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ARTICLES
ANTIMICROBIAL SUSCEPTIBILITY AND RESISTANCE GENES IN SALMONELLA ENTERICA SEROVAR ENTERITIDIS ISOLATED FROM TURKEYS
Türkyılmaz, S.1,3*, Hazımoğlu Ş.2 and Bozdoğan B.3 Adnan Menderes Üniversitesi, Aydın, TURKEY
1
Veteriner Fakültesi, Mikrobiyoloji AD 2 Sağlık Bilimleri Enstitüsü 3 ADU BILTEM Epidemiyoloji Birim
*Corresponding author: Süheyla Türkyılmaz, Tel.: +90 256 247 07 00, Fax: +90 256 247 07 20 E-mail: suhturkyilmaz@yahoo.com
Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis), a cause of food poisoning in humans was isolated from turkeys slaughtered in Izmir, Turkey. It was identified by classical techniques and polymerase chain reaction (PCR), Its antimicrobial susceptibility was determined, and the common resistance genes in antibiotic-resistant strains were demonstrated by PCR. Of the 587 cloacal swabs, 86 (14.7%) were Salmonella positive and of these, 11 (1.9%) were serovar Enteritidis. Antimicrobial susceptibilities were tested by agar dilution MICs. All strains were susceptible to cefoxitin, ciprofloxacin and amikacin. Of the 11 strains tested 9 (81.8%), 6 (54.6%), 6 (54.6%), and 3 (27.3%) were susceptible to gentamicin, ampicillin, chloramphenicol, and tetracycline respectively. Among 11 S. Enteritidis isolates 2 were susceptible to all antimicrobials tested, and 7 strains were multi-drug resistant. Of 8 tetracycline resistant strains, 5 and 3 carried tet(A), tet(B) genes, respectively. Of 5 ampicillin resistant strains, 3 had blaTEM, and 2 had blaOXA. Of 5 chloramphenicol-resistant strains one had cat3, 3 had floR, and known resistance genes were negative by PCR in one resistant strain. Of the 2 gentamicin-resistant strains, one had aadB, and one had aacC genes. This study verified the presence of S. Enteritidis among turkey flocks in Izmir. Antibacterial resistance among isolated strains was relatively high. Further surveys are necessary to detect presence of salmonellae in poultry and turkey meat products that could present a potential health threat. Keywords: Salmonella Enteritidis, turkey, multiplex-PCR, antimicrobial susceptibility profiles, resistance genes
ABSTRACT
INTRODUCTION
Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) is an important cause of human salmonellosis and food poisoning (1). S. Enteritidis has become prevalent in humans and on poultry farms as a result of vertical and horizontal transmission in and between large poultry organisations (2). Antimicrobial resistance is a growing public health problem which causes increased morbidity and mortality among humans and animals. Antimicrobials are used for treatment of infected animals , to protect them from infectious diseases, and to provide a faster growth rate. Overuse of antimicrobials may cause the
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selection, emergence and dissemination of resistant pathogens that are transmitted to humans through various routes, including the consumption of contaminated food. The resistance determinants of these organisms can be transferred also to the human gut bacterial flora, so becoming a reservoir of resistance genes for pathogenic bacteria. Treatments of infections due to resistant pathogens are more difficult and food borne diseases associated with antibiotic resistant bacteria have become a major public health issue (3, 4). Turkeys are a common source of Salmonella infection for the consumer. The isolation rates of Salmonella from turkeys and their environment are often higher than those from layers and broilers (5).
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The pathogenic ability of Salmonella depends on many virulence factors which are either chromosomal or plasmidic. Among these virulence factors, the chromosomally located gene, invA, encodes an inner membrane protein and provides the bacteria with the capability to invade epithelial cells as seen in cultured epithelial cells (6). There is little published information on the antimicrobial susceptibility profiles and antimicrobial resistance patterns of indigenous S. Enteritidis isolates. This information could help limit the spread of infections and provide data about the best choice for treatment. Resistance genes of antibiotic-resistant Salmonella spp. isolated from food animals have not been studied in Turkey. The aim of this study was to isolate salmonellae from turkeys slaughtered in İzmir province, to identify S. Enteritidis by classical techniques and polymerase chain reaction (PCR), to determine antimicrobial susceptibility profiles and antimicrobial resistance genes in antibiotic-resistant isolates.
MATERIALS AND METHODS
Bacteria A total of 587 cloacal swab samples were taken from turkeys at different ages (110-130 day old) from a turkey abattoir in Izmir province from March 2006 through March 2007. The cloacal swabs were transported in Carry-Blair transport medium (Oxoid) to the laboratory and cultured on the same day. All isolates identified as Salmonella were frozen for further studies. For all experiments, reference strain S. Typhimurium (ATCC 14028) served as positive control and quality control strains. Escherichia coli strain (ATCC 25922) and Staphylococcus aureus (ATCC 29213) as negative control were included. Isolation and Identification Salmonellae were isolated by standard methods (7). Nine ms of buffered peptone water (Oxoid) were inoculated with swabs. Samples were incubated for 18 h at 37°C, then 0.1 ml of the pre-enriched cultures were transferred to Rappaport-Vassiliadis (Oxoid) and Selenite broths (Difco) and incubated at 42 °C and 37 °C, respectively. After 24 and 48 h of incubation, 10µl of culture from each enriched broth was streaked on Salmonella Shigella (SS) agar (Difco) and xylose lysine deoxycholate (XLD) agar (Difco) and incubated at 37 °C for 24 h. The plates were examined for the presence of typical Salmonella colonies on SS and XLD agars (8). Suspected colonies were confirmed by conventional biochemical methods and also with a commercial kit (API 20E Kit System, Bio-Mérieux, France), following standard procedures for confirmation of genus level identifications. All colonies were tested serologically using specific Salmonella polyvalent O, monovalent O:9, and type-specific H antisera (Bio-Rad Laboratories, Inc. Richmond, CA, USA) (9).
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Extraction of DNA, Primers and Multiplex PCR For DNA extraction, a few colonies grown on selective agar was transferred to a 1.5 ml tube containing 100µl sterile lysis solution (1 ml 1M Tris-HCl, 22.5 µl IGEPAL CA-630 –Sigma-, 22.5 µl Tween 20 –Biorad-, 220 µl Protease K (10mg/ml), 8,8 µl DiH2O). The tubes were vortexed and incubated for 10 min at 60 °C, 10 min at 95 °C and cooled to 4 °C. Molecular identification was done by PCR. Salmonella spp. (invA) and S. Enteritidis (sefA) specific primer sets used in the multiplex-PCR assay were designed by Cortez et al. The sequences of forward and reverse primers were as follows: invA-1 (5’-TTGTTACGGCTATTTTGACCA-3’) invA-2 (5’-CTGACTGCTACCTTGCTGATG-3’), sefA-1 (5’-GCAGCGGTTACTATTGCAGC-3’), and sefA-2 (5’-TGTGACAGGGACATTTAGCG-3’), respectively. Multiplex PCR were done as described previously. This primer set generated amplicons of 521 bp and 330 bp, from invA and sefA, respectively using the method described (10). Detection of Minimal Inhibition Concentration Minimal inhibitory concentration (MIC) tests were carried out on Mueller-Hinton agar (Acumedia, USA) containing serial two-fold serial dilutions of the antibiotics. Twelve concentrations of tetracycline, ampicillin, chloramphenicol, gentamicin, cefoxitin, ciprofloxacin, and amikacin ranging from 0.06 to 128 mg/L were tested. The inocula were prepared by diluting overnight Brain Hearth Infusion Broth (Difco, USA) cultures to yield ~104 cfu per spot before testing. The plates were spot-inoculated with a Steers-type multipoint inoculator (AQS Manufacturing, UK) and incubated for 18 h at 37°C. Breakpoints indicated by CLSI (11) used for determination of susceptibility and resistance were assessed daily. Quality control strains, were assessed daily (each strain three times a week) from pure subcultures, and the inhibition zones were within the limits as defined by the CLSI. Resistance Genes Common resistance genes were detected for tetracycline, ampicillin, chloramphenicol and gentamicin by PCR using the conditions described previously. All primers used are shown in Table 1 (12, 13, 14, 15, 16, 17).
RESULTS
Isolation, Identification and PCR Salmonella strains were isolated from 86 out of 587 (14.7 %) cloacal swab samples. All strains were positive for the invA gene , specific for Salmonella genus. Of 86 isolates, 11 (1.9 %) were positive for the sefA gene, and were designated as serovar Enteridis. The presence of these genes was tested by multiplex PCR as shown in Figure 1. These strains were also positive by invA PCR
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All 86 Salmonella isolates were positive by Salmonella polyvalent O antisera.. Salmonella isolates were tested with monovalent O:9 antisera and 11 isolates were positive, and all were in serogroup D1. All 11 strains reacted also with “g,m” from phase 1 antisera and with “1,7” from phase 2 antisera in the agglutination test. Thus, the 11 strains had the following antigenic formula: “O9, 12:g,m:1,7” which corresponds to S. Enteridis serovar. These 11 isolates were also positive by sefA PCR. Antimicrobial susceptibility profiles All strains were susceptible to cefoxitin, ciprofloxacin and amikacin. Of 11 strains tested 9 (81.8%), 6 (54.6%), 6 (54.6%), and 3 (27.3%) were susceptible to gentamicin, chloramphenicol, ampicillin, and tetracycline respectively. Of the tetracycline resistant strains, 3, 1, 3, and 1 strain had MICs of 16, 32, 64 and 128 mg/L respectively. Five ampicillin resistant strains all had MICs 128 mg/L. Five were resistant to chloramphenicol with MICs 32 mg/L (4 strain), and 128 mg/L (1 strains). Two were resistant to gentamicin with MICs 16 mg/L (Table 2). The distribution of resistance and susceptibility rates of isolated Salmonella strains are shown in Figure 2. Of 11 strains 9 (81.8%) were antibiotic resistant isolates. Two strains were found to be resistant to only one antimicrobial, while 7 were multi-drug resistant. Two strains were susceptible to all antibiotics used. Resistance genes Presence of known resistance genes was studied among resistant strains. Of 8 tetracycline resistant strains, 5 and 3 carried tet(A), and tet(B) genes, respectively. Of 5 ampicillin resistant strains 3 had blaTEM and 2 had blaOXA. Of 5 chloramphenicol resistant strains one had cat3, 3 had floR .Known resistance genes were undetected by PCR in one resistant strain. Of the two gentamicin resistant strains, 1 had aadB and 1 had aacC genes (Table 2).
DISCUSSION
Salmonellae can be isolated from numerous animal species including turkeys because these strains are part of the normal flora in these species. For this reason the intestinal tract is the primary reservoir of zoonotic Salmonella. S. Enteritidis is now the most common cause of salmonellosis. Many studies show different rates of Salmonella isolates in cloacal swabs and stool samples in turkeys world-wide, with a wide range of rates reported. In some studies the rates are as low as 3.3% (18) and in others as high as 53.8 % (19). In Turkey only a few studies have shown the presence of Salmonella in turkeys, Aksakal et al. found isolation rates of 5% in eastern Turkey (20). In another study from north-eastern Turkey, no Salmonella was isolated from 42 samples (21). In this study, the isolation rate of Salmonella serovars from cloacal swabs was 14.7%. The
differences in isolation rates may be due to differences in isolation and sampling methods used, and geographic characteristics of regions where the studies were done. Increasing rates of antimicrobial resistance among bacteria including Salmonella is a growing health care problem that needs continuous survey. Antimicrobial resistance of Salmonella has double importance. Firstly for the treatment of poultry in which they cause infections, and also for human infections that they cause (1,2). One of the main factors for development of antimicrobial resistance is the over-use of antimicrobials. Of the most common antimicrobial resistance in Salmonella, quinolone, aminoglycoside, beta-lactam and tetracycline resistance can be cited. The resistance levels show wide differences among reports. Blackburn et al. reported that all Salmonella strains isolated from turkeys were resistant to gentamicin (22). Resistance rates were low among Salmonella isolated in Denmark. Pederson et al. stated that 1.7 %, 1.7 %, 8.7 %, and 9 % of strains isolated from Danish turkeys between 1995 and 2000, were resistant to gentamicin, trimethoprim/sulphamethoxazole, tetracycline and streptomycin, respectively (23). However high level resistance data were reported from Canada. Poppe et al. found that 25.8 %, 38 %, 14.3 %, 1.7 %, 2.4 %, and 27.7 % of isolated strains in Canada from turkeys were resistant to gentamicin, tetracycline, ampicillin, trimethoprim/ sulphamethoxazole, chloramphenicol, and kanamycin, respectively (24). Aksakal also reported that 20.0 %, 25.0 %, 35.0 %, 35.0 %, 65.0 % of Salmonella strains isolated from turkey stools in the Van province of Turkey were resistant to trimethoprim/sulphamethoxazole, ampicillin, gentamicin, tetracycline, and penicillin G, respectively (20). In the study of 11 S. Enteritidis strains tested 2 (18.2%), 5 (45.5%), 5 (45.5%), and 8 (72.7%) were resistant to gentamicin, chloramphenicol, ampicillin, and tetracycline respectively. None of the isolate was resistant to ciprofloxacin, cefoxitin and amikacin. Also an analysis of multidrug resistance levels was seen to be important among Salmonella in our study, since 7 (63.6%) strains were multi-drug resistant. Tetracycline resistance genes were detected among all 8 tetracycline-resistant isolates. The distribution was as follows: 5 tet(A), 3 tet(B). Roberts reported tet(B) and tet(C) tetracycline resistance genes to occur most frequently in salmonellae (25). However Pierano et al. reported that they found 25 tet(A), 23 tet(D) and 10 tet(C) (26). The most common tetracycline resistance determinant was also tet(A) in chickens (27, 28, 29) as well as in humans (28). Tetracycline resistance was also the most common resistance in our study like previous reports. The high level resistance to tetracycline may be due to this antibiotic being one of the most commonly used antibiotic for animal production. Among 5 ampicillin resistant isolates, 3 contained blaTEM and 2
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strains carried blaOXA. None of the isolates was positive for blaPSE-1. Resistance to ampicillin in Salmonella is usually mediated by TEM type β-lactamases which was the most common gene found among turkey isolates in the present study (26). Of the 5 chloramphenicolresistant isolates, the resistance genes, cat3 and floR were found in 1 and 3 isolates, respectively. One isolates were negative for cat1, cat2 and cat3 genes as well as for floR gene. Gentamicin resistance is usually due to inactivation of these antibiotics by enzymes. In this study of the 2 gentamicin-resistant isolates, 1 carried aadB and the other carried the aacC gene. In most studies aadBassociated gentamicin resistance is more common (30, 31).
REFERENCES
1. Seo, K.H., Holt, P.S., Brackett, R.E., Gast, R.K. and Stone, H.D. Mucosal humoral immunity to experimental Salmonella enteritidis infection in chicken crop. Avian Dis. 46: 1015–1020, 2002. 2. O’Brien, J.P.D. Aspects of Salmonella enteritidis control in poultry. World’s Poultry Sci. Journal 46: 119-124, 1990. 3. Molla, B., Mesfin, A. and Alemayehu, D. Multiple antimicrobial-resistant Salmonella serotypes isolated from chicken carcasses and giblets in Debre Zeit and Addis ababa, Ethiopia. Ethiop. J. Health. Dev. 17: 131-149, 2003. 4. Schwarz, S. and Chaalus-Dancla, E. Use of antimicrobials in veterinary medicine and mechanisms of resistance. Vet. Res. 32: 201-225, 2001. 5. Irwin, R.J., Poppe, C., Messier, S., Finley, G.G. and Oggel, J.A. National survey to estimate the prevalence of Salmonella sp. among Canadian registered commercial turkey flocks. Can. J. Vet. Res. 58: 263-267, 1994. 6. Darwin, K.H. and Miller, V.L. Molecular basis of the interaction of Salmonella with the intestinal mucosa. Clin. Microbiol. Rev. 12: 405-428, 1999. 7. ISO 6579: Microbiology General Guidance on Methods for the Detection of Salmonella. International Organization of Standardization, Geneva, Switzerland, 1993. 8. Antunes, P., Reu, C., Sousa, J.C., Peixe, L.and Pestana N. Incidence of Salmonella from poultry products and their susceptibility to antimicrobial agents. Int.J. Food Microbiol. 82: 97-103, 2003. 9. Koneman, E.W., Allen, S.D., Janda, W.M. and Schreckenberger, P.C.: Color Atlas and Textbook of Diagnostic Microbiology. 5th ed. Philadelphia, Lippincott, USA, 1997. 10. Cortez, A.L.L., Carvalho, A.C.F.B., Ikuno, A.A., Burger, K.P. and Vidal-Martins, A.M.C. Identification of Salmonella spp. isolates from chicken abattoirs by
multiplex-PCR. Res.Vet. Sci. 81: 340–344, 2006. 11. Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing: Seventeenth Informational Supplement M100-S17. Wayne, PA, USA: CLSI, 2007. 12. Carlson, S.A., Bolton, L.F., Briggs, C.E., Hurd, H.S. Sharma, V.K., Fedorka-Cray, P.J. and Jones, B.D. Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR. Mol. Cell. Probes 13: 213–222, 1999. 13. Fonseca, E.L., Mykytczuk, O.L., Asensi, M.D., Reis, E. M. F., Ferraz, L. R., Paula, F.L., L.K. , Ng. And Rodrıgues, D.P. Clonality and antimicrobial resistance gene profiles of multidrug-resistant Salmonella enterica serovar infantis isolates from four public hospitals in Rio de Janeiro, Brazil. J. Clin. Microbiol. 44: 2767-2772, 2006. 14. Gallardo, F., Ruiz, J., Marco F., Towner, K.J. and Vila, J. Increase in incidence of resistance to ampicillin, chloramphenicol and trimethoprim in clinical isolates of Salmonella serotype Typhimurium with investigation of molecular epidemiology and mechanisms of resistance. J. Med. Microbiol. 48: 367–374, 1999. 15. Lynee, A.M., Rhodes-Clark, B.S., Bliven, K., Zhao, S. and Foley, S.L. Antimicrobial resistance genes associated with Salmonella enterica serovar Newport isolates from food animals. Antimicrob. Agents Chemother. 52: 353-356. 2008. 16. Randall, L.P., Cooles, S.W., Osborn, M.K., Piddock, L.J.V. and Woodward, M.J. Antibiotic resistance genes, integrons and multiple antibiotic resistances in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK. J. Antimicrob. Chemother. 53: 208-216, 2004. 17. Smith, M.S., Yang, R.K., Knapp, C.W., Niu, Y., Peak, N., Hanfelt, M.M., Galland, J.C. and Graham, D.W. Quantification of tetracycline resistance genes in feedlot lagoons by real-time PCR. Appl. Environ. Microbiol. 70: 7372–7377, 2004. 18. Arora, A.K., Gupta, S.C. and Kaushik, R.K. Isolation on Salmonella gallinarum from turkeys. Indian Vet. J. 65: 171, 1988. 19. Hoover, N.J., Kenney, P.B., Amick, J.D. and Hypes, W.A. Preharvest sources of Salmonella colonisation in Turkey production. Poultry Sci. 76: 1232-1238, 1997. 20. Aksakal, A.: The occurance and prevalence Salmonella species in faeces of certain fowls and their susceptibility to antibiotics (In Turkish). YYÜ Vet. Fak. Derg. 14: 95-101, 2003. 21. Genç, O. and Otlu S. Salmonella isolations from different avian species in Kars distinct of Turkey. (In Turkish). Kafkas Üniv. Vet. Fak. Derg. 11: 25-27,
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2005. 22. Blackburn, O.B., Sclater, L.K. and Swanson, M.R. Antibiotic resistance of members of THA genus Salmonella isolated from chicken, turkeys, cattle, and swine in the United States during October 1981 through September. Am. J.Vet Res. 45: 1245-1249, 1982. 23. Pederson, K., Hansen, H.C., Jorgensen, J.C. and Borck B. Serovars of Salmonella isolated from Danish turkeys between 1995 and 2000 and their antibiotic resistance. Vet. Rec. 150: 471-474, 2002 24. Poppe, C., Kolar, J.J., Demckzuk, W.H.B. and Harris, J.E. Drug resistance and biochemical characteristics of salmonellae from turkeys. Can. J. Vet. Res. 1995, 59: 241-248. 25. Roberts, M.C. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 245: 195-203, 2005. 26. Pierano, G., Agerso, Y., Aarestrup, F.M. and Rodrigues, D.P. Occurrence of integrons and antimicrobial resistance genes among Salmonella enterica from Brazil. J. Appl. Antimic. Chemot. 58: 305-309, 2006. 27. Asai, T., Itagaki, M., Shiroki Y., Yamada M., Tokoro M., Kojima A., Ishihara K., Esaki H., Tamura Y. and Takahashi T. Antimicrobial resistance types and
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genes in Salmonella enterica Infantis isolates from retail raw chicken meat and broiler chickens on farms. J. Food Prot. 69: 214–216, 2006. Hamada, K., Oshima, K. and Tsuji, H. Drug resistance genes encoded in integrons and in extraintegrons: their distribution and lateral transfer among pathogenic enterobacteriaceae including enterohemorrhagic Escherichia coli and Salmonella enterica serovars Typhimurium and Infantis. Jpn. J. Infect. Dis.56: 123–126, 2003. Shahada F., Chuma T., Tobata T., Okamoto K., Sueyoshi M. and Takase K. Molecular epidemiology of antimicrobial resistance among Salmonella enterica serovar Infantis from poultry in Kagoshima, Japan. Int. J. Antimicrob. Agents 28: 302–307, 2006. Wang, Z. and Rossman T.G. Large-scale super coiled plasmid 14 preparation by acidic phenol extraction. Biotechniques 16: 460-463, 1994. Zhao, S., Qaiyumi, S., Friedman, S., Singh, R., Foley, S.L., White, D.G., Mcdermott, P.F., Donkar, T., Bolin, C., Munro, S., Baron, E.J. and Walker R.D. Characterization of Salmonella enterica serotype newport isolated from humans and food animals. J. Clin. Microbiol. 41: 5366-5371, 2003.
Table 1: Primers used in PCR for the detection of resistance genes
Gene Primer* tetA tetB tetC tetG tetS blaTEM blaPSE-1 blaOXA aadB aacC cat1 cat2 cat3 floR F R F R F R F R F R F R F R F R F R F R F R F R F R F R Nucleotide Sequence (5’ to 3’) GCTACATCCTGCTTGCCTTC CATAGATCGCCGTGAAGAGG TTGGTTAGGGGCAAGTTTTG GTAATGGGCCAATAACACCG CTTGAGAGCCTTCAACCCAG ATGGTCGTCATCTACCTGCC GCTCGGTGGTATCTCTGCTC AGCAACAGAATCGGGAACAC CATAGACAAGCCGTTGACC ATGTTTTTGGAACGCCAGAG CATTTCCGTGTCGCCCTTAT TCCATAGTTGCCTGACTCCC AATGGCAATCAGCGCTTCCC GGGGCTTGATGCTCACTACA ACCAGATTCAACTTTCAA TCTTGGCTTTTATGCTTG GAGCGAAATCTGCCGCTCTGG CTGTTACAACGGACTGGCCGC GGCGCGATCAACGAATTTATCCGA CCATTCGATGCCGAAGGAAACGAT CCTATAACCAGACCGTTCAG TCACAGACGGCATGATGAAC CCGGATTGACCTGAATACCT TCACATACTGCATGATGAAC CCCACAATTCACCGTATTCC GAACCTGTACTGAGAGCGGC AACCCGCCCTCTGGATCAAGTCAA CAAATCACGGGCCACGCTGTATC Annealing Temperature 55 53 56 59 58 55 59 55 61 58 56 56 58 60 Resistance Mechanism Efflux Efflux Efflux Efflux Ribosomal protection β-lactamase β-lactamase β-lactamase Aminoglycoside adenyltransferase Aminoglycoside acetyltransferase Chlorampenicol acetyltransferase Chlorampenicol acetyltransferase Chlorampenicol acetyltransferase Efflux Size (bp) 210 659 417 468 667 793 586 590 319 488 491 567 310 548 Encoded Resistance** TET TET TET TET TET AMP AMP AMP GEN GEN CHL CHL CHL CHL Reference 13 13 13 12 17 16 12 14 16 15 16 16 EU715370.1 16
*F, forward; R, reverse; **TET: Tetracycline, AMP: Ampicillin GEN: Gentamicin CHL: Chloramphenicol
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Table 2: Distribution of resistant strains and resistance genes among S. Enteritidis strains
Drug** No of isolates having MIC(mg/L) 0,06 0 0 0 0,125 0,25 0 0 0 0 0 0 0,5 0 0 0 1 0 0 0 2 1 0 1 4 2 3 2 8 0 1 3 16 32 3 2 0 1 0 4 Breakpoint 64 128 (mg/L) 3 0 0 1 5 1 > 16 > 32 > 32 No of resistant Genes tested isolates and (%) tetA tetB tetC 8 (72.7) tetG tetS blaTEM 5 (45.4) blaOXA blaPSE-1 floR cat3 5 (45.4) cat2 cat1 unknown GEN CEF CIP AMI 0 9 9 0 0 2 1 0 0 0 1 0 0 0 0 6 0 0 0 4 2 0 0 1 7 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 > 16 > 32 >4 > 64 2 (18.2) 0 0 0 aadB aacC No of isolates 5 3 3 2 3 1 1 1 1
TET AMP CHL
**TET: Tetracycline, AMP: Ampicillin, CIP:Ciprofloxacin, AMI: Amikacin
GEN: Gentamicin, CHL: Chloramphenicol, CEF: Cefoxitin,
Figure 1. Salmonella specific genes invA and S. Enteritidis specific sefA were determined by PCR (10). Salmonella spp. is all positive for invA and only Salmonella Enteritidis isolates were positive for sefA. Lane M = 100 bp Marker Lane 1 = Salmonella Typhimurium (positive control) Lanes 2-9 = sefA positive S. Enteritidis strains from our study. Lane 10. E. coli (negative control) Lane 11= Negative control without DNA.
Figure 2. The distribution of resistance and susceptibility rates of isolated S. Enteritidis strains
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