Use of restriction fragment length polymorphism to characterize methicillinresistant Staphylococcus aureus in dairy products. Biology and Medicine

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1 eissn: 0989 Use of restriction fragment length polymorphism to characterize methicillinresistant Staphylococcus aureus in dairy products Biology and edicine Research Article Volume, Issue, Pages 0, 0

2 Research Article Biology and edicine, (): 0, 0 Use of restriction fragment length polymorphism to characterize methicillin-resistant Staphylococcus aureus in dairy products Salehi *, V Razavilar, H irzaei, A Javadi, S Banan Khojasteh Department of Food Hygiene, Faculty of Specialized Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran. Department of Food Hygiene, Faculty of Veterinary edicine, Tabriz Branch, Islamic Azad University, Tabriz, Iran. Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran. *Corresponding Author: Accepted: th Aug 0, Published: 0 th Aug 0 Abstract Staphylococcus aureus is one of the frequent causes of animal and human infections. In the present study, methicillinresistant S. aureus (RSA) was investigated in dairy products in Iran. From coagulase-positive S. aureus isolates, samples had RSA gene. Amplification of RSA gene produced polymerase chain reaction product with the size of 0 bp. By analyzing the restriction fragment length polymorphism (RFLP) of RSA gene with sa restriction enzyme, there was no restriction pattern. The results showed that there was no genetic diversity in RSA gene in S. aureus isolates from dairy products of different areas in East-Azerbaijan Province, Iran. Keywords: RFLP; ethicillin-resistant; Staphylococcus aureus; Dairy products. Introduction Staphylococcus (S.) aureus is worldwide the most important pathogen in food poisoning and also is involved in another wide variety of infections found in human beings and animals. It can cause gastrointestinal symptoms like nausea, emesis, abdominal cramps, and diarrhea in humans (Scherrer et al. 00; orandi et al. 00). Also S. aureus is a major nosocomial pathogenic agent that causes endocarditis, osteomyelitis, pneumonia, toxic shock syndrome, carbuncles, and boils (Shopsin et al. 00). Food poisoning due to S. aureus is related to the production of enterotoxins by microorganism in food stuffs. S. aureus can access milk through direct excretion from udders suffering clinical and sub-clinical staphylococcal mastitis and by environmental contamination during the milk handling and processing (orandi et al. 00). In 99, methicillin, synthetic penicillin, was introduced. However, by 90, methicillinresistant S. aureus (RSA) strains were identified, the direct result of S. aureus acquiring the eca gene, which encodes for an altered penicillin-binding protein gene (PBP a ) (Shopsin et al. 00). RSA is one of the important pathogenic microorganisms in the hospital and community settings, with substantial rates in morbidity and mortality (Shittu et al. 00). In the United States, RSA is responsible for approximately % of the nosocomial infections, and reports of community acquired RSA infection are increasing (Shopsin and Kreiswirth 00). Because published reports on the use of restriction fragment length polymorphism (RFLP) to characterize RSA in dairy products in Iran are rare and little information is available in this regard, the present study was conducted as one of the first reports. aterials and ethods Bacterial strains A group of isolates were selected from dairy products in East-Azerbaijan province, Iran. Of these isolates, were obtained from raw milk (arand and Tabriz cities), from traditionally produced cheese (Tasouj, Bonab, and Lighvan) and from ice cream (Tabriz). Biochemical profile All the strains were subcultured three times in brain-heart infusion (BHI) broth at C for hr. BID: AR0-B

3 Research Article Biology and edicine, (): 0, 0 All the isolates were tested twice and retested in case of discrepancies. Extraction of DNA Bacterial DNA extraction was carried out according to Rodrigues da Silva and da Silva (00). PCR technique PCR was performed in a -μl reaction mixture containing μl of template DNA,. μl of master kit, 0 μl of HO, and 0. μl of each primer ( -AAA, ATC, GAT, GGT, AAA, GGT, TGG, C- ; -AGT, TCT, GGA, GTA, CCG, GAT, TTG, C- ). The PCR reaction was performed according to the following cyclic condition: Initial denaturation at 9 C for min, cycles of min in each with denaturation at 9 C, min annealing at C, min extension at C, a final 0 min extension at C. Restriction fragment length polymorphism The PCR products were digested with Sma for restriction analysis. Reaction mixture containing -μl of PCR products,.-μl of enzyme, -μl of HO and.-μl restriction buffer, and then incubated at C overnight. Agarose gel electrophoresis The resulting fragments were separated by electrophoresis in % agarose gel. The gels were stained with ethidium bromide and visualized on an UV transluminator. The size of the fragment was determined by comparison with the 00 bp marker. Results The findings showed that only there were RSA in samples. The size of PCR products of RSA was 0 bp (Figure ). Digestion of RSA PCR products with Sma yielded no different restriction profile. So, it was observed that no restriction site is produced with Sma for methicillin-resistant genes (Figure ) in examined dairy products. Figure : Agarose gel electrophoresis of RSA gene of Staphylococcus aureus PCR Product. Lane : 00 bp marker. Lanes : approximately 0 bp long S. aureus RSA PCR product. Figure : PCR amplified RSA gene digested with the DNA restriction endonuclease of Sma. Lane : 00 bp marker. Lanes : have no RFLP pattern. After treatment of RSA PCR product with Sma endonuclease. BID: AR0-B 8

4 Research Article Biology and edicine, (): 0, 0 Discussion ethicillin-resistant S. aureus isolates were once confined largely to hospitals, other healthcare environments, and patients frequenting these facilities. Community-associated RSA strains have rapidly disseminated among the general population in many countries (Chowdhury, 0). Restriction endonuclease digestion analysis has been used to determine RSA isolates. The results of this study suggested that no RFLP pattern is produced in isolates yielding methicillin-resistant S. aureus. But Wei et al. (99) from clinical isolates of RSA collected from Australian hospitals, reported bands of 00 kb with Sma digestion. Nafisi et al. (008) researched on the isolates of coagulase-positive S. aureus among 0 clinical staff from different areas of Shahrekord city, Iran, reported that phenotypically, cases (%) of the isolates and genotypically (eca), cases (%) of the isolates were resistant to methicillin. As RSA may be present in raw milk and traditional dairy products, insufficiently hygienic handling of these contaminated foods may lead to transmission of RSA to human and possible colonization in nostrils, skin, and gastrointestinal tract irzaei et al. (0). Findings of irzaei et al. (0) showed that (0%) of the pasteurized milk isolates and (%) of the traditional cheese isolates collected from Sarab city, Iran, contained eca gene (RSA). Chu et al. (0) reported the appearance of RSA strains in mastitic goats for the first time in Taiwan. eca gene of Staphylococcus aureus isolated from raw water buffalo milk and dairy products in Turkey were determined by Pamuk et al. (0). All strains showed resistance to at least one antibiotic. Of the 9 S. aureus strains, (.%) harbored eca gene. Overall,.% of the samples were contaminated with RSA. Study of Grady et al. (000) in RSA isolated with fluorescent amplified-fragment length polymorphism (FAFLP technique) showed another approach to the epidemiological study of RSA. They found several clusters of strains and isolates with that technique. Hookey et al. (999) reported that in typing isolates and reference strains of RSA with FAFLP technique, there were from 0 to 0 fragments, 0 0 bp in size. Janwithayanuchit et al. (00) from a total of 9 RSA, achieved different genotypes by PCR-RFLP pattern, however, these results are in contrast with our findings. It is concluded that although no restriction pattern was seen in the present study in Iran, East-Azerbaijan province, laboratories are encouraged to investigate strains with restriction sites and RFLP pattern. This is the first report on the molecular typing of the RSA based on RFLP technique. easures to prevent contamination and growth of RSA in food, including the use of microbiological criteria should be equal to those that are valid for S. aureus in general. Conflict of Interests There is no conflict of interest. References Chowdhury A, 0. Evolving antibiotic resistance: a great threat to medical practice. Bangladesh Journal of edical Science, :. Chu C, Yu C, Lee Y, Su Y, 0. Genetically divergent methicillin-resistant Staphylococcus aureus and secdependent mastitis of dairy goats in Taiwan. BC Veterinary Research, 8: 0. Grady R, O Neill G, Cookson B, Stanley J, 000. Fluorescent amplified-fragment length polymorphism analysis of the RSA epidemic. FES icrobiology Letters, 8: 0. Hookey JV, Edwards V, Patel S, Richardson JF, Cookson BD, 999. Use of fluorescent amplified fragment length. Journal of icrobiological ethods, :. Janwithayanuchit I, Ngam-Ululert S, Paungmonung P, Rangsipanuratn W, 00. Epidemiologic study of methicillin-resistant Staphylococcus aureus by coagulase gene polymorphism. Science Asia, :. irzaei H, Tofighi A, Karimi Sarabi H, Farajli, 0. Prevalence of methicillin-resistant Staphylococcus aureus in raw milk and dairy products in Sarab by culture and PCR techniques. Journal of Animal and Veterinary Advances, 0: 0. orandi S, Brasca, Lodi R, Brusetti L, Andrighetto C, Lombardi A, 00. Biochemical profiles, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (LVA) for typing Staphylococcus aureus isolated from dairy products. Research in Veterinary Science, 88:. BID: AR0-B 9

5 Research Article Biology and edicine, (): 0, 0 Nafisi R, Kalhor H, Zamanzad B, Karimi A, Farokhi A, Validi, 008. Comparison of agar screen and duplex-pcr in determination of methicillin-resistant Staphylococcus aureus (RSA) strains isolated from nose of personnel in Hajar hospital of Shahrekord. Arak University edical Sciences Journal, : 9 0. Pamuk S, Yildirim Y, Seker A, Gurler Z, Kara R, 0. A survey of the occurrence and properties of methicillinresistant Staphylococcus aureus and methicillinresistant Staphylococcus intermedius in water buffalo milk and dairy products in Turkey. International Journal of Dairy Technology, :. Rodrigues da Silva E, da Silva N, 00. Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in Southern Brazil. Canadian Journal of Veterinary Research, 9: 0. Scherrer D, Corti S, uehlherr JE, Zweifel C, Stephan R, 00. Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from raw bulk-tank milk samples of goats and sheep. Veterinary icrobiology, 0: 0 0. Shittu A, Lin J, orrison D, 00. olecular identification and characterization of manitolnegative methicillin-resistant Staphylococcus aureus. Diagnostic icrobiology and Infectious Disease, : 9 9. Shopsin B, Kreiswirth BN, 00. olecular epidemiology of methicillin-resistant Staphylococcus aureus. Emerging Infectious Diseases, :. Wei Q, Grubb WB, 99. Typing of Australian methicillin-resistant Staphylococcus aureus strains by pulsed field gel electrophoresis. Journal of edical icrobiology, : 8 9. Wei Q, Wang F, Grubb WB, 99. Use of contour-clamped homogeneous electric field (CHEF) electrophoresis to methicillin-resistant Staphylococcus aureus. Journal of edical icrobiology, :. BID: AR0-B 0