Molecular Detection Of Toxins And Disinfectant Resistance Genes Among Staphylococcus Aureus Isolated From Dairy Cattle In Egypt | ||
| Journal of Applied Veterinary Sciences | ||
| Article 5, Volume 5, Issue 1, January 2020, Pages 35-45 PDF (738.9 K) | ||
| Document Type: Original Article | ||
| DOI: 10.21608/javs.2020.75411 | ||
| Authors | ||
| Ebtsam Kotb* 1; Jehan Gafer2 | ||
| 1Animal Reproduction Research Institute, Harm, Giza, Egypt | ||
| 2Animal reproduction research Institute, Haram, Giza, Egypt | ||
| Abstract | ||
| The objectives of the study were to detect toxins and antiseptic resistance genes in Staphylococcus aureus isolated from cows with subclinical mastitis in Egypt. A total of 400 quarter milk samples (QMS) were collected from different dairy herds in which quaternary ammonium compounds (QAC) had been used as a disinfectant for more than 3years. The collected samples were subjected to bacterial investigation. S. aureus was successfully isolated confirmed by duplex PCR targeting 16S rRNA and nuc genes. It also determined their antibiogram and sensitivity to disinfectant. Genes of QAC(qacA/B), enterotoxins (Sea, Seb) and exfoliative toxins (ETB) were detected by simplex and multiplex PCR. Results of bacterial investigation revealed 103 (25.75%) S. aureus isolates. Results of the antibiogram demonstrate that most microbial antibiotic resistance were recorded for Penicillin G (85.7%) and Tetracycline (54.2%). While Gentamycin, Neomycin and Amoxicillin+ clavulanic acid show moderate resistance (21.4%, 10%, and 7.1%) respectively, although Norfloxacin and Cephradine exhibited seldom resistance with high sensitivity of 95% and 94.3% respectively. Regarding the results of QAC sensitivity, only 8 isolates (7.76%) were resistant to benzalkonium chloride (BC) versus to 13 isolates (12.62%) harbour QAC gene could be detected by PCR with specific amplicon of 220bp corresponding to qacA/B. The results revealed Positive amplification of 102 bp specific for Sea gene in 19(18.44%) isolates and 164bp specific for Seb gene in 13(12.62%) isolates while there is no amplification was detected for etb gene. In conclusion, an Antibiogram, as well as the identification of toxigenic and QAC genes in this study, may open another perspective in planning some alternative therapeutic strategies against multi resistances S. aureus mastitis. Monitoring cross-resistance between antibiotics and antiseptic should be further investigated. | ||
| Keywords | ||
| mastitis; quaternary ammonium compounds ( QAC ); QMS; PCR; S.aureus | ||
| References | ||
|
ABBASI M., MAJID B.S., NIMA B. AND MOROVAT T. 2017. Antibiotic Resistance Patterns and Virulence Determinants of Different SCCmec and Pulsotypes of Staphylococcus Aureus Isolated from a Major Hospital in Ilam, Iran. The Open Microbiology Journal, 11, 211-223
ABD EL TAWAB, A.A., AHMED M. A., FATMA I. EL-HOFY, HODA A. A. AND EMAN A. H. 2016. Bacteriological and molecular studies on toxigenic Staphylococcus aureus in milk and some milk products. Benha Veterinary Medical Journal, Vol. 31, No. 2:202-209
AKINDOLIRE, M.A., BABALOLA, O.O. AND ATEBA, C.N. 2015. Detection of Antibiotic-Resistant Staphylococcus aureus from Milk: A Public Health Implication.Int. J. Environ. Res. Public Health 12, 10254–10275.
ALAM M.M., KOBAYASHI N., UEHARA N. AND WATANABE N. 2003. Analysis of distribution and genomic diversity of high-level antiseptic resistance genes qacA and qacB in human clinical isolates of Staphylococcus aureus. Microbe Drug Resist.;9:109–121
ANGELES M. ARGUDÍN, NADINE C., OLIVIER S., JEAN LE GUENNEC STÉPHANIE N. AND PATRICK B. 2013. Genotyping and antimicrobial resistance of Staphylococcus aureus isolated from diseased turkeys. Avian Pathology, Vol. 42, No. 6, 572–580
ARGUDIN M.A., MENDOZA M.C. AND RODICIO M.R. 2010. Food poisoning and Staphylococcus aureus enterotoxins. Toxin, 2: 1751-1773.
ASHRAF, A.; ABD EL-TAWAB, ASHRAF, M.; NABIH, MOHSIEN, A. AGAG AND MARWAH, H., ABD ALI .2017 Molecular studies of virulence genes of Salmonella Typhimurium causing clinical mastitis in dairy cattle, Benha Veterinary Medical Journal Vol. 33, No 2: 27 – 37, December
BAGCIGIL A.F., TAPONEN S., KOORT J., BENGTSSON B., MYLLYNIEMI A. AND PYORALA S. 2012. The genetic basis of penicillin resistance of S. aureus isolated in bovine mastitis. Acta Veterinaria Scandinavia; 54:69
BANGER Y.C. SINGH B., DOHARE A.K. AND VERMA M.R. 2015. A systematic review and meta-analysis of the prevalence of subclinical mastitis in dairy cows in India. Trop. Animal Health Prod.; 47: 291-297.
BJORLAND J., MARIANNE SUNDE, AND STEINAR WAAGE 2001. Plasmid-Borne smr Gene Causes Resistance to Quaternary ammonium Compounds in Bovine S aureus. Journal Of Clinical Microbiology, Nov, p. 3999–4004
BJORLAND J., STEINUM T., KVITLE B., WAAGE S., SUNDE M. AND HEIR E. 2005. Widespread distribution of disinfectant resistance genes among staphylococci of bovine and caprine origin in Norway. J. Clin. Microbiol., 43, 4363−4368.
BRAKSTAD O., AASBAKK G.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. CANTEKIN1 Z., ERGUN Y., SOLMAZ H. AND TEK E.2019. Detection of slime genes and antiseptic/antibiotic resistance genes in Staphylococcal isolates from Damascus goats with subclinical mastitis. Revue Méd. Vét., 170, 7-9, 174-178
CERVINKOVA D., BABAK V., MAROSEVIC D., KUBIKOVA I. AND JAGLIC Z. 2013. The role of the qacA gene in mediating resistance to quaternary ammonium compounds. Microbial Drug Resistance 19, (3): 160–167.
CHAPMAN S. 2003. Disinfectant resistance mechanisms, cross-resistance and coresistance. Int Biodeterior Biodegrad 51:271-276.
CLINICAL AND LABORATORY STANDARDS INSTITUTE.2014. Performance standards for antimicrobial susceptibility testing; Twenty-Fourth Informational Supplement. Wayne,PA: Clinical and Laboratory Standards Institute. CLSI document; M100- S24
COUTO, N., BELAS A., KADLEC K., SCHWARZ S. AND POMBA C. 2015. Clonal diversity, virulence patterns and antimicrobial and biocide susceptibility among human, animal and environmental MRSA in Portugal. J. Antimicrobe. Chemother. 70, 2483–2487.
DAMAVANDI M.S., DEHKORDI M.S., DEHGHAN A., HEIBATI F., TAGHADDOSI R. AND GHOLIPOUR A. 2017. Detection of Antiseptic Resistance Genes among Staphylococcus aureus Colonizing Nurses and Coagulase-Negative Staphylococci Isolated from Clinical Specimens at Teaching Hospitals in the Southwest of Iran. Jundishapur J. Microbiol. 10(1): 1-7.
DE JONG A., GARCH F.E., SIMJEE S., MOYAERT H., ROSE M. AND YOUALA M. 2018. Monitoring of antimicrobial susceptibility of udder pathogens recovered from cases of clinical mastitis in dairy cows across Europe: vet path results. Vet. Microbiol. 213, 73–81.
EBTSAM E.Z. KOTB 2001.Detection of bacterial antigens in milk from clinical cases of bovine mastitis master degree Cairo university
EBTSAM E.Z. KOTB; RAGHIB, R.W. AND OLA A. ABDEL FATTAH 2014.The economic impact of mastitis in some dairy farms in Egypt. J Egypt. Vet. Med. Assoc.4.579-595
EBTSAM E.Z. KOTB; EL-SHAFAIE M.A. AND SAMEH A. IBRAHEM. 2018. Molecular characterization of toxigenic and antibiotic-resistant of staphylococcus aureus of recurrent bovine mastitis. Assiut Vet. Med. J. Vol. 64 No. 158 July.
EL-JAKEE J.K., NAGWA S. ATTA, SAMY A.A., BAKRY M.A., ELGABRY E.A., MAI M. KANDIL AND GAD EL-SAID W.A. 2011. Antimicrobial Resistance in Clinical Isolates of Staphylococcus aureus from Bovine and Human Sources in Egypt. Global Veterinaria 7 (6): 581-586
ERGUN Y. Z., CANTEKIN1 K., GURTURK H., SOLMAZ I.H. AND EKIN, D. OZTURK 2017. Distribution of antiseptic resistance genes in Staphylococcus spp. from bovine mastitis Veterinarni Medicine, 62, (04): 200–203
HAVERI M.A., ROSLO¨F L.RANTALA AND PYO¨RA¨LA¨.S. 2007. Virulence genes of bovine Staphylococcus aureus from persistent and nonpersistent intramammary infections with different clinical characteristics. J. Appl. Microbiol. 103:993–1000.
HO J., BOOST M. AND DONOGHUE M. O. 2015. Does the extensive use of qac disinfectants select for enterotoxigenic staphylococcus aureus? the 7th International Congress of the Asia Pacific Society of Infection Control, Taipei, Taiwan, March 26-29
IGNAK, S., YASAR N. AND BULENT G. 2017. Frequency of antiseptic resistance genes in clinical staphylococci and enterococci isolates in Turkey. Antimicrobial resistance and infection control. 6:88, 1-7
ITO T., OKUMA K., MA X.X., YUZAWA H. AND HIRAMATSU K. 2003. Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC. Drug Resist. Update. 6, 41–52.
JAGLIC Z. AND CERVINKOVA D. 2012. The genetic basis of resistance to quaternary ammonium compounds – the qac genes and their role: a review. Vet Med.;57: 275–281.
JØRGENSEN, H.J.; MORK, T.; CAUGANT, D.A.; KEARNS, A. AND RORVIK, L.M. 2005. Genetic Variation among Staphylococcus aureus Strains from Norwegian Bulk Milk. Appl. Environ. Microbiol. 71: 8352–8361.
KALOREY D.R., SHANMUGAM Y., KURKURE N.V., CHOUSALKAR K.K. AND BARBUDDHE S.B. 2007. Detection of gene encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases. J. Vet. Sci; 8: 151-154.
LONCARIC I., ALEXANDER T., SILVIA H., MICHAEL P.S., MAREIKE T., MAGDA D.E., JOACHIM S. AND FRANK KÜ. 2019. Prevalence of Methicillin-Resistant Staphylococcus sp. (MRS) in Different Companion Animals and Determination of Risk Factors for Colonization with MRS. Antibiotics, 8, 36; 1-9.
LONGTIN J., SEAH C., SIEBERT K., MCGEER A., SIMOR A., LONGTIN Y., LOW D.E. AND MELANO R.G.2011. Distribution of antiseptic resistance genes qacA, qacB, and smr in methicillin-resistant Staphylococcus aureus isolated in Toronto, Canada, from 2005 to 2009. Antimicrobe Agents Chemother 55, 2999–3001.
MARQUEZ. 2018. Staphylococcus aureus Isolates from Bovine Mastitis in Eight Countries: Genotypes, Detection of Genes Encoding Different Toxins and Other Virulence Genes Toxins, 10, 247; 1-22.
MARTINEAU F., PICARD F.J., LANSAC N., MÉNARD C., ROY P.H., OUELLETTE M., BERGERON M.G. 2000. Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrobe Agents Chemother, 231-238.
MAŠLANKOVÁ J., PILIPINO I., TKÁČIKOVÁ L. 2009. Pheno and genotyping of Staphylococcus aureus isolates of sheep origin. Acta Vet. Brno. 78, 345–352.
MEHROTRA, M.; WANG, G. AND JOHNSON, W.M. 2000. Multiplex PCR for Detection of Genes for Staphylococcus aureus Enterotoxins, Exfoliative Toxins, Toxic Shock Syndrome Toxin 1, and Methicillin Resistance. Journal of clinical microbiology. Vol. 38, No. 3.
MONISTERO V., HANS U.G., CLAUDIA P., PAOLA C., BIANCA C., ENRIQUETA B. AND ALEJANDRO CEBALLOS-MONDAY S.R. AND BOHACH G.A. 2018. Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates. J. Clin. Microbiol, 37: 3411-3414.
NAHER N. I., ZINAT F.A.M., MASUDUL A.C., ADNAN M., KAMARUDDIN K. M., ZONAED A.M.A.M. AND INKEYAS U. 2014. Characterization of bovine subclinical mastitis caused by staphylococcus aureus in Southern Bangladesh by bacteriological and molecular approaches. Asian Journal of Biological Sciences 7 (1): 1-12
NAKIPOĞLU Y., IĞNAK S., GÜRLER N. AND GÜRLER B. 2012. The prevalence of antiseptic resistance genes (qacA/B and smr) and antibiotic resistance in clinical Staphylococcus Aureus strains. Mikrobiyol Bul.; 46:180–189.
NMC − NATIONAL MASTITIS COUNCIL. 1999. National Mastitis Council recommended protocol for determining the efficacy of a post milking barrier teat dip based on reduction of naturally occurring new intramammary infections. In: Proceedings of the 38th Annual Meeting of the National Mastitis Council, Arlington, 1999. 239−242.
NOOR S., NUHA J.K. AND HEBA A. M. 2019. Detection a New Antiseptic Resistant Variant of QAC Gene in Some Multi-Drug Resistant Staphylococcus aureus Isolated from Different Clinical Sources. Baghdad Science Journal Vol.16 (3) 571-579
PAULSEN I.T., BROWN M.H., LITTLEJOHN T.G., MITCHELL B.A. AND SKURRAY R.A. 1996. Multidrug resistance proteins QacA and QacB from staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity. Proc. Natl. Acad. Sci. USA 93: 3630-3635.
PÉREZ M.M., PRENAFETA A AND VALLE J. 2009. Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl beta-1, 6 glucosamine specific antibody production using biofilm-embedded bacteria. Vaccine; 27(17): 2379-2386.
PEREYRA E.A., PICECH F., RENNA M.S., BARAVALLE C., ANDREOTTI C.S. AND RUSSI R. 2016. Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells. Vet. Microbiol. 183, 69–77.
PESAVENTO G., DUCCI B., COMODO N. AND NOSTRO A. LO. 2007. Antimicrobial resistance profile of Staphylococcus aureus isolated from raw meat: research for methicillin-resistant Staphylococcus aureus (MRSA). Food Control 18:196–200.
PICCININI R., BORROMEO V. AND ZECCONI A. 2010. Relationship between Staphylococcus aureus gene pattern and dairy herd mastitis. Vet. Microbiol., 145, 100–105.
PIECHOTA M., KOT B., ZDUNEK E., MITRUS J., WICHA J., WOLSKA M.K. AND SACHANOWICZ K. 2014. Distribution of classical enterotoxin genes in staphylococci from the milk of cows with- and without mastitis and the cowshed environment. Pol. J. Vet. Sci., 17, 407–411.
PRAG G., FALK-BRYNHILDSEN K., JACOBSSON S., HELLMARK B., UNEMO M. AND SÖDERQUIST B. 2014. Decreased susceptibility to chlorhexidine and prevalence of disinfectant resistance genes among clinical isolates of Staphylococcus epidermidis. APMIS.;122:961–7.
RASHA M. E. 2018. Genetic Characterization of Enterotoxigenic Strains of Methicillin-Resistant and Susceptible S. aureus Recovered from Bovine Mastitis. Asian J. Biol. Sci., 11 (1): 1-8
REISCHL U., PLUZ M., EHRET W. AND WOLF H. 1994. PCR-based detection of mycobacteria in sputum samples using a simple and reliable DNA extraction protocol. Bio. Techniques, RUSSELL A.D. 2004. Bacterial adaptation and resistance to antiseptics, disinfectants and preservatives is not a new phenomenon. Journal of Hospital Infections 57, 97−104.
SAINI V., MCCLURE J.T., LEGER D., KEEFE G.P., SCHOLL D. T., MORCK D.W. AND BARKEMA H. W. 2012. Antimicrobial resistance profile of common mastitis pathogens on Canadian dairy farm. J. Dairy Sci.; 95: 4319-4332.
SCHWEIZER H.P. TRICLOSAN. 2001. a widely used biocide and its link to antibiotics. FEMS Microbiol Lett 202:1-7.
SONG M., BAI Y., XU J., CARTER M. Q., SHI C. AND SHI X. 2015. Genetic diversity and virulence potential of Staphylococcus aureus isolated from raw and processed food commodities in Shanghai. Int. J. Food Microbiol. 195, 1–8.
TENNET J.M., LYON B.R., MIDGLEY M., JONES I.G., PUREWAL A.S. AND SKURRAY R.A. 1989. Physical and biochemical characterization of QacA gene encoding antiseptic and disinfectant resistance in staphylococcus aureus. J. Gen. Microbiol 135: 1-10.
THOMAS L., RUSSELL A.D. AND MAILLARD J.Y. 2005. Antimicrobial activity of chlorhexidine diacetate and benzalkonium chloride against Pseudomonas aeruginosa and its response to biocide residues.J. Appl Microbiol 98, 533–543.
UCUNCU M. 2015. Dairy Products and Technologies (in Turkish). Meta Press. Bornova, Izmir. 94–96
VALI L., DAVIES S.E., LAI L.L., DAVE J. AND AMYES S.G. 2008. Frequency of biocide resistance genes, antibiotic resistance and the effect of chlorhexidine exposure on clinical methicillin-resistant Staphylococcus aureus isolates. J Antimicrobe Chemother.;61: 524–532.
VALI L., DASHTI A.A., MATHEW F. AND UDO E.E. 2017. Characterization of heterogeneous MRSA and MSSA with reduced susceptibility to chlorhexidine in Kuwaiti hospitals. Front Microbiol 20, 1359.
VIJAYAKUMAR R. AND SANDLE T. 2018. review on biocide reduced susceptibility due to plasmid-borne antiseptic-resistant genes—special notes on pharmaceutical environmental isolates. Journal of Applied Microbiology. 126, pp.1011—1022.
WANG FEI, YANG HONGIUM, HIS HONG-BIN AND WANG CHANGFA. 2011. Study on hemolysin phenotype and genotype distribution of Staphylococcus aureus caused bovine mastitis in Shandong dairy farms. Int.J.Appl.Res.Vet. Med. Vol. 9, No.4 PP 416 - 421.
WANG J.T., SHENG W.H. AND WANG J.L. 2008. Longitudinal analysis of chlorhexidine susceptibilities of nosocomial methicillin-resistant Staphylococcus aureus isolates at a teaching hospital in Taiwan. J. Antimicrobe Chemother. 62:514–517.
WASSENAAR T.M., USSERY D., NIELSEN L.N. AND INGMER H. 2015. Review and phylogenetic analysis of qac genes that reduce susceptibility to quaternary ammonium compounds in Staphylococcus species. Eur J. Microbiol Immunol (Bp).;5:44–61.
YAMAMOTO T., HUNG W.C., TAKANO T. AND NISHIYAMA A. 2013. Genetic nature and virulence of community-associated methicillin-resistant S. aureus.Biomedicine 3, 2–18
YANG FENG; WANG, Q.,I.; WANG, X.; RONG, U.; WANG LING; XIN-PU1, L.I.; LUO JIN-YIN, L.U.O.; ZHANG SHI-DONG AND HONG-SHENG, L.I. 2016. Genetic characterization of antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis cases in Northwest China. Journal of Integrative Agriculture, 15(12): 2842–2847.
YU-CHENG CHIANG, A.; WAN-WEN LIAO, A.; CHIN-MING FAN, A.; WAN-YU PAI, A.; CHIEN-SHUN CHIOU, C. AND HAU-YANG TSEN. 2008. PCR detection of Staphylococcal enterotoxins (SEs) N, O, P, Q, R, U, and survey of SE types in S. aureus isolate from food-poisoning cases in Taiwan. International Journal of Food Microbiology 121, 66–73.
ZHANG M., O’DONOGHUE M.M., ITO T., HIRAMATSU K. AND BOOST M.V. 2011. Prevalence of antiseptic-resistance genes in Staphylococcus aureus and coagulase-negative staphylococci colonizing nurses and the general population in Hong Kong. J Hosp Infect.;78:113–117.
ZMANTAR T., BOCHRA K., HANENE M. AND AMINA B. 2011. Detection of macrolide and disinfectant resistance genes in clinical Staphylococcus aureus and coagulase-negative staphylococci. BMC Research Notes. 4:453 1-9
| ||
|
Statistics Article View: 1,101 PDF Download: 822 |
||
