First Report of Foodborne Klebsiella pneumoniae Coharboring bla_VIM-1, bla_NDM-1, and mcr-9

LETTER

Carbapenem and colistin antibiotics are often the last-resort treatment for severe infections caused by multidrug-resistant bacteria (1–3). Fortunately, most carbapenemase producers are susceptible to colistin, and many colistin-resistant strains have been reported to be sensitive to carbapenems (2–5). The present study is the first report of a Klebsiella pneumoniae strain LM22-1 harboring bla_NDM-1, bla_VIM-1, and mcr-9, which was recovered from food in Japan.

Strain LM22-1 was isolated in February 2016 from unfrozen chicken obtained from a local grocer in Higashihiroshima City, Hiroshima, Japan. The chicken sample was confirmed to originate from Japan. The isolate showed resistance to a wide range of antibiotics, including carbapenems, by broth microdilution assay (Table 1) (6). Carbapenemase-producing K. pneumoniae strains are uncommon in Japan but have been documented in Hiroshima hospitals (7, 8). To fully understand the genetic characteristics of K. pneumoniae LM22-1, the complete genome sequence was determined by whole-genome sequencing (9). The LM22-1 chromosomal DNA is 5,293,597 bp in size and harbors different antibiotic resistance genes (Table 2). The isolate also contains six plasmids, two of which are large and harbor different antibiotic and heavy metal resistance genes.

TABLE 1.

MICs of antimicrobials for NDM-1- and VIM-1-producing Klebsiella pneumoniae strain LM-22-1 and its E. coli transconjugant

Isolate/transconjuganta	MIC (μg/ml) of:
	CTXb	CRO	IPM	MEM	NAL	CIP	CHL	CST	GEN	TET	DOR	ETP	KAN	FOM	TGC
LM22-1	512	512	16	16	8	1	4	0.125	4	0.5	16	32	256	>1024	1
LM22-1-TC1	512	ND	8	16	4	0.5	4	0.25	4	0.5	8	16	256	ND	1
LM22-1-TC5	256	ND	4	4	4	0.5	4	0.125	0.25	0.5	2	4	128	ND	0.25
E. coli J53	0.0625	ND	0.25	0.0312	2	0.015	4	0.125	0.125	0.5	≤0.0078	≤0.0156	1	ND	0.125
E. coli ATCC25922	0.0625	ND	0.125	0.0156	1	0.0078	4	0.5	0.25	0.5	≤0.0078	0.0156	2	ND	0.125

^aLM22-1-TC1 is a transconjugant containing both pLM22-1-VIM-1 and pLM22-1-NDM-1 while LM22-1-TC5 is a transconjugant containing pLM22-1-NDM-1 only.

^bAbbreviations: CTX, cefotaxime; CRO, ceftriaxone; IPM, imipenem; MEM, meropenem; NAL, nalidixic acid; CIP, ciprofloxacin; CHL, chloramphenicol; CST, colistin; GEN, gentamicin; TET, tetracycline; DOR, doripenem; ETP, ertapenem; KAN, kanamycin; FOM, fosfomycin; TGC, tigecycline; ND, not determined.

TABLE 2.

Location of detected antimicrobial resistance genes

Location	Antimicrobial resistance genes
LM22-1 chromosome	blaSHV-71, oqxAB, and fosA
pLM22-1-VIM-1	blaVIM-1, mcr-9, blaTEM-1B, blaCTX-M-9, aadA24, ant(2'')-Ia, aadA2, aph(3')-Ia, sul1, and dfrA1
pLM22-1-NDM-1	blaNDM-1, aac(6')-Ib, aadA1, blaCTX-M-15, blaOXA-9, blaTEM-1A, qnrS1, and aac(6')-Ib-cr

The bla_VIM-1-harboring plasmid pLM22-1-VIM-1 is an IncHI2A plasmid that is 281,251 bp in size with 304 open reading frames (ORFs) and 47% GC content (Fig. 1A). Genomic analysis of pLM22-1-VIM-1 confirmed the coexistence of mcr-9 and bla_VIM-1 in addition to a wide range of other antibiotic resistance genes (Table 2). The mcr-9 gene has been recently reported to be associated with IncHI2 plasmids in both colistin-resistant Escherichia coli in France (10) and colistin-susceptible Enterobacter hormaechei in the United States and Egypt (4, 5). Interestingly, the organization of plasmid pLM22-1-VIM-1 was very similar (99.87% nucleotide sequence identity with a query coverage of 89%) to a portion of the Salmonella enterica subsp. enterica serovar Newport strain 0307-213 chromosome (GenBank accession number CP012599.1).

FIG 1.

Analysis of the IncHI2A plasmid pLM22-1-VIM-1 carried by the foodborne carbapenemase-producing K. pneumoniae strain LM-22-1. (A) Circular map of pLM22-1-VIM-1. (B) The genetic environment of the bla_VIM-1 gene, which lies within a class 1 integron. (C) Genetic context of the mcr-9 gene.

The bla_NDM-1-harboring plasmid pLM22-1-NDM-1 is a large plasmid of 124,214 bp with 161 ORFs and 52% GC content (Fig. 2A). It is an IncFII(K) plasmid that harbors different antibiotic resistance genes in addition to the bla_NDM-1 gene (Table 2). Plasmid pLM22-1-NDM-1 had a similar organization (99.28% nucleotide sequence identity, 76% query coverage) to K. pneumoniae bla_NDM-1 IncF-plasmid p2 (GenBank accession number CP009115.1; Fig. 2B), suggesting that both plasmids might have evolved from a single ancestor or one might have evolved from the other.

FIG 2.

Analysis of the IncFII(K) plasmid pLM22-1-NDM-1 carried by the foodborne carbapenem-producing K. pneumoniae strain LM-22-1. (A) Circular map of pLM22-1-NDM-1. (B) Comparative sequence analysis of the bla_NDM-1-encoding plasmid pLM22-1-NDM-1 to a related bla_NDM-1 IncF plasmid p2 (GenBank accession number CP009115.1) harbored by another K. pneumoniae strain.

The genetic context of bla_VIM-1, bla_NDM-1, and mcr-9 was examined. Our results showed that bla_VIM-1 is located in a unique, 7,073-bp class 1 integron [bla_VIM-1-aac(6′)-Il-dfrAI-ΔaadA24-smr-ISPa21] (Fig. 1B). Genetic mapping of bla_NDM-1 revealed that the insertion sequence ISAba125 was interrupted by the insertion of an ISSpu2-like (IS630 family) element, and a bleomycin resistance gene, ble_MBL, was upstream and downstream of bla_NDM-1, respectively. Genetic mapping of mcr-9 revealed that it is flanked by an IS903 insertion element upstream and by an IS1 insertion element downstream (Fig. 1C). For mcr-9, similar genetic patterns were observed in other sequenced IncH12 plasmids, including pME-1a (GenBank accession number NZ_CP041734.1), pMRVIM0813 (GenBank accession number KP975077), and pCTXM9_020038 (accession number CP031724) (4). Unlike our mcr-9-harboring plasmid, an IncHI2 plasmid from a colistin-resistant E. coli 68A isolate in France was also shown to harbor wbuC, qseC, and qseB as well as an ATPase gene downstream of the region containing the mcr-9 gene (10). These findings explain the colistin susceptibility of the LM22-1 strain and confirm the significant role of the qseB/qseC system in the induction of mcr-9-mediated colistin resistance (4, 10).

Filter-mating experiments were performed at 37°C with an azide-resistant E. coli strain J53. The results showed that pLM22-1-NDM-1 could be transferred but pLM22-1-VIM-1 could not be transferred. Genomic comparison of pLM22-1-VIM-1 with plasmid R478 (GenBank accession number BX664015) confirmed that both have similar intact conjugative transfer loci, located in the Tra1 and Tra2 regions (Fig. S1 in the supplemental material). Therefore, the filter-mating experiments were repeated at 25 to 30°C as previously described (5), and both plasmids were successively transferred (Table 1). Multilocus sequence typing of LM22-1 confirmed that it is a sequence type (ST) 30 strain. K. pneumoniae ST30 strains have been identified in infected patients in the United States and China (11, 12). Therefore, this strain might have been transmitted from food handlers. Although it is unlikely that this bacterial strain is spreading in Japan, the discovery of such a highly resistant strain in food in Japan—a country with a low level of antimicrobial resistance and high hygiene standards—is of grave concern, as an epidemic could have potentially disastrous consequences.

Data availability. The complete genome sequence of K. pneumoniae strain LM22-1 has been deposited at DDBJ/ENA/GenBank under BioProject no. PRJDB9227 (SRA accession no. DRA009528).