die from listeriosis after eating deli meat sliced by a retailer than in a federal (inspected) plant.” (Johnston, 2009). Current L. monocytogenes risk assessment models have identified RTE deli meats as posing one of the highest risks for consumers to contract listeriosis. The USDA/FDA/ CDC collaborative risk assessment has declared that RTE deli meats pose the single greatest danger for listeriosis. The public spends billions of dollars each year eating from delis, where sliced meats account for almost 40% of deli sales (Anonymous, 2000). In 1998, consumers were found to purchase deli foods an average of 2.5 times per week, an increase from

spectrum implementation in the deli retail industry. Almost half (43%) of deli employees are between the ages of 16 and 24, earning an average of only $215 / week, which ranks these employees right at the bottom pay rate of all industries (Bureau of Labor Statistics, 2009). One in 8 front line supervisors are Hispanic, more than ¼ of the employees speak a language other than English at home, and 1/5 of adults read at or below the 5th grade (Sneed and Strohbehn, 2008). There is potential to significantly improve this situation by conducting culturally appropriate education in partnership with deli retailers and health departments. Only a small number of studies have investigated the behaviors of employees working in potentially L. monocytogenes contaminated retail delis. Furthermore, none of these have focused on comparative evaluation of deli training materials, targeting known L. monocytogenes harborages, developing new risk assessment modeling information, or deli training specific for minority employees.

1.2 times per week just 5 years before in 1994 (Anonymous, 2000). However, FSIS lacks both the legislative authority and staff to inspect the approximately 1 million deli and food service establishments employing an estimated 9 million workers.

puter based training platforms. For example, Frash et al. (2006) measured how well current face-to-face food safety training for food service managers was being transferred to their employees in 1,000 stores across 8 states. The extent of the transfer of food

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Effectiveness of current training Although there have been previous evaluations of training materials, none have been as complete as is possible with more recently developed com-

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safety training was assessed by tracking the number of health code violations reported by individual instore inspections from local health inspectors. They hypothesized that greater knowledge and more training would translate into fewer violations. Unfortunately they found no significant reduction in the number of violations in stores where the manager had greater food safety knowledge. In a second study published more recently, Noble et al. (2009) surveyed more than 150 stores in Canada comparing 1) those that required food safety training for their managers and employees to 2) stores without required food safety training for managers. They also categorized 863 health code violations from 1,417

first thing to evaluate is whether there is a universally accepted set of standards by which the current knowledge base that food service and especially deli employees are being trained. The Model Food Code provides generally accepted minimums, but there are multitudes of varying health department and company regulations.

health department inspections. Once again, there was no significant difference between retail stores requiring food safety training and those not requiring training. They concluded that food handlers did not put their knowledge into practice, as evidenced by the numerous violations of health code regulations that were specifically covered in the training. It can be assumed that the training reported in this research of both the managers and the deli workers was in the traditional face-to-face manager training new employee. What is known is that in these Canadian stores all the trained managers and food handlers had passed an exam and obtained a Food Handler Certification. Consequently there is a need contribute additional understanding to enhancing the “transfer of training” to the target audience in the retail deli.

Whether evaluating face-to-face or computer based training, all training has at least two principal instructional elements that must be considered: 1) the information, the facts that get transmitted to the knowledge realm of the employee and 2) the presentation, the way in which the knowledge is trans-

ning with the 1993 edition (FDA, 2009). The latest revision, due out in early 2011, requires documentation by management of the managers’ food safety training of employees. This complex regulatory situation is well illustrated by the Texas Department of Health Guidelines for Food Establishments (2009), which contains almost 70 references to various health departmental requirements at that must be part of deli employee training and their certification exams. The Retail Food Service Food Safety Consortium was formed to assist in this area and is composed of faculty from 5 universities and 3 national associations. In addition, third party auditors, such AIB, have their own food safety employee training manuals, which are more than 100 pages long. Third party auditors are instructed to “observe employee behavior and ask questions” as the basis of determining the effectiveness of employee food safety training (O’Bryan, 2010). This morass of regulations and lack of an accepted method to verify learning on the part of the employee may contribute to putting consumers at greater risk. Secondly, there is a need to know how food employees are being trained. An informal poll of a group of food processor HACCP managers meeting in a focus group setting, conducted by the authors, revealed several important

mitted. For a training program to be effective, it must not only transmit the appropriate knowledge that is needed, but must also present the knowledge in a learner appropriate manner. When performing an evaluation of existing food training programs, the

points to be considered. They were asked “How do you make the time and how do you train rank and file employees plus new employees when their turn-over rate exceeds 150%?” For the most part, there was consensus that training had to be constant with con-

Training requirements

Need for more targeted training approaches As of 2005, 48 of 56 U.S. state and territories’ health department food safety rules were based on one of the five versions of FDA’s Food Code, begin-

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current explanations provided on correct safety procedures. In addition, group meetings, “hands-on” approaches and belonging to a team were deemed as important. In short, all of these solutions were all traditional face-to-face approaches. While these face-to-face methods are appropriate, they are not always feasible with increased time pressures and dwindling training resources (McEvoy and Buller, 1990). However, utilizing this established training information together with training methods that are supported by technologies may result in better training in a more time efficient manner.

Potential barriers for current training approaches Immigrants are a substantial and growing segment of the U.S. labor force. In 2008, 24.1 million persons (15.6%) of the U.S. labor force were foreignborn (Bureau of Labor Statistics, 2009). In 2000, 25 million of the 31 million foreign-born people residing in the U.S. indicated that they spoke a language other than English at home. (Shin and Bruno, 2003). Despite the language barrier, millions of non-English speaking immigrants work in U.S. labor force and many of them in retail delis. Currently, the foodservice industry is one of the largest employers of Non-English Speaking Individuals (NESI) in the U.S. (National Restaurant Association, 2006). In New York City, home to the most restaurants in the U.S., 67.5% of restaurant workers were immigrants (Restaurant Opportunities Center of New York, 2003). Language barriers are but one of the great impediments to smooth integration of immigrants into a workforce (Loosemore and Lee, 2001; Victor, 1992). The process of communication has multiple variables, including culture, norms, attitudes, social organizations, non-verbal behavior and language (Victor, 1992). Foodservice managers often struggle to communicate with Non-English speakers (Lee and Chon, 2000).

Food safety training platforms Education is considered the teaching of facts and 167

Table 2. Selected computer based food safety employee training programs. System

Website

Alchemy Systems

http://www.alchemysystems.com

ServSafe

http://www.servsafe.com/foodsafety

FMI Super Safe Mark program

http://www.fmi.org/supersafemark/

Training Achievement http://www.tapseries.com/ Program, TAP

knowledge, in contrast to training, which actually allows the employee to experience “hands-on” interaction with the knowledge (Yiannas, 2008). Therefore, the first task in successful training is to evaluate the content (knowledge) contained in current retail food service training platforms. The four most commonly used computer-based food safety employee training programs are listed in Table 2. These four retail food safety training platforms are considered representative of currently used training programs. A complete summary of the characteristics for each platform, including contact information, price, training target audience, numbers of modules/units, certifications, and other parameters, are being reviewed by a panel of experts in training . Alchemy Systems has a training library focused on HACCP for foodservice and frontline workers in food processing plants. They also design and build eLearning modules for their clients and already have several food processors as customers. ServSafe offers a $15 online training program, Starters Training and Assessment, which is advertised as a complete solution to deliver food safety training. The Food Marketing Institute (FMI) in cooperation with Learnovation, LLC developed SuperSafeMark for training employees of supermarkets and food warehouses; the employees’ text and on-line training is $15, and a complete kit for trainers, including slides, videos, and manuals is $200 for non-FMI members. The Training Achievement Program, TAP, offers online training for food

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Table 3. Food Safety Training Programs and Opportunities for Deli Workers Categories

Current Training Available

Potential training opportunities

Programs

Computer based, more general food safety training platforms

Delphi techniques to identify specific training/education objectives specific for deli workers.

Evaluation

Limited to available platforms with delispecific information, some integrate standards and permits ranking in an evaluation matrix

New training modules suitable for deli workers designed in cooperation with commercial suppliers and professional multi-media staff

Knowledge Base

Limited knowledge of experts, deli managers and employees

Usability testing by new employees for the deli specific training

Outcome

Minimal deli-specific knowledge and under-motivated usability by new food service students

Motivation of short-term learning and long-term behavior change

handlers at $15 per student. Several of these training platforms have been accepted by U.S. state and county health departments as meeting their requirements for Food Handler Certification. To adapt these general food safety training platforms specifically for deli operations for ideal implementation: 1) an Evaluation Matrix should be developed that is specific for individual delis, which identifies the amount of knowledge, including the elements of the FDA’s Model Food Code, plus details from the best from the training sites. Developing an individual Evaluation Matrix allows the incorporation of generally accepted practices and the local cultural norms of “this is how we do that around here”. Once the evaluation is complete, the current training regime needs to be compared against this newly developed Evaluation Matrix. In addition, the content should be classified using the revised Bloom’s Taxonomy (Anderson and Krathwohl, 2001) to determine the instructional level of the content in relation to each standard that will be taught. Some of the supporting research that deli managers will need has already been done. Pisik (1997) normalized an instrument for evaluating on-line courses for: content, appropriate for learners (appropriate exam-

retail establishment can be garnered from 1) the list of standards for knowledge content that regulatory bodies require enhanced with 2) additional knowledge content determined to be critical by the resident deli subject matter experts. This list of standards could be incorporated in the Evaluation Matrix to compare what experts feel must be included in the training versus what is actually included in each of the current training programs. For example, to our knowledge, none of the current training utilizes the most recent research findings that have shown that persistent strains of L. monocytogenes reside in specific niches in deli environments (Sauders et al. 2004, 2009). These L. monocytogenes harborages must be identified and addressed in deli specific food safety training programs (Table 2). To identify missing or more specific standards, holding Delphi panels composed of experts in deli operations to identify food safety knowledge specific to individual retail deli operations may be needed. The Delphi technique is a means for obtaining group input for ideas to develop a number of alternatives for a situation (Linstone and Turoff, 1975). In order to solicit participation from a variety of subject experts, a modified Delphi technique using the World-

ples), transfer of learning to job, design / packaging and usability issues such as ease of navigation and logical layout of the materials. The imperative knowledge that must be included for complete deli employee training in a particular

Wide-Web is a possible approach. A prospective Delphi panel for delis would be composed of experts including corporate supermarket personnel, supermarket deli managers, hourly supermarket deli employees, independently owned deli managers,

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hourly deli employees, health department inspectors and food safety professionals. An appropriate number of content experts currently working in delis are considered to be 15 to 20 to complete each round (Linstone and Turoff, 1975). A three-round Delphi Panel Design can be used to better define panel member views of what food safety topics are specific to deli operations. Initially, the process involves supplying a list of deli specific trainings in addition to general sanitation training (Gapud, 2009), and asking the Delphi panel members for other factors that may not have been listed. Online survey instruments can be a useful to rapidly facilitate this process.

It is important in this process to assess the delivery and usability of these retail food safety training platforms to potential deli managers and their employees. Computer based training software, such as those listed in Table 2, would be an ideal place to start. However, “hands-on” evaluation by individuals actually working in the food service industry should be an essential follow up step. Prior to evaluating the knowledge content of the food safety training platforms, participants would have to be given a pretest to determine their current level of food safety knowledge. Short-term learning could be assessed by completing a post-test after these participants have used various training platforms. Once com-

Secondly, panel members could be required to narrow the list to the top 10 training foci and then rank those 10 according to importance when examining food safety priorities for delis. Finally, panel members would be asked whether they agree with the rankings in Round 2 or re-rank order the factors again. They will be asked if they feel any of the top 10 items would reflect differently depending on individual needs of the deli where this training will take place. The Delphi panel results can also be integrated into the general food safety and sanitation training to create integrated standards, which could be built into the Evaluation Matrix to identify the breadth and depth of knowledge found in the existing training programs. This integrated standards list could also be used to identify gaps in content and inform the development of new deli specific online training materials. Once expert investigators identify which integrated content standards are addressed, to what extent and at which level of learning for each of the four online training platforms, the Evaluation Matrix can be used to assess the knowledge content presented in the online training programs that are under consideration for purchase, against the integrated standard developed after being reviewed by

pleted, the training platform could be further evaluated using the checklist for evaluating online courses described previously. In addition, several of these platforms are fairly versatile in also having programs in Spanish, Vietnamese, Chinese, Korean or Asian (Urdu, Hindi or Bengali). In summary, evaluating current and computer based training and measuring learning outcomes addresses two of Kirkpatrick’s (1998) four levels of evaluation (Reaction, Learning, Behavior, and Results). Achieving thorough evaluation should result in critical comparative data that food safety instructors and deli managers could use to make informed choices in selecting training materials suitable for their particular needs.

deli managers and employees. This would include reviewing all programs and calculating inter-rater reliabilities to determine the level of agreement among experts and also would allow outliers to be reassessed.

learners and that all have similar reading levels. In addition, young people entering the workforce may be more familiar with computers and online training. By combining the best practices for training NonEnglish Speaking Individuals (NESI) workers with on-

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Developing deli specific training The U.S. Census Bureau noted that by 2015, the Hispanic population will be double the size it was in 1990 (U.S. Census Bureau, 2009). As one of the largest employers of Hispanics, the restaurant industry must focus its efforts on training non-English speakers in food safety. Offering training material in Spanish may be a limited start but may not be a complete solution. By doing so, the assumption is made that all workers (not just Hispanic workers) are visual

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line training, deli operations can be more successful in their food safety training, therefore reducing the risk of foodborne illness from L. monocytogenes. Usability testing is defined as the degree of success a user has in learning and using a product to achieve their goals, as well as satisfaction of the user with the product (Dumas and Redish, 1993). Usability testing is not a test of the specific training materials, but rather an evaluation of the interactions between the user-learner and the training materials. Rubin (1994) suggested that the most logical approach for deli managers with sufficient lead time is to conduct a literature search, write and review training ideas along the way and then create learning objectives

test knowledge survey covering basic knowledge of retail deli food safety, given brief instructions for launching the web-based learning and provided with a task list. Participants are encouraged to talk out loud (think out loud) during the entire time they are using the learning module. Their conversations can be observed by a test monitor and/or videotaped during their entire session. After completion of the training modules, participants are usually given a written post-test knowledge survey and a profile questionnaire to obtain demographic information. After the post-test, participants are asked their opinions of how usable the computer based module was. The test monitor also provides a way

that are specific for the current retail deli operation. Each of the new training modules are typically broken down into clear, concise plans for testing. The major concepts pertinent for testing deli workers include: (1) short-term learning by the user, as measured using a questionnaire prior to and after completion, (2) user’s completion times, as measured by overall completion time for each module, the number of videos used by each user and number of links properly accessed, (3) user completion of tasks, as outlined in the virtual orientation and (4) overall user satisfaction with each of the design elements, as measured by an oral question and answer session of each user after completion of the virtual tour. Rubin’s (1994) design for a usability test is focused on user’s reactions to the major elements. Data collection methods include “think aloud” user testing (Travis, 2003), which consists of recording users talking out loud as they perform prescribed tasks. The usability data collection can be combined with a contemporaneous record of the users’ thoughts from the think aloud sessions while they are completing their virtual tours. O’Bryan et al. (2009, 2010) have demonstrated that this combination gives a complete evaluation by each user with minimal crossover among users and minimal external influ-

for these learners to offer their opinions and suggestions for improvement. By giving participants a pretest and post-test assessment of learning, in addition to the usability testing, two of Kirkpatrick’s four levels of evaluation (Reaction, Learning, Behavior, and Results) can be addressed, giving a more robust and accurate evaluation. Objective data can be complied on completion times, number of tasks successfully completed and overall user satisfaction with the design elements can be recorded. The summary of the think-aloud comments into similar groupings can be compiled and analyzed for common trends or themes that can be used to inform revisions to the training program. These suggested approaches can yield production of new online training programs targeted specifically at diverse deli workers and incorporating the latest methods and knowledge concerning effective sanitation techniques for the reduction of L. monocytogenes harborages. The Delphi Panel provides a ranked list of factors that can be used for developing deli food safety modules. The evaluation of the new training materials is focused on two of Kirkpatrick’s four levels of evaluation (Reaction and Learning). While it is desirable to address all four levels, the timeframe required to address all four levels is

ences. Nielsen (2000) determined that 5 users representing the target population will discover 85% of the usability flaws of a web site design, with 12 to 15 users finding close to 100% of problems. Typically, each participant is given a written pre-

usually beyond the scope of ongoing program development. However, data obtained from this evaluation still provides a foundation to facilitate future assessment of the remaining two levels (Behavior and Results).

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Certified training programs There are also advantages of having a certification program associated with any training. However, new certification programs are usually not needed because 1) there already are several nationally (and internationally) recognized certification programs for food safety trainers and managers and 2) many of these certifications have already met the current legal requirements imposed by local and state health officials. The recent review by Gapud (2009) covered these. Sites such as ServSafe’s give an overview of these varying legal and certification requirements.

the principal factors in reducing risk, minimizing or eliminating the hazard. Short term assessment of training for deli employees to minimize behaviors that cause cross-contamination is fairly achievable. However, long-term behavior changes are difficult to measure in employees where there are high-turnover rates, such as in a deli. Deli specific training with pre-test and post-test assessment of learning, in addition to the usability testing at the Reaction and Learning levels, could make these newly developed training modules part of the solution.

ACKNOWLEDGEMENTS

Secondly, consumer in-home preparation is the final link in the food safety chain. However, existing programs, such as Partnership for Food Safety Education – Food Marketing Institute and Fightbac have extensive cooperative networks and a track record of proven successes in educating consumers in safe food handling practices.

This review was supported by a USDA National Integrated Food Safety Initiative Grant to the authors, an American Meat Institute grant to Drs. Phil Crandall and Steve Ricke and a USDA CREES Food Safety Consortium grant to Steve Ricke.

CONCLUSIONS A carefully planned training program that includes assessing impact of training on both short and long term changes in behavior can lead to a decrease in the number of consumers contracting listeriosis. Noble et al. (2009) have developed methods to compare the public health inspection records, especially looking for violations of hand hygiene before and after training of employees to see if increased inspection without additional training decreased the number of critical violations. If sanitation techniques are better able to clean, sanitize and thermally treat potential L. monocytogenes harborages on food contact surfaces, equipment and environmental surfaces, then it can be assumed that employees in these environments will be less likely to cross-contaminate RTE foods with L. monocytogenes. Again, fewer L.

Anderson, L. W. and D. R. Krathwohl. 2001. A taxonomy for learning, teaching and assessing: A revision of Bloom’s Taxonomy of educational objectives. Longman, New York, NY. 384 p. Anonymous. 2000. Dollars from the Deli. Findarticles. com. http://findarticles.com/p/articles/mi_m3289/ is_2_169/ai_60071462/ Accessed Nov, 2009. Bricher, J. L. 2007. Protecting your customers while safeguarding your business. Food Saf. 13:1-40. Buchholz, A. L., Z. Yan, E. T. Ryser. 2008. Glo-germ as a cross contamination indicator during processing of leafy greens. IAFP 95th Annual Meeting, p. 14 (Abst. T4-11). Buchholz, A. L., G. R. Davidson, B. P. Marks, E. C. D. Todd, E. T. Ryser. 2009. Transfer of Escherichia coli O157:H7 from equipment surfaces to iceberg and romaine lettuce during simulated commercial processing., IAFP 95th Annual Meeting, p. 17 (Abst.

monocytogenes harborages and minimal cross-contamination will decrease the hazard and decrease the risk of listeriosis. Extending these findings to impact deli specific training targeting Hispanics and other minority deli employees can decrease one of

T5-04). Buchholz, A. L., G. R. Davidson, D. T. Campos, B. P. Marks, E. C. D. Todd, E. T. Ryser. 2009. Quantification of Escherichia coli O157:H7 transfer to equipment during commercial production of fresh-cut

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Survival of Salmonella in Organic and Conventional Broiler Feed as Affected by Temperature and Water Activity A. Petkar1, W. Q. Alali1*, M. A. Harrison1,2, and L. R. Beuchat1 Center for Food Safety and Department of Food Science and Technology, University of Georgia, Griffin, Georgia 30223-1797 2 Athens, Georgia 30602-2610

ABSTRACT The objective of this study was to compare the ability of Salmonella to survive in organic and conventional broiler feeds as affected by temperature (11, 25, and 38°C), water activity (aw 0.75, 0.55, and 0.43) and storage time (up to 80 days). Feeds were inoculated with a mixture of five Salmonella serotypes at high and low populations (6 and 3 log CFU/g, respectively), and populations and presence (by enrichment) were monitored over time. Although the number of Salmonella in organic feed for the majority of temperatureby-aw combinations was significantly lower (P ≤ 0.05) compared to the number in conventional feed over the 80-day storage period, differences in mean populations were less than 1 log CFU/g. The odds-ratio (OR) for presence of Salmonella was significantly higher (P ≤ 0.05) in conventional feed than in organic feed containing high and low inocula (OR = 4.76 and 2.92, respectively). Based on these findings, we generally conclude that there were no biologically significant differences in survival of Salmonella in organic and conventional poultry feeds. Keywords: Salmonella, conventional poultry feed, organic poultry feed, storage temperature, water activity, aw

Agric. Food Anal. Bacteriol. 1: 175-185, 2011

INTRODUCTION Salmonella is one of the most common pathogens known to cause foodborne disease in the United States and worldwide. According to Scallan et al. (2011), an estimated 1.03 million people suffer from Correspondence: Walid Q. Alali , walali@uga.edu Tel: +1 - 770-467-6066 Fax: +1-770-229-3216

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salmonellosis annually in the U.S. Nontyphoidal Salmonella is considered to be the leading cause of foodborne illness-related hospitalizations and death in the U.S. Poultry and poultry products are considered to be an important source of Salmonella (Tauxe, 1991; Bryan and Doyle, 1995) and contaminated feed is considered to be one of the main sources of Salmonella infection in broiler birds (Jones et al., 1991; Maciorowski et al., 2004).

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The presence of Salmonella in poultry feed as well as feed ingredients such as grain, oilseed meal, feathers, fishmeal, and meat by-products has been documented (Williams 1981a; Cox et al., 1983; Stuart 1984; Veldman et al., 1995). After contaminated feed is consumed by broilers, Salmonella can multiply in the gastrointestinal tract of the bird and potentially be shed in the feces during grow out (Cason et al. 1994). Several intervention methods, including heating and pelleting at 70°C and 90°C, irradiation (gamma rays), and addition of chemicals (organic acids, organic salts, formaldehyde, and bacterial membrane disruptors such as terpenes and essential oils) have been applied to poultry feed and feed ingredi-

survive in USDA-certified organic broiler feed versus conventional broiler feed stored at different temperatures and aw over an 80-day period.

ents to control Salmonella (Wilder, 1969; Hinton and Linton, 1988). Despite the use of these interventions, poultry feed can be recontaminated with Salmonella post-production, e.g., during storage at the feed mill, transportation, and storage at the farm (Hinton and Linton, 1988). Storage of poultry feed under various environmental conditions may influence the survival and growth of Salmonella. Reports from several studies show that survival of Salmonella is influenced by factors such as the presence of antimicrobials, moisture content, and storage temperature (Himathongkham et al., 1996; Halls and Tallentire 1978; Furuta et al., 1980; McCapes et al., 1989). Juven et al. (1984) reported that survival of Salmonella is greater at a water activity (aw) of 0.43 than at 0.75. The survival and heat resistance of Salmonella in poultry feed has been reported to be inversely related to moisture content and relative humidity (% relative humidity = aw x 100), except at a moisture content that allows growth (Liu et al., 1969; Carlson and Snoeyenbos, 1970; Juven et al., 1984). In a recent study, it was found that conventional poultry feed was contaminated with Salmonella, whereas USDA-certified organic feed was Salmonella-free (Alali et al., 2010). It is unclear whether the

at 37°C. A loopful of TSB culture of each serotype was streaked on a double-layered agar medium consisting of tryptic soy agar (TSA) (Difco, BD) and xylose lysine tergitol (XLT4) agar (Difco, BD), and incubated at 37°C for 24 h. The TSA-XLT4 agar was prepared as described in Kang and Fung (2000). This agar has been used for direct plating to support recovery and enumeration of injured Salmonella. The agar is composed of a bottom layer of XLT4 agar as a selective medium and thin top layer of nonselective TSA.

organic feed was contaminated with Salmonella which subsequently did not survive or was present at a level below the limit of detection, or whether the feed was not contaminated. The objective of this study was to determine the ability of Salmonella to

MATERIALS AND METHODS Salmonella serotypes Five Salmonella enterica serotypes were obtained from the Poultry Diagnostic Research Center, University of Georgia, Athens, GA. These serotypes were S. Typhimurium, S. Heidelberg, S. Enteritidis, S. Montevideo, and S. Gaminara. All serotypes were grown in tryptic soy broth (TSB) (Difco, BD; Sparks, MD) for 24 h

Salmonella inoculum To prepare the Salmonella inoculum, 6-ml quantities of nutrient broth (NB) (Difco, BD) were inoculated with cells from single colonies of Salmonella formed on TSA-XLT4 agar plates incubated at 37°C for 24 h. Each serotype was cultured separately. The NB inoculum concentration was adjusted to approximately 8 log CFU/ml using a spectrophotometer (Spectronic 20; Bausch and Lomb, Rochester, NY) at an optical density of 0.5 to 0.6, as described by Kaiser et al. (2002). This suspension was used to prepare a highpopulation dry chalk powder inoculum. Ten-fold serial dilutions were made in Nutrient Agar (NA) to prepare a low-population chalk inoculum.

Preparation of dry chalk inocula Chalk was used as a carrier for preparing a dry Salmonella inoculum (Okelo et al., 2008). Prior to use,

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Table 1. Composition of conventional and organic broiler-grower feeds.

Corn meal

72.79

65.80

Soybean meal

15.80

23.77

Alfalfa meal (dehydrated)

1.20

Poultry meat & bone meal

6.00

Fat

2.70

5.00

Dicalcium phosphate

0.20

1.24

Limestone

0.66

1.55

Salt

0.39

0.31

Methionine

0.29

0.23

Crayola chalk (Code #51-0320, Binney and Smith, Easton, PA) was sterilized by autoclaving. The chalk did not possess antimicrobial properties. The pH of the chalk was 7.0, as measured by a pH meter (Fisher Scientific, Pittsburgh, PA). Chalk (4.3-g pieces) was submerged in the five-serotype mixture of high- or low-population NB suspension for 12 h at 37°C, then placed in sterile petri dishes and dried for 72 h at 37°C. The inoculated, dried chalk was pulverized using a food processor (Hamilton Beach Food Processor, model 70590, type FP 11, Southern Pines, NC) in a laminar air flow chamber to obtain powdered inocula containing Salmonella at approximately 7 log CFU/g (high inoculum) and 4 log CFU/g (low inoculum) of

Vitamin premix2

0.25

0.12

chalk. The aw of chalk powder inocula was 0.22.

0.65

0.70

Trace mineral premix

0.08

Coban4

0.05

BMD5

0.05

L-Threonine

0.07

TBCC5

0.02

Conventional Organic feed (%) feed (%)6

Ingredient

Lysine 3

Vitamin mix contained the following: vitamin A, vitamin D3, vitamin E, vitamin B12, riboflavin, niacin, d-Pantothenic acid, choline, menadione, folic acid, thiamine, pyridoxine, biotin, and ethoxyquin. 1

Trace mineral mix contained the following: calcium, iron, magnesium, manganese, zinc, copper, iodine, selenium. 2

Coban: coccidiostat

Enumeration of Salmonella in dry chalk inocula Enumeration of viable Salmonella was done by adding 10 g of high- or low-population chalk inoculum to 90 ml of phosphate buffered saline (PBS), vigorously shaking the suspension, and making 10fold serial dilutions in PBS. Samples (0.1 ml) were surface plated in duplicate on TSA-XLT4 agar plates and incubated at 37°C for 24 h before colonies were counted.

BMD: bacitracin methylene disalycilate

TBCC: tribaic copper chloride

Specific ingredients listed for organic broiler feed: monosodium phosphate, organic kelp meal, diatomaceous earth, , ferrous sulfate, organic apple cider vinegar, , zinc sulfate, , organic potato starch, organic dehydrated eggs, organic dried tomato pomace, organic dried whole milk, organic linseed meal, organic aloe vera gel concentrate, organic soybean oil, organic oat flour, lecithin, organic wheat middlings, organic sugar, potassium chloride, attapulgite clay, pyridoxine hydrochloride, folic acid, ferric choline citrate complex, zinc choline citrate complex, carotene, ascorbic acid, yeast culture, cobalt sulfate, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium thermophilum, Enterococcus faecium, organic sources of (cayenne pepper, peppermint, garlic, parsley, dandelion root extract, elder flowers, dandelion extract, ginger extract, German chamomile, lemon grass extract, thyme, sweet fennel extract, sweet basil, sage, cloves), and natural tocopherols. 6

177

Feeds used Conventional pelleted broiler feed formulated for grower birds (Table 1) was purchased from two conventional poultry companies (companies A and B). The formulations listed for these feeds were similar. Organic mash feed formulated for grower birds (Table 1) was obtained from two organic poultry companies (companies C and D). Prior to inoculation, feed samples were tested (as described later under Salmonella analysis-selective enrichment) to ensure that they were negative for Salmonella.

Preparation of feeds with desired aw Saturated salt solutions (potassium carbonate, sodium bromide, and sodium chloride) were placed

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inside each cabinet to attain the desired aw (Rockland, 1960). Three cabinets at three different relative humidities were each kept at three different temperatures (11, 25, and 38°C). Conventional and organic feeds were stored inside secador desiccator cabinets (Structure Probe, Inc., West Chester, PA) for 3 weeks to reach target aw values of approximately 0.43, 0.55 and 0.75 prior to inoculating with Salmonella. The aw of feeds was measured over time using an Aqualabaw meter (Decagon Devices, Inc., Pullman, WA).

(0.1 ml) from each dilution (10-1 to 10-3) were spread plated on TSA-XLT4 agar plates. After incubating plates for 24 h at 37°C, colonies presumptive for Salmonella were enumerated. For the low-inoculum samples, the procedure for enumerating Salmonella was similar with the exception that suspensions were not diluted. Undiluted LB/feed homogenates were surface-plated on TSA-XLT4 agar. The LB/feed suspensions were incubated for 24 h at 37°C.

Selective enrichment

Experimental design All experiments were replicated twice. Feeds from companies A and C (conventional and organic, respectively) were used in the first trial, whereas feeds from companies B and D were used in the second trial. A 3 x 3 factorial design was used to conduct the study. For each feed containing either high- or low inoculum, three storage temperatures (11, 25, and 38°C) and three aw levels (0.75, 0.55 and 0.43) were tested to determine their effects on survival of Salmonella in conventional and organic broiler feeds over a 80-day period. For each temperature/aw combination, triplicate 9-g samples of conventional and organic feed in sterile glass test tubes were inoculated with 1 g of powdered chalk inoculum to obtain populations of approximately 6 log (high) and 3 log (low) CFU/g. The inoculated feeds were mixed, deposited in tubes, and placed inside the cabinets.

When direct plating of diluted feed samples was negative for Salmonella, 1 ml and 0.1 ml of preenriched LB/feed mixture were inoculated into 10 ml of tetrathionate (TT) broth (Difco, BD) and 10 ml of Rappaport Vassiliadis broth (RV) broth (Difco, BD), respectively. After incubating broths for 24 h at 35°C (TT broth) and 42°C (RV broth), a loopful of the culture was streaked on TSA-XLT4 agar. After incubating plates for 24 h at 37°C, cells from colonies presumptive for Salmonella were inoculated into triple sugar iron (TSI) agar slants (Difco, BD), incubated with caps loosened at 35°C for 18 to 24 h, and examined for carbohydrate fermentation, gas production, and hydrogen sulfide production. Based on these observations, feed samples were judged as positive or negative for Salmonella.

Statistical analysis

Triplicate 10-g samples of inoculated feed stored for 0, 3, 7, 14, 21, 28, 35, 50, 65, and 80 days at each temperature/aw combination were analyzed for populations and presence (by enrichment) of Salmonella. For the high-inoculum samples, 10 g were suspended in a 90 ml of Luria Bertani (LB) (Sigma Al-

Populations of Salmonella (log CFU/g) determined by direct plating samples of conventional and organic broiler feeds were compared among each temperature/aw combination, by inoculum level, and at each storage day using repeated measures of analysis of variance ANOVA in General Linear Model (GLM) in SAS software version 9.1.3 (GLM procedure, SAS Inst., Inc., Cary, NC). Salmonella counts were logarithmically transformed by use of log base 10 to

drich Corp., St. Louis, MO) broth in a 250-ml flask, followed by shaking for 1 h on a rotary shaker (New Brunswick Scientific, Edison, NJ). Ten-fold serial dilutions of the suspension were made in sterile microcentrifuge tubes containing 0.9 ml of (PBS). Aliquots

approximate normality. Data from the two replicate experiments were tested using Levene’s test for homogeneity of variances. The variances between the two replicate experiments were not significantly different (P > 0.05). Based on these findings, data from

Analysis of feeds for Salmonella

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Table 2. Mean Salmonella populations (log10 CFU/g) in conventional and organic poultry feed initially containing high or low inoculum and at different temperature and aw combinations over an 80-day storage period. Salmonella (log CFU/g) Inoculum level

Temp (C )

Conventional feed

Organic feed

P-valuea

High

0.43

6.17

5.93

<0.001

5.29

5.05

0.140

2.69

2.52

0.600

6.21

5.93

0.055

5.55

5.09

0.030

2.86

2.44

0.150

6.31

6.16

0.030

5.67

4.79

0.001

2.44

2.22

0.380

4.29

4.12

0.020

3.26

2.64

<0.001

1.10

0.80

0.009

4.58

4.24

<0.001

3.89

3.15

<0.001

1.39

1.06

0.011

4.43

4.08

<0.001

2.96

1.86

<0.001

0.93

0.81

0.003

0.55

0.75

Low

0.43

0.55

0.75

P-values were considered significant at ≤ 0.05 at different temperatures and aw.

both replicate trials were pooled to obtain a set of six observations for each sampling day. In addition, the proportion of samples positive for Salmonella was compared among each temperature-by-aw combination, by inoculum level, and at each sampling time using a GLM with a binomial distribution and a logit link in SAS (GENMOD procedure). The reported odds ratios (OR) from GLM model was comparing the odds of Salmonella in one type of feed a group to the odds of Salmonella in the other type of feed (Dohoo et al., 2003). A sample was considered positive if direct plating was positive or if enrichment was positive when direct plating was negative.

RESULTS All uninoculated feed samples were negative for Salmonella. Salmonella populations in inoculated 179

conventional and organic broiler feeds were determined over an 80-day period. Although the differences between populations in the two types of feeds held at various temperatures/aw conditions were small (generally < 1 log CFU/g), these differences were statistically significant (P ≤ 0.05) within both inoculum levels. For the feeds containing a high inoculum, mean log CFU of Salmonella/g over the 80-day storage period were 4.71 ± 0.09 and 4.36 ± 0.09 for conventional and organic feed, respectively. For feeds containing the low inoculum, the mean log CFU of Salmonella/g were 2.88 ± 0.08 and 2.38 ± 0.08 for conventional and organic feed, respectively. We considered differences in Salmonella populations between organic and conventional feeds to be biologically meaningful if they were significantly different and >1 log CFU/g. At day 0, the mean populations of Salmonella re-

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Figure. 1. Mean number of Salmonella (log CFU/g) in conventional and organic poultry feeds [high inoculum level (106 CFU/g)] over a storage period of 80 days at various temperature/aw conditions: A to C = 11oC, aw 0.43, 0.55, and 0.75; D to F = 25oC, aw 0.43, 0.55, and 0.75; and G to I = 38oC, aw 0.43, 0.55, and 0.75. Each data point represents the mean of values from three replicate samples for two experiments (n = 6) per treatment. 0.55

0.75

11˚C

0.43

38˚C

25˚C

covered from inoculated conventional and organic feeds containing high and low inocula levels were approximately 6 and 3 log CFU/g, respectively, indicating an approximate 1-log CFU/g reduction based on the number of Salmonella added to the feeds via chalk inocula. The effects of different temperature/ aw combinations by feed type and inoculum level on survival of Salmonella over an 80-day storage period are shown in Table 2. The mean Salmonella populations in organic and conventional feeds initially con-

0.75) stored at 25°C for 50 and 65 days (Fig. 1, F). There were significant differences (> 1 log CFU/g) in low-inoculum feeds stored at 25°C and aw 0.43, 0.55, and 0.75 during the study period (Fig. 2, D-F). Regardless of inoculum level, Salmonella in organic and conventional feeds decreased to an undetectable level (2 log CFU/g by direct plating; 1 CFU/10 g by enrichment) in feeds stored at 38°C, regardless of the aw (Fig. 1, G-I; Fig. 2, G-I). Considering all temperature/aw combinations, the

taining high and low inocula and stored at different temperature/aw combinations for up to 80 days are shown in Figures 1 and 2, respectively. There were statistically significant differences (P ≤ 0.05) and > 1 log CFU/g difference in high-inoculum feeds (aw

OR for the presence of Salmonella in conventional feed was significantly (P ≤ 0.05) higher than that in organic feed containing a high inoculum (OR = 4.76 [95% confidence interval {CI}], 2.66 to 22.57). The OR for presence of Salmonella in low-inoculum conven-

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tional feed was significantly higher (P ≤ 0.05) than for organic feed (OR = 2.92 [95% CI, 2.16 to 4.53]). The GLM model comparing Salmonella populations in conventional feed versus organic feed (as the reference category) failed to converge due to the sparse amount of data (no or few positives) for feed stored at most temperature/aw combinations, regardless of the inoculum level. The percentage of Salmonellapositive conventional feed samples containing a high inoculum level decreased by 50% at the end of 80-day storage period as compared to 43% in organic feed (Fig. 3). In contrast, at a low inoculum level, the percentage of Salmonella-positive conventional feed samples decreased 58% by the end of the 80-

ter and fall, 25°C in spring, and 38°C in summer) in southeastern U.S. These conditions were selected to simulate environmental conditions for handling and storage of poultry feed along the productionto-consumption chain. Furthermore, to investigate differences in contamination levels, poultry feeds were inoculated with high and low populations of Salmonella. Since poultry feeds are generally stored for less than 8 to 10 weeks from production to consumption, the period of 80 days was chosen to evaluate the survivability of Salmonella. Although most of the differences in mean Salmonella populations between the two types of feed, at each temperature/aw combination, and by inoculum

day storage period as compared to 40% in organic feed (Fig. 3).

The ability of Salmonella to survive in organic versus conventional broiler feed as affected by storage temperature and aw has not been reported, although in the past decades, poultry feed has been considered to be an important vehicle for transmission of Salmonella to broiler birds. Salmonella has been detected in poultry feed and feed ingredients (Williams, 1981a; Williams, 1981 b; Cox et al., 1983; Stuart, 1984; Veldman et al., 1995). In our experiments, a dry inoculation technique rather than a liquid-suspension inoculation was used to distribute Salmonella in feeds. The dry inoculum did not substantially alter the aw of the feed. Compared to a wet inoculum, distribution of Salmonella from dry inocula is homogenously distributed and changes in background microbiota are expected to be minimum (Hoffmans and Fung, 1993). In addition, dry inoculation more closely mimics contamination that might occur via dust and other dry materials in growout facilities. We evaluated three temperatures and three aw

population were statistically significant (Table 2), the numerical differences were too small (<1.0 log CFU/g) to be considered biologically meaningful. The proportion of samples positive for Salmonella was significantly higher in conventional feed compared to organic feed for both inoculum levels over all temperature/aw combinations. Differences in composition of organic and conventional feeds (Table 1) did not appear to impact the survival of Salmonella; however, feed composition may have impacted the percentage of samples positive for Salmonella in the two types of feed. A possible explanation for this difference may be attributable to the absence of animal protein meals (blood and bone) and presence of natural antimicrobials such as garlic, clove, ginger, and basil in organic feed. Leuschner and Zamparini (2002) tested the effect of different natural antimicrobials such as garlic, ginger, mustard, and cloves on growth and survival of Escherichia coli O157 and S. enterica serotype Enteritidis in broth model systems. Garlic and clove showed bacteriostatic and bacteriocidal effects on both foodborne pathogens. Clove was found to be more effective than garlic. Mustard and ginger also exhibited bacteriostatic activities against both bacteria. When examining survival of Salmonella as affect-

values covering ranges existing in storage facilities at feed mills, during transportation, and on-farm during different seasons. Poultry feeds are marketed at aw ranging from about 0.45 to 0.75 and exposed to various storage temperatures, (e.g., 11°C in win-

ed by storage time (Figs. 1 and 2), a few differences >1 log CFU/g were observed, mainly in feed containing a low inoculum and stored at 25°C. Salmonella in both high- and low-inoculum feeds stored at 38°C decreased to populations below the limit for detection

DISCUSSION

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Figure. 2. Mean number of Salmonella (log CFU/g) in conventional and organic poultry feeds [low inoculum level (103 CFU/g)] over a storage period of 80 days at various temperature/aw conditions: A to C = 11oC, aw 0.43, 0.55, and 0.75; D to F = 25oC, aw 0.43, 0.55, and 0.75; and G to I = 38oC, aw 0.43, 0.55, and 0.75. Each data point represents the mean of values from three replicate samples for two experiments (n = 6) per treatment. 0.55

0.75

11˚C

0.43

38˚C

25˚C

by direct plating (2 log CFU/g). Similar observations in conventional poultry feed were reported by Williams and Benson (1978), where S. Typhimurium survived much longer at 11 and 25°C than at 38°C. In our study, in feed containing a high inoculum, the mean number of Salmonella recovered from both types of feed ranged from 4 to 5 log CFU/g after storage for 80 days at 11 and 25°C, regardless of aw. This observation con-

of cells, thus immediately lowering the number of viable Salmonella as compared to what may occur in the feed to which a dry inoculum is added. The inoculation procedure and aw of feeds have been reported to affect the survivability of Salmonella (Liu et al., 1969; Carlson and Snoeyenbos, 1970; Juven et al., 1984). Previous studies show that Salmonella does not grow but survives well in low-aw foods such as peanut

curs with the findings of Davies and Wray (1996) showing that S. Typhimurium declined to 3 log CFU/g of conventional poultry feed over a period of 3 months. These researchers inoculated feed using a suspension of Salmonella that may have resulted in osmotic shock

butter, infant formula, cereals, and dry aniseed (GMA, 2009). These and other low-aw products have been associated with outbreaks of salmonellosis. Investigations suggest that factors such as poor sanitation practices, poor equipment design, improper mainte-

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Figure. 3. Percentage of Salmonella-positive samples in conventional and organic poultry feeds initially containing a high- and low inocula and stored up to 80 days.

and 0.55, Salmonella survived at higher levels in both types of feed as compared to survival at aw 0.75. Reductions were more rapid as the storage temperature was increased.

ACKNOWLEDGEMENTS We thank Dr. John Maurer from Poultry Diagnostic Research Center, University of Georgia, Athens, GA for providing the Salmonella isolates used in this study. We also thank Rebekah Turk for her technical assistance in the laboratory.

REFERENCES

nance, and poor ingredient control were contributing factors in these outbreaks. During storage, reduction in Salmonella populations occurs but is dependent on multiple factors, including temperature and aw. Feeds and foods containing different ingredients but similar moisture content can have different aw values, depending on the type and amount of solute present, leading to different rates of inactivation (Duncan and Adams, 1972). In our study, there was no biologically meaningful difference in the number of Salmonella recovered from conventional and organic feeds stored at most temperature/aw combinations, regardless of the inoculum level, throughout the 80-day storage period. Several researchers have shown that survival of Salmonella in dried products (e.g., fish meals, dry milk, poultry feed, cocoa powder, pecans, and meat and bone meal) is influenced by aw (Beuchat and Mann, 2010; Doesburg et al., 1970; Juven et al., 1984). Interactions between aw and environmental factors such as temperature may play an important role in affecting the survival of Salmonella (Banwart and Ayres, 1956;

Alali, W. Q., S. Thakur, R. D. Berghaus, M. P. Martin, and W. Gebreyes. 2010. Prevalence and distribution of Salmonella in organic and conventional broiler poultry farms. Foodborne Path. Dis. 7:13631371. Banwart, G. J. and J. C Ayres. 1956. The effect of high temperature on the content of Salmonella and on functional properties of dried egg white. Food Technol. 10:68-73. Beuchat, L. R. and D. A. Mann. 2010. Factors affecting infiltration and survival of Salmonella in in-shell pecans and nutmeats. J. Food Prot. 73:1257-1268. Bryan, F. L. and M. P. Doyle. 1995. Health risks and consequences of Salmonella and Campylobacter jejuni in raw poultry. J. Food Prot. 58:326-344. Carlson, V. L. and G. H. Snoeyenbos. 1970. Effect of moisture on Salmonella populations in animal feeds. Poult. Sci. 49:717-725. Cason, J. A., N. A. Cox, and J. S. Bailey. 1994. Transmission of Salmonella Typhimurium during hatching of broiler chicks. Avian Dis. 38:583–588. Corry, J. E. L. 1976. The safety of intermediate moisture foods with respect to Salmonella. In: R. Davies, G. G. Birch, and K. J. Parker. Ed. Intermediate moisture foods. Applied Science Publishers, Lon-

Corry, 1976; Doesburg et al., 1970). Juven et al. (1984) showed that Salmonella can survive at higher populations in feeds at aw 0.43 and 0.53 compared to feeds at aw 0.75. We observed a similar pattern for Salmonella in conventional and organic broiler feeds. At aw 0.43

don. p 215-238. Cox, N. A., J. S. Bailey, J. E. Thomson, and B. J. Juven. 1983. Salmonella and other Enterobacteriaceae found in commercial poultry feed. Poult. Sci. 62:2169-2175.

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Davies, R. H. and C. Wray. 1996. Persistence of Salmonella Enteritidis in poultry units and poultry food. British Poult. Sci. 37:589–596. Doesburg, J. J., E. C. Lamprecht, and M. Elliot. 1970. Death rates of Salmonellae in fish meals with different water activities. I. During storage. J. Sci. Food Agric. 21:632- 635. Dohoo, I. R., W. Martin, and H. Stryhn. 2003. Veterinary Epidemiologic Research. Charlottetown, Canada: University of Prince Edward Island. Duncan, M. S. and A.W. Adams. 1972. Effects of a chemical additive and of formaldehyde-gas fumigation on Salmonella in poultry feeds. Poult. Sci. 51:797–802.

Lamont. 2002. Salmonella enterica serovars Enteritidis burden in broiler breeder chicks genetically associated with vaccine antibody response. Avian Dis. 46:25-31 Kang, D. H. and D. Y. C. Fung. 2000. Application of thin agar layer method for recovery of injured Salmonella Typhimurium. Int. J. Food Microbiol. 54:127-132. Leuschner, R. G. K. and J. Zamparini. 2002. Effects of spices on growth and survival of Escherichia coli O157 and Salmonella enterica serovar Enteritidis in broth model systems and mayonnaise. Food Control. 13:399-404. Liu, T. S., G. H. Snoeyenbos, and V. L. Carlson.1969.

Furuta, K., I. Oku, and S. Sato. 1980. Bacterial contamination in feed ingredients, formulated chicken feed and reduction of viable bacteria by pelleting. Lab. Anim. 14:221-224. Grocery Manufacturers Association. 2009. Control of Salmonella in low moisture foods. Gmaonline. org. http://www.gmaonline.org/science/SalmonellaControlGuidance.pdf. Accessed May, 2009. Halls, N. A. and A. Tallentire. 1978. Effects of processing and gamma radiation on microbiological contaminants of a laboratory animal diet. Lab Anim. 12:5-10. Hinton, M. and A. H. Linton. 1988. Control of Salmonella infections in broiler chickens by the acid treatment of their feed. Vet. Rec. 123:416–421. Himathongkham, S., M. Das Gracas Pereira, and H. Riemann. 1996. Heat destruction of Salmonella in poultry feed: effect of time, temperature and moisture. Avian Dis. 36:625-631. Hoffmans, C. M. and D. Y. C. Fung. 1993. Effective method for dry inoculation of bacterial cultures. J. Rapid. Meth. Auto. Microbiol. 1:287-294. Jones, F. T., R. C. Axtell, D. V. Rives, S. E. Scheideler, F. R. Tarver, R. L. Walker, and M. J. Wineland. 1991. A survey of Salmonella contamination in modern broiler production. J. Food Prot. 54:502-507.

Thermal resistance of Salmonella Senftenberg 775 W in dry animal feeds. Av. Dis. 13:611-631. Maciorowski, K. G., F. T. Jones, S. D. Pillai, and S. C. Ricke. 2004. Incidence, sources, and control of food-borne Salmonella spp. in poultry feeds. W. Poult. Sci. J. 60:446-457. McCapes, R. H., H. E. Ekperigin, W. J. Cameron, W. L. Ritchie, J. Slagter, V. Stangeland, and K.V. Nagraja. 1989. Effect of new pelleting process on level of contamination of poultry mash by Escherichia coli and Salmonella. Av. Dis. 41:58-61. Okelo, P. O., S. W. Joseph, D. D. Wagner, F. W. Wheaton, L. W. Douglass, and L. E. Carr. 2008. Improvements in reduction of feed contamination: An alternative monitor of bacterial killing during feed extrusion. J. Appl. Poult. Res. 17:219-228. Rockland, L. B. 1960. Saturated salt solutions for static control of relative humidity between 5° and 40°C. Anal. Chem. 32:1375. Scallan, E., R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, M. A. Widdowson, S. L. Roy. 2011. Foodborne illness acquired in the United States—major pathogens. Emerging Infect. Dis. 17:7-15. Stuart, J. C. 1984. The introduction of Salmonella into poultry flocks. J. Sci. Food Agric. 35:632-633. Tauxe, R. V. 1991. Salmonella: A postmodern patho-

Juven, B. J., N. A. Cox, J. S. Bailey, J. E. Thomson, O. W. Charles, and J. V. Shutze. 1984. Survival of Salmonella in dry food and feed. J. Food Prot. 47:445-448. Kaiser, M. G., N. Lakshmanan, T. Wing, and S. J.

gen. J. Food Prot. 54:563-568. Veldman, A., H. A. Vahl, G. J Borggreve, D. C. and Fuller. 1995. A survey of incidence of Salmonella species and Enterobacteriaceae in poultry feeds and feed components. Vet. Res. 136:169-172.

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Wilder, O. H. M. 1969. A method of destroying Salmonella. J. Am. Oil Chem. Soc. 46:233–234. Williams, J. E. and S. T. Benson. 1978. Survival of Salmonella Typhimurium in poultry feed and litter at three temperatures. Avian Dis. 22:742-747. Williams, J. E. 1981a. Salmonella in poultry feedsa worldwide review. Part I: Introduction. World’s Poult. Sci. J. 37:6-19. Williams, J. E. 1981b. Salmonella in poultry feeds - a worldwide review. Part III. Methods in control and elimination. World’s Poult. Sci. J. 37:97-105.

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BRIEF COMMUNICATION Isolation and Initial Characterization of Plasmids in an Acetogenic Ruminal Isolate R. S. Pinder1,2 , and J. A. Patterson1 Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-1026 2 Current address: 7855 South 600 East, Brownsburg, IN 46112

ABSTRACT Two of nine acetogenic ruminal isolates screened for plasmids were found to contain plasmid DNA. Five plasmids ranging in size from 4.5 to 32 kilobase pairs (kb) were observed in isolate H3HH while a single 35 kb plasmid was observed in isolate H4. The smallest of the plasmids from isolate H3HH, estimated at 4.5 kb, was isolated using gel electrophoresis followed by electroelution. Of the 13 restriction endonucleases tested, this plasmid was cut once by EcoRV, SinI and HindIII and cut twice BglII. The physiological functions of the individual plasmids are unknown. However, a plasmidfree derivative (H3HP) of isolate H3HH displayed increased sensitivity to several antibiotics. Keywords: Acetogens, plasmids, rumen, genetics, bacteria, isolation, characterization Agric. Food Anal. Bacteriol. 1: 186-192, 2011

INTRODUCTION Interest in acetogenic bacteria stems from their possible utilization as competitors against methanogens as a hydrogen sink in the ruminal microbial community, for acetate production from inexpensive feedstocks, and their ability to utilize methoxylated phenolics and phenolic acrylates (Drake, 1992; Hespell, 1987; Ragsdale, 1991). Methane production represents a 5 to 15% loss of digestible feed energy to ruminants (Blaxter and Clapperton, 1965). Shifting any portion of the hydrogen used to form methane towards acetate production could result in a concomitant increase in animal productivity.

Correspondence: J. Patterson, jpatters@purdue.edu Tel: +1 -765-494-4826 Fax: +1-765-494-9347

Although isolation and characterization of plasmids in other ruminal bacteria (e.g., Prevotella ruminicola, Butyrivibrio fibrisolvens, Selenomonas ruminantium) has been reported (Asmundson and Kelly, 1987; Dean et al., 1989; Flint et al., 1988; Flint and Stewart, 1987; Mann et al., 1986; Martin and Dean, 1989; Ricke et al., 1996; Teather, 1982;) the genetic systems of ruminal acetogenic bacteria remain unknown (Forsberg et al., 1986). Analysis of plasmid DNA in acetogens isolated from ruminal contents is of interest because plasmids provide a means towards developing understanding of acetogen physiology and potentially for genetically manipulating acetogens (Dean et al., 1989). This report describes the isolation and initial characterization of plasmid DNA from a ruminal acetogenic bacterium.

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MATERIALS AND METHODS Organisms and Media Nine H2-utilizing, acetate producing isolates (H1 through H9) that had been isolated previously from an H2 limited continuous culture system (Boccazzi et al., 2011) were grown anaerobically in acetogen medium which contained: (mg/L) K2HPO4, 300; KH2PO4, 300; (NH4)2SO4, 300; NaCl, 600; MgSO4 · 7 H2O, 123; CaCl2· H2O, 80; NH4Cl, 540; HSeO3, 0.2; NiCl2 · 6 H2O, 20; hemin, 0.01; resazurin, 1; clarified rumen fluid, 5 ml; trace mineral solution (Greening and Leedle, 1989), 10 ml; yeast extract, 500. After the pH of the medium was adjusted to 6.8 with HCl, the medium was prepared using basic anaerobic techniques. The medium was boiled in an appropriately sized reagent bottle while being gassed with oxygen-free CO2. The bottle was sealed with a rubber stopper, wired in place and autoclaved (121ºC, 20 min). After cooling, the flask was transferred into an anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI) and the following sterile anaerobic reagents were added aseptically (in mg/liter): Na2CO3, 4000; cysteine · HCl, 250; Na2S · 7 H2O, 250 and vitamin solution (Greening and Leedle, 1989), 10 ml. Twenty ml of media was aseptically dispensed into sterile serum bottles (125 ml) which were then capped with butyl stoppers (Bellco Glass Co., Vineland, NJ).

Plasmid Isolation Plasmid DNA was extracted from isolates by the procedure of Sanders and Klaenhammer (1983), with slight modifications: Ten ml of culture were transferred to sterile polypropylene centrifuge tubes (17 x 100 mm, Fisher Scientific Co., Pittsburgh, PA). The cells were harvested by centrifugation (6,000 x g, 7 min, 0ºC), washed once with 10 ml of TES buffer (30 mM Tris, pH 8; 5 mM EDTA; 50 mM NaCl), recentrifuged and resuspended in 1 ml of sucrose buffer (50 mM Tris, pH 7.5; 5mM EDTA; 25% (w/v) sucrose). Freshly prepared lysozyme solution (10 mg/ml in 50 mM tris, pH 8) was added to a final concentration of 1 mg/

When H2-grown cells were desired, a 10% inoculum (from a culture at mid-log phase growing on H2/ CO2) was aseptically added to serum bottles which were then flushed (for 30 sec) and pressurized to 200 kPa with a 80:20 mixture of H2/CO2. These bottles were incubated at least 48 h with vigorous shaking at 37ºC. If cells were to be used for plasmid isolation, the medium was supplemented with 0.5% glu-

ml followed by incubation (1 h, 0ºC). Sphaeroplasts were separated from cell wall debris by centrifugation (4,500 x g, 10 min, 0ºC) and resuspended in 0.5 ml of glucose lysis buffer (50 mM Tris; 5 mM EDTA; 50 mM glucose; 3% (w/v) sodium dodecyl sulfate; mixed with 4.3 μl of 10 N NaOH immediately prior to use). After adding proteinase K (final conc. 0.1mg/ml) the pellet was disrupted using a plastic disposable pipette and the lysate incubated (1 h, 62ºC). The lysate was slowly cooled to room temperature, 70 μl of 2 M Tris, pH 7 was added, followed by 70 μl of 5 M NaCl. The lysate was transferred to a 1.5 ml microcentrifuge tube along with 0.5 ml of phenol (saturated with 3 % NaCl), emulsified with a vortexer (Genie mixer; Scientific Products, Inc., McGaw Park, IL) for 3 sec and incubated (5 min, RT). To aid in separation of the phases, 0.3 ml of chloroform was mixed in and the emulsion centrifuged (13,000 x g, 5 min, RT). The upper phase (approx 0.6 ml) was removed to a second microcentrifuge tube and 0.6 ml of chloroform:isoamyl alcohol (24:1) added. After a second centrifugation, 0.5 ml of the upper phase was removed to a third tube. DNA was precipitated by adding 2 vol of ice-cold (-20ºC) ethanol and incubating (>1 h, -60ºC). The precipitated DNA was recovered by centrifugation (13,000 x g, 5 min, RT) and dissolved in 30 μl of sterile distilled water. To reduce RNA inter-

cose, inoculated (10% v/v), and incubated overnight at 37ºC with gentle shaking (100 rpm). When larger amounts of plasmid DNA were needed, the isolates were grown anaerobically in brain heart infusion broth (Difco Laboratories, Detroit, MI) overnight.

ference, 1 μl of DNAse free RNAse (10 mg/ml) solution was added, followed by incubation (20 min, 37ºC). When larger quantities (greater than 1 mg) of plasmid DNA were required (i.e., for isolation of individual plasmids), a procedure similar to that described by

Cultivation

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Anderson and McKay (1983) was used. Three liters of culture were centrifuged (8,000 x g, 7 min, 0ºC). Pelleted cells were resuspended in 120 ml of STE buffer (6.7% (w/v) sucrose; 50 mM Tris, pH 8; 1 mM EDTA, pH 8). The cell suspension was divided into four 250-ml polypropylene centrifuge bottles. After adding 7.5 ml of fresh lysozyme solution (20 mg/ml in 50 mM Tris, pH 8) to each bottle, the cells were incubated on ice for 1 h, then at 37ºC for 10 min. Next, 3.75 ml of chelating buffer (0.25 M EDTA, pH 8; 50 mM Tris, pH 8) was added to each bottle and the cell suspension swirled for 10 sec. Cell lysis was initiated by adding 2.25 ml of lysing solution (20 % SDS, 50 mM Tris; 20 mM EDTA; pH 8) to each bottle

phases were mixed together, followed by centrifugation (6,000x g, 10 min, 0ºC). The upper aqueous layer was removed and DNA was precipitated by adding 2 volumes of ice-cold (-20ºC) ethanol and incubation overnight at -20ºC. The precipitated DNA was recovered by centrifugation (9,000 x g, 45 min, -5ºC) and resuspended in sterile distilled water and pooled. The plasmid DNA preparation was kept at 4ºC until used. Individual plasmids from isolate H3HH were isolated as follows: DNA was electrophoresed through 1 % agarose in TAE buffer (40mM tris-acetate, pH 7.8; 2mM EDTA, pH 7.8) in a horizontal electrophoresis apparatus (Model H5, BRL, Inc., Gaithersburg,

followed by vigorous mixing by hand for 15 sec. Nuclease activity in the cell suspensions was reduced by adding 0.5 ml of proteinase K solution (10 mg/ml in 50 mM Tris, pH 8) to each bottle and incubating for 10 min at 37ºC. To complete cell lysis and irreversibly denature chromosomal DNA, 0.6 ml of 10 N NaOH was added slowly while swirling the bottles by hand. The bottles were swirled for an additional 10 min using a gyratory shaker (Model G76; New Brunswick Scientific Co., New Brunswick, NJ). In order to neutralize the pH of the lysates, 4 ml of 2 M Tris, pH 7 was added to each bottle and swirling continued for 3 additional minutes. After mixing in 5.7 ml of 5 M NaCl, the preparations were centrifuged (9,000 x g, 15 min, 0ºC). The supernatant from each bottle was transferred to a second 250-ml centrifuge bottle along with 55 ml of phenol (saturated with 3% NaCl in distilled H2O). The preparations were swirled for 10 minutes using the gyratory shaker, after which 55 ml of chloroform was added into each bottle. After centrifugation (6,000 x g, 10 min, 0ºC), the upper aqueous phase was removed to a clean 250-ml centrifuge bottle with an inverted 25 ml pipette and set aside. Twenty-five ml of TES buffer was added to the phenol:chloroform mixture and vigorously mixed for 30 sec to extract additional DNA from the organic

MD). The plasmid bands were visualized by incubating the gel in an ethidium bromide solution (0.5 μg/ ml) for 10 min and briefly illuminating the gel with ultraviolet light. The gel areas containing the desired plasmid DNA were separated from the rest of the gel with a razor blade. Plasmid DNA was extracted from these gel strips by electroelution as follows: The gel strip was inserted into dialysis tubing (Spectro-por, 12000 to 14000 MWCO, Spectrum Medical Systems, Houston, TX). After removing most of the buffer surrounding the gel slice, the tubing was clamped shut and placed in the electrophoresis unit filled with TAE buffer. The tubing was arranged so that the gel slice was parallel to the electrodes. The plasmid DNA was moved out of the agarose gel slice by applying 5 V/cm (100 V) for 2 h. Following a 1 min polarity reversal of the electric current, the tubing was removed from the unit, unclamped and the gel slice carefully removed. The contents of the dialysis tubing were transferred to a polypropylene centrifuge tube (17x 100 mm, Fisher Scientific Co., Pittsburgh, PA). The inside of the dialysis tubing was rinsed with a small amount of sterile distilled water which was then transferred to the centrifuge tube containing the plasmid DNA. After an initial centrifugation (13,000 x g, 5 min, 0ºC) to remove agarose

phase. Following centrifugation (6,000 x g, 10 min, 0ºC), the upper aqueous phase was removed with an inverted 25 ml pipette and mixed with the aqueous fraction previously set aside. After adding an equal volume of chloroform:isoamyl alcohol (24:1), the two

particles, the plasmid DNA was transferred to a microcentrifuge tube, precipitated with ethanol, and pelleted by centrifugation (13,000 x g, 5 min, -5ºC). The DNA was dissolved in 50 ul of sterile distilled water and stored at 4ºC until used.

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Restriction enzyme analysis Plasmid DNA was digested by restriction endonucleases under the conditions (e.g., salt concentration) specified by the supplier of each enzyme. The plasmid preparations were incubated for 3 h with the appropriate amount of enzymes. When digestions with two different enzymes were performed simultaneously, conditions were adjusted appropriately to optimize activity of both restriction endonucleases. The size of plasmid DNA fragments was estimated by electrophoresis through horizontal 1 % agarose gels using TAE buffer. A 1 kb linear DNA ‘ladder’ (Gibco BRL, Inc, Gaithersburg, MD) served as the molecular weight marker and was used to estimate the size of plasmid DNA fragments. After electrophoresis, plasmid DNA bands were visualized by soaking the agarose gel in ethidium bromide solution for 20 minutes. The gels were illuminated with a UV transiluminator and photographed with a Polaroid MP-4 Land camera system using type 57 Polaroid film.

Antibiotic sensitivity The sensitivity of isolates H3HH and H3HP to various antibiotics was determined as follows: 0.1 ml of an overnight culture of isolate H3HH was spread across the surface of 15 cm diameter petri dishes containing brain heart infusion broth (Difco Laboratories, Detroit, MI) solidified with 2% agar. Once the surface had dried, the appropriate antibiotic diffusion disks (Difco Laboratories, Detroit, MI) containing one of the following: novobiocin, vancomycin, kanamycin, chloramphenicol, penicillin G, rifampicin, polymixin B, streptomycin, erythromycin, tetracycline, gentamycin or nalidixic acid were aseptically placed on the agar surface separated by at least at least 3 cm. The plates were incubated anaerobically for 24 h at 37ºC and the diameter of the zones of inhibition measured.

Reagents Restriction endonucleases were obtained from Boeringer Mannheim (Indianapolis, IN) and Prome189

ga, Inc (Madison, WI). Tris, phenol, proteinase K, lysozyme, and ethidium bromide were obtained from Sigma Chemical Co. (St Louis, MO). SeaKem ME agarose was obtained from FMC Corp (Rockland, ME). Gases (H2, H2/CO2, N2, N2/CO2, CO2) were obtained from Matheson Gas Products (Joliet, IL). All other reagents were of the highest purity commercially available.

RESULTS AND DISCUSSION The underlying objective for determining if ruminal acetogenic isolates contain plasmid DNA was to obtain the genetic machinery (i.e. origins of replication) necessary for construction of genetic transfer systems (i.e., shuttle vectors). Although shuttle vectors have been inserted into a non-ruminal acetogen, we felt that use of replicons obtained from plasmids found in acetogens isolated from ruminal contents would enable construction of stable and efficient shuttle vectors between the acetogens isolated from ruminal contents and organisms with wellresearched genetic systems such as E. coli. Several procedures, including those described by Birnboim and Doly (1979), Kado and Liu (1981), and Dean et al., (1989) were tested but resulted in poor recovery (both quantitative and qualitative) of acetogen plasmid DNA (data not shown). The methods described herein were adapted from published procedures (Anderson and McKay, 1983; Sanders and Klaenhammer, 1983) designed for use with streptococci and lactococci. Plasmid DNA was difficult to obtain from these isolates due to high nuclease activity as well as a very tough cell membrane. Deoxyribonuclease activity is not unusual among ruminal bacteria and could pose a barrier to development of gene transfer systems (Flint and Thomson, 1990; Javorsky and Vanat, 1992). However, in isolate H3HH, the deoxyribonuclease activity could be reduced by treating the lysate with proteinase K. Alternatively, when the lysate was heated to 95ºC for 10 min deoxyribonuclease activity was reduced, but the reproducibility was variable and at times plasmid DNA could not be visually detected in ethidium bromide (EtBr) stained agarose gels. Incu-

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Figure 1. Physical map of pRSP5 obtained from

isolate H3HH. Plasmid DNA was electrophoresed through a 0.7% agarose gel and briefly stained with ethidium bromide (0.5 μg/ml). The appropriate band was excised from the gel and the DNA electroeluted out of the agarose. Aliquots of the isolated plasmid DNA were subjected to restriction enzyme digestion and the resulting fragments electrophoresed through a 1% agarose gel , stained with ethidium bromide (0.5 μg/ml) and photographed. The length of the fragments was estimated by comparison with a 1 kb DNA ladder that was also electrophoresed through the gel. The fragments were aligned based on the digestion pattern obtained by simutaneous digestion with two restriction enzymes.

bation of the cell suspension with lysozyme was very important as only minimal lysis was achieved if this step was omitted. A 1 h incubation at 0ºC was used for both small- and large-scale preparations but an additional 10 min incubation at 37ºC was necessary during the large scale preparation to increase DNA yields. Incubation with mutanolysin, another cellwall degrading enzyme, was not effective (data not shown). Isolate H3HH contained plasmid DNA ranging in size from 32 to 4.5 kilobase (kb) (data not shown) while a second isolate (H4) contained a single 35 kb plasmid (data not shown). The presence of plasmid DNA suggests that these isolates may be capable of maintaining foreign DNA, a prerequisite for the exploration of the genetic systems of these organisms. Because of the number of plasmid bands obtained from isolate H3HH (which could be covalently closed circular (CCC), open circular (OC) and linear forms of a few plasmids or CCC form of many plasmids), it was impossible to determine the actual number of plasmids using conventional (unidimensional) agarose gel electropheresis. However, using the tech-

from isolate H3HH (data not shown). The presence of multiple plasmids in one organism is not unusual and has been reported previously in other bacterial species including: Escherichia coli V517 (Macrina et al., 1978), Lactococcus lactis (Sanders and Klaenhammer, 1983). Because of apparent high copy number (estimated by the relative band brightness of EtBr stained gels) and relatively small size, the smallest plasmid of isolate H3HH (named pRSP5) was selected for characterization . A restriction map of the plasmid was constructed by performing single and double digestions with 13 restriction endonucleases (Fig. 1). Although this plasmid was cut by SinI, EcoRV, HindIII and BglI, this plasmid did not appear to contain cleavage sites for PstI, SalI, EcoRI, PvuI, PvuII, BamHi, XhoI, SphI, and EcoRI. Analysis of the digested DNA

nique of Hintermann et al. (1981) where plasmid DNA was subjected to two dimensional agarose gel electrophoresis with ultraviolet light irradiation preceeding the second dimension electrophoresis, five plasmid bands were identified in DNA obtained

H3HH is repeatedly transferred in rich media (such as brain heart infusion). A plasmid-free derivative (H3HP) of isolate H3HH (obtained after more than 10 transfers in brain heart infusion) displayed increased sensitivity to antibiotics, suggesting the presence of

fragments suggested that pRSP5 is a circular molecule of approximately 4.5 kb. The function of the individual plasmids has not been established because sub-isolates containing a single plasmid band have not been isolated. However, the plasmids are unstable and disappear if isolate

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Table 1. Antibiotic sensitivity of isolates H3HH and H3HP Growth inhibition zone (mm)

sis of the genes that encode enzymes as well as accessory proteins) of these bacteria is needed. Most, if not all, of the enzymes involved in the acetyl-CoA pathway have been purified to homogeneity and characterized (Ragsdale, 1991). More recently, the complete sequence has become available for certain nonruminal acetogens such as Moorella thermoacetica (f. Clostridium thermoaceticum) which should aid genetic modifications (Pierce et al., 2008).

amount / disk

isolate H3HH

isolate H3HP

Chloramphenicol

30 μg

29a

Erythromycin

15 μg

Gentamycin

10 μg

Kanamycin

30 μg

Nalidixic acid

30 μg

Novobiocin

30 μg

Penicillin G

2 units

Anderson, D. G. and L. L. McKay. 1983. Simple and

Polymixin B

300 units

Rifampicin

5 μg

Streptomycin

10 μg

Tetracycline

30 μg

Vancomycin

30 μg

antibiotic resistance genes on these plasmids (Table 1). However, isolate H3HP is capable of growth on H2/CO2 at rates similar to isolate H3HH (data not shown), suggesting that these plasmids do not carry genes essential for chemolithoautotrophic growth. The work described here may open the door for the potential genetic manipulation of acetogens isolated from the rumen. Further research will be needed to determine the genetic information (i.e., origin of replication, promoters, open reading frames, and other components) encoded in pRSP5. The restriction map of pRSP5 will prove helpful for further analysis of this plasmid as well as providing sites for insertion of genes, antibiotic resistance markers or

rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46:549-552. Asmundson, R. V. and W. J. Kely. 1987. Isolation and characterization of plasmid DNA from Ruminococcus. Curr. Microbiol. 16:97-100. Birnboim, H. C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. Blaxter, K. L. and J. L. Clapperton. 1965. Prediction of the amount of methane produced by ruminants. Br. J. Nutr. 19:511-522. Boccazzi, P., and J. A. Patterson. 2011. Using hydrogen limited anaerobic continuous culture to isolate low hydrogen threshold ruminal acetogenic bacteria. Agric. Food Anal. Bacteriol. 1:33-44. Dean, R. G., S. A. Martin, and C. Carver. 1989. Isolation of plasmid DNA from the ruminal bacterium Selenomonas ruminantium HD4. Lett. Appl. Microbiol. 8:45-48. Drake, H.L. 1992. Acetogenesis and Acetogenic bacteria. In: J. Lederberg. Ed. Encyclopedia of Microbiology. Academic Press, Inc. San Diego, CA. p 1-15. Flint, H. J. and C. S. Stewart. 1987. Antibiotic resistance patterns and plasmids of ruminal strains of

other vectors. Nevertheless, before further work on preparation of shuttle vectors for acetogenic bacteria is performed, a more thorough understanding of the physiology (i.e., determination of control points of the acetyl CoA pathway) and genetics (i.e., analy-

Bacteroides ruminicola and Bacteroides multiacidus. Appl. Microbiol. Biotechnol. 26:450-455. Flint, H. J., S. H. Duncan, J. Bisset and C. S. Stewart. 1988. The isolation of tetracycline-resistant strains of strictly anaerobic bacteria from the rumen. Lett.

antibiotic

Overnight broth cultures (0.1 ml) were spread on 100 mm diameter plates containing brain heart infusion medium + 1% agar. Antibiotic disks were placed on the dried surface. Plates were incubated for 48 h inside an anaerobic incubator at 37oC. Values reported are mean (in mm) of two different zones, each in a different plate. a

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REFERENCES

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Appl. Microbiol. 6:113-115. Flint, H. J. and A. M. Thomson. 1990. Deoxyribonuclease activity in rumen bacteria. Lett. Appl. Microbiol. 11:18-21. Forsberg, C. W., B. Crosby, and D. Y. Thomas. 1986. Potential for manipulation of the rumen fermentation through the use of recombinant DNA techniques. J. Anim. Sci. 63:310-325. Greening, R. C., and J. A. Z. Leedle. 1989. Enrichment and isolation of Acetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen. Arch. Microbiol. 151:399-407 Hespell, R. A. 1987. Biotechnology and modifications of the rumen microbial ecosystem. Proc. Nutr. Soc. 46:401-413. Hintermann, G., H. M. Fischer, R. Crameri, and R. Hutter. 1981. Simple procedure for distinguishing CCC, OC, and L forms of plasmid DNA by agarose gel electrophoresis. Plasmid 5:371-373. Javorsky, P. and I. Vanat. 1992. Deoxyribonuclease activity in Streptococcus bovis. Lett. Appl. Microbiol. 14:108-110. Kado, C. I. and S.-T. Liu. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:1365-1373. Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J. Ayers, and S. M. McCowen. 1978. A multiple plasmidcontaining Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1:417-420. Mann, S. P. ,G. P. Hazlewood, and C. G. Orpin. 1986. Characterization of a cryptic plasmid (pOM1) in Butyrivibrio fibrisolvens by restriction endonuclease analysis and its cloning in Escherichia coli. Curr. Microbiol. 13:17-22. Martin, S. A. and R. G. Dean. 1989. Characterization of a plasmid from the ruminal bacterium Selenomonas ruminantium. Appl. Environ. Microbiol. 55:3035-3038. Pierce, E., G. Xie, R. D. Barabote, E. Saunders, C.

Ragsdale, S. W. and L. G. Ljundahl. 1984. Hydrogenase from Acetobacterium woodii. Arch. Microbiol. 139:361-365. Ricke, S. C., S. A. Martin, D. J. Nisbet. 1996. Ecology, metabolism, and genetics of ruminal selenomonads. Crit. Rev. Microbiol. 22:27-56. Sanders, M. E. and T. R. Klaenhammer. 1983. Characterization of phage-sensitive mutants from a phage-insensitive strain of Streptococcus lactis: evidence for a plasmid determinant that prevents phage absorption. Appl. Environ. Microbiol. 46:1125-1133. Teather, R. M. 1982. Isolation of plasmid DNA from Butyrivibrio fibrisolvens. Appl. Environ. Microbiol. 43:298-302.

S. Han, J. C. Detter, P. Richardson, T. S. Brettin, A. Das, L. G. Ljungdahl, and S. W. Ragsdale. 2008. The complete genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum). Environ. Microbiol. 10:2550-2573. Agric. Food Anal. Bacteriol. â&#x20AC;˘ AFABjournal.com â&#x20AC;˘ Vol. 1, Issue 2 - November 2011

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CONTENT OF MANUSCRIPT We invite you to consider submitting your research and review manuscripts to AFAB. The journal serves as a peer reviewed scientific forum for to the latest advancements in bacteriology research on Agricultural and Food Systems which includes the following fields:

•

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•

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•

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•

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•

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•

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•

Analytical microbiology

•

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Examples: Chase, G. and L. Erlandsen. 1976. Evidence for a complex life cycle and endospore formation in the attached, filamentous, segmented bacterium from murine ileum. J. Bacteriol. 127:572-583. Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van Doesburg, and A. J. M. Stams. 2009. A typical one-carbon metabolism of an acetogenic and hydrogenogenic Moorella thermioacetica strain. Arch. Microbiol. 191:123-131. Book: Examples: Author(s) [or editor(s)]. Year. Title. Edition or volume (if relevant). Publisher name, Place of publication. Number

Inclusive pages of chapter.

Examples: O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010. Assessing consumer concerns and perceptions of food safety risks and practices: Methodologies and outcomes. In: S. C. Ricke and F. T. Jones. Eds. Perspectives on Food Safety Issues of Food Animal Derived Foods. Univ. Arkansas Press, Fayetteville, AR. p 273-288. Dissertation and thesis: Author. Date of degree. Title. Type of publication, such as Ph.D. Diss or M.S. thesis. Institution, Place of institution. Total number of pages.

Maciorowski, K. G. 2000. Rapid detection of Salmonella spp. and indicators of fecal contamination in animal feed. Ph.D. Diss. Texas A&M University, College Station, TX. Donalson, L. M. 2005. The in vivo and in vitro effect of a fructooligosacharide prebiotic combined with alfalfa molt diets on egg production and Salmonella in laying hens. M.S. thesis. Texas A&M University, College Station, TX. Van Loo, E. 2009. Consumer perception of ready-toeat deli foods and organic meat. M.S. thesis. University of Arkansas, Fayetteville, AR. 202 p. Web sites, patents: Examples: Davis, C. 2010. Salmonella. Medicinenet.com. http://www.medicinenet.com/salmonella /article. htm. Accessed July, 2010.

of pages.

Hungate, R. E. 1966. The rumen and its microbes. Academic Press, Inc., New York, NY. 533 p. 199

Afab, F. 2010, Development of a novel process. U.S. Patent #_____

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Abstracts and Symposia Proceedings: Fischer, J. R. 2007. Building a prosperous future in which agriculture uses and produces energy efficiently and effectively. NABC report 19, Agricultural Biofuels: Tech., Sustainability, and Profitability. p.27 Musgrove, M. T., and M. E. Berrang. 2008. Presence of aerobic microorganisms, Enterobacteriaceae and Salmonella in the shell egg processing environment. IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10) Vianna, M. E., H. P. Horz, G. Conrads. 2006. Options and risks by using diagnostic gene chips. Program and abstracts book , The 8th Biennieal Congress of the Anaerobe Society of the Americas. p. 86 (Abstr.)

Data Presentation in Tables and Figures Figures and tables to be published in AFAB must be constructed in such a fashion that they are able to “stand alone” in the published manuscript. This

means that the reader should be able to look at the figure or table independently of the rest of the manuscript and be able to comprehend the experimental approach sufficiently to interpret the data. Consequently, all statistical analyses should be very carefully presented along with variation estimates and what constitutes an independent replication and the number of replicates used to calculate the averages presented in the table or figure. Each table and figure must be on a separate page in the submitted paper. If your manuscript is accepted for publication, you will need to submit all data for charts, tables and figures in Excel spreadsheet format. All figures should be clearly presented with well defined axis and units of measurement. Symbols, lines, and bars must be made distinct as “stand alone” black and white presentations. Stippling, dashed lines etc. are encouraged for multiple comparison but shades of gray are discouraged. Color images, micrographs, pictures are recommended and there is no additional fee for their submission.

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