MOLECULARCHARACTERISATIONOFStaphylococcusaureus SUBTYPES INFECTINGPOST-SURGICAL WOUNDSOFPATIENTSREPORTING TOKAKAMEGACOUNTYTEACHINGANDREFERRALHOSPITAL

Abstract

Post-surgical wounds are progressively becoming hotspots for multi-drug resistant (MDR) Staphylococcus aureus infections globally. S. aureus has been attributed to more than 1.1 million hospital deaths as of 2019, marking hospitals as favorable environment for its survival and transmission. Reports indicate that the success of S. aureus stems from genetic diversity, attributed to the development of drug resistance and genetic modifications in its genome. However, the molecular structure and evolution of S. aureus to unravel its genetic diversity remains geographically limited. Information on dominant lineages of the pathogen in Western Kenya is needed. Therefore, the present cross-sectional study sought to molecularly characterise S. aureus sub-types infecting post-surgical sites of patients reporting to Kakamega County General Teaching and Referral Hospital.This study was based on patients in the surgical wards of Kakamega County General Teaching and Referral Hospital (KCGTRH). The study recruited seventy (70) consented patients from the surgical wards, presenting with post-surgical wound infections. Wounds were phenotypically examined and classified based on CDC scheme, and swabs taken aseptically. Standard microbiological cultures and biochemical tests were carried out on the swabs for microbial recovery. Antimicrobial sensitivity tests were carried out on the recovered isolates using the selected 15 antibiotics. Associations between antibiotic history, wound class, and organism isolated. was determined at p < 0.05 (Spearman Chi-square). Zymogen DNA extraction kit was used for DNA extraction from all the six strains which were resistant to Oxacillin and Ceftriaxone, a 3rd generation cephalosporin used as a prophylactic surgical treatment at the facility. The genomes were sequenced by HiSeq Illumina platform (NoVo gene Co., Ltd). Raw reads were trimmed (Unipro UGENE v.44.0), assembled (SPAdes and bowtie2). Sequences of each cluster of orthologous genes were aligned (MUSCLE), and locations of horizontally acquired genomic islands (GIs) in bacterial chromosomes mapped (Seward Gene Island Sniffer). Moreover, subtypes were determined (multi-locus sequence typing, MLST), and genetic diversity determined by sequence alignment and phylogenetic analysis (Mauve). Drug resistant genes (DRGs) and virulence genes were determined (Comprehensive Antibiotic Resistance Database- Resistant Gene Identifier, CARD-RGI), and the virulence factor database (VDFB) respectively. For modifications in DRGs, respective protein sequences were aligned (ClustalW-BioEdit v7.0.5.3) to identify mismatches in amino acid sequences. Among the 70 isolates, 24 (34.4%) was S. aureus. Class III wounds were predominated by Pseudomonas aeruginosa, while class IV wounds by S. aureus (p=0.001), and between wound class and previous history of exposure to antibiotics (p = 0.001). Sequence type (ST) 152 was the most predominant sub-type in circulation within the community and hospital environment. ST8 had an extended antimicrobial resistance, mostly due to mutated target proteins and regulators of efflux pumps. Five of the six isolates contained a 20.7-kb circular plasmid carrying blaZ (associated with resistance to -lactam antibiotics). These five strains all belonged to the same sequence type, ST152. Despite the similarity of the plasmids of these isolates, whole genome sequencing revealed that the strains differed, depending on whether they were associated with hospital-acquired or community-acquired infections. Genomic islands contributed significantly to their virulence potential. Among the 24 S. aureus isolates, 25% were multi-drug resistant. Several antimicrobial resistant and virulence genes were discovered. The most striking evolutionary changes were observed in the UvrAB gene complex. This complex was conserved in the hospital-acquired isolates of ST152 but varied significantly in the community-acquired isolates of ST152 and in ST8.The presents a restricted cluster of drug resistance genes circulating within the study site, with a predominant subtype 152 among hospital and community settings and therefore, calls for urgent control measures.