Complete genome sequencing revealed novel genetic contexts of themcr-1gene inEscherichia colistrains

Sir, Polymyxin-based treatment is often considered as the last-line therapy for MDR Gram-negative bacterial infections. Resistance to polymyxins, in particular colistin, was previously linked to adaptive or mutational mechanisms involving chromosomally encoded genes before the discovery in late 2015 of a plasmid-mediated mobile colistin-resistance determinant (mcr-1).1 The widespread dissemination of mcr-1 was subsequently discovered as this resistance determinant was found in at least 20 countries from various continents within a span of 3 months.2 A retrospective study has shown that the mcr-1 gene can be traced back to the early 1980s, which coincides with the introduction of colistin into animal husbandry in China,3 and hence the current distribution of MCR-1-producing Enterobacteriaceae may only represent the tip of the iceberg. Comparative analysis of genetic structures of mcr-1 in (a) various plasmids and (b) the chromosome of EC590. Not drawn to scale. pEC5-1 (CP016185); pEC13-1 (CP016186); pS2.14-2 (CP016187); pmcr-1_IncI2 (KU761326); pHNSHP45 (KP347127); pEC2-4 (CP016184); pEC2_1-4 (CP016183); EC590 (CP016182). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. pEC5-1, pEC13-1 and pS2.14-2 were IncI2 plasmids that shared over 96% of their sequences (at 99% nucleotide identity) with pmcr1_IncI2 (KU761326)—an mcr-1-harbouring plasmid that was isolated from a clinical ESBL-producing E. coli strain in China (Figure S1).5 The genetic context of mcr-1 in pEC5-1, pEC13-1 and pS2.14-2 was identical to that of pmcr1_IncI2 (nikB–mcr-1–pap), whereas, in pHNSHP45 (KP347127), an ISApl1 mobile element was inserted between nikB and mcr-1. The upstream ISApl1 insertion in mcr-1-bearing precursor plasmids such as pEC5-1, pEC13-1, pS2.14-2 and pmcr1_IncI2 was hypothesized to be the key genetic event underlying the rapid mobilization and acquisition of this colistin-resistance gene.6 In IncHl1 megaplasmids pEC2-4 and pEC2_1-4 the mcr-1-associated cassettes interrupted a gene that encodes a putative DNA repair protein and the pap elements were also found to be truncated. The ISApl1–mcr-1–Δpap–ISApl1 in pEC2-4 was bracketed by a 2 bp (TG) target site duplication, while, in pEC2_1-4, a 2961 bp deletion surrounding the downstream ISApl1 element resulted in a different genetic arrangement of ISApl1–mcr-1–Δpap–Δorfunknown–int. Both plasmids also harboured other clinically relevant antibiotic resistance genes, including aadA1 (aminoglycoside), qnrS1 (fluoroquinolone), floR (florfenicol/chloramphenicol) and sul3 (sulphonamide), whereas only pEC2-4 carried an additional fosfomycin resistance determinant (fosA). pEC2-4 and pEC2_1-4 bear the closest resemblance to pB71 (KP899806; 63% query coverage; 99% nucleotide identity) and p109/9 (KP899805; 70% query coverage; 99% nucleotide identity), respectively, from Salmonella Typhimurium (Figure S1). The finding of mcr-1-carrying IncHl1 plasmids in addition to previously reported classes of mcr-1-positive plasmids that include IncI2, IncF, IncX4, IncHl2 and IncP incompatibility groups1,7–10 provided further evidence that transmission of this resistance gene occurred via distinct plasmids. An ISApl1-mediated translocation of mcr-1 into the chromosome of E. coli was recently proposed by Veldman et al.,9 but the site of integration and the mcr-1-associated genetic structures were not reported. In the present study, multiple copies of ISApl1–mcr-1–Δpap that were arranged in tandem were found to be inserted between nhaA and sokC genes in the chromosome of clinical isolate EC590 resulting in a unique genetic arrangement of nhaA–ISApl1–mcr-1–Δpap–ISApl1–mcr-1–Δpap–ISApl1–mcr-1– Δpap–ISApl1–sokC, which was further confirmed by long-range PCR and Sanger sequencing. The presence of a 2 bp (TC) target site duplication flanking this genetic structure indicated that the triplicated mcr-1 gene locus was inserted via a transposition or recombination process. Sequences flanking the insertion site were high in AT content, which corresponded to the observations of Tegetmeyer et al.11 The information gathered from the complete genome sequencing of our E. coli strains and other studies showed that the mcr-1 gene is associated with diverse genetic structures and localizations. ISApl1 is most likely an important factor responsible for mcr-1 plasticity as well as for the integration of the resistance gene into various classes of plasmids and chromosomes. The insertion element could have played a role in the triplication of the mcr-1 locus, but the underlying mechanism remains unknown. The different STs of the mcr-1-positive E. coli isolates that were sequenced in this study supports the hypothesis that multiple horizontal transmission pathways may be responsible for the dissemination of this resistance gene.8 Immediate revision regarding the use of colistin in agriculture is highly warranted to prevent the further spread of mcr-1-mediated colistin-resistant strains. The nucleotide sequences described in this study were deposited in GenBank under the accession numbers CP016182–CP016187. This study was supported by a PPP grant (no. PG094-2015B) to P. S. C. and HIR Grants, University of Malaya (H-50001-A000027; A000001-50001) awarded to K.-G. C. None to declare.