{'fraction_non_alphanumeric': 0.04436711613745106, 'fraction_numerical': 0.03610265332753371, 'mean_word_length': 5.308641975308642, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 1, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Complete genome sequencing revealed novel genetic contexts of the mcr-1 gene in Escherichia coli strains

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). 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. 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, and hence the current distribution of MCR-1-producing Enterobacteriaceae may only represent the tip of the iceberg. The mcr-1 gene has attracted significant interest due to its potential for horizontal transfer and impact on clinical treatment options. In order to gain insights into the localization and genetic organization of the mcr-1 gene, we sequenced six different STs of mcr-1-positive Escherichia coli strains (Table S1, available as Supplementary data at JAC Online) that were previously isolated from various sources in Malaysia. Genomic DNAs were extracted using a MasterPure DNA Purification Kit (Epicenter, Madison, WI, USA) and long-read sequencing was performed on a PacBio RS II instrument (Pacific Biosciences, Menlo Park, CA, USA). The singlemolecule sequencing reads were de novo assembled using Hierarchical Genome Assembly Process (HGAP) v. 3.0, and overlapping regions were assessed with Gepard followed by circularization using Minimus2 pipeline in the AMOS software package. The genomes were annotated via RAST and replicon sequences in the plasmids were analysed using PlasmidFinder (https://cge.cbs.dtu.dk/ services/). The genetic contexts of the mcr-1-harbouring plasmids and chromosome are summarized in Figure 1. 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). 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. 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–Dpap–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–Dpap– Dorfunknown–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-1carrying IncHl1 plasmids in addition to previously reported classes of mcr-1-positive plasmids that include IncI2, IncF, IncX4, IncHl2 and IncP incompatibility groups 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., 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–Dpap 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–Dpap–ISApl1–mcr-1–Dpap–ISApl1–mcr1–Dpap–ISApl1–sokC, which was further confirmed by long-range