Open Access
Research (Published online: 25-06-2017)
21. Tetracycline resistance phenotypes and genotypes of coagulase-negative staphylococcal isolates from bubaline mastitis in Egypt
K. A. Abd El-Razik, A. A. Arafa, R. H. Hedia and E. S. Ibrahim
Veterinary World, 10(6): 702-710

K. A. Abd El-Razik: Department of Animal Reproduction, Veterinary Division, National Research Center, Dokki, Giza, Egypt.
A. A. Arafa: Department of Microbiology and Immunology, Veterinary Division, National Research Center, Dokki, Giza, Egypt.
R. H. Hedia: Department of Microbiology and Immunology, Veterinary Division, National Research Center, Dokki, Giza, Egypt.
E. S. Ibrahim: Department of Microbiology and Immunology, Veterinary Division, National Research Center, Dokki, Giza, Egypt.

doi: 10.14202/vetworld.2017.702-710

Share this article on [Facebook] [LinkedIn]

Article history: Received: 21-01-2017, Accepted: 08-05-2017, Published online: 25-06-2017

Corresponding author: K. A. Abd El-Razik


Citation: El-Razik KAA, Arafa AA, Hedia RH, Ibrahim ES (2017) Tetracycline resistance phenotypes and genotypes of coagulase-negative staphylococcal isolates from bubaline mastitis in Egypt, Veterinary World, 10(6): 702-710.

Aim: This study was devoted to elucidate the tetracycline resistance of coagulase-negative staphylococci (CNS) derived from normal and subclinical mastitic (SCM) buffaloes' milk in Egypt.

Materials and Methods: A total of 81 milk samples from 46 normal buffalo milk samples and 35 SCM buffalo milk samples at private dairy farms of Egypt were used in this study. CNS were identified using phenotypic and molecular methods (polymerase chain reaction [PCR]). CNS isolates were tested for tetracycline resistance using routine methods and multiplex PCR targeting tetracycline (tet) resistance genes followed by sequencing of positive PCR products and phylogenetic analysis.

Results: Isolation and identification of 28 (34.5%) CNS from normal and SCM buffaloes' milk, namely, Staphylococcus intermedius (39.2%), Staphylococcus xylosus (25.0%), Staphylococcus epidermidis (10.7%), Staphylococcus hominis (10.7%), and 3.5% to each of Staphylococcus sciuri, Staphylococcus hyicus, Staphylococcus lugdunensis, and Staphylococcus simulans. Using nested PCR, all the 28 CNS isolates revealed positive for 16srRNA gene specific for genus staphylococci and negative for thermonuclease (nuc) gene specific for Staphylococcus aureus species. The presence of tetracycline resistance-encoding genes (tetK, tetL, tetM, and tetO) was detected by multiplex PCR. All isolates were negative for tetL, M, and O genes while 14 (50%) CNS isolates were positive for tetK gene, namely, S. lugdunensis (100%), S. hominis (100%), S. epidermidis (66.6%), S. intermedius (45.4%), and S. xylosus (42.8%). Nucleotide sequencing of tetK gene followed by phylogenetic analysis showed the high homology between our CNS isolates genes of tetracycline resistance with S. aureus isolates including Egyptian ones. This proves the transfer of the tetracycline resistance encoding genes between coagulase-negative and coagulase-positive Staphylococcus spp.

Conclusion: CNS isolates have distinguishingly high resistance to tetracycline. Abundant tetracycline usage for mastitis treatment leads to the spread of genetic resistance mechanisms inside CNS strains and among all Staphylococcus spp. Consequently, tetracycline is not effective anymore.

Keywords: buffaloes, coagulase-negative staphylococci, mastitis, tetracycline resistance, tetK gene.


1. Bogni, C., Odierno, L., Raspanti, C., Giraudo, J., Larriestra, A., Reinoso, E., Lasagno, M., Ferrari, M., Ducros, E., Frigerio, C., Bettera, S., Pel-legrino, M., Frola, I., Dieser, S. and Vissio, C. (2011) War against mastitis: current concepts on controlling bovine mastitis pathogens. In: Mendez-Vilas A, editor. Science against Microbial Pathogens: Communicating Current Research and Technological Advances. Zaragoza, Espana: Formatex Research Center. p483-94.

2. Park, Y., Fox, L., Hancock, D., McMahan, W. and Park, Y. (2012) Prevalence and antibiotic resistance of mastitis pathogens isolated from dairy herds transitioning to organic management. J. Vet. Sci., 13: 103-105. [Crossref] [PubMed] [PMC]

3. Supre, K., Haesebrouck, F., Zadoks, R.N., Vaneechoutte, M., Piepers, S. and de Vliegher, S. (2011) Some coagulase-negative Staphylococcus species affect udder health more than others. J. Dairy Sci., 94: 2329-2340. [Crossref] [PubMed]

4. Oliver, S.P., Gillespie, B.E., Headrick, S.J., Moorehead, H., Lunn, P., Dowlen, H.H., Johnson, D.L., Lamar, K.C., Chester, S.T. and Moseley, W.M. (2004) Efficacy of extended ceftiofur intramammary therapy for treatment of subclinical mastitis in lactating dairy cows. J. Dairy Sci., 87: 2393-2400. [Crossref]

5. Schwarz, S., Roberts, M.C., Werckenthin, C., Pang, Y. and Lange, C. (1998) Tetracycline resistance in Staphylococcus spp. From domestic animals. Vet. Microbiol., 63: 217-227. [Crossref]

6. Roberts, M.C. (1996) Tetracycline resistance determinants: Mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol. Rev., 19: 1-24. [Crossref] [PubMed]

7. Irlinger, F. (2008) Safety assessment of dairy microorganisms: Coagulase-negative staphylococci. Int. J. Food Microbiol., 126: 302-310. [Crossref] [PubMed]

8. Osman, K.M., Abd El-Razik, K.A., Marie, H.S.H. and Arafa, A. (2016) Coagulase-negative Staphylococci collected from bovine milk: Species and antimicrobial gene diversity. J. Food Saf., 36: 89-99. [Crossref]

9. Thorberg, B.M., Kuhn, I., Aarestrup, F.M., Brandstrom, B., Jonsson, P. and Danielsson-Tham, M.L. (2006) Pheno-and genotyping of Staphylococcus epidermidis isolated from bovine milk and human skin. Vet. Microbiol., 115: 163-172. [Crossref] [PubMed]

10. Sampimon, O.C., Barkema, H.W., Berends, I.M.G, Sol, J. and Lam, T.J.G. (2009) Prevalence and herd-level risk factors for intramammary infection with coagulase-negative staphylococci in Dutch dairy herds. Vet. Microbiol., 134: 37-44. [Crossref] [PubMed]

11. FAO/WHO/OIE. (2008) Joint FAO/WHO/OIE Expert Meeting on Critically Important Antimicrobials. Report of a Meeting Held in FAO, Rome, Italy, and WHO, Geneva, Switzerland, November. 26-30.

12. WHO. (2009) World health organization critically important antimicrobials for human medicine 2nd Revision. WHO Advisory Group on Integrated Surveillance of Antimicrobial Resistance (AGISAR), Department of Food Safety and Zoonoses.

13. CLSI. (2012) Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Third Informational Supplement, CLSI, Wayne, PA.

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

15. Monday, S.R. and Bohach, G.A. (1999) Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates. J. Clin. Microbiol., 37: 3411-3414. [PubMed] [PMC]

16. 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]

17. Ng, L.K., Martin, I., Alfa, M. and Mulvey, M. (2001) Multiplex PCR for the detection of tetracycline resistant genes. Mol. Cell. Probes, 15: 209-215. [Crossref] [PubMed]

18. Felsenstein J. (1985) Phylogenies and the comparative method. Am. Nat., 125: 1-15. [Crossref]

19. Vanderhaeghen, W., Piepers, S., Leroy, F., van Coillie, E., Haesebrouck, F. and de Vliegher, S. (2014) Invited review: Effect, persistence, and virulence of coagulase negative Staphylococcus species associated with ruminant udder health. J. Dairy Sci., 97: 5275-5293. [Crossref]

20. Pyorala, S. and Taponen, S. (2009) Coagulase-negative staphylococci-emerging mastitis pathogens. Vet. Microbiol., 134: 3-8. [Crossref] [PubMed]

21. Osman, K.M., Abd El-Razik, K.A., Marie, H.S.H. and Arafa, A. (2015) Relevance of biofilm formation and virulence of different species of coagulase-negative Staphylococci to public health. Eur. J. Clin. Microbiol. Infect. Dis., 34: 2009-2016. [Crossref] [PubMed]

22. El-Ashker, M., Gwida, M., Tomaso, H., Monecke, S., Ehricht, R., El-Gohary, F. and Hotzel, H. (2015) Staphylococci in cattle and buffaloes with mastitis in Dakahlia Governorate, Egypt. J. Dairy Sci., 98: 1-10. [Crossref] [PubMed]

23. Soares, L.C., Pereira, I.A., Pribul, B.R., Oliva, M.S., Coelho, S.M.O. and Souza, M.M.S. (2012) Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis. Pesqui. Vet. Bras., 32: 692-696. [Crossref]

24. Booth, N. and Mcdonald, L.E. (1992) Farmacologiae Terapeuticaem Veterinaria. 6th ed. Guanabara Koogan, Rio de Janeiro. p. 997.

25. Jamali, H., Paydarb, M., Radmehrc, B., Salmah, I. and Dadrasniaa, A. (2015) Prevalence and antimicrobial resistance of Staphylococcus aureus isolated from raw milk and dairy products. Food Control, 54: 383-388. [Crossref]

26. Zadoks, R. and Watts, J. (2009) Species identification of coagulase-negative Staphylococci: Genotyping is superior to phenotyping. Vet. Microbiol., 134: 20-28. [Crossref]

27. Levy, S.B., McMurray, L.M., Barbosa, T.M., Burdett, V., Courvalin, P. and Hillen, W. (1999) Nomen-clature for new tetracycline resistance determinants. Antimicrob. Agents Chemother., 43: 523-524.

28. Jamali, H., Radmehrc, B. and Salmah, I. (2014) Short communication: Prevalence and antibiotic resistance of Staphylococcus aureus isolated from bovine clinical mastitis. J. Dairy Sci., 97: 2226-2230. [Crossref]

29. Walther, C. and Perreten, V. (2007) Methicillin-resistant Staphylococcus epidermidis in organic milk production. J. Dairy Sci., 90: 5351. [Crossref] [PubMed]

30. Thorberg, B.M., Danielsson-Tham, M.L., Emanuelson, U. and PerssonWaller, K. (2009) Bovine subclinical mastitis caused by different types of coagulase-negative Staphylococci. J. Dairy Sci., 92: 4962-4970. [Crossref] [PubMed]

31. Buttner, S., Flechtner, O., Muntener, C. and Overesch, G. (2011) Berichtuber den Vertrieb von Antibiotika in der Veterinarmedizin und das Antibiotika resistenz monitoring bei Nutztieren in der Schweiz(ARCH-VET 2010). Federal Veterinary Office and Swissmedic, Bern, Switzerland. Available from: Last accessed on 26-12-2016.

32. 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]

33. Klimiene, I., Virgailis, M., Pavilonis, A., Siugzdiniene, R., Mockeliunas, R. and Ruzauskas, M. (2016) Phenotypical and genotypical antimicrobial resistance of coagulase-negative Staphylococci isolated from cow mastitis. Pol. J. Vet. Sci., 19: 639-646. [Crossref] [PubMed]

34. Cengiz, S., Dinc, G. and Cengiz, M. (2015) Evaluation of antimicrobial resistance in Staphylococcus spp. Isolated from subclinical mastitis in cows. Pak. Vet. J., 35: 334-338.