A novel multidrug resistant Klebsiella pneumoniae strain harboring three co-occurring colistin resistance genes

Colistin represents the last-resort antibiotics used for treating carbapenem-resistant Enterobacteriaceae-related infections. Colistin resistance has been reported in Saudi Arabia in multidrug resistant (MDR) Gram-negativebacteria in up to 40% of the cases. Though, only a handful of colistin resistance cases were genetically investigated due to the lack of an approved clinical microbiology method for resistance detection. In this study, we aimed tocharacterize and survey the presence of mobilized colistin resistance (mcr) genes in MDR Klebsiella pneumoniae clinical isolates using whole-genome sequencing (WGS) as part of hospital-wide surveillance for antimicrobial resistance genes in Gram-negative MDR bacterial pathogen isolates (n=1,633) during 2014-2018 in Saudi Arabia. One of the isolates (NGKP-54) was cultured from the sputum of a female patient with pneumonia in her late-60s who was being treated for Non-Small-Cell Lung Carcinoma in 2015. We produced the complete genome of theNGKP-54 strain using a combination of short and long reads WGS and in silico multilocus sequence typing (MLST). The NGKP-54 isolate was assigned a novel sequence type (ST3513) that is a single-locus variant of ST1425. The ST1425 variant has been reported in the Middle East and known to be involved in the production of carbapenemases. The NGKP-54 isolate was resistant to aminoglycoside, cephalosporin, and fluoroquinolone,as determined by conventional microbiological protocols and colistin broth microdilution (BMD). The NGKP-54strain conferred resistance to colistin with MIC of 4 µg/ml. Analysis of the complete genome revealed the presence of three colistin-resistance determining genes including mcr-1, mcr-8, and a partial mcr-8 gene. We observed that allthree mcr-genes contributed to the colistin resistance of NGKP-54 in complementation experiments. Expression of mcr-1 and mcr-8 genes in E. coli DH5a independently conferred colistin resistance at higherconcentration than the original NGKP-54 isolate. Our phylogenetic analysis of all mcr variants revealed MCR-8 and MCR-5 are the closest orthologues to EptA. We have identified for the first time, the presence of mcr-8 in a colistinresistant K. pneumoniae strain in Saudi Arabia and this is the first global report of the co-occurrence of three mcr-encoding genes in the same strain. Saudi Arabia is a hub for annual mass-gathering events, thus increasing the chances that multi-resistant K. pneumoniae can potentially spread globally unless closely monitored for the presence of resistant-determinant loci using WGS-based protocols.