Molecular Analysis of Coagulase Gene Polymorphism in Clinical Isolates of Methicilin Resistant Staphylococcus aureus by Restriction Fragment Length Polymorphism Based Genotyping

Abstract

Problem statement: To investigate coagulase gene variants through Restriction Fragment Length Polymorphism (RFLP) amongst clinical isolates of Methicillin-Resistant Staphylococcus Aureus (MRSA) collected from Chennai based hospitals. Approach: A total of 85 MRSA clinical isolates collected from various clinical sources were studied for the molecular typing purpose. To perform coagulase gene typing, the repeated units encoding hypervariable regions of the Staphylococcus aureus coagulase gene were amplified by the PCR followed by AluI and HaeIII restriction enzyme digestion of the PCR product and analyzed for RFLP. Results: Cluster analysis performed with Un-weighted Pair Grouping Using Arithmetic Mean Analysis (UPGMA) at >80% similarity level classified all strains into 31 and 21 genotypes based upon AluI and HaeIII RFLP patterns respectively. AluI RFLP patterns gave a high discriminatory index and proved to be better than that of HaeIII in typing of MRSA strains. Conclusion: Method investigated in this study, proved to be quick, reliable and useful for typing large number of MRSA strains from various clinical isolates with high fidelity. Grouping of strains based on RFLP patterns of both enzymes, improved the discriminatory index. Finally, using this methodology group of epidemiological strains can be typed reliably and effectively using both enzymes. Significance and impact of the study: This efficient and reliable typing procedure is beneficial to develop efficient infection control measures in hospitals for staphylococcal infection. This typing procedure could be used to analyze large number of strains with in a short period of time and thus useful for epidemiological investigations.

High incidence rate of methicillin-resistant Staphylococcus aureus (MRSA) among healthcare workers in Saudi Arabia

“…These findings clearly indicate that medical students, being constantly in touch with hospitals and inpatients, do stand a higher risk of colonization, further proving that it is absolutely essential to develop rapid and regular screening programs for transmission of MRSA in healthcare settings. In most cases, culture methods are used to identify MRSA, but the results take at least two days [25]. It is necessary to develop faster and more precise methods for identifying MRSA.…”

Characterization of Coa Gene and Antimicrobial Profiles of Staphylococcus Aureus Isolated from Bovine Clinical and Subclinical mastitis

“…Pcr assay: PCR assay was performed for the detection of the nuc (encoding for the S. aureus-specific thermonuclease) gene and coa gene encoding for coagulase as previously described by Sallam et al (2015) and Himabindu et al (2009), respectively. The primers sequences (Hokkaido System Science Co.Ltd., Hokkaido, Sapporo, Japan and Metabion, Germany) and its amplicons size are listed in Table 1 12.5 µl master mix (Thermo scientific, USA); 1 µl of each primer; 2 µl DNA template and volume of the reaction mixture was completed to 25 µl using DNase/RNase-free water.…”

Characterisation of the Virulence Factors and Genetic Types of Methicillin Susceptible Staphylococcus aureus from Patients and Healthy Individuals

“…PCR-RFLP of coa gene is based on the restriction digest of the heterogeneity of the coagulase region that contains the 81 bp tandem repeats at 3 0 coding region [11] whereas PFGE is based on digestion of chromosomal DNA with the restriction enzyme followed by agarose gel electrophoresis [12]. On the other hand, spa typing is based on the sequence analysis of polymorphic region X of S. aureus protein A is reported to be a highly effective tool in subtyping S. aureus [12].…”