Co-existence of plasmid-mediated tigecycline and colistin resistance genes tet(X4) and mcr-1 in a community-acquired Escherichia coli isolate in China

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Sir, Tigecycline and colistin are considered as the ‘last-resort’ antibiotics used to treat severe infections caused by MDR bacteria, particularly carbapenem-resistant Enterobacteriaceae.1 However, the emergence and widespread nature of plasmid-borne mcr-1 and tet(X4) genes, which confer resistance to tigecycline and colistin, represent a significantly increasing threat to public health.2,3 Since 2015, the mcr-1 gene, which confers colistin resistance, has been frequently reported from clinical, veterinary and environmental sources.4,5 Later, the family of mobile tet(X) tetracycline-inactivating variants, which can mediate high-level resistance to the entire family of tetracycline antibiotics, including the newly US FDA-approved eravacycline and omadacycline, was reported in multiple ecological niches.6,7 Despite the fact that an alarming number of tet(X4)-carrying Enterobacteriaceae isolates have been identified in livestock and the environment, the prevalence of tet(X4)-positive isolates in clinical specimens is still rare.2 Here, we report the first, to the best of our knowledge, identification of a colistin- and tigecycline-resistant Escherichia coli isolate recovered from a patient with a urinary tract infection in China, co-harbouring mcr-1, tet(X4) and blaCTX-M-14 genes. WGS and microbiological analysis were performed to elucidate the genomic characteristics and potential transmission mechanisms of tet(X4) and mcr-1. During our routine surveillance of antimicrobial resistance, a total of 1864 E. coli isolates were collected from a tertiary hospital in Yiwu, Zhejiang province, China during the period 2015–19. The retrospective PCR screening analyses confirmed the presence of 36 (1.9%) isolates carrying mcr-1. Meanwhile, only one (<0.1%) isolate harboured tet(X4), with the others mostly susceptible to tigecycline (MIC90 ≤0.5 mg/L; range ≤0.5–2 mg/L). The tet(X4)-carrying isolate EC931 also encoded mcr-1. EC931 was recovered from a urine sample of an elderly female patient in February 2017, who was admitted to the Department of Nephrology with symptoms of low back pain and diagnosed with a urinary tract infection, and the isolate was collected within 24 h after admission. The patient resided in the countryside, without a history of travel, hospitalization or use of antibiotics in the last 3 months, and thus the infection was considered to be community-acquired. The patient was successfully treated with a 6 day administration of piperacillin/tazobactam and was linked to a good prognosis. Results of antimicrobial susceptibility testing showed that EC931 was resistant not only to tigecycline and colistin but also to quinolones and third-generation cephalosporins (Table S1, available as Supplementary data at JAC Online). In addition, the conjugation assay confirmed that the tet(X4) and mcr-1 genes were both located on a conjugative plasmid(s), since the transconjugant strain was resistant to tigecycline and colistin only and no longer to quinolones and third-generation cephalosporins (Figure S1 and Table S1). Hybrid genome sequencing using both Illumina HiSeq and the Nanopore MinION platforms was performed on E. coli EC931, allowing its complete genome sequence to be obtained.8In silico MLST analysis showed that EC931 belongs to ST773, a sporadic and even rare ST. The phylogenetic relationship between EC931 and 34 other ST773 strains deposited in the public database suggests that the closest relative of EC931 was recovered from poultry in Vietnam, which differed by only 19 core genome MLST (cgMLST) loci. The Vietnamese strain also carried several antimicrobial resistance genes, i.e. blaTEM-1B, aac(3)-IId, mdf(A), tet(B) and catA1, but not the tet(X4) gene. This suggests that the MDR trait was laterally acquired by EC931 or that the trait was initially present in the Vietnamese strain but later lost without selection pressure. The chromosome of EC931 encoded blaCTX-M-14 along with several mutations in the QRDRs, such as S83L and D87N in GyrA and S80I in ParC. These findings explained the chromosome-mediated resistance to quinolones and third-generation cephalosporins. The tet(X4) gene was located on a 50 626 bp plasmid designated as pEC931_tetX of incompatibility group IncX1. This plasmid also carried other resistance genes, including aadA2, lnu(F), floR and tet(A). To investigate the genetic contexts of tet(X4), a detailed comparison of the tet(X4)-bearing plasmids of clinical, livestock and environmental origins was performed. The most similar plasmid to pEC931_tetX in the public database was E. coli plasmid pYY76-1-2, which showed 90% coverage and 95% identity. This isolate was, however, recovered from the faeces of a cow in 2018 in Hainan, a province in China over 2000 km away from Zhejiang province.9 The only difference between the two plasmids were blaTEM-1B and its adjacent Tn3-like transposon in pEC931_tetX being replaced by an IS26-mediated transposon carrying blaSHV-12 in pYY76-1-2; both structures were commonly present in clinical specimens (Figure 1). The gene tet(X4) was flanked by ISCR2 and IS26 in both plasmids, but this surrounding structure was different from that of the other tet(X4)-carrying plasmids. The latter was bracketed by ISCR2 at both ends (Figure S2 and Table S2). Except for pYY76-1-2, no other plasmids shared over 50% of gene content with pEC931_tetX. (a) Phylogenetic relationship between E. coli EC931 and 34 other ST773 E. coli strains retrieved from NCBI GenBank, NCBI SRA and Enterobase, generated using the cgMLST approach. Clonal relationships among different isolates are depicted by the line length connecting each circle. The numbers given in square brackets indicate the number of isolates recovered from each country. (b) Phylogenetic relationship between E. coli EC931 and five ST773 E. coli strains recovered from other cities in China. (c) Comparison of the homologous regions shared by two tet(X4)-bearing plasmids: pEC931_tetX and pYY76-1-2. The arrows represent coding sequences [blue arrows, tet(X4); red arrows, other antimicrobial resistance genes; yellow arrows, conjugation-related genes] and indicate transcriptional directions of the ORFs. The Δ symbol indicates that the gene is truncated. (d) Linear genetic context of the mcr-1-carrying plasmid pEC931_mcr. The arrows represent coding sequences (blue arrow, mcr-1; yellow arrows, conjugation-related genes). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC . The gene mcr-1 was located on another plasmid, pEC931_mcr, which is 33 858 bp in length and belongs to the IncX4 group. This plasmid showed >99% coverage and >99% nucleotide identity with other 33 kb mcr-1-bearing IncX4 plasmids in China.10 Unlike other types of mcr-1-bearing plasmids, these 33 kb plasmids are characterized by a conserved gene content and lack of an ISApl1 element flanking the mcr-1 gene. Accordingly, a plasmid instead of a transposon functions as the vector to help the dissemination of mcr-1. This study reports for the first time (to the best of our knowledge) a clinical E. coli isolate co-carrying mobile tigecycline and colistin resistance determinants tet(X4) and mcr-1 in China. Despite their currently low prevalence rate, the plasmid-borne nature of the two genes represents a potential threat for the clinical usage of the last-resort antibiotics. Given the reported high rate of tet(X4) and mcr-1 from the veterinary sector, a possible flow of these resistance genes from livestock to humans is postulated. Continuous surveillance of these genes in humans, animals and environments is therefore urgently needed, with the aim of identifying reservoirs and monitoring the transmission dynamics in the community and healthcare settings. The nucleotide sequences of the chromosome and plasmids of E. coli isolate EC931 have been deposited in the NCBI GenBank under accession numbers CP049118–CP049131. This work was supported by the National Natural Science Foundation of China (31670132 and 81702042). None to declare. Tables S1 and S2 and Figures S1 and S2 are available as Supplementary data at JAC Online.