Various Sequence Types of Enterobacteriaceae Isolated from Commercial Chicken Farms in China and Carrying the bla_NDM-5 Gene

A total of 108 meropenem-resistant Enterobacteriaceae isolates were obtained from 1,658 rectal swabs collected from 15 unrelated commercial chicken farms in China between 2014 and 2016. These samples yielded 16 Escherichia coli and 2 Klebsiella pneumoniae isolates of diverse sequence types carrying a bla_NDM-5-bearing IncX3 plasmid.

ABSTRACT

A total of 108 meropenem-resistant Enterobacteriaceae isolates were obtained from 1,658 rectal swabs collected from 15 unrelated commercial chicken farms in China between 2014 and 2016. These samples yielded 16 Escherichia coli and 2 Klebsiella pneumoniae isolates of diverse sequence types carrying a bla_NDM-5-bearing IncX3 plasmid. K. pneumoniae strain sequence type 709 (ST709) has two bla_NDM-5-carrying plasmids that were transferred together to E.coli.

TEXT

Multidrug-resistant organisms, including those that are carbapenemase-producing Enterobacteriaceae, are becoming a challenging threat to public health worldwide (1). Gene bla_NDM-5, a variant of bla_NDM, was first identified in 2011 in an Escherichia coli sequence type 648 (ST648) isolate from a patient in the United Kingdom (2). Since then, it has been reported in various parts of the world, including South Korea (3), Denmark (4), Algeria (5), and China (6–8). The cooccurrence of NDM-5 and other carbapenemase enzyme isolates from the same patient is extremely worrisome because it might lead to therapeutic failure and death. The first cooccurrence of NDM-4- and NDM-5-producing Klebsiella pneumoniae in the same patient was reported in 2016 (9). In 2017, a carbapenem-resistant K. pneumoniae ST147 isolate harboring bla_NDM-5 and bla_OXA-181 from a hospitalized patient was found in the United States (10). In 2018, a bla_NDM-5- and bla_OXA-48-like-coproducing E. coli strain was first isolated in South Korea (11). Meanwhile, the first case of a clinical Klebsiella michiganensis isolate producing KPC-2, NDM-1, and NDM-5 was reported in China (12). A fusion plasmid (IncX3 and IncFIB) recoverable from an NDM-5-producing clinical E. coli isolate was recently characterized, and these types of recombination events presumably play a potential role in the development of new plasmids with extended resistance profiles (13). In this study, we identified the presence of various sequence types of Enterobacteriaceae carrying bla_NDM-5 in chickens from multiple farms across seven Chinese provinces.

A total of 108 nonrepeated meropenem-resistant Enterobacteriaceae (97 E. coli and 11 K. pneumoniae) isolates (6.5%) were obtained from 1,658 rectal swabs collected from 15 unrelated commercial chicken farms in China between 2014 and 2016 (see Fig. S1 in the supplemental material). All of the Enterobacteriaceae isolates were selected on MacConkey agar plates supplemented with 2 μg/ml meropenem. Species identification was performed with the BD Phoenix-100 system (Becton Dickinson) and confirmed by 16S rRNA gene sequencing. Antimicrobial susceptibility testing was performed on Mueller-Hinton agar plates testing for 16 antimicrobials according to CLSI guidelines (14), except polymyxin B, for which European Committee on Antimicrobial Susceptibility Testing breakpoints were used (15).

These Enterobacteriaceae isolates were then subjected to screening for the presence of bla_NDM and mcr genes by PCR assay (see Table S1 in the supplemental material) as previously described (16). Of 108 meropenem-resistant Enterobacteriaceae strains, 52 (48 E. coli and 4 K. pneumonia; 3.14%) were found to harbor the bla_NDM-5 gene. Multilocus sequence typing (MLST) was performed as previously described (http://bigsdb.pasteur.fr/); 16 different sequence types of E. coli isolates and 2 sequence types of K. pneumoniae isolates were found. Three of the 16 strains of E. coli were found to coharbor the mcr-1 gene. Multidrug-resistant E. coli strain ZT614 ST132 was resistant to all 16 antimicrobials. Phylogenetic relationship, antimicrobial resistance phenotypes, and sequence types of the strains are shown in Fig. 1.

FIG 1.

Phylogenetic relationship, antimicrobial resistance phenotypes, source of isolate, and sequence types of the 18 different Enterobacteriaceae: (a) Escherichia coli and (b) Klebsiella pneumoniae. A phylogenetic tree based on a maximum-likelihood method was built by MEGA6 with nucleotide sequences of 8 MLST genes to reveal a more detailed relationship among the analyzed strains. Bootstrap values (percentages of 1,000 replications) of >50% are shown at each node. 1, Failure to find any corresponding ST type with MLST database blasting. S, susceptible; I, intermediate; R, resistant; PB, polymyxin B; IPM, imipenem; CTX, cefotaxime; CAZ, ceftazidime; FOX, cefoxitin; AMC, amoxicillin-clavulanate; AK, amikacin; CIP, ciprofloxacin; DO, doxycycline; FOS, fosfomycin; CHL, chloramphenicol; SXT, trimethoprim- sulfamethoxazole; CRO, ceftriaxone; CN, gentamicin; ATM, aztreonam; MEM, meropenem.

Conjugation experiments were performed between 18 different isolates (Fig. 1) and E. coli J53 Az^r as the recipient. Transconjugants were selected on Mueller-Hinton agar (MHA; Oxoid) plates that contained 200 μg/ml sodium azide with 2 μg/ml imipenem. All of them could successfully transfer their carbapenem resistance genes to the recipient strain E. coli J53 Az^r. There was no cotransfer of carbapenem and colistin resistance phenotype transconjugant. The total plasmid DNA from 18 transconjugants was extracted using a Qiagen plasmid minikit following manufacturer's recommendations (Qiagen, Hilden, Germany). Whole-genome sequencing was performed on the Illumina MiSeq platform (Majorbio, Shanghai) using a 350-bp paired-end TruSeq library with a 2 × 300 run. A draft assembly of the plasmids was made with plasmidSPAdes (17). Predicted gaps were closed by PCR and Sanger sequencing the using specifically designed primers listed in Table S1. Identification of antibiotic resistance genes was done by ResFinder 3.0 (http://www.genomicepidemiology.org/), and plasmid replicon types were determined by using the PlasmidFinder tool (http://genomicepidemiology.org/).

Sequence analysis revealed that all of the transconjugants harbored a 46-kb bla_NDM-5-bearing IncX3 plasmid. BLASTN results showed that all 18 IncX3 plasmids had almost 100% identity to the 46,161-bp plasmid pBJ114-46 (GenBank accession no. MF679143) with only 1 to 4 single-base changes (data not shown), indicating that the bla_NDM-5-bearing IncX3 plasmid was an important vector responsible for the dissemination of NDM-5 among Enterobacteriaceae isolates originating from chicken farms in China. Recently, a study identified the occurrence of similar IncX3 plasmids carrying bla_NDM-5 in pigs originating from multiple farms across China (18), confirming that this mobile NDM vector is widespread in animal production.

Interestingly, a transconjugant of the ST709 K. pneumoniae strain named SCKLB138 harbored two bla_NDM-5-bearing plasmids, pSCKLB-1 and pSCKLB-2 (see Fig. S2 in the supplemental material). Plasmid pSCKLB-1 was found to comprise the IncFIB and IncFII replicons; the bla_NDM-5, bla_OXA-10, rmtB, aadA1, and bla_TEM-1 genes; and some other resistance gene cassettes bounded by various insertion sequences. bla_NDM-5 together with the bleomycin resistance gene ble_MBL, tat, and tnpF form part of the transposon Tn125 (ΔTn125). Upstream of the ISAba125, IS26 and ISCfr1 were identified bracketing bla_TEM-1 and the 16S rRNA methylase gene rmtB, conferring resistance to aminoglycosides. The same arrangement of ΔTn125 and rmtB was found in ST147 K. pneumoniae plasmid pCC1409-1 (GenBank accession no. KT725789), except that the upstream of the bla_TEM-1 gene of pCC1409-1 was truncated by Tn2 resolvase rather than ISCfr1 (Fig. 2a). The bla_OXA-10 gene was localized downstream of bla_NDM-5 in a class 1 integron with the dfrA14-arr-2-cmlA7-bla_OXA-10-aadA1 cassette array and the ISCR1 element behind the 3′-conserved segments. The region bracketed by bla_NDM-1 and tnpR in plasmid pHN-NDM0711 exhibited 99% identity to the corresponding region of pSCKLB-2 (Fig. 2a). Plasmid pSCKLB-2 was a 46-kb IncX3 bla_NDM-5-bearing plasmid. An IS5 was inserted with ISAba125 upstream of bla_NDM-5 and the ble, trpF, and tat genes downstream from bla_NDM-5. Comparison of the genetic characteristics of pSCKLB-2 and pUCLAOXA232-2 (GenBank accession no. NZ_CP012563) showed that an ISL3 was inserted downstream of the resolvase gene, leading to the flanking 8-bp direct repeats (ATATGCAT). The bla_NDM-5-carrying region bracketed by IS26 and tnpA was inserted into the umuD gene, resulting in a pair of 3-bp direct repeats (TGT). The ISAba125 gene was interrupted by IS5 and split into two fragments, resulting in a pair of 4-bp direct repeats (CTAA) (Fig. 2b).

FIG 2.

The genetic context of bla_NDM-5 on pSCKLB138-1 and pSCKLB138-2 compared with other plasmids. (a) Genetic structure of bla_NDM-5 gene on pSCKLB138-1 compared with pCC1409-1 and pNDM15-1078. (b) Genetic structure of bla_NDM-5 gene on pSCKLB138-2 compared with pUCLAOXA232-2.

In conclusion, this study identified a self-transmissible IncX3 plasmid carrying bla_NDM-5 that was an important vector responsible for the dissemination of NDM-5 among Enterobacteriaceae isolates originating from chicken farms in China. The cooccurrence of bla_NDM-5 and other resistance rmtB and mcr-1 genes in Enterobacteriaceae isolated in chicken farms strongly suggests a potential food chain dissemination pathway, which warrants further attention. To the best of our knowledge, this is the first report of two bla_NDM-5-carrying plasmids coexisting in a K. pneumoniae strain isolated from commercial chicken farms in China. The results highlight that the chicken farms are an important reservoir of Enterobacteriaceae carrying bla_NDM-5 gene.

Accession number(s).

The complete nucleotide sequences of plasmids pSCKLB-1 and pSCKLB-2 characterized in this study were submitted to the GenBank database and assigned accession numbers MH161191 and MH161192.