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Colistin is considered as a last resort antibiotic. The re-use of this antibiotic highlighted the emergence of colistin resistance mediated by chromosomal and plasmidic resistance mechanisms. Five colistin-resistant  Klebsiella pneumoniae  strains from Laos and Thailand were analyzed by Next Generation Sequencing (NGS) approaches to determine their colistin resistance mechanisms. Antimicrobial susceptibility testing, conjugation and transformation were performed on these strains. Moreover, whole genome sequencing (WGS) combining Illumina (MiSeq) and Oxford Nanopore technologies (MinION) was realized to obtain closed genomes and plasmids. Resistome analyses as well as location of  mcr  genes and its genetic environments were done  in silico . All five strains had colistin MIC of 32 mg/L and were positive for  mcr-3  variants including additionally positive for a  mcr-8  variant gene. The novel variants were named  mcr - 3.21 ,  mcr - 3.26, mcr - 3.28 , and  mcr-8.3  genes. The  mcr-3  variants genes were located on plasmids IncP1, IncFII, and IncI1 type, while  mcr-8.3  gene was found on an IncFII type plasmid. The genetic environment of  mcr - 3.21  and  mcr-3.26  genes were composed of a composite transposon IS Kpn40 -  mcr - 3 -dgkA- IS Kpn40.  Concerning  mcr-8.3  gene, a similar genetic environment of  mcr - 8.1  gene surrounded by ISIX 2  and IS 903B  was observed. To the best of our knowledge, this is the first description of the novel variants  mcr - 3.21 ,  mcr - 3.26, mcr-3.28  and  mcr - 8.3  genes as well as the first study on co-occurrence of  mcr - 3  and  mcr-8  genes. Spread and evolution of  mcr  genes should be monitored.

Co-occurrence of Variants of mcr-3 and mcr-8 Genes in a Klebsiella pneumoniae Isolate From Laos