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www.gbtrp.com International Journal of Biological Technology (2010) 1(3):50-55 ISSN: 0976 - 4313

Original Article

Prevalence, antibiotic resistance and RAPD analysis of food isolates of Salmonella species D.Jegadeeshkumar1*, V. Saritha1, K. Moorthy2, B.T. Suresh kumar2, 1 Chromopark Research centre, 62-B, Near N.K.R. College, Trichy Main Road, Namakkal- 637001 2 Vivekanandha college of Arts & Science, Tiruchengode,South India Address for corresponding author E- mail jegadeeshkumar2@gmail.com, Mobile: + 91 9940292388. Received: 15.09.2010; Revised:25.10.2010; Accepted: 11.11.2010; Published: 01.12.2010 Abstract A study was conducted to determine the prevalence, antibiotic resistance and RAPD patterns of Salmonella species on different food samples. A totally 30 foods samples were collected from in around area of Namakkal town. In our findings, out off 30 samples, 14 isolates (46.6%) were isolated from 3 types of food samples and highest prevalence occurred in fish samples (90%). Four types of species were isolated from among the 3 types of samples such as S.typhi (55.5%) S.paratyphi A (48.1%), S.paratyphi B (25.9%) and S.typhimurium (22.2%). All isolates were subjected into antibiotic stability test. Present study all isolates showed multiple drug resistance, and 100 % resistant to Vancomycin, Novobiocin, Nitrofurantoin, Ciproflaxacin, and Methicillin. The increasing resistance was observed for Amoxiclave (92.55 %) and Bacitracin (78.57 %). The highest antibiotic resistance patterns were observed in fruits (80.5%) second most fish isolates (72.2%). Random amplified polymorphic DNA (RAPD) analysis was applied for molecular characterization of Salmonella species. A total of 3 primers were used and only single primer showed good discriminatory power for all isolates. Dendrogram showed heterogeneity present in all isolates. Key words: Antibiotic resistance, food samples, Salmonella spp, RAPD analysis.

Introduction Every year million kilograms of antibiotics are used to the treatment of people, animals and agriculture purpose. But microbes acquire resistant property by different mechanisms. For Example the resistance genes can be transferred to other bacteria by through mobile genetic elements. Antibiotic resistance is one of the public health problems in world wide. In recent years problems related to salmonella have increased significantly. Salmonella causes one of the most common enteric (intestinal) infections in the world wide. Salmonella is the second most common bacterial foodborne illness after Campylobacter infection. Salmonella species are important zoonotic pathogens in humans and animals (Winokur etal.,2000) salmonella species cause a wide range of human disease such as enteric fever, gastroenteritis, Bacteremia etc,. (Bennasar etal., 2000) Salmonella infection occurs when bacteria are ingested, typically from food derived from infected animals. Infection can also occur by ingesting the faeces of an infected animal or person. Food sources include raw or undercooked eggs/egg products, raw milk or raw milk products, contaminated water, meat and meat products, and poultry. Š Gayathri Teknological Publication

Raw fruits and vegetables contaminated during slicing have been implicated in several foodborne outbreaks, as have foods contaminated by food handlers who did not adhere to proper hygienic standards and practice proper hand washing techniques. Ingested strains multiple in the stomach containing low PH (Acidic) condition then strains enter to intestinal epithelium of lymphoid follicles and affects the lymph node, spleen and liver where further multiply take place (Jones,1997) The emergence of antimicrobial resistance salmonella strains is of great concern worldwide Typhoid fever is a global problem with an estimated 12-33 million people occurring in worldwide (Kohinur etal.,2010). In India more than 3, 00,000 cases are reported every year and are associated with significant morbidity and mortality. Drug resistant Salmonella typhi has been reported in India since 1960 and outbreaks by these strains occur at intervals in various parts of India (Surg Capt etal.,2004). The previous reported incidences of multidrug resistant strains have ranged from 11% in Vellore and 94.7% in Bangalore (Rathish etal.,1994). In 1990 multidrug resistant Salmonella typhi with reduced susceptibilities to fluroquiolnes were 50


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isolated in Vellore in India. The nalidix acid resistance Salmonella typhi with decreased susceptibility to ciprofloxacin is now endemic in India (Philippa etal.,1998) To investigate the origins and the relationships among different isolates, more precise methods than serotyping are needed. A variety of DNA-based typing methods have been applied to identify Salmonella species, including plasmid profile biotyping ribotyping PFGE (Antonie etal.,2008). Each of these approaches has provided useful insights into evolutionary and epidemiological relationships of several Salmonella serovars. The variety of molecular subtyping approaches are available, from a practical standpoint, when several isolates are to be compared over the course of few days; the most general procedure for the comparison of genomes is random amplified polymorphic DNA (RAPD) analysis (Del Tufo and Tingey , 1994). In the present study, we analyzed strains of Salmonella isolated from different foods in Chennai. This study was conducted to identify the antimicrobial resistance pattern of the Salmonella and differentiate the isolates by RAPD methods.

Materials and Methods Different food samples such as Banana, Grapes, Meat, and Fish (Sankara, Kelangan, Parai, Mathi and Prawn) were collected from Namakkal town. The samples were collected in polythylene bags. The food samples processing and inoculation of samples for bacteriological analysis were completed within 2 hrs of collection. The samples were rinsed with sterile water to remove the adhering particles. A 25 g of flesh was cut with the use of sterile knife and homogenized in 9 ml of alkaline peptone water (Maripandi and Al- salamah, 2010). Then loopful of sample inoculated into selenite F broth and incubated the medium at 37°C for 24 hrs. After incubation period loop full of culture was taken from selenite F broth and directly plated on the surface of SS agar and incubated at 37°C for 24 hrs. The primary identification of the bacterial isolates was made based on colonial appearance, pigmentation and Biochemical tests. Characterization and identification of the isolates was done using the methods of Cheesbrough, 2004. Antibiotic stability test The Kirby Bauer disc diffusion method using nutrient agar plate was used for

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detecting the susceptibility or resistant pattern of isolates. In this stability test 12 different antibiotic disc were used including Ampicillin, Amoxiclave, Chloramphenicol, Bacitracin, Methicillin, Kanamycin, Ciprofloxacin, Norfloxacin, Nitrofurantoin, Novobiocin, Vancomycin and Tetracycline, which are commonly used for the treatment of typhoid fever. DNA preparation DNA samples were prepared from bacterial strains using standard protocol (Un– Ho Jin etal., 2000 and Un–Ho Jin etal., 2004). The bacterial cells are heated with Tris, EDTA and SDS solution. Then nucleic acid extracted by Phenol: chloroform: isoamylalcohol (24:25:1). Finally the DNA was precipitated by 70% ethanol. PCR Anaysis In the RAPD fingerprinting analysis we selected three different primers such as: 1 5’GCG GAA ATA G 3’; 2 - 5’AAC GCG CAA C 3’ and 3 - 5’AGC GTC ACT C 3’ (Bianca ramlho Quintaer etal., 2004). PCR was performed in 20µl reaction mixtures consists of 0.5 µl of 1 U of Taq DNA polymerase, 2µl of 10 mM Tris HCl, 50 mM KCL, 2.0 mM MgCl2, 1 µl 200 mM of each dNTP mix, 1 µl 20 pm of primer and 2µl of Template DNA then these mixtures make up to 13.5 µl with molecular grade water. Following conditions are applied for the amplification such as initial denaturation at 940C for 1 min, 30 cycles of denaturation at 940C for 1 min, anneling of primer at 360C for 1 min and extention at 720C for 2 min, final extention at 720C for 7 min. After amplification the products were separated by EtBr stained 1.5% Agarose Gel electrophoresis.

Result A total of 14 (46.66%) Salmonella spp. was isolated from 30 different food samples. The isolates were found to be (Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A and Salmonella paratyphi B) depending on the colony morphology on selective media (Salmonella shigella agar) and biochemical characteristics. The incidence of salmonella was recorded as shown in Table 1. In our current studies the highest percentage of incidence were occurred in fish (90%) samples. Among the 4 types of isolates Salmonella paratyphi A were occurred in all samples but highest incidence in meat samples (Table-2). 51


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Table-1: Percentage incidence of Salmonella isolates in food samples Samples

Isolates S.typhi S.typhimurium S.paratyphi A S.paratyphi B

Seafoods (Fish) 55.5% 22.2% 11.1% 11.1%

Fruits

Meat

0% 0% 33.3% 66.6%

0% 0% 100% 0%

highest resistance was observed from fruit samples (80.5%) followed by fish samples (72.2%).

Meat, 66.60%

Fruit, 80.50%

Table- 2: Occurrence of Salmonella species from different food samples Samples Fish fruits Meat

No. of Samples taken 10 10 10

No of isolates obtained 9 3 2

% incidence 90 30 20

Fish, 72.20%

Fish

Fig.1: Antibiotic Salmonella species

Fruit

Meat

resistance

patterns

of

PCR Analysis

Table -3: Antibiotic Resistance patterns of Salmonella isolates S.No

Isolates

1.

S.typhi 1

2.

S.typhi 2

3.

S.typhi 3

4.

S.typhi 4

5.

S.typhi 5

6.

S.typhimurium 1 S.typhimurium 2 S.paratyphi A 1 S.paratyphi B 1 S.paratyphi A 2 S.paratyphi B 2 S.paratyphi A 3 S.paratyphi A 4 S.paratyphi B 3

7. 8. 9. 10. 11. 12. 13. 14.

Source Fish Fish Fish Fish Fish Fish Fish Fish Fish Fruit Fruit Meat Meat Fruit

Resistance patterns A Am B M K Cf Nf Nv Va T A Am B M K Cf Nf Nv Va A Am B M K Cf Nf Nv Va T A Am B M K Cf Nf Nv Va T A Am B M K Cf Nf Nv Va M K Cf Nf Nv Va A Am M K Cf Nf Nv Va A Am B M Cf Nf Nv Va A Am B M Cf Nf Nv Va A Am B M K Cf Nf Nv Va T A Am B M Cf Nf Nv Va T A Am B M Cf Nf Nv Va T A Am M Cf Nf Nv Va A Am B M K Cf Nf Nv Va T

The antibiotic resistant pattern of the isolates was recorded and tabulated in Table 3 and Figure 1. The isolates were 100 % resistant to Vancomycin, Novobiocin, Nitrofurantoin, Ciproflaxacin, and Methicillin. The increasing resistance was observed for Amoxiclave (92.55 %) and Bacitracin (78.57 %). The intermediate resistance was observed to Tetracycline (57.14 %). In our present study the isolates were 100 % sensitive to Norflaxacin and Chloramphenicol. Among the 3 samples the © Gayathri Teknological Publication

Lane 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15

Isolates sample S.typhi 1 Fish S.typhi 2 Fish S.typhi 3 Fish S.typhi 4 Fish S.typhi 5 Fish S.typhimurium 1 Fish S.typhimurium 2 Fish S.paratyphi A 1 Fish S.paratyphi B 1 Fish S.paratyphi A 2 Fruit S.paratyphi B 2 Fruit S.paratyphi A 3 Meat S.paratyphi A 4 Meat S.paratyphi B 3 Fruit M-100 bp DNA Markers

Fig.2: RAPD analysis of Salmonella species isolate from different food samples

The RAPD patterns were obtained from the DNA of Salmonella spp. In this study random primers were used to determine the genetic diversity among Salmonella species by PCR amplification. All the primers were subjected to optimized conditions for amplification to all strains but 3 -5’AGC GTC ACTC 3’ primer provided good discriminatory power among different isolates. The amplified fragments ranging from 100 bp to above 1000bp. Out of the 14 samples 3 isolates of Salmonella produced distinct bands patterns. 52


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(Lane 1, 5, and 7). Number of common bands was observed to be present in large number of the isolates. The molecular weights of the common bands are 200,400 bp, 1000 bp and above 1000 bp (Figure 2). Three isolates has number of bands compare to other isolates and two isolates has very few number of bands (lane 4, 9 and 10). The dendrogram for cluster analysis of all the isolates were constructed by using NTSYS software by UPGMA. The cluster analysis reveals that the phylogenetic relationship among the isolates exists irrespective of their sources (Figure 3). The current finding reveals that there was heterogeneity in our isolates of salmonella species that expression was may also be affected by source of isolates.

Fig.3: Dendrogram analysis of Salmonella species isolate from different food samples

Discussion The incidence of Salmonella species 90 %, 30% and 20 % from fish, fruits & meat samples respectively. The percentage incidence of salmonella (20%) from meat samples are contrary to the results of Chen et al,2004 who reported 70% of the Salmonella strains isolated from retail meats purchased in the Washington. The prevalence of Salmonella was comparatively higher in fish (90 %) when compared to meat (20 %) & fruit samples (30 %). In this study, the prevalence from fish was 90% which is in disagreement to the results of Hatha and Lakshmanaperumalsamy,1997 from India, who reported 20 % prevalence from seafood. The differences between the results of the present paper and those of other researchers, may be explained when several factors, such as differences in origin, time Š Gayathri Teknological Publication

period of collection and sampling procedure (Bryan and Dole , 1995). The results of this study indicate that the fish foods are the major source of infection that harbors Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A and Salmonella paratyphi B. The fruit samples & meat samples harbors only Salmonella paratyphi A and Salmonella paratyphi B. The antibiotic resistance pattern of the isolates indicates that all isolates are from the highly pathogenic group and as all the isolates were resistant to atleast 6 antibiotics. In our current study all isolates were resistant to Methicillin, Ciprofloxacin, Nitrofurantoin, Novobiocin, Vancomycin. The results of this study were agreement with Cardoso et al. (18) who reported 100 % resistance to Novobiocin and 95 % resistance to Nitrofurantoin of the salmonella isolates from broiler carcasses in Brazil. He also reported the isolates were 100 % sensitive to Norfloxacin which is also agreement with this study. The results of the present study were contrary to the result of Martha Oliveira and Cardoso etal,2006, who reported 100 % resistance towards tetracycline & 100 % sensitivity towards ciprofloxacin. From this study it is also seen that the ciprofloxacin resistant strains were emerging. Our findings are similar to those described in previous studies showing that Salmonella isolates in retail meats are commonly resistant to multiple antimicrobials, including Tetracycline (Manie etal.,1998 and white etal.,2001). The emergence of drug resistance salmonella species are challenges to the treatment and prevention typhoid. Mainly this infection occurred by poor personal hygienic and inadequate food handling (Senthilkumar and Prabakran etal.,2005). The food handles prominently play a role in spread of typhoid fever. Those peoples isolated the pathogenic multidrug resistance salmonella from food handlers. Therefore, government agencies must adopt measures to educate the vendors about food safety and hygienic. In this study, a RAPD fingerprinting method for the detection of genetic diversity of Salmonella based on short DNA primers was developed. RAPD fingerprinting using these primers produced characteristic band patterns for different isolates. Out of the 14 samples 3 isolates of Salmonella produced distinct bands patterns. Among the 14 isolates 3 had low 53


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number of bands such as Salmonella typhi, Salmonella paratyphi A and Salmonella paratyphi B. The amplified fragments ranging from 100 bp to above 1000bp and very few isolates had similar band patterns. This result was clearly indicated the diversity of isolates. RAPD does not require any specific knowledge of the DNA sequences of the target organism. This makes it a tool of great power and general applicability. In this paper we report the RAPD technique to the differentiation of strains of salmonella. The previous reported also RAPD has good discriminatory power for the differentiation of Salmonella strains (Chansiripornchai etal.,2000 and De Cesare etl.,2001) and some investigators have also reported that RAPD analysis has greater discriminatory power than PFGE for the differentiation of Salmonella strains (Hudson etal., 2001 and Muresu etal.,2001). Conclusion In this study, three different food samples such as fish, fruits and meat samples were investigated for the prevalence of Salmonella spp. The isolates when subjected to Antibiotic Sensitivity testing, the isolates showed multidrug resistant pattern. All the isolates were resistant to atleast 6 antibiotic. These highly pathogenic, multidrug resistant Salmonella species from foods may cause illness in humans. Continuous up to date monitoring and control methodologies, which should be applied in fruit cultivating, poultry farms and slaughterhouses and by retailers, for the prevention or reduction/total elimination of this pathogen, where possible, are strongly recommended. Acknowledgements: We are grateful to Dr. P. Rajen (Srimath Andavan College, Trichy and Dr. B. Senthilkumar (Muthiammal College of arts and science, Rasipuram, for comments on the manuscript.

Reference Antoine, S.T. Annaelle, K. and Anne, B. 2008. Epidemiological analysis of Salmonella enterica from beef sampled in the slaughterhouse and retailers in Dakar (Senegal) using pulsed-field gel electrophoresis and antibiotic susceptibility testing. J. Food Microbiol.,123: 191-197. Bennasar, A., Gloria de Luna, B., Cabrer, L., and Lalucat, J. 2000. Rapid identification of S. typhimurium, S. enteritidis and S. virchow isolates by Polymerase Chain Reaction based © Gayathri Teknological Publication

fingerprinting Methods. Iternatl microbial.,3:31–38. Bianca Ramalho Quintaes, Nilma Cintra Leal, Eliane Moura, Falavina Reis and Ernesto Hofer. 2004. Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi. Brazilian Society for Tropical Medicine., 37:143-147. Bryan, F.L., and Dole, M.P. 1995. Health risks and consequences of Salmonella and Campylobacter jejuni in raw poultry. J. Food Protec., 58: 326- 344. Chansiripornchai, N., Ramasoota, P., Bangtrakulnonth, A., Sasipreeyajan, J., and Svenson, S.B. 2000. Application of randomly amplified polymorphic DNA (RAPD) analysis for typing Avian Salmonella enterica subsp. enterica. FEMS Immunology and Medical Microbiology., 29, 221–225. Cheesbrough, M. 2004. Morphology and characterization of E. coli and S. aureus. District Laboratory practice in tropical countries part II. Cambridge University .pp. 157-179. Chen, S., Shaohua Zhao, David, G., White Carl, M., Schroeder, Ran Lu, Hanchun Yang, Patrick, F., McDermott, Sherry Ayers., and Meng, J. 2004. Characterization of MultipleAntimicrobial-Resistant Salmonella Serovars Isolated from Retail Meats. Applied and environmental microbiology, 70: 1–7. De Cesare, A., Manfreda, G., Dambaugh, T. R., Guerzoni, M. E., and. Franchini, A.2001. Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella Enteritidis and Salmonella Typhimurium strains isolated in Italy. J. Appl. Microbiol., 91:780-785. Del Tufo, J. P., and Tingey, S., 1994. RAPD assay. Methods Mol. Biol., 28:237–241. Hatha, A.A.M., and Lakshmanaperumalsamy, P. 1997. Prevalence of Salmonella in fish and crustaceans from markets in Coimbatore, South India. Food Microbiology., 14: 111–116. Hudson, C. R., M., Garcia, R. K., Gast, and Maurer, J. J, 2001. Determination of close genetic relatedness of the major Salmonella Enteritidis phage types by pulse field gel electrophoresis and DNA sequence analysis of several Salmonella virulence genes. Avian Dis., 45:875-886. Jones, B.D.1997, Host responses to pathogenic Salmonella infection. Genes and development., 11:679-679.

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Kohinur begum, Tanvir Ahmed Reza, Margia Haque, Akil Hossain, F. M., Kabirul Hassan, Shaik Nahid Hasan, Nargis Akhter, Aliza Ahmed and Utpal Barua,2010. Isolation, identification and antibiotic resistance pattern of Salmonella Spp. from chicken eggs, intestines and environmental samples. Bangladesh Pharmaceutical Journal. 13:2327. Manie, T., Khan, S., Brozel, V. S., Veith, W. J., and Gouws P. A., 1998 Antimicrobial resistance of bacteria isolated from slaughtered and retail chickens in South Africa. Lett. Appl. Microbiol., 26:253–258. Maripandi, A., and Al-Salamah, A.A. 2010. Multiple-antibiotic resistance and plasmid profiles of Salmonella enteritidis isolated from retail chicken meats. Am. J. Food Technol., 5: 260-268. Martha Oliveira Cardoso, Aldemir Reginato Ribeiro, Luciana Ruschel dos Santos, Fernando Pilotto, Hamilton, L.S., de Moraes, Carlos Tadeu Pippi Salle, Silvio Luís da Silveira Rocha, Vladimir Pinheiro do Nascimento,2006. Antibiotic resistance in salmonella enteritidis isolated from broiler carcasses. Brazilian Journal of Microbiology., 37:368-371. Muresu, E., Piana, A., Azara, A., Maida, I., Nastasi, A., Sajid, S. D., and Rubino, S. 2001. Clonal relations among Salmonella enteritidis phage type 3 outbreaks isolates traced by DNA fingerprinting. New Microbiol., 24:371-377. Philippa, M. A., Shanahan, Mary, Jesudason, V., Christopher, J., Thomson, and Sebastian, G. B.1998. Molecular Analysis of and Identification of Antibiotic Resistance Genes in Clinical Isolates of Salmonella typhi from India. Journal of clinical microbiology., 36: 1595–1600. Rathish, K.C., Chandrashekar, M.R., and Nagesha, C.N.1994. Multidrug resistant Salmonella typhi in Bangalore, South India. Indian J Med Sci., 48:85-88. Senthilkumar, B., and Prabakaran, G.2005. Multiple drug resistant Salmonella typhi in asymptomatic typhoid carriers among food handlers in Namakkal district Tamilnadu. Indian J.Med. Microbial., 23: 92-94. Surg Capt, R.N,. Misra., Surg Capt, K.S., Bawa Col, S.K., Magu, Surg Cdr S., Bhandari Col Nagendra, Wg Cdr Menon. P.K. 2004. Outbreak of Multi-drug Resistant Salmonella Typhi Enteric fever in Mumbai Garrison. MJAFI., 61: 148-150.

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Un-Ho Jin, Sung-Hak Cho, Min-Gon Kim, Sang-Do Ha Keun-Sung Kim, Kyu-Ho Lee, Kwang-Yup Kim, Duck Hwa Chung, YoungChoon Lee, and Cheorl-Ho Kim, 2004. PCR Method Based on the ogdH Gene for the Detection of Salmonella spp. from Chicken Meat Samples. The Journal of Microbiology., 42: 216-222. Un-Ho Jin, Tae-Wook Chung, June-Ki Kim, Kyung-Soo Nam, Sang-Do Ha, and Cheorl-Ho Kim. 2000. Differentiation of Salmonella typhimurium from Gram-negative Intestinal Microbes by Randomly Amplified Polymorphic DNA (RAPD) Fingerprinting. The Journal of Microbiology, 38:8-10. White, D. G., Zhao, S., Sudler, R., Ayers, S., Friedman, S., Chen, S., McDermott, P.F., McDermott, S., Wagner, D. D., and Meng. J.,2001. The isolation of antibiotic-resistant Salmonella from retail ground meats. N. Engl. J. Med., 345:1147–1154. Winokur, P.L., Brueggemann, A., DeSalvo, D.L., Hoffmann, L., Apley, M.D., Uhlenhopp, E.K., Pfaller, M.A., and Doern, G.V., 2000. Animal and human multidrug-resistant, cephalosporin-resistant Salmonella isolates expressing a plasmidmediated CMY-2 AmpC beta-lactamase. Antimicrob. Agents Chemother., 44: 2777-2783.

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