Co-occurrence ofmcr-1and ESBL on a single plasmid inSalmonella enterica

Sir, Liu et al.1 recently reported the first transferrable colistin resistance gene mcr-1 with prevalence in animals, foodstuffs and human beings in China. The mcr-1 gene found in Escherichia coli,2,3Klebsiella pneumoniae4 and Salmonella spp.5 has been proved subsequently to be disseminated worldwide. The present study investigated the presence of the mcr-1 gene among 53 ESBL-positive Salmonella spp. isolates, and obtained the complete sequence of an IncI2-type conjugative plasmid co-harbouring mcr-1 and blaCTX-M-55 genes in a Salmonella enterica isolate. Fifty-three ESBL-positive Salmonella spp. isolates were collected between 2014 and 2015 from diseased chickens in eight provinces in China (Anhui, Hebei, Heilongjiang, Henan, Hubei, Ningxia, Shandong and Sichuan). All isolates collected from different samples were identified by using an automated system (BD Diagnostic Systems, Sparks, MD, USA). The mcr-1 and blaCTX-M genes were detected by PCR amplification, and the corresponding primers amplifying the whole mcr-1 gene are listed in Table S1 (available as Supplementary data at JAC Online). We found a total of four (7.5%) mcr-1-positive isolates with colistin MICs ranging from 4 to 8 mg/L (Supplementary Data), and DNA sequencing of the whole mcr-1 gene revealed 100% identity compared with the originally published gene sequence.1 Conjugation experiments and S1-PFGE demonstrated the co-occurrence of mcr-1 and blaCTX-M-55 on a conjugative plasmid of about 65 kb (Supplementary Data) in one isolate named SC23. SC23 belongs to the ST292 S. enterica serovar Albany, which was isolated from the intestinal contents of a sick chicken in Sichuan province on 13 January 2014. The plasmid was successfully transferred from S. enterica to E. coli J53 Azr with a frequency of 3 × 10−4 per recipient cell. The MICs of colistin and cefotaxime for transconjugants were 8 and >128 mg/L, respectively, showing that the plasmid conferred a >32-fold and >256-fold increase in MIC compared with the recipient E. coli J53 Azr (Supplementary Data). The plasmid co-harbouring the mcr-1 and blaCTX-M-55 genes was extracted from the positive transconjugant with a Qiagen Midi kit (Qiagen, Hilden, Germany), and sequencing was carried out with an Illumina MiSeq using a 350 bp paired-end library (Majorbio Company, Shanghai, China). The sequences obtained were assembled into a unique scaffold and the existing gaps were closed by PCR using specific primers listed in Supplementary Data. The complete sequence of the 65 419 bp plasmid, designated pSCS23, was found to encode 57 ORFs and the overall GC content was 42.26% (Figure 1). A BLAST comparison of our plasmid and pHNSHP451 revealed a 99% similarity (86% query coverage), and both plasmids share the same ISApl1 transposable element (3676 bp) that contains mcr-1 and tnpA genes. In contrast, other studies showed that the mcr-1 gene is flanked by either a truncated ISApl1 element without the tnpA gene6 or sequences without the whole ISApl1 element.7 This indicates that the genetic environment of the mcr-1 gene is more mobile than we expected. Notably, pSCS23 harbours the blaCTX-M-55 gene, which is flanked by the ISEcp1 mobile element within the structure of ISEcp1–tnpA–blaCTX-M-55 (2600 bp) (Supplementary Data). Furthermore, two recent studies demonstrated a plasmid co-harbouring either blaCTX-M-18 or tetA–tetR–sul1–aadA1–dfrA1–strA–strB6 accompanied by the mcr-1 gene in E. coli. For other species, a recent study identified a plasmid carrying mcr-1 and blaCTX-M-15 and blaTEM-1 in Enterobacter aerogenes from a patient in China.9 It seems that the mcr-1 gene easily exists together with blaCTX-M genes, and we speculate that the selection pressure on the mcr-1 gene may select for broad-spectrum cephalosporin resistance, especially by ISEcp1–tnpA–blaCTX-M. The coexistence of mcr-1 and blaCTX-M genes on a single plasmid presents more severe challenges in terms of controlling the election and transmission of mcr-1. Another characteristic of pSCS23 is that it lacks the IS683 mobile element (2703 bp) that exists on pHNSHP45, which is consistent with other plasmids harbouring the mcr-1 gene but lacking IS683.6 We postulate that the IS683 element should be present on pHNSHP45 occasionally, although complete sequences of plasmids carrying the mcr-1 gene are very scarce at present. Moreover, pSCS23 showed a 99% similarity (83% query coverage) to plasmid pSTH21 identified in S. enterica isolated from a human in China, which also carries the blaCTX-M-55 gene flanked by ISEcp1.10 Circular genetic map of pSCS23. Sequence comparison of pSCS23 (KU934209) with other completely sequenced plasmids: pHNSHP45, KP347127; and pSTH21, LN623683. The mcr-1 and blaCTX-M-55 genes are highlighted by red boxes. The map was drawn using BRIG (http://sourceforge.net/projects/brig/). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Here we report for the first time the co-occurrence of mcr-1 and blaCTX-M-55 on a single plasmid in S. enterica. Strong evidence exists for the ongoing transmission of mcr-1 on a global scale and its co-occurrence on plasmids with blaCTX-M genes. This evidence is of grave concern and merits immediate action to limit the selection and transmission of mcr-1 in order to preserve the efficacy of polymyxins. This work was supported by ‘973’ National Basic Research Program of China (project number 2013CB127200) and the General Program of National Natural Science Foundation of China (grant numbers 31572547 and 31572548). None to declare. We thank Dr Frank Riehle for his excellent assistance with revision of the manuscript.