Application of nanopore sequencing to identify antimicrobial resistance genes, mobile genetic elements and virulence factors in clinical isolates

Abstract The global health challenge posed by the emergence of antibiotic resistance pathogen is further exacerbated in African countries by the indiscriminate use of antibiotics, poor surveillance and lack of stewardship programs. To address this issue, we employed the Oxford Nanopore Technologies (ONT) to sequence 17 clinical isolates from a referral hospital in Kenya. Our comprehensive bioinformatics approach facilitated the assembly, identification of sequence types and prediction of antimicrobial resistance genes, mobile genetic elements (plasmids and integrons) and virulence genes. Of the 17 isolates, five were A. baumannii , four E. coli , three S. haemolyticus , three were E. cloacae, while S. aureus and E. faecalis were single isolates. For the detection of AMR genes, A. baumannii isolates harbored genes such as blaOXA-23 which mediates resistance to carbapenems, E. coli and E. cloacae carried blaCTX-M-15 which confers resistance to cephalosporins and S. haemolyticus harbored blaZ, responsible for resistance against penicillins . S. aureus co-haboured mecA and blaZ genes. In addition,, various other different AMR genes to chloramphenicol, macrolides, aminoglycosides, tetracycline were also observed. For plasmid replicons, E. coli carried the most number of plasmids and shared ColRNAI_1 and IncFIB(pB171)_1_pB171 with A. baumannii and IncR_1 with E. cloacae. Many genes encoding various virulence factors including fimA-I and ompA , senB were identified in E. coli, hlgA-C and hla/hly, hlb, hld in S. aureus and efaA , ebpA-C in E. faecalis. In conclusion, most isolates contained a combination of different AMR genes harbored in plasmids and integrons and virulence genes. This study provides significant information on genetic determinants of antibiotic resistant pathogens in clinical isolates and could assist in developing strategies that improve patient treatment. Author Summary Antimicrobial resistance remains a major health challenge across the globe. The continued misuse and lack of proper monitoring has worsened the problem of antibiotic resistant infections. In this study, we sought to use nanopore sequencing to identify antibiotic resistance genes, mobile genetic elements and virulence factors from clinical isolates which showed resistance against commonly used antibiotics. We found the presence of resistance genes to multiple different antibiotics including beta-lactams, macrolides, tetracycline and aminoglycosides across multiple bacterial species. We also identified plasmid replicons and class I integrons which facilitate the spread of antimicrobial resistant genes. Furthermore, several virulence factors that help resistant bacteria to survive were identified. Overall, this study highlights the widespread issue of antibiotic resistance, factors contributing to its persistence in clinical isolates and utility of nanopore sequencing for monitoring genetic determinants of antimicrobial resistance.