Meta-Analysis and Systematic Review of Phenotypic and Genotypic Antimicrobial Resistance and Virulence Factors in Vibrio parahaemolyticus Isolated from Shrimp

This systematic review and meta-analysis investigates the prevalence of Vibrio parahaemolyticus, its virulence factors, antimicrobial resistance (AMR), and its resistance determinants in shrimp. This study was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, to identify and select relevant peer-reviewed articles published between January 2020 and December 2022. The search strategy involved multiple online databases, including Google Scholar, PubMed, ScienceDirect, and Scopus. The inclusion criteria focused on studies that examined V. parahaemolyticus prevalence, virulence factors, and AMR in shrimp from farms to retail outlets. A total of 32 studies were analyzed, revealing a pooled estimate prevalence of V. parahaemolyticus in shrimp at 46.0%, with significant heterogeneity observed. Subgroup analysis highlighted varying prevalence rates across continents, emphasizing the need for further investigation. Virulence factor analysis identified thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) as the most common. Phenotypic AMR analysis indicated notable resistance to glycopeptides, nitrofurans, and beta-lactams. However, the correlation between antimicrobial usage in shrimp farming and observed resistance patterns was inconclusive. Funnel plots suggested potential publication bias, indicating a need for cautious interpretation of findings. This study underscores the urgency of coordinated efforts to address AMR in V. parahaemolyticus to safeguard public health and to ensure sustainable aquaculture practices.


Introduction
Antimicrobial resistance (AMR) has emerged as a critical global health threat, fundamentally reshaping the management of infectious diseases and posing unprecedented challenges to healthcare systems worldwide [1].The increasing resistance of a diverse array of bacteria responsible for infections in both human and animal populations to previously effective antimicrobial treatments has become a pressing concern [2].This alarming phenomenon could imperil human health and undermine the sustainability of animal production, and this requires swift and coordinated actions to avert an imminent global health crisis.
Vibrio parahaemolyticus, a halophilic bacterium in the family Vibrionaceae, thrives within a temperature range from 10 • C to 44 • C, with an optimal growth temperature between 35 • C and 37 • C within marine and estuarine environments [3].Studies have highlighted the role of this bacterium as a primary causative agent of acute hepatopancreatic necrosis disease in shrimp populations [4].Further research suggests that V. parahaemolyticus can rapidly spread within shrimp tissues, affecting vital organs such as the gills, hepatopancreas, intestine, muscles, and hemolymph, exacerbating the challenges faced in shrimp farming [5].V. parahaemolyticus has the potential to trigger devastating vibriosis outbreaks in shrimp aquaculture, leading to substantial economic losses for farmers worldwide [6].Historically linked to seafood, such as shrimp, oysters, clams, and cockles, V. parahaemolyticus received recognition for its pathogenic potential in humans in 1950 following a major outbreak in Japan in which contaminated sardine with the bacterium led to 20 fatalities and 270 hospitalizations [7].Subsequently, outbreaks stemming from the consumption of raw or undercooked seafood tainted with V. parahaemolyticus have been documented in various countries, including the USA, Thailand, Vietnam, China, Spain, and Chile [8][9][10][11][12][13].The disease pathogenesis of V. parahaemolyticus infections is caused by virulence factors such as thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH), encoded by tdh and trh genes, respectively.These two factors are the main factors causing infections in shrimps as well as seafood-borne bacterial gastroenteritis in humans [14].This emphasizes the significance of comprehending and managing V. parahaemolyticus infections to safeguard both the aquaculture industry and the general health of seafood consumers.
Currently, the primary approach to the treatment of V. parahaemolyticus infections involves the administration of antimicrobial agents.However, alarming reports have surfaced, highlighting the emergence of AMR strains of V. parahaemolyticus and increasing public health concerns [15].The consequences of infections caused by pathogenic and resistant V. parahaemolyticus extend beyond the scope of healthcare, affecting economic and social aspects as well.Outbreaks involving resistant strains can disrupt the seafood industry, erode consumer trust, trigger trade restrictions, and cause economic losses.The interconnectedness between AMR, seafood trade, and economic stability necessitates comprehensive investigations to guide policy interventions and mitigate potential consequences.
The rise of AMR within V. parahaemolyticus strains adds further complexity to managing infections, requiring immediate action to mitigate potential impacts on human health and the sustainability of aquaculture systems.The consequences extend to both patient well-being and healthcare systems, leading to prolonged illnesses, treatment failures, and more severe clinical outcomes.Despite its importance, there remains a lack of comprehensive understanding regarding the prevalence of V. parahaemolyticus, its virulence factors, and AMR in shrimp, which serve as crucial reservoirs for this pathogen.This study seeks to bridge this gap by conducting a meta-analysis and systematic review of existing literature.The objective of this meta-analysis and comprehensive systematic review was to investigate the prevalence of V. parahaemolyticus, along with its virulence factors and AMR phenotype and genotype.This study offers valuable insights into the global occurrence and patterns of AMR in V. parahaemolyticus found in shrimp, thereby enhancing the international understanding of foodborne pathogens and AMR.Therefore, the findings from this study can collectively help formulate effective strategies involving various stakeholders to mitigate the impact of AMR in V. parahaemolyticus, safeguard public health, and establish resilient socioeconomic systems.
For  (O-F) test, salt tolerance test, sulfur reduction (cysteine desulfurase) test, utilization of alpha ketoglutarate, urease test, triple sugar iron agar (TSI), as well as the commercial API 20E biochemical test kits (bioMérieux, Marcy-l'Etoile, France) were applied.In addition to PCR, MALDI-TOF and VITEK2 were employed by the studies for bacterial identification.Furthermore, 16S rRNA sequencing along with species-specific genes (tlh, tl, and toxR) detection were also utilized.

Prevalence of V. parahaemolyticus and Virulence Factors
The prevalence of V. parahaemolyticus in different anatomical parts of shrimp, such as hepatopancreas, hemolymph, gill, intestine, midgut, stomach, tissue or flesh, whole shrimp, and swab samples, were considered in the analysis.In addition to shrimp, some studies included other aquatic animals such as marine fish, oysters, crabs, shellfish, clams, mussels, tilapia, rui, and squid for examination.A subset of studies also included environmental samples, such as sediment, pond water, and reservoir water.
This comprehensive approach allowed for a robust examination of V. parahaemolyticus prevalence, considering both the random-effects model and the visualizations of study weights and bias assessment.The prevalence of V. parahaemolyticus was determined among 22 studies (22/32 = 68.8%)from 9018 samples/isolates, and the pooled estimate of V. parahaemolyticus prevalence in shrimp was 46.0% with a 95% C.I. (33.4-58.6%)and Qstatistic 2747.10 (Figure 2A).The overall heterogeneity was identified with tau 2 = 0.088, I 2 = 99.43%,H 2 = 175.09,and p-value < 0.0001.
Although our study primarily provided descriptive presentations of the prevalence, virulence factors, and AMR of V. parahaemolyticus in shrimp populations, we recognize the potential advantages of conducting further analyses to extract additional insights from the dataset.As such, we have performed subgroup analyses to explore the relationship between variables more comprehensively and to identify potential determinants of V. parahaemolyticus prevalence and AMR in shrimp across different continents.Subgroup analysis was assessed based on continents-except China due to availability of several publications.The prevalence of V. parahaemolyticus was high in Asia (63.1% with C.I. = 46.7-79.5%),while China exhibited the lowest prevalence at 19.0% (C.I. = 12.0-26.0%).A funnel plot was employed for bias assessment, contributing to a comprehensive evaluation of the prevalence data (Figure 2C).

Antimicrobial Susceptibility Testing (AST)
Among 32 studies used (n = 29/32, 90.6%), the phenotypic antimicrobial susceptibility of V. parahaemolyticus was mostly tested by disk diffusion test (n = 23), broth microdilution method (n = 5), and both disk diffusion and broth microdilution (n = 1).The methods used for AST were followed by the Clinical and Laboratory Standards Institute (CLSI) (EP17, M45, and M100), European Committee on Antimicrobial Susceptibility Testing (EUCAST), and National Committee for Clinical Laboratory Standards (NCCLS).The interpretive The analysis of virulence genes in V. parahaemolyticus within shrimp was conducted in 15 out of 32 articles (46.9%) from 3644 bacterial isolates.Virulence factors were limited to a specified set that included tdh, trh, pvuA, pvsA, wza, lafA, tcp, zot, nanH, pirA, and pirB.Among these, tdh and trh were identified as the most frequently detected virulence genes.The estimated prevalence of virulence factors was determined as 22.5% with a 95% C.I. of 11.4-33.6%(Figure 2B).The heterogeneity was observed using a Q value of 6236.45 with tau 2 = 0.104, I 2 = 99.96%,H 2 = 2228.49,and p-value < 0.0001 (Figure 2B), and the bias assessment was indicated in a funnel plot (shown here in Figure 2D).

Antimicrobial Susceptibility Testing (AST)
Among 32 studies used (n = 29/32, 90.6%), the phenotypic antimicrobial susceptibility of V. parahaemolyticus was mostly tested by disk diffusion test (n = 23), broth microdilution method (n = 5), and both disk diffusion and broth microdilution (n = 1).The methods used for AST were followed by the Clinical and Laboratory Standards Institute (CLSI) (EP17, M45, and M100), European Committee on Antimicrobial Susceptibility Testing (EUCAST), and National Committee for Clinical Laboratory Standards (NCCLS).The interpretive criteria were as indicated according to the CLSI breakpoints and epidemiological cut-off values (ECOFFs) set by EUCAST.

Publication Bias (Reporting Bias)
In this meta-analysis, publication bias was graphically assessed using visual funnel plots by the regression-based test of Egger at p < 0.05.On visual inspection, the funnel plot showed slight asymmetrical distribution in meta-analysis for V. parahaemolyticus, virulence factors, AMR, and their determinants (Figure 2C,D, Figures S1 and S2).

Discussion
This study comprehensively investigated the overall prevalence of V. parahaemolyticus, virulence factors, and AMR isolates in shrimp retrieved from 32 published articles.Given the considerable diversity in study designs and population characteristics across the included studies, a random-effects model was employed for meta-analysis.This study involved the inclusion of equal proportions of both retail and agricultural farms.The examination of subgroup meta-analysis was conducted with a focus on continents, revealing a heightened prevalence of V. parahaemolyticus in Asia at 57.0%, with China exhibiting the lowest prevalence at 19.0%.Nevertheless, there was a scarcity of studies from Europe and the USA.Shrimp presented a higher risk, with a prevalence rate of 3.2%, when contrasted with other decapod crustaceans such as lobster and crab, which had prevalence rates of 3.0% and 3.1%, respectively [63].Consequently, it is imperative to conduct additional research to evaluate the prevalence of V. parahaemolyticus and its virulence factors, enhancing the representativeness of findings across these continents.The assessment of virulence factors in shrimp revealed that the predominant virulence factors detected were tdh and trh.Additionally, five studies performed serotyping for V. parahaemolyticus isolates, and the distribution of serotypes O3:K6, O5:KUT, O11:KUT, O3:KUT, O1:KUT, O3:K20, O1-O8, O10-O12, K25, K31, K64, and K68 was observed [33,42,44,48,49].
The primary technique used for AST involved a disk diffusion test, followed by a broth microdilution test.Notably, in South Korea, an automatic system such as Sensititre™ was used to perform broth microdilution [30,31].The criteria for choosing antimicrobials for AST were not clearly outlined.However, it is recommended that antimicrobials should be selected based on AMR monitoring and surveillance guidelines by following CLSI standards for aquatic animals, humans, and animals and the OIE list of antimicrobial agents of veterinary importance [64][65][66].While E. coli ATCC 25922 serves as the primary control strain for AST, about one-third of the studies did not explicitly mention the use of reference strains.This lack of clarity raises concerns about the reliability of the results in those studies, emphasizing the importance of proper reference controls in ensuring the accuracy of antimicrobial susceptibility assessments.In all the studies reviewed, the primary molecular method used for identifying resistance genes was predominantly PCR.However, the use of Whole-Genome Sequencing (WGS) was limited and not extensively utilized for this purpose.
This study observed that V. parahaemolyticus predominantly exhibited phenotypic resistance to glycopeptides, nitrofurans, and beta-lactams, a pattern similar to findings in a study on AMR Vibrio in marine bivalves [67].Notably, these antimicrobials are not commonly used in shrimp farming practices.Contrasting results were found in studies on shrimp aquaculture in China and Vietnam, where oxytetracycline emerged as the primary antimicrobial and frequently used antimicrobial classes included tetracyclines, sulfonamides, and quinolones [68,69].The inconsistency in these observations tentatively suggests that phenotypic AMR may not necessarily arise because of the selective pressure of antimicrobial applications on farms.Moreover, V. parahaemolyticus isolates with high resistance were not detectable in shrimp feed, implying a potential contribution from external sources of antimicrobials entering the farms.In human medicine, glycopeptides, nitrofurans, and beta-lactams are widely prescribed, and their resistance is observed in hospital wastewater worldwide [70].To address the potential consumer risk associated with shrimp consumption, further studies are needed to investigate the sources of AMR contamination in shrimp farms, particularly focusing on environmental water.Funnel plots were performed to evaluate publication bias and heterogeneity.Larger sample studies are clustered near the mean effect size at the top of the graph, contrasting with smaller-sample studies positioned at the bottom.The variability in effect size estimates within these smaller studies was influenced by sampling differences, evident in the graph's distribution.Asymmetry in funnel plots indicated potential publication bias for the prevalence estimate of V. parahaemolyticus, virulence factors, and AMR.Notably, the funnel plot for V. parahaemolyticus prevalence suggests more heterogeneity with varied sample sizes than that from the occurrence of virulence factors.Regarding the funnel plot for V. parahaemolyticus prevalence, smaller studies with significant results were more likely to be published than those without significant virulence factors exhibited.
The previous studies referred to the CLSI standard for AST.Most of these studies adopted CLSI criteria originally developed for human and animal sources.Notably, only one CLSI document suggested nine antimicrobial classes for susceptibility tests specific to V. cholerae and other Vibrio spp.These classes included aminoglycosides, beta-lactams, carbapenems, cephalosporins, folate pathway inhibitors, macrolides, phenicols, quinolones, and tetracyclines with limited reference ranges [71].Recent CLSI guidelines for bacteria isolated from aquatic animals do not provide any reference criteria for Vibrio spp.[65].One study specifically examined the resistance of V. parahaemolyticus to medemycin, a newly developed macrolide antibiotic.This was because of the increased use of this drug due to its higher potency and low gastrointestinal irritation compared with other macrolides [72].Results in our study suggested that harmonized protocols for antimicrobial resistance testing in aquaculture should be promptly initiated to achieve a valid global AMR assessment and prioritize urgent needs.
The limitations of this study were classified into two primary aspects: the review process and the evidence included in the review.The comprehensiveness of this study may be influenced by the chosen search strategies and time constraints.The potential introduction of bias could occur if other articles were not included in the study.Regarding the evidence incorporated in this study, the reliability of the included articles may exhibit variation concerning study design, sampling methods, and the approach to bacterial identification and selection criteria for performing AST.These variations could influence the overall robustness of the review and present challenges in the analysis of the data.Therefore, it is important to expand the pool of studies and continuously monitor the trends in V. parahaemolyticus and its resistant isolates.Further investigation in this regard is necessary to enhance our understanding and inform ongoing efforts to address potential public health implications.
In future research, it is essential to monitor and survey the trends in resistant V. parahaemolyticus isolates.This is crucial for evaluating the effectiveness of national and international frameworks or policies addressing AMR within the aquaculture sector.Such trend data derived from epidemiological studies will also contribute to a better understanding of the situation.

Study Selection
This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines (Table S1) [67].Different peerreviewed journal articles published between 1 January 2020 and 31 December 2022 were selected based on inclusion and exclusion criteria.The prevalence of V. parahaemolyticus was calculated as the proportion of positive samples to the total number of samples, and the occurrence of virulence factors and AMR was expressed as the proportion of positive isolates to the total number of bacterial isolates.The primary focus of this study was on determining the prevalence of V. parahaemolyticus in shrimp from farms through retail outlets.Moreover, because of the significant losses in shrimp production caused by V. parahaemolyticus, only analyses involving this organism were taken into account, and data on any other aquatic animals were disregarded.This investigation specifically characterized the phenotypic and genotypic profiles of AMR, virulence factors, and resistance determinants in V. parahaemolyticus isolates obtained from these shrimp samples.To ensure the inclusion of publications, we implemented control measures involving cross-referencing between Web of Science (https://mjl.clarivate.com/search-results,accessed on 13 April 2024) and other databases.Furthermore, the words used in identification algorithms were clear and unique, increasing the likelihood of retrieving overlapping publications.

Eligibility Criteria
The articles that were considered eligible were peer-reviewed research papers published in reputable scientific journals listed in Google Scholar (https://scholar.google.com/,accessed on 13 April 2024), PubMed (https://pubmed.ncbi.nlm.nih.gov/,accessed on 13 April 2024), ScienceDirect (https://www.sciencedirect.com/,accessed on 13 April 2024), and Scopus (https://www.scopus.com/,accessed on 13 April 2024).All articles focused on pathogenicity and AMR characterization in V. parahaemolyticus as significant aspects of the study.The studies concerned strains of V. parahaemolyticus isolated from shrimp with exposure to AMR patterns, particularly phenotypic (e.g., disk diffusion and dilution methods) and genotypic (e.g., detection of resistance genes and determinants) assessments.Finally, the articles that were considered eligible were accessible for detailed analysis and reporting standards, including providing proper bacterial isolates; clear descriptions of the methods for bacterial isolation and confirmation, the method of antimicrobial susceptibility testing (AST), and the detection of virulence factors; and the results, discussions, and conclusions could be followed.These criteria were consistently applied during the article selection process.

Inclusion Criteria
This systematic review specifically targeted shrimp as the animal species of interest, with a primary focus on examining the presence of V. parahaemolyticus in shrimp.Included studies were those that examined the prevalence of virulence genes, AMR, or/and the associated resistance determinants in V. parahaemolyticus isolates derived from shrimp.Various methods were employed for the detection and confirmation of V. parahaemolyticus, including bacterial culture, biochemical tests, analytical profile index (API20E), and molecular techniques such as polymerase chain reaction (PCR) and Whole-Genome Sequencing.The AST method for detecting AMR phenotypes was also considered.Additionally, molecular techniques for identifying virulence genes, resistance genes, and their determinants were incorporated.The studies were limited to studies published in English.

Exclusion Criteria
Other aquatic animals, such as bivalves, fish, mollusks, and aquatic plants, were excluded from this study.Studies that did not report sample size, number of isolates, the method for bacterial isolation and confirmation, and AST were excluded from this study.Antimicrobials, including antifungals, antiseptics, and disinfectants, were also excluded from this study.Questionnaires, survey studies, and epidemiological studies without aquatic animals were also excluded.Studies that were not primary or original research but were based on review articles, abstracts, proceedings, short communications, correspondence, editorials, opinion articles, research theses, and book chapters were excluded.In addition, studies that were not published in the English language and were non-peer reviewed were not included.

Search Strategy
The search strategy was to explore the prevalence of bacteria, virulence factors, AMR, and resistant determinants among V. parahaemolyticus isolated from shrimp samples.Either the presence or absence of virulence factors, AMR, and resistant determinants of V. parahaemolyticus isolates was considered possible in the eligible criteria.The online databases searched were Google Scholar, PubMed, ScienceDirect, and Scopus.Searches on com-mercial websites, including Google and Yahoo, were performed using Boolean operators such as AND, OR, and NOT in conjunction to combine keywords in database searches (Table S2) [73].
The identification algorithm for the relevant literature was listed using the following keywords: V. parahaemolyticus, antimicrobial resistance, AMR, antibiotic resistance, AST, resistance profile, virulence factors (TDH, TRH, and other pathogenicity), and resistance determinants (integrons, integrative and conjugative elements (ICEs), and plasmid), specifically in shrimp, from farm to retail.Several keyword combinations were also used for each topic area to ensure as many articles as possible were captured within the specified timeframe and subject area.
This systematic review followed the PRISMA guidelines and a completed PRISMA 2020 checklist is provided (Table S1).Complete publications were reviewed by three veterinarians and one microbiologist.Any discrepancies or uncertainties were resolved through discussion and consensus among the reviewers.Additionally, we ensured the verification and credibility of the selected articles by cross-referencing them with reputable scientific journals listed in the databases.Subsequently, all articles that met the inclusion criteria were consolidated, and duplication detection was performed.Publications that met the inclusion criteria were classified by bacterial isolation and confirmation of V. parahaemolyticus from shrimp, number of total and positive samples and isolates, country, method of AST, and molecular detection of resistance genes and their determinants.All data with full details of the search strategy and results were independently extracted from materials and methods, results, discussions, figures, and tables by two individuals (V.T. and S.A.) and double-checked by the third investigator (S.J.), and the qualified articles were compiled in Microsoft Excel for further data analysis.

Data Collection and Assessment of Data Quality
Inclusion and exclusion criteria were used to detect the possible publications relevant to this study.The primary outcome measures were based on the prevalence of V. parahaemolyticus and contained virulence factors, phenotypic and genotypic AMR, and other genetic determinants.All eligible published articles underwent extraction, encompassing essential details such as the article title, author names, publication year, country, duration of data collection, shrimp species, shrimp status, total samples/bacterial isolates, number of positive samples/isolates, virulence genes, resistance phenotypes and genotypes, as well as their determinants.Additionally, other variables included were the detection and confirmation of V. parahaemolyticus, method for AST, bacterial control strains, and molecular techniques used for detection of resistance genes and their genetic determinants.

Statistical Analyses
The extracted data were stored in a Microsoft Excel spreadsheet and imported into STATA program version 18.0 (StataCorp, College Station, TX, USA).An observational study for one-sample binary outcome summaries of the prevalence of V. parahaemolyticus, virulence factors, AMR, and their determinants isolated from shrimp was examined.The prevalence was calculated based on the proportion of positive samples/isolates to number of total samples/isolates.The data are expressed as a pooled estimate of the prevalence of V. parahaemolyticus, virulence factors, resistance to each antimicrobial class, and determinants.Random-effects meta-analyses were conducted, and the summary weighted average proportion (effect size) was calculated based on the individual effect sizes and 95% confidence intervals (C.I.).Heterogeneity between studies of prevalence estimates among the studies was investigated using Q-statistics with I 2 Index [74,75].A possible source of heterogeneity among observed studies was assessed through subgroup analysis based on the continent of the source of samples.
The visual assessment of publication or dissemination bias was performed using a funnel plot for asymmetry with a pooled estimate.Egger's tests were employed for detecting publication bias.Studies were included in the meta-analyses if they met the criteria, including at least 20 isolates for all analyzed studies and at least three studies for each meta-analysis.All analyses were two-tailed tests, with statistical significance set at a p-value <0.05.The meta-analyses utilized a random-effects model in STATA software.

Conclusions
In conclusion, this comprehensive systematic review and meta-analysis has presented the prevalence of V. parahaemolyticus in shrimp, along with its associated virulence factors and AMR profiles.The study revealed a significant prevalence of V. parahaemolyticus in shrimp samples globally, with Asia exhibiting particularly high rates.Notably, virulence genes, such as tdh and trh, were frequently identified, indicating the potential pathogenicity of V. parahaemolyticus.Our study highlighted the levels of phenotypic AMR, revealing significant resistance against glycopeptides, nitrofurans, and beta-lactams.Furthermore, the investigation identified gaps in AMR surveillance and control measures, emphasizing the need for harmonized protocols and enhanced monitoring efforts.This finding showed the importance of ongoing research and collaborative interventions to address the growing threat of AMR in V. parahaemolyticus, to ensure public health safety, and to promote sustainable aquaculture practices.

Figure 1 .
Figure 1.A PRISMA flow chart of the study selection process and the literature search.Different databases were assessed to search for eligible studies of V. parahaemolyticus using predefined search strategies.

Figure 1 .
Figure 1.A PRISMA flow chart of the study selection process and the literature search.Different databases were assessed to search for eligible studies of V. parahaemolyticus using predefined search strategies.
(A) Estimated prevalence of V. parahaemolyticus.

Figure 2 .
Figure 2.The meta-analysis of prevalence of V. parahaemolyticus and virulence factors in shrimp using random-effects model with a 95% C.I.A forest plot of (A) prevalence of V. parahaemolyticus[19,25- 29,31,33-44,46-49,51,53]  and (B) prevalence of virulence factors[4,24,28,33-36,38-40,42,44,45,47,49,51- 53]  representing the results in meta-analysis with size squares proportional to the weight assigned to the study; a funnel plot of the prevalence estimates of V. parahaemolyticus (C) and virulence factors (D) to assess potential bias.A red vertical line represents the overall prevalence from meta-analysis, and the diagonal lines provide 95% C.I.The prevalence with ranges of individual virulence factors is represented by the light blue square with line, while the average prevalence is presented by the red diamond.The overall heterogeneity of virulence factors is indicated by the green diamond.

Table 1 .
Descriptive summary of prevalence of V. parahaemolyticus and virulence genes among relevant studies (n = 32).

Table 2 .
Summary of pooled prevalence of AMR V. parahaemolyticus stratified by antimicrobial classes.
NA: data not available; *: the observed data based on a single study.