Prevalence, bacterial load, and genomic characterization of Vibrio parahaemolyticus in freshwater-farmed Litopenaeus vannamei from China

Shrimp was the leading food vehicle for Vibrio parahaemolyticus outbreaks in China, yet studies on V. parahaemolyticus in globally predominant farmed shrimp (Litopenaeus vannamei) - particularly in freshwater culture remain limited. This study aimed to investigate and evaluate the contamination of V. parahaemolyticus in freshwater-farmed marine shrimp (L. annamei) in China. The prevalence and bacterial load of V. parahaemolyticus in shrimps were assessed by China China National Standard GB 4789.7-2013 and isolates were obtained for genomic analysis. The prevalence of V. parahaemolyticus in shrimp samples was 39.1% and the bacteria load ranged from 3.6 MPN/g to 24,000 MPN/g. Analysis on isolates demonstrated that 75.8% (94/124) were assigned to 28 sequence types (STs), whereas 24.2% STs were unknown. All isolates harbored 24 antibiotic resistance genes (ARGs), of which tet(34), tet(35), and bla_CARB were harbored in all isolates. There was a significant correlation between bla_CARB types and STs, with isolates sharing identical STs carried the same blaCARB variant. The trh+ isolates (9.7%, 12/124) was simultaneously coexisted with hlyB and hlyC. All four ST79 strains harbored trh/hlyB/hlyC and bla_{CARB_46}. Our findings elucidated the contamination of V. parahaemolyticus in freshwater-farmed L. vannamei, with heavy bacteria load in Fujian province. The emergence of specific ST co-harboring critical virulence genes and ARGs indicating the necessity for targeted surveillance and specified control in aquaculture systems.

1 Introduction

Vibrio parahaemolyticus can cause acute gastroenteritis in humans through contamination of raw or undercooked aquatic products (Ceccarelli et al., 2013; Ghenem et al., 2017; Su and Liu, 2007). The primary implicated food for V. parahaemolyticus outbreaks in America and Europe is shellfish (Cabello et al., 2007; Centers for Disease Control and Prevention, 1998, 1999; Fearnley et al., 2024; Lozano-León et al., 2003; Raszl et al., 2016; Taylor et al., 2018). Meanwhile, according to China national foodborne disease surveillance, the primary food is shrimp. According to FAO (2024) and China Fisheries Statistical Yearbook (2023), Litopenaeus vannamei ranked top in shrimp aquaculture production globally, including China. Although L. vannamei is usually produced via marine aquaculture, its farming differs in China. More than 36% of total L. vannamei production was transferred to freshwater farming in China. Although studies on V. parahaemolyticus contamination in marine-farmed L. vannamei are not rare, studies investigating V. parahaemolyticus contamination in freshwater-farmed L. vannamei i are scarce, studies on their genetic traits are even rarer.

Although V. parahaemolyticus has a wide array of virulence genes, including hemolysins and type III secretion systems (T3SS), the profile of virulence genes varies significantly among different source isolates. Notably, the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), which are encoded by tdh and trh genes, respectively, have clinical significance and serve as key virulence markers (Nichuchi and Kaper, 1995; Raghunath, 2015). In addition, V. parahaemolyticus develops resistance to multiple antibiotics owing to the misuse of antibiotics in aquaculture production (Elmahdi et al., 2016; Hossain et al., 2022). Studies from regions such as Bulgaria, South Korea, Mexico, China, Vietnam, and Malaysia have demonstrated that V. parahaemolyticus isolates from aquatic products are resistant to multiple antibiotics, including ampicillin, ciprofloxacin, ceftazidime, cefotaxime, and tetracycline. Among these, the ampicillin-resostant strains accounted for the highest proportion (Flores-Villaseñor et al., 2024; Jeong et al., 2020; Li et al., 2020; Stratev et al., 2023; Tan et al., 2020; Vu et al., 2022).

Therefore, this study aimed to investigate the contamination status and assess the virulence and antimicrobial resistance potential of V. parahaemolyticus in freshwater-farmed marine shrimp (L. vannamei) in China.

2 Materials and methods

2.1 Sample collection

Samples were collected from nine sampling sites across freshwater L. vannamei farms in the Shandong and Fujian provinces of China, including L. vannamei (184), freshwater (42), feed (18), and aquaculture worker’s feces (6). From each sampling pool, approximately 100 g of L. vannamei shrimp samples and 450 mL of culture water samples were collected. Water temperature and salinity were simultaneously measured. Detailed information are presented in Supplementary material.

2.2 Detection and isolation

V. parahaemolyticus was qualitatively and quantitatively detected in samples following the Chinese national standard GB 4789.7 - 2013 “Microbiological Examination of Food - Detection of Vibrio parahaemolyticus.” All samples underwent primary enrichment in 3% sodium chloride alkaline peptone water with incubation at 37 °C for 12 h. Subsequent V. parahaemolyticus isolation was achieved by streaking onto selective thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates and incubated at 37 °C for 24 h. Typical colonies were cultured onto 3% sodium chloride tryptic soy agar plates at 37° C for 24 h. Presumptive V. parahaemolyticus isolates were confirmed through Vitek 2 Compact. Afterwards, the confirmed V. parahaemolyticus strains were then taken for whole genome sequencing.

2.3 Whole genome sequencing

Genomic DNA was extracted using a Bacterial Genomic DNA Extraction Kit (Beijing Tiangen Biotech Co., Ltd.). The extracted DNA was sent to Novogene for whole-genome sequencing on an Illumina NovaSeq 6000 platform using the Illumina HiSeq protocol. Genome assembly used SPAdes v3.15.4.¹ Quality control of the assembled sequences was performed using CheckM.² Duplicate strains were screened by integrating Average Nucleotide Identity (ANI) analysis³ and QUAST.⁴ The reference strain used for comparison was V. parahaemolyticus ATCC17802.

2.4 Bacterial species identification and multi-locus sequence typing

Bacterial species were identified using KmerFinder 3.2.⁵ Multi-locus sequence typing (MLST) of the strains was conducted based on seven housekeeping genes (dnaE, recA, dtdS, gyrB, pntA, tnaA and pryC) using PubMLST.⁶ A minimum spanning tree was constructed using GrapeTree’s MSTree algorithm⁷ based on multi-locus sequence typing (MLST).

2.5 Screening of ARGs, virulence genes, and plasmids

ARGs, virulence genes and plasmids were identified using the ResFinder,⁸ Virulence Factor Database (VFDB⁹) and PlasmidFinder.¹⁰ For all analyses, minimum thresholds of ≥80% coverage and ≥80% identity were applied.

2.6 Phylogenetic analysis

Genome annotation was performed using Prokka v1.14.6. Core genome analysis was perforemed using Roary v3.13.0 and Mafft v7.525. The core SNPs were identified using Snippy v4.6.0.¹¹ Gubbins v2.4.1 was used to filter SNP recombination.¹² A maximum-likelihood phylogenetic tree was generated using FastTree v2.1.10 with parameters “nt -gtr.” Phylogenetic tree of the genomes was visualized using FigTree v1.4.4.¹³

3 Results

3.1 Characteristics of sampling sites

The sampling months and environmental parameters (e.g., water temperature and salinity) of aquaculture farms in Shandong and Fujian are shown in Table 1. The average water temperature and salinity in aquaculture farms in Shandong and Fujian provinces were 32.8 °C, 0.53‰ and 24.2 °C, 3.65‰, respectively.

Table 1

Table 1. Basic information on sampling of Litopenaeus vannamei farms.

3.2 Prevalence and bacterial load of Litopenaeus vannamei

A total of 250 samples were collected, comprising 184 shrimp samples, 42 aquaculture freshwater samples, six worker’s feces samples, and 18 feed samples. The overall detection rate of V. parahaemolyticus was 32.8% (82/250), with detection rates of 39.1% (72/184) in shrimp and 23.8% (10/42) in freshwater samples. V. parahaemolyticus was not detected in workers’ feaces or feed samples. There was a statistically significant difference in V. parahaemolyticus detection rates among the sample types (χ² = 23.958, p < 0.05) (Figure 1a).

Figure 1

Figure 1. Detection and bacterial load of V. parahaemolyticus in samples. (a) Detection of V. parahaemolyticus in different type samples types. (b) Detection of V. parahaemolyticus in samples of shrimp from different provinces. (c) Contamination levels of V. parahaemolyticus in positive samples of shrimp from different provinces. ‘**’ means P < 0.01, ‘***’ means P < 0.001, ‘ns’ means P > 0.05.

This study was conducted in Shandong and Fujian provinces. The overall detection rate of V. parahaemolyticus was 26.7% (54/202) in Shandong and 58.3% (28/48) in Fujian, and the difference was statistically significant (χ² = 17.571, p < 0.05). For shrimp samples, the detection rates of V. parahaemolyticus were 35.3% (53/150) in Shandong and 55.9% (19/34) in Fujian, with a statistically significant difference (χ² = 4.914, p < 0.05). The median (P₂₅, P₇₅) pollution levels of V. parahaemolyticus-positive shrimp samples in Shandong and Fujian were 43 (23, 120) MPN/g and 2,400 (2,400, 24,000) MPN/g respectively, with a statistically significant difference in pollution levels (Z = − 6.145, p < 0.05) (Figures 1b,c).

3.3 MLST and the minimum spanning tree

Of the 124 V. parahaemolyticus strains, 94 were assigned to 28 STs, with 16 STs containing at least two isolates each, whereas 12 STs were represented by single isolates only. Predominant STs were ST2671 (19.6%, 18/92), ST2165 (14.1%, 13/92), and ST1196 (9.8%, 9/92) (Figure 2a). The minimum spanning tree based on provincial origins and allelic profiles revealed two distinct clusters with clear genetic structures (Figure 2b). The distribution of sequence types in the shrimp and water isolates is shown in Supplementary material.

Figure 2

Figure 2. MLST and minimum spanning tree of 124 V. parahaemolyticus isolates. (a) Distribution of MLST in 94 isolates. (b) Minimum spanning tree of 94 isolates.

3.4 Distribution of ARGs and plasmids

In this study, a total of 24 ARGs were identified in 124 V. parahaemolyticus strains and classified into four antimicrobial categories, namely β-lactam, tetracycline, quinolone, and sulphonamide (Figure 3a). Notably, tet(34), tet(35), and bla_CARB, responsible for tetracycline and β-lactam resistance, were present in all the 124 isolates. Additionally, only a small proportion of the isolates carried resistance genes against quinolone and sulphonamide antibiotics, with detection rates of 12.9% (16/124) and 0.8% (1/124), respectively. A lncC-type plasmid was identified in one shrimp-derived isolate; however, no known antibiotic resistance genes were detected in this plasmid sequence.

Figure 3

Figure 3. Distribution and statistical analysis of ARGs in 124 V. parahaemolyticus isolates. (a) Distribution of ARGs in 124 V. parahaemolyticus isolates. (b) Differences in the number of ARGs in isolates across different provinces. (c) Differences in isolates across number of ARGs in different sources. ‘**’ means P < 0.01, ‘***’ means P < 0.001, ‘ns’ means P > 0.05.

The 124 strains were classified by provinces (77 from Shandong and 44 from Fujian) and sources (110 from shrimp and 14 from aquaculture water). The Mann–Whitney U test revealed a statistically significant difference in the number of ARGs between different provinces (Z = −2.769, p < 0.05). In contrast, there was no significant difference between shrimp and aquaculture water isolates (Z = −1.287, p = 0.198) (Figures 3b,c).

3.5 Distribution of virulence genes

Among the 124 V. parahaemolyticus strains analyzed, a total of 52 virulence genes encoding five pathogenic factors were identified. Among these, 50 genes related to hemolysin, Type III Secretion System (T3SS), adhesion factors, and flagella have the potential to pose risks to human health. Notably, the pirA and pirB genes, encoding the toxins PirA and PirB, respectively, are strongly linked to acute hepatopancreatic necrosis disease (AHPND) in shrimp. Four hemolysin-related genes were detected, with all the strains harboring the tlh gene and 9.7% (12/124) of the strains harboring the trh, hlyB, and hlyC genes. All the strains possessed T3SS1-related genes, with 99.2% (123/124) encoding four T3SS1 effector proteins (vopR, vopS, vopQ, and VPA450). Additionally, two adhesion genes (mam7 and vpadF) and five flagella genes (fliG, cheY, cheW, flgB, fliN) were also identified (Figure 4a).

Figure 4

Figure 4. Distribution and statistical analysis of virulence genes in 124 V. parahaemolyticus isolates. (a) Distribution of virulence genes in 124 V. parahaemolyticus isolates. (b) Differences in the number of virulence genes in isolates across different provinces. (c) Differences in the number of virulence genes in isolates across different sources. ‘**’ means P < 0.01, ‘***’ means P < 0.001, ‘ns’ means P > 0.05.

In this study, most of the strains (87.1%, 108/124) harbored 45 or 46 virulence genes. The median numbers of virulence genes carried by strains from Shandong and Fujian provinces was 45 and 46, respectively, and the difference in the number of virulence genes harbored by strains from the two provinces was statistically significant (Z = −4.774, p < 0.05). In addition, the differences in the number of virulence genes harbored by strains from different sources remained significant (Z = −2.824, p < 0.05) (Figures 4b,c).

3.6 Phylogenetic analysis

Phylogenetic analysis demonstrated that ST significantly correlated with the 17 distinct bla_CARB gene variants, whereas isolates of the same ST harbored identical bla_CARB gene variants. Furthermore, this study identified four V. parahaemolyticus strains isolated from shrimps; the strains harbored the trh gene and all belonged to ST79, with the bla_CARB type being 46. Our study demonstrated that hlyB and hlyC genes encoding α-hemolysin were exclusively detected in trh+ strains and trh+ was coexisted with hlyB and hlyC (Figure 5).

Figure 5

Figure 5. Phylogenetic tree and heatmap of sequence types, ARGs and virulence genes in 124 V. parahaemolyticus isolates. The different colors of branches and strain names represent the provincial origins and sample types of the strains, respectively. Different colors of squares were used to indicate the STs, ARGs and virulence genes.

4 Discussion

V. parahaemolyticus is an important foodborne pathogen, which is widely distributed in seawater and various seafood products, including shellfish and shrimp (Li et al., 2019). With increasing global demand for L. vannamei, freshwater aquaculture of this species has gained significant momentum in China. Study has demonstrated that V. parahaemolyticus is more prevalent in freshwater food than in seafood (Li et al., 2023). Therefore, the present study investigated the contamination characteristic of V. parahaemolyticus in freshwater-farmed L. vannamei from selected regions of China and characterized the genetic features of the isolates.

The results of the present study revealed a detection rate of 39.1% of V. parahaemolyticus in freshwater-farmed L. vannamei samples, which is considerably lower than the prevalence reported in most marine shrimp farming countries (Mok et al., 2021; Narayanan et al., 2020; Siddique et al., 2021; Tran et al., 2018), including India (80.0%), South Korea (54.5%), Bangladesh (69.4%), and Vietnam (87.5%), which rate was significantly higher than that observed in Zhejiang Province (3.33%) (Jiang et al., 2020), where shrimps are farmed in freshwater. However, the detection rate of V. parahaemolyticus in shrimp samples from Fujian Province (55.9%) was significantly higher than that in shrimp samples from Shandong Province (35.3%). This discrepancy may be attributed to the relatively higher salinity of aquaculture water in Fujian (3.65‰), compared to that in Shandong (0.53‰). V. parahaemolyticus abundance rises with increased salinity in freshwater, additionally, the abundance is associated with temperature and pH (León Robles et al., 2013). Furthermore, MLST based on seven housekeeping genes (González-Escalona et al., 2008) was performed on 94 V. parahaemolyticus isolates, identifying 28 STs. This demonstrates the genetic diversity of the strains from freshwater aquaculture sources. Notably, 24.3% of the strains (30/124) belonged to novel sequence types, suggesting potential genetic divergence of V. parahaemolyticus from freshwater aquaculture.

In the present, all V. parahaemolyticus isolates carried tetracycline resistance genes tet(34) and tet(35), as well as β-lactam resistance gene bla_CARB. Antibiotic resistance gene that tet(34) and bla_CARB are present in two Vibrio parahaemolyticus strains isolated from cultured shrimp in Bangladesh (Ahmmed et al., 2019). The detection of bla_CARB gene in this study is higher than that in isolates from farmed L. vannamei in Ningde (18.37%) (Zhang et al., 2024). Studies (Bai et al., 2024; Zheng et al., 2025) have demonstrated that the isolates from seafood harboring tet(34), tet(35) and bla_{CARB _46} were susceptible to tetracycline but resistant to ampicillin and cefazolin. Furthermore, this study revealed a strong correlation between bla_CARB gene variants and STs, whereby isolates sharing identical STs carried the same bla_CARB variant. Consistent with previous reports (Li et al., 2016), bla_{CARB_17} demonstrated higher conservation than the commonly used V. parahaemolyticus-specific marker tlh in PCR detection, exhibiting 100% specificity. Based on these findings, we hypothesized that bla_CARB gene may serve as potential target for subtyping of V. parahaemolyticus, as an useful complementary method for MLST. Besides tetracycline and β-lactam resistance genes, there have quinolone and sulphonamide resistance genes absenced in aquaculture isolates, which may be its extensive use in human medicine and aquaculture systems (Lei et al., 2020). The application of biofertilizers increases fluoroquinolone resistance (Zhao et al., 2020).

The pathogenic factors of V. parahaemolyticus include hemolysins and T3SS and so on. Studies have shown that tdh and trh, which are closely associated with pathogenicity, are present in more than 80% of clinical isolates, whereas their carriage rates in food and environmental isolates generally range from 0 to 10% (León-Sicairos et al., 2022; Tewawong et al., 2024; Zhong et al., 2022). In this study, none of the strains carried the tdh gene, the trh positively rate among the shrimp-derived strains was 9.1% (10/110), which was lower than that in India saline water shrimps (9.5, 14.8%) (Sudan et al., 2023; Narayanan et al., 2020). Study (Paria et al., 2021) had revealed that the tdh and trh positive isolates were resistant to β-lactam antibiotics. Besides, our investigation revealed that the α-hemolysin genes hlyB and hlyC were exclusively present in trh-positive strains. This finding agrees with that of Zha et al. (2023), who identified a pathogenic V. parahaemolyticus strain lacking tdh and trh but carrying hlyA-hlyD. Combined with our results, we speculate that hlyB and hlyC represent unique hemolysins with potential pathogenic significance. Furthermore, all four trh+ strains belonged to ST79, which originated from shrimp samples collected from two distinct farms in Shandong Province. Because ST79 has also been reported in clinical isolates from China in PubMLST, it is important to confirm the potential risks of this ST through further investigation.

T3SS encompasses T3SS1 and T3SS2 clusters (Matsuda et al., 2020). Almost all the clinical and environmental strains of V. parahaemolyticus encode T3SS1, and T3SS2 is closely associated with its pathogenicity. In the present study all strains harbored T3SS1, which is consistent with the findings of Siriphap A (Siriphap et al., 2024). In contrast, none of the strains harbored the T3SS2 gene. Notably, one shrimp-derived strain from Fujian Province harbored genes encoding PirA and PirB toxins, which have been shown to cause acute hepatopancreatic necrosis disease (AHPND) in shrimp, with mortality rates of 70 ~ 100% (Lin et al., 2017), potentially leading to substantial economic losses in aquaculture. In total, our strains harbored 52 virulence-associated genes. The presence of these genes not only poses a potential public health threat but also threatens the sustainability of aquaculture.

In conclusion, the high detection rate of V. parahaemolyticus in Fujian freshwater-farmed L. vannamei. and the presence of virlence gene trh and β-lactam resistance genes in isolates have raised significant public health concerns. Besides, V. parahaemolyticus isolates in this study showed the emergence of ST79 co-harboring critical virulence genes trh and ARGs. Therefore, it is strongly recommended that targeted surveillance and specified control measures are being implemented in aquaculture systems in the future.

Data availability statement

The data presented in the study are deposited in the China National Center for Bioinformation repository, accession number is PRJCA051154.

Author contributions

FS: Data curation, Formal analysis, Visualization, Writing – original draft. WL: Investigation, Writing – original draft. ZC: Investigation, Resources, Writing – review & editing. PH: Investigation, Resources, Writing – review & editing. NS: Investigation, Resources, Writing – review & editing. RL: Investigation, Resources, Writing – review & editing. XL: Investigation, Resources, Writing – review & editing. WC: Investigation, Resources, Writing – review & editing. LZ: Investigation, Resources, Writing – review & editing. SY: Data curation, Methodology, Writing – review & editing. SC: Conceptualization, Supervision, Writing – review & editing. YG: Methodology, Supervision, Writing – review & editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This research was supported by the National Key Research and Development Program of China (No. 2022YFC2303905).

Acknowledgments

We would like to thank all the growers who allowed us to collect samples from their farms.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The authors declare that no Gen AI was used in the creation of this manuscript.

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Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2025.1713237/full#supplementary-material

Footnotes

1. ^https://github.com/ablab/spades

2. ^https://github.com/Ecogenomics/CheckM

3. ^https://github.com/ParBLiSS/FastANI

4. ^https://quast.sourceforge.net/

5. ^https://cge.food.dtu.dk/services/KmerFinder/

6. ^https://pubmlst.org/organisms/vibrio-parahaemolyticus

7. ^https://github.com/achtman-lab/GrapeTree/releases

8. ^http://genepi.food.dtu.dk/resfinder

9. ^https://www.mgc.ac.cn/VFs

10. ^https://cge.food.dtu.dk/services/PlasmidFinder/

11. ^https://github.com/tseemann/snippy

12. ^https://github.com/nickjcroucher/gubbins

13. ^http://tree.bio.ed.ac.uk/software/figtree/

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