Research Article of American Journal of Microbiology and Immunology
Detection of methicillin resistant and slime factor production of coagulase negative Staphylococcus spp. in bovine clinical mastitis by using PCR
S. M. El-Berbawy*; S.M. Sayed*;El-Toukhy, E. I. ** and Amal, A. Megahed***
* Assiut Lab. (Bacteriology Department)
** AHRI (Biotechnology Depart.) Dokki
*** Port Said Lab. (Bacteriology Depart.)
This study aims to investigate the slime production of Coagulase negative staphylococci (CoNS) isolates by phenotypic method on Congo Red Agar plates (CRA) and Genotypic detection of icaA, icaD and mecA genes by polymerase chain reaction (PCR). Out of 105 milk samples obtained from clinical bovine mastitis, 101samples (96.2%) were positive for bacterial growth. CoNS isolates was detected in 20 isolates with a percentage of 19.8%. Their ability to form biofilm as one of the most important virulence factors of the organisms using Congo Red Agar (CRA) method was investigated in which 13 out of 17 CoNS isolates (76.47%) were found to be slime producers. By PCR, mecA gene was found in threeout of 6 CoNS isolates (50%). Also six (100%) and three (50%) isolates were positive for icaA gene and icaD gene, respectively. In addition one isolate out of the six CoNS isolates (16.67%) was positive for the presence of icaA, icaD and mecA genes and also has the ability to form biofilm. The in vitro activities of CoNS against 11 selected antimicrobial agents referred that the highest resistance rate of CoNS observed to Lincomycin (100%), followed by Cefotaxime (94.41%), Oxacillin (58.82%), Ampicillin (47.06%) and Penicillin (41.18%), while the highest rate of sensitivity observed to Enrofloxacin and Gentamicin (100%, for each), followed by Doxycycline (94.11%).Conclusion, the findings of the present study demonstrated the ability of CoNS isolated from bovine clinical mastitis to form biofilms. This must be considered as an alarming situation, and so attention must be paid toward implementation of new ways for effective prophylaxis, control, and treatment of such infections in the dairy farms. The prudent use of antibiotics and rapid and continuous screening for resistant microorganisms should be more focused to prevent the emergence and spread methicillin resistant coagulase negative staphylococci, because these strains can cause severe damage to infected sites and may be widespread in the environment.
Keywords: Cows, clinical mastitis, coagulase-negative staphylococci, slime factor, mecA, icaA, icaD genes.
How to cite this article:
S. M. El-Berbawy, S.M. Sayed, El-Toukhy, E. I. , Detection of methicillin resistant and slime factor production of coagulase negative Staphylococcus spp. in bovine clinical mastitis by using PCR. American Journal of Microbiology and Immunology, 2016,1:4. DOI: 10.28933/ei-berbawy-ajmi-08-2016
1. Hend, M. S. El-Damaty. Study on the contagious and environmental bovine mastitis with special emphasis to subclinical form. Ph.D. Thesis, infectious diseases, Fac. of Vet. Med. ZagazigUnvi., 2013.
2. Pyörälä, S. and Taponen S. Coagulase-negative staphylococci emerging mastitis pathogens. Vet Microbiol., 2009; 134:3–8.
3. Idriss, Sh. E.; Foltys, V.; Tančin,V. et al. Mastitis pathogens in milk of dairy cows in Slovakia. Slovak J. Anim. Sci., 2013; 46(3): 115-119.
4. Bochniarz M. and Wawron, W. Haemolytic and proteolytic activity of coagulase-negative staphylococci isolated from mastitis cows. Pol. J. Vet. Sci.,2012; 15: 61-65.
5. Oliveira, M.; Bexiga, R.; Nunes, S. F. et al. Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidismastitis isolates. Vet. Microbiol., 2006;118:133-140.
6. Tormo, M. Á.; Knecht, E.; Götz, F.; et al. Bap-dependent biofilm formation by pathogenic species of Staphylococcus: evidence of horizontal gene transfer? Microbiology, 2005; 151(7): 2465–2475.
7. Cucarella, C.; Tormo, M. A.; Ubeda, C. et al. Role of biofilm-associated protein Bap in the pathogenesis of bovine Staphylococcus aureus. Infection and Immunity, 2004; 72:2177-2185.
8. Melchior, M. B.; Vaarkamp, H. and Fink-Gremmels, J. Biofilms: A role in recurrent mastitis infections? Vet. J., 2006; 171:398-407.
9. Stevens, N. T.; Tharmabala, M.; Dillane, T.; et al. Biofilm and the role of the icaoperon and aap in Staphylococcus epidermidisisolates causing neurosurgical meningitis. Clin.Microbiol.Infect.,2008; 14:719-722.
10. Chambers, H. F. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clinical Microbiology Reviews, 1997; 10:781-791.
11. Bogado, I.; Sutich, E.; Krapp, A. et al. Methicillin resistance study in clinical isolates of coagulase-negative staphylococci and determination of their susceptibility to alternative antimicrobial agents. J. of Applied Microbiology, 2001; 91: 344-350.
12. De Lencastre, H.; Figueiredo, A.; Urban, C. et al. Multiple mechanisms of methicillin resistance and improved methods for detection in clinical isolates of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 19991; 35: 632-639.
13. Unal, S.; Hoskins, J.; Flokowitsh, J. E. et al. Detection of methicillin-resistant staphylococci by using the polymerase chain reaction.Journal of Clinical Microbiology, 1992; 30: 1685-1691.
14. Archer, G. L., and Niemeyer, D. M. Origin and evolution of DNA associated with resistance to methicillin in staphylococci. Trends Microbiol.,1994; 2:325–347.
15. Quinn, P. J.; Carter, M. E.; Markey, B. et al. Clinical veterinary microbiology. 6th ed., 2004; Mosby, Edinburgh, London, New York, Philadelphia, St. Louis, Sydney, Toronto
16. National Committee For Clinical Laboratory Standards (NCCLS). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. Approved Standard.NCCLS Document M31-A2, 2002; Wayne, PA.
17. Freeman, D. J.; Falkiner, F. R. and Keane, C. T. New method for detecting slime roduction by coagulase negative staphylococci.J. Clin. Pathol .,1989; 42:872-874.
18. Martín, L.; Díez, G.; Morales, M. et al. Simultaneous PCR detection of icacluster and methicillin and mupirocin resistance genes in catheter-isolated Staphylococcus. Inter. Microbiol., 2004; 7: 63-66.
19. Shusheng, Z.; Xiaoguang, C.; Mingming, F. et al. Analysis of S. epidermidisicaAand icaD by polymerase chain reaction and slime production : a case control study. Infect. Dis., 2013; 13: 242.
20. Iorio, N., Azevedo, M., Frazão, V. et al. Methicillin-resistant Staphylococcus epidermidis carrying biofilm formation genes: detection of clinical isolates by multiplex PCR. Inter. Microbiol., 2001;14: 13-17.
21. Bochniarz, M.; Wawron, W. and Szczubiał, M. Coagulase-negative staphylococci (CNS) as an aetiological factor of mastitis in cows. Polish Journal of Veterinary Sciences, 2013; 16(3): 487–492.
22. Kudinha, T. andSimango, C. Prevalence of coagulase-negative staphylococci in bovine mastitis in Zimbabwe. J. S. Afr. Vet. Ass., 2002; 73(2): 62–65.
23. Kurjogi, M. M. andKaliwal, B. B. prevalence and antimicrobial susceptibility of bacteria isolated from bovine mastitis. Advances in Applied Sci. Research, 2011; 2 (6):229-235.
24. Vasiľ, M.; Elečko, J.; Zigo, F. and Farkašová, Z. Occurrence of some pathogenity factors in coagulase negative staphylococci isolated from mastitis milk in dairy cows. Potravinárstvo, 2012; 6(2):60-63.
25. Pitkälä, A.; Haveri, M.; Pyörälä, S. et al. Bovine mastitis in Finland 2001–prevalence, distribution of bacteria, and antimicrobial resistance. J. Dairy Sci., 2004; 87: 2433-2441.
26. Moniri, R.; Dastehgoli, K. and Akramian, A. Increasing resistant coagulase negative staphylococci in bovine clinical mastitis. Pakistan J. of Biological Sci., 2007; 10(15):2465-2469.
27. Kalmus, P.; Viltrop, A.; Aasmae, B. et al. Occurrence of clinical mastitis in primiparous Estonian dairy cows in different housing conditions. Acta Vet. Scand., 2006; 48:21.
28. Larissa, A.; Zeni, C.; Jeroen, D. B. et al. Clinical mastitis caused by coagulase-negative Staphylococci in Canadian dairy herds. WCDS Advances in Dairy Technology, 2013; 25: 373.
29. Baba, E.; Fukata, T. and Matsumoto, H. Ecological studies on coagulase-negative staphylococci in and around bovine udder. Bull Univ. Osaka Pref. Ser. B., 1980; 32: 69-75.
30. Jarp, J. Classification of coagulase-negative staphylococci isolated from bovine clinical and subclinical mastitis. Vet. Microbiol., 1991; 27: 151-158.
31. Birgersson, A.; Jonsson, P. and Holmberg, O. Species identification and some characteristics of coagulase-negative staphylococci isolated from bovine udders. Vet. Microbiol., 1992; 31: 181-189.
32. Malinowski, E.; Lassa, H.; Kłossowska, A.; et al. Etiological agents of dairy cows’ mastitis in western part of Poland. Pol. J. Vet. Sci., 2006; 9: 191-194.
33. Moon, J.S.; Lee, A.R.; Kang, H.M.; et. Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea. J. Dairy Sci., 2007; 90: 1176–1185.
34. Lee, J. H. Methicillin (oxacillin)-resistant Staphylococcus aureus strains isolated from major food animals and their potential transmission to humans. Appl. Environ. Microbiol., 2003; 69:6489–6494.
35. Gianneechini, R. E.; Concha, C. and Franklin, A. Antimicrobial susceptibility of udder pathogens isolated from dairy herds in the west littoral region of Uruguay. Acta Vet. Scand., 2002; 43:31-41.
36. Bouman, M,.;Irigoyen, D. and Bertón, A. Analisis de los resultados de 427 muestrasremitidasparaaislamiento de bacterias de mastitis y antibiograma. (Study of results from 427 milk samples remitted for bacteriologic cultures and susceptibility testing against antimicrobial agents). Jornadas de Salud de Ubre, Nva.Helvecia, Uruguay, 1999; 59-68.
37. Arslan, S. and Özkardes, F. Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples. MemInstOswaldo Cruz, Rio de Janeiro, 2007; 102(1): 29-33.
38. Idriss, Sh. E.; Foltys, V.; Tančin, V. et al. Mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Nitra, Slovakia. Slovak J. Anim. Sci., 2014; 47(1): 33-38.
39. Kaliwal, B. B.; Sadashiv, S. O.; Kurjogi, M, M. et al. prevalence and antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitis. Veterinary World, 2011; 4(4):158-161.
40. Nagappa, S. K. and S. P. Singh Isolation and antibiogram of coagulase negative Staphylococci from bovine mastitic milk. Journal of Foodborne and Zoonotic Diseases, 2013; 1(1): 21-23.
41. Odd, G. B. and Maeland, J. A. Mechanism of methicillin resistance in staphylococci. APMIS, 1997; 105:264–276.
42. Türkyilmaz, S. and Eskііzmіrlіler, S. Detection of slime factor production and antibiotic resistance in staphylococcus strains isolated from various animal clinical samples. Turk. J. Vet. Anim. Sci., 2006; 30: 201-206.
43. Samah, F. D. and Hanaa A. E. A. Investigation of biofilm forming ability in staphylococci causing bovine mastitis using phenotypic and genotypic assays. Scientific World J., Article ID 378492, 2013; 9.
44. Mohan, U.; Jindal, L. and Aggarwal, P. Species distribution and antibiotic sensitivity pattern of coagulase negative staphylococci isolated from various clinical specimens. Indian J. Med.Microbiol., 2002; 20: 45-46.
45. Vasudevan, P.; Nair, M. K. M.; Annamalai, T. et al. Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation,” Veterinary Microbiology, 2003; 92 (1-2): 179–185.
46. Gotz, F. Microreview on Staphylococcus and biofilms.Molecular Microbiology, 2002; 43: 1367–1378.