Vegetable-Derived Carbapenemase-Producing High-Risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 Clones in Japan: Coexistence of bla_NDM-1, bla_OXA-66, bla_OXA-72, and an AbaR4-Like Resistance Island in the Same Sample

Carbapenemase-producing Gram-negative bacteria (CPGNB) cause severe health care-associated infections and constitute a major public health threat. Here, we investigated the genetic features of CPGNB isolated from fresh vegetable samples in Japan and found CPGNB, including Klebsiella pneumoniae and Acinetobacter baumannii, with dissimilar carbapenemases.

ABSTRACT

This study was conducted to characterize carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumannii isolated from fresh vegetables in Japan. Two K. pneumoniae isolates (AO15 and AO22) and one A. baumannii isolate (AO22) were collected from vegetables in the city of Higashihiroshima, Japan, and subjected to antimicrobial susceptibility testing, conjugation experiments, and complete genome sequencing using Illumina MiniSeq and Oxford Nanopore MinION sequencing platforms. The two K. pneumoniae isolates were clonal, belonging to sequence type 15 (ST15), and were determined to carry 19 different antimicrobial resistance genes, including bla_NDM-1. Both the isolates carried bla_NDM-1 on a self-transmissible IncFII(K):IncR plasmid of 122,804 bp with other genes conferring resistance to aminoglycosides [aac(6′)-Ib, aadA1, and aph(3′)-VI], β-lactams (bla_CTX-M-15, bla_OXA-9, and bla_TEM-1A), fluoroquinolones [aac(6′)-Ib-cr], and quinolones (qnrS1). A. baumannii AO22 carried bla_OXA-66 on the chromosome, while bla_OXA-72 was found as two copies on a GR2-type plasmid of 10,880 bp. Interestingly, A. baumannii AO22 harbored an AbaR4-like genomic resistance island (GI) of 41,665 bp carrying genes conferring resistance to tetracycline [tet(B)], sulfonamides (sul2), and streptomycin (strAB). Here, we identified Japanese carbapenemase-producing Gram-negative bacteria isolated from vegetables, posing a food safety issue and a public health concern. Additionally, we reported a GR2-type plasmid carrying two copies of bla_OXA-72 and an AbaR4-like resistance island from a foodborne A. baumannii isolate.

IMPORTANCE Carbapenemase-producing Gram-negative bacteria (CPGNB) cause severe health care-associated infections and constitute a major public health threat. Here, we investigated the genetic features of CPGNB isolated from fresh vegetable samples in Japan and found CPGNB, including Klebsiella pneumoniae and Acinetobacter baumannii, with dissimilar carbapenemases. The NDM carbapenemase, rarely described in Japan, was detected in two K. pneumoniae isolates. The A. baumannii isolate identified in this study carried bla_OXA-66 on the chromosome, while bla_OXA-72 was found as two copies on a GR2-type plasmid. This study indicates that even one fresh ready-to-eat vegetable sample might serve as a significant source of genes (bla_NDM-1, bla_OXA-72, bla_CTX-M-14b, and bla_CTX-M-15) encoding resistance to frontline and clinically important antibiotics (carbapenems and cephalosporins). Furthermore, the detection of these organisms in fresh vegetables in Japan is alarming and poses a food safety issue and a public health concern.

INTRODUCTION

The emergence and international dissemination of carbapenemase-producing Gram-negative bacteria (CPGNB) constitute a major public health threat (1). Due to the excessive use of antibiotics in the livestock sector, food might serve as a reservoir of multidrug-resistant bacteria with transferable antibiotic resistance genes (1). Contamination of vegetables with microorganisms could be a result of direct contact with the soil, the use of wastewater and manure as fertilizers, or even human or animal contact during growth and collection (2). Additionally, the discharge of multidrug-resistant Gram-negative bacteria (MDRGNB) into the environment could occur through wastewater and feces contamination of plants, soil, surface water, rural environments, and food products (3). Therefore, the One Health approach was introduced and is defined as a strategy focusing on prevention of the spread of antimicrobial resistance between different local ecosystems (i.e., hospitals, wastewater treatment plants, and natural environments, including food and vegetables) (4).

The acquired carbapenemase genes in CPGNB belong to three different Ambler classes: (i) the class A serine carbapenemases KPC and FRI; (ii) the class B metallo-β-lactamases (MBLs) of the NDM, VIM, and IMP types; and (iii) the class D OXA-48-like, OXA-23-like, and OXA-24-like oxacillinases (5). CPGNB have been isolated from fresh ready-to-eat vegetables from different countries, including Algeria, China, Germany, Myanmar, Italy, Lebanon, and Switzerland (2, 6–13). Escherichia coli strains coproducing NDM-1 and KPC-2 or the colistin resistance protein Mcr-1 and NDM have been previously identified from vegetables in China, posing a public health threat (7, 10). Alarmingly, a German nosocomial outbreak of illness caused by bla_VIM-carrying Citrobacter freundii was linked to the consumption of vegetable salads (6). We recently reported the first foodborne Klebsiella pneumoniae strain coharboring mcr-9, bla_VIM-1, and bla_NDM-1 (14). Therefore, surveillance of these organisms in fresh vegetables is needed, as they could lead to hospital outbreaks.

Multidrug-resistant ESKAPE bacteria (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are some of the most challenging hospital-acquired pathogens and cause severe bacterial infections among hospitalized immunocompromised and critically ill patients globally (3, 15). This group of bacteria could disseminate antimicrobial resistance genes through horizontal transfer using mobile genetic elements (MGEs) (i.e., plasmids, insertion sequences, and genomic islands) (16). Important examples of MGEs are (i) NDM-1- and VIM-1-carrying plasmids in K. pneumoniae (14) and (ii) the AbaR3 and AbaR4 genomic islands (GI), identified in A. baumannii, with Tn6019 and Tn2006 as the backbone transposons, respectively (17). Notably, Tn2006 harboring the class D carbapenemase gene bla_OXA-23 was identified interrupting an AbaR4-like GI named AbaR25 (17).

Despite the absence of reports about CPGNB isolated from vegetables in Japan, this study was conducted to investigate the possible detection and characterization of these organisms. Also, we report the isolation of two different carbapenemase-producing ESKAPE bacteria, namely, NDM-1-producing K. pneumoniae and OXA-66- and OXA-72-producing A. baumannii strains, from fresh vegetables in Japan. The genetic features of the strains were analyzed by generating the complete genome sequences using the Illumina MiniSeq and Oxford Nanopore MinION sequencing platforms.

RESULTS AND DISCUSSION

Characterization of K. pneumoniae AO15, K. pneumoniae AO22, and A. baumannii AO22.

Three carbapenemase-producing isolates, two NDM-1-producing K. pneumoniae isolates (AO15 and AO22) and one OXA-66- and OXA-72-coproducing A. baumannii isolate (AO22), were identified from two different vegetable samples (2/48; 4.17%) (Table 1). The three isolates exhibited multidrug-resistant phenotypes (5). The two K. pneumoniae isolates (AO15 and AO22) were resistant to cefotaxime, imipenem, meropenem, ertapenem, kanamycin, tetracycline, and fosfomycin but susceptible to gentamicin, ciprofloxacin, chloramphenicol, colistin, and tigecycline. In addition, A. baumannii AO22 was resistant to all antimicrobials tested except colistin and fosfomycin (Table 2). PCR and DNA sequencing confirmed the presence of bla_NDM-1 in K. pneumoniae AO15 and K. pneumoniae AO22 and bla_OXA-24-like and bla_OXA-51-like in A. baumannii AO22. The two K. pneumoniae isolates were clonal, belonging to sequence type 15 (ST15), and were found to carry 19 different antimicrobial resistance genes, including bla_NDM-1, bla_CTX-M-15, and bla_CTX-M-14b, located on the chromosome and three different plasmids (Table 3).

TABLE 1.

Features of carbapenemase-producing K. pneumoniae and A. baumannii isolates identified in this study

Strain	Source	Date purchased (YYYY/MM/DD)	Retail shop (shop location; crop origin)
K. pneumoniae AO15	Organic Italian parsley	2015/02/12	A (Hiroshima; Hiroshima)
K. pneumoniae AO22	Organic baby leaf mix	2015/02/16	B (Hiroshima; Kumamoto)
A. baumannii AO22	Organic baby leaf mix	2015/02/16	B (Hiroshima; Kumamoto)

TABLE 2.

MICs for carbapenemase-producing K. pneumoniae and A. baumannii isolates identified in this study

Antibiotic	MIC (μg/ml) fora:
	K. pneumoniae AO15	K. pneumoniae AO15-TC1b	K. pneumoniae AO22	K. pneumoniae AO22-TC5b	A. baumannii AO22	E. coli J53
Cefotaxime	256	256	256	512	512	0.0625
Doripenem	0.5	4	4	4	64	≤0.0078
Meropenem	8	4	4	4	128	0.0312
Imipenem	16	16	8	16	128	0.25
Ertapenem	16	8	16	4	512	≤0.0156
Kanamycin	512	64	512	128	512	1
Gentamicin	<1	<1	<1	<1	128	<1
Ciprofloxacin	1	0.5	1	0.5	4	0.0625
Chloramphenicol	4	4	4	4	128	4
Tetracycline	128	<2	128	<2	64	<2
Tigecycline	<0.5	<0.5	<0.5	<0.5	8	<0.5
Colistin	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5
Fosfomycin	256	8	256	4	128	8

^aInterpretation was done according to the Clinical and Laboratory Standards Institute guidelines (37). Values that indicate resistance are in bold.

^bTC, transconjugant.

TABLE 3.

Features of chromosome and the plasmids identified from K. pneumoniae AO15, K. pneumoniae AO22, and A. baumannii AO22 from fresh vegetables in Japan^a

Sample	Size (bp)	GC%	No. of CDSs	MLST or pMLST	Incompatibility group	Antimicrobial resistance gene(s)
K. pneumoniae AO15
Chromosome	5,273,076	57.5	4,860	ST15	ND	blaSHV-28, fosA, oqxAB
pKpnAO15-1	201,745	52.5	228	K2:A−:B−	IncFIB(K):IncFII(K)	blaSHV-1, blaTEM-1A, tet(D)
pKpnAO15-2	122,804	52	156	K2:A−:B−	IncFII(K):IncR	aac(6′)-Ib, aadA1, aph(3′)-VI, blaCTX-M-15, blaNDM-1, blaOXA-9, blaTEM-1A, aac(6′)-Ib-cr, qnrS1
pKpnAO15-3	67,119	50.7	84	ND	IncM1	aph(6)-Id, aph(3′)-VIb, aph(3′')-Ib,b blaCTX-M-14b
pKpnAO15-4	24,265	62.4	27	ND	ND	ND
pKpnAO15-5	9,294	55.2	9	ND	ColRNAI	ND
K. pneumoniae AO22
Chromosome	5,270,888	57.5	4,882	ST15	ND	blaSHV-28, fosA, oqxAB
pKpnAO22-1	201,745	52.5	228	K2:A−:B−	IncFIB(K):IncFII(K)	blaSHV-1, blaTEM-1A, tet(D)
pKpnAO22-2	122,804	52	156	K2:A−:B−	IncFII(K):IncR	aac(6′)-Ib, aadA1, aph(3′)-VI, blaCTX-M-15, blaNDM-1, blaOXA-9, blaTEM-1A, aac(6′)-Ib-cr, qnrS1
pKpnAO22-3	67,119	50.7	84	ND	IncM1	aph(6)-Id, aph(3′)-VIb, aph(3′')-Ib,b blaCTX-M-14b
pKpnAO22-4	9,294	55.2	9	ND	ColRNAI	ND
A. baumannii AO22
Chromosome	3,954,640	39	3,712	ST2 (Pasteur); ST1808 or ST348 (Oxford)	ND	blaADC-25, blaOXA-66, sul2, tet(B), aac(3)-Ia, aac(6′)-Ip, aph(3′')-Ib, aph(6)-Id
pAbaAO22-1	110,967	41.6	119	ND	ND	ND
pAbaAO22-2	10,880	34.5	15	ND	GR2	blaOXA-72b

^aCDSs, coding sequences; ND, not determined.

^bThis gene was found in two copies.

Although carbapenemase-producing K. pneumoniae strains occur infrequently in Japan (18), we reported the first NDM-1- and VIM-1-producing strain from unfrozen chicken (14). Furthermore, the ST15 K. pneumoniae clone appears to be a very successful international clone because of its association with bla_NDM-1 in Spain, Croatia, Thailand, Canada, China, France, and Morocco, bla_KPC-2 in Bulgaria, bla_VIM-4 in Hungary, and other ESBLs (bla_CTX-M, bla_SHV, and bla_TEM) (19–21). ST15 CTX-M-producing K. pneumoniae isolates have been found to be disseminated within and between hospitals among companion animals in Japan (20). Additionally, the same clone was observed in 20% of the isolates collected in Nagasaki University Hospital (21). Moreover, ST15 NDM-1-carrying K. pneumoniae was reported from patients from Syria, Greece, the Netherlands, and Albania (22–26). Therefore, the K. pneumoniae isolates (AO15 and AO22) might have been transmitted from food handlers. Notably, NDM-1-producing Enterobacterales of vegetable origin have been previously reported in China and Myanmar (7–10).

OXA-72-producing A. baumannii human clinical isolates have been previously documented in Japan (27). Only one report described the detection of OXA-72-producing Acinetobacter calcoaceticus from vegetables in Lebanon (13). According to the multilocus sequence typing (MLST) scheme of the Pasteur Institute, A. baumannii AO22 belonged to ST2 (belonging to the global clonal lineage II [European clone II]), which has been previously recognized in clinical isolates in Japan (27). Therefore, it is essential to survey CPGNB, especially of vegetable origin, in Japan, as these organisms could lead to hospital outbreaks.

By combining the short reads of Illumina MiniSeq and the long reads of Oxford Nanopore sequencing, high-quality assemblies were obtained satisfactorily for closing the genomes and the plasmids contained in the isolates (Table 3). K. pneumoniae AO15, K. pneumoniae AO22, and A. baumannii AO22 were found to carry five, four, and two plasmids, respectively (Table 3). Although K. pneumoniae AO15 and K. pneumoniae AO22 were isolated from two different vegetables on two different dates from two different supermarkets (Table 1), pairwise genome comparisons using JspeciesWS showed the clonality between them. For K. pneumoniae AO15, 100% (5,273,076 bp/5,270,888 bp) aligned nucleotides showed 100% average nucleotide identity (ANI) to K. pneumoniae AO22.

Identification of a bla_NDM-1/IncFII(K):IncR plasmid in Japanese K. pneumoniae strains collected from vegetables.

The bla_NDM-1 gene was located on the plasmids pKpnAO15-2 and pKpnAO22-2 from isolates K. pneumoniae AO15 and K. pneumoniae AO22, respectively. Both the plasmids were IncFII(K):IncR type, were 122,804 bp (Fig. 1A) in length, and shared >99.99% nucleotide identity as detected by pairwise comparisons using JspeciesWS (Table 3). In addition, both the plasmids showed >99.27% sequence identity to K. pneumoniae plasmid p2 (76% coverage; CP009115.1) and plasmid pKp15-T2 (68% coverage; MN657248.1), which were isolated from patients in the United States and Germany, respectively, and harbored bla_NDM-1 (Fig. 1B). Interestingly, both the plasmids showed >99.99% ANI to the K. pneumoniae plasmid pLM22-1-NDM-1, identified from unfrozen chickens in our study in 2016 from the same city (98.86% [122,804 bp/124,214 bp] aligned nucleotides), as detected by pairwise comparisons using JspeciesWS (Fig. 1B) (14).

FIG 1.

Plasmid structure of pKpnAO15-2, and pKpnAO22-2 identified in this study and comparison with other similar plasmids. Both the plasmids were the IncFII(K):IncR type, had 122,804 bp, and shared >99.99% nucleotide identity. (A) Genes and open reading frames (ORFs) are shown as arrows with their transcriptional orientations indicated by arrowheads. This figure was generated using the BRIG tool (http://brig.sourceforge.net/). (B) The whole sequence of pKpnAO15-2 was used as a reference. The plasmids were included in the following order: pKpnAO15-2 (identified in this study), pKpnAO22-2 (identified in this study), pLM22-1-NDM-1, plasmid p2 (CP009115.1), and plasmid pKp15-T2 (MN657248.1) (B).

The bla_NDM-1 genetic context, aph(3′)-III-ΔISAba125-ISSpu2-bla_NDM-1-ble_MBL-trpF, was observed in pKpnAO15-2, pKpnAO22-2, plasmid p2 (CP009115.1), and pLM22-1-NDM-1 (14). The plasmids pKpnAO22-2 and pKpnAO15-2 were successfully transferred by conjugation to the azide-resistant strain E. coli J53. The transconjugants carrying both the plasmids showed resistance to imipenem, meropenem, doripenem, and ertapenem (Table 2).

Identification of a GR2-type plasmid carrying two copies of bla_OXA-72 from foodborne A. baumannii.

The A. baumannii AO22 carried plasmids pAbaAO22-1 and pAbaAO22-2 (Table 3). Interestingly, pAbaAO22-2 was a GR2-type plasmid of 10,880 bp, carrying five XerC/XerD-like binding sites flanking two copies of bla_OXA-72 and a putative toxin-antitoxin system enabling plasmid maintenance. XerC/XerD site-specific tyrosine recombinases might be involved in the mobilization of bla_{OXA-24/40-like} between Acinetobacter spp. (23, 24). A BLASTn search using the whole pAbaAO22-2 sequence query identified that it has high similarity to other A. baumannii plasmids (Fig. 2), for example, pAB120 identified from patients with necrotizing fasciitis in the United States in 2012 (100% coverage and 99.97% sequence identity; CP031446.1), pAB120 identified from Lithuanian hospitals in 2010 (28) (100% coverage and 99.96% sequence identity; JX069966.1), and plasmid pAC1-BRL identified from a migratory bird in Brazil in 2012 (29) (99.93% identity with 86% coverage; MN266872.1). Interestingly, pAbaAO22-2 showed 99.85% identity with 85% coverage to the A. baumannii plasmid pAB-NCGM253 (AB823544.1) identified from a patient in Japan (27). Transformation of such OXA-72-carrying plasmids (pAB120 or pAC1-BRL) to carbapenem-susceptible A. baumannii strains resulted in an increase in the MIC of imipenem (32 μg/ml) and meropenem (32 to 64 μg/ml) (23, 24). Here, we report the detection of pAbaAO22-2 carrying two copies of bla_OXA-72 isolated from a foodborne A. baumannii isolate.

FIG 2.

Comparison of the bla_OXA-72-carrying pAbaAO22-2 and other related GR2-type plasmids. Genes and open reading frames (ORFs) are shown as arrows with their transcriptional orientations indicated by arrowheads. The figure was drawn using the EasyFig tool (http://mjsull.github.io/Easyfig/). bla_OXA-72 was detected as two copies in pAbaAO22-2 (identified in this study), pAB120 (CP031446.1), and pAB120 (JX069966.1), while pAB-NCGM253 (NC_021489.1) and pAC1-BRL (MN266872.1) carried only one copy of bla_OXA-72.

Identification of an AbaR4-like GI from foodborne A. baumannii.

Analysis of the chromosome of A. baumannii AO22 identified the presence of an AbaR4-like GI, designated AbaR4-AO22, of 41,665 bp, carrying antimicrobial resistance genes against tetracycline [tetA(B)], sulfonamides (sul2), and streptomycin (strAB); sul2 was flanked by ISAba1, which might increase its expression. The backbone of AbaR4-AO22 consists of sets of transposition genes, tniC-tniA-tniB, uspA (encoding universal stress protein), sulP (encoding sulfate permease), and xerC (encoding tyrosine integrase). AbaR4-AO22 was integrated into the chromosomal comM gene and was flanked by a 5-bp target site duplication of 5′-GCGGT-3′. A BLASTn search using the whole AbaR4-AO22 sequence query revealed that it is highly similar to other A. baumannii chromosomal AbaR4-like GI (Fig. 3), for example, those of strain XH386, isolated in China (100% coverage and 100% sequence identity; CP021326.1), and strain CMC-CR-MDR-Ab66, isolated from a patient in the United States (30) (100% coverage and 99.99% sequence identity; CP016300.1). Compared to AbaR25, identified in Latvia in 2008, AbaR4-AO22 showed deletion of Tn2006 carrying bla_OXA-23 (Fig. 3) (17). Here, we report the detection of an AbaR4-like resistance island from a foodborne A. baumannii isolate.

FIG 3.

Genetic structures of AbaR4-AO22 and other related AbaR4-like GIs. Genes and open reading frames (ORFs) are shown as arrows, with their transcriptional orientations indicated by arrowheads. The figure was drawn using the EasyFig tool (http://mjsull.github.io/Easyfig/). AbaR4-AO22 was integrated into the chromosomal comM gene and was flanked by a 5-bp target site duplication of 5′-GCGGT-3′. AbaR4-AO22 was identical to AbaR4 from A. baumannii strain XH386 (CP021326.1) but lacking Tn2006 (ISAba1-bla_OXA-23-ΔATPase-ΔDead-yeeA-ISAba1) identified in AbaR25 (JX481978).

Analysis of the virulome and heavy metal resistance genes.

Several virulence factors have been identified, illustrating the potential for pathogenicity associated with the consumption of organic vegetables contaminated with these bacterial pathogens (Table 4). The mrk and fim operons, encoding type 3 and type I fimbriae, respectively, were identified from the two K. pneumoniae isolates in this study (31, 32). The mrk operon was previously detected from carbapenemase-producing K. pneumoniae isolates from Italy (32). The wzi gene (allele 215; associated K type KL117), associated with the attachment of the capsule to the host cell surface, was identified from the two K. pneumoniae isolates (32). Concerning the iron uptake systems, genes encoding the aerobactin system (iuc), Ent siderophore (ent), salmochelin (iro), the yersiniabactin system (ybt), and its receptor (fyuA) have been identified from the two K. pneumoniae isolates (32). Additionally, the toxin colibactin (clb operon), which induces chromosomal instability and DNA damage in eukaryotic cells, has been detected from the two K. pneumoniae isolates (33). The type VI secretion system (T6SS), which acts as an injection machinery perforating eukaryotic and prokaryotic target membranes, has also been detected (34). A. baumannii AO22 was determined to carry genes for outer membrane protein (ompA), iron uptake acinetobactin (bar, bas, and bau operons), quorum sensing (abaI and abaR), biofilm-associated protein (bap) and csu pili. Several heavy metal resistance genes against mercury (mer operon), arsenite (ars operon), copper (pco operon), and silver (sil operon) were detected in the two K. pneumoniae isolates (Table 5). Coexistence of antibiotic resistance and heavy metal has been identified previously (35). Heavy metal resistance genes might be a potential factor for maintenance and spreading of antibiotic resistance due to possible coselection (35). Other features of the two K. pneumoniae isolates are found in Table 5.

TABLE 4.

The virulome of the carbapenemase-producing isolates K. pneumoniae AO15, K. pneumoniae AO22, and A. baumannii AO22 from fresh vegetables in Japan according to the virulence factor database (http://www.mgc.ac.cn/VFs/)^a

Virulence gene category	Protein(s) (gene[s]) in:
	K. pneumoniae AO15	K. pneumoniae AO22	A. baumannii AO22
Adherence	Type I fimbriae (fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK); type 3 fimbriae (mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ)	Type I fimbriae (fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK); type 3 fimbriae (mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ)	Outer membrane protein (ompA)
Antiphagocytosis	Capsule	Capsule	NA
Efflux pump	AcrAB (acrA, acrB)	AcrAB (acrA, acrB)	NA
Iron uptake	Aerobactin (iucA, iucB, iucC, iucD, iutA); Ent siderophore (entA, entB, entC, entD, entE, entF, fepA, fepB, fepC, fepD, fepG, fes); salmochelin (iroB, iroC, iroD, iroE, iroN); yersiniabactin (fyuA, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX)	Aerobactin (iucA, iucB, iucC, iucD, iutA); Ent siderophore (entA, entB, entC, entD, entE, entF, fepA, fepB, fepC, fepD, fepG, fes); salmochelin (iroB, iroC, iroD, iroE, iroN); yersiniabactin (fyuA, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX)	Acinetobactin (barA, barB, basA, basB, basC, basD, basF, basG, basH, basI, basJ, bauA, bauB, bauC, bauD, bauE, bauF, ent)
Nutritional factors	Allantoin utilization (allA, allB, allC, allD, allR, allS)	Allantoin utilization (allA, allB, allC, allD, allR, allS)	NA
Regulation	RcsAB (regulation of capsule synthesis) (rcsA, rcsB)	RcsAB (regulation of capsule synthesis) (rcsA, rcsB)	Quorum sensing (autoinducer-receptor mechanism) (abaI, abaR); two-component system (bfmR, bfmS)
Secretion system	T6SS-I (clpV/tssH, dotU/tssL, hcp/tssD, icmF/tssM, impA/tssA, sciN/tssJ, tle1, tli1, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC); T6SS-II (clpV, dotU, icmF, impF, impH, impJ, ompA, sciN, vasA/impG, vgrG); T6SS-III (dotU, icmF, impA, impF, impG, impH, impJ, lysM, ompA, sciN, vgrG)	T6SS-I (clpV/tssH, dotU/tssL, hcp/tssD, icmF/tssM, impA/tssA, sciN/tssJ, tle1, tli1, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC); T6SS-II (clpV, dotU, icmF, impF, impH, impJ, ompA, sciN, vasA/impG, vgrG); T6SS-III (dotU, icmF, impA, impF, impG, impH, impJ, lysM, ompA, sciN, vgrG)	NA
Serum resistance	LPS rfb locus	LPS rfb locus	PbpG (penicillin-binding protein)
Toxin	Colibactin (clbA, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbI, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, clbS)	Colibactin (clbA, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbI, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, clbS)	NA
Biofilm formation	NA	NA	AdeFGH efflux pump (adeF, adeG, adeH); biofilm-associated protein (bap); Csu pili (csuA, csuA/B, csuB, csuC, csuD, csuE); PNAG (pgaA, pgaB, pgaC, pgaD)
Enzymes	NA	NA	Phospholipase C (plc); phospholipase D (plcD)
Immune evasion	NA	NA	Capsule; LPS (lpsB, lpxA, lpxB, lpxC, lpxD, lpxL, lpxM)

^aNA, not applicable; LPS, lipopolysaccharide; PNAG, poly-N-acetylglucosamine.

TABLE 5.

Features of the carbapenemase-producing K. pneumoniae isolates identified in this study

Feature(s)	Characteristic(s) in:
	AO15	AO22
Efflux systems and regulators	acrA, acrB, acrR, marA, marR, soxS, soxR, ramA, ramR, rob, sdiA, fis, envR, oqxA, oqxB, oqxR, rarA	acrA, acrB, acrR, marA, marR, soxS, soxR, ramA, ramR, rob, sdiA, fis, envR, oqxA, oqxB, oqxR, rarA
Wzi allele	215	215
Associated K type	KL117	KL117
Aerobactin	Closest iutA allele, 28	Closest iutA allele, 28
Heavy metal resistance genes	arsA, arsB, arsC, arsD, arsR, merC, merP, merR, merT, pcoA, pcoB, pcoC, pcoD, pcoE, pcoR, pcoS, silA, silC, silE, silF, silG, silP, silR, silS	arsA, arsB, arsC, arsD, arsR, merC, merP, merR, merT, pcoA, pcoB, pcoC, pcoD, pcoE, pcoR, pcoS, silA, silC, silE, silF, silG, silP, silR, silS
Salmochelin siderophore synthesis	Closest iroN allele, 11	Closest iroN allele, 11
Urease-nickel cluster	Closest ureC2 allele, 8	Closest ureC2 allele, 8
hvK2 multiplex PCR	KpI50233a allele 4	KpI50233a allele 4

Evolutionary relatedness of ST15 K. pneumoniae isolates.

Whole-genome sequencing data for these two isolates and those from the NCBI Assembly database were used to generate a core genome single-nucleotide-polymorphism (cg-SNP)-based phylogenetic tree of the K. pneumoniae strains isolated in Japan from clinical sources, including blood (Fig. 4). The tree clustered the two ST15 K. pneumoniae isolates with another highly similar ST15 K. pneumoniae (Assembly accession number GCA001950675) strain isolated from a human clinical sample. Additionally, the tree showed the clonal relationship between these two ST15 isolates from vegetables and other K. pneumoniae isolates from human blood samples with different STs (ST3822, ST327, ST997, ST107, and ST540) (Fig. 4). These results strongly suggested the contamination of the two vegetable samples from a human source (probably a food handler).

FIG 4.

Maximum-likelihood phylogeny of the two K. pneumoniae strains isolated in this study and other K. pneumoniae strains isolated from Japan. Registered genomes of K. pneumoniae isolated in Japan were collected from the NCBI Assembly database. Sequence type (ST) is given as a suffix on each NCBI assembly number. The phylogeny was constructed from SNPs from 113 K. pneumoniae core genomes, using generalized time-reversible (GTR), subtree-prune-regraft (SPR) moves, and maximum-likelihood nearest-neighbor interchanges (NNIs). The phylogenetic tree was visualized using iTOL (https://itol.embl.de), and the string color represents the sample source: red, green, and yellow correspond to human blood, human source other than blood, and vegetable, respectively.

Conclusions.

Here, we report the detection of (i) CPGNB collected from vegetables in Japan, (ii) the coexistence of two different CPGNB from the same vegetable sample, and (iii) a GR2-type plasmid carrying two copies of bla_OXA-72 and an AbaR4-like resistance island from a foodborne A. baumannii. This study illustrated that a single fresh, ready-to-eat vegetable might serve as a significant source of carbapenemase-producing and multidrug-resistant ESKAPE bacteria, some of the most problematic hospital-acquired pathogens, with plasmids and GI carrying genes (bla_NDM-1, bla_OXA-66, bla_OXA-72, bla_CTX-M-14b, and bla_CTX-M-15) encoding resistance to frontline and clinically important antibiotics (carbapenem and cephalosporin). Irrigation water, soil, and animal or human contact might also be possible sources of contamination to the environment, resulting in the emergence of such multidrug-resistant organisms. Using the animal waste from farms as an organic fertilizer exacerbates the problem by contaminating the soil and the vegetables with antibiotic-resistant bacteria (36). Additionally, the slurry discharged from these farms contaminates irrigational water, leading to the same problem (36). Excessive use of antimicrobials (including biocides) along the food chain and in food animals might contribute to this phenomenon. Furthermore, the detection of these organisms in fresh vegetables in Japan, a country with high hygienic standards and relatively low levels of antimicrobial resistance, is upsetting and poses a food safety issue and a public health threat, as the resistant organisms might be transferred to humans. From One Health and Global Health perspectives, surveillance plans are needed at the local and international levels to provide information for risk mitigation strategies to minimize resistance spread.

MATERIALS AND METHODS

Bacterial isolates, antimicrobial susceptibility testing, and detection of carbapenemase-encoding genes.

A total of 48 different fresh vegetable samples were randomly collected between October 2014 and February 2015 from 12 different supermarkets in the city of Higashihiroshima, Japan. Briefly, 25 g of each vegetable sample was homogenized using a stomacher blender and enriched in tryptic soy broth (225 ml) for 24 h at 37°C with shaking, followed by direct spreading on MacConkey agar supplemented with 100 μg/ml ampicillin. Three carbapenemase-producing strains, two NDM-1-producing K. pneumoniae isolates (AO15 and AO22) and one OXA-66- and OXA-72-coproducing A. baumannii isolate (AO22), were identified and confirmed by PCR and DNA sequencing as reported previously (5, 31). K. pneumoniae AO22 and A. baumannii AO22 were isolated from the same baby leaf mix sample (Table 1). These vegetables were local crops from Kumamoto and Hiroshima prefectures in Japan (Table 1). The MICs of various antimicrobial agents were determined using the broth microdilution (BMD) method according to the Clinical and Laboratory Standards Institute guidelines (37) (Table 2). For all experiments, antibiotic solutions were prepared by following the CLSI recommendations. E. coli ATCC 25922 was used for quality control. PCR and DNA sequencing were used to detect the presence of carbapenemase-encoding genes, including the class D oxacillinases, as reported previously (5, 38).

Complete genome sequencing, assembly, and analysis.

Total genomic DNA was extracted using the Qiagen Genomic-tip 20/G kit (Qiagen) following the manufacturer’s recommendations. For Illumina MiniSeq sequencing, the DNA library was prepared using a Nextera XT library prep kit and a Nextera XT index kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. For Oxford Nanopore sequencing, the library was constructed using the SQK-RBK004 rapid barcoding kit, loaded onto a FLO-MIN106 R9.4 flow cell, and sequenced with the MinION device (Oxford Nanopore Technologies, Oxford, UK) for 48 h. A hybrid assembly of Illumina short reads and MinION long reads was performed using Unicycler (39). The annotation was performed using DFAST (https://dfast.ddbj.nig.ac.jp/). The whole-genome sequences of the three strains were analyzed at the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/) using ResFinder-3.2 (threshold for gene predictions was 90%), MLST 2.0, plasmid MLST (pMLST), and PlasmidFinder. Genomic comparisons were performed using the BRIG tool (http://brig.sourceforge.net/) and EasyFig tool (http://mjsull.github.io/Easyfig/). The BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and ISfinder (https://isfinder.biotoul.fr/) were used to analyze the plasmids and AbaR4-Like GI. Pairwise comparison of the two K. pneumoniae genomes was performed with JSpeciesWS (http://jspecies.ribohost.com/jspeciesws/) using average nucleotide identity (ANI) calculation based on BLAST+ (ANIb). Screening of K. pneumoniae strains for heavy metal resistance genes, efflux systems and regulators, wzi alleles, and their associated K types was performed using K. pneumoniae typing software offered by Institut Pasteur (https://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html). Identification of the virulome was performed using the virulence factor database (VFDB; http://www.mgc.ac.cn/VFs/) (40).

Filter-mating conjugation.

A mating-out assay was performed using NDM-1-producing K. pneumoniae isolates as the donors and the azide-resistant E. coli strain J53 as the recipient (5). The transconjugants were selected on Luria-Bertani agar plates containing 100 μg/ml ampicillin and 100 μg/ml sodium azide.

Evolutionary relatedness and phylogenetic tree construction.

K. pneumoniae genomes were collected from the NCBI Genome Assembly database. The strains isolated in Japan were selected, and finally, 111 K. pneumoniae genomes were applied for phylogenetic analysis. The NCBI BioSample database was referenced to identify the sample source of each selected K. pneumoniae strain. The ST was investigated using MLST (https://github.com/tseemann/mlst) based on BIGSdb (41). Core genomes and SNPs were analyzed using Roary (https://github.com/sanger-pathogens/Roary) (42) and SNP sites (https://github.com/sanger-pathogens/snp-sites) (43). The phylogeny was constructed using FastTree 2 (44) with default settings. The Newick file produced by FastTree 2 was visualized using iTOL (https://itol.embl.de).

Data availability.

The complete genome sequences of K. pneumoniae AO15, K. pneumoniae AO22, and A. baumannii AO22 were submitted to DDBJ/ENA/GenBank under BioProject ID PRJDB10009 (SRA accession numbers DRA010326, DRA010325, and DRA010324, respectively).