A novel disrupted mcr-1 gene and a lysogenized phage P1-like sequence detected from a large conjugative plasmid, cultured from a human atypical enteropathogenic Escherichia coli (aEPEC) recovered in China

Sir, (a) Schematic representation of the flanking regions of the mcr-1 cassettes in plasmids pSLK172-1, pMR0516mcr (accession number KX276657) and pHNSHP45-2 (accession number KU341381). Areas shaded in grey indicate homologies between the corresponding genetic loci on each plasmid. ORFs are shown with arrows [blue arrows, ISApl1; red arrows, mcr-1; grey arrow, terF; green arrows, replication-related ORF; pink arrows, membrane protein (MP); white arrows, hypothetical protein (HP)], the orientation of which indicates the direction of transcription. Alignment of plasmid pSLK172-1 along with a homologous region in pMR0516mcr lacking the mcr-1 insert and depicting the dinucleotide (GT) constituting a TSD at this locus is shown. (b) A schematic illustration showing the comparison of structural features between plasmid pSLK172-1 and the sequences of plasmid pHNSHP45-2 (accession number KU341381), phage P1 (accession number AF234172), denoted by the thick blue line shown in the figure, and plasmid pMR0516mcr (accession number KX276657). Areas shaded in grey indicate homologies between the corresponding genetic loci on each plasmid. Antibiotic resistance-encoding genes are indicated by red boxes and denoted by thick (continuous or broken) red lines. The individual conjugation-related genes (associated with tra and trh) are indicated with capital letters inside the yellow boxes. Blue boxes denote transposon- and integron-associated genes. The heavy metal resistance genes are indicated by grey boxes. The replication-associated genes are shown in green. The maintenance and stability functioning genes are indicated in purple. The tRNAs on phage P1 are indicated by orange boxes, and white boxes indicate hypothetical proteins. The figure is not drawn to scale. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. The E. coli SLK172 study isolate was cultured from a faecal sample obtained from a male who presented to a clinic in Beijing in 2015 with symptoms indicative of diarrhoea and hypogastric pain. The bacterial isolate was assessed for the presence of the mcr-1 gene by PCR, as described previously.1 S1-nuclease PFGE and conjugation experiments were carried out to characterize the plasmids. WGS was performed using a PacBio RS II platform to assess the genomic background. Antimicrobial susceptibility testing assays showed that the isolate was resistant to several antimicrobial compounds used in human medicine, including cephalosporins and fosfomycin. Interestingly, it was susceptible by broth microdilution to colistin (MIC 0.06 mg/L), but appeared to contain a novel mcr-1 gene as determined by PCR. Plasmid profiling of E. coli SLK172 identified two plasmids, ∼370 and 120 kbp in size. Both were transferable by conjugation. WGS revealed that the E. coli SLK172 chromosome (accession number CP017631) harboured the eae gene, but not the bfp gene, suggesting the isolate could be classified as an aEPEC of ST 189. A uniquely disrupted mcr-1 gene and two ESBL genes, blaCTX-M-14 and blaCTX-M-55, were identified. Furthermore, the isolate also carried 22 other antibiotic resistance genes that were located on two plasmids, pSLK172-1 (accession number CP017632) and pSLK172-2 (accession number CP017633) (Table S1, available as Supplementary data at JAC Online). In the latest reports, mcr-1-containing sequences identified to date possess a common 2600 bp DNA segment that in most cases is flanked at one or both ends by ISApl1.2,4 There is increasing evidence that ISApl1 plays a pivotal role in the mobilization of mcr-1.4 Notably, in plasmid pSLK172-1 the mcr-1 gene was flanked on the proximal side by one of two copies of ISApl1 orientated in the same direction, with the second element being inserted directly into the mcr-1 gene itself (Figure 1a). ISApl1 contains a characteristic dinucleotide (-GT-) located within the coding sequence that acts as a potential hot spot for recombination. This sequence is a known target site for duplication (TSD), suggesting that the resistance-encoding gene can be mobilized as a composite transposon that contains 2 bp of the original flanking DNA abutting the 3′ end of the proximally located ISApl1 element, a feature that is repeated for the distally located element. This gives rise to two 2 bp TSDs (Figure 1a).4 The complete sequence of plasmid pSLK172-1 was identified as a 369 298 bp circular episome belonging to multi-replicon types HI2, HI2A, Y and N. Comparative analysis indicated that plasmid pSLK172-1 presented a backbone structure similar to that of pHNSHP45-2 (an mcr-1-carrying plasmid that originated from an intensive pig farm in China) (accession number KU341381), along with additional features including a phage P1-like sequence (accession number AF234172) (Figure 1b).5,6 Furthermore the mcr-1-containing cassette together with the ter and tra loci exhibited 99% nucleotide identity to that of pMR0516mcr (accession number KX276657) when excluding the truncated ISApl1 on pSLK172-1.7 Novel regions identified in this new arrangement included phage P1-like sequences together with 12 antimicrobial resistance-encoding genes, including blaCTX-M-14, fosA3 and floR (Table S1). In pSLK172-1, the phage P1-like sequences were shown to lysogenize into its antibiotic resistance encoding domain (Figure 1b). Interestingly, this phage P1-like region had earlier been reported to be located on a blaCTX-M-15-carrying plasmid, pKP12226 (accession number KP453775), that was identified in a Klebsiella pneumoniae isolate of ST 11.8 When compared, three tRNAs (including tRNA-Asn, tRNA-Thr and tRNA-Ile) of phage P1 found to be present on plasmid pKP12226 were absent on plasmid pSLK172-1, and it is speculated that these are sites of genetic recombination in the former.8 Additionally, the ref (encoding the recombination enhancement function) and cre (encoding a recombinase) genes in the phage P1-like region were associated with Tn21 and IS5075, respectively, in plasmid pSLK172-1, which could play a role in facilitating recombination in this scenario.6 The second plasmid, pSLK172-2, was 120 528 bp in size and a comparison of its structural features is presented in Figure S1. To conclude, this study reports the characterization of a novel disrupted mcr-1 co-located with blaCTX-M-14 on a 370 kbp plasmid. Whether future exposure to colistin will result in the recovery of an intact and functional mcr-1 gene, following the deletion of ISApl1 from plasmid pSLK172-1, remains to be determined. This work was supported by the National Natural Science Foundation of China (grant number 81402685 to L. B.) and the Beijing Talents Fund (grant number 2015000021223ZK35 to L. B.). The Irish Department of Agriculture, Food & the Marine (DAFM) FIRM programme is also acknowledged for providing financial support (grant number 11-F-051). None to declare. Table S1 and Figure S1 are available as Supplementary data at JAC Online.