Open Access
Research (Published online: 05-06-2017)
5. Molecular characterization of antibiotic-resistant Staphylococcus aureus from livestock (bovine and swine)
Asima Zehra, Randhir Singh, Simranpreet Kaur and J. P. S. Gill
Veterinary World, 10(6): 598-604

Asima Zehra: School of Public Health and Zoonoses, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
Randhir Singh: School of Public Health and Zoonoses, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
Simranpreet Kaur: School of Public Health and Zoonoses, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
J. P. S. Gill: School of Public Health and Zoonoses, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.

doi: 10.14202/vetworld.2017.598-604

Share this article on [Facebook] [LinkedIn]

Article history: Received: 15-12-2016, Accepted: 15-04-2017, Published online: 05-06-2017

Corresponding author: Asima Zehra

E-mail: assimazehra@gmail.com

Citation: Zehra A, Singh R, Kaur S, Gill JPS (2017) Molecular characterization of antibiotic-resistant Staphylococcus aureus from livestock (bovine and swine), Veterinary World, 10(6): 598-604.
Abstract

Aim: The aim of this study was to figure the prevalence, phenotypic and genotypic antibiotic resistance (AR) pattern of Staphylococcus aureus isolated from bovine and swine nares.

Materials and Methods: Colonies with typical morphology on Baird-Parker agar supplemented with egg-yolk tellurite emulsion were selected and biochemically/genotypically identified as S. aureus. These strains were further subjected to epsilometer test for their sensitivity to various clinically important antibiotics and antibiotic susceptibility testing for amoxicillin/clavulanic acid, and double-disk diffusion testing was performed by the standard disc diffusion method following CLSI guidelines. S. aureus strains were also tested for the presence of AR genes, viz., blaZ, mecA, aacA-aphD, erm (ermA, ermB, ermC), tet (efflux genes tetK and tetL, tetM and tetO of the ribosomal protection family), and vanA.

Results: The nasal cavities of 17 out of 47 randomly selected bovine and 20 out of 28 randomly selected swine were positive for S. aureus, representing the prevalence of 36.2% (95% confidence interval [CI]: 22.5-49.9) and 71.4% (95% CI: 54.7-88.1), respectively. Most of the S. aureus strains showed higher resistance to penicillin (94.6%, minimal inhibitory concentration [MIC] =1.5 μg/ml) followed by ciprofloxacin (56.7%, MIC =32 μg/ml) and tetracycline (18.9%, MIC =32 μg/ml). About 10-15% of the strains were resistant to gentamicin (MIC 16 μg/ml) and oxacillin (MIC 6-8 μg/ml). None of the strains were resistant to vancomycin (MIC 0.25-1.5 μg/ml). In this study, 32.4% strains were resistant to three or more than three antibiotics and prevalence of this multi-drug resistant S. aureus was 45% (95% CI: 26.6-63.4) and 17.6% (95% CI: 6.7- 28.5) in swine and bovine nasal samples, respectively. Four strains from pigs were borderline oxacillin-resistant S. aureus MIC 6-8 μg/ml, but none were mecA positive. Two of these strains were β-lactamase hyperproducers. Among the resistance genes blaZ, tetK, tetL, tetM, ermB, and aacA-aphD were found.

Conclusion: Our results demonstrated the absence of mecA and pvl gene, but the presence of multi-drug resistant S. aureus in the nares of healthy animals which has a potential to spread in a community.

Keywords: antibiotic resistance genes, epsilometer test, livestock nasal swabs, multidrug resistance, Staphylococcus aureus.

References

1. Ogata, K., Narimatsu, H., Suzuki, M., Higuchi, W., Yamamoto, T. and Taniguchi, H. (2012) Commercially distributed meat as a potential vehicle for community-acquired methicillin-resistant Staphylococcus aureus. Appl. Environ. Microbiol., 78: 2797-2802. [Crossref] [PubMed] [PMC]

2. Larsen, R.A. (2014) ESCMID Online presentation on the Emergence and Spread of MRSA CC398. Event: Molecular typing methods for pathogens, Lyon, france. PGEC lecture Diagnostic Bacteriology and General Microbiology. https://www.escmid.org/escmid_publications/escmid_elibrary/?q=Larsen&id=2173&L=0&x=0&y=0, Accessed on 24-08-2016.

3. Bennett, R.W. and Lancette, G.A. (2001) Staphylococcus aureus. In: FDA Bacteriological analytical manual. 8th ed. Ch. 12. AOAC International, Gaithersburg, MD.

4. Clinical and Laboratory Standards Institute. (2011) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard, M100-S21. Vol. 31. Clinical and Laboratory Standards Institute, Wayne, PA, 2011.

5. Strommenger, B., Kettlitz, C., Werner, G. and Witte, W. (2003) Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J. Clin. Microbiol., 41(9): 4089-4094. [Crossref] [PMC]

6. Martineau, F., Picard, J.F., Lansac, N., Menard, C., Roy, H.P., Ouellette, M. and Bergeron, G.M. (2002) Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob. Agents Chemother., 44(2): 231-238. [Crossref]

7. Huys, G., D'Haene, K., Van Eldere, J., von Holy, A. and Swings, J. (2005) Molecular diversity and characterization of tetracycline-resistant Staphylococcus aureus isolates from a poultry processing plant. Appl. Environ. Microbiol., 71: 574-579. [Crossref] [PubMed] [PMC]

8. Saha, B., Singh, AK., Ghosh, A. and Bal, M. (2008) Identification and characterization of a vancomycin-resistant Staphylococcus aureus isolated from Kolkata (South Asia). J. Med. Microbiol., 57(1): 72-79. [Crossref] [PubMed]

9. Hookey, J.V., Richardson, J.F. and Cookson, B.D. (1998) Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene. J. Clin. Microbiol., 36(4): 1083-1089. [PubMed] [PMC]

10. Brakstad, O.G., Aasbakk, K. and Maeland, J.A. (1992) Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol., 30: 1654-1660. [PubMed] [PMC]

11. Kadlec, K., Febler, T.A., Hauschild, T. and Schwarz, S. (2012) Novel and uncommon antimicrobial resistance genes in livestock associated methicillin-resistant Staphylococcus aureus. Clin. Microbiol. Infect., 18: 745-755. [Crossref] [PubMed]

12. Ugwu, C.C., Gomez-Sanz, E., Agbo, C.I., Torres, C. and Brazilian, F.K. (2015) Characterization of mannitol-fermenting methicillin-resistant Staphylococci isolated from pigs in Nigeria. J. Microbiol., 46(3): 885-892. [Crossref]

13. Centre for Science and Environment (CSE). (2014) The Latest Study by CSE's Pollution Monitoring Lab Finds Antibiotic Residues in Chicken. Food Safety and Toxins. Available from: http://www.cseindia.org/node/5487, Accessed on 14-08-2016.

14. Frye, J.G. and Fedorka-Cray, P.J. (2007) Prevalence, distribution and characterization of ceftiofur resistance in Salmonella enterica isolated from animals in the USA from 1999 to 2003. Int. J. Antimicrob. Agents, 30: 134-142. [Crossref] [PubMed]

15. Duran, N., Ozer, B., Duran, G.G., Onlen, Y. and Demir, C. (2012) Antibiotic resistance genes and susceptibility patterns in Staphylococci. Indian J. Med. Res., 135: 389-396. [PubMed] [PMC]

16. Morcillo, A., Castro, B., Rodriguez-Alvarez, C., Abreu, R., Aguirre-Jaime, A. and Arias, A. (2015) Descriptive analysis of antibiotic-resistant patterns of methicillin-resistant Staphylococcus aureus (MRSA) st398 isolated from healthy swine. Int. J. Environ. Res. Publ. Health, 12: 611-622. [Crossref] [PubMed] [PMC]

17. de Neeling, A.J., van den Broek, M.J., Spalburg, E.C., van Santen-Verheuvel, M.G., Dam-Deisz, W.D., Boshuizen, H.C., van de Giessen, A.W., van Duijkeren, E., Huijsdens, X.W. (2007) High prevalence of methicillin resistant Staphylococcus aureus in pigs. Vet. Microbiol., 122: 366-372. [Crossref] [PubMed]

18. Zhang, C., Song, L., Chen, H., Liu, Y., Qin, Y. and Ning, Y. (2012) Antimicrobial susceptibility and molecular subtypes of Staphylococcus aureus isolated from pig tonsils and cow's milk in China. Can. J. Vet. Res., 76: 268-274. [PubMed] [PMC]

19. Schmithausen, R.M., Schulze-Geisthoevel, S.V., Stemmer, F., El-Jade, M., Reif, M., Hack, S., Meilaender, A., Montabauer, G., Fimmers, R., Parcina, M., Hoerauf, A. and Exner, M. (2015) Analysis of transmission of MRSA and ESBL-E among pigs and farm personnel. PloS One, 10: e0138173. [Crossref]

20. Frey, Y., Rodriguez, J.P., Thomann, A., Schwendener, S. and Perreten, V. (2013) Genetic characterization of antimicrobial resistance in coagulase-negative Staphylococci from bovine mastitis milk. J. Dairy Sci., 96: 2247-2257. [Crossref] [PubMed]

21. Yang, F., Wang, Q., Wang, X., Wang, L., Xiao, M., Li, X., Luo, J., Zhang, S. and Li, H. (2015) Prevalence of blaZ gene and other virulence genes in penicillin-resistant Staphylococcus aureus isolated from bovine mastitis cases in Gansu, China. Turk. J. Vet. Anim. Sci., 39: 634-636. [Crossref]

22. Croes, S., Deurenberg, R.H., Boumans, M.L.L., Beisser, P.S., Neef, C. and Stobberingh, E.E. (2009) Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. Aureus lineage. BMC Microbiol., 9: 229. [Crossref] [PubMed] [PMC]

23. Shore, A.C. and Coleman, D.C. (2013) Staphylococcal cassette chromosome mec: Recent advances and new insights. Int. J. Med. Microbiol., 303: 350-359. [Crossref] [PubMed]

24. Laurent, F., Chardon, H., Haenni, M., Bes, M., Reverdy, M.E., Madec, J.Y., Lagier, E., Vandenesch, F. and Tristan, A. (2012) MRSA harboring mec A variant gene mec C in France. Emerg. Infect. Dis., 18: 1465-1467. [Crossref] [PubMed] [PMC]

25. Pereira, V., Lopes, C., Castro, A., Silva, J., Gibbs, P. and Teixeira, P. (2009) Characterization for enterotoxin production, virulence factors, and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal. Food Microbiol., 26: 278-282. [Crossref] [PubMed]

26. Balslev, U., Bremmelgaard, A., Svejgaard, E., Havstreym, J. and Westh, H. (2005) An outbreak of borderline oxacillin-resistant Staphylococcus aureus (BORSA) in a dermatological unit. Microb. Drug Resist., 11: 78-81. [Crossref] [PubMed]

27. Krupa, P., Bystron, J., Podkowik, M., Empel, J., Mroczkowska, A. and Bania, J. (2015) Population Structure and Oxacillin Resistance of Staphylococcus aureus from Pigs and Pork Meat in South-West of Poland. Bio. Med. Res. Int. DOI: 10.1155/2015/141475. [Crossref]

28. Oppliger, A., Moreillon, P., Charriere, N., Giddey, M., Morisset, D. and Sakwinska, O. (2012) Antimicrobial resistance of Staphylococcus aureus strains acquired by pig farmers from pigs. Appl. Environ. Microbiol., 78: 8010-8014. [Crossref] [PubMed] [PMC]