Sir, The emergence and spread of carbapenem-resistant isolates, especially New Delhi MBL (NDM)-producing Enterobacteriaceae, has become a global concern. Although NDM-producing Enterobacteriaceae have been mostly observed in clinical cases, they have also been identified in food-producing animals1,2 and wildlife.3 Recently, XDR bacteria harbouring both blaNDM and mcr-1 genes were observed in isolates from animals,4,5 posing a potential threat to public health. However, reports on the coexistence of blaNDM and mcr-1 in bacteria isolated from animals at slaughter remains sporadic.1,6 Here, we report two Escherichia coli strains, SD133 and SD138, co-producing NDM and MCR-1, isolated from chickens at slaughter in July 2015 in China. Fifty faecal samples were collected from chickens at slaughter in a poultry slaughterhouse located in Qingdao, China. Faecal samples were inoculated on antibiotic-free MacConkey plates. Suspected E. coli colonies were randomly selected and identified by MALDI-TOF MS. Only one E. coli strain was selected per faecal sample. A total of 33 E. coli isolates were recovered from the 50 faecal samples. MICs of 19 antimicrobial agents were determined by the agar dilution method or broth microdilution method (limited to colistin) and were interpreted according to CLSI M100-S27.7 Two (6.1%) E. coli strains, SD133 and SD138, showed resistance to imipenem and had an XDR pattern (Table 1). PCR and Sanger sequencing confirmed that the two strains harboured both blaNDM (SD133, blaNDM-1; SD138, blaNDM-9) and mcr-1 (Table 1). WGS results showed that SD133 belonged to novel ST7506 and harboured 18 resistance genes, whereas SD138 belonged to ST48 (ST10 complex) and harboured 14 resistance genes (Table 1). Characterization of blaNDM-positive strains and their transformants Novel ST of SD133 was obtained by analysing WGS results with an online tool (http://enterobase.warwick.ac.uk/) and ST of SD138 was obtained using an online tool (https://cge.cbs.dtu.dk/services/MLST/). WGS and plasmid sequencing was analysed via online database ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/). WGS and plasmid sequencing was analysed by online database PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/). MICs of 19 antibiotics were evaluated using the agar dilution method or the broth microdilution method (colistin) and were interpreted according to CLSI M100-S27. Breakpoints of neomycin (>8 mg/L) and florfenicol (>16 mg/L) were interpreted according to EUCAST (http://mic.eucast.org/Eucast2/). Characterization of blaNDM-positive strains and their transformants Novel ST of SD133 was obtained by analysing WGS results with an online tool (http://enterobase.warwick.ac.uk/) and ST of SD138 was obtained using an online tool (https://cge.cbs.dtu.dk/services/MLST/). WGS and plasmid sequencing was analysed via online database ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/). WGS and plasmid sequencing was analysed by online database PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/). MICs of 19 antibiotics were evaluated using the agar dilution method or the broth microdilution method (colistin) and were interpreted according to CLSI M100-S27. Breakpoints of neomycin (>8 mg/L) and florfenicol (>16 mg/L) were interpreted according to EUCAST (http://mic.eucast.org/Eucast2/). S1-PFGE and Southern hybridization indicated that, in SD133, blaNDM-1 and mcr-1 were located on an ∼130 kb plasmid and an ∼60 kb plasmid, respectively; in SD138, blaNDM-9 and mcr-1 were located on an ∼100 kb plasmid and on the chromosome, respectively. Furthermore, blaNDM-1 in SD133 can be transferred to E. coli DH5α via transformation and blaNDM-9 in SD138 can be transferred to E. coli C600 via conjugation at a frequency of 2.44 × 10−5 transconjugants/donor (Table 1). Plasmids were sequenced by PacBio platform (pHNSD133T1 carrying blaNDM-1) or Illumina HiSeq 2500 (pHNSD138-1 carrying blaNDM-9). pHNSD133T1 was a 129 713 bp IncY plasmid encoding 195 ORFs. It mainly consisted of a phage P7-like backbone and three mobile elements: IS1294, IS186 and a 32.8 kb MDR region (Figure S1A, available as Supplementary data at JAC Online). pHNSD133T1 was found to be 98% identical to Enterobacteria phage P7 (GenBank accession number: AF503408) with 62% coverage, and phage P1 mod749::IS5 (AF234172) with 57% coverage. However, three P1 phage-related tRNA (tRNA-Asn, tRNA-Thr and tRNA-Met) target sites for genetic recombination,8 were absent in pHNSD133T1. blaNDM-1 was observed in an 11 738 bp complex class 1 integron, which was sequentially organized as a 5′-CS (intI1), variable region 1 (VR1), 3′-CS1 (qacEΔ1 and sul1), ISCR1, VR2 and 3′-CS2 (qacEΔ1 and sul1). VR1 comprised four sequentially arranged genes: aac(6′)-Ib-cr, arr-3, dfrA27 and aadA16; VR2 contained a blaNDM-1 unit (ΔISAba125-blaNDM-1-bleMBL-trpF), catB3 and arr-3. The sequence of VR2 was found to be identical to those of the Proteus mirabilis plasmid pNDM-PM58 (KP662515) and the E. coli Y5 chromosome (CP013483). Furthermore, despite the 157 bp deletion between aadA16 and dfrA27 in VR2, the complex class 1 integron in pHNSD133T was found to be 99% identical to the E. coli Y5 chromosome (Figure S1B). Interestingly, E. coli Y5 was from a patient in Yantai,9 which is ∼178 km away from Qingdao. To the best of our knowledge, this study identified for the first time the presence of blaNDM-1 gene in phage-like IncY plasmid, which has been associated with mcr-110 and blaVIM-1.11 Although phage-like plasmid is non-conjugative, it can be integrated into conjugative plasmids via recombination.10 In addition, mcr-1 was found in an IncI2 plasmid in strain SDX5C133, namely pHNSD133-MCR, which was almost 99% identical (99% coverage) to pWF-5-19C_mcr-1 (KX505142, Cronobacter sakazakii, chicken, China) (Figure S2). pHNSD138-1 was a 103 506 bp circular plasmid belonging to IncK2, which was associated with the spread of blaCMY-2/412 and mcr-113 in E. coli isolated from retailed chicken in European countries. The backbone of pHNSD138-1 was highly similar to plasmid pHNTH02-1 (MG196294), which was previously detected in E. coli THSJ02 isolated from retail chicken meat in Guangzhou, China,14 with 95% coverage and 99% identity (Figure S3A). It was also highly homologous to pDV45 (KR905384), pMbl488 (KY565558) and pMbl536 (KY689635), which were found in E. coli isolated from chicken meat samples in Switzerland (75% coverage and 97%–99% identity). The MDR regions of pHNSD138-1 and pHNTH02-1 were both bounded by IRR of ΔTn1721 and ΔTn5393 (Figure S3B). In pHNTH02-1, blaNDM-9 was embedded in an ISCR1 complex class 1 integron with two copies of IS26, characterized by the transposition module IS26-ΔISAba125-blaNDM-9-bleMBL-trpF-tat-ΔcatA-ISCR1-qacEΔ1-sul1-aadA2-gcuF-dfrA12-intI1-IS26. The transposition module was similar to that of pC629 (CP015725, Salmonella enterica serovar Indiana, chicken carcass, China), pWF-5-19C_NDM (KX505143, C. sakazakii, chicken, China), pKPGJ-2a (CP017850, Klebsiella variicola, river water, Korea) and p5CRE51-NDM-9 (CP021177, E. coli, human, Taiwan). The structure was inverted in pHNSD138-1, which was possibly owing to an insertion of IS1294 within the tat gene and the truncation of intI1 by the IS26-aac(3)-IV-aph(4)-Ia-ISEc59 transposition unit. We proposed that the transfer and spread of blaNDM-9 was associated with the IS26-ΔISAba125-blaNDM-9-bleMBL-trpF-tat-ΔcatA-ISCR1-qacEΔ1-sul1-aad2-gcuF-dfrA12-intI1-IS26 unit. Competition assay and plasmid stability were performed as previously described.15 The loss rates of pHNSD133T1 and pHNSD138-1 were 16% and 0%, respectively. Further study yielded relative fitness <0.82 ± 0.09 to 0.63 ± 0.05 for E. coli DH5α carrying pHNSD133T1 (Figure S4), indicating a significant biological cost. Relative fitness of E. coli DH5α containing pHNSD138-1 was 1.06 ± 0.05–0.98 ± 0.02, indicating a small fitness cost. The stability and low fitness cost of pHNSD138-1 suggest its potential to spread widely. Our study identified the coexistence of blaNDM and mcr-1 in two E. coli strains isolated from chickens at slaughter. Multiple resistance genes were observed in blaNDM-bearing plasmids, making selection through the use of other antibiotics possible, since carbapenems are not typically used in animal production. IncY and IncK2 are carriers of not only blaNDM but also mcr-1. Thus, the simultaneous acquisition of mcr-1 and blaNDM by IncY or IncK2 plasmids is possible. Further investigation is needed to determine the evolutionary characteristics of these plasmids. This work was supported in part by grants from the National Natural Science Foundation of China (no. 31625026 and no. 81661138002). None to declare. Figures S1–S4 are available as Supplementary data at JAC Online.
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