Identification of a Novel Metallo-β-Lactamase, VAM-1, in a Foodborne Vibrio alginolyticus Isolate from China

ABSTRACT

A multidrug-resistant Vibrio alginolyticus isolate recovered from a shrimp sample with reduced carbapenem susceptibility produced a novel metallo-β-lactamase (MBL), VAM-1. That carbapenemase shared 67% to 70% amino acid identity with several VMB family subclass B1 MBLs, which were recently reported among some marine bacteria including Vibrio, Glaciecola, and Thalassomonas. The bla_VAM-1 gene was located in a novel conjugative plasmid, namely, pC1579, and multiple copies of bla_VAM-1 via an unusual mechanism of gene amplification were detected in pC1579. These findings underline the emergence of marine organisms acting as natural reservoirs for MBL genes and the importance of continuous bacterial antibiotic resistance surveillance.

INTRODUCTION

Metallo-β-lactamases (MBLs) are zinc-dependent enzymes that can effectively hydrolyze all β-lactam antibiotics except monobactams. In addition, MBLs are resistant to β-lactamase inhibitors, such as clavulanate, tazobactam, and avibactam (1–3). Thus, MBL-producing bacteria are generally resistant to penicillins, cephalosporins, and carbapenems. MBLs belong to Ambler class B and can be further categorized into three subclasses, B1, B2, and B3, among which the subclass B1 enzymes comprising IMP-1, VIM-1, and NDM-1 are the most important since they are widely identified in clinically relevant species such as Enterobacterales, Acinetobacter spp., and Pseudomonas spp. (4–6).

Vibrio alginolyticus, a Gram-negative halophilic bacterium, is ubiquitous in marine environments all over the world (7). As a common opportunistic pathogen with a type III secretion system (T3SS), V. alginolyticus can cause not only serious vibriosis in marine aquatic animals but also soft tissue infection, gastroenteritis, and septicemia in humans after consumption of contaminated sea products, which has posed a great threat to human health and economy (8). Several studies have shown that V. alginolyticus are usually sensitive to antibiotics, including cephalosporins, tetracyclines, and fluoroquinolones, which are commonly used to treat zoonotic infections (7). However, the prolonged and indiscriminate use of such antibiotics has led to the emergence and evolution of multidrug-resistant (MDR) V. alginolyticus strains in the past few years (9–11). Although, carbapenems are often considered last-line drugs for treatment of bacterial infections, production of MBLs has been previously observed among V. alginolyticus strains harboring acquired MBL-encoding genes, such as bla_NDM-1 and bla_VIM-1 (12, 13). Moreover, a novel MBL, VMB-1, was recently identified in an environmental V. alginolyticus isolate (14). The rapid development of carbapenem resistance in V. alginolyticus should be given more attention and investigation.

Here, we analyzed a multidrug-resistant V. alginolyticus isolate Vb1579 that had been recovered from a shrimp sample from a free market in Shenzhen, China in August 2016 during a survey aimed to study the spread of multidrug-resistant Gram-negative bacteria among food-producing animals in China. Samples were processed as previously described (15). The isolate Vb1579 was identified as V. alginolyticus by multiplex PCR assays and DNA sequencing and further confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker) (16). Antimicrobial susceptibility of this isolate was determined using the standard agar dilution method and interpreted in accordance with the CLSI standard (17). The results showed that V. alginolyticus Vb1579 was resistant to most β-lactam antibiotics tested, including ampicillin, amoxicillin-clavulanic acid, ceftriaxone, and cefotaxime. However, Vb1579 did not exhibited resistance to meropenem (0.12 mg/liter) or imipenem (0.06 mg/liter). Regarding non-β-lactam antibiotics, the isolate Vb1579 was found to be resistant to tetracycline but susceptible to ciprofloxacin, chloramphenicol, amikacin, and gentamicin (Table 1).

TABLE 1.

MICs for transconjugants and transformants harboring bla_VAM-1 and their parental strains

Antibiotic	MICs (mg/liter) for:
	Vb1579	C1579	E. coli J53	DH5α-blaVAM-1	DH5α-pBackZero	DH5α
Ampicillin	>256	>256	8	>256	4	4
Ampicillin-sulbactam	128	128	4	256	4	4
Amoxicillin-clavulanic acid	32	>64	8	32	8	8
Benzylpenicillin	>256	>256	32	>256	32	32
Piperacillin	64	64	2	>256	2	2
Piperacillin-tazobactam	32	32	1	256	1	1
Cephalothin	>128	>128	16	>128	16	16
Ceftriaxone	64	32	0.06	>128	0.06	0.06
Cefotaxime	64	16	0.06	128	0.06	0.06
Ceftazidime	128	>128	0.5	>128	0.5	0.5
Ceftazidime-avibactam	0.06	>128	0.125	>128	0.125	0.125
Cefepime	16	2	0.06	32	0.06	0.06
Aztreonam	1	0.25	0.125	0.25	0.125	0.125
Meropenem	0.12	1	0.015	32	0.0075	0.0075
Imipenem	0.06	0.25	0.015	8	0.0075	0.0075
Ertapenem	0.25	1	0.015	64	0.0075	0.0075
Ciprofloxacin	0.25	0.015	0.015	0.03	0.03	0.03
Chloramphenicol	4	32	4	2	4	4
Tetracycline	32	32	2	0.5	1	1
Amikacin	2	1	1	1	0.5	0.5
Gentamicin	2	0.5	0.25	0.25	0.125	0.125

To determine the transferability of the antibiotic resistance property, a conjugation assay by filter mating was performed using the azide-resistant Escherichia coli strain J53 as the recipient strain (18), with the result showing that the cephalosporin and tetracycline resistance phenotype could be transferred to E. coli J53. Compared with the recipient strain E. coli J53, the transconjugant designated C1579 exhibited relatively higher MICs of meropenem and imipenem (Table 1). Interestingly, the transconjugant C1579 also presented slightly higher MICs of meropenem and imipenem than the parental strain Vb1579. A similar observation was also reported from previous studies (13, 19), which implied that the level of carbapenemase production in V. alginolyticus Vb1579 could be lower than that in the E. coli background and was likely related to the species of its host. Such discrepancy warrants further studies. For carbapenemase detection, a modified carbapenem inactivation method (mCIM) was performed for meropenem on Mueller-Hinton agar. In addition, a combined disc test with EDTA (eCIM) was conducted for MBL detection (20). Both V. alginolyticus Vb1579 and the transconjugant C1579 had identical positive results for the mCIM/eCIM test, indicating expression of a metallo-β-lactamase (see Fig. S1 in the supplemental material).

Further analysis of V. alginolyticus Vb1579 and the corresponding transconjugants in a multiplex PCR assay (21) for KPC, OXA-48, NDM, VIM, and IMP carbapenemase genes did not yield any positive result. To identify the gene responsible for carbapenemase activity in V. alginolyticus Vb1579, the genomic DNA of the isolate Vb1579 was extracted, and whole-genome sequencing (WGS) was performed on the Illumina HiSeq X10 and MinION sequencing platforms in accordance with the workflow reported previously (22). DNA contigs were assembled using Unicycler (23). WGS analysis using the RAST tool and the Resistance Gene Identifier tool on the CARD website revealed V. alginolyticus Vb1579 harbored bla_CARB-12 and bla_VEB-18, which encode a PSE family carbenicillin-hydrolyzing class A β-lactamase and a minor extended-spectrum β-lactamase recently identified in Vibrio spp., respectively (24). In addition, a 741-bp open reading frame that coded for a 246-amino-acid peptide sequence could be identified as a potential MBL gene. The novel MBL was named VAM-1 (Vibrio alginolyticus metallo-β-lactamase).

Subsequently, the VAM-1 protein sequence was used as a query and a BLASTP search was performed in both the β-lactamase database (BLDB) and nonredundant protein sequence (nr) databases. A phylogenetic tree about MBLs including VAM-1 was constructed using 31 protein sequences (Fig. 1; see also Table S2 in the supplemental material). VAM-1 was found to show moderate sequence identity with amino acid homology of 70.21%, 68.60%, and 68.29% with a Vibrio alginolyticus strain Vb1796 (VMB-1), Glaciecola sp. strain KUL10 (VMB-1 like), and Alteromonadaceae bacterium strain Bs3, respectively, and the first two MBLs have been reported in our previous studies (14). In addition, VAM-1 exhibits over 51.28% similarity with SHN-1, a metallo-β-lactamase isolated from Shewanella denitrificans. Interestingly, Alteromonadaceae sp. Bs3, Vibrio alginolyticus, Glaciecola sp., and Shewanella denitrificans OS217 were all marine bacteria, which suggests that, although marine microorganisms have been thought of as a new resource of therapeutics, they may also become a major reservoir of novel metallo-β-lactamases that pose a threat to human health (25). VAM-1 was close to VMB-1 in the same leaf node of two branches, which is consistent with VAM-1 showing high similarity to VMB-1 in the amino acid profile (Fig. 1). Multiple sequences alignment (MSA) analysis of VAM-1 with several typical metallo-β-lactamases in subclass B1 MBLs indicated that these enzymes shared highly conserved active sites, including the amino acid residues coordinating the zinc ions (Fig. 2), suggesting that VAM-1 might belong to subclass B1 MBLs.

FIG 1.

Phylogenetic tree of VAM-1 with other subclass B1 MBLs. The evolutionary history was inferred by using the maximum likelihood method and the LG+G+I model. A discrete gamma distribution was used to model evolutionary rate differences among sites (4 categories [+G, parameter = 1.380]). This analysis involved 31 amino acid sequences (see Table S2 in the supplemental material). There was a total of 323 positions in the final data set. Evolutionary analyses were conducted in PhyML 3.0 and displayed on iTOLv6. Internal nodes are depicted as green solid circle. Connections between nodes are curves and are shown in black. Leaf lines are gray. VAM-1 is highlighted in red.

FIG 2.

Amino acid of VAM-1 alignment with representative class B1 MBLs. The strictly conserved amino acid residues are boxed in red. Physicochemically similar amino acids are shown in red. Black asterisks (*) indicate amino acids involved in binding zinc ions. The figure was prepared using CLUSTALΩ and ESPript 3.0.

In order to gain insight into the resistance phenotype conferred by VAM-1, the full-length gene of bla_VAM-1 was amplified from V. alginolyticus Vb1579 by designed primers (see Table S1 in the supplemental material) and cloned into pBackZero-T. This plasmid, pBackZero-T-bla_VAM-1, was transformed into E. coli DH5α for antimicrobial susceptibility testing. Susceptibility testing showed that E. coli harboring bla_VAM-1 exhibited high-level resistance to all β-lactam antibiotics, including ampicillin, amoxicillin-clavulanic acid, benzylpenicillin, piperacillin, piperacillin-tazobactam, cephalothin, ceftriaxone, cefotaxime, ceftazidime, ceftazidime-avibactam, cefepime, ertapenem, meropenem, and imipenem (Table 1). For expression and purification of VAM-1, the gene bla_VAM-1 without the signal peptide-coding region (residues) was cloned into pET28 and transformed into E. coli BL21(DE3). VAM-1 protein was purified as previously described (14).

To understand substrate binding and catalytic efficiency for VAM-1, the enzyme kinetic assays toward four different kinds of β-lactam antibiotics were performed and followed a previous study (Table 2) (14). Results showed that VAM-1 exhibited similar behavior to NDM-1, which has a higher affinity (low K_m) for meropenem than imipenem, but the catalytic efficiency of VAM-1 toward meropenem and imipenem was maintained at the same level, with the values being 4.26 × 10⁴ M⁻¹ s⁻¹ and 1.20 × 10⁴ M⁻¹ s⁻¹, respectively. Compared to both NDM-1 and VIM-1, although VAM-1 displayed high hydrolytic efficiency toward tested β-lactam substrates, its k_cat/K_m value is slightly lower (Table 2) (26, 27), indicating that the resistance activity of VAM-1 against meropenem and imipenem is lower than that of NDM-1 and IMP-1.

TABLE 2.

Kinetic parameters of purified VAM-1, NDM-1, and VIM-1

Substrate	Metallo-β-lactamase	Kinetic parameter
		Km (μM)	kcat (s−1)	kcat/Km (M−1 s−1)
Ampicillin	VAM-1	639.9 ± 47.1	6.85 ± 1.04	1.07 × 104
	NDM-1	305.8 ± 32.5	386.1 ± 24.7	1.31 × 106
	VIM-1	224.0 ± 14.5	165.4 ± 4.7	7.38 × 105
Benzylpenicillin	VAM-1	131.1 ± 7.1	121.3 ± 2.5	9.25 × 105
	NDM-1	50.1 ± 5.1	234.2 ± 6.9	4.67 × 106
	VIM-1	164.6 ± 7.7	124.0 ± 2.4	7.53 × 105
Cephalothin	VAM-1	8.9 ± 0.4	20.3 ± 0.4	2.28 × 106
	NDM-1	5.8 ± 0.4	38.7 ± 0.9	6.67 × 106
	VIM-1	100.2 ± 7.3	175.0 ± 6.9	1.75 × 106
Cefotaxime	VAM-1	10.3 ± 0.5	1.01 ± 0.02	9.81 × 104
	NDM-1	18.1 ± 2.2	24.3 ± 1.6	1.34 × 106
	VIM-1	61.3 ± 8.2	120.1 ± 9.2	1.96 × 106
Ceftazidime	VAM-1	8.5 ± 0.4	2.4 ± 0.1	2.82 × 105
	NDM-1	33.8 ± 3.1	52.4 ± 2.1	1.55 × 106
	VIM-1	75.9 ± 9.4	2.5 ± 0.2	3.29 × 104
Cefepime	VAM-1	30.4 ± 3.7	5.5 ± 0.3	1.81 × 104
	NDM-1	27.9 ± 3.4	16.1 ± 0.8	5.77 × 105
	VIM-1	89.9 ± 10.9	8.7 ± 0.6	9.68 × 104
Meropenem	VAM-1	15.5 ± 1.7	0.66 ± 0.03	4.26 × 104
	NDM-1	30.7 ± 3.9	22.1 ± 1.8	7.19 × 105
	VIM-1	52.5 ± 2.3	18.9 ± 0.4	3.61 × 105
Imipenem	VAM-1	42.4 ± 4.8	0.51 ± 0.03	1.20 × 104
	NDM-1	29.0 ± 2.4	23.6 ± 0.9	8.14 × 105
	VIM-1	6.2 ± 0.6	4.1 ± 0.3	6.61 × 105

WGS analysis of V. alginolyticus Vb1579 revealed that bla_VAM-1 was found to be located on a 236-kb contig that could be circularized in silico, which was consistent with the plasmid profile observed by S1-pulsed-field gel electrophoresis (PFGE) on V. alginolyticus Vb1579 and the transconjugant C1579 (data not shown). The complete sequence of this plasmid from the transconjugant C1579 was obtained by using both Illumina and Nanopore sequencing platforms and a de novo hybrid assembly strategy utilizing Unicycler (23). The plasmid, designated as pC1579 (GenBank accession no. MN865127), was found to consist of 236,774 bp, exhibit an average G+C content of 44.5%, and comprise 221 predicted coding sequences (CDSs). After removing the mobile elements and the multidrug resistance (MDR)-encoding region, NCBI BLAST analysis revealed that the backbone of pC1579 shared a high degree of genetic similarity with plasmid p345-185 (GenBank accession no. CP025539) from Vibrio harveyi strain 345 and p2 (GenBank accession no. CP030801) from Vibrio owensii strain 20160513VC2W. Easyfig and BRIG were used in comparative analyses of these three plasmids (Fig. 3a; see also Fig. S2 in the supplemental material) (28, 29). Notably, these two plasmids and pC1579 could not be classified into any of the known incompatibility groups by PlasmidFinder, implying that they probably belong to a novel incompatibility group. Further studies are therefore needed to investigate the putative rep gene, while some other backbone genes involved in plasmid partitioning (parA and parB) and DNA metabolism (tus and pri) were found in pC1579. Moreover, the genes (tra) involved in plasmid conjugative transfer were identified in two regions in these three plasmids. One transfer region, downstream of the parAB module, was a 30-kb region containing 13 genes (traIDLEKBVACFWUN) and the other one (traFHG) was located within another 6-kb region (Fig. 3a). Sequence comparison analysis showed that the set of 16 tra genes in pC1579 exhibited 50 to 82% identity to the type IV secretion system usually harbored by IncC plasmids (30). Moreover, pC1579 also shares the same organization of the 16 tra genes as IncC plasmids (data not shown).

FIG 3.

Genetic environment of bla_VAM-1. (a) Sequence alignment between pC1579 (MN865127), p345-185 (GenBank accession no. CP025539), and p2 (GenBank accession no. CP030801). The plasmid pC1579 was used as a reference to compare with the other two plasmids. The outer circle with red arrows signifies annotation of the reference sequence. Gaps in the circle refer to plasmid regions that are missing when compared to the reference. (b) Schematic of the predicted translocatable unit (TU) harboring bla_VAM-1. The TUs are present on pC1579.

The bla_VAM-1 gene was found to be located in a 4,834-bp region that contains an ISCR1 element, followed by two resistance genes aadA1 and bla_CARB-12, which encode the aminoglycoside nucleotidyltransferase and a PSE family carbenicillin-hydrolyzing class A β-lactamase, respectively (Fig. 3b). The ISCR1 element is an unusual insertion sequence that demonstrates IS91-like characteristics and could mobilize adjacent DNA sequences via a rolling-circle replication (31). The presence of ISCR1 indicated its important role in the original mobilization of bla_VAM-1. However, sequence analysis of this region did not detect the 3′-conserved sequence (CS) region of qacE1-sul1 resistance genes, which was generally found downstream of the transposable element mediated by ISCR1. Moreover, the bla_VAM-1-harboring region was flanked by two copies of ISShfr9 in the same orientation. Coincidentally, this organization is a representative composite transposon structure, suggesting that this gene array has the potential to undertake horizontal transfer (Fig. 3b). However, sequence analysis did not reveal the presence of any distinct direct repeats (DRs), which was regarded as the target site duplication signals for transposition (32). The transposase encoded by ISShfr9 belonged to the Tn3 family, while the transfer events mediated by ISShfr9 were little reported to date. It is necessary to further assess the mobility of ISShfr9. Interestingly, this gene array (ISShfr9-aadA1-bla_CARB-12-bla_VAM-1-orf459-ISCR1) formed an entire unit and created a tandem array in the MDR region of pC1579, which increased the copy number of these resistance genes (Fig. 3a). Recently, Hubbard et al. (33) reported a similar observation and revealed a novel model presented for gene movement that was mediated by insertion sequence IS26. Thus, it would be interesting to validate if ISShfr9 has the same capacity to transpose resistance genes as IS26 and further affect the production level of VAM-1.

To investigate the level of dissemination of bla_VAM-1, we then screened all environmental strains from our laboratory collection from 2015 to 2017, which include E. coli, Salmonella, and Vibrio. The result of PCR screening showed that only 15 Vibrio isolates carried the bla_VAM-1 gene. Species identification was further performed, with results indicating that 13 of 15 bla_VAM-1-bearing Vibrio isolates belonged to V. alginolyticus and that the remaining 2 were Vibrio parahaemolyticus (1 ST1800 and 1 ST2257). PFGE of these 13 bla_VAM-1-positive V. alginolyticus isolates was performed to examine their relationships, with results showing that the majority of isolates belonged to different clonal lineages (data not shown). Hence, it is supposed that bla_VAM-1 only remains detectable in environmental strains of Vibrio at present, and it would take time to transfer the bla_VAM-1 gene from the environmental strains to clinical pathogens. While, the limited genomic data impede us from tracking the source and origin of bla_VAM-1, conducting continuous surveillance of bla_VAM-1 worldwide is recommended in the future.

In this work, we characterize a novel subclass B1 MBL, VAM-1, possessing effective hydrolytic activity against β-lactam antibiotics, such as carbapenems and cephalosporins, consequently conferring antimicrobial resistance on the bla_VAM-1 carriers. Sequence analysis and conjugation assays suggest that bla_VAM-1 may have emerged from a marine reservoir and could be efficiently transferred from natural reservoirs to other pathogens like Enterobacterales via horizontal transfer mediated by a novel conjugative plasmid. So far, little is known about the MDR marine microorganism and the diversity of marine plasmids, although they seem to play an important role in the dissemination of resistance genes in the natural environment. In order to combat antibiotic resistance, monitoring the spread of resistance genes is an important strategy. Furthermore, identification of new carbapenem resistance determinants is necessary for fulfilling these strategies and may aid the development of effective control measures.

Data availability.

The complete nucleotide sequence of pC1579 has been assigned GenBank accession no. MN865127. The protein sequence of VAM-1 has been assigned GenBank accession no. QTJ60982.