AFAB-Volume2-Issue3 by cielo shabatura

ISSN: 2159-8967 www.AFABjournal.com

Volume 2, Issue 3 2012

158 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

EDITORIAL BOARD Sooyoun Ahn

W.K. Kim

University of Florida, 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

Oklahoma State 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

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

EDITORS

MANAGING AND LAYOUT EDITOR Ellen J. Van Loo Ghent, Belgium

TECHNICAL EDITOR

Todd R. Callaway FFSRU, USADA-ARS, USA

Jessica C. Shabatura Fayetteville, Arkansas, USA

Cesar Compadre University of Arkansas for Medical Sciences, USA

ONLINE EDITION EDITOR C.S. Shabatura Fayetteville, Arkansas, USA

Philip G. Crandall University of Arkansas, USA

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160 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

TABLE OF CONTENTS ARTICLES 162

Age and Diet Effects on Fecal Populations and Antibiotic Resistance of a Multi-drug Resistant Escherichia coli in Dairy Calves T. S. Edrington, R. L. Farrow, B. H. Carter, A. Islas, G. R. Hagevoort, T. R. Callaway, R. C. Anderson, and D. J. Nisbet

175 Sugar Yields from Dilute Acid Pretreatment and Enzymatic Hydrolysis of Sweetgum A. C. Djioleu, E. M. Martin, M. H. Pelkki, and D. J. Carrier

187 Microbiological Quality Assessment of Raw Meat and Meat Products, and Antibiotic Susceptibility of Isolated Staphylococcus aureus S. Datta, I. G. Shah, A. Akter, K. Fatema, T. H. Islam, A. Bandyopadhyay, Z. U.M. Khan, and D. Biswas

195 Effect of Stressors on the Viability of Listeria During an in vitro Cold-Smoking Process J. R. Pittman, T. B. Schmidt, A. Corzo, T. R. Callaway, J. A. Carroll, and J. R. Donaldson

209 Antibacterial Activity of Plant Extracts on Foodborne Bacterial Pathogens And Food Spoilage Bacteria

N. Murali, G. S. Kumar-Phillips, N. C. Rath, J. Marcy, and M. F. Slavik

222 Prevalence of foodborne pathogens and effectiveness of washing or cooking in reducing microbiological risk of contaminated Red amaranth

Md. A. A. Mamun, H. A. Simul, A. Rahman, N. N. Gazi, and Md. L. Bari

Introduction to Authors 233 Instructions for Authors

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Age and Diet Effects on Fecal Populations and Antibiotic Resistance of a Multi-drug Resistant Escherichia coli in Dairy Calves† T. S. Edrington1, R. L. Farrow1, B. H. Carter2, A. Islas2, G. R. Hagevoort3, T. R. Callaway1, R. C. Anderson1, and D. J. Nisbet1 Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, USDA - ARS, College Station, TX 77845 2 Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 88003 3 Agricultural Experiment Station, New Mexico State University, Clovis, NM 88101 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 Dairy calves are colonized at a very young age by a multi-drug resistant Escherichia coli (MDR EC) and research studies indicate that the prevalence is not related to recent use of antimicrobials, but that diet and other environmental factors are likely involved. To further investigate the occurrence of this bacterium, we sampled dairy calves on southwestern United States farms at one week of age through 6 months, and determined not only prevalence, but fecal concentrations of the MDR EC. The influence of feeding pasteurized (PWM) versus non-pasteurized (NPWM) waste milk was examined, and the effect of weaning was investigated. The number of fecal samples positive for MDR EC as well as their populations decreased (P < 0.01) with increasing calf age. Slight differences were observed when comparing PWM and NPWM feeding, with MDR EC concentration and prevalence in the latter group generally decreasing at younger ages. No significant differences were observed in the fecal concentrations of MDR EC due to weaning. No clear differences were observed in resistance when comparing calves fed PWM or NPWM. Approximately 41% of the MDR EC isolates collected throughout the study were resistant to 10 or more antibiotics, with two primary phenotypes: ACSSuT and MDR-AmpC. Based on the results herein, it appears that neither pasteurization of the waste milk or weaning, has a significant effect on the prevalence or concentration of MDR EC, and based on the age-associated decline in prevalence, they survive in an immature digestive system with limited bacterial diversity and competition for resources. Keywords: E. coli, multi-drug resistance, dairy calves, age, weaning Agric. Food Anal. Bacteriol. 2: 162-174, 2012 Correspondence: T.S. Edrington tom.edrington@ars.usda.gov Tel: +1-979-260-3757 Fax: +1-979-260-9332

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INTRODUCTION Antimicrobial resistant bacteria are a growing concern worldwide for both veterinary and human medicine (National Academy of Science, 1999). While the increased resistance in pathogenic bacteria is of utmost concern, commensal bacteria can also be highly resistant to a wide variety of antimicrobials and are considered by some as a potential reservoir of resistance elements for the pathogenic strains (Shoemaker et al., 2001; Summers, 2002). More specifically, the presence of multi-drug resistant (MDR) non-pathogenic commensal bacteria such as Escherichia coli on dairy farms could theoretically provide

in younger animals (Howe and Linton, 1976; Hinton et al., 1985). Mature dairy cows sampled in 21 states and cultured for E. coli and Salmonella found that the majority of isolates (greater than 80%) were susceptible to all antibiotics examined (Lundin et al., 2008). Houser and colleagues (2008) reported that 62% of the E. coli isolated from healthy lactating dairy cows were susceptible to all antibiotics examined and 21% were resistant to only one antibiotic, ampicillin. We reported similar results when examining dairy cattle of various ages for MDR Salmonella (Edrington et al., 2008). In this research we found that young calves, prior to weaning, were more likely to harbor MDR Salmonella than all other classes of dairy animals

a pool of transferable resistance genes for important pathogens such as Salmonella and E. coli O157:H7 (Schmieger and Schicklmaier, 1999; Winokur et al., 2001; Oâ&#x20AC;&#x2122;Brien 2002; Hoyle et al., 2004). The general consensus is that antimicrobial-resistant bacteria, to include commensals in humans and animals, are produced, maintained and disseminated due to the selection pressure induced by exposure to antimicrobial drugs (van den Bogarrd and Stobberingh, 2000). Research examining E. coli in calves reported that exposure to antibiotic in the feed resulted in the development of not only resistance to the fed antibiotic but several other antibiotics as well (Wierup et al., 1975). Others have reported that the discontinuation of feeding an antibiotic-medicated milk replacer to dairy calves resulted in an increase in tetracycline susceptibility in E. coli and Salmonella isolates during the first three months that a non-medicated milk replacer was fed (Kaneene et al., 2008). While exposure to antibiotics certainly contributes to resistant bacteria, other nonantibiotic influences have been reported (Sogaard 1973; Smith 1975; Gellin et al., 1989; Gilliver et al., 1999). Younger animals generally harbor more resistant enteric flora than older animals (Wierup, 1975; Mar-

(heifers, lactating and dry cows) examined. The primary exception was cows in the hospital pen, as they also exhibited significant levels of MDR Salmonella. We hypothesized that the reasons for the high incidence of MDR Salmonella in these two groups was a result of previous antimicrobial treatment, as these two groups of cattle are the most likely to receive antibiotic therapy, and/or due to a disturbed or underdeveloped gastrointestinal microflora. In the case of young calves, their intestinal microflora is developing and changing with the introduction of new feedstuffs, weaning, environmental exposure, and other factors, whereas the cows in the sick pen are generally off-feed resulting in a disturbed microflora vulnerable to competition from new bacterial species. Several studies have documented the prevalence of a highly resistant E. coli in dairy calves; however, the results did not provide for a complete description of the early temporal shifts or compare calves from different geographic regions and management systems (Wierup, 1975; Howe and Linton, 1976; Hinton et al., 1984; Khachatryan et al., 2004). Interestingly, these MDR E. coli do not appear to be specific to a geographic region or management practice, having been reported in Washington (DeFrancesco et al., 2004), Pennsylvania (Houser et al., 2008), and the SW

tel and Coudert, 1993). Pre-weaned calves have been reported with higher MDR levels in enteric flora, possibly a result of increased fecal-oral transmission, higher strain turnover within the gastrointestinal tract, or higher levels of antimicrobial drug use

United States (Edrington, unpublished data). Others have documented that pre-weaned calves had the greatest prevalence of resistant E. coli, with levels decreasing with increasing animal age (Khachatryan et al., 2004). Healthy dairy calves were reported to

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be rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth (Donaldson et al., 2006) with the highest prevalence observed in 2-week old calves. Calves were reported to shed MDR bacteria resistant to 9 and 10 antibiotics as early as one day of age (Donaldson et al., 2006) with similar observations reported by others (Orden et al., 2000; Werckenthin et al., 2002). The question then arises: Are the levels of resistance in these calves a result of previous/current antibiotic exposure? Berge and co-workers (2006) reported higher levels of MDR E. coli in calves fed antimicrobials compared to those on non-medicated feed. Isolates cultured from older calves not fed antimicrobials (14 and 28 d old),

This research was conducted on several large commercial dairies (greater than 3000 head) in the southwestern United States. Four collections were made for this research project. The first sampled calves on two farms representing six age groups (1 week, 2 weeks, 1, 2, 4 and 6 months of age). Fecal samples (approximately 20 g) were collected from freshly voided, undisturbed fecal pats from 15 animals per age group on each farm (n = 90 samples/ farm; 180 total samples). Both farms utilized waste milk to feed the calves prior to weaning, one farm pasteurizing the milk prior to feeding, the other using non-pasteurized waste milk. A second similar collection was made, the only difference being that

had higher levels of resistance compared to day old animals with 14-day old calves most likely to shed increasingly resistant bacteria (Berge et al., 2006). In contrast to this, others have reported that the maintenance of the E. coli SSuT resistance phenotype in dairy calves was due to environmental components independent of antibiotic selection (Khachatryan et al., 2006a). Further research by this same group (Khachatryan et al., 2006b) reported that the antimicrobial resistant genes are not responsible for the greater fitness advantage of antimicrobial-resistant E. coli in calves, but that the farm environment and the diet clearly exert critical selective pressures responsible for the maintenance of antimicrobial resistance genes. Others have also reported that housing and dietary changes, occurring at weaning, may affect the prevalence of antibiotic-resistant strains by altering the calfâ&#x20AC;&#x2122;s exposure to other animal stock and bacterial strains that in turn change the E. coli composition of their gut microflora (Hoyle et al., 2004). Therefore, the objectives of the current research were to evaluate the effect of age, diet (pasteurized or non-pasteurized waste milk), weaning and farm origin on fecal populations and prevalence of MDR E. coli in dairy calves. Antimicrobial susceptibility patterns were also examined.

a different farm utilizing pasteurized waste milk was sampled. A total of 360 samples were collected and cultured for multi-drug resistant E. coli (MDR EC). A third collection was made in order to evaluate the influence of weaning on the prevalence of MDR EC in dairy calves and was part of a larger study examining the role of weaning on the prevalence of a number of important bacteria (Edrington et al., 2011). Two groups of calves were utilized, the first weaned at approximately 12 weeks of age (avg. BW = 122 kg) and the second group at approximately 10 weeks of age (were not weighed at weaning; estimated BW = 110 kg). Fecal samples were collected from all calves via rectal palpation on two occasions, two days preand again two days post-weaning for bacterial culture described below. The fourth collection sampled newborn calves (1 to 3 days of age) from four different dairies during their first week of arrival at a central calf rearing facility. Rectal fecal samples were collected into sterile palpation sleeves from 38, 45, 69 and 40 calves (n = 192 total samples) representing each of the four farms over a four-week period.

MATERIALS AND METHODS Animals and Sample Collection

Bacterial Culture and Isolation All fecal samples were collected into sterile palpation sleeves, placed on ice and shipped to our laboratory in College Station, Texas for processing the day following collection. For culture and quantitation of MDR EC populations, 10 g of fecal material was diluted in 90 mL of tryptic soy broth

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and plated on MacConkey’s agar containing 32 µg/ mL chloramphenicol, using a commercially available spiral plater. Following incubation (24 h, 37º C), colonies exhibiting typical E. coli morphology were manually counted to determine colony forming units (CFU)/g feces. This was converted to CFU (log10)/g feces for statistical analysis and data presentation below. A portion of the isolates from each collection were confirmed as E. coli using the API 20E test kit (BioMerieux, Durham, NC). Isolates were stored as glycerol stocks (10% v/v) in TSB at - 80ºC. All media and agar were from Difco Laboratories (Detroit, MI). Reagents and antibiotics were obtained from Sigma Chemical Co. (St. Louis, MO).

Determination of Antimicrobial Susceptibility Antimicrobial susceptibility was determined using the Sensititre automated antimicrobial susceptibility system according to the manufacturer’s directions (Trek Diagnostic Systems, Westlake, OH). Broth microdilution was used according to methods described by the National Committee for Clinical Laboratory Standards (CLSI 2005) using the NARM’s panel for gram-negative isolates. Resistance breakpoints were determined using the CLSI (CLSI 2005) interpretive standards unless unavailable, in which case breakpoints in the NARMS 2000 Annual Report (FDA 2000) or those provided by Trek Diagnostic were

Table 1. Fecal prevalence of MDR EC (number and populations) in dairy calves of multiple ages, housed on two commercial dairy farms and feeding pasteurized (PWM) or non-pasteurized (NPWM) waste milk through weaning

Item

Calf Age 1 wk

2 wks

1 mo

2 mos

4 mos

6 mos

no. positive

15/15

13/15

9/15

CFU(log10)/g feces

2.7bB

5.4A

5.2aA

3.7aB

2.4B

no. positive

15/15

10/15

8/15

4/15

CFU(log10)/g feces

5.1aA

5.2A

5.8A

3.1bB

2.2bBC

1.8C

14/15

15/15

14/15

5.3B

6.3aA

5.4aB

5aB

3.8aC

no. positive

15/15

10/15

14/15

9/15

CFU(log10)/g feces

5.4AB

5.9bA

4.9B

2.9bC

2.2bC

Collection 1 Farm A - PWM

Farm B - NPWM

Collection 2 Farm A - PWM no. positive CFU(log10)/g feces Farm B - NPWM

CFU within collection and age column with different superscripts differ (P < 0.05). CFU within collection and farm row with different superscripts differ (P < 0.05). Culture negative samples assigned value of 1.0. ab

ABC

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used. Escherichia coli ATCC 25922, E. coli ATCC 35218, and Enterococcus faecalis ATCC 29212 were used as quality control organisms.

Data were analyzed using SAS Version 8.02 (SAS Inst. Inc., Cary, NC, USA). Quantitative data expressed as CFU (log10)/g feces were subjected to analysis of variance appropriate for a completely randomized design. A value of 1.0 was assigned to all negative samples for statistical analysis. Pen prevalence was subjected to Chi-square analysis using the PROC FREQ procedure. Means were consid-

isolates were susceptible to ciprofloxacin and ceftriaxone. The number of isolates resistant to all other antibiotics examined decreased with increasing calf age at each collection time (Table 2). Multi-drug resistance and resistance phenotypes are presented in Table 3. One isolate was resistant to two antibiotics with all other isolates resistant to four or more antimicrobials. Thirty-eight percent of the isolates were resistant to 10 or more antibiotics, the majority of which were cultured in the November collection. Primary resistance patterns observed were ACSSuT and MDR-AmpC, the first of which was more prevalent in the second collection and the frequency of the MDR-AmpC pattern similar among

ered different at a 5% level of significance.

collections (Table 3).

RESULTS

Influence of Weaning on Prevalence and Antimicrobial Susceptibility of MDR EC

Statistical Analysis

Influence of Age on Prevalence and Antimicrobial Susceptibility of MDR EC The prevalence and concentration of MDR EC is presented by age and by farm [feeding pasteurized (PWM) or non-pasteurized waste milk (NPWM)] in Table 1 for the two collections. The number of fecal samples positive for MDR EC decreased with increasing calf age during both collections, with the decrease being more pronounced when comparing the farm feeding NPWM versus the two farms feeding PWM. Fecal concentration of MDR EC likewise decreased (P < 0.01) with increasing age on all farms for both collections (Table 1). When comparing type of waste-milk fed, MDR EC concentration decreased more rapidly with increasing age in the farms feeding NPWM (Table 1). Antimicrobial susceptibility was examined in MDR EC isolates (six isolates/age group/collection; n = 72 total MDR EC isolates). In general, during the first collection, more resistance was observed in the farm using NPWM compared to collection 2, when

Samples were collected from two groups of calves immediately prior to and following weaning and cultured for MDR EC (Table 4). No significant differences were observed in the fecal concentrations or in the number of MDR EC positive pens in either group or when data was combined across groups. There was a tendency (P = 0.06) for fewer MDR EC positive pens in the second group of calves post-weaning. Twenty MDR EC isolates were examined for antimicrobial susceptibility (five per group pre- and postweaning). All of the isolates were susceptible to amikacin, ceftriaxone, ciprofloxacin and naladixic acid, and all but one isolate susceptible to amoxicillin/clavulanic acid, cefoxitin and ceftiofur. All isolates were resistant to kanamycin, sulfisoxazole and tetracycline and all but one resistant to chloramphenicol (data not shown). Half of the isolates were resistant to four or five antibiotics and most of the remaining half of the isolates (nine isolates) resistant to six, seven, or eight antibiotics (Table 5). One isolate was resistant to 10 antibiotics. Several patterns of resistance were

the opposite trend was observed, therefore the data was pooled across farm and presented by collection date in Table 2. All isolates were resistant to chloramphenicol and tetracycline and all but one were resistant to sulfisoxazole, whereas the majority of the

observed, the most prevalent being ACSSuT. One isolate demonstrated the MDR-AmpC pattern of resistance (Table 5). Weaning did not appear to have any influence on antimicrobial resistance in these isolates.

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Table 2. Antimicrobial resistance profiles of MDR EC isolates cultured from fecal samples, by collection, from dairy calves of multiple ages on commercial dairy farms. Data represents the number of isolates resistant to the minimum inhibitory concentration (MIC) listed for each antibiotic

Item

MIC

Collection

1 wk

2 wks

No. isolates examined

Calf Age 1 mo 2 mos

Combined

Ages

4 mos

6 mos

Antibiotic Amikacin Gentamicin Kanamycin Streptomycin Ceftiofur Ceftriaxone Cefoxitin Ampicillin Chloramphenicol Ciprofloxacin Nalidixic acid Sulfisoxazole Tetracycline

> 64 > 16 > 64 > 64 >8 > 64 > 32 > 32 > 32 >4 > 32 > 256 > 16

Trimethoprim/ sulfamethoxazole

> 4/76

Amoxicillin/ clavulanic acid

> 32/16

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Table 3. Multi-drug resistance and patterns of resistance in MDR EC isolates by collection, cultured from fecal samples of dairy calves of multiple ages on commercial dairy farms Item

Calf Age

Combined

Ages

Collection

1 wk

2 wks

1 mo

2 mos

4 mos

6 mos

No. isolates examined

in each animal class

Resistant to: 2 antibiotics 4 to 6 antibiotics 7 to 9 antibiotics >10 antibiotics At least: ACSSuTa MDR-AmpC

ACSSuT = resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline.

MDR-AmpC = resistant to ACSSuT plus amoxicillin/clavulanic acid and ceftiofur, and a decreased susceptibility to ceftriaxone (MIC > 2 µg/ml). b

Table 4. MDR EC [fecal concentration = FC; CFU (log10)/g feces] and pen prevalence [% pens with calf culture positive for MDR EC (% Pens)] in two groups of dairy calves on a commercial dairy farm, sampled two days pre- and post-weaning (by group and combined)

Group

No. samples

Pre-weaning

Post-weaning

No. Pens

% Pens

3.3

3.8

3.7

2.9

Combined

144

3.5

3.4

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Table 5. Multi-drug resistance and patterns of resistance in fecal MDR EC isolates cultured from dairy calves. Data combined from two groups of dairy calves on a commercial dairy farm, two days pre- and post-weaning.

Item

Time Pre-wean

No. isolates examined

Post-wean

0 - 3 antibiotics

4 or 5 antibiotics

6 - 10 antibiotics

No. isolates resistant to:

Phenotypes

DISCUSSION A few years ago, while investigating a suspected outbreak of salmonellosis, we cultured MDR EC from a relatively large number of young dairy calves. Subsequent examination of the literature revealed that the occurrence of MDR EC had been documented in young dairy calves in other regions of the United States (DeFrancesco et al., 2004; Houser et al., 2008) and that this particular E. coli, or the maintenance of resistance in this species, was thought to be restricted to very young calves. The prevalence of resistant organisms is typically higher in younger animals (Brophy et al., 1977; Hinton et al., 1985; Zhang

In general, farms were similar in regards to susceptibility/resistance to individual antibiotics. The majority of all MDR EC isolates (greater than 80%) were resistant to chloramphenicol, streptomycin, sulfisoxasole, and tetracycline, while approximately half displayed resistance to amoxicillin/clavulanic acid, cefoxitin, ceftiofur, ceftriaxone, gentamicin, kanamycin, naladixic acid, and trimethoprim/sulfisoxasole (Table 6). Multi-drug resistance (2 to 14 antibiotics) was observed in all 192 isolates examined with most (69%) resistant to 8 or more antibiotics (Table 7). The most prevalent resistance phenotypes were ACSSuT

et al., 1998; Mathew et al., 1999). This at first would seem counter-intuitive if the development of antimicrobial resistance is related to previous antibiotic therapy. However, young animals are typically more susceptible to disease and receive antibiotics for the treatment or prevention of such diseases. Even so, it would stand to reason that as age increases, exposure to antibiotics would also increase, and therefore the prevalence of resistant isolates would be greater in older animals. However, as this is not the case in dairy cattle (Edrington et al., 2008; Houser et al., 2008; Lundin et al., 2008), researchers have speculated that perhaps this increased resistance in dairy calves is due to their exposure to more antibiotics for medication and/or growth promotion compared to mature cows. Khachatryan and coworkers (2004) reported just the opposite however, in that the resistant E. coli demonstrated a greater fitness in the calf intestinal tract environment that was independent of exposure to antimicrobial drugs and that drug use was not required to maintain a high prevalence of this resistant strain of E. coli. Others reported that the clustering of MDR EC in calves 2 to 4 weeks of age, on both dairies and calf ranches, suggest there are host-specific factors influencing the emergence of resistance that may not be associated with anti-

and MDR Amp-C, both found in 36% of the isolates. Multi-drug resistance was similar among farms with the exception of Farms A and C, in which fewer ACSSuT and more MDR AmpC phenotypes were observed on Farm A (Table 7).

biotic use (Berge et al., 2005). Taken together, this suggests that the development or maintenance of the resistance of E. coli in dairy calves is not dependent on exposure to antibiotics, but was an environmental or diet induced phenomenon.

ACSSuT

MDR-AmpC

ACSSuT=resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. a

MDR-AmpC = resistant to ACSSuT plus resistant to amoxicillin/clavulanic acid and ceftiofur and decreased susceptibility to ceftriaxone (MIC > 2 µg/mL). b

Farm Origin and Influence on Antimicrobial Susceptibility of MDR EC

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Table 6. Antimicrobial resistance profiles of fecal MDR EC isolates from dairy calves originating from multiple dairy farms upon arrival at a central heifer raising facility. Data represents the number of isolates resistant to the minimum inhibitory concentration (MIC) listed for each antibiotic.

Item

Farm of Origin MIC

No. isolates examined

Combined

Ages (%)

192

Antibiotic Amikacin

> 64

4 (2.1)

Gentamicin

> 16

94 (49)

Kanamycin

> 64

125 (65)

Streptomycin

> 64

149 (78)

Ceftiofur

79 (41)

Ceftriaxone

> 64

94 (49)

Cefoxitin

> 32

87 (45)

Ampicillin

> 32

179 (93)

Chloramphenicol

> 32

191 (99)

Ciprofloxacin

65 (34)

Naladixic acid

> 32

97 (51)

Sulfisoxazole

> 256

191 (99)

Tetracycline

> 16

191 (99)

Trimethoprim/sulfamethoxazole

> 4/76

97 (51)

Amoxicillin/clavulanic acid

> 32/16

86 (45)

Table 7. Multi-drug resistance and patterns of resistance (number of isolates and percentage in parentheses) in fecal MDR EC isolates cultured from newborn calves, originating from four different dairies, upon arrival at a central heifer raising facility

Farm of Origin

Across

Item

Farms

No. isolates examined

192

0 - 3 antibiotics

1 (1.5)

1 (0.5)

4 - 7 antibiotics

11 (29)

8 (18)

30 (43)

9 (23)

58 (30)

8 - 14 antibiotics

27 (71)

37 (82)

38 (55)

31 (78)

133 (69)

10 (26)

16 (36)

28 (41)

15 (38)

69 (36)

17 (45)

19 (42)

18 (26)

15 (38)

69 (36)

No. isolates resistant to:

Phenotypes ACSSuTa MDR-AmpC

ACSSuT=resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline MDR-AmpC = resistant to ACSSuT plus resistant to amoxicillin/clavulanic acid and ceftiofur and decreased susceptibility to ceftriaxone (MIC > 2 µg/mL) a

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Dairy calves experience a number of changes during a relatively short time frame that may explain the age related decrease for this bacterium. Adaptation and eventual weaning from a liquid, milk based diet to a diet composed of hay and grain, and the associated changes in gastrointestinal microflora could explain these age-related changes. Results of the current research demonstrated an age-related change in fecal populations and prevalence of MDR EC in dairy calves as reported by others and discussed above. We did however culture MDR EC from a substantial number of calves at 6 months of age, older that most of the calves examined in previous research. Examination of calves pre- and post-weaning found

have reduced the bacterial species that are more able to compete with the MDR EC, thus providing MDR EC a competitive advantage in the calves fed PWM. Some researchers have hypothesized that the presence of MDR EC in calves fed waste milk is due to a selection pressure maintained through the feeding of low concentrations of antibiotics contained in the milk (Berge et al., 2005). Subsequent examination of the waste milk failed to confirm the presence of antibiotics in the milk and led to the conclusion that feeding hospital milk had no observable impact on antibiotic resistance in E. coli. In the current research, if antibiotics in the milk were responsible for the MDR EC, then we would expect to see higher

no significant differences in MDR EC prevalence or populations. Taken together, these results suggest that the disappearance of MDR EC in dairy calves is a gradual process that is not strongly influenced by changing diet or other animal husbandry factors as we originally hypothesized. If these changes were in fact a result of changing diet and maturation of the digestive system, then we would expect to see a more substantial decline prior to six months of age, as diet changes significantly early in age but are very subtle later (4 and 6 months). Pasteurization of the waste milk used to feed the calves appeared to have slight influence on MDR EC populations in these dairy calves. Both the number of MDR EC positive samples and the concentration of MDR EC were lower in calves fed the NPWM compared to PWM. Significant reductions (90 to 95%) in total bacterial counts as well as for specific pathogens such as Salmonella have been reported following pasteurization of waste milk (Stabel et al., 2004; Ruzante et al., 2008). However, milk that is not properly chilled following pasteurization provides a warm environment for rapid bacterial growth, increasing the number of cells as much as 8-fold per hour. Overall bacterial counts in PWM prior to feeding, were reported to range from 500,000 to 100 million CFU/

levels in calves fed NPWM, assuming the pasteurization process affected antibiotic residues in the milk. On the other hand, if pasteurization had no affect on the antibiotics in the milk, then we would expect to see similar levels among the feeding groups, not the subtle differences we observed. Possibly the differences we observed were due to some other farm related factor and not pasteurization of the waste milk. This is certainly plausible and a drawback from the experimental design. Unfortunately, conducting research on commercial dairy farms, while providing for “real-world” settings, does have short-comings; in this case the dairyman pasteurizing waste milk was not willing to feed some of the calves on his farm non-pasteurized milk due to health concerns and labor issues. Therefore the next best scenario was to sample calves on different farms, similar in most all aspects, except for pasteurization of the waste milk. While other factors may have influenced the results, the widespread dissemination of MDR EC among dairy calves and similarity of resistance phenotypes, as observed in the first three collections as well as the fourth collection, comparing calves from four different farms, suggests this is unlikely and the differences are likely due to handling of the waste milk.

ml, which was not different from 60% of the farms pasteurizing the milk (Ruzante et al., 2008). Possibly the differences that were observed in this research are a result of competitive exclusion as influenced by the pasteurization process. Pasteurization may

Contrary to the research of Khachatryan et al. (2004), who reported a greater prevalence of SSuT resistance in milk-fed calves, Hinton et al. (1984) found that fecal E. coli from calves were more likely to develop MDR resistance during and immediately

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after weaning from a medicated milk replacer. In our research, inclusion of the MDR EC isolates collected pre- and post-weaning in this discussion confounds the interpretation. The MDR EC isolates cultured from the weaning study were resistant to fewer different antibiotics (11) and displayed two patterns of resistance (ACSSuT and MDR-AmpC) than isolates from younger calves in the first collection. However, in comparing these two groups of isolates, it must be taken into account that they were collected from different farms with different management techniques and at different times of the year. Results of this research indicate that the persistence of MDR EC in dairy calves is a function of age.

the bacteria, we suggest that while the gut is undeveloped in terms of bacterial diversity, the MDR EC is able to successfully compete, however as the bacterial flora diversifies and increases in numbers, the MDR EC loses its competitive advantage due, at least in part, to being MDR and is slowly removed from the gastrointestinal tract. Khachatryan and colleagues (2004) presented a similar explanation. Their research suggested a direct benefit of the resistance genes themselves or linkage to other genes that are adaptive in this environment. However, they went on to say that relative absence of a diverse bacterial fauna, due in part to the milk diet, is indicative that the MDR EC compete effectively only when significant

Furthermore, the decline in populations and prevalence does not appear to directly correspond to changes in diet and may be a more subtle indication of gastrointestinal maturation or other factors yet to be determined. While E. coli is present in mature cows, it is not reported to be MDR, indicating that maternal transfer is not responsible for its presence in calves but some other environmental factor(s). The gradual disappearance with age, suggest diet may be a limiting factor, although if entirely responsible for the presence and/or disappearance of the bacteria then we might expect bigger decreases in its populations when diet is significantly changed, such as at weaning, and not the steady decline we observed when diet was not changed. We hypothesize that the survival and disappearance is simply a matter of the competitive fitness of this species within the developing gastrointestinal microflora of the calf. Results of this research and of others support this conclusion. Berge and colleagues (2005) suggested that in the young calf-gastrointestinal environment, E. coli with multiple antibiotic resistance exhibits a higher fitness compared to susceptible E. coli. The intestinal microbiota is very different in a young milk fed calf compared to an adult animal, which the MDR EC appear to find more suitable for

competition is lacking and as the animal ages and the gut matures, the resistance becomes a burden and the MDR EC is excluded from the system. Previous research examining MDR Salmonella in dairy calves supports this idea. Similar to these results, we found MDR Salmonella only in young calves or sick cows, suggesting that its ability to compete within the gastrointestinal tract depends on an immature or disturbed microflora (Edrington et al., 2008). The impact of this population of MDR EC on overall calf health appears to be minimal if any, however the potential transfer of resistance elements to pathogenic bacteria such as Salmonella cannot be ruled out. Research into the origin or transmission source of this bacteria as well as methods to hasten the elimination from the gastrointestinal tract of the calf could theoretically reduce the potential development of MDR pathogenic bacteria, leading to improved calf health and in the long term, improved herd health. Reducing the “load” of pathogenic bacteria in the production setting has significant food safety implications.

survival (Khachatryan et al., 2004). This would suggest that the presence of resistance elements may give the MDR EC a survival advantage over susceptible strains in the developing gastrointestinal tract. However, as resistance generally comes at a cost to

Portions of the above research were funded by the Food Animal Concerns Trust.

ACKNOWLEDGEMENTS

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REFERENCES Berge, A.C.B., E.R. Atwill, and W.M. Sischo. 2005. Animal and farm influences on the dynamics of antibiotic resistance in faecal Escherichia coli in young dairy calves. Prev. Vet. Med. 69:25-38. Berge, A.C.B., D.A. Moore, and W.M. Sischo. 2006. Field trial evaluating the influence of prophylactic and therapeutic antimicrobial administration on antimicrobial resistance of fecal Escherichia coli in dairy calves. Appl. Environ. Microbiol. 72:38723878. Brophy, P.O., P.J. Caffrey, and J.D. Collins. 1977. Sensitivity patterns of Escherichia coli isolated

on. 1989. Antibiotic resistance of gram-negative enteric bacteria from pigs in three herds with different histories of antibiotic exposure. Appl. Environ. Microbiol. 55:2287-2292. Gilliver, M.A., M. Bennett, M. Begon, S.M. Hazel, and C.A. Hart. 1999. Antibiotic resistance found in wild rodents. Nature. 401:233-234. Hinton, M., A.H. Linton, and A.J. Hedges. 1985. The ecology of Escherichia coli in calves reared as dairycow replacements. J. Appl. Bacteriol. 58:131-138. Hinton, M., P.D. Rixson, V. Allen, and A.H. Linton. 1984. The persistence of drug resistant Escherichia coli strains in the majority faecal flora of calves. J. Appl. Bacteriol. 58:131-138.

from calves during and following prophylactic chlortetracycline therapy. Br. Vet. J. 133:340-345. Clinical and Laboratory Standards Institute. 2005. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement. CLSI/NCCLS document M100-S15. Wayne, PA. DeFrancesco, K.A., R.N. Cobbold, D.H. Rice, T.E. Besser, and D.D. Hancock. 2004. Antimicrobial resistance of commensal Escherichia coli from dairy cattle associated with recent multi-resistant salmonellosis outbreaks. Vet. Micro. 98:55-61. Donaldson, S.C., B.A. Straley, N.V. Hegde, A.A. Sawant, C. DebRoy, and B.M. Jayarao. 2006. Molecular epidemiology of ceftiofur-resistant Escherichia coli isolates from dairy calves. Appl. Environ. Microbiol. 72:3940-3948. Edrington, T.S., T.R. Callaway, R.C. Anderson, and D.J. Nisbet. 2008. Prevalence of multi-drug resistant Salmonella on commercial dairies utilizing a single heifer raising facility. J. Food Prot. 71:27-34. Edrington, T.S., B.H. Carter, R.L. Farrow, A. Islas, G.R. Hagevoort, T.H. Friend, T.R. Callaway, R.C. Anderson, and D.J. Nisbet. 2011. Influence of weaning on fecal shedding of pathogenic bacteria in dairy calves. Foodborne Path. Dis. 8:395-401. Food and Drug Administration. 2000. National An-

Houser, B.A., S.C. Donaldson, R. Padte, A.A. Sawant, C. DebRoy, and B.M. Jayarao. 2008. Assessment of phenotypic and genotypic diversity of Escherichia coli shed by healthy lactating dairy cattle. Foodborne Path. Dis. 5:41-51. Howe, K., and A.H. Linton. 1976. A longitudinal study of Escherichia coli in cows and calves with special reference to the distribution of O-antigen types and antibiotic resistance. J. Appl. Bacteriol. 40:331-340. Hoyle, D.V., H.I. Knight, D.J. Shaw, K. Hillman, M.C. Pearce, J.C. Low, G.J. Gunn, and M.E. Woolhouse. 2004. Acquisition and epidemiology of antimicrobial-resistant Escherichia coli in a cohort of newborn calves. J. Antimicrob. Chemother. 53:867871. Kaneene, J.B., L.D. Warnick, C.A. Bolin, R.J. Erskine, K. May, and R.A. Miller. 2008. Changes in tetracycline susceptibility of enteric bacteria following switching to nonmedicated milk replacer for dairy calves. J. Clin. Microbiol. 46:1968-1977. Khachatryan, A.R., D.D. Hancock, T.E. Besser, and D.R. Call. 2004. Role of calf-adapted Escherichia coli in maintenance of antimicrobial drug resistance in dairy calves. Appl. Environ. Microbiol. 70:752-757.

timicrobial Resistance Monitoring System Annual Report. Available: http://www.cdc.gov/narms/annual/2000/tables/table2.htm. Accessed January 5, 2007. Gellin, G., B.E. Langlois, K.A. Dawson, and D.K. Aar-

Khachatryan, A.R., T.E. Besser, D.D. Hancock, and D.R. Call. 2006a. Use of a nonmedicated dietary supplement correlates with increased prevalence of streptomycin-sulfa-tetracycline-resistant Escherichia coli on a dairy farm. Appl. Environ. Micro-

Agric. Food Anal. Bacteriol. â&#x20AC;˘ AFABjournal.com â&#x20AC;˘ Vol. 2, Issue 3 - 2012

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biol. 72:4583-4588. Khachatryan, A.R., D.D. Hancock, T.E. Besser, and D.R. Call. 2006b. Antimicrobial drug resistance genes do not convey a secondary fitness advantage to calf-adapted Escherichia coli. Appl. Environ. Microbiol. 72:443-448. Lundin, J.I., D.A. Dargatz, B.A. Wagner, J.E. Lombard, A.E. Hill, S.R. Ladely, and P.J. Fedorka-Cray. 2008. Antimicrobial drug resistance of fecal Escherichia coli and Salmonella spp. isolates from United States dairy cows. Foodborne Path. Dis. 5:7-19. Martel, J.L., and M. Coudert. 1993. Bacterial resistance monitoring in animals: the French national experiences of surveillance schemes. Vet. Micro-

biol. Lett. 170:251-256. Smith, H.W. 1975. Persistence of tetracycline resistance in pig E. coli. Nature. 258: 628-630. Sogaard, H. 1973. Incidence of drug resistance and transmissible R factors in strains of E. coli isolated from faeces of healthy pigs. Acta Vet. Scand. 14:381-391. Stabel, J.R., S. Hurd, L. Calvente, and R.F. Rosenbusch. 2004. Destruction of Mycobacterium paratuberculosis, Salmonella spp., and Mycoplasma spp. in raw milk by a commercial on-farm hightemperature, short-time pasteurizer. J. Dairy Sci. 87:2177-2183. Summers, A.O. 2002. Generally overlooked funda-

biol. 35:321-338. Mathew, A.G., A.M. Saxton, W.G. Upchurch, and S.E. Chattin. 1999. Multiple antibiotic resistance patterns of Escherichia coli isolates from swine farms. Appl. Environ. Microbiol. 65:2770-2772. National Academy of Science. 1999. The use of drugs in food animals: benefits and risks. National Academy Press, Washington, D.C. O’Brien, T.F. 2002. Emergence, spread, and environmental effect of antimicrobial resistance: how use of an antimicrobial anywhere can increase resistance to any antimicrobial anywhere else. Clin. Infect. Dis. 34(Suppl. 3):S78-S84. Orden, J.A., J.A. Ruiz-Santa-Quiteria, S. Garcia, D. Cid, and R. de la Fuenta. 2000. In vitro susceptibility of Escherichia coli strains isolated from diarrhoeic dairy calves to 15 antimicrobial agents. J. Vet. Med. B 47:329-335. Ruzante, J.M., I.A. Gardner, J.S. Cullor, W.L. Smith, J.H. Kirk, and J.M. Adaska. 2008. Isolation of Mycobacterium avium subsp. paratuberculosis from waste milk delivered to California calf ranches. Foodborne Path. Dis. 5:681-686. Shoemaker, N.B., H. Vlamakis, K. Hayes, and A.A. Salyers. 2001. Evidence for extensive resistance gene transfer among Bacteroides spp. and among

mentals of bacterial genetics and ecology. Clin. Inf. Dis. 34:S85-92. Van den Bogaard, A.E., and E.E. Stobberingh. 2000. Epidemiology of resistance to antibiotics. Links between animals and humans. Int. J. Antimicrob. Agents. 14:327-335. Weirup, M., K. Larsson, P. Holtenius, S.O. Jacobsson, and I. Meansson. 1975. The effect of antibiotic supplementation on antibiotic resistance, transferable antibiotic resistance, morbidity, and growth in calves. Nord. Vet. Med. 27:253-265. Werckenthin, C., S. Seidl, J. Riedl, E. Kiossis, G. Wolf, R. Stolla, and Q.R. Kaaden. 2002. Escherichia coli isolates from young calves in Bavaria: in vitro susceptibilities to 14 anti-microbial agents. J. Vet. Med. B 49:61-65. Winokur, P.L., D.L. Vonstein, L.J. Hoffman, E.K. Uhlenhopp, and G.V. Doern. 2001. Evidence for transfer of CMY-2 AmpC beta-lactamase plasmids between Escherichia coli and Salmonella isolates from food animals and humans. Antimicrob. Agents Chemother. 45:2716-2722. Zhang, X.L., F. Wang, D.M. Zhu, S. Wu, P.C. Wu, Y.D. Chen, Y.Q. Wang, and L Zhou. 1998. The carriage of Escherichia coli resistant to antibiotics in healthy populations in Shanghai. Biomed. Environ. Sci.

Bacteroides and other genera in the human colon. Appl. Environ. Microbiol. 67:561-568. Schmieger, H., and P. Schicklmaier. 1999. Transduction of multiple drug resistance of Salmonella enterica serovar Typhimurium DT 104. FEMS Micro-

11:314-320.

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Sugar Yields from Dilute Acid Pretreatment and Enzymatic Hydrolysis of Sweetgum (Liquidambar styraciflua L.) A. C. Djioleu1, E. M. Martin1, M. Pelkki2, D. J. Carrier1 Department of Biological and Agricultural Engineering, University of Arkansas, 203 Engineering Hall, Fayetteville, AR 72701 2 School of Forest Resources, University of Arkansas, Monticello, Monticello, AR 71656 1

ABSTRACT The possibility of using sweetgum from southern pine dominated forests as a biobased refinery feedstock was investigated. Sweetgum wood and bark were pretreated with 0.98% (v/v) sulfuric acid at 140˚C for 30, 40, 50, 60 or 70 min and at 160˚C for 30, 40, 50 or 60 min. The water insoluble solid (WIS) fraction was hydrolyzed with a cellulase enzyme cocktail. Maximum xylose and glucose yields from the wood were 82 and 86%, respectively. Similarly, the respective maximum yields of xylose and glucose from the bark were 93 and 24%. Acid based pretreatment also produced fermentation inhibitory compounds such as furfural, hydroxymethylfurfural (HMF), formic acid and acetic acid in concentrations ranging from 0.1 to 32.3 g/ 100 g of raw dry biomass. Sweetgum bark was more recalcitrant to enzymatic hydrolysis than wood and also led to higher concentrations of formic acid. Sweetgum wood could be a good source of carbohydrate for a biobased refinery, but the removal of bark might be necessary to achieve better yields. Keywords: Dilute acid pretreatment, Enzymatic hydrolysis, Xylose, Glucose, Yields, Inhibitors, Sweetgum Agric. Food Anal. Bacteriol. 2: 175-186, 2012

INTRODUCTION Southern pine forests produce nearly 60% of the softwood lumber in the U. S.; in Arkansas, nearly 75% of all produced timber is from pine-dominated forests (Arkansas Forestry Commission, 2008). However, hardwood competition in the pine forest understory is a major impediment to pine forest growth. ThereCorrespondence: D. J. Carrier - carrier@uark.edu Tel: +1 -479-575-2542; Fax: +1-479-575-2689.

fore, southern pine forests are managed intensively (Wear and Greis, 2002). Annually, more than $150 million are spent reducing or eliminating competition in southern pine forests, primarily through the use of herbicides (Siry, 2002). The hardwood understory is composed of sweetgum (Liquidambar styraciflua L.), among others, which are competitors with pine for site resources. In Arkansas, the quantity of logging residue ranges from 1.71 to 2.03 million dry tons annually, and total

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forest based biomass resources are approximately 50 million dry tons annually (Gan and Smith, 2006; Jackson, 2007). Instead of being a nuisance, this hardwood understory growth could become an important source of biomass for biobased refineries, especially because sweetgum is a fast-growing hardwood. Capturing biomass from fuel-reduction thinning and understory harvests could raise forest based biomass production from 2.3 million to 5 million dry tons annually in the state of Arkansas alone (Pelkki, 2007). In a standard biorefinery, lignocellulosic biomass is deconstructed into simple sugars that can be used to produce either biofuels or other biochemical

citrant to subsequent enzymatic attack; the percent digestibility remained at 25% throughout the duration of enzymatic treatment (Torget et al., 1991). Although sweetgum has been investigated as a biorefinery feedstock, explicit data on sugar yields obtained with cutting edge saccharification enzyme cocktails and the formation of inhibitory compounds have not been reported. The purpose of this effort was to study the deconstruction of sweetgum wood and bark using dilute acid pretreatment and enzymatic hydrolysis. Xylose and glucose yields, as percentages of the theoretical amount in non-pretreated (raw) dry biomass, were investigated, as well as the formation of inhibitory products such as furfural,

products (Wyman, 1994). The deconstruction process consists of a pretreatment step, which is important in altering biomass structure and facilitates subsequent enzymatic hydrolysis of pretreated biomass. There are several methods for pretreatment, but dilute acid pretreatment has gained considerable importance over the years (Sannigrahi et al., 2011). In most studies involving dilute acid pretreatment, the efficiency of pretreatment is measured by the digestibility of the pretreated biomass, and less attention is paid to the other attributes of a good pretreatment process such as limiting the formation of compounds, which could inhibit subsequent enzymatic hydrolysis, or fermentation steps. In addition, for a long time, glucose was the primary sugar of interest. However, to improve the overall biorefinery operation costs, it is necessary to consider hemicellulose sugars, especially xylose, which can also be fermented into ethanol by the appropriate microorganism (Chung et al., 2005; Saha et al., 2005). Investigations focusing on the digestibility of sweetgum biomass after dilute acid pretreatment have been reported by Torget et al. (1990; 1991). An 80% digestibility of cellulose was obtained with a 60 min 140˚C dilute acid (pH 1.35 to 1.45) pretreatment of debarked sweetgum wood. Sweetgum bark also

hydroxymethylfurfural (HMF), formic acid and acetic acid.

was investigated as a feedstock; dilute acid pretreatment did not improve enzymatic digestibility (Torget et al., 1991). At 140˚C, dilute acid (pH 1.35 to 1.45) pretreatments released 40% of the sweetgum bark carbohydrates, but the remaining biomass was recal-

MATERIALS AND METHODS Raw biomass Chipped wood and bark from mature sweetgum trees were obtained from Matthew Pelkki and Philip Tappe, School of Forest Resources, University of Arkansas, Monticello, AR. The biomass was milled to pass through a 20 mesh (0.84 mm) screen using a Wiley Mini Mill (Thomas Scientific, Swedesboro, NJ) and samples were dried in a 105˚C oven until sample moisture was less than 5% as determined using an MB45 Moisture Analyzer (Ohaus Corporation, Pine Brook, NJ). Analyses for structural carbohydrate and acid insoluble lignin (AIL) content of the raw biomass were conducted as described in NREL LAP/TP-51042618 (Sluiter et al., 2008), except the biomass was subjected to a 24 h ethanol extraction rather than a water extraction followed by an ethanol extraction (Table 1).

Pretreatment One g of raw biomass (wood or bark) was soaked in 10 mL of 0.98% (v/v) sulfuric acid (H2SO4) in a 50 mL centrifuge tube for 12 h. The mixture was placed in a 32 mL stainless steel pretreatment tube (14.22

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Table 1. Composition of raw sweetgum biomass in % dry basis

Biomass

Glucan

Xylan

AILa

Wood

42.2

19.9

22.5

Bark

18.2

6.0

31.9

Acid insoluble lignin

mm inner diameter, 5.59 mm wall thickness, 200 mm length) with an additional 10 mL of H2SO4. Pretreatment tubes containing raw biomass and acid were heated in a fluidized sand bath (Techne Incorporated, Burlington, NJ) at 140˚C for 30, 40, 50, 60 or 70 min and at 160˚C for 30, 40, 50 or 60 min. After pretreatment, tubes were immediately submerged into cold tap water for 1 min; slurry contents were poured into 15 mL centrifuge tubes for separation into liquid fraction (prehydrolysate) and solid fractions (pretreated biomass). The pretreated biomass was washed by stirring in 30 mL of Millipore filtered water on a stir plate, set at 300 rpm for 30 min. The water-insoluble-solid (WIS) fraction was separated from the wash water by vacuum filtration through a Büchner funnel containing Whatman No. 1 filter paper. The WIS fraction was stored at 4˚C for a maximum of 3 days until used for enzymatic hydrolysis. Prehydrolysate and wash water were recovered and stored for a maximum of 3 days at 4˚C before xylose, glucose, and degradation compounds determination. Each pretreatment experiment was performed in triplicate.

Enzymatic hydrolysis An industrialized enzyme cocktail, Accellerase®1500, provided by Genencor (Danisco US Inc., Rochester, NY) was used to hydrolyze the WIS fraction. The enzyme cocktail had an endoglucanase activity of 2200 to 2800 CMC U/g and a ß-glucosidase activity of 525 to 775 pNPG U/g (provided by the manufacturer). The WIS fraction was mixed in a 50 mL amber bottle with 5 mL of citrate buffer (pH = 4.8), 0.5 mL of enzyme and 4.5 mL of Millipore filtered water. The amber bottle was placed in a shaking water

bath (Thermo Electron Corporation, Winchester, VA) at 55˚C and 100 rpm for 24 h. The resulting slurry was poured into a 15 mL centrifuge tube, submerged in boiling water to stop the reaction, and centrifuged at 3000 g for 2 min. The volume of the supernatant (enzymatic hydrolysate) was measured and the liquid was stored at 4˚C for a maximum of 3 days until analyzed for sugar content; the pellet was discarded. Each enzymatic hydrolysis experiment was performed in triplicate.

Analysis Five mL aliquots of prehydrolysate, wash water and the enzymatic hydrolysate were neutralized with calcium carbonate and filtered through a 0.2 µm filter for xylose and glucose analyses with a Waters 2695 Separations module (Milford, MA) equipped with Shodex precolumn (SP-G, 8 µm, 6 x 50 mm) and Shodex column (SP0810, 8 µm x 300 mm). Millipore filtered water (0.2 mL/min) was the mobile phase and the column was heated to 85˚C with an external heater. Sugars were detected with a Waters 2414 Refractive Index Detector (Milford, MA). Aliquots of the prehydrolysate and wash water were analyzed for degradation compounds with a Waters 2695 Separations module equipped with a Bio-Rad Aminex HPX-87H Ion Exclusion 7.8 mm X 30 mm column, heated to 55˚C. The mobile phase was 0.005 M H2SO4 flowing at 0.6 mL/min. Compounds were detected with a UV index using the Waters 2996 Photodiode Array detector. Furfural and HMF were detected at 280 nm; whereas, formic acid and acetic acid were detected at 210 nm.

RESULTS AND DISCUSSION Sweetgum wood Dilute acid pretreatment Figure 1 represents the yields of xylose and glucose recovered in the prehydrolysate and wash water using various pretreatment times at 140˚C and 160˚C.

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Figure 1. Prehydrolysates of sweetgum wood: xylose and glucose yields. Pretreatment occurrureed at (A): 140˚C and (B): 160˚C with 0.98% (v/v) H2SO4. Yields represent the amount of xylose and glucose recovered as a percentage of the theoretical amount in the raw biomass. Error bars are 95% confidence interval. 100 90

(A)

Yields (%)

70 60 50

Xylose

Glucose

30 20 10 0 30

Pretreatment time (min) 90

(B)

Yields (%)

70 60 50 40

Xylose

Glucose

20 10 0 30

Pretreatment time (min) Although the two liquid streams were analyzed separately, their carbohydrate contents were combined to calculate xylose and glucose yields as percentages of the theoretical amount in the dried raw biomass. Xylose was the primary sugar recovered in the prehydrolysate and wash water, indicating hydrolysis of

within 40 min of pretreatment, at which time xylose yield decreased. Glucose also was detected in the prehydrolysate and wash water. Pretreatment time did not affect glucose recovery at 140˚C, with less than 5% of the glucose recovered. However, at 160˚C glucose yield increased with pretreatment time.

the hemicellulosic fraction of wood during pretreatment. At 140˚C (Figure 1A), xylose yield increased with pretreatment time up to a maximum value of 79% after 60 min. Conversely, at 160˚C (Figure 1B) hemicellulose hydrolysis released its maximum (71%)

Pretreatment at lower temperatures is ideal in achieving a high xylose recovery. More elevated temperatures, especially for prolonged periods of time, will result in considerable loss of xylose and premature hydrolysis of the cellulosic fraction, which

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can result in glucose degradation. These findings are in agreement with studies performed on other feedstock with dilute acid pretreatment (Cara et al., 2008; Lloyd and Wyman, 2005; Torget et al., 1990). An inherent and undesirable property of dilute acid pretreatment is the production of sugar and lignin degradation compounds which are inhibitory to enzymatic hydrolysis and detrimental to microorganisms used in sugar fermentation (Palmqvist and Hahn-Hagerdal, 2000). Furfural, HMF, formic acid and acetic acid were detected in prehydrolysate and wash water from the wood pretreatment (Table 2). Furfural and HMF result from xylose and glucose degradation, respectively, and both can fur-

ery. Even though there was a slight degradation of xylose at 140˚C, xylose recovery did not decline with pretreatment time because, at lower temperature, the rate of xylan hydrolysis is higher than its degradation rate (Lloyd and Wyman, 2005).

ther degrade into formic acid; acetic acid is released from the acetyl group of the hemicellulose polymer (Palmqvist and Hahn-Hagerdal, 2000). Concentrations (g/100 g of dried raw biomass) of degradation products increased with time and severity of pretreatment (Table 2). Sugar degradation was less severe at 140˚C than at 160˚C; the increase in degradation compounds, especially furfural and formic acid, at 160˚C coincided with a decrease in xylose recov-

from the cellulose digestibility reported in the study done by Torget et al. (1990). As expected, most of the glucose was solubilized during enzymatic hydrolysis for both pretreatment temperatures; however, biomass pretreated at 140˚C (Figure 2A) was less responsive to enzymatic attack than the one pretreated at 160˚C (Figure 2B), shown here by a higher glucose recovery at 160˚C than at 140˚C. Although most of the xylose was solubilized during pretreat-

Enzymatic hydrolysis The effects of pretreatment time on xylose and glucose yields from the enzymatic hydrolysis of sweetgum wood pretreated at 140˚C and 160˚C are depicted in Figure 2. Xylose and glucose yields were calculated as percentages of the theoretical amount in the dried raw biomass and should be differentiated

Table 2. Degradation compounds (g/100g of raw biomass dry basis) produced from 0.98% (v/v) sulfuric acid pretreatment of sweetgum wood Pretreatment conditions

Prehydrolysate

Te m p . Time (˚C) (min)

Acetic Acid

140

Wash water a Formic Acid

HMF

Acetic Acid

Furfural

Formic Acid

HMF

4.6 ± 1.6 0.1 ± 0.1

1.8 ± 0.6

0.0 ± 0.0

2.5 ± 0.4

0.1 ± 0.1

0.8 ± 0.4

0.0 ± 0.0

2.9 ± 0.8 0.2 ± 0.2

1.8 ± 1.0

0.0 ± 0.0

2.7 ± 0.5

0.1 ± 0.1

0.9 ± 0.5

0.0 ± 0.0

140

2.9 ± 0.4 0.2 ± 0.0

1.8 ± 0.3

0.0 ± 0.0

3.1 ± 0.5

0.1 ± 0.0

0.9 ± 0.1

0.0 ± 0.0

140

3.6 ± 0.4 0.3 ± 0.2

3.4 ± 2.1

0.0 ± 0.0

2.5 ± 0.9

0.2 ± 0.2

1.5 ± 1.4

0.0 ± 0.0

140

3.0 ± 1.2 0.3 ± 0.1

3.1 ± 0.7

0.0 ± 0.0

3.3 ± 1.1

0.4 ± 0.3

3.0 ± 2.0

0.0 ± 0.0

160

4.8 ± 3.5 0.7 ± 0.3

8.1 ± 4.4

0.1 ± 0.0

3.2 ± 0.7

0.6 ± 0.2

5.4 ± 2.8

0.0 ± 0.0

160

4.3 ± 0.1 1.2 ± 0.2

10.4 ± 1.4

0.1 ± 0.0

2.7 ± 0.1

0.7 ± 0.1

4.8 ± 0.3

0.0 ± 0.0

160

5.0 ± 0.7 1.3 ± 0.2

10.6 ± 1.4

0.2 ± 0.1

3.2 ± 0.3

1.0 ± 0.1

5.6 ± 0.4

0.1 ± 0.0

160

3.7 ± 1.3 1.6 ± 0.7

6.6 ± 2.2

0.2 ± 0.1

3.7 ± 1.1

1.8 ± 0.6

5.5 ± 2.5

0.2 ± 0.1

Furfural

Means ± standard deviation of three replications a Water used for washing biomass after pretreatment Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

179

Figure 2. Enzymatic hydrolysates of pretreated sweetgum wood: xylose and glucose yields. Pretreatment occurred at (A): 140˚C and (B): 160˚C with 0.98% (v/v) H2SO4. Yields represent the amount of xylose and glucose recovered as a percentage of the theoretical amount in the raw biomass. Error bars are 95% confidence interval

(A)

Yields (%)

20 15 Xylose

Glucose

5 0 30

Pretreatment time (min)

(B)

Yields (%)

70 60 50 40

Xylose

Glucose

20 10 0 30

Pretreatment time (min) ment at 140˚C, when it comes to sweetgum wood, nearly complete removal of the hemicellulose during pretreatment does not translate to a highly digestible biomass. It is possible that performing the enzymatic hydrolysis for more than 24 h could improve the glucose yield; however, we showed that glucose yield increased only 10% after 48 h of enzymatic hy-

drolysis. Moreover, 24 h was the time recommended by the enzyme manufacturer for maximum activity of the enzyme. Seventy four percent of glucose was recovered in the enzymatic hydrolysate of the biomass pretreated at 160˚C (Figure 2B) and better digestibility of the pretreated wood was observed with an increasing

180 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

pretreatment time. Obtaining more digestible material from pretreatment conducted at harsher conditions has been observed and explained in the literature (Foston and Ragauskas, 2010); hydrolysis of the amorphous section of the cellulose, observed in this work, resulted in higher glucose concentrations during prolonged pretreatment at 160˚C. Kabel et al. (2007) attribute the relationship between high temperature and cellulose degradability to the disruption of the lignin structure during pretreatment; however, the lignin structure in raw and pretreated sweetgum wood was not analyzed in our work. Overall yields The dilemma between maximizing xylose recovery during pretreatment and producing a highly digestible cellulosic material occurred because the conditions for maximum xylose recovery do not correspond to the condition for maximum glucose recovery. Similar results had been observed (Lloyd and

Wyman, 2005). One solution to this issue could be to maximize the yields of total fermentable sugars (TFS = xylose + glucose) from pretreatment and enzymatic hydrolysis of the biomass as reported by Lloyd and Wyman (2005). Yields of xylose, glucose and TFS expressed as percentages of theoretical amounts in the dried raw wood (sugar yields) or as the amount of sugars (g) produced from 100 g of dried raw biomass (raw biomass yields) are depicted in Table 3. In general, at 140˚C xylose, glucose, and TFS yields increased with pretreatment time. Up to 47% of TFS was recovered after 70 min of pretreatment; these pretreatment conditions yielded maximum xylose recovery of 82%. Any sugar cocktail (xylose + glucose) obtained at 140˚C contained mainly xylose and, for a fermentation process, this is not the ideal sugar stream. Pretreatment at 160˚C yielded a maximum TFS of 72% after 60 min of pretreatment; these pretreatment conditions also gave maximum glucose recovery of 86%. At 160˚C, an increase in pretreatment time did not have an effect on TFS yields; how-

Table 3. Sugars produced from 0.98% (v/v) sulfuric acid pretreatment and enzymatic hydrolysis of sweetgum wood Pretreatment conditions Temp. Time (˚C) (min)

Sugar yieldsa Xylose

Glucose

Raw biomass yieldsb TFSc

Xylose

Glucose

TFSc

140

68.9 ± 8.8

13.4 ± 7.8

31.5 ± 5.2

15.6 ± 2.0

6.3 ± 3.7

21.9 ± 3.6

140

74.1 ± 7.6

20.8 ± 3.6

38.2 ± 4.7

16.8 ± 1.7

9.8 ± 1.7

26.5 ± 3.3

140

71.3 ± 9.4

23.1 ± 1.2

38.8 ± 3.7

16.1 ± 2.1

10.8 ± 0.6

27.0 ± 2.6

140

82.1 ± 6.8

27.4 ± 1.7

45.2 ± 3.2

19.9 ± 2.4

15.8 ± 5.5

35.7 ± 7.2

140

82.0 ± 3.6

30.4 ± 0.7

47.2 ± 1.6

18.6 ± 0.8

14.3 ± 0.3

32.8 ± 1.1

160

71.4 ± 5.6

55.0 ± 5.5

64.6 ± 8.7

16.2 ± 1.3

28.7 ± 6.6

44.9 ± 6.1

160

72.1 ± 12.1

66.8 ± 3.9

68.5 ± 1.4

16.3 ± 2.7

31.3 ± 1.8

47.7 ± 1.0

160

54.0 ± 14.1

74.8 ± 3.9

68.1 ± 1.9

13.1 ± 2.1

35.1 ± 1.8

47.3 ± 1.4

160

41.9 ± 9.8

86.2 ± 1.0

71.8 ± 3.8

9.5 ± 2.2

40.4 ± 0.5

49.9 ± 2.6

Mean ± standard deviation of three replications a Percentage of the amount of individual sugar present in one g of the raw biomass. Xylose: 0.23 g; glucose: 0. 47 g; TFS: 0.7 g. b Yields in g/100 g of raw material c Total fermentable sugars Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

181

Figure 3. Prehydrolysates of sweetgum bark: xylose and glucose yields. Pretreatment occurred at (A): 140˚C and (B): 160˚C with 0.98% (v/v) H2SO4. Yields represent the amount of xylose and glucose recovered as a percentage of the theoretical amount in the raw biomass. Error bars are 95% confidence interval. 100

(A)

90 80

Yields (%)

70 60 Xylose

Glucose

40 30 20 10 0 30

Pretreatment time (min)

100

(B)

90 80

Yields (%)

70 60 50

Xylose

Glucose

30 20 10 0 30

Pretreatment time (min) ever, the sugar stream obtained at times before 40 min had a higher percentage of xylose than streams obtained after 40 min, which had a higher percentage of glucose. This occurred because the xylose concentration in the sugar stream decreased while the glucose concentration increased with pretreatment time.

Sweetgum Bark Dilute acid pretreatment In assessing biomass as feedstock for a biorefinery, tree bark usually is not considered an ideal candidate, mainly because it is not a substantial source of carbohydrate when compared to tree wood.

182 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

However, using the whole tree would simplify supply chain processing. The sweetgum bark used for this study contained on a dry basis 18.2% glucan and 6.0% xylan (Table 1). Even though bark should be integrated in the biomass conversion process, wood will dictate process parameters; therefore, sweetgum bark in this study was submitted to the same pretreatment and enzymatic hydrolysis conditions as sweetgum wood. The effects of pretreatment time on xylose and glucose yields from the prehydrolysate and wash water of the bark pretreated at 140˚C and 160˚C are shown in Figure 3. Sugar recovery from sweetgum bark pretreatment did not follow the same trend as

xylose or glucose yield was nonexistent with the bark because the maximum recovery for both sugars occurred at 160˚C. Moreover, at 160˚C the pretreatment time did not affect TFS or glucose yields; therefore, maximizing xylose recovery could be the only factor dictating the pretreatment conditions for sweetgum bark. Furfural, HMF, formic acid and acetic acid were present in the prehydrolysate and wash water from the bark pretreatment (Table 4). Concentrations of these by-products in pretreatment liquid streams were lower at 140˚C than at 160˚C. It was expected concentration of furfural and formic acid would be higher at 140˚C than at 160˚C given that a higher loss

for sweetgum wood pretreatment. Xylose loss occurred faster at 140˚C (Figure 3A) than at 160˚C (Figure 3B). More xylose was recovered at 160˚C than at 140˚C; these results were in contrast to results obtained for sweetgum wood because harsher pretreatment conditions of the wood yielded lower xylose recovery. Table 5 includes the sugar recoveries from the pretreatment and the enzymatic hydrolysis of sweetgum bark. The issue between maximizing

of xylose occurred at 140˚C. Concentrations of formic acid in the bark prehydrolysate and wash water, especially at 160˚C, were over 11 g per 100 g of raw biomass. When combining formic acid recovery in the prehydrolysate and wash water obtained from pretreatment at 160˚C for 40 min, formic acid yield was 43% of the raw biomass. Thus, for sweetgum bark, reactions other than sugar degradation are responsible for xylose loss and formation of formic

Table 4. Degradation compounds (g/100g of raw biomass) produced from 0.98% (v/v) sulfuric acid pretreatment of sweetgum bark Pretreatment conditions

Prehydrolysate

Wash watera

Temp (˚C)

Time (min)

Acetic Acid

Furfural

Formic Acid

HMF

Acetic Acid

140

1.4 ± 0.3

0.0 ± 0.0

16.1 ± 0.7

0.0 ± 0.0

1.6 ± 0.2 0.0 ± 0.0 4.8 ± 1.1 0.0 ± 0.0

140

3.0 ± 2.1

0.0 ± 0.0

19.7 ± 3.8

0.0 ± 0.0

1.5 ± 0.2 0.0 ± 0.0 3.9 ± 1.5 0.0 ± 0.0

140

1.6 ± 0.8

0.1 ± 0.0

14.5 ± 6.8

0.0 ± 0.0

2.0 ± 0.6 0.1 ± 0.0 7.6 ± 3.4 0.0 ± 0.0

140

1.5 ± 0.4

0.1 ± 0.0

12.5 ± 3.7

0.0 ± 0.0

2.0 ± 0.7 0.1 ± 0.1 7.7 ± 3.0 0.0 ± 0.0

140

1.1 ± 0.1

0.1 ± 0.0

9.0 ± 0.4

0.0 ± 0.0

2.6 ± 0.3 0.2 ± 0.0 11.9 ± 1.1 0.0 ± 0.0

160

3.9 ± 2.1

0.1 ± 0.1

21.6 ± 5.1

0.1 ± 0.0

2.6 ± 0.5 0.2 ± 0.1 14.7 ± 3.3 0.0 ± 0.0

160

7.9 ± 4.9

0.4 ± 0.1

32.3 ± 11.1 0.1 ± 0.0

2.0 ± 0.8 0.2 ± 0.1 11.2 ± 4.2 0.0 ± 0.0

160

6.3 ± 2.9

0.5 ± 0.1

26.9 ± 2.2

0.1 ± 0.0

2.2 ± 0.5 0.3 ± 0.0 10.8 ± 1.7 0.0 ± 0.0

160

2.6 ± 0.9

0.6 ± 0.3

16.7 ± 8.2

0.1 ± 0.0

2.5 ± 0.9 0.5 ± 0.1 10.8 ± 5.3 0.1 ± 0.0

Furfural

Formic Acid

HMF

Mean ± standard deviation of three replications a Water used for washing biomass after pretreatment Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

183

Table 5. Sugars produced from 0.98% (v/v) sulfuric acid pretreatment and enzymatic hydrolysis of sweetgum bark Pretreatment conditions

Sugar yields a

Raw biomass yields b

Temp (˚C)

Time (min)

Xylose

Glucose

TFS c

Xylose

Glucose

TFS c

140

60.0 ± 7.5

15.9 ± 2.1

29.5 ± 3.7

5.3 ± 0.7

3.2 ± 0.4

8.5 ± 1.1

140

81.4 ± 11.1 16.9 ± 2.4

36.7 ± 5.0

7.2 ± 1.0

3.4 ± 0.5

10.6 ± 1.5

140

73.6 ± 5.7

16.9 ± 0.8

34.3 ± 2.2

6.5 ± 0.5

3.4 ± 0.2

9.9 ± 0.6

140

65.3 ± 12.5 16.9 ± 2.2

31.8 ± 3.7

5.8 ± 1.1

3.4 ± 0.4

9.2 ± 1.1

140

60.2 ± 3.5

13.9 ± 0.6

28.1 ± 0.8

5.3 ± 0.3

2.8 ± 0.1

8.1 ± 0.2

160

88.2 ± 5.8

17.8 ± 1.7

40.3 ± 2.1

7.8 ± 0.5

3.6 ± 0.3

11.6 ± 0.6

160

93.5 ± 11.3 21.4 ± 1.7

44.5 ± 3.3

8.3 ± 1.0

4.3 ± 0.3

12.9 ± 1.0

160

91.8 ± 12.7 22.4 ± 2.4

44.9 ± 4.4

8.1 ± 1.1

4.4 ± 0.5

13.0 ± 1.3

160

72.7 ± 1.8

39.3 ± 1.3

6.4 ± 0.2

4.9 ± 0.2

11.3 ± 0.4

24.5 ± 1.2

Mean ± standard deviation of three replications a Percentage of the amount of individual sugar present in one g of the raw biomass. Xylose: 0.09 g; glucose: 0. 2 g; TFS: 0.29 g. b Yields in g/100 g of raw material00 c Total fermentable sugars acid during pretreatment. The presence of those inhibitory compounds at such elevated concentrations in the pretreatment liquid streams could be another reason why bark is not an ideal candidate as a feedstock for a biorefinery. Enzymatic hydrolysis The enzymatic hydrolysis of the sweetgum bark (Figure 4) was not as successful as the hydrolysis of the sweetgum wood. A maximum of 11% of glucose was recovered in bark enzymatic hydrolysate compared to 74% for wood. An increase in pretreatment time or temperature did not improve glucose yields. This resistance to enzymatic attack after pretreatment has been reported to be inherent to sweetgum bark (Torget et al., 1991). Specifically, hot dilute acid pretreatment promotes lignin condensation, causing the formation of a lignin barrier that can possibly impede the access of enzyme binding sites to cellu-

lose bonds (Torget et al., 1991). Moreover, Cantarella et al. (2004) showed that formic acid concentrations of 11 mg/mL inhibited the cellulose enzymatic cocktail; therefore, the formic acid detected in the bark prehydrolysates of our study could contribute to the recalcitrance observed in the bark. Insufficient washing of the pretreated pellet could exacerbate this recalcitrance. A better understanding of sweetgum bark structure and composition needs to be established to design optimum processing conditions to maximize saccharification of this feedstock system.

CONCLUSIONS Dilute acid pretreatment at 160˚C for 60 min coupled with enzymatic hydrolysis of sweetgum wood yielded a maximum of 72% total fermentable sugars. Sweetgum wood could be a potential feedstock for a biochemical based refinery, especially because it is

184 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

Figure 4. Enzymatic hydrolysates of pretreated sweetgum bark: xylose and glucose yields. Pretreatment occurred at (A): 140˚C and (B): 160˚C with 0.98% (v/v) H2SO4. Yields represent the amount of xylose and glucose recovered as a percentage of the theoretical amount in the raw biomass. Error bars are 95% confidence interval. 9

(A)

Yields (%)

7 6 5 4

Xylose

Glucose

2 1 0 30

Pretreatment time (min)

(B)

Yields (%)

10 8 Xylose

Glucose

4 2 0 30

Pretreatment time (min) a fast growing, voluntary tree. However, the addition of sweetgum bark with the wood could render the use of the whole tree problematic because it pro-

ACKNOWLEDGEMENTS

duces high concentrations of inhibitory compounds causing resistance to enzymatic hydrolysis. Bark is removed in pulp and paper operations; the removal of this component also may be necessary in biorefinery operations.

sion of Agriculture, and the Department of Biological and Agricultural Engineering for financial assistance. The authors also acknowledge South Central Sun Grant award # DTOS59-07-G-00053 for financial support to A. C. Djioleu, Department of Energy

The authors thank the University of Arkansas, Divi-

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

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award #08GO88035 for pretreatment equipment and CSREES National Research Initiative award # 2008-01499 for the HPLC instrument.

Arkansas Forestry Commission. 2008. Timber severance tax report for 2007. Arkansas Forestry Commission, Little Rock, AR. Cantarella, M., L. Cantarella, A. Gallifuoco, A. Spera, and F. Alfani. 2004. Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF. Bio-

stover followed by enzymatic hydrolysis of the remaining solids. Bioresour. Technol. 96:1967-1977. Palmqvist, E. and B. Hahn-Hagerdal. 2000. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 74:25-33. Pelkki, M. 2007. Statewide In-Forest Residue Supply for Arkansas. Presentation for the Arkansas Department of Economic Development August 29, 2007, Little Rock, AR. Saha, B. C., L. B. Iten, M. A. Cotta, and Y. V. Wu. 2005. Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol. Proc. Biochem. 40:3693-3700.

technol. Prog. 20:200-206. Cara, C., E. Ruiz, J. M. Oliva, F. Saez, and E. Castro. 2008. Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification. Bioresour. Technol. 99:1869-1876. Chung, Y., A. Bakalinsky, and M. H. Penner. 2005. Enzymatic saccharification and fermentation of xylose-optimized dilute acid-treated lignocellulosics. Appl. Biochem. Biotechnol. 121-124:947-961. Foston, M. and A. J. Ragauskas. 2010. Changes in lignocellulosic supramolecular and ultrastructure during dilute acid pretreatment of Populus and switchgrass. Biomass Bioenergy 34:1885-1895. Gan J. and C. Smith. 2006. Availability of logging residues and potential for electricity production and carbon displacement in the USA. Biomass Bioenergy 30:1011-1020. Jackson, S. 2007. Southeastern Biomass/Bioenergy Overview. In: W. Hubbard, L. Biles, C. Mayfield, and S. Ashton. Eds. Sustainable Forestry for Bioenergy and Bio-based Products: Trainers Curriculum Notebook. Southern Forest Research Partnership Inc, Athens, GA. p 45-475. Kabel, M. A., G. Bos, J. Zeevalking, A. G. J. Voragen, and H. A. Schols. 2007. Effect of pretreatment se-

Sannigrahi, P., D. Kim, S. Jung, and A. Ragauskas. 2011. Pseudo-lignin and pretreatment chemistry. Energy Environ. Sci. 4:1306-1310. Siry, J. 2002. Intensive Timber Management Practices. In: D. N Wear and J. G. Greis. Eds. Southern forest resource assessment. Gen. Tech. Rep. SRS53, USDA For. Serv. So. Res. Sta., Asheville, NC. p 327-340. Sluiter, A., B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton, and D. Crocker. 2008. Determination of Structural Carbohydrates and Lignin in Biomass. Laboratory Analytical Procedure (LAP). National Renewable Energy Laboratory, Golden, CO. Torget, R., P. Werdene, M. Himmel, and K. Grohmann. 1990. Dilute acid pretreatment of short rotation woody and herbaceous crops. Appl. Biochem. Biotechnol. 24/25:115-126. Torget, R., P. Werdene, M. Himmel, and K. Grohmann. 1991. Dilute acid pretreatment of hardwood bark. Bioresour. Technol. 35:239-246. Wear, D. and J. Greis. 2002. Southern forest resource assessment: summary report. Gen.Tech. Rep. SRS54, USDA For.Serv.So.Res.Sta., Asheville, NC. p 103. Wyman, C.E. 1994. Ethanol from lignocellulosic biomass: technology, economics, and opportunities.

REFERENCES

verity on xylan solubility and enzymatic breakdown on the remaining cellulose from wheat straw. Bioresour. Technol. 98:2034-2042. Lloyd, T. A., and C. E. Wyman. 2005. Combined sugar yields for dilute sulfuric acid pretreatment of corn

Bioresour. Technol. 50:3-16.

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Microbiological Quality Assessment of Raw Meat and Meat Products, and Antibiotic Susceptibility of Isolated Staphylococcus aureus S. Datta1, A. Akter1, I. G. Shah1, K. Fatema1, T. H. Islam1 , A. Bandyopadhyay2, Z. U.M. Khan1, D. Biswas3 Department of Microbiology, Primeasia University, 9 Banani, Dhaka-1213, Bangladesh 2 DOEACC Centre, Jadavpur University Campus, Kolkata-700032, India 3 Department of Animal and Avian Sciences, University of Maryland College Park, MD, USA 1

ABSTRACT The objective of this study was to assess the microbiological quality of raw meat and processed meat products in Dhaka city and test the antibiotic susceptibility of the Staphylococcus isolates. A total number of 79 meat samples were categorized into two groups, viz., Group-1 meat (raw meat), collected from different slaughter yards and meat stalls located in the commercial areas of Dhaka city and Group-2 meat (processed meat products), collected from ready-to-eat foods. Microbiological quality of the samples was determined by Total Viable Bacterial Count (TVBC), Total Coliform Count (TCC), Total Salmonella and Shigella Count (TSSC), Total Staphylococcus aureus Count (TSAC) and Total Fungal Count (TFC). Heterotrophic bacteria were recovered from all the meat samples but no Salmonella, Shigella were detected. As expected, some of the samples were found positive with Staphylococcus spp. and coliform. The statistical analysis showed that the mean TVBC (log value/g) was significantly greater (P<0.05) in raw meat from Kawranbazar than all other meat samples studied. TVBC and TSAC exhibited regional significant variation (P<0.05), whereas TCC did not show any remarkable regional variation. Our present study reveals that the TVBC, TCC and TSAC of the meat samples were high in those commercial areas and pose potential risk for public health. From seventy nine samples, 35 isolates of S. aureus were obtained and identified by standard biochemical tests. All these isolates were tested for their sensitivity against common antibiotics used in Bangladesh. Percentage resistance of the S. aureus samples to penicillin, ampicillin, streptomycin, tetracycline, amoxicillin and neomycin were found to be 85.71%, 71.42%, 100%, 71.42%, 100% and 85.71%, respectively. But no resistance to vancomycin, bacitracin, cefaclor and ciprofloxacin was found in these isolates. The percentage of multidrug resistant (MDR, resistant against more than three antibiotics) staphylococci was 20%. Keywords: Raw meat, meat products, contamination, microbiological quality, antibiotic, susceptibility, resistance, Staphylococcus, Bangladesh Agric. Food Anal. Bacteriol. 2: 187-194, 2012 Correspondence: Suvamoy Datta, suvamoy.datta@primeasia.edu.bd Tel: +880-17-5554-8324

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INTRODUCTION Radical dietary shifts in many developed and developing nations are supplanting traditional patterns of eating with a western diet high in animal meat products and refined carbohydrates and low in whole grains, fruits, and vegetables (Frank et al., 2008). There are considerable human health consequences with foodborne infections ranging from protracted illness to death and patients with impaired immunity are at greater risk. Microbiological food borne diseases are typically caused by bacteria or their metabolites, parasites, viruses or toxins. The importance of different food borne diseases varies

chain (Norrung et al., 2009). The high prevalence of diarrheal diseases in many developing countries suggests major underlying food safety problems (Food safety and food borne illness, 2009). These food items can cause serious problems when they are contaminated with harmful microorganisms due to lack of proper sanitary condition, hygiene practices, and proper storage and mishandling (WHO, 2009). Due to unawareness and non-enforcement of laws often consumers buy meat and meat product that failed to protect consumersâ&#x20AC;&#x2122; right and possess a potential risk. In Bangladesh, beef rolls, chicken fries, sandwiches are gradually becoming the popular ready-to-eat

between countries depending on foods consumed, food processing, preparation, handling, storage techniques employed, and sensitivity of the population (ICMSF, 2002). Meat is not only highly susceptible to spoilage, but also frequently implicated in the spread of food borne illness. Contaminated raw meat is one of the main sources of foodborne illness (Bhandare et al., 2007; Podpecan et al., 2007). During slaughter and processing, all potentially edible tissues are subjected to contamination from a variety of sources within and outside animal. In living animals, those surfaces in contact with the environment, harbor a variety of microorganisms. The contaminating organisms are derived mainly from the hide of the animal and also comprise organisms that originate from both feces. In addition, processed meat foods are more prone to contamination with pathogenic microorganisms during the various stages of processing. Meat and meat products are important sources of human infections with a variety of foodborne pathogens, i.e. Salmonella spp., Campylobacter jejuni/coli, Yersinia enterocolitica, verotoxigenic Escherichia coli and, to some extent, Listeria monocytogenes. Some pathogens in meats (eg. Salmonella spp., Campylobacter spp.) are most efficiently controlled by the main in-

foods and there is also a rapid growth in local production of chicken fries in recent years. After 1996, the large foreign franchises were launched especially Pizza Hut, KFC (Kentucky Fried Chicken). This trend was followed by local producers and many franchise companies were formed. However, there are major differences between local chicken fries and those franchised. The quality of locally produced and franchise chicken fries should be monitored from time to time to ensure that the products meet the minimum requirements of standards and specifications, and are of acceptable quality to the consumers. Considerable studies have been carried out in different countries of the world on fast foods and fast food restaurants with respect to the outbreak of many gastrointestinal and other diseases (Easa et al., 2010). The indiscriminate use of antimicrobial drugs in food animals may result in transfer of resistance to human, it is unlikely that the so called reverse-antimicrobial drug will be restricted to use in human medicine (Schwartz and Chaslus Dancla, 2001). The problem may be due to the natural resistance of species to certain antibiotics, possible transfer of antibiotics resistance among species and the use of subtherapeutic doses of antibiotics in animal feeds to improve animal productivity could also select for re-

terventions applied in the primary production combined with the optimization of the slaughter hygiene. For organisms like, L. monocytogenes, Staphylococcus aureus and Clostridium spp., the main control measures are focused on later stages of the meat

sistance strains. Recently, a dramatic increase in the resistance against antibiotics routinely used in human as well as in veterinary medicine has been recorded in the members of the genus Staphylococcus. Development of resistant or multi resistant Staphylococcus

188 Agric. Food Anal. Bacteriol. â&#x20AC;˘ AFABjournal.com â&#x20AC;˘ Vol. 2, Issue 3 - 2012

strains causes considerable therapeutic problems. Although, it is not inevitable to prove a direct role of drug resistance in bacteria contaminating food items with increased clinical cases of resistant infections, the presence of such bacteria in food items and their related environment could play a role on the spread of antimicrobial resistance amongst food borne pathogens (Farzana et al., 2009). Therefore, this study was conducted to investigate the microbial quality of raw meat and meat products available in most commercial areas in Dhaka City and to determine the antibiotic resistant pattern of the isolated S. aureus.

MATERIALS AND METHODS Meat Sample Collections Samples of raw meat were collected from different butcher open shops. The samples collected from Mohakhali bazar, Kawran bazaar, Badda Rampura bazaar and Khilkhet bazaar were marked as ‘1’, ’2’, ’3’ and’4’, respectively. Fast food samples named ready packet Bangla meat products, chicken sandwiches, beef rolls, chicken cutlets, chicken shawarmas and chicken fries, were collected from six different locations including Farmgate, Motijheel, Malibagh, Mouchak, Banani and Uttara, and samples collected from Farmgate, Motijheel, Malibagh, Mouchak, Banani and Uttara were marked as ‘5’, ‘6’, ’7’, ’8’, ’9’ and ‘10’, respectively. The collected samples were immediately transported in insulated ice containers to the laboratory for microbial analysis.

Meat sample preparation Ten gram of the solid sample was weighed and aseptically taken into a sterile jar containing 90 ml sterile normal saline. It was homogenized with sterile blender (Retsch, GM 200, Australia) at 3000 rpm for 5-10 min. A 1mL aliquot of homogenate was transferred to a test tube containing 9 mL sterile distilled water to make 10-2 dilution and shaken well with vortex mixer (Digosystem, VM-1000, Taiwan). Serial dilu-

tions up to 10-5 were prepared for the microbiological analysis.

Microbiological analysis The microbiological quality and safety of meat and meat products were assessed on the basis of Total Viable Bacterial Count (TVBC), Total Coliform Count (TCC), Total Staphylococcus aureus Count (TSAC) and Total Salmonella and Shigella Count (TSSC), and Total Fungal Count (TFC) using Plate count agar (PCA, Himedia, India), MacConkey agar (MCA, HiMedia, India), Mannitol Salt agar (MSA, HiMedia, India), Salmonella-Shigella agar (SSA, HiMedia, India) and Potato Dextrose agar (PDA, HiMedia, India), respectively. Diluted meat samples in normal saline were spread onto these plates and incubated at 37°C for 24 hr except detection of fungi, which were incubated at 25°C for 5 days. Staphylococcus isolates were confirmed by microscopic, cultural and standard biochemical tests (motility, catalase, coagulase, oxidase, urease, citrate utilization, indole, gelatin hydrolysis, MR-VP, TSI test) according to Bergey’s Manual of Determinative Bacteriology, (9th Edition, 1994) for further analysis.

Antibiotic Susceptibility Testing The antibiotic susceptibility of the Staphylococcus isolates was determined using the standard disc-diffusion (Kirby–Bauer, 1997) method. Overnight grown cultures were used for the test. The antibiotic discs (Oxoid®, UK) used in this study were: amoxicillin (10 μg), ampicillin (25 μg), bacitracin (10 μg), cefaclor (30 μg), ciprofloxacin (5 μg), neomycin (30 μg), penicillinG, (10 units), streptomycin (10 μg), tetracycline (30 μg) and vancomycin (30 μg).

RESULTS AND DISCUSSION Microbiological analysis The present study evaluated the microbiological quality of raw meat and meat products in Dhaka,

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Bangladesh. The microbiological condition of safety and hygiene were then assayed using the methods recommended by ICMSF (International Commission on Microbiological Specifications for Foods). The total viable counts of raw meat and meat products were determined by standard method. We found that all samples were contaminated with microorganisms (Table 1).

Total Viable Bacterial Count (TVBC) (log mean value/g) The TVBC mean log value in sample 1 to sample 10 were 6.47, 8.65, 7.75, 7.63, 7.54, 6.57, 6.29, 5.42, 6.74 and 5.59, respectively. Viable counts of raw meat were significantly higher (P<0.05) in raw meat sample ‘2’ followed by sample ‘3’, ‘4’, ‘5’ and significantly lower mean value was observed in chicken cutlets and chicken fries. However, TVBC of sample 3, 4 and 5 did not differ significantly among them. Similarly, sample 1, 6, 7 and 9; and sample 8 and 10, did not differ among them. TVBC found in meat samples of the present study indicated a remarkable increase. Hoque et al. (2008) studied raw meat samples and found 6.03 from slaughter yards and 6.53 from meat stalls. Another study was carried out by Waliullah et al. (2011), on the meat based fast foods from Dhaka University campus areas. The standard plate count revealed that in that meat based fast food chicken sandwiches were 5.12, chicken burgers 6.23 and hot dogs 6.42. Since chicken fries and chicken shawrmas contain fried meat, low TVBC was observed and our results agreed with their results.

Total Coliform Count (TCC) (log mean value/g)

coliform counts from samples 1, 3, and 8. However, no significant difference was found between the mean TCC of sample 4 and 6. These counts indicate the aseptic techniques of food processing.

Total Staphylococcus aureus (TSAC) (log mean value/g)

Count

A total of 35 out of 79 meat samples were found positive in total S. aureus counts (TSAC). TSAC of samples 1, 2, 4, 5, 6, and 7, were 5.48, 5.75, 4.13, 5.95, 4.34, and 5.97, respectively but no S. aureus was found in the samples 3, 9 and 10 (Table 1). The mean staphylococcal counts of raw meat sample 2, sample 5 and beef roll were significantly high (P<0.05) than other samples. However, there was no significant difference among sample 4 and 6. Anawar et al. (2004) isolated 90.63% Staphylococcus spp. from dressed broilers. Our results showed that 73.68% S. aureus were isolated from raw meat samples.

Total Salmonella-Shigella Count (TSSC) (log mean value/g) None of the samples contained Salmonella and Shigella (Table 1); this is in accordance with the results of Selvan et al. (2007) who did not recover Salmonella from samples of retail meat products. The absence of Salmonella in the meat product samples indicate the quality of raw meat and other hygienic processing including the quality of the water used in processing.

Total Fungal Count (TFC) (log mean value/g)

The total coliform count (TCC) in meat samples 1, 3, 4, 6 and 8 were 4.31, 4.42, 5.5, 5.58, and 4.8, respectively (Table 1). None of TCC pathogens were

No fungus was found in the sample of meat and meat products except sample ‘2’ and sample ‘4’. However, total fungus counts of Kawran Bazar and Khilkhet were 4.65 and 3.65, respectively (Table 1).

detected in samples 2, 5, 7, 9 and 10. It was found that chicken sandwiches contained significantly (P<0.05) higher numbers of coliform compared to other open meat samples and meat products. Analysis of variance revealed significantly lower (P<0.05)

It is evident from Table 1, sample 2, i.e. the raw meat sample collected from Kawran Bazar was not safe as compared to the other samples with high TVBC, TSAC, and TCC. Among meat products, Bangla packet products, followed by beef rolls and

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Table 1. Mean (±SE) and analysis of bacterial counts (log value/g) of different samplesA Total Count

Meat and Meat Products Raw meat1 (n=5)

Raw meat2 (n=7)

Raw meat3 (n=3)

Raw meat4 (n=4)

Bangla meat5 (n=10)

TVBC

6.47±0.2101c

8.65±0.065a

7.45±0.093b

7.63±0.088b

7.54±0.101b

TCC

4.31±0.1002b

4.42±0.05b

5.5±0.047a

TSAC

5.48±0.0675

5.75±0.0607

4.13±0.0132

5.95±0.0408a

TSSC

TFC

4.65±0.0612

3.65±0.09

Chicken Sandwich6 (n=10)

Beef Roll7 (n=10)

Chicken cutlet8 (n=10)

Chicken Shawarma9 (n=10)

Chicken fry10 (n=10)

TVBC

6.57±0.097c

6.29±0.08c

5.42±0.07d

6.74±0.07c

5.59±0.09d

TCC

5.58±0.024a

4.8±0.3007b

TSAC

4.34±0.0655

5.97±0.0599

TSSC

TFC

these number indicates the sample numbers

1-10

Data represent the mean values obtained from ten 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 row are not significantly different (P<0.05). A

chicken cutlets were significantly (P< 0.05) contaminated as compared to the other meat products. According to statistical analysis, it was noted that the microbiological quality of most of the raw meat and meat products of Dhaka city was significantly poor. Our finding showed that raw meat and meat products, which were collected from different location, were contaminated with pathogenic microorganisms. As observed in the course of the study, the method of slaughtering of animals is responsible for the microbial contamination .The study indicated that the count of enumerated bacteria in raw meat was higher than acceptable values, making the prod-

Multidrug resistant strains of S. aureus will be a risk factor for the public health of a developing country such as Bangladesh. To find the prevalence of drug resistant Staphylococci, assays for susceptibility profiles were performed. High level resistance of staphylococci isolates to various classes of antibiotics was observed. As expected, the staphylococcal isolates from the meat samples were found to be resistant to many of the antibiotics tested, particularly the ones that are generally used as initial line of treatment. Our susceptibility results of the isolated staphylococci from seven different locations showed that they are highly resistant to penicillin, ampicillin,

uct a potential public health hazard.

amoxicillin, tetracycline, streptomycin, and neomycin. Percentage resistance of the S. aureus samples to penicillin, ampicillin, amoxicillin, streptomycin and neomycin were 85.71, 71.42, 100, 100, and 85.71, respectively. As evident from Table 2, all S. aureus

Antibiotic sensitivity of the Staphylococcus isolates

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Table 2. Antibiotic Resistance patterns of isolated S. aureusB Sample

Number of Antibiotic resistant isolates from each sample Pen

Amp

Amo

Tet

Strp

Neo

Cef

Cipro

Bac

Van

1(n=5)

2(n=5)

4(n=4)

5(n=5)

6(n=6)

7(n=4)

8(n=6)

Total

Percentage (%)

85.71

71.42

100

71.42

100

85.71

Sample ‘3’,’9’ and ‘10’ were omitted since S. auerus was absent in these samples

isolates showed no resistance to vancomycin, bacitracin, cefaclor and ciprofloxacin, i.e., they are susceptible, thus giving us some way of treating any infection caused by the same strains of the S.aureus isolates. Thirty five of the tested isolates of staphylococci (47 %) were resistant to only two antibiotics, 25 (33 %) to three antibiotics, and multidrug resistance (MDR) was confirmed in 15 isolates (20 %) (Figure 1). Regecová et al. (2009) found 47 % to two antibiotics, and multidrug resistance was 25 % from fish meat Staphylococci isolates. In Korea, Heo et al. (2008)

Figure 1. Percentage of Multidrug Resistance in S. aureus isolates (against two or more than two drugs)

found 7.8% MDR S. aureus isolates from domestic and imported meats. Waters et al. (2011) found 52% MDR (resistant against 3 or more drug) S. aureus from poultry and meats.

CONCLUSIONS The presence of bacteria in meat has been widely reported from different parts of the world (Holds et al., 2007; Kinsella et al., 2008). Some groups recognized the presence of bacteria especially gramnegative organisms as an indicator of open air meat spoilage, while others argued this assertion and considered the presence of a high number of background organisms as pathogen-reduction strategy due to the organisms antagonistic effect against pathogenic bacteria and thus safe for meat quality. Our result indicated that the gram negative coliforms and gram-positive bacteria were present predominantly, and the fungus was the least frequent in the meat and meat samples. In view of the microbial implication in handling, slaughtering, dressing, processing and distribution of meat and meat products which may endanger human health, the study was undertaken to determine the extent of microbial contamination of meat in the commercial areas of

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Dhaka city in Bangladesh. Contamination prevention rather than end-product testing to ensure the safety of meat is needed. As raw meats were heavily contaminated with microorganisms and are potential sources of food borne infections, therefore raw meat handlers should receive education in food hygiene. Meat and poultry processors and regulators should use process control techniques to ensure that performance standards for meat and poultry are met. Vaccination, immuno-modulation and pre and probiotics need to be considered as alternatives for combating bacterial infection as far as possible, although they are unlikely to completely replace antimicrobial drugs (Schwarzt et al., 2001). We therefore, suggest

ence. 8(10). Farzana, K., S. Akhter, and F. Jabeen. 2009. Prevalence and antibiotic resistance of bacteria in two ethnic milk based products. Pak. J. Blot. 41:935943. Frank, B. H. 2008. Globalization of Food Patterns and Cardiovascular Disease Risk. Circulation. 118:19131914. Heo, H. J., B. K. Kyung, D. H. Bae, C. K. Park, and Y. Ju Lee. 2008. Antimicrobial resistance of Staphylococcus aureus isolated from domestic and imported raw meat in Korea. Korean J Vet Res. 48:75-81. Holds, G., A. Pointon, M. Lorimer, A. Kiermeier, G. Raven, and J. Sumner. 2007. Microbial profiles of

the application of stringent hygiene practices along the food chain and prudent use of antibiotics in animal husbandry which are essential for the control of further emergence of multidrug resistance.

Anower, A. K. M. M., M. M. Rahman, M. A. Ehsan, M. A. Islam, M. R. Islam, G. C. Shil, and M. S. Rahman. 2004. Bacteriological Profile of Dressed Broilers and Its Public Health Implications. Bangl. J. Vet. Med. 2:69-73. Bergey, D. H., J. G Holt, and N. R Krieg. 1994. Bergey’s Manual of Determinative Bacteriology, 9th Edition. Lippincott Williams & Wilkins. MI, USA. Bhandare, S. G., A. T. Sherikarv, A. M. Paturkar, V. S. Waskar, and R. J. Zende. 2007. A comparison of

carcasses and minced meat from Kangaroos processed in South Australia. Int. J. Food. Microbiol. 123:88-92. ICMSF (International Commission on Microbiological Specifications for Foods). 2002. Microorganisms in Foods 7. Microbiological Testing in Food Safety Management. New York: Kluwer Academic/ Plenum Publishers. Kinsella, K. J., D. M. Prendergast, M. S. McCann, I. S. Blair, D. A. McDowell, and J. Sheridan. 2008. The survival of Salmonallea enteric serovar Typhimurium DT 104 and total viable counts on beef surfaces at different relative humidities and temperatures J. App. Microbiol. 106:171-180. Kirby-Bauer Method. 1997. Disk Diffusion Susceptibility Testing. Newsletter of Animal Disease Diagnostic Laboratory. http://www.addl.purdue.edu/ newsletters/1997/spring/dds.shtml. Hoque, M. A., M. P. Siddique, M. A. Habib, V. Sarker, and K.A. Choudhury. 2008. Evaluation of sanitary quality of goat meat obtained from slaughter yards and meat stalls at late market hours. Bangl. J. Vet. Med. 6:87-92. Norrung, B., J. K. Anderson, and S. Buncic. 2009. Main Concerns of Pathogenic Microorganisms in Meat. In Safety of Meat and Processed Meat

microbial contamination of sheep/goat carcasses in a modern Indian abattoir and traditional meat shops. Food. Contr. 18:854-868. Easa, S. M. H. 2010. The Microbial Quality of Fast Food and Traditional Fast Food. Nature and Sci-

(Toldra F.), Part I, Springer New York, pp 3-29. Selvan, P., R. Narendra Babu, S. Sureshkumar, and V. Venkataramanujam. 2007. Microbial Quality of Retail Meat Products Available in Chennai City. Am. J. Food Technol. 2:55-59.

ACKNOWLEDGEMENTS A study on the microbiological quality of raw meat and processed meat products was carried out in the Department of Microbiology, Primeasia University, Banani, Dhaka, Bangladesh.

REFERENCES

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Podpecan, B., A. Pengov, and S. Vadnjal. 2007. The source of contamination of ground meat for production of meat products with bacteria Staphylococccus aureus. Slov. Vet. Res. 44:24-30. Regecová, I., M. Pipová, P. Jevinová, P. Popelka, and I. Kožárová. 2009. Determination of sensitivity of Staphylococcal isolates from fish meat against selected antibiotics. Folia Veterinaria 53:37-39. Schwarz, S., and D. E. Chaslus. Use of antimicrobials in veterinary medicine and mechanism if resistance. Vet Res 2001 32:201-225. Schwarzt, S., C. Kehrenberg, and T. R. Walsh. 2001. Use of antimicrobials in veterinary medicine and food animal production. Intern J. Antimicrob Agen. 17:431-437. Waliullah, S., and C. R. Ahsan. 2011. Assessment of microbiological quality of some meat-based fast foods collected from street vendors. J. Innov. Strategy 5:44-46. Waters, A. E., T. Contente-Cuomo, J. Buchhagen, C. M. Liu, L. Watson, K. Pearce, J. T. Foster, J. Bowers, E. M. Driebe, D. M. Engelthaler, P. S. Keim, and L. B. Price. 2011. Multidrug-Resistant Staphylococcus aureus in US Meat and Poultry. Clin Infect Dis. 52:1227–1230. WHO. 2009. Food safety and food borne illness. Factsheets of the Programmes and Projects of WHO. http://www.who.int/mediacentre/factsheets/fs237/en/.

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Effect of Stressors on the Viability of Listeria During an in vitro Cold-Smoking Process† J. R. Pittman1, T. B. Schmidt2, A. Corzo3, T. R. Callaway4, J. A. Carroll5, and J. R. Donaldson1 Department of Biological Sciences, Mississippi State University, Mississippi State, MS, 2 Animal Science Department, University of Nebraska, Lincoln, NE 3 Department of Poultry Science, Mississippi State University, Mississippi State, MS 4 Food and Feed Safety Research Unit, U. S. Department of Agriculture, Agricultural Research Service, College Station, TX 5 Livestock Issues Research Unit, U. S. Department of Agriculture, Agriculture Research Service, Lubbock, TX 1

† Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual’s income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity provider and employer.

ABSTRACT Listeria monocytogenes is a dangerous food-borne pathogen and is a frequent contaminant found in the cold-smoked fish industry. To identify strategies to eliminate this bacterium from the cold-smoking processing environment, it is imperative to understand how this microorganism tolerates the conditions encountered. The aim of this study was to determine whether exposure to conditions likely to be encountered during the cold-smoking process differentially impacts various strains of Listeria monocytogenes and Listeria innocua. Viability of L. monocytogenes (EGDe, F2365, HCC7, ATCC 15313, and HCC23) and L. innocua in exponential or stationary growth phase were analyzed following a sequential exposure to conditions that mimic those utilized in the cold-smoking process: freeze (-20°C)-thaw (25°C), elevated salt, liquid smoke, and anaerobic storage (2°C). Viability for stationary phase cells exposed to the mock process decreased (P<0.05) for all strains except EGDe. Viability for exponential phase cells also decreased (P<0.05) for all strains except for EGDe and HCC7 treated cells. The cell envelope of the avirulent strain HCC23 was altered by all treatments examined, while the cell envelope of the virulent strain HCC7 was altered only after exposure to liquid smoke and anaerobic storage. Results indicate that both virulent and avirulent strains in this study, whether in exponential or stationary phase, can tolerate the conditions encountered during the cold-smoking process, and that virulent strains are more resistant than avirulent strains. Collectively, these data strongly suggest that differences exist in the mechanisms utilized by virulent and avirulent strains to adapt to conditions encountered in the cold-smoking process. Keywords: Listeria monocytogenes, Listeria innocua, cold-smoking, salmon, stress-response, salt, liquid smoke, cold, transmission electron microscopy, L-forms, protoplasts Correspondence: Janet R. Donaldson, donaldson@biology.msstate.edu Tel: +1 -662-325-9547 Fax: +1-662-325-7582

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INTRODUCTION Listeria monocytogenes is a gram-positive, foodborne bacterium that causes 2,500 cases of listeriosis and nearly 500 deaths annually in the United States, making it one of the most deadly food-borne pathogens (Ben Embarek, 1994; Mead et al., 1999). The costs associated with the annual cases of listeriosis in the United States is estimated at $8.8 billion USD for health care alone (Scharff, 2010). Consumption of food contaminated with L. monocytogenes poses a serious health risk to the elderly, pregnant women, immunocompromised individuals, and human neonates, and can result in life-threatening medical conditions such as meningitis, meningoencephalitis, spontaneous abortive pregnancies, as well as febrile gastroenteritis (Cossart and Toledo-Arana, 2008; Posfay-Barbe and Wald, 2009; Sleator et al., 2009). Ready-to-eat foods, such as smoked finfish, are the most common sources of listeriosis (Gilbreth et al., 2005; Gombas et al., 2003). The prevalence of L. monocytogenes in cold-smoked fish products is typically between 15-20% (Uyttendaele et al., 2009). Various aspects within finfish smoking facilities can serve as sources of contamination, such as contaminated raw materials entering the plant and contaminated food-processing equipment (Fonnesbech Vogel et al., 2001; Rorvik, 2000; Vaz-Velho et al., 2001). Persistent L. monocytogenes strains present in biofilms or L-forms can also lead to contamination of the processed food products (Gandhi and Chikindas, 2007; Moretro and Langsrud, 2004). The ubiquitous and recalcitrant nature of L. monocytogenes makes removal or exclusion of this microorganism from the processing environment difficult (Wulff et al., 2006). For instance, L. monocytogenes can survive conditions encountered during the cold-smoking process, such as decreased temperatures (2°C), elevated concentrations of salt (3.5 to 6%, w/v), and phenols encountered in cold smoke or through the addition of liquid smoke (Gandhi and Chikindas, 2007; Hwang, 2007; Porsby et al., 2008). Additionally, Listeria is capable of continued growth even under anaerobic storage conditions (Cortesi et al., 1997; Guyer and Jemmi, 1991; Rorvik et al., 1991).

While previous studies have provided vital information on the response of L. monocytogenes to conditions encountered during the cold-smoking process, most of these studies have only included the analysis of one or two strains. Additionally, none of these prior studies examined the effect of sequential exposure to each of the steps in the cold-smoking process on the viability of L. monocytogenes. Therefore, the goal of this study was to determine whether exposure to conditions typically encountered during the cold-smoking process differentially impacts various serovars of Listeria of different pathogenic potential.

MATERIALS AND METHODS Bacterial strains and growth conditions Virulent and avirulent strains of Listeria analyzed in this study are listed in Table 1. All strains were routinely cultivated under aerobic conditions in brain-heart infusion (BHI) medium at 37°C in an orbital shaking incubator. The viability of each strain was monitored throughout the study by viable plate counts on BHI agar; plates were incubated for 24 h at 37°C prior to enumeration. All assays were performed with bacteria in either stationary phase or exponential (mid-logarithmic) phase. To obtain cells in stationary phase, colonies from each strain were grown in BHI broth for 8 h, diluted 1:100 in 5 mL BHI, then allowed to grow at 37°C for 16 h to an OD600 1-1.5. To obtain cells in exponential phase, colonies from each strain were grown in BHI broth for 16 h, diluted 1:100 in 5 mL BHI, then allowed to grow at 37°C to an OD600 0.3-0.4.

Mock cold-smoking procedure Stationary and exponential phase cells were exposed to a mock cold-smoking procedure to determine the effect of the sequential process on cell viability; this procedure was developed based upon information obtained through consultations with individuals from two cold-smoking facilities located in

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Table 1. Strains utilized Strain/ Source Virulent EGDe/ ATCC BAA-679 F2365/ CDC

Serovar

Isolation Information

References

1/2a

Murray et al., 1926

Guinea Pig Mexican-style soft cheese Catfish Brain

4a 1

Catfish Brain Guinea Pig

Wang et al., 1998 Barber, 1939; Murray et al., 1926

Dairy Product

Danielsson-Tham et al., 1993

HCC7/ MSU CVM Avirulent HCC23/ MSU CVM1 ATCC 15313 1

Listeria innocua ATCC BAA-680

Linnan et al., 1988 Wang et al., 1998

HCC7 and HCC23 were isolated from catfish specimens at Mississippi State University’s College of Veterinary Medicine ; isolates were serotyped and characterized (Wang et al., 1998) 1

Figure 1. Experimental design of mock cold-smoking process. Cells in either mid-log or stationary phase were initially frozen for 2 h, then thawed for 1 h. Cells were then exposed to 6% NaCl for 1 h, 0.6% liquid smoke for 1 h, and then finally to 2°C anaerobic conditions for 16 h. To control for affects within each condition tested, aliquots of cells were exposed to 2°C anaerobic conditions following the initial freeze/thaw or the salt treatment.

Alaska, United States. Specifically, cells (5 mL) were frozen at -20°C for 2 h and then thawed at room temperature (RT) for 1 h. Following the freeze-thaw

NaCl for 1 h at 30°C, pelleted by centrifugation and re-suspended in BHI supplemented with 0.6% liquid smoke to mimic exposure to phenolic compounds

treatment, 1 mL of cells was pelleted by centrifugation for 2 min at 10,000 x g (TOMY MX 301, TOMY TECH USA, Freemont, CA) and then re-suspended in 1 mL BHI supplemented with 6% NaCl (w/v) to mimic the brining process. Cells were treated with

encountered during the smoking step as previously described (Faith et al., 2007). Cells were treated with liquid smoke for 1 h at 30°C, after which cells were pelleted by centrifugation at 10,000 x g and resuspended in 1 mL of BHI broth. Cells were then vacu-

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um-sealed and stored anaerobically at 2°C for 16 h; anaerobic conditions were verified by a Mitsubishi anaerobic indicator. Briefly, a single anaerobic indicator packet was placed in each container immediately before being sealed and the absence of a color change indicated that the condition was anaerobic. Growth was monitored by viable plate counts following exposure to each of the conditions examined: prior to freezing and after thawing, salt treatment, liquid smoke treatment, and refrigerated storage (Figure 1). Three independent replicates were performed for each strain. The viable plate counts were used to calculate the mean log10 proportion reduction values [log10(Ntreated CFU/mL)/(Noriginal CFU/mL)].

Statistical analysis Viability data were analyzed as a completely random design with repeated measures using the mixed procedures of SAS to test the effects of the coldsmoking process on the resistance of six strains of Listeria to storage conditions (SAS version 9.2, 2008, SAS Institute Inc., Cary, NC). When F-tests were significant (P< 0.05), treatment means were separated using the method of least significant difference.

Transmission electron microscopy Exponential phase cultures of the L. monocytogenes strains HCC7 and HCC23 were exposed to the mock cold-smoking process as described above. For each culture 2 mL of cells were collected and processed for analysis by transmission electron microscopy (TEM) prior to the freezing condition and after thawing, salt treatment, liquid smoke treatment, and storage as previously described (Merritt et al., 2010). Briefly, cells were pelleted by centrifugation at 10,000 x g for 2 min, washed with 1X PBS, and fixed in 2.5% (v/v) glutaraldehyde in 0.1M cacodylate buffer overnight at 2°C. Samples then were washed with 0.1M cacodylate buffer, post-fixed in 1% (v/v) osmium tetraoxide in 0.1M cacodylate buffer, washed with distilled water, dehydrated in an ethanol series, treated in a stepwise resin/acetone series, and embedded overnight in resin at 68-70°C. Samples were

sectioned to 60-80nm in thickness using an ultramicrotome (Reichert-Jung Ultracut E); sections were double stained with uranyl acetate and lead citrate and viewed under a transmission electron microscope (JEOL JEM-100CXII, JEOL Ltd., Tokyo, Japan). The widths of the cell membrane, cell wall, and the entire cell envelope were measured for 20 individual cells from two independent experiments. The mean average of the cells at mid-log phase (control) was compared to the mean average of cells exposed to each condition using a student-paired t-test. A P-value < 0.05 indicated that changes in the thickness of the structures examined were statistically significant.

RESULTS AND DISCUSSION Previous studies examining the effects of the coldsmoking process on L. monocytogenes are conflicting, as some studies suggest the process leads to a reduction in cell viability while others report that cellular concentrations increase following storage conditions (Guyer and Jemmi, 1991; Porsby et al., 2008). To determine whether these differences could be attributed to strain-to-strain variation, six different strains were exposed to a sequential series of conditions that mimic the cold-smoking fish process to determine if cell viability was affected differently. An in vitro model (Figure 1) was used in the present study in order to determine the direct effect that exposure to these conditions has on the viability of L. monocytogenes, as opposed to inoculating a single strain or microbial consortium onto raw salmon fillets as has been performed by others (Hwang, 2007; Neunlist et al., 2005; Porsby et al., 2008). The in vitro model was tested on cells in both stationary and exponential growth phases to determine whether differences exist in the resistance capability when cells are in the more sensitive state of exponential growth as opposed to the more resilient stationary growth phase.

Effects of the cold-smoking process on the viability of Listeria in stationary phase

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Table 2. Log10 proportion reduction values for stationary phase cells exposed to a mock coldsmoking procedure Strain

Stationary Cells

FreezeThaw

Salt

Liquid Smoke

Storage

EGDe

0.000 a

0.026 a

0.196 b, c

0.185 a

0.271 a

F2365

0.000 a

0.252 a, b

0.220 b, c

0.298 a, c

-0.209 b

HCC7

0.000 a

0.109 a

0.132 a, b, c

0.170 a

-0.261 b

HCC23

0.000 a

-0.046 a

-0.185 a, b, c

-0.353 b

-0.776 c

ATCC 15313

0.000 a

0.127 a

0.018 a

-0.118 a, b

-0.733 c

L. innocua

0.000 a

-0.104 a, c

-0.149 a, b

-0.376 b

-0.701 c

a, b, c

Least significant means within column lacking common subscripts differ (P <0.05)

The process of cold-smoking finfish requires an

strains tested (P> 0.21).

initial freezing of the fish in order to remove parasitic pathogens. The fish are then subjected to a salt brining process, utilizing 4-6% NaCl and exposure to smoking conditions. Since the products are not fully preserved, it is critical that storage temperature conditions are 4°C or less. To determine whether the sequential exposure to these conditions affects the viability of cells when in stationary phase, the mean log10 proportion reduction values of the virulent strains EGDe, F2365, and HCC7 and the avirulent strains HCC23, ATCC 15313, and L. innocua were analyzed following the cold-smoking procedure presented in Figure 1. The first step of the challenge involved exposing cells in stationary phase to a freeze-thaw condition. Consistent with previous studies, the psychrotolerant nature of Listeria allowed for viability to remain relatively unchanged following freeze-thaw (Azizoglu et al., 2009; Wemekamp-Kamphuis et al., 2002a) (Table 2). None of the changes exhibited following freeze-thaw were significant between the strains tested in this study. Following the freeze-thaw condition, cells were exposed to 6% NaCl at 30°C for 1 h. Exposure to salt as a means to simulate brining conditions encountered during the process has been shown to

Following the sequential exposure of stationary phase cells to a freeze-thaw then salt condition, cells were then treated with liquid smoke for 1 h at 30°C. The application of liquid smoke as an alternative method of smoking fish products has been frequently used to mimic exposure to the phenols, carbonyls, and organic acids present during the smoking process; it has also been reported to have a detrimental effect on the viability of L. monocytogenes (Faith et al., 2007; Gedela et al., 2007; Guilbaud et al., 2008; Hwang, 2007; Sunen, 1998; Thurette et al., 1998; Vitt et al., 2001). In general, exposure to liquid smoke led to an increase in viability (as indicated by an increase in log proportion reduction values) in virulent strains and a decrease in viability in avirulent strains in the present study. Within the treatment, strain F2365 had the highest viability in comparison to strains HCC23 and L. innocua (Table 2). In comparison to non-treated controls, virulent strain F2365 (P= 0.0253) increased in viability and the avirulent strains L. innocua (P= 0.0052) and HCC23 (P= 0.0085) decreased. However, the change in viability exhibited by F2365, L. innocua, and HCC23 was not significant in regards to the affect already induced by the salt treatment. This indicates that viability remained stable in these strains following the salt treatment.

result in a significant decrease in cell survival of L. monocytogenes (Neunlist et al., 2005). However, in the present study, exposure of stationary-phase cells to high osmolarity following a previous freeze-thaw did not significantly alter the viability of any of the

The final condition in the sequential exposure to stressors involved in the smoking process was exposure to an anaerobic cold storage (2°C) for 16 h. The viability of EGDe increased (P= 0.04) in comparison to non-treated cells, while the viability of both F2365

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and HCC7 decreased (P= 0.11 and 0.05). Surprisingly, the viability of all three avirulent strains decreased (P< 0.0001; Table 2). Following anaerobic storage conditions, EGDe had the least log10 CFU/ml proportion reduction (0.271) and was greater (P< 0.05) than HCC23, which had the greatest log10 proportion reduction (-0.776; Table 2). Since viability was not altered following the cold exposure portion (first step) of the cold-smoking procedure, these data suggest that one of the stressors encountered in this challenge may actually precondition Listeria to be more resistant to the cold storage conditions encountered at the conclusion of the process. To determine whether exposure to the stressors

or to storage conditions resulted in higher (P< 0.05) viability of EGDe and F2365 in comparison to the avirulent strains HCC23, ATCC 15313, and L. innocua (Figure 2). The decreased viability exhibited by the avirulent strains in response to osmotic stress could be due to a defective capability to uptake compatible osmolytes (e.g., peptides glycine betaine) from the BHI medium (Amezaga et al., 1995). These data suggest that overall the virulent strains examined were more resistant to the cold-smoking process than the avirulent strains when in stationary phase.

Effects of the cold-smoking process on the viability of Listeria in mid-log phase

individually (as opposed to sequentially) were responsible for the significant decrease in viability observed among the avirulent strains, the effect that a pre-treatment with either freeze-thaw or salt had on viability following the anaerobic 2°C storage was examined (Figure 2). With the pretreatment of freezethaw conditions, F2365 had a greater (P= 0.04) increase in population growth than all other strains tested (Figure 2). However, the exposure to salt pri-

Variations were evident in the viability of virulent and avirulent strains following exposure to the mock cold-smoking process when cells were in stationary phase. Therefore, the next objective of this study was to determine whether variations in viability also existed between the strains when in the more susceptible state of exponential growth (mid-log growth phase). The sequential series of conditions tested were the

Figure 2. Proportion reduction values (log10 CFU/mL) of stationary phase cells following treatment with either freeze-thaw or salt, followed by anaerobic 2°C conditions for 16 h. Cells in stationary phase were pre-treated with either freeze-thaw (green filled) or salt (orange fill) prior to exposure to 2°C anaerobic conditions. Viability was expressed as the mean log10 proportion reduction values [log10 (Ntreated CFU/mL)/(Noriginal CFU/mL)]. Values represent the average log10 CFU/ mL proportion reduction values ± standard error from (n = 3) independent replicates. Within each pretreatment condition, values with different superscripts are significantly different (P < 0.05). 0.4 a a

b a

Log10 (Nt/N0 )

-0.4

-0.8

-1.2

EGDe

F2365

HCC7

HCC23

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ATCC 15313 L. innocua

Table 3. Log10 proportion reduction values for mid-log phase cells exposed to a mock coldsmoking procedure

a, b, c

Strain

Mid-log Cells

FreezeThaw

Salt

Liquid Smoke

Storage

EGDe

0.000 a

0.085 a

-0.043 a

-0.241 a

-0.261 a

F2365

0.000 a

-0.049 a

-0.462 b

-0.464 a, c

-0.703 b

HCC7

0.000 a

0.018 a

0.111 a

0.178 b

-0.037

HCC23

0.000 a

0.122 a

-0.693 b, c

-0.238 a

-0.354 a, b, c

ATCC 15313

0.000 a

0.068 a

0.004 a

0.157 a, b

-0.567 a, b

L. innocua

0.000 a

0.155 a

-0.180 a, b

-0.594 a, c

-0.682 b

a, c

Least significant means within column lacking common subscripts differ significantly (P <0.05)

same as the conditions presented in Figure 1. The

ing that this strain is more susceptible to these con-

first step of the cold-smoking process entailed exposure to the freeze-thaw condition. The least significant mean log10 proportion reduction values (CFU/ mL) of virulent strains EGDe, F2365, and HCC7 and the avirulent strains HCC23, ATCC 15313, and L. innocua in mid-log phase following the cold-smoking process are presented in Table 3. Viability remained relatively unchanged after exposure to the freezethaw stress and no differences (P > 0.05) were found to exist between the strains (Table 3), which is consistent with previous studies (Wemekamp-Kamphuis et al., 2002a) and also the present data obtained when cells were in stationary phase (Table 2). Subsequent exposure to high salt conditions resulted in a decrease in viability in HCC23 (P= 0.0002) and F2365 (P= 0.0103) in comparison to control cells (Table 3). These data are in contrast to results obtained from stationary phase cells exposed to these conditions, which indicated that none of the strains tested exhibited a significant change in viability following exposure to salt. This suggests that these strains are actually more susceptible to a salt treatment when in exponential growth as compared to when in stationary phase. When stationary phase cells were sequentially exposed to liquid smoke, viability remained unaltered

ditions encountered in the cold-smoking process. HCC23 significantly increased in viability in comparison to cells following the salt exposure (P= 0.0115). The viability of F2365 remained stable following exposure to liquid smoke, yet decreased (P= 0.01) in regards to mid-log phase control cells (Table 3). These data indicate that exposure to liquid smoke did not affect this strain any more than the exposure to salt. Viability of F2365, HCC23, ATCC 15313, and L. innocua significantly decreased after the sequential exposure to the anaerobic storage in comparison to non-treated cells. Of all strains tested, only the viability of ATCC 15313 decreased (P< 0.0001) following storage in comparison to viability prior to this treatment (log10 proportion reduction values of 0.157 after liquid smoke decreased to -0.567 following storage). To determine which individual condition of the cold-smoking process impacted Listeria the most when in mid-log phase, viability following a pretreatment with either freeze-thaw or salt followed by anaerobic storage was analyzed. The log10 proportion reduction values (CFU/mL) for each strain with statistical analysis specific within each pretreatment category were analyzed (Figure 3). The effect of the

in the virulent strains (Table 2). However, much variation was observed in the viability of mid-log cells following the subsequent exposure to liquid smoke (Table 3). L. innocua had a decrease (P= 0.0012) in viability in comparison to non-treated cells, suggest-

freeze-thaw pre-treatment on viability following storage conditions resulted in a significant decrease in viability of L. innocua in comparison to only HCC7 and HCC23. A pretreatment with salt prior to anaerobic storage did not significantly alter the viability

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Figure 3. Proportion reduction values (log10 CFU/mL) of mid-log phase cells following exposure to freeze-thaw or salt, followed by anaerobic 2°C conditions. Cells in mid-log phase were pretreated with either freeze-thaw (green filled) or salt (orange fill) prior to exposure to 2°C anaerobic conditions. Viability was expressed as the mean log10 proportion reduction values [log10 (Ntreated CFU/mL)/(Noriginal CFU/mL)]. Values represent the average log10 CFU/m proportion reduction values ± standard error from (n = 3) independent replicates. Within each pretreatment condition, values with different superscripts are significantly different (P < 0.05).

0.2 ac ab a

Log10 (Nt/N0)

abc ab

abc a

b ab

-0.2

-0.4

-0.6

-0.8 EGDe

F2365

HCC7

HCC23

ATCC 15313 L. innocua

Table 4. Average thickness (nm) of the cell membrane, cell wall, and cell envelope of HCC7 and HCC23 cells following exposure to the mock cold-smoking process Mid-log Phase HCC7 HCC23

Freeze-Thaw

6% NaCl

HCC7 HCC23 HCC7

HCC23

0.6% Liquid Smoke HCC7

HCC23

Storage HCC7

HCC23

Cell membrane

7.22

8.70

7.96*

6.26*

7.24

5.74*

6.60

4.43*

7.17

5.93*

Cell wall

17.84

17.78

17.40

18.20

17.60

14.69*

17.10

19.80*

22.86*

18.04

Cell envelope

24.92

26.48

25.36

24.46*

24.84

20.43*

23.7*

24.23*

30.14*

23.98*

* Indicates significant changes (P < 0.05) in cells exposed to freeze-thaw, salt, liquid smoke, or storage treatments compared to mid-log phase cells

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Figure 4. Transmission electron micrographs of HCC7 and HCC23 following exposure to stressors encountered during the cold-smoking process. Micrographs of HCC7 (A) and HCC23 (B) in mid-log phase; HCC7 (C) and HCC23 (D) following freeze-thaw. Retraction of the cell membrane from the cell wall of HCC7 following exposure to 6% NaCl is depicted in (E). Abnormal swollen morphology of HCC23 after exposure to 6% NaCl is depicted in (F). Normal appearing cells of HCC7 following exposure to 0.6% liquid smoke is depicted in (G). (H) is representative of protoplasts observed in HCC23 following exposure to liquid smoke. The presence of filamentous cells of HCC7 following 2°C anaerobic conditions is indicated by (I). Damage to the cell wall of HCC23 following anaerobic storage is depicted in (J). Scale bars represent 0.5um.

Freeze-Thaw 6% NaCl

0.5 µm

Mid-log Phase

0.6% Liquid Smoke

HCC23

Anaerobic Storage

HCC7

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between any strains examined (Figure 3; P> 0.19). This suggests that exposure to either of these conditions prior to anaerobic storage may lead to the decrease in viability among populations of Listeria strains.

Changes in the width of the cell membrane, cell wall, and the entire cell envelope in response to the stresses of the cold-smoking process were examined by TEM. Only mid-log phase cells were analyzed, as the affect of the process on log proportion reduc-

discovered that YneA is involved in cell elongation and inhibition of cell division following induction of the SOS response (van der Veen et al., 2010). This could potentially lead to the formation of filamentous cells following exposure to 6% NaCl as observed in this study and others (Hazeleger et al., 2006). Exposure to elevated salt concentrations caused almost no change in the thickness of the cell envelope of HCC7, but led to a significant decrease in the thickness of HCC23â&#x20AC;&#x2122;s cell membrane and cell wall (P<0.01) in comparison to non-treated cells (Table 4). Decreased expression or activity of penicillinbinding proteins (PBP) following exposure to high salt concentrations could result in reduced pepti-

tion was generally higher when cells were in mid-log phase as opposed to stationary phase. The TEM analysis was limited to HCC7 and HCC23 because HCC23 exhibited a significant decrease in viability following the exposure to the process, while HCC7 remained unaltered, suggesting that the two strains possess different adaptive mechanisms. Additionally, these two strains were isolated from fish (Wang et al., 1998), making the strains appropriate model microorganisms for this analysis. Representative micrographs from each treatment for each strain are presented in Figure 4. Following the initial freezing conditions, a majority of HCC7 and HCC23 cells remained undamaged (65%); the remainder of these cells was elongated (Figure 4C and D). Freeze-thaw conditions resulted in a thickening of the cell membrane of HCC7 (P= 0.0033), while the cell membrane and cell envelope (P< 0.01) of HCC23 decreased in thickness (Table 4). The increase in the thickness of the cell membrane of HCC7 could potentially be due to changes in the lipid membrane composition in order to maintain membrane fluidity (Annous et al., 1997). Following exposure to 6% NaCl, the majority of HCC7 and HCC23 cells remained unchanged (63% and 60%, respectively). However, detachment of the

doglycan cross-links and an abnormal morphology similar to what was observed in this study (Guinane et al., 2006), potentially altering the susceptibility of HCC23 to osmotic stress (Piuri et al., 2005; Popham and Young, 2003). It has been postulated that one reason for the increased incidence of listeriosis in humans is the reduced salt content of food products, allowing for survival and growth of the organism and potential likelihood of infection following consumption by susceptible individuals (Goulet et al., 2008). It will be critical to further examine the expression of peptidoglycan-associated proteins following exposure to a salt stress. Following the sequential exposure of mid-log HCC7 and HCC23 cells to freeze-thaw and salt, cells were treated with 0.6% liquid smoke. A major proportion (84%) of HCC7 cells appeared normal (Figure 4G), but the remainder of the cells (16%) exhibited detachment of the cell membrane from the cell wall. In HCC23 about 39% of the cells were elongated, 8% exhibited loss of cytoplasmic material, and 11% had a deformed morphology. Liquid smoke exposure caused a decrease in the thickness of the cell envelope due to a decrease in the cell membrane of both HCC7 (P= 0.0179) and HCC23 (P< 0.01) (Table 4). Cell wall thickness of HCC23 increased following

cell membrane from the cell wall was observed in 14% of the HCC7 cells and 7% of the HCC23 cells (Figure 4E, F). Interestingly, 23% of the HCC7 cells and 25% of the HCC23 cells became elongated following exposure to salt (Figure 4F). It was recently

liquid smoke treatment (P< 0.01). Damage to the cell envelope of HCC23 observed in this study is consistent with the ability of liquid smoke to damage the cell membrane (Guilbaud et al., 2008), causing the cell membrane to become thinner and the cell wall

Effects of the cold-smoking process on cell wall integrity

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to become thicker in comparison to untreated cells. The increased thickness of the cell wall could be accounted for by changes in the composition of the cell wall to increase chemical resistance as observed by others (Santos et al., 2004). Exposure to liquid smoke also resulted in the potential production of protoplasts (4.5%) (Figure 4H), indicating either serious damage to the cell wall occurred or the cells were transitioning into L-forms. L. monocytogenes L-forms have been shown to have an increased expression of stress-response genes and are capable of growth and cell division (Dell’Era et al., 2009), thus providing an adaptive mechanism to survive environmental stressors (Markova et al.,

of HCC7 increased in thickness due to a thicker cell wall (P< 0.01), while the thickness of the cell membrane and cell envelope of HCC23 decreased (P< 0.01; Table 4). One potential explanation for a decrease in the cell membrane thickness of HCC23 is that under anaerobiosis and low temperature conditions, synthesis of unsaturated fatty acids and branched-chain fatty acids could be inhibited due to reduced levels of NADH oxidation (de Sarrau et al., 2012; Van Der Voort and Abee, 2009). This would lead to reduced membrane fluidity and an inability to adapt to cold temperatures.

2010). It is therefore possible that L-forms of L. monocytogenes are generated during the cold-smoking process. Because cultivation of L-forms of L. monocytogenes requires the use of specialized media and a prolonged incubation time, they may go undetected in the food-processing environment. Therefore, it is necessary to further determine whether this is the point in the processing procedure where Listeria transition into an L-form state. A previous study reported that exposure of L. monocytogenes to 7% NaCl for 1 h could provide cross-protection against 0.1% H2O2 (Lou and Yousef, 1997). Thus the salt treatment in the present study could have protected the bacterial cells from liquid smoke damage via a similar mechanism. Although exposure of Shewanella putrefaciens to increasing concentrations of NaCl increased sensitivity to liquid smoke (Leblanc et al., 2000), a longer or altered treatment with smoking conditions might produce altered results. The final step of the sequential exposure was an anaerobic incubation at 2°C for 16 h. HCC7 had an increased thickness of the cell envelope as indicated by a thickening of the cell wall (P< 0.01), and 33% of the cells formed chains of cells with a visible septum (Figure 4I); the remainder of the cells appeared to be

CONCLUSIONS

normal. Following anaerobic storage, 46% of HCC23 cells showed signs of damage to the cell wall, 48% were elongated or filamentous, and ~16% formed a division septum towards the pole of the cell. Following anaerobic storage conditions, the cell envelope

sippi State University for his assistance with this project. This project was supported through the Office of Research, the Department of Biological Sciences, and the Research Initiation Program at Mississippi State University.

In conclusion, the virulent L. monocytogenes strains examined in this study were more resistant to storage conditions following salt than the avirulent strains regardless of the growth phase. This resistance could be explained by expression of similar mechanisms required for survival during exposure to salt and low temperatures. Previous studies have reported an overlap in the expression of genes required for the transport of osmolytes following cold or osmotic stress in L. monocytogenes (WemekampKamphuis et al., 2004; Wemekamp-Kamphuis et al., 2002b), suggesting freeze-thaw conditions are required to increase resistance to salt. The ability of the sub-lethally injured cells to survive these conditions suggests that these strains may be undetected during sampling in the food-processing environment and could continue to grow following storage conditions. Further testing is needed to determine whether this affect is primarily governed by the exposure to salt.

ACKNOWLEDGEMENTS We would like to thank Kendrick Currie at Missis-

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REFERENCES Amezaga, M. R., I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth. 1995. The role of peptide metabolism in the growth of Listeria monocytogenes ATCC 23074 at high osmolarity. Microbiol. 141 ( Pt 1):41-9. Annous, B. A., L. A. Becker, D. O. Bayles, D. P. Labeda, and B. J. Wilkinson. 1997. Critical role of anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures. Appl. Environ. Microbiol. 63:3887-94. Azizoglu, R. O., J. Osborne, S. Wilson, and S. Kathariou. 2009. Role of growth temperature in freeze-

Faith, N. G., A. E. Yousef, and J. B. Luchansky. 2007. Inhibition of Listeria monocytogenes by liquid smoke and isoeugenol, a phenolic component found in smoke. J. Food Safety 12:303-314. Fonnesbech Vogel, B., H. H. Huss , B. Ojeniyi, P. Ahrens, and L. Gram. 2001. Elucidation of Listeria monocytogenes contamination routes in coldsmoked salmon processing plants detected by DNA-based typing methods. Appl. Environ. Microbiol. 67:2586-95. Gandhi, M., and M. L. Chikindas. 2007. Listeria: A foodborne pathogen that knows how to survive. Int. J. Food Microbiol. 113:1-15. Gedela, S., R. K. Gamble, S. Macwana, J. R. Escou-

thaw tolerance of Listeria spp. Appl. Environ. Microbiol. 75:5315-20. Barber, M. 1939. A comparative study of Listerella and Erysipelothrix. J. Pathol. Bacteriol. 48:11-23. Ben Embarek, P. K. 1994. Presence, detection and growth of Listeria monocytogenes in seafoods: a review. Int. J. Food Microbiol. 23:17-34. Cortesi, M. L., T. Sarli, A. Santoro, N. Murru, and T. Pepe. 1997. Distribution and behavior of Listeria monocytogenes in three lots of naturally-contaminated vacuum-packed smoked salmon stored at 2 and 10 degrees C. Int. J. Food Microbiol. 37:20914. Cossart, P., and A. Toledo-Arana. 2008. Listeria monocytogenes, a unique model in infection biology: an overview. Microbes Infect. 10:1041-50. Danielsson-Tham, M. L., J. Bille, R. Brosch, C. Buchriesser, K. Persson, A. Schwarzkopf, W. Tham, and J. Ursing. 1993. Characterization of Listeria strains isolated from soft cheese. Int. J. Food Microbiol. 18:161-6. de Sarrau, B., T. Clavel, C. Clerte, F. Carlin, C. Ginies, and C. Nguyen-The. 2012. Influence of Anaerobiosis and Low Temperature on Bacillus cereus Growth, Metabolism, and Membrane Properties. Appl. Environ. Microbiol. 78:1715-23.

bas, and P. M. Muriana. 2007. Effect of inhibitory extracts derived from liquid smoke combined with postprocess pasteurization for control of Listeria monocytogenes on ready-to-eat meats. J. Food Prot. 70:2749-56. Gilbreth, S. E., J. E. Call, F. M. Wallace, V. N. Scott, Y. Chen, and J. B. Luchansky. 2005. Relatedness of Listeria monocytogenes Isolates recovered from selected ready-to-eat foods and listeriosis patients in the United States. Appl. Environ. Microbiol. 71:8115-22. Gombas, D. E., Y. Chen, R. S. Clavero, and V. N. Scott. 2003. Survey of Listeria monocytogenes in ready-to-eat foods. J. Food. Prot. 66:559-69. Goulet, V., C. Hedberg, A. Le Monnier, and H. de Valk. 2008. Increasing incidence of listeriosis in France and other European countries. Emerg. Infect. Dis. 14:734-40. Guilbaud, M., I. Chafsey, M. F. Pilet, F. Leroi, H. Prevost, M. Hebraud, and X. Doussett. 2008. Response of Listeria monocytogenes to liquid smoke. J. Appl. Microbiol. 104:1744-53. Guinane, C. M., P. D. Cotter, R. P. Ross, and C. Hill. 2006. Contribution of penicillin-binding protein homologs to antibiotic resistance, cell morphology, and virulence of Listeria monocytogenes

Dell’Era, S., C. Buchrieser, E. Couve, B. Schnell, Y. Briers, M. Schuppler, and M. J. Loessner. 2009. Listeria monocytogenes L-forms respond to cell wall deficiency by modifying gene expression and the mode of division. Mol. Microbiol. 73:306-22.

EGDe. Antimicrob. Agents Chemother. 50:2824-8. Guyer, S., and T. Jemmi. 1991. Behavior of Listeria monocytogenes during fabrication and storage of experimentally contaminated smoked salmon. Appl. Environ. Microbiol. 57:1523-7.

206 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

Hazeleger, W. C., M. Dalvoorde, and R. R. Beumer. 2006. Fluorescence microscopy of NaCl-stressed, elongated Salmonella and Listeria cells reveals the presence of septa in filaments. Int. J. Food Microbiol. 112:288-90. Hwang, C.A. 2007. Effect of salt, smoke compound, and storage temperature on the growth of Listeria monocytogenes in simulated smoked salmon. J. Food Prot. 70:2321-8. Leblanc, L., F. Leroi, A. Hartke, and Y. Auffray. 2000. Do stresses encountered during the smoked salmon process influence the survival of the spoiling bacterium Shewanella putrefaciens? Lett. Appl. Microbiol. 30:437-42.

V. Besnard, M. Federighi, and F. Leroi. 2005. Effect of salting and cold-smoking process on the culturability, viability, and virulence of Listeria monocytogenes strain Scott A. J. Food Prot. 68:85-91. Piuri, M., C. Sanchez-Rivas, andS. M. Ruzal. 2005. Cell wall modifications during osmotic stress in Lactobacillus casei. J. Appl. Microbiol. 98:84-95. Popham, D. L. and K. D. Young. 2003. Role of penicillin-binding proteins in bacterial cell morphogenesis. Curr. Opin. Microbiol. 6:594-9. Porsby, C. H., B. F. Vogel, M. Mohr, and L. Gram. 2008. Influence of processing steps in cold-smoked salmon production on survival and growth of persistent and presumed non-persistent Listeria monocyto-

Linnan, M. J., L. Mascola, X. D. Lou, V. Goulet, S. May, C. Salminen, D. W. Hird, M. L. Yonekura., P. Hayes, and R. Weaver. 1988. Epidemic listeriosis associated with Mexican-style cheese. N. Engl. J. Med. 319:823-8. Lou, Y., and A. E. Yousef. 1997. Adaptation to sublethal environmental stresses protects Listeria monocytogenes against lethal preservation factors. Appl. Environ. Microbiol. 63:1252-5. Markova, N., G. Slavchev, L. Michailova, and M. Jourdanova. 2010. Survival of Escherichia coli under lethal heat stress by L-form conversion. Int. J. Biol. Sci. 6:303-15. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresse, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-Related Illness and Death in the United States. Emerg. Infect. Dis. 5:607-625. Merritt, M. E., A. M. Lawrence, and J. R. Donaldson. 2010. Comparative Study of the Effect of Bile on the Listeria monocytogenes Virulent Strain EGDe and Avirulent Strain HCC23 Arch. Clinical Micro. 1:4-9. Moretro, T., and S. Langsrud. 2004. Listeria monocytogenes: biofilm formation and persistence in food-processing environments. Biofilms 1:107-121. Murray, E. G. D., R. A. Webb, and M. B. R. Swann.

genes. Int. J. Food Microbiol. 122:287-95. Posfay-Barbe, K. M., and E. R. Wald. 2009. Listeriosis. Semin. Fetal Neonatal. Med. 14:228-33. Rorvik, L. M. 2000. Listeria monocytogenes in the smoked salmon industry. Int. J. Food Microbiol. 62:183-90. Rorvik, L. M., M. Yndestad, and E. Skjerve. 1991. Growth of Listeria monocytogenes in vacuumpacked, smoked salmon, during storage at 4 degrees C. Int. J. Food Microbiol. 14:111-7. Santos, P. M., D. Benndorf, and I. Sa-Correia. 2004. Insights into Pseudomonas putida KT2440 response to phenol-induced stress by quantitative proteomics. Proteomics 4:2640-52. Scharff, R. L. 2010. Health-related costs from foodborne illness in the United States. Georgetown University, Washington, DC. Sleator, R. D., D. Watson, C. Hill, C. G. and Gahan. 2009. The interaction between Listeria monocytogenes and the host gastrointestinal tract. Microbiol. 155:2463-75. Sunen E. 1998. Minimum inhibitory concentration of smoke wood extracts against spoilage and pathogenic micro-organisms associated with foods. Lett. Appl. Microbiol. 27:45-8. Thurette, J., J. M. Membre, L. H. Ching, R. Tailliez,

1926. A disease of rabbit characterised by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus: Bacterium monocytogenes (n. sp.). J. Pathol. Bacteriol. 29:407-439. Neunlist, M. R., M. Ralazamahaleo, J. M. Cappelier,

and M. Catteau. 1998. Behavior of Listeria spp. in smoked fish products affected by liquid smoke, NaCl concentration, and temperature. J. Food Prot. 61:1475-9. Uyttendaele, M., P. Busschaert, A. Valero, A. H.

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Geeraerd, A. Vermeulen, L. Jacxsens, K. K. Goh, A. De Loy, J. F. Van Impe, and F. Devlieghere. 2009. Prevalence and challenge tests of Listeria monocytogenes in Belgian produced and retailed mayonnaise-based deli-salads, cooked meat products and smoked fish between 2005 and 2007. Int. J. Food Microbiol. 133:94-104. van der Veen, S., S. van Schalkwijk, D. Molenaar, W. M. de Vos, T. Abee, and M. H. Wells-Bennik. 2010. The SOS response of Listeria monocytogenes is involved in stress resistance and mutagenesis. Microbiol. 156:374-84. Van Der Voort, M. and T. Abee. 2009. Transcriptional regulation of metabolic pathways, alternative

BetL, Gbu, and OpuC of Listeria monocytogenes affect virulence and growth at high osmolarity. Appl. Environ. Microbiol. 68:4710-6. Wulff, G., L. Gram, P. Ahrens, and B. F. Vogel. 2006. One group of genetically similar Listeria monocytogenes strains frequently dominates and persists in several fish slaughter- and smokehouses. Appl. Environ. Microbiol. 72:4313-22.

respiration and enterotoxin genes in anaerobic growth of Bacillus cereus ATCC 14579. J. Appl. Microbiol. 107:795-804. Vaz-Velho, M., G. Duarte, J. McLauchlin, and P. Gibbs. 2001. Characterization of Listeria monocytogenes isolated from production lines of fresh and coldsmoked fish. J. Appl. Microbiol. 91:556-62. Vitt, S. M., B. A. Himelbloom, and C. A. Crapo. 2001. Inhibition of Listeria innocua and L. monocytogenes in a laboratory medium and cold-smoked salmon containing liquid smoke. J. Food Safety 21:111-125. Wang, C., L. F. Zhang, F. W. Austin, and C. R. Boyle. 1998. Characterization of Listeria monocytogenes isolated from channel catfish (Ictalurus punctatus). Am. J. Vet. Res. 59:1125-8. Wemekamp-Kamphuis, H. H., A. K. Karatzas, J. A. Wouters, and T. Abee. 2002a. Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure. Appl. Environ. Microbiol. 68:456-63. Wemekamp-Kamphuis, H. H., R. D. Sleator, J. A> Wouters, C. Hill, and T. Abee. 2004. Molecular and physiological analysis of the role of osmolyte transporters BetL, Gbu, and OpuC in growth of Listeria monocytogenes at low temperatures. Appl. Environ. Microbiol. 70:2912-2918. Wemekamp-Kamphuis, H. H., J. A. Wouters, R. D. Sleator, C. G. Gahan, C. Hill, and T. Abee. 2002b. Multiple deletions of the osmolyte transporters 208 Agric. Food Anal. Bacteriol. â&#x20AC;˘ AFABjournal.com â&#x20AC;˘ Vol. 2, Issue 3 - 2012

Antibacterial Activity of Plant Extracts on Foodborne Bacterial Pathogens and Food Spoilage Bacteria N. Murali1, G. S. Kumar-Phillips1, N.C. Rath1,2, J. Marcy1 and M. F. Slavik1 Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701 2 USDA/ARS

ABSTRACT Bacterial foodborne diseases are caused by consumption of foods contaminated with bacteria and/ or their toxins. In this study, we evaluated antibacterial properties of twelve different extracts including turmeric, lemon and different kinds of teas against four major pathogenic foodborne bacteria including Campylobacter jejuni, Escherichia coli O157:H7, Salmonella Enteritidis and Staphylococcus aureus and food spoilage bacteria Pseudomonas aeruginosa and Pseudomonas putida. Of the twelve extracts, lemon extract was found to be most antibacterial and killed all the bacteria within 24 h of incubation. Among the bacterial pathogens, E. coli O157:H7 was most susceptible to lemon extract and C. jejuni was the least susceptible. Turmeric was found to kill all the C. jejuni isolates and MRSA within 36 h but killed E. coli and S. Enteritidis only after 48 h of incubation. However, turmeric showed maximum activity against P. putida which was killed within 24 h of incubation, but failed to kill P. aeruginosa even after 48 h of incubation. Among the different teas tested, green and white tea extracts were found to be the most antibacterial and white tea killed all the bacteria except C. jejuni 81176 within 48 h of incubation. Other tea varieties including Rose of Suzhou, Sweet Fruit Garden and Silver Needle had various degrees of bactericidal effects. These results demonstrate the potential for using plant extracts, especially lemon extracts, as successful antibacterial agents. These extracts could be used as food additives to certain foods to reduce or eliminate foodborne bacterial pathogens and food spoilage bacteria.

Keywords: Foodborne bacteria, Tea, Turmeric, Lemon, Plant Extract, Flavonoids, Campylobacter, E. coli, Salmonella, MRSA, Pseudomonas Agric. Food Anal. Bacteriol. 2:209-221, 2012

Correspondence: Geetha S. Kumar-Phillips, gskumar@uark.edu Tel: +1-479-856-5079

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INTRODUCTION Some foodborne bacteria responsible for disease and spoilage are resistant to existing treatments, generating a need for different methods to eliminate or reduce these bacteria (Sirsat et al., 2009). Recently, medicinal plants and their extracts have gained importance as potential antibacterial agents. Secondary metabolites of plants including tannins, flavonoids and alkaloids have been found to possess antimicrobial properties in vitro (Dahanukar et al., 2000). Many medicinal plants including Clausena anisata (Rutaceae), Ocimum tenuiflorum (tulsi), Curcuma longa (turmeric), Azardirachta indica (neem),

2008). A recent study reported that tea polyphenols altered the integrity of the outer and inner bacterial membranes as evidenced by transmission electron microscopy (TEM) and disrupted the cell walls (Yi et al., 2010). Green, black and white teas were used in the study since they are the most common varieties of teas consumed by people around the world. Other teas like Silver Needle, Rose of Suzhou, and oolong were used since they are specialty teas and we wanted to compare their antibacterial properties with the common teas consumed Most plants contain flavonoids in varying amounts. Some plants like lemon (Citrus limon) and turmeric (Curcuma longa) contain high amount of polyphe-

Eugenia caryophyllata (clove) have been shown to possess antibacterial effects in vitro (Sher, 2009). In a recent study, it was shown that flavonoids (or phytochemicals), if used in combination with antibiotics, were not only effective against Pseudomonas infection, but also decreased the toxicity of the antibiotics (Jayaraman et al., 2010). Flavonoids are secondary plant metabolites used in nutraceutical industries (Srinivas et al., 2009) as antioxidants and antimicrobial agents and are gaining importance in the food industry (Srinivas et al., 2010). Of the flavonoids, tea flavonoids have been extensively studied for almost a decade. The most common tea flavonoids are catechins and epicatechins. Different types of teas (Camellia sinensis) contain varying amounts of flavonoids. Increased enzymatic oxidation of tea results in a decrease in catechin concentration and formation of complexes like theaflavins. Black tea undergoes complete fermentation, oolong tea undergoes partial fermentation, while green and white teas are unfermented (Chou et al., 1999). Black tea has approximately 9% catechin and 4% theaflavin, while green teas contain as high as 30% catechins w/v (Wiseman et al., 1997). Researchers have shown that Asian black teas and green teas (we have specified Asian teas since black

nols. While fruits like lemon contain essential oils and eriocitrin (a flavonoid) which is bactericidal, turmeric contains a flavonoid curcumin, known for its antioxidant, anti-inflammatory and antibacterial properties. Lemon juice contains 5 to 6% citric acid, has a sour taste and a pH from 2 to 3. This property of lemon juice can be exploited to make lemon a good antibacterial agent. In one study conducted by Conte and coworkers (2007) lemon extracts were found to successfully inhibit vegetative cells and spores of some food spoilage microorganisms including yeast, Bacillus species and lactic acid bacteria. Naz and coworkers (2010) have shown that curcumin and essential oils of turmeric varieties were effective against Bacillus and Azotobacter species. Other researchers have shown that inhibition of the FtsZ assembly dynamics (FtsZ is a protein shown to play a critical role in bacterial cytokinesis) in the Zâ&#x20AC;&#x201C;ring is a possible antibacterial mechanism of action of curcumin (Rai et al., 2008). These in vitro studies show that lemon extracts and curcumin have potential antibacterial properties against both Gram positive as well as Gram negative bacteria. In the present study, we evaluated the antibacterial activity of plant extracts including lemon, turmeric and different varieties of teas including black,

and green teas are mostly cultivated in asia) exhibit antibacterial activity in vitro against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Enterococcus fecalis (Bancirova, 2010) and spoilage bacteria in fish and meat (Wenjiao et al.,

green, white, oolong, mint, Rose of Suzhou, Sweet Fruit Garden, Silver Needle, Emerald Princess and rooibos against foodborne bacteria including C. jejuni, E. coli O157:H7, S. Enteritidis, S. aureus and food spoilage bacteria including P. aeruginosa and

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P. putida. Although there have been various studies reporting the effects of tea flavonoids, this study tests the activities of different kinds of teas including specialty teas like Rose of Suzhou, Sweet Fruit Garden, Silver Needle, Rooibos and Emerald Princess. Viable cell counts were performed and viability percentages were calculated for each extract to assess the efficacy of the extracts. Due to the importance of foodborne bacterial pathogens and food spoilage bacteria, plant extracts could prove to be efficient and practical antibacterial treatments.

MATERIALS AND METHODS Bacterial strains used Pathogenic bacteria included four different isolates of Campylobacter jejuni including human isolate 81176, chicken isolates from a pre–chilled chicken carcass (PRCC), from a post–chilled chicken carcass (POCC), and from a retail chicken carcass (RECC); Escherichia coli O157:H7 ATCC 43888; Salmonella Enteritidis (T1B4E); methycillin resistant Staphylococcus aureus ATCC 43300. Pseudomonas aeruginosa ATCC 17485 and Pseudomonas putida ATCC 10145 were used as the food spoilage bacteria.

Media used Campylobacter Enrichment Broth (CEB) (Acumedia®, Lansing, MI) was used for initial culturing of C. jejuni strains and Nutrient Broth (NB) (DIFCO®, Franklin Lakes, NJ) was used for culturing E. coli, S. Enteritidis, MRSA, P. aeruginosa and P. putida. Mueller Hinton Agar (MHA) (DIFCO®, Franklin Lakes, NJ) was used for plating for all the bacteria after serial dilution. For enumeration of C. jejuni, in addition to MHA, Campylobacter Enrichment Agar (CEA) supplemented with 5% horse blood was used.

Turmeric Extraction Dried and powdered turmeric was purchased from

a local supermarket (Fayetteville, AR). Ten grams were boiled in 100 mL sterile water for 20 minutes and filtered through sterile gauze. After neutralizing the pH to 7.0±0.2 with 10N NaOH, the filtrate was autoclaved at 121°C for 15 minutes. The autoclaved turmeric extract was stored in a dark bottle at -20°C for further use (Weerasekera et al., 2008).

Lemon Extraction Fresh lemon fruits were purchased from a local supermarket (Fayetteville, Arkansas). The lemons first were washed with tap water and then with distilled water. Twenty grams of cut fruits including the rind and flesh were immersed in 100 ml of 96% ethanol for 30 mins and ground with a mortar and pestle to extract soluble material. The ground extracts were held at room temperature for 48 h, filtered through Whatman No. 1 paper and placed in sterile petri dishes for 48h at room temperature to evaporate the ethanol. Dried extracts were re-suspended in 15 ml of phosphate buffered saline (PBS) and maintained at 4°C until used (Valtierra- Rodríguez et al., 2010).

Tea Extraction Ten different varieties of teas (Camellia sinensis) were included in the study including, black tea, green tea, white tea, oolong tea, mint tea, Rose of Suzhou tea, Sweet Fruit Garden tea, Silver Needle tea, Emerald Princess tea and Rooibos tea (Aspalathus linearis). Dried and powdered tea leaves were purchased from various sources. Ten grams of tea leaves were suspended in sterile phosphate buffered saline (PBS) 20% concentrations (w/v), held at room temperature for 3h and centrifuged at 15,000 rpm for 10 minutes. After transferring the supernatants to another sterile tube, the pH was adjusted to 7.0 ± 0.2 and the extracts were stored in a dark bottle at 4°C (Diker et al., 1991). In order to evaluate boiling as an extraction method, green tea, oolong tea, Silver Needle tea and Rose of Suzhou tea leaves were boiled for 5 minutes each, cooled and centrifuged at 15,000 rpm for 10 minutes. The supernatant was transferred to a sterile tube and pH neutralized.

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Measurement of bactericidal activity Bacterial strains of C. jejuni were grown in Campylobacter Enrichment Broth (CEB) for 18 h at 42°C. E. coli, S. Enteritidis, MRSA, P. putida and P. aeruginosa strains were grown in Nutrient Broth (NB) for 18 h at 37°C. The cultures were centrifuged at 8000 x g for 2 minutes at 25°C. The supernatants were discarded and the pellets were reconstituted in fresh enrichment broth media. Five ml of each bacterial suspension were mixed with 5 mL of the above extracts and incubated as follows: 1. 42°C microaerobic conditions for C. jejuni strains and; 2. 37°C for E. coli, S. Enteritidis, MRSA, P. putida and P. aeruginosa. Five mL of the bacterial suspensions were mixed with 5 mL of the growth media and were used as controls. Viable cell counts of the above incubated test samples and controls were determined by serial dilution in PBS and plated onto MHA at regular intervals. The results were expressed in terms of log CFU vs. time

for each isolate and each extract.

Statistical Analysis All tests were done three times to establish statistical significance. Statistical analysis was performed using JMP 8.0 provided by University of Arkansas, Fayetteville. The results were considered statistically significant with p<0.001.

RESULTS AND DISCUSSION Antibacterial activity of lemon Citrus limon contains approximately 5 to 6% citric acid and the pH of the juice is 2 to 3. The lemon peel is rich in essential oils. The lemon extract used for this study contained both the juice and the essential oil. The extract killed all the bacteria within 24

Figure 1. Mean Log CFU vs. time of five different extracts against E. coli O157:H7. Lemon extract was the most effective of the extracts killing the bacteria within 24 h of incubation (p<0.001) 10 9 8

Mean Log CFU

7 Lemon extract

Turmeric 5

Green Tea White Tea

Silver Needle tea

3 2 1 0 00h

01h

06h

12h

24h

Time 212 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

36h

48h

Table 1. Antibacterial activity of twelve different plant extracts against four major foodborne bacterial pathogens and two spoilage bacteria. PLANT

C. ejuni 81176

C. jejuni PRCC

C. jejuni POCC

C. jejuni RECC

E.coli

S.Enteritidis

MRSA

+++

Turmeric

+++

Black tea

Emerald Princess tea

Sweet Fruit Garden tea

Rooibos tea

Mint tea

White tea

Green tea

Silver Needle tea

Oolong tea

Rose of Suzhou tea

EXTRACTS

Lemon

P. aeruginosa P. putida

Legend: +++ indicates that the extract killed the bacteria within 24 h of incubation; ++ indicates death within 36 h of incubation; + indicates bacteria were killed only after 48 h of incubation while – indicates that the bacteria were not killed even after 48 h of incubation. PRCC-pre chilled chicken carcass; POCC-post chilled chicken carcass; RECC-retail chicken carcass; MRSA-methycillin resistant S. aureus.

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Figure 2. Mean Log CFU vs. time of plant extracts against poultry strain of C. jejuni RECC (retail chicken carcass). Green tea was more effective than white tea killing the strain within 36 h of incubation. White tea killed the bacteria only after 48 h of incubation (p<0.001) 11 10 9 8

Mean Log CFU

7 Lemon extract

Turmeric Green Tea

White Tea Silver Needle tea

4 3 2 1 0 00h

01h

06h

12h

24h

36h

48h

Time

h of incubation (Table 1). E. coli O157:H7 (Figure 1) was the most susceptible to the extract followed by MRSA, Campylobacter jejuni, Salmonella Enteritidis, P. aeruginosa and P. putida. Previous experiments by Fischer and Phillips (2006) have shown that the zone of inhibition of commercially available lemon essential oils is greater against Staphylococcus aureus than E. coli O157:H7 and Campylobacter jejuni. The difference in this study is probably either due to a difference in the strains used or because the current study uses lemon juice, rich in citric acid, in addition to lemon essential oils. In our study, C. jejuni and MRSA showed approximately a 2-log reduction after 1h of incubation, while E. coli O157:H7 showed a 3–log reduction. Among C. jejuni strains, the RECC isolate showed a greater susceptibility than POCC strain, with a 5-log reduction after 6 hours as com-

pared to 3-log reduction after 6 h of incubation, respectively. Salmonella Enteritidis also showed a 3-log reduction after 1 h of incubation which was similar to the results reported by Xiong and coworkers (1999). Furthermore, lemon extract killed both spoilage bacteria P. aeruginosa and P. putida within 24 h on incubation. P. aeruginosa and P. putida also showed a 6–log decrease in growth within 1 h of incubation with lemon extract. These results were similar to those by Adedeji and coworkers (2007) who tested the effect of lemon and lime juice on clinical isolates of P. aeruginosa. Another study also reported that lemon essential oils showed significant antibacterial activity against Pseudomonas (Prabuseenivasan et al., 2006).

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Figure 3. Mean Log CFU vs. time S. Enteritidis with various tea extracts. Rooibos and mint tea extracts killed the isolate within 36 hours of incubation while oolong and green tea extracts killed the isolates after 48 hours of incubation. (p<0.001) 11 10 9 8

Mean Log CFU

7 Rooibos Tea

Mint Tea Green Tea

Oolong Tea 4

Silver Needle tea

3 2 1 0 00h

01h

06h

12h

24h

36h

48h

Time

Antibacterial activity of different types of teas Teas are aromatic beverages containing large amounts of flavonoids, which are known antioxidants and antibacterial agents. Green, white, oolong, black and herbal teas were tested for their antibacterial activity against different bacteria. Green and white teas showed highest antibacterial effects probably because green and white teas contain the largest amount of flavonoids among the varieties of teas (Table 1). White tea killed all the C. jejuni isolates except C. jejuni 81176 in 36 h, while black tea killed all the bacteria only after 48 h of incubation. C. jejuni POCC and RECC isolates showed a 4-log decrease with green and white teas (Figure 2) after 12 h incubation. Lee and coworkers (2009) reported that

green teas have potential anti-adhesive properties. In an experiment conducted in-vitro with human and mouse epithelial cell lines, it was shown that green tea extracts inhibited adhesion of S. aureus and Helicobacter pylori but not E. coli. In another study by Cho and coworkers (2008), there was a synergistic effect between green tea and antibiotics. This combination was highly effective against various strains of MRSA. As shown in Figure 3, specialty teas including rooibos and mint killed S. Enteritidis and C. jejuni, E. coli, and MRSA isolates (data not shown) within 36 hours of incubation. Rooibos tea showed a 5-log decrease within 12 h of incubation, while mint tea showed 4-log decrease within 12 h of incubation with S. Enteritidis. In addition, with rooibos and oolong teas all bacteria showed an initial decrease of approximately 2-log CFU after 1 h of incubation and

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Figure 4. Mean Log CFU vs. time with C. jejuni human strain 81176. Turmeric was effective against 81176 and killed C. jejuni within 36 h of incubation. (p<0.001)

11 10 9 8

Mean Log CFU

7 Lemon extract

Turmeric Green Tea

White Tea Silver Needle tea

4 3 2 1

0 00h

01h

06h

12h

24h

36h

48h

Time

rooibos tea killed all the bacteria within 36 h, while Emerald Princess showed a 1-log decrease within 1 h of incubation and killed all the bacteria within 48 h. However, with Rose of Suzhou, Sweet Fruit Garden and Silver Needle after an initial decrease in growth, a 1-log increase in growth of S. Enteritidis was seen between 24 h and 36 h of incubation. With spoilage bacteria, green tea did not kill P. aeruginosa and P. putida strains even after 48 h of incubation, but showed a 9–log reduction in count after 48 h. These findings were consistent with those of Vandeputte and coworkers (2010). Although many studies have reported that green tea and green

To evaluate boiling as a method of extraction, green, oolong, Silver Needle and Rose of Suzhou teas were boiled and their antibacterial activities were compared (Table 2). Silver Needle tea, a type of white tea, showed little antibacterial activity in the current study, until the boiling extraction method was used. It was found that boiling significantly increased the antibacterial efficacy of Silver Needle tea and resulted in a 4-log reduction in growth of P. putida after 6 h of incubation and killed all the bacteria within 48 h of incubation. It was also found that boiling increases the efficacy of antibacterial effect of green tea which killed all the isolates within 24 h of incuba-

tea catechins possess bacteriostatic effects against Pseudomonas species, there have been other reports which indicated little or no effect (Vandeputte et al., 2010; Yi et al., 2010).

tion, while green tea extracted without boiling killed the isolates only after 36 h. While boiled green tea extract showed a 6-log reduction in growth within 12 h of incubation, green tea extracted without boiling showed only a 4-log reduction with E. coli O157:H7.

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Table 2. Comparison of antibacterial activity of different tea extracts with and without boiling against four major foodborne bacterial pathogens and two spoilage bacteria.

Plant Extract

Green tea

C. jejuni C. jejuni 81176 PRCC

C. jejuni POCC

C. jejuni RECC

E. coli O157:H7

S. Enteritidis

MRSA

P. aeruginosa

P. putida

Boiled Green tea

+++

Silver Needle

Boiled Silver Needle

Sweet Fruit Garden

Boiled Sweet Fruit Garden

Rose of Suzhou

Boiled Rose of Suzhou

+++ indicates that the extract killed the bacteria within 24 h of incubation; ++ indicates death within 36 h of incubation; + indicates bacteria were killed only after 48 h of incubation while – indicates that the bacteria were not killed even after 48 h of incubation. PRCC-pre chilled chicken carcass; POCC-post chilled chicken carcass; RECC-retail chicken carcass; MRSAmethycillin resistant S. aureus.

Efficacy of Turmeric Turmeric was found to be the most bactericidal against MRSA followed by S. Enteritidis, C. jejuni and, finally, E. coli O157:H7 (Table 1). Turmeric completely killed all the bacteria within 36 h of incubation with a 2–log decrease within 6 h after incubation with C. jejuni 81176. C. jejuni 81176 (Figure 4) showed maximum sensitivity, while C. jejuni PRCC showed least sensitivity to turmeric. Tajbhaksh and coworkers (2008) also reported similar results using curcumin, indium curcumin, indium diacetylcurcum-

With respect to P. aeruginosa (Figure 5) and P. putida (Figure 6), turmeric was able to produce approximately a 5–log decrease after 24 h of incubation with P. aeruginosa, and killed P. putida within 24 h. These results were also similar to Tajbaksh and coworkers (2008), who reported that indium curcumin and indium diacetylcurcumin was effective against P. aeruginosa with a minimum inhibitory concentration of 23.4 mg/mL. Another recent study also reported similar results, where curcumin nanoparticles were tested for their antibacterial efficacy against Pseudomonas. The same study also showed that curcumin

in, and diacetyl curcumin against S. aureus and E. coli. Another recent study by Moghaddam and coworkers (2009) showed that curcumin when coupled with antibiotics was more effective against S. aureus than curcumin alone.

nanoparticles inhibited 80% of P. aeruginosa while curcumin alone inhibited only 60%. They went on to prove that curcumin nanoparticles were more effective than curcumin itself since they are more soluble in water (Bhawana et al., 2011). These studies

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Figure 5. Mean Log CFU vs. time with P. aeruginosa. Lemon extract was found to be effective against P. aeruginosa killing the isolate within 24 h of incubation. White tea killed the organism within 48 h of incubation, while turmeric and boiled Silver Needle tea failed to kill even after 48 h. (p<0.001)

12 11 10 9

Mean Log CFU

8 7

Lemon extract Turmeric

White tea

Silver Needle tea Boiled Silver Needle tea

4 3 2 1 0 00h

01h

06h

24h

48h

Time

showed turmeric could be used as an antibacterial agent worldwide. Curcumin the active ingredient in turmeric is a polyphenolic compound with many therapeutic uses including, but not limited to, potential anti-HIV, anti-tumor, anti-amyloid and antiinflammatory properties, in addition to being antimicrobial.

CONCLUSIONS

the bacteria within 24 h of incubation, with a 5-log decrease in count of C. jejuni strains after 6 h of incubation and a 6-log reduction of P. putida, followed by green tea extract, white tea extract, turmeric extract and black tea extract. However, three specialty teas including Rose of Suzhou, Sweet Fruit Garden and Silver Needle teas were ineffective against all the organisms and failed to kill the bacteria even after 48h of incubation. When the teas were boiled to extract flavonoids and the extraction methods were

A total of 12 extracts were tested against four different bacterial foodborne pathogens and two food spoilage bacteria. Of these plant extracts, lemon extract was found to be the most effective and killed all

compared, it was found that Silver Needle tea killed all the isolates except P. aeruginosa within 48 h of incubation with a 3-log reduction in growth of C. jejuni, E. coli and S. Enteritidis and a 4-log reduction of P. putida isolates within 6 h of incubation. It was also

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Figure 6. Mean Log CFU vs. time with P. putida. Lemon proved to be the most effective against P. putida. Although Silver Needle tea proved to be the least effective and failed to kill the bacteria even after 48 h of incubation; turmeric and white tea were found to be quite effective, killing the isolate within 24 h and 48 h respectively. (p<0.001)

11 10 9 8

Mean Log CFU

Lemon extract Turmeric

Green tea 5

White tea

Silver Needle tea

Boiled Silver Needle tea 3

2 1 0 00h

01h

06h

24h

48h

Time

found that boiling increased the antibacterial efficiency of green tea extract which killed all the bacterial isolates within 24 h of incubation as compared to 36 h with green tea extracts without boiling. Based on this research, it can be concluded that plant extracts have great potential to be used as effective antibacterial agents against foodborne pathogens and spoilage bacteria.

Afr. J. Biomed. Res. 10:183–187. Bancirova, M. 2010. Comparison of the antioxidant capacity and the antimicrobial activity of black and green tea. Food Res. Int. 43:1379-1382. Bhawana, R., K. Basniwal, H. S. Buttar, V. K. Jain, and N. Jain. 2011. Curcumin nanoparticles: Preparation, characterization, and antimicrobial study. J. Agric. Food Chem. 59:2056-2061. Cho, Y. S., N. L. Schiller, and K. H. Oh. 2008. Antibacterial effects of green tea polyphenols on clinical

REFERENCES

isolates of methicillin-resistant Staphylococcus aureus. Curr. Microbiol. 57:542-546. Chou, C. C., L. L. Lin, and K. T. Chung. 1999. Antimicrobial activity of tea as affected by the degree of fermentation and manufacturing season. Int. J.

Adedeji, G. B., O. E. Fagade, and A. A. Oyelade. 2007. Prevalence of Pseudomonas aeruginosa in clinical samples and its sensitivity to citrus extract.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

219

Food Microbiol. 48:125-130. Conte, A., B. Speranza, M. Sinigaglia, and M. A. Del Nobile. 2007. Effect of lemon extract on foodborne microorganisms. J. Food Prot. 70:1896-1900. Dahanukar, S.A., R. A. Kulkarni, and N. N. Rege. 2000. Pharmacology of medicinal plants and natural products. Indian J. Pharmacol. 32:S8-S118. Diker, K. S., M. Akan, G. Hascelik, and M. Yurdakok. 1991. The bactericidal activity of tea against Campylobacter jejuni and Campylobacter coli. Lett. Appl. Microbiol. 12:34-35. Fisher, K., and C. A. Phillips. 2006. The effect of lemon, orange and bergamot essential oils and their components on the survival of Campylobacter

ucts from medicinal plants. Gomal J. Med. Sci. 7:72-78. Sirsat, S. A., A. Muthaiyan, and S. C. Ricke. 2009. Antimicrobials for foodborne pathogen reduction in organic and natural poultry production. J. Appl. Poult. Res. 18:379-338. Srinivas, K., J. W. King, J. K. Monrad, L. R. Howard, and C. M. Hansen. 2009. Optimization of subcritical fluid extraction of bioactive compounds using Hansen solubility parameters. J. Food Sci. 74: E342-54. Srinivas, K., J. W. King, L. R. Howard, and J. K. Monrad. 2010. Solubility and solution thermodynamic properties of quercetin and quercetin dihydrate in

jejuni, Escherichia coli O157, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus in vitro and in food systems. J. Appl. Microbiol. 101:1232-1240. Jayaraman, P., M. K. Sakharkar, C. S. Lim, T. H. Tang, and K. R. Sakharkar. 2010. Activity and interactions of antibiotic and phytochemical combinations against Pseudomonas aeruginosa in vitro. Int. J. Biol. Sci. 6:556-568. Lee, J. H., J. S. Shim, M. S. Chung, S. T. Lim, and K. H. Kim. 2009. In vitro anti-adhesive activity of green tea extract against pathogen adhesion. Phytother. Res. 23:460-466. Moghaddam, K.M., M. Iranshahi, M. C. Yazdi, and A. R. Shahverdi. 2009. The combination effect of curcumin with different antibiotics against Staphylococcus aureus. Int. J. Green Pharm. AprJun:141-143. Naz, S., S. Jabeen, S. Iliyas, F. Manzoor, F. Aslam, and A. Ali. 2010. Antibacterial activity of Curcuma longa varieties against different strains of bacteria. Pak. J. Bot. 42:455-462. Prabuseenivasan, S., M. Jayakumar, and S. Ignacimuthu. 2006. In vitro antibacterial activity of some plant essential oils. BMC Complement. Altern. Med. 6:39.

subcritical water. J. Food Eng. 100:208-218. Tajbakhsh, S., K. Mohammadi, I. Deilami, K. Zandi, M. Fouladvand, E. Ramedani, and G. Asayesh. 2008. Antibacterial activity of Indium curcumin and Indium diacetylcurcumin. Afr. J. Biotechnol. 7:3832-3835. Valtierra- Rodríguez, D., N. L. Heredia, S. Garcia, and E. Sanchez. 2010. Reduction of Campylobacter jejuni and Campylobacter coli in poultry skin by fruit extracts. J.Food Prot. 73:477-482. Vandeputte, O. M., M. Kiendrebeogo, S. Rajaonson, B. Diallo, A. Mol, M. El Jaziri, and M. Baucher. 2010. Identification of catechin as one of the flavonoids from Combretum albiflorum bark extract that reduces the production of quorum-sensing-controlled virulence factors in Pseudomonas aeruginosa PAO1. Appl. Environ. Microbiol. 76:243-253. Weerasekera, D., N. Fernando, L. B. A. E. Bogahawatta, R. Rajapakse-Mallikahewa, and D. J. Naulla. 2008. Bactericidal effect of selected spices, medicinal plants and tea on Helicobacter pylori strains from Sri Lanka. J. Natn. Sci. Foundation Sri Lanka. 36:91-94. Wenjiao, F., C. Yuanlong, and Z. Shuo. 2008. The use of a tea polyphenol dip to extend the shelf life of silver carp (Hypophthalmicthys molitrix) during

Rai, D., J. K. Singh, N. Roy, and D. Panda. 2008. Curcumin inhibits FtsZ assembly: An attractive mechanism for its antibacterial activity. Biochem. J. 410:147-155. Sher, A. 2009. Antimicrobial activity of natural prod-

storage in ice. Food Chem. 108:148–153. Wiseman, S. A., D. A. Balentine, and B. Frei. 1997. Antioxidants in tea. Crit. Rev. Food Sci. Nutr. 37:705-718. Xiong, R., G. Xie, and A. S. Edmondson. 1999. The

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fate of Salmonella enteritidis PT4 in home-made mayonnaise prepared with citric acid. Lett. Appl. Microbiol. 28:36-40. Yi, S. M., J. L. Zhu, L. L. Fu, and J. R. Li. 2010. Tea polyphenols inhibit Pseudomonas aeruginosa through damage to the cell membrane. Int. J. Food Microbiol. 144:111-117.

APPENDIX - DIFFERENT VARIETIES OF TEAS USED IN THE STUDY AND THEIR SCIENTIFIC NAMES

S.No Tea Varieties

Scientific Name

Composition

Green Tea

Camellia sinensis

Fresh tea leaf → Withering → Curling → minimal Oxidation → Drying → Green tea

Black Tea

Camellia sinensis

Fresh tea leaf→ Withering → Curling → complete Oxidation → Drying → Black tea

White Tea

Camellia sinensis

Fresh tea leaf → Withering → Drying (air drying, solar drying or mechanical drying) → White tea

Silver Needle Tea

Camellia sinensis

It is a type of white tea. For the production of Silver Needle, only the leaf shoots, i.e. the leaf buds before opening, are plucked, and the buds undergo light oxidation – hence classified as white tea.

Rooibos Tea

This tea is usually classified as ‘black’ tea since it undergoes complete oxidation. Recently ‘green’ vaAspalathus linearis rieties of Rooibos are produced with partial oxidative processing.

Mint Tea

Camellia sinensis

Herbal tea infused with peppermint, Mentha piperita.

Oolong Tea

Camellia sinensis

Traditional Chinese tea produced through a unique process including withering under the strong sun and oxidation before curling and twisting.

Enerald Princess Tea

Camellia sinensis

A blend of green tea with pineapple, citrus pieces, coconut, cornflowers and rose petals.

Rose of Suzhou Tea

Camellia sinensis

This is a Suzhou (the Venice of China) white tea variety comprised of jasmine flowers, marigold blossoms and a globe amaranth flower.

10.

Sweet Fruit Garden Tea Herbal tea

Herbal tea with sweet and sour Morello cherries & tart raspberries and a hint of hibiscus, apple, pineapple and papaya.

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Prevalence of Foodborne Pathogens and Effectiveness of Washing or Cooking in Reducing Microbiological Risk of Contaminated Red Amaranth Md. Arafat Al Mamun1, Hasina Akther Simul2, Asma Rahman3, N. N. Gazi2, and Md. Latiful Bari1 Food Analysis and Research Laboratory 2 Environmental Chemistry laboratory, 3 Drug Laboratory, Center for Advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh 1

ABSTRACT Red amaranth (lal shak) is one of the main vegetables consumed extensively in Bangladesh. This study was conducted to monitor the prevalence of microorganisms, heavy metal contents and vitamins in raw red-amaranth and the impact of cooking on microorganisms, heavy metal contents and vitamins of red amaranth. The averaged viable bacterial load and coliform count in raw amaranth samples was recorded as > log 8.0 CFU/g, and approximately 7.0 log CFU/g, respectively throughout this study. Higher prevalence of pathogenic microorganisms such as Salmonella spp, Escherichia coli, Listeria spp. and Yersinia spp. were recorded in raw red amaranth samples. Washing raw amaranth samples with tap water removed some soil and other debris, but did not reduce the bacterial load. However, washing these vegetables with scallop powder followed by a distilled water wash could reduce 1.0-1.5 log CFU/g of viable bacterial load and coliform bacterial load. Washing these vegetables with 200 ppm chlorine water was able to reduce additional 2.0 log CFU/g. However, cooking these vegetables at a boiling temperature (90°C, for 15 min) did reduce by more than 5.0 log CFU/g the viable bacterial load but was unable to inactivate them. In contrast, cooking successfully eliminated coliform and all the pathogenic microorganisms including Salmonella spp., E. coli, Listeria spp. and Yersinia spp. from all the samples tested. In addition, cooking reduced almost 90% vitamin-C content from 14.2 mg/100 g to 1.5 mg/100 g. In contrast, some metal (Ca, Fe and Zn) contents were increased significantly after cooking. Therefore, these study results demonstrated that cooking could reduce the microbiological risk of red amaranth and improve food safety for human consumption. However, cooking reduced the vitamin-C content and increased some of the metal contents, coming from the supply water. Keywords: Prevalence pathogens, Red amaranth, microbial risk, scallop powder, washing and cooking Agric. Food Anal. Bacteriol. 2: 222-231, 2012

Correspondence: Md. Latiful Bari, latiful@univdhaka.edu Tel: 8802-9661920-59 Ext 4721 Fax: 8802-8615583

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INTRODUCTION Amaranth (Amaranthus tricolor L.) plays an important role in nutrition among the leafy vegetables grown in Bangladesh. Among the leafy types, Amaranthus tricolor L. is the most commonly cultivated species in Bangladesh. It is cultivated all over the country in any season due to its adaptability to a wide range of soil and climate (Alam et al., 2007). Red amaranth (lal shak) is grown in homestead gardens using indigenous technologies and ground water is often used for irrigation and is subsequently harvested and sold in local markets without any processing steps between harvest and market. Consumers

with other disinfectants is partially effective in removing disease-causing microorganisms from the surface of raw fruits and vegetables (Beuchat et al., 2001). Washing produce with sodium-chlorinated water (NaClO) is the most commonly used method to remove pathogens from fruit and vegetable surfaces. The active hypochlorite is believed to lose its effectiveness by reacting with nitrogen-containing compounds in foods, resulting in halogenated organic compounds (Wei et al., 1985). Concern over the carcinogenicity and toxicity of these compounds, particularly trihalomethanes, has prompted consideration of alternative disinfectants. Inatsu et al. (2005) reported that acidified sodium chloride solution could be useful as a

buy these vegetables from local markets, bring them home along with other vegetables in a basket, wash with tap water, cut into pieces and cooked or stir fry with spices and consume. During the past decade, increased frequency of fresh produce associated outbreaks was reported. E. coli O157:H7, Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica is of particular food safety concern, because they are widespread in the environment (IFT, 2004), grow under refrigeration conditions (ILSI, 2005), and are frequent residents in certain food processing establishments (Brandl, 2006). These microorganisms have been isolated from soil, sewage sludge, vegetation, and water (NACMCF, 1999) and, therefore, have the potential to contaminate produce surfaces. Many vegetables, including bean sprouts, cabbage, cucumber, potatoes, and radishes, have been reported to be contaminated with pathogenic microorganisms (Beuchat, 1996). The pathogen has been reported to survive longterm storage on leafy vegetables, has been responsible for numerous product recalls of salads (Wong et al., 2000), and was identified as being responsible for an outbreak of foodborne disease due to coleslaw prepared from contaminated raw cabbage. Many sanitizers have been evaluated for their ef-

sanitizer for surface washing of fresh produce. In this study scallop powder-a new biodegradable and natural alkaline sanitizer was used to see its effectiveness in reducing microorganisms on the produce surface. The level of sanitation and the populations of microorganisms are of primary importance to the quality, shelf stability, and safety of fresh produce (NACMCF, 1999). Based on a comprehensive survey produce outbreaks accounted for 13% (713/5,416) of total outbreaks and 21% (34,049/161,089) of associated illnesses from 1990 through 2005, according to data from the Center for Science in the Public Interest (CSPI) (Smith DeWaal and Bhuiya, 2009). However, in Bangladesh, unlike other developing countries, such databases for fresh produce do not exist. Outbreak data is necessary to improve the hazard analysis for various food commodities, and to ensure produce safety. Nevertheless arsenic contamination of groundwater for irrigation in the homestead gardens is a widely based concern (Rahman and Hasan, 2007). Therefore, use of this contaminated water during vegetable production, washing and cooking may introduce arsenic in the final product. In addition, as there is a lack of hygiene education in the rural population, therefore, there are frequent opportunities for microbial con-

fectiveness in killing pathogenic microorganisms on different produce (Beuchat et al., 2001). Chlorine as sodium or potassium salt and calcium hypochlorite commonly has been used to eliminate microorganisms from food surfaces. Treatment of raw produce

tamination of pond and well water. Use of this water in any stage of vegetables chain may contribute to contamination of the final products. Based on the above mentioned facts this study was designed: 1) to determine the prevalence of mi-

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croorganisms and pathogens in raw red amaranth 2) to determine the effectiveness of scallop powder and widely used chlorinated water for controlling the natural microflora and environmental foodborne pathogens including Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7, Yersinia enterocolitica in raw red amaranth; 3) to determine the effectiveness of cooking in reducing microbiological risk of heavily contaminated market Red amaranth; and 4) to determine the heavy metal content and vitamin-C in fresh and cooked red amaranths.

MATERIALS AND METHODS Samples collection Commercial red amaranth samples were purchased from the Kawranbazar market of Dhaka city, Bangladesh and were used within 24 h of collection. Raw vegetable samples were collected aseptically in sterile polyethylene bags and transported to the laboratory. Cracked or dirty red amaranth samples were discarded.

perforated tray to drain off the excessive water and placed in laminar flow bio-safety cabinet to facilitate drying for 2 hours.

Cooking of samples and cooling down to room temperature As the consumer will usually cook or stir fry the red amaranth samples with spices and subsequently consumed. Therefore, an experiment was designed to see the effectiveness of cooking on microorganisms. Washed or non-washed commercial red amaranths samples were boiled at approximately 90°C for 15 minutes in a bowl and after boiling, the red amaranth samples were placed on a sterile perforated tray to drain off the excessive water and placed in laminar flow bio-safety cabinet to facilitate cooling down to room temperature.

Sample processing and microbiological analysis

Scallop powder solutions and chlorine water were used as washing solutions and were prepared immediately prior to application. The final concentration of scallop powder solutions was adjusted to 0.01% (w/v) in deionized water. The chlorine solution was prepared using sodium hypochlorite (Wako Chemical Co. Ltd, Osaka, Japan) solution to distilled water (v/v), and concentration was adjusted to 200 ppm. Distilled water (DW) was used for control experiments. Tap water/supply water was also used to wash raw red amaranths. For each experimental condition, 50 g of samples was washed with 250 mL of the washing solution in a sterile beaker (1 L). Wash-

After washing and/or cooking, the 25 g red amaranth samples were placed in a stomacher bag with 225 mL of sterile saline water. The mixture was pummeled for 60 s and serial decimal dilutions were prepared with sterile saline water. The diluted and undiluted samples (0.1 mL) were then surface plated on both selective and nonselective agar media. Tryptic soy agar (TSA; Oxoid) was used as a nonselective media for determination of viable cells number. Coliform agar; Sorbitol MacConkey agar (SMAC) supplemented with cefixime (0.05 mg/liter) and potassium tellurite (2.5 mg/liter) (CT23 selective supplement, Oxoid); Bismuth sulfite agar (BSA; Oxoid); Listeria selective agar supplemented with SR0227E and Yersinia selective agar supplemented with Yersinia Selective Supplement were used as the selective media for the determination of coliform bacteria, E. coli O157:H7, Salmonella spp., L. monocytogenes and

ing was carried out for 5 min at room temperature with gentle agitation using a glass rod. After washing, the solutions were decanted, and the samples were rinsed with 250 mL of distilled water. After that the red amaranth samples were placed in a sterile

Y. enterocolitica respectively, in the red amaranths samples. All the plates were then incubated at 37°C for 24 to 48 h and counted. After incubation from the respective selective media at least five presumptive

Washing solution and sample washing

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colonies of Salmonella, E. coli O157:H7, L. monocytogenes and Y. enterocolitica were subjected to confirmation tests using a direct immunoassay test kit (Universal Health Watch, Columbia, MD, USA and/ or API diagnostic Kits, Oxoid, UK). All experiments were repeated five times to confirm the reproducibility.

Red amaranth samples were brought from a local market. The samples were washed with water and half of the samples were subsequently cooked and

uncooked samples were then freeze dried and kept at room temperature until use. Freeze Dryer (Ilshin Lab Co, Ltd. Seoul, Korea), Microwave oven (CEM Corporation, NC, USA, Model Marss Express), and microwave Teflon sealed vessels were used. A Perkin Elmer Atomic Absorption Spectrophotometer (Model A Analyst 800; Illinois, USA) was used for metal analysis in red amaranth vegetables. Digested samples were mechanically ground and weighted approximately 0.125 g into a Teflon vessel; 10 mL of concentrated nitric acid (Sigma pure chemical Industries, ltd, USA) was added and digested in a microwave oven. The condition of microwave digestion was: powder 800W; rate 10 min; temp 100°C;

the excess water was drained off. The cooked and

time 10 min and 800W; 10 min; 200°C for 10 min.

Sample preparation, Metal Analysis

Digestion

and

Table 1. Prevalence of viable bacteria, coliform bacteria, and other foodborne pathogens in preand post-washing / cooking Red amaranths.

Population (log CFU/g) a

Treatment/conditions

Total viable Total coliform bacteria bacteria

E. coli

Salmonella spp.

Listeria spp.

Yersinia spp.

1. No treatment

8.7 ± 0.5C

6.9 ± 0.1A

4.6 ± 0.2A

5.8 ± 0.2A

5.7 ± 0.1A

2. Washing with tap water follow

8.5 ± 0.1A

6.1 ± 0.4B

4.0 ± 0.1A

5.7 ± 0.3B

5.9 ± 0.3B

5.7 ± 0.1A

3. Dipped in scallop powder water for 5 min with periodic stirrer

7.6 ± 0.2A

5.5 ± 0.2A

3.1 ± 0.1A

4.9 ± 0.1A

4.3 ± 0. 2A

4.9 ± 0.1A

4. Dipped in Scallop Powder Water followed by a 2nd wash with DW

7.4 ± 0.3B

4.9 ± 0.1A

3.1 ± 0.1A

4.8 ± 0.1A

4.4 ± 0.1A

4.9 ± 0.2B

5. Washing with 200 ppm chlorine water for 5 min

6.4 ± 0.2B

5.1 ± 0.1A

3.3 ± 0.2A

4.8 ± 0.2A

3.27 ± 0.1A

4.9 ± 0.1A

6. Washing with 200 ppm chlorine water followed by 2nd wash with DW

6.2 ± 0.3C

5.1 ± 0.4B

3.2 ± 0.3B

4.7 ± 0.3B

BDL

4.5 ± 0.1A

7. Boiling and Cooling

3.2 ± 0.5C

n=10 for each data, count= Average of ‘∑n’ ± SD; BDL= Below Detection limit. The detection limit was < 1.0 log CFU/g. The mean values in columns with different letters are significantly (P < 0.05) different, while mean values with the same letter are not significantly different a

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After digestion the content of Teflon vessel was dissolved in de-ionized water and filtered into 25 mL volumetric flask quantitatively and brought up to the mark with de-ionized water. The digested sample solutions were subsequently analyzed for the metals Ca, Cd, Cu, Fe, Mn, Na, Pb and Zn by an automatic sampler and analyzed by using an air acetylene flame in combination with single element hollow cathode lamps into an atomic absorption spectrophotometer, having a detection limit as described in Table 2, respectively. However, the arsenic (As) was analyzed by GF-AAS (Graphite Furnace Atomic Absorption Spectrophotometer; Perkin Elmer, Illinois, USA) with electrode less discharge lamp of having a detection

ASEAN Manual of Nutrient Analysis, 2011 and Lakshanasomya, 1998 as described below:

limit 0.5 μg/L of arsenic. The wavelength, correlation coefficient and detection limit of each metal is listed in Table 1.

phase: 0.3 mM potassium dihydrogen phosphate in 0.35% (v/v) ortho- phosphoric acid, Diluent: 3% metaphosphoric acid, Flow rate: 0.5 mL/min., Detection: 248 nm., Injector: 20 μL.

Instrumentation and Chromatographic conditions A Prominence HPLC system (Shimadzu Scientific Instrument, Tokyo, Japan) equipped with two pumps (LC-20AD), an auto sampler (SIL 20AC HT), UV-Visible detector (SPD 20A), column oven (CTO-20AC) and communication bus module (CBM 20A) was used for this analysis. The data was processed using LC- solutions software. Stationary phase: Analytical reversed phase C-18, Luna 5μ, 250 x 4.6 mm, Phenomenex, Inc., Mobile

Determination of Vitamin C by HPLC The cooked and fresh Red Amaranth samples were analyzed for vitamin-C (ascorbic acid) content using High Performance Liquid Chromatography (HPLC) according to the method as described in the

Preparation of standard solutions and method calibration The stock solution of L-ascorbic acid was prepared by diluents having a concentration of 1.0 mg/ mL. The appropriate volume from this stock solution

Table 2. The wavelength, correlation coefficient and detection limit of each metal Wavelength

Linear Range

Detection Limit

(mg/ L)

Correlation Coefficient

( hollow Cathode Lamp)

(mg/ L)

422.7

0-5.0

0.997050

0.10

228.8

0-2.0

0.999616

0.03

324.8

0-5.0

0.998286

0.02

248.3

0-6.0

0.999012

0.02

279.5

0-2.0

0.999877

0.05

589.0

0-1.0

0.997632

0.01

283.3

0-20.0

0.999520

0.45

213.9

0-1.0

0.999461

0.02

Metals

226 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

was further diluted with the same diluent to prepare standards 60, 70, 80, 90 and 100 μg/mL of ascorbic acid. Each of the five different concentrations mentioned previously were injected for three times to obtain average peak area. The average peak areas were plotted against concentrations to construct a linear curve from which the correlation coefficients, slopes and interception values were calculated (Figure 1A). Preparation of sample solution Fresh or cooked samples (2.5 g) were pulverized with 3% meta phosphoric acid in a 200 mL volumetric flask and vortexed for 5 minutes to uniformly mix and the volume was adjusted to 100 mL with same diluent. The contents were subsequently filtered through a 0.45 μm membrane and injected into the system. The chromatogram of standard, fresh and boiled Red amaranth is shown in Figure 1 (B, C, D), respectively.

pathogenic bacteria. Washing raw amaranth samples with tap water removed some soil and other debris, but did not reduce the bacterial load. However, washing these vegetables with scallop powder followed by a distilled water wash did reduce by 1.0 to 1.5 log CFU/g of the viable bacterial load and coliform bacterial load. However, washing these vegetables with 200 ppm chlorine water reduced by an additional 2.0 log CFU/g the viable bacterial load and coliform count. However, scallop powder and 200 ppm chlorine water was unable to inactivate pathogenic bacteria. Vegetables can act as a vector for transporting pathogenic bacteria from the farm (Beuchat, 2001).

The averaged viable bacterial loads and coliform counts in the raw amaranth samples were recovered at greater than log 8.0 CFU/g, and approximately 7.0 log CFU/g, respectively throughout this study.

Although washing produce with tap water may remove some soil and other debris, it cannot be relied upon to remove microorganisms and may result in cross-contamination of food preparation surfaces, utensils, and other food items (Bari et al., 2005). To reduce the risk of food poisoning caused by contaminated vegetables, effective sanitation of the raw produce is required. Washing raw produce with water containing sodium hypochlorite (NaClO) is the most commonly used method for removing pathogens from the surfaces of vegetables (Wei et al., 1985). In this study, 200 ppm of chlorinated water was used, which is the maximum limit permitted for washing of raw vegetables by the United States Food and Drug Administration (FDA). The active hypochlorite is believed to lose its effectiveness after reacting with nitrogen compounds in foods, resulting in halogenated organic compounds (Odabasi, 2008). Concern over the carcinogenicity and toxicity of these compounds, particularly of trihalomethanes, has prompted consideration of alternative disinfectants (Wei et al., 1985). In this study, scallop powder-a new biodegradable alkaline sanitizer is used. The inner portion of the scallop (Patinopecten yessoensis) shell was baked at 200˚C and then exposed to a heat treat-

Higher prevalence of pathogens such as Salmonella spp, E. coli, Listeria spp and Yersinia spp were observed in the raw red amaranth samples. This finding demonstrated that the red amaranth sold in the local market of Dhaka city is heavily contaminated with

ment of 1000°C, and then passed through a micro sieve to obtain 5 to 15 um particle size of powder. This powder said to have bactericidal action against Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and Bacillus subtilis (vegetative cells).

Statistical analysis All trials were replicated five times. Reported plate count data represented the mean values obtained from five 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.

RESULTS AND DISCUSSION

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Figure 1. A) Linearity of curve for standard ascorbic acid and HPLC chromatogram of B) standard ascorbic acid C) fresh red amaranth and D) cooked red amaranth.

12000000

y = 107449x + 460448 R² = 0.9907

Area

10000000 8000000 6000000 4000000 2000000 0 0

60 Concentration

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100

120

The bactericidal action is due to calcium oxide that is converted by heat treatment from calcium carbonate, which is the main component of the shell powder. As this powder is produced from natural sources these do not pose any hazard to the environment, and biodegradable (Sawai et al., 2001). However, cooking these vegetable at a boiling temperature (90Ë&#x161;C, for 15 min) was able to reduce by more than 5.0 log CFU/g of the viable bacterial load and coliform bacterial load but was unable to inactivate them. In contrast, cooking successfully eliminated all pathogens including Salmonella spp, E. coli, Listeria spp. and Yersinia spp. from all samples tested. The average vitamin C content in raw red amaranths was recorded as 14.2 mg/100g, and after cooking, the vitamin-C content was reduced significantly and recorded as 1.5 mg/100g, which is approximately 90% lower than from fresh samples (Table 3 and Figure 1). This finding suggested that the eating habit could lead to a lower intake of micronutrients even though microbiologically safe. In addition, as there is a lack of hygiene education in rural population, therefore, there is every chance of microbial contamination in any stage of food chain. In addition, lack of processing facilities after the harvest may also contribute to cross contamination of transport utensils, storages boxes, and finally

display utensils. Furthermore, consumer purchase these vegetables from local market, bring them home with other vegetables in a basket, and store them in the refrigerator (vegetable boxes) with other perishable vegetables, consequently cross-contaminating the entire process from farm to freeze. These production and storage practices may contribute to contaminating other foods in the refrigerator. To ensure the microbiological quality and safety of fresh vegetables, processing steps must be introduced to reduce the cross contamination during the entire process. In addition, good agricultural practices (GAP) must be introduced in the field/agricultural land when producing fresh vegetable intended for human consumption.

ACKNOWLEDGEMENTS This research work is a coordinated research of different laboratories of the Center for Advanced Research in Sciences (CARS), University of Dhaka. The authors express their sincere gratitude to Dr. Zakir Sultan for his advice and technical support on HPLC. The authors also express their sincere gratitude to Md. Ashraful Islam, Kanik Kumar Sharker, Md. Harun-or-rashid and Md. Abul-Kalam Azad for their technical and all-out support and cooperation

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Table 3. The metal and vitamin-C content of Red amaranth samples before and after cooking. Cooked sample (mg/100g)

Uncooked sample (mg/100g)

127.20

88.10

0.15

0.18

0.46

15.22

5.12

0.78

0.42

34.00

37.60

BDL*

2.26

1.60

0.002

0.001

Vit-C

1.5

14.2

Metals

Vitamin C

during this work.

Alam, M.N., M. S. Jahan, M. K. Ali, M. S. Islam, and S. M. A. T. Khandaker. 2007. Effect of Vermicompost and NPKS Fertilizers on Growth, Yield and Yield Components of Red Amaranth. Austr. J. Basic Appl. Sci. 1:706-716. ASEAN Manual of Nutrient Analysis 2011. Editors, P. Puwastien, T. E. Siong, J. Kantasubrata, G. Craven, R. R. Feliciano, K. Judprasong. ASEANFOODS published by Institute of Nutrition, Mahidol University, Thailand, 141-144. Bari, M. L., D. O. Ukuku, T. Kawasaki, Y. Inatsu, K. Is-

Prot. 68:1381-1387. Beuchat, L. R. 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59:204– 216. Beuchat, L. R., L. J. Harris, T. E. Ward, and T. M. Kajs. 2001. Development of a proposed standard method for assessing the efficacy of fresh produce sanitizers. J. Food Prot. 64:1103–9. Brandl, M. T. 2006. Fitness of Human Enteric Pathogens on Plants and Implications for Food Safety. Annual Review of Phytopathology 44: 367-392. IFT. 2004. Institute of Food Technologists: Scientific Status Summary. August 2004. Bacteria Associated with foodborne diseases, p. 1-25.

shiki and S. Kawamoto. 2005. Combined effcacy of nisin and pediocin with Sodium Lactate, Citric Acid, Phytic Acid and Potassium Sorbate and EDTA in Reducing Listeria monocytogenes Population of Inoculated Fresh-cut Produce. J. Food

Inatsu, Y., M. L. Bari, S. Kawasaki, K. Isshiki, and S. Kawamoto. 2005. Efficacy of acidified sodium chlorite treatments in reducing Escherichia coli O157:H7 on Chinese cabbage. J. Food Prot. 68:251-255.

REFERENCES

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ILSI. 2005. ILSI Research Foundation/Risk Science Institute. 2005. Achieving continuous improvement in reductions in foodborne listeriosis - A risk based approach. J. Food Prot. 68:1932-1994. Lakshanasomya, N. 1998. Determination on Vitamin C in Some Kinds of Food by HPLC. Bull. Dept. Med. Sci. 40:347-357. NACMCF (National Advisory Committee on Microbiological Criteria for Foods). 1999. Microbiological safety evaluation and recommendations on fresh produce. Food Control 10:117–143. Odabasi, M. 2008. Halogenated volatile organic compounds from the use of chlorine-bleach-containing household products. Environ. Sci. Technol. 42:1445-51. Rahman, I.M.M., and Hasan M.T. 2007. Arsenic Incorporation into Garden Vegetables Irrigated with Contaminated Water. J. Appl. Sci. Environ. Manage. 11:105–112. Sawai, J., M. Satoh, M. Horikawa, H. Shiga, and H. Kojima. 2001. Heated scallop-shell powder slurry treatment of shredded cabbage. J. Food Prot. 64:1579–1583. Smith DeWaal, C., and F. Bhuiya. 2009. Center for Science in the Public Interest (CSPI), Washington, DC, Available at http://www.cspinet.org/foodsafety/IAFPPoster.pdf accessed on 4th March 2012. Wei, C.-I., D. L. Cook, and J. R. Kirk. 1985. Use of chlorine compounds in the food industry. Food Technol. 39:107–115. Wong, S., D. Street, S. I. Delgado, and K. C. Klontz. 2000. Recalls of foods and cosmetics due to microbial contamination reported to the U.S. Food and Drug Administration. J. Food Prot. 63:1113–1116.

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INSTRUCTIONS TO AUTHORS MANUSCRIPT SUBMISSION

CONTENT OF MANUSCRIPT

Authors must submit their papers electronically (submit@afabjournal.com). According to instructions provided online at our site: www.afabjournal. com. Authors who are unable to submit electronically should contact the editorial office for assistance by email at editor@afabjournal.com.

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:

• • • • • • • • • • • • • • • •

Aerobic microbiology Aerobiology Anaerobic microbiology Analytical microbiology Animal microbiology Antibiotics Antimicrobials Bacteriophage Bioremediation Biotechnology Detection Environmental microbiology Feed microbiology Fermentation Food bacteriology Food control

• • • • • • • • • • • • • • • •

Foodborne pathogens Gastrointestinal microbiology Microbial education Microbial genetics Microbial physiology Modeling and microbial kinetics Natural products Phytoceuticals Quantitative microbiology Plant microbiology Plant pathogens Prebiotics Probiotics Rumen microbiology Rapid methods Toxins

• • •

Food microbiology Food quality Food Safety

• • •

Veterinary microbiology Waste microbiology Water microbiology

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With an open access publication model of this journal, all interested readers around the world can freely access articles online. AFAB publishes original papers including, but not limited to the types of manuscripts described in the following sections. Papers that have been, or are scheduled to be, published elsewhere should not be submitted and will not be reviewed. Opinions or views expressed in papers published by AFAB are those of the author(s) and do not necessarily represent the opinion of the AFAB or the editorial board.

MANUSCRIPT TYPES Full-Length Research Manuscripts AFAB accepts full-length research articles containing four (4) figures and/or tables or more. AFAB emphasizes the importance of sound scientific experimentation on any of the topics listed in the focus areas followed by clear concise writing that describes the research in its entirety. The results of experiments published in AFAB must be replicated, with appropriate statistical assessment of experimental variation and assignment of significant difference. Major headings to include are: Abstract, Introduction, Materials and Methods, Results, Discussion (or Results and Discussion), Conclusion, Acknowledgements (optional), Appendix for abbreviations (optional), and References. Manuscripts clearly lacking in language will be returned to author without review, with a suggestion that English editing be sought before the paper is reconsidered. AFAB offers a fee based language service upon request. Please contact language@afabjournal.com for more information about our fees and services.

Rapid Communications Under normal circumstances, AFAB aims for receipt-to-decision times of approximately one month or less. Accepted papers will have priority for publication in the next available issue of AFAB. However, if an author chooses or requires a much more rapid

peer review, the journal editorial office has the capability to shorten the review timing to one week or less. Any type of manuscript whether it be a full length manuscript, brief communication or review paper can be submitted as a rapid communication. There will be additional costs for processing and page charges will be double the normal rate. Authors who choose this option must select Rapid Communications as the paper type when submitting the paper and the editors will judge whether a rapid review is possible and let the author know immediately.

Brief Communications Brief communications are short research notes giving the results of complete experiments but are considered less comprehensive than full-length articles with three (3) figures and/or tables or less. Manuscripts should be prepared with the same subheadings as full length research papers. The running head above the title of the paper is “Brief Communications.”

Unsolicited Review Papers Review papers are welcome on any topic listed in the focus section and have no page limits. Reviews are assessed the same pages charges as all other manuscripts. All AFAB guidelines for style and form apply. Major headings to include are: Abstract, Introduction, Main discussion topics and appropriate subheadings, Conclusions, Acknowledgements (optional) and References. Review papers shorter than 20 pages of double spaced text and references will be considered mini-reviews with the subheading above the title on the first page. The running head above the title of the paper is either “Review” or “Mini-review”.

Solicited Review Papers Solicited reviews will have no page limits. The editor-in-chief will send invitations to the authors and then review these contributions when they are submitted. Nominations or suggestions for potential timely reviews are welcomed by the editors or edito-

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rial board members and should be sent to submit@ afabjournal.com. There will be no page charges for solicited review papers but the solicitation must originate from the editor-in-chief or one of the editors. Requests from authors will automatically be classified as unsolicited review papers. The running head above the title of the paper will be “Invited Review.”

Conference and Special Issues Reviews AFAB welcomes opportunities to publish papers from symposia, scientific conference, and/or meetings in their entirety. Conference organizers need simply to contact AFAB at submit@afabjournal.com and a rapid decision is guaranteed. If in agreement, the conference organizers must guarantee delivery of a set number of peer reviewed manuscripts within a specified time and submitted in the same format as that described for unsolicited review papers. Conference papers must be prepared in accordance with the guidelines for review articles and are subject to peer review. The conference chair must decide whether or not they wish to serve as Special Issue Editor and conduct the editorial review process. If the conference chair/organizer chooses to serve as special issue editor, this will involve review of the papers and, if necessary, returning them to the authors for revision. The conference organizer then submits the revised manuscripts to the journal editorial office for further editorial examination. Final revisions by the author and recommendations for acceptance or rejection by the chair must be completed by a mutually agreed upon date between the editor and the conference organizer. Manuscripts not meeting this deadline will not be included in the published symposium proceedings but if submitted later can still be considered as unsolicited review papers. Although offprints and costs of pages are the same as for all other papers, the symposium chair may be asked to guarantee an agreed upon number of hard copies to be purchased by conference attendees. If the decision is not to publish the symposium as a special issue, the individual authors retain the right to submit their papers for consideration for the journal as ordinary unsolicited review manuscripts.

Book Reviews AFAB publishes reviews of books considered to be of interest to the readers. The editor-in-chief ordinarily solicits reviews. Book reviews shall be prepared in accordance to the style and form requirements of the journal, and they are subject to editorial revision. No page charges will be assessed solicited reviews while unsolicited book reviews will be assigned the regular page charge rate.

Opinions and Current Viewpoints The purpose of this section will be to discuss, critique, or expand on scientific points made in articles recently published in AFAB. Drafts must be received within 6 months of an article’s publication. Opinions and current perspectives do not have page limits. They shall have a title followed by the body of the text and references. Author name(s) and affiliation(s) shall be placed between the end of the text and list of references. If this document pertains to a particular manuscript then the author(s) of the original paper(s) will be provided a copy of the letter and offered the opportunity to submit for consideration a reply within 30 days. Responses will have the same page restrictions and format as the original opinion and current viewpoint, and the titles shall end with “Opinions.” They will be published together. Letters and replies shall follow appropriate AFAB format and may be edited by the editor-in-chief and a technical editor. If multiple letters on the same topic are received, a representative set of opinions concerning a specific article will be published. A disclaimer will be added by the editorial staff that the opinion expressed in this viewpoint is the authors alone and does not necessarily represent the opinion of AFAB or the editorial board.

COPYRIGHT AGREEMENT The copyright form is published in AFAB as space permits and is available online (www.afabjournal.com). AFAB grants to the author the right of re-publication in any book of which he or she is the author or edi-

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tor, subject only to giving proper credit to the original journal publication of the article by AFAB. AFAB retains the copyright to all materials accepted for publication in the journal. If an author desires to reprint a table or figure published from a non-AFAB source, written evidence of copyright permission from an authority representing that source must be obtained by the author and forwarded to the AFAB editorial office.

PEER REVIEW PROCESS Authors will be requested to provide the names and complete addresses including emails of five (5) potential reviewers who have expertise in the research area and no conflict of interest with any of the authors. Except for manuscripts designated as Rapid Communication each reviewer has two (2) weeks to review the manuscript, and submit comments electronically to the editorial office. Authors have three (3) weeks to complete the revision, which shall be returned to the editorial office within six (6) weeks after which the authors risk having their manuscript removed from AFAB files if they fail to ask the editorial office for an extension by email. Deleted manuscripts will be reconsidered, but they will have to be processed as new manuscripts with an additional processing fee assessed upon submission. Once reviewed, the author will be notified of the outcome and advised accordingly. Editors handle all initial correspondence with authors during the review process. The editor-in chief will notify the author of the final decision to accept or reject. Rejected manuscripts can be resubmitted only with an invitation from the editor or editor-in chief. Revised versions of previously rejected manuscripts are treated as new submissions.

PRODUCTION OF PROOFS Accepted manuscripts are forwarded to the editorial office for technical editing and layout. The manuscript is then formatted, figures are reproduced, and author proofs are prepared as PDFs. Author proofs of all manuscripts will be provided to the corresponding author. Author proofs should be read carefully and checked against the typed manuscript, because the

responsibility for proofreading is with the author(s). Corrections must be returned by e-mail. Changes sent by e-mail to the technical editor must indicate page, column, and line numbers for each correction to be made on the proof. Corrections can also be marked using “track changes” in Microsoft Word or using e-annotation tools for electronic proof correction in Adobe Acrobat to indicate necessary changes. Author alterations to proofs exceeding 5% of the original proof content will be charged to the author. All correspondence of proofs must be agreed to by the editorial office and the author within 48 hours or proof will be published as is and AFAB will assume no responsibility for errors that result in the final publication.

PUBLICATION CHARGES AFAB has two publication charge options: conventional page charges and rapid communication. The current charge for conventional publication is $25 per printed page in the journal. There is no additional charge for the publication of pages containing color images, micrographs or pictures. For authors who wish to have their papers processed as a rapid communication, authors will pay the rapid communication fee when proofs are returned to the editorial office in addition to twice the conventional page charges. Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one week and $100 per journal page.

HARD COPY OFFPRINTS If you are wishing to obtain a physical hard copy of the AFAB journal, offprints are available in any quantity at an additional charge: $100/page for black-white and $150/page for color prints. You may order your offprints at any time after publication on our website. Scientific conference organizers may be expected to agree to a set number of offprints as a part of their agreement with AFAB.

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MANUSCRIPT CONTENT REQUIREMENTS Preparing the Manuscript File Manuscripts must be written in grammatically correct English. AFAB offers a fee based language service upon request (language@afabjournal.com). Manuscripts should be typed double-spaced, with lines and pages numbered consecutively. All documents must be submitted in Microsoft Word (.doc or .docx, PC or Mac). All special characters (e.g., Greek, math, symbols) should be inserted using the symbols palette available in this font. Tables and figures should be placed in separate sections at the end of the manuscript (not placed in the text). Failure to follow these instructions will cause delays of the processing and review of the manuscript.

Title Page At the very top of the title page, include a title of not more than 100 characters. Format the title with the first letter of each word capitalized. No abbreviations should be used. Under the title, the authors names are listed. Use the author’s initials for both first and middle names with a period (full-stop) between initials (e.g., W. A. Afab). Underneath the authors, a list affiliations must be listed. Please use numerical superscripts after the author’s names to designate affiliation. If an authors address has changed since the research was completed, this new information must be designated as “Current address:”. The corresponding author should be indicated with an asterisk e.g., * Corresponding author. The title page shall include the name and full address of the corresponding author. Telephone and e-mail address must also be provided for the corresponding author, and emailaddresses must be provided for all authors.

at the beginning of the manuscript. In vivo, in vitro and bacterial names must be italicized (obligatory). Authors must avoid single sentence paragraphs and merge such paragraphs appropriately. Authors must not begin sentences with “Figure or Table shows…” as these are inanimate objects and cannot “show” anything. When number are reported in text or in tables, always put a zero in front of decimal numbers: “0.10” instead of “.10”.

MANUSCRIPT SECTIONS Abstract The abstract provides an abridged version of the manuscript. Please submit your abstract on a separate page after the title page. The abstract should provide a justification of your work, objectives, methods, results, discussion and implications of study or review findings . Your abstract must consist of complete sentences without references to other work or footnotes and must not exceed 250 words. On the same page as your abstract, please provide at least ten (10) keywords to be used for linking and indexing. Ideally, these keywords should include significant words from the title.

Introduction The introduction should clearly present the foundation of the manuscript topic and what makes the research or the review unique. The introduction should validate why this topic is important based on previously published literature, and the relevance of the current research. Overall goals and project objectives must be clearly stated in the final sentence of the last paragraphs of the introduction.

Materials and Methods Editing Author-derived abbreviations should be defined at first use in the abstract and again in the body of the manuscript. If abbreviations are extensive authors may need to provide a list of abbreviations

Information on equipment and chemicals used must include the full company name, city, and state (country if outside the United States or Province if in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].

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Variability, Replication, and Statistical Analysis To properly assess biological systems independent replication of experiments and quantification of variation among replicates is required by AFAB. Reviewers and/or editors may request additional statistical analysis depending on the nature of the data and it will be the responsibility of the authors to respond appropriately. Statistical methods commonly used in the bacteriology do not need to be described in detail, but an adequate description and/or appropriate references should be provided. The statistical model and experimental unit must be designated when appropriate. The experimental unit is the smallest unit to which an individual treatment is imposed. For bacterial growth studies, the average of replicate tubes per single study per treatment is the experimental unit; therefore, individual studies must be replicated. Repeated time analyses of the same sample usually do not constitute independent experimental units. Measurements on the same experimental unit over time are also not independent and must not be considered as independent experimental units. For analysis of time effects, assess as a rate of change over time. Standard deviation refers to the variability in the biological response being measured and is presented as standard deviation or standard error according to the definitions described in statistical references or textbooks.

Results Results represent the presentation of data in words and all data should be described in same fashion. No discussion of literature is included in the results section.

Discussion The discussion section involves comparing the current data outcomes with previously published work in this area without repeating the text in the results section. Critical and in-depth dialogue is encouraged.

Results and Discussion Results and discussion can be under combined or separate headings.

Conclusions State conclusions (not a summary) briefly in one paragraph.

Acknowledgments Acknowledgments of individuals should include institution, city, and state; city and country if not U.S.; and City or Province if in Canada. Copies being reviewed shall have authors’ institutions omitted to retain anonymity.

References a) Citing References In Text Authors of cited papers in the text are to be presented as follows: Adams and Harry (1992) or Smith and Jones (1990, 1992). If more than two authors of one article, the first author’s name is followed by the abbreviation et al. in italics. If the sentence structure requires that the authors’ names be included in parentheses, the proper format is (Adams and Harry, 1982; Harry, 1988a,b; Harry et al., 1993). Citations to a group of references should be listed first alphabetically then chronologically. Work that has not been submitted or accepted for publication shall be listed in the text as: “G.C. Jay (institution, city, and state, personal communication).” The author’s own unpublished work should be listed in the text as “(J. Adams, unpublished data).” Personal communications and unsubmitted unpublished data must not be included in the References section. Two or more publications by the same authors in the same year must be made distinct with lowercase letters after the year (2010a,b). Likewise when multiple author citations designated by et al. in the text have the same first author, then even if the other authors are different these references in the text and the references section must be identified by a letter. For example

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“(James et al., 2010a,b)” in text, refers to “James, Smith, and Elliot. 2010a” and “James, West, and Adams. 2010b” in the reference section. b) Citing References In Reference Section In the References section, references are listed in alphabetical order by authors’ last names, and then chronologically. List only those references cited in the text. Manuscripts submitted for publication, accepted for publication or in press can be given in the reference section followed by the designation: “(submitted)”, “(accepted)’, or “(In Press), respectively. If the DOI number of unpublished references is available, you must give the number. The year of publication follows the authors’ names. All authors’ names must be included in the citation in the Reference section. Journals must be abbreviated. First and last page numbers must be provided. Sample references are given below. Consult recent issues of AFAB for examples not included in the following section. Journal manuscript: Author(s). Year. Article title. Journal title [abbreviated]. Volume number:inclusive pages.

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 Chapter: Author(s) of the chapter. Year. Title of the chapter. In: author(s) or editor(s). Title of the book. Edition or volume, if relevant. Publisher name, Place of publication. 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.

Examples: 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:

Book: Author(s) [or editor(s)]. Year. Title. Edition or volume (if relevant). Publisher name, Place of publication. Number of pages.

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

Examples: Davis, C. 2010.

Salmonella. Medicinenet.com.

http://www.medicinenet.com/salmonella /article. htm. Accessed July, 2010. 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, and 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. In addition, you will need to submit all data for charts, tables and figures in native format when possible (e.g., Microsoft Excel, Powerpoint). Photographs should be submitted as high-resolution (600 dpi) .jpg or tif. files. 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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Online Publication: www.AFABjournal.com