Prevalence and antimicrobial susceptibility of foodborne V. parahaemolyticus isolates

A total of 230 non-duplicate V. parahaemolyticus strains were isolated from 195 fresh seafood samples collected in different markets in Changsha, Hunan Province in China. These included 50 abalone, 21 clam, 36 razor clam, 76 shrimp, and 12 other types of seafood samples, including 1 sea snail, 7 fish, and 4 scallop samples purchased from January 2022 to March 2023 (Table S1). The contamination rate of V. parahaemolyticus in seafood products was 62.1% (Table 1). Among these V. parahaemolyticus strains, 42 were isolated from abalone, 38 were from clam, 61 were recovered from the razor clam, 75 were obtained from shrimp, and 14 were from other types of seafood samples. The isolation rate of V. parahaemolyticus was the highest in clam (90.5%), followed by razor clam (80.6%), shrimp (56.5%), and abalone (50.0%), with the lowest rate being observed in other types of seafood samples (41.7%) (Table 1). Antimicrobial susceptibility tests revealed that these strains exhibited resistance to most of the test antibiotics, with a resistance rate of 100% to ampicillin, followed by cefotaxime (71.3%), kanamycin (36.1%), chloramphenicol (30.0%), meropenem (7.4%), ciprofloxacin (5.7%), amikacin (5.7%), and tetracycline (0.9%). However, all the strains remained susceptible to colistin and tigecycline (Table S2). PCR screening for the carbapenemase gene revealed that 5.6% (13 out of 230) of the V. parahaemolyticus isolates tested positive for the blaNDM-1 gene. These isolates were subsequently subjected to analysis by conjugation experiments. The results indicated that the blaNDM-1-encoding plasmid was conjugative in 92.3% (12 out of 13) of the test strains, with conjugation frequencies ranging from 8.6 × 10⁻5 to 1.3 × 10⁻3 (Fig. 1).

Table 1 The prevalence and antimicrobial susceptibility of V. parahaemolyticus strains of food originFig. 1

Genomic analysis of 48 foodborne V. parahaemolyticus isolates. A maximum likelihood phylogenetic tree was constructed using the core genome SNPs and midpoint rooted. The host, year of collection, AMR phenotype, and antimicrobial resistance genes are shown. ND, not detected; +, the blaNDM-1 gene was transferred successfully; -, the blaNDM-1 gene was not transferrable

Whole-genome sequencing and phylogenetic analysis of foodborne V. parahaemolyticus isolates

Among the 230 foodborne V. parahaemolyticus isolates tested, 47 cefotaxime-resistant strains were subjected to whole-genome sequencing. The phenotypic characteristics of these 47 cefotaxime-resistant strains are shown in Fig. 1; it should be noted that 17 of the strains also exhibited carbapenem resistance. BLASTn results showed that the primary mechanism conferring third-generation cephalosporin resistance in foodborne Vibrio is dissemination of β-lactam resistance genes (blaCARB, n = 47; blaCMY-4, n = 1; blaCTX-Ms, n = 10; blaNDM-1, n = 13; blaOXA-10, n = 1; blaPER-1, n = 1; blaVEB-1, n = 1; and blaVMB-1, n = 4), with the blaNDM-1 gene being the most prevalent. The other resistance genes identified in these strains were those which conferred resistance to aminoglycosides (aac(3)-IId, n = 7; aac(6′)-Ia, n = 1; aadA16, n = 7; ant(2″)-Ia, n = 7; ant(3″)-Ia, n = 1; aph(3′)-Ia, n = 9; strA, n = 22; and strB, n = 22), florfenicol (cmlA, n = 1 and floR, n = 18), quinolones (aac(6′)-Ib-cr, n = 11; qnrD1, n = 4; qnrS, n = 2; and qnrVC, n = 5), tetracyclines (tet(34), n = 44; tet(35), n = 7; tet(A), n = 20; tet(B), n = 1; tet(E), n = 4 and tet(M), n = 1), sulfonamides (sul1, n = 21 and sul2, n = 23), trimethoprim (dfrA, n = 24), macrolides (mph(A), n = 10), and rifampin (ARR-3, n = 8) (Fig. 1). No QRDR point mutations were detected in the entire strain collection. Phylogenetic analysis revealed that the foodborne V. parahaemolyticus strains were genetically diverse and not clonally disseminated, indicating that the blaNDM-1-bearing strains have evolved through multiple pathways. Multilocus sequence typing (MLST) identified 19 distinct sequence types (STs). Specifically, ST114, ST1776, ST2602, ST532, and ST1847 were each found in two strains. The following sequence types were each found in one strain: ST1152, ST3063, ST2792, ST918, ST2989, ST12, ST108, ST1354, ST2726, ST843, ST1061, ST1363, ST970, and ST864. Additionally, 23 isolates were assigned to unknown STs based on cluster analysis. The MLST diversity corroborates the findings of the phylogenetic analysis, indicating that the V. parahaemolyticus strains originated from different sources (Fig. 1).

The genetic context of the bla NDM-1 gene in V. parahaemolyticus

To investigate genetic characteristics and transmission mechanisms of the carbapenem resistance in foodborne V. parahaemolyticus, a total of 13 strains harboring the blaNDM-1 gene were subjected to long-reads Nanopore Sequencing. BLASTn analysis revealed that in 12 out of the 13 strains, blaNDM-1 genes were located in IncC-type plasmids, indicating that the blaNDM-1 gene was extensively distributed in seafood and aquatic products through transmission of IncC-type plasmids (Fig. 1). However, in strain VP288, the blaNDM-1 gene was found to be chromosomally located. We next determined how the blaNDM-1 gene and the associated mobile elements were integrated into the backbone of IncC plasmids. Comparative analysis of the AMR region of such plasmids revealed a high degree of similarity with that of Aeromonas enteropelogenes strain VA118; the genetic environment surrounding the blaNDM-1 gene in strains VP288 and VA118 was nearly identical, with an ISCR1 element being located downstream and flanked by two sul1 genes (Figure S1). Linear sequence alignment of the 12 blaNDM-1-bearing plasmids indicated that these plasmids could be categorized into four distinct groups (I–IV) on the basis of genetic variations in their genetic backbones (Figure S2). The plasmids pVP181-NDM (group I), pVP156-NDM and pVP209-NDM (group II), pVP148-NDM and pVP205-NDM (group III), and pVP228-NDM and pVP14-NDM (group IV) were selected as representative plasmids from each group for further comparative analysis. The linear sequence alignment of these IncC-type plasmids also revealed the presence of classical genes associated with replication (repA), partitioning (parAB and stbA), transferability (tra and trh), and DNA metabolism (topB, dsbC, ssb, bet, exo, dcm3, nuc, pri, rhs1, int, yacC, krA, uvrD, acr12, acaCD) within the backbone of all seven complete plasmids. However, it was observed that the resistance-encoding regions exhibit a great variability among these plasmids (Fig. 2, 3, 4, and 5). The fundamental genetic characteristics of these seven plasmids are summarized in Table 2.

Fig. 2

Linear sequence alignment of plasmid pVP181-NDM (PP860827), pHNAH8161B (CP104606) and P2-NDM-1(OL348378). Gray shading indicates homology between the corresponding genetic loci in each plasmid. Arrows indicate CDSs, with arrowheads indicating the direction of transcription

Fig. 3

Linear sequence alignment of plasmid pVb1282-tet (OQ622010), pVP156-NDM (PP860828), pVP209-NDM (PP860833), and pKp55(KY887594). Gray shading indicates homology between the corresponding genetic loci in each plasmid. Arrows indicate CDSs, with arrowheads indicating the direction of transcription

Fig. 4

Linear sequence alignment of plasmid pVP205-NDM(PP860829), pVP148-NDM(PP860830), pCf53 (KY887593), and pVb2175 (OK067239). Gray shading indicates homology between the corresponding genetic loci in each plasmid. Arrows indicate CDSs, with arrowheads indicating the direction of transcription

Fig. 5

Linear sequence alignment of IncC-type plasmids pVP228-NDM(PP860831), pCf75(CP047308), pVP14-NDM(PP860832), and p1504-2(CP019053). Gray shading indicates homology between the corresponding genetic loci in each plasmid. Arrows indicate CDSs, with arrowheads indicating the direction of transcription

Table 2 Genetic features of IncC type blaNDM-1-bearing plasmids recovered from V. parahaemolyticus

The plasmid pVP181-NDM (group I) was identified to be the largest among the seven plasmids; BLASTn analysis against the NCBI database revealed that pVP181-NDM shared a remarkably high degree of genetic similarity with pHNAH8161B (CP104606) and P2-NDM-1 (OL348378). Notably, the plasmid pVP181-NDM harbored a resistance region of 38,277 bp, which was punctuated by six IS26 elements that were known to facilitate the dissemination of antimicrobial resistance genes (Liu et al. 2022). Within this region, the class 1 integron In1021 (Int1-aac(6′)-Ib-cr-ARR3-dfrA27-aadA16-sul1) was identified in pVP181-NDM. The blaNDM-1 gene was found located downstream of the ISCR1 mobile element and was flanked by the catB3, ARR-3, sul1, and ChrA genes, a configuration identical to that observed in other IncC-type plasmids such as pHNAH212861 (CP104923) in Citrobacter portucalensis and pC1-MCR-NDM (OL348380) in E. coli strains (Wang et al. 2022). The tnsA, tnsB, tnsC, tnsD, and tnsE genes, which encode proteins essential for Tn7 transposition, were inserted into the pVP181-NDM backbone flanked by two ISSheE3 mobile elements, suggesting that the genomic region adjacent to the ISSheE3 elements was a hot spot for Tn7 transposon insertion (Fig. 2).

The group II plasmid pVP156-NDM and pVP209-NDM was found to harbor two resistance regions, with one being 13,250 bp in length and the other one is the blaNDM-1-bearing region with a size of 28,375 bp and 25,430 bp, respectively. The first resistance-encoding region of the plasmid pVP156-NDM and pVP209-NDM harbors genes that confer resistance to chloramphenicol (floR), tetracycline (tet(A), tet(R)), aminoglycoside (strA, strB), and sulfonamide (sul2). Comparative analysis of the linear sequence of pVP156-NDM and pVP209-NDM indicated that the two plasmids exhibited a high degree of similarity with pKp55 (KY887594), which was recovered from another Klebsiella pneumoniae strain, Kp55 (Fig. 3). It should be noted that the resistance region harboring blaNDM-1 gene exhibits a high degree of sequence variation among the plasmids pVb1282-tet, pVP156-NDM, pVP209-NDM, and pKp55. Specifically, the class I integron In469 (intI-ARR-3-dfrA27-aadA16), In79 (intI-qacE), and In1197(intI-blaIMP-26-aac(6′)-Ib-cr-catB3-sul1) were identified in pVb1282-tet, pVP156-NDM, and pKp55, respectively. However, the integron was not found in pVP209-NDM (Fig. 3). The blaNDM-1 gene was bracketed by two copies of ISCR1 insertion elements flanked by a 117 bp truncated ISAba125 element; this finding strongly indicates that a preferential integration of the blaNDM-1 gene into the IncC-type plasmid backbone and that this process is facilitated by the presence of the ISAba125 and ISCR1 elements.

The backbones of pVP205-NDM and pVP148-NDM (group III) shared a common resistance-encoding region of 13,250 bp. BLAST analysis indicated that pVP205-NDM and pVP148-NDM exhibited a high degree of similarity to pCf53 (KY887593), which was recovered from a Citrobacter freundii strain Cf53 isolated from gulls in New South Wales, Australia (Papagiannitsis et al. 2017), and to pVb2175 (OK067239) recovered from a Vibrio alginolyticus strain (Vb2175) isolated from shrimp samples in Shenzhen, China (Zheng et al. 2022). Notably, both pVP205-NDM and pVP148-NDM harbored two blaNDM-1 genes flanked by three copies of ISCR1 insertion elements oriented in the same direction. The genetic context surrounding the blaNDM-1 gene in these plasmids differed markedly from that of pNDM2310 and p21OH12SH02B-1, which showed the highest sequence coverage with pVP205-NDM and pVP148-NDM. This highlights the structural heterogeneity of these plasmids and suggests that the blaNDM-1-carrying resistance region is relatively novel (Figure S3). As depicted in Fig. 4, although pVP205-NDM and pVP148-NDM possess an IncC type backbone similar to that of plasmids pCf53 and pVb2175, their resistance profiles are remarkably different. The classical genetic context of blaNDM-1 gene in Tn125 is present in pVb2175; however, the groEL, groES, cutA, and dsbC genes are notably absent in pVP205-NDM and pVP148-NDM, suggesting that these plasmids have undergone recombination events that altered their genetic architecture (Figure S4).

The group IV plasmid pVP228-NDM and pVP14-NDM demonstrated a remarkable genetic similarity with plasmid pCf75 found in Citrobacter freundii strain L75 isolated from a urine sample in Guangzhou, China, and also with plasmid p1540-2 carried by an Escherichia coli strain CRE1540 isolated from a clinical rectal swab sample in Hong Kong (Fig. 5) (Li et al. 2020b). The plasmid pVP228-NDM contains a similar MDR region to that of pVP181-NDM, except that the orientation of the IS26-floR-IS26 module was different (Figure S4). Plasmid pCf75 shared a highly similar backbone with pVP228-NDM but lacked a 13.4 kbp of the resistance module (IS26-aac(3′)-IId-IS26-ISCR1-floR-tet(A)-tet(R)-IS26-aph(3′)-Ia). Plasmid pVP14-NDM contained a backbone that was almost structurally identical to that of pCf75, except for the absence of the aac(6′)-Ib-cr gene. Moreover, the blaNDM-1-encoding AMR regions in the seven plasmids were compared with the archetype of Tn125 and plasmid pVb2175, which was previously characterized in our laboratory. The linear sequence maps revealed that the AMR regions harboring the blaNDM-1 gene in these plasmids displayed marked differences from each other and that their genetic contexts were notably different from that observed in Tn125 (Figure S4). These observations imply that the heterogeneity observed in the resistance regions is likely the result of complex interactions between various integrons, transposases, and mobile genetic elements, suggesting that multiple molecular mechanisms are responsible for driving the evolution of AMR in V. parahaemolyticus.