Standardization of mPCR for Biocide Resistance Genes and their Prevalence in Staphylococcus aureus

Tawheed Ahmad Shafi; Baljinder Kumar Bansal; Shukriti Sharma; Dhiraj Kumar Gupta; Swaran Singh Randhawa

Authors

Tawheed Ahmad Shafi Department of Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University Ludhiana, Punjab, INDIA
Baljinder Kumar Bansal Department of Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University Ludhiana, Punjab, INDIA
Shukriti Sharma Department of Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University Ludhiana, Punjab, INDIA
Dhiraj Kumar Gupta Department of Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University Ludhiana, Punjab, INDIA
Swaran Singh Randhawa Department of Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University Ludhiana, Punjab, INDIA

Keywords:

Biocides resistance, Bovine mastitis, sepA, norA, mepA, qacA/B

Abstract

Biocides are commonly used in infection control programs to prevent the new infections of the udder and colonization of microorganisms on other contact surfaces. In the present study, a multiplex PCR (mPCR) assay simultaneously amplifying 04 biocide resistance genes was standardised. The mPCR was applied on 30 S. aureus isolates, isolated from milk of mastitis affected cows and prevalence of these genes in S. aureus was studied. High resistance mediated by sepA (83.33%), norA (80%) and mepA (80%) resistance genes was observed while resistance mediated by qacA/B (3.33%) gene was found very low. Genetic resistance against biocides is a widespread problem that has been extensively studied in food industry and hospitals particularly targeting methicillin resistant S. aureus, however, limited studies on biocide resistance in S. aureus of animal origin have been undertaken.

References

Bansal, B., Gupta, D., Sharma, S., Shafi, T., Filia, G. (2019). Phenotypic antibiotic resistance pattern and presence of mecA in Staphylococcus aureus isolated from bovine mastitis. International Journal of Livestock Research, 9(9): 65-79. doi: 10.5455/ijlr.20180516043453

Berg, T., Firth, N., Apisiridej, S., Hettiaratchi, A., Leelaporn, A., Skurrayet, R.A. (1998). Complete nucleotide sequence of pSK41: evolution of staphylococcal conjugative multi-resistance plasmids. Journal of Bacteriology, 180: 4350-4359.

Brakstad, O.G., Aasbakk, K., Maeland, J.A. (1992) Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. Journal of Clinical Microbiology, 30(7): 1654-1660.

Cervinkova, D., Babak, V., Marosevic, D., Kubikova, I., Jaglic, Z. (2013). The role of the qacA gene in mediating resistance to quaternary ammonium compounds. Microbial Drug Resistance, 19(3): 160-167.

Conceição, T., Coelho, C., de Lencastre, H., Aires-de-Sousa, M. (2016). High prevalence of biocide resistance determinants in Staphylococcus aureus isolates from three African countries. Antimicrobial Agents and Chemotherapy, 60: 678-681. doi:10.1128/AAC.02140-15.

Costa, S.S., Viveiros, M., Amaral, L., Couto, I. (2013). Multidrug efflux pumps in Staphylococcus aureus: an update. The open Microbiology Journal, 7: 59-71. doi: 10.2174/1874285801307010059

Couto, I., Costa, S.S., Viveiros, M., Martins, M., Amaral, L. (2008). Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide. Journal of Antimicrobial Chemotherapy, 62: 504-513. doi: 10.1093/jac/dkn217

Couto, N., Belas, A., Couto, I., Perreten, V., Pomb, C. (2015). Genetic relatedness, antimicrobial and biocide susceptibility comparative analysis of methicillin-resistant and susceptible Staphylococcus pseudintermedius from Portugal. Microbial Drug Resistance, 20(4): 364-371. doi: 10.1089/mdr.2013.0043

Ergun, Z., Cantekin, K., Gurturk, H., Solmaz, I., Ekin, H., Ozturk, D. (2017). Distribution of antiseptic resistance genes in Staphylococcus spp. from bovine mastitis Y. Veterinarni Medicina, 62(04): 200-203. doi: 10.17221/265/2015-VETMED

Hassan, K.A., Skurray, R.A., Brown, M.H. (2007). Active export proteins mediating drug resistance in staphylococci. Journal of Molecular Microbiology and Biotechnology, 12: 180-196. doi: 10.1159/000106673

Ho, C.M., Li, C.Y., Ho, M.W., Lin, C.Y., Liu, S.H., Lu, J.J. (2012). High rate of qacA and qacB positive methicillin-resistant Staphylococcus aureus isolates from chlorhexidine-impregnated catheter-related bloodstream infections. Antimicrobial Agents and Chemotherapy, 56(11): 5693-5697.

Horner, C., Mawer, D., Wilcox, M. (2012). Reduced susceptibility to chlorhexidine in staphylococci: is it increasing and does it matter? Journal of Antimicrobial Chemotherapy, 67: 2547-2559. doi: 10.1093/jac/dks284

Jaglic, Z., and Cervinkova, D. (2012). Genetic basis of resistance to quaternary ammonium compounds--the qac genes and their role: a review. Veterinarni Medicina, 57(6): 275-281. doi: 10.17221/6013-VETMED

Kaatz, G.W., McAleese, F., Seo, S.M. (2005). Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion (MATE) transport protein. Antimicrobial Agents and Chemotherapy, 49: 1857-1864. doi: 10.1128/AAC.49.5.1857-1864.2005

Kosmidis, C., Schindler, B.D., Jacinto, P.L., Patel, D., Bains, K., Seo, S.M., Kaatz, G.W. (2012). Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of Staphylococcus aureus. International Journal of Antimicrobial Agents, 40(3): 204-209. doi: 10.1016/j.ijantimicag.2012.04.014

LaBreck, P.T., Rice, G.K., Paskey, A.C., Elassal, E.M., Cer, R.Z. Law, N.N., Schlett, C.D., Bennett, J.W., Millar, E.V., Ellis, M.W., Hamilton, T., Bishop-Lilly, K.A., and Merrell, D.S. (2018). Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, qacA. Frontiers in Microbiology, 9: 1-17. doi: 10.3389/fmicb.2018.02664

Mayer, S., Boos, M., Beyer, A., Fluit, A.C., Schmitz, F.J. (2001). Distribution of the antiseptic resistance genes qacA, qacB and qacC in 497 methicillin-resistant and susceptible European isolates of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy, 47: 896-897. doi: 10.1093/jac/47.6.896

McGann, P., Kwak, Y.I., Summers, A., Cummings, J.F., Waterman, P.E., Lesho, E.P. (2011). Detection of qacA/B in clinical isolates of methicillin-resistant Staphylococcus aureus from a regional healthcare network in the eastern United States. Infection Control and Hospital Epidemiology, 32: 1116-1119. doi: 10.1136/bmj.327.7425.1177

Narui, K., Noguchi, N., Wakasugi, K., Sasatsu, M. (2002). Cloning and characterization of a novel chromosomal drug efﬂux gene in Staphylococcus aureus. Biological and Pharmaceutical Bulletin, 25: 1533-1536. doi: 10.1248/bpb.25.1533

National Mastitis Council (1999).. Laboratory Handbook on Bovine Mastitis. Revised ed. National Mastitis Council, Verona: WI.

Noguchi, N., Suwa, J., Narui, K., Sasatsu, M., Ito, T., Hiramatsu, K., Song, J.H. (2005). Susceptibilities to antiseptic agents and distribution of antiseptic-resistance genes qacA/B and smr of methicillin-resistant Staphylococcus aureus isolated in Asia during 1998 and 1999. Journal of Medical Microbiology, 54: 557-565.

Paulsen, I.T., Brown, M.H., Dunstan, S.J., Skurray, R.A. (1995). Molecular characterization of the staphylococcal multidrug resistance export protein QacC. Journal of Bacteriology, 177: 2827-2833. doi: 10.1128/jb.177.10.2827-2833.1995

Senn, L., Clerc, O., Zanetti, G., Basset, P., Prod’hom, G., Gordon, N.C., Sheppard, A.E., Crook, D.W. (2016). The stealthy superbug: the role of asymptomatic enteric carriage in maintaining a long-term hospital outbreak of ST228 methicillin-resistant Staphylococcus aureus. MBio, 2016; 7(1): e02039-15. doi:10.1128/mBio.02039-15

Shrivastava, N., Shrivastav, A. (2019). MRSA in Dairy Cattle: A Review. International Journal of Livestock Research, 9(11): 38-55. doi: 10.5455/ijlr.20190611070925

Sidhu, M.S., Heir, E., Leegaard, T., Wiger, K., Holck, A. (2002). Frequency of disinfectant resistance genes and genetic linkage with beta-lactamase transposon Tn552 among clinical staphylococci. Antimicrobial Agents and Chemotherapy, 46: 2797-2803.

Sierra, J.M., Ruiz, J., Jimenez De Anta, M.T., Vila, J. (2000). Prevalence of two different genes encoding NorA in 23 clinical strains of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy, 46: 145-146.

Strommenger, B., Kettlitz, C., Werner, G., Witte, W. (2003). Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. Journal of Clinical Microbiology, 41(9): 4089-4094.

Thomas, L., Maillard, J.Y., Lambert, R.J.W., Russell, A.D. (2000). Development of resistance to chlorhexidine di acetate in Pseudomonas aeruginosa and the effect of a ‘residual’ concentration. Journal of Hospital Infection, 46: 297-303. doi: 10.1053/jhin.2000.0851

Ucuncu, M. (2015). Dairy Products and Technologies (in Turkish). Meta Press. Bornova, Izmir. 94-96.

Wang, C., Cai, P., Zhan, Q., Mi, Z., Huang, Z., Chen, G. (2008b). Distribution of antiseptic-resistance genes qacA/B in clinical isolates of methicillin resistant Staphylococcus aureus in China. Journal of Hospital Infection, 69: 393-394.

Wang, J.T., Sheng, W.H., Wang, J.L., Chen, D., Chen, M.L., Chen, Y.C., Chang, S.C. (2008a). Longitudinal analysis of chlorhexidine susceptibilities of nosocomial methicillin-resistant Staphylococcus aureus isolates at a teaching hospital in Taiwan. Journal of Antimicrobial Chemotherapy, 62: 514-517.

Zhang, M., O’Donoghue, M.M., Ito, T., Hiramatsu, K., Boost, M.V. (2011). Prevalence of antiseptic-resistance genes in Staphylococcus aureus and coagulase-negative Staphylococci colonising nurses and the general population in Hong Kong. Journal of Hospital Infection, 78: 113-117. doi: 10.1016/j.jhin.2011.02.018