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

Abstract

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 ISKpn40- mcr-3-dgkA- ISKpn40. Concerning mcr-8.3 gene, a similar genetic environment of mcr-8.1 gene surrounded by ISIX2 and IS903B 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.

Keywords: colistin; epidemiology – descriptive; mcr; resistance; whole genome sequencing.

FIGURE 1

(A) Amino acid sequence alignments of the new MCR-3 and MCR-8 variants using Clustal W on NPS@ website. (B) Phylogenetic tree of MCR variants, including MCR-3 and MCR-8 variants. Sequences were aligned using MUSCLE and phylogenetic interferences were obtained using the neighbor-joining method within the MEGA 7 software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 500 times to generate a majority consensus tree. Evolutionary distances were computed using the Poisson correction method and are expressed in units of the number of amino acid substitutions per site.

FIGURE 2

Circular representation of the five complete mcr-3 plasmids (left) and the mcr-8 plasmid (right). Figure constructed using CGView software.

FIGURE 3

(Not to scale) (A) Comparison of the genetic environment of the mcr-3 gene in plasmids: pWJ1 (KY924928), pLH102-A (CP035195), pECSC102 (MG552133), pLH375-3 (CP035200), pTH114-3 (CP035208), pLH94-3 (CP035205), and pTH164-3 (CP035213). (B) Comparison of the genetic environment of the mcr-8 gene in plasmid pK91 (MG736312) and in the plasmid of strain LH94 carrying mcr-8.3 gene: pLH94-8 (CP035204). HP, hypothetical protein, IS, insertion sequence, Tn, transposon.