Vancomycin MIC Creep and Identification of Clonal Types of mecA and mecC Positive Methicillin Resistant Staphylococcus aureus

Abstract

The discovery of mecC gene shows an ongoing evolutionary process in MRSA strains. Vancomycin MIC creep is a regional problem. The identification and characterization of MRSA clones especially those with high vancomycin MIC values are thereby required to be done by rapid and reliable typing methods. It was a cross-sectional descriptive study, conducted in the Department of Microbiology and Immunology at the University of Health Sciences, Lahore, Pakistan during a period of 5 years (2016 to 2020). A total of 3305 S. aureus isolates were reconfirmed by using standard methods. Molecular confirmation of MRSA strains was performed by mecA and mecC genes identification using PCR after the phenotypic screening with 30μg cefoxitin disk. Antimicrobial susceptibility testing by Kirby- Bauer disk diffusion method and identification of Penton valentine leukocidin gene by PCR was carried out for all MRSA isolates. Vancomycin MICs for MRSA strains were determined by E- Test for assessment of creep. Isolates having MIC values >1.5 μg/ml, were further subjected for SCCmec typing (I-V and XI) and multiple-locus variable number tandem repeat analysis. Out of 507 cefoxitin resistant S. aureus strains, 437 (86.2%) isolates were mecA gene positive while 70 (13.8%) isolates were negative for mecA gene. Among these mecA negative isolates, 3 (4.3%) strains were positive for mecC gene. The majority of the specimen sources of mecA and mecC positive MRSA were from pus (43%-50%). Overall, 50.0% of the samples were received from the surgery department. None of the MRSA isolates was resistant to linezolid. For erythromycin, there were 77.0% resistant isolates, followed by trimethoprim-sulfamethoxazole (73.9%), ciprofloxacin (63.9%), xx gentamicin (50.5%), clindamycin (39.8%), and doxycycline (36.6%). The percentage of MRSA isolates harbouring pvl gene was 31.6%. In this study, there were 295 (67%) HA-MRSA strains and 145 (33.0%) community-acquired MRSA isolates. In HAMRSA strains there were 16.9% pvl positive and 83.1% pvl negative isolates. In CAMRSA isolates, 61.4% pvl positive and 38.6% pvl negative strains were found. A rising trend of high vancomycin MIC values (creep phenomenon) was observed that may lead towards vancomycin resistance in the future. Geometric mean MIC was on the continuous rise with a value of 1.58μg/ml in 2020. There were 78.2%MRSA isolates that exhibited vancomycin MIC ≥1.50 μg/ml and 21.8 % isolates with MIC < 1.50μg/ml. Isolates with MIC “1.0”μg/ml had a clear decline from 2016 to 2020 (28.6%-14.8%). The percentage of isolates with MIC “2.0”μg/ml increased from 19.0% to 39.8%. SCCmec type III was most commonly present in 38.3% of MRSA isolates. A high percentage of SCCmec type III (35%) and IIIa (12.5%) was present in HAMRSA strains while SCCmec IV subtypes 28.3% were most commonly present in CAMRSA strains. High pvl positivity 73.4% was recorded among SCCmec IV and V while 13.4% pvl positivity was observed among SCCmec types I, II, III, and IIIA. SCCmec XI was identified in two mecC MRSA strains. For MLVA, dendrograms were formed based on the clustering of strains according to the similarity index. One hundred and twenty MRSA isolates were clustered into 60 genotypic groups due to slight variations in VNTRs. By MST all 60 clusters of isolated MLVA strain formed a phylogenetic tree. To conclude, vancomycin MIC creep was observed was observed for mecA and mecC positive MRSA isolates over a period of 5 years. A high prevalence of MRSA isolates harboring pvl gene was observed. SCCmec XI was identified in mecC MRSA isolates and SCC mec III was the most prevalent type. A similarity index above 92% xxi indicated by MLVA analysis revealed that genetic diversity among the MRSA population was very low and strains were related to each other.