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Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR
Published on: November 4, 2016
Identification of Variable Traits among the Methicillin Resistant and Sensitive Coagulase Negative Staphylococci in
Sudipta Mahato1, Hiral U Mistry1, Shalini Chakraborty1
1National Institute of Animal BiotechnologyHyderabad, India.
Abstract:
Methicillin resistant Staphylococcus aureus causing bovine mastitis has been very well investigated worldwide. However, there are only limited reports on the characterization of methicillin resistant and sensitive coagulase negative staphylococci (CoNS) across the globe. Hence, in the present study, we aim to determine the phenotypic traits based on antimicrobial susceptibility profile and genotypic characterization by verifying the presence of resistance determinants, virulence and toxin genes present in the CoNS causing clinical mastitis. We obtained 62 CoNS isolates from 167 mastitic milk samples collected from three different states of India. The 62 isolates comprises of 10 different CoNS species S. sciuri, S. haemolyticus, S. chromogenes, S. saprophyticus, S. xylosus, S. simulans, S. agnetis, S. epidermidis, S. gallinarum, and S. cohinii. Susceptibility screening against 11 antibiotics determined 45.16% isolates as multidrug resistant (resistant to more than two class of antibiotic), 46.74% resistant (one or two antibiotic class) and 8.06% isolates were pan-sensitive (sensitive to all drugs). High resistance was observed against oxacillin and cefoxitin, whereas all isolates were susceptible toward vancomycin and linezolid. Fifty three isolates were methicillin resistant and 9 isolates were sensitive as determined by oxacillin susceptibility assay. The methicillin resistance gene, mecA was found in 95.16% isolates and staphylococcal cassette chromosome mec (SCCmec) typing predominantly revealed Type III (n = 34) and Type V (n = 18). Interestingly, 11.9% of mecA positive isolates were oxacillin susceptible and referred as oxacillin susceptible mecA positive staphylococci (OS-MRS). Additionally, genes encoding for enterotoxin, (sea, seb, seh, see) toxic shock syndrome (tsst), exfoliatin (eta, etb, etd) and virulence (pvl, Y-hlg) were also screened. Of all the genes examined, 67.74% of isolate were positive for the Y-hlg gene, followed by the sea gene in 25.8% whereas in none of the isolates the eta and the etb gene was amplified. The study also highlights the incidence of clinical isolates of CoNS, which are harboring the toxin and the virulence genes rendering them as a more potential threat. This is the first report of animal origin OS-MRS from India, which emphasizes on the inclusion of both the genetic and phenotypic test for proper characterization of CoNS and preventing resistant strain misidentification.
Insights
Coagulase-negative staphylococci (CoNS) causing bovine mastitis in India exhibit significant antimicrobial resistance. This study characterized CoNS, revealing high prevalence of methicillin resistance genes and virulence factors, highlighting the need for accurate identification methods.
Area of Science:
- Veterinary Microbiology
- Antimicrobial Resistance
- Bovine Health
Background:
- Methicillin-resistant *Staphylococcus aureus* (MRSA) in bovine mastitis is well-studied, but methicillin-resistant and sensitive coagulase-negative staphylococci (CoNS) remain under-characterized globally.
- CoNS are increasingly recognized as significant pathogens in bovine mastitis, posing challenges to treatment and herd management.
Purpose of the Study:
- To phenotypically and genotypically characterize CoNS isolates from clinical mastitis in India.
- To determine antimicrobial susceptibility profiles, identify resistance determinants, and screen for virulence and toxin genes in bovine CoNS.
Main Methods:
- Isolation and identification of 62 CoNS strains from 167 mastitic milk samples across three Indian states.
- Antimicrobial susceptibility testing against 11 antibiotics.
- Detection of the *mecA* gene and Staphylococcal Cassette Chromosome *mec* (SCC*mec*) typing.
- Screening for enterotoxin, toxic shock syndrome, exfoliatin, and virulence genes.
Main Results:
- 45.16% of isolates were multidrug-resistant, with high resistance to oxacillin and cefoxitin, but susceptibility to vancomycin and linezolid.
- The *mecA* gene was detected in 95.16% of isolates; SCC*mec* types III and V were predominant.
- 11.9% of *mecA*-positive isolates were oxacillin-susceptible (OS-MRS), a novel finding in Indian animal isolates.
- The *Y-hlg* (67.74%) and *sea* (25.8%) genes were frequently detected, while *eta* and *etb* were absent.
Conclusions:
- Bovine CoNS in India possess significant antimicrobial resistance and harbor virulence genes, posing a potential threat to animal and public health.
- The identification of OS-MRS highlights the importance of integrating both genotypic and phenotypic testing for accurate CoNS characterization.
- These findings underscore the need for robust diagnostic strategies to prevent misidentification and manage resistant strains effectively.
