Emergence of Escherichia coli co-producing NDM-1 and KPC-2 carbapenemases from a retail vegetable, China

Sir, Carbapenemase-producing Enterobacteriaceae have been increasingly reported worldwide and have become an urgent public health threat.1 Co-production of New Delhi metallo-β-lactamase-1 (NDM-1) and Klebsiella pneumoniae carbapenemase 2 (KPC-2) in a single bacteria species has so far been described in clinical Enterobacteriaceae (K. pneumoniae, Enterobacter hormaechei, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa) in China, India and other countries.2–6 Here, we report a carbapenem-resistant Escherichia coli strain co-producing NDM-1 and KPC-2 from a retail vegetable in China. E. coli isolate TS79e was obtained from fresh lettuce from a market in Guangzhou, China in October 2015. The MICs of 12 antimicrobial agents were determined by the agar dilution method or broth microdilution method (limited to colistin) and interpreted by CLSI breakpoints7 or EUCAST epidemiological cut-off values (http://mic.eucast.org/Eucast2/). This isolate was highly resistant (>128 mg/L) to imipenem, ceftazidime, cefotaxime, cefoxitin, cefquinome, amikacin, tetracycline and fosfomycin, but susceptible to colistin and tigecycline (Table 1). PCR and Sanger sequencing with primers listed in Table S1 (available as Supplementary data at JAC Online) confirmed that this strain harboured blaNDM-1, blaKPC-2, fosA3 and floR. MLST showed that E. coli TS79e belonged to ST877 (www.warwick.ac.uk/mlst/). Characteristics of E. coli TS79e and its transconjugant/transformanta Resistance genes identified by plasmid sequencing are underlined. Characteristics of E. coli TS79e and its transconjugant/transformanta Resistance genes identified by plasmid sequencing are underlined. S1-PFGE and Southern hybridization revealed that blaNDM-1 and blaKPC-2 were located on ∼118 and ∼64 kb plasmids, respectively, designated as plasmids pHNTS79-NDM and pHNTS79-KPC, respectively. Additionally, blaKPC-2 was able to transfer from E. coli TS79e to E. coli C600 by conjugation and blaNDM-1 was successfully transferred to E. coli DH5α by transformation. The blaKPC-2-carrying transconjugant showed greatly elevated MICs of imipenem, ceftazidime and cefotaxime (Table 1). The blaNDM-1-harbouring transformant exhibited significantly increased MICs of imipenem, ceftazidime, cefotaxime, cefoxitin, cefquinome, amikacin and tetracycline. Notably, although isolate TS79e showed a high MIC of imipenem, the transconjugant/transformant showed low imipenem MIC values of 2 mg/L. This result is in agreement with a previously described K. pneumoniae strain from India that co-produced NDM-1 and KPC-2,4 indicating that other mechanisms such as porin loss and overexpression of efflux systems might also be responsible for the high-level imipenem resistance of strain TS79e.8 Plasmid pHNTS79-NDM was classified as IncA/C-ST1 (http://pubmlst.org/plasmid/), whereas the replicon type of pHNTS79-KPC could not be determined by PCR-based replicon typing.9 IncA/C plasmids have been described as major carriers responsible for the global spread of blaNDM among Enterobacteriaceae, particularly in humans.10,11 To investigate the genetic elements of blaNDM-1 and blaKPC-2, we used Illumina Miseq Technology (Illumina, San Diego, CA, USA) to sequence plasmids pHNTS79-NDM and pHNTS79-KPC extracted from transformant/transconjugant. Sequence reads were assembled into contigs by SOAPdenovo version 2.04 (http://soap.genomics.org.cn/), yielding 14 and 19 contigs, respectively. Resistance genes were annotated using ResFinder 2.1 (https://cge.cbs.dtu.dk//services/ResFinder/). The results revealed that aacA4, tet(D), sul1, armA, mph(E), msr(E), erm(B) and ΔblaDHA-1 were co-located with blaNDM-1 on pHNTS79-NDM, whereas streptomycin resistance genes strA and strB were also located on blaKPC-2-carrying plasmid pHNTS79-KPC. A 6078 bp blaNDM-1-associated fragment with genetic content IS26-ΔISAba125-blaNDM-1-bleMBL-ΔtrpF-ΔblaDHA-1-ampR-qacEΔ1-sul1 was obtained from pHNTS79-NDM. It was identical to the corresponding region of plasmids pNDM-VF (GenBank accession number KR733543, Vibrio fluvialis, India), pNDM-OM (JX988621, K. pneumoniae, Oman) and pNDM-HK (HQ451074, E. coli, Hong Kong) obtained from humans (Figure S1). As commonly described in blaNDM-1-carrying plasmids, such as pHNEC46 (KX503323, E. coli, pig, China) and pNDM-BJ01 (JQ001791, Acinetobacter lwoffii, human, China), ISAba125 was upstream of blaNDM-1, and ble and trpF were present downstream of blaNDM-1, although ISAba125 was truncated by an IS26 element in pHNTS79-KPC. Interestingly, trpF was followed by an incomplete blaDHA-1 and its associated regulatory gene ampR, and part of the 3′-CS containing qacEΔ1 and sul1, which was similar to genomic island 1 (PGI1-PmPEL) in a clinical Proteus mirabilis strain (KF856624, France), as well as to IncA/C2 plasmid pEC2-NDM-3 (KC999035, E. coli, human, Australia), harbouring blaNDM-3 with a single nucleotide difference from blaNDM-1 (Figure S1). Additionally, a 6095 bp region in plasmid pHNTS79-KPC was obtained. The 3’-end of Tn3 was interrupted by ISKpn27, flanked by 6 bp DRs, and then it was connected with blaKPC-2-ΔISKpn6, including 74 bp sequences upstream of blaKPC-2, which may originate from Tn4401 (EU176011) (Figure S1). Various remnants of Tn3 were identified downstream of ISKpn27 in different blaKPC-2-bearing plasmids, such as 206 bp in p112298-KPC (KP987215, Citrobacter freundii, human, China), 877 bp in pKPC2 (KR014106, Aeromonas hydrophila, human, China) and only 32 bp in pHNTS79-KPC. Furthermore, this 6 kb fragment with linear structure ΔTn3-ISKpn27-blaKPC-2-ΔISKpn6-korC-klcA-ΔrepB was commonly observed in other plasmids from different species, such as plasmids p112298-KPC, pKPC2, pBK34397 (KU295132, E. coli, human, USA) and pKP048 (FJ628167, K. pneumoniae, human, China) (Figure S1). The nucleotide sequences of 6078 and 6095 bp fragments of plasmids pHNTS79-NDM and pHNTS79-KPC were deposited in GenBank under accession numbers MF383355 and MF383354, respectively. Carbapenemase-producing (OXA-48 and OXA-181) Enterobacteriaceae isolates have been previously described in vegetables.12,13 To the best of our knowledge, this is the first report of either the blaNDM or blaKPC genes in bacteria from vegetables and also the first report of the coexistence of blaNDM-1 and blaKPC-2 in E. coli. Although the origin of blaNDM-1 and blaKPC-2 in vegetables is unclear, hospital wastewater or human sewage contamination might be sources. The isolate TS79e co-producing two carbapenemases showed extremely high-level resistance to imipenem and also carried several other important resistance genes (armA and fosA3), consistent with the results reported for clinical Enterobacteriaceae isolates that cause a significant challenge to clinical treatment.2,5 Most importantly, the emergence of E. coli co-producing NDM-1 and KPC-2 in fresh vegetables is alarming, as vegetables such as lettuce are often consumed raw and might be a possible source from which humans could acquire clinically relevant resistance genes, such as blaNDM, blaKPC, armA and fosA3, thus posing a serious threat to public health. Appropriate measures, such as improving sewage treatment protocols and protecting irrigation water from faecal pollution, need to be taken to improve food safety. This work was supported in part by the grants from the National Natural Science Foundation of China (NSFC) (31625026 and 81661138002) and the National Key Basic Research Program of China (no. 2013CB127200). None to declare. Table S1 and Figure S1 are available as Supplementary data at JAC Online.