Introduction
Aquaculture environments are considered to be reservoirs of various biological pollutants, including infectious agents and antimicrobial resistance genes (ARGs) [
1]. ARGs, which are contaminants of emerging concern [
2], encode antimicrobial resistance and play an essential role in the process of resistance prevalence and proliferation [
3]. In the environment, ARGs can be transferred from one microorganism to another, including from non-pathogenic to pathogenic species, through horizontal gene transfer. Pathogenic microorganisms that carry ARGs within aquaculture settings represent one of the most significant global health hazards, with millions of individuals succumbing to foodborne and waterborne diseases annually [
4].
Vibrio parahaemolyticus is a halophilic marine bacterium that belongs to the family Vibrionaceae and is widely distributed in brackish water and marine environments [
5]. This bacterium is a facultative human pathogen and is responsible for approximately 25% of all foodborne diseases closely associated with the consumption of raw seafood [
6]. The ingestion of contaminated raw fish and shellfish, or seafood that has not been sufficiently heat-treated, can lead to symptoms such as diarrhea, abdominal pain, vomiting, chills, and low-grade fever in humans [
7]. The significance of infections caused by this pathogen is increasing in public health due to a steady rise in its incidence worldwide over recent decades.
V. parahaemolyticus is considered an emerging pathogen with a global distribution. Its infectious capability is mediated by various virulence factors, including hemolysin, urease, 2 type III secretion systems, and 2 type VI secretion systems, as well as the formation of persister cells [
8]. To survive during infections and in seafood,
V. parahaemolyticus cells may also form biofilms, which shield the cells from host defenses and external factors, including antibiotics, thereby contributing to antimicrobial resistance.
Antibiotics are widely used in aquaculture for both disease prevention and the treatment of infections potentially caused by
V. parahaemolyticus [
4]. However, the long-term use of antibiotics, particularly at incorrect dosages, can lead to the development of antimicrobial resistance in marine bacteria, including
V. parahaemolyticus [
4]. Moreover, antibiotics present in aquatic products and seafood can be ingested by animals and humans, potentially leading to the development of resistance in microorganisms that cause diseases in both animals and humans. This resistance can diminish the effectiveness of these antibiotics in treating infections.
To date, hundreds of
V. parahaemolyticus strains have been isolated from seafood and shrimp farming environments in Vietnam [
9–
11]. However, no previous study has investigated the molecular resistance mechanisms and biofilm formation of
V. parahaemolyticus in marine fish environments in Vietnam. Cat Ba Island is a significant marine fish production area and a well-known tourist destination in the northeast of Vietnam. To mitigate outbreaks caused by the consumption of raw or undercooked fish contaminated with
V. parahaemolyticus, and to prevent potential risks to human health, it is necessary to monitor the prevalence of
V. parahaemolyticus in fish mariculture rearing water. Additionally, identifying antimicrobial resistance and ARGs, virulence factor genes, and biofilm formation is of paramount importance. The aim of this study was to improve our understanding of the current state of antimicrobial resistance in the marine environment and the risks posed by the virulence factor genes and ARGs of
V. parahaemolyticus, which can cause foodborne diseases in aquatic animals and humans.
Materials and Methods
Sampling
The seawater samples were collected following the method described by Zhao et al. [
12], with slight modifications. In brief, water from the fish mariculture environment was collected from a depth of 10 to 20 cm below the seawater surface. A total of 150 samples were collected from 10 grouper mariculture farms in Lan Ha Bay (Cat Ba Island), Hai Phong (
Figure S1 and
Table S1), during the survey period between February 2021 and August 2021.
Bacteria Isolation
V. parahaemolyticus was isolated as described by Mok et al. [
13]. Briefly, 5 mL of seawater was added to a flask containing 95 mL of peptone water with 1.5% NaCl and then incubated at 28 °C for 24 hours. Subsequently, 100 μL aliquots were spread on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Merck) and incubated for 48 hours at 28 °C. Colonies that grew on TCBS were characterized by their color, shape, and size. The blue colonies were subsequently inoculated onto CHROMagar medium (Titan). Purple presumptive colonies were further confirmed through biochemical and polymerase chain reaction (PCR) assays.
Genomic DNA Extraction and Identification of V. parahaemolyticus
Colonies of each suspected isolate were cultured in TSB medium (Merck) supplemented with 1.5% NaCl at 28 °C. After 24 hours of incubation, 1.5 mL of the bacterial culture was harvested by centrifugation at 13,000 rpm for 5 minutes. The total genomic DNA from each suspected isolate was then extracted using the Favorgen Tissue Genomic DNA Extraction Mini Kit (Favorgen), following the manufacturer’s instructions.
V. parahaemolyticus was identified by a PCR assay using
toxR-specific primers [
14]. The genomic DNA of
V. parahaemolyticus from the collection of the Department of Fisheries, University of Agriculture and Forestry, Hue University was used as a positive control, while nuclease-free water was used as a negative control.
The presence of virulence genes was identified using the primers listed in
Table S2. PCR reactions were performed in PTC200 thermal cyclers (Marshall Scientific). Each 25-µL reaction mixture contained 12.5 µL of MyTaq PCR master mix (Bioline), 1 µL of DNA template (50 ng/µL), 1 µL of each primer (10 pmol), and 9.5 µL of distilled water. The PCR program was as follows: initial denaturation at 95 °C for 5 minutes, followed by 35 cycles of 95 °C for 30 seconds, an annealing temperature of 50 to 60 °C for 30 seconds (varying based on the primer used), and extension at 72 °C for 1 minute, with a final elongation step at 72 °C for 5 minutes. PCR products were visualized by agarose gel electrophoresis using Safe DNA stain (AAT Bioquest).
Antimicrobial Susceptibility Profiling and PCR Detection of ARGs
The antimicrobial susceptibility of each
V. parahaemolyticus isolate was assessed using the disc diffusion method, following the Clinical and Laboratory Standards Institute (CLSI) M45 guidelines [
15]. Initially, colonies from each isolate were suspended in 1% NaCl and adjusted to match the 0.5 McFarland standard. This suspension was then spread onto Mueller-Hinton agar plates (HiMedia). Subsequently, discs impregnated with 9 antimicrobial agents (Mast Diagnostics)—ampicillin (10 μg), ciprofloxacin (5 μg), kanamycin (30 μg), gentamicin (10 μg), tetracycline (30 μg), chloramphenicol (30 μg), erythromycin (15 μg), ceftazidime (30 μg), and ceftiofur (30 μg)—were placed equidistantly on the surface of the agar. The plates were then incubated at 28 °C for 18 to 24 hours.
Escherichia coli ATCC 25922 served as the quality control strain. The resistance profile of
V. parahaemolyticus was determined by measuring the zones of inhibition, in accordance with the CLSI M45 guidelines for
Vibrio spp. and the CLSI M100 guidelines [
15,
16]. The multiple antibiotic resistance (MAR) index was calculated using the formula a/b, where “a” is the number of antibiotics to which resistance was detected in a single isolate, and “b” represents the total number of antibiotics tested [
17].
Concurrent with antimicrobial resistance testing, genotype screening was performed using PCR assays to detect the presence of ARGs for ampicillin, gentamicin, ciprofloxacin, erythromycin, and tetracycline. The primers for the targeted ARGs and the PCR conditions are detailed in
Table S1.
Biofilm Formation Assay
The biofilm production capability of
V. parahaemolyticus isolates was assessed using a previously described method [
18], with some modifications. In brief, overnight cultures of
V. parahaemolyticus grown in TSB with 1.5% (w/v) NaCl at 30 °C were diluted to an optical density at 600 nm (OD600) of 0.1. Then, 150 μL of the diluted cell suspension was aliquoted in triplicate into 96-well plates (SPL Life Science). After 24 hours of incubation, non-adherent cells and culture medium were removed, and the attached cells were washed 3 times with a 2 mM CaCl
2/MgCl
2 buffer. The cells were then stained with 0.01% crystal violet (Merck) for 15 minutes. Subsequently, the plates were washed 3 more times with the 2 mM CaCl
2/MgCl
2 buffer. To facilitate the measurement of absorbance, 70% ethanol was added to each well before the contents were transferred to a new 96-well plate. Absorbance was measured at a wavelength of 540 nm. The criteria for interpreting the biofilm formation capacity of
V. parahaemolyticus were as follows, based on a previous report [
19]. Briefly, OD>4×ODc means strong biofilm formation; 2×ODc<OD≤4×Odc, moderate biofilm formation; ODc<OD≤2×Odc, weak biofilm formation; and negative biofilm formation when OD<ODc. ODc refers to the control measurement, which was performed in a microtiter plate without cells.
Statistical Analysis
The statistical analysis was carried out using IBM SPSS ver. 20.0 (IBM Corp.). The significance of differences in biofilm formation, the prevalence of V. parahaemolyticus, and the distribution of ARGs was examined using the chi-square test. A p-value ≤0.05 was considered as significant.
Results
Prevalence of V. parahaemolyticus in Fish Farming Water
The distribution of
V. parahaemolyticus in farming water is presented in
Table 1. In total, 51 suspected
V. parahaemolyticus isolates were obtained from a total of 150 samples, using TCBS agar and CHROMagar. These isolates were further verified by a PCR assay (
Figure 1A) with
toxR primers, confirming that 37 of them were
V. parahaemolyticus. Of these, 11 isolates were collected from 76 samples obtained in the winter, while 26 were identified from 74 samples obtained in the summer. Consequently, the isolation rate of
V. parahaemolyticus was significantly higher in the summer (35.1%) than in the winter (14.5%) (
p<0.05, chi-square test).
Detection of Virulence-Related Genes
The presence of the
tdh and
trh genes in all 37
V. parahaemolyticus isolates was examined. The results, as shown in
Figure 1 and
Table 2, indicated that 3 (8.1%) isolates were positive for the
tdh gene and 8 (21.6%) isolates were positive for the
trh gene. However, none of the isolates carried both the
tdh and
trh genes. Additionally, biofilm-associated genes were highly prevalent, with 83.8% of isolates harboring the
VP0952 gene, and the
VP0950 and
VP0962 genes were present in 81.08% of isolates.
Antimicrobial Resistance Pattern of the V. parahaemolyticus Isolates
The antibiotic resistance profile of 37 isolates against 9 antibiotics is presented in
Table 3. None of the 37 isolates exhibited resistance to ceftazidime, cefotaxime, or chloramphenicol. A lower resistance rate was observed for ciprofloxacin (18.9%, 7/37), kanamycin (13.5%, 5/37), and gentamicin (10.8%, 4/37). In contrast, a majority of the isolates were resistant to ampicillin (86.5%, 32/37), erythromycin (75.7%, 28/37), and tetracycline (73.0%, 27/37).
The multidrug resistance profile of 37
V. parahaemolyticus strains was also examined (
Table 2). All 37
V. parahaemolyticus isolates were found to be resistant to at least 1 antibiotic, among which 2 (5.4%), 10 (27.0%), 16 (43.2%), 7 (18.9%), and 2 (5.4%) isolates were resistant to 1, 2, 3, 4, and 5 antibiotics, respectively. Overall, the MAR index ranged from 0.11 to 0.55 (
Table 2), with 18 different resistance patterns. The highest frequencies of multidrug-resistant phenotypes were observed for ampicillin, tetracycline, and erythromycin (AMP TCN ERY) with 24.32%. Additionally, 10.81% and 8.1% of isolates exhibited the (AMP TCN) and (AMP GEN TCN ERY) as well as (AMP CIP TCN ERY) phenotypes, respectively.
ARGs in V. parahaemolyticus Isolates
The prevalence of 8 ARGs in
V. parahaemolyticus isolates was examined. As shown in
Table 4,
aac(3)-IV,
qnrA, and
tetA were detected in 16.2%, 27.0%, and 62.2% of isolates, whereas blaTEM, blaOXA, blaSHV, ermA, and ermB were undetected in all isolates tested. Six isolates were
aac(3)-IV-positive, 4 were susceptible to gentamicin, and 2 had intermediate resistance to gentamicin. All 10
qnrA-positive isolates were resistant to ciprofloxacin. Of 27 isolates exhibiting resistance to tetracycline, 23 isolates harbored the
tetA gene.
Biofilm Formation of V. parahaemolyticus Isolates
The ability of
V. parahaemolyticus isolates to form biofilms was investigated, as depicted in
Figure 2. Out of the isolates tested, 31 (83.8%) were capable of producing biofilms, whereas 6 (16.2%) could not form biofilms. Within the group of 25 multidrug-resistant isolates, 4 were identified as strong biofilm producers, adhering robustly to polystyrene. Additionally, 11 isolates were classified as moderate biofilm producers, 9 as weak biofilm producers, and only 1 isolate (VA37) was unable to produce biofilm. Conversely, among the 12 non-multidrug-resistant isolates, a single isolate demonstrated moderate biofilm formation, 6 were weak biofilm producers, and 5 were incapable of forming biofilms.
Discussion
V. parahaemolyticus is an opportunistic pathogen and a major causative agent of food-borne illnesses worldwide [
7]. This bacterium causes gastroenteritis following the consumption of contaminated raw or inadequately cooked seafood. Additionally, exposure to water containing
V. parahaemolyticus can lead to wound infections and septicemia, which can be life-threatening for individuals with certain medical conditions, such as diabetes or immune deficiency [
7]. The wide distribution of
V. parahaemolyticus in tropical seawater and mariculture environments in different countries has been reported [
20–
23] and its presence depends on the water temperature [
13]. In this study,
V. parahaemolyticus was present in 24.7% (37/150) of samples, and it was more frequently identified in summer (35.1%) than in winter (14.5%). Previous studies have also noted a higher prevalence of
V. parahaemolyticus in summer and autumn than in winter and spring [
13,
24]. This finding supports the hypothesis that the presence of
V. parahaemolyticus is associated with a seasonal pattern, with outbreaks in the warmer season and lower prevalence in the cold season [
25]. Since
V. parahaemolyticus is cold-susceptible, its growth may be inhibited at lower temperature in winter [
7], while higher temperatures in summer promote
V. parahaemolyticus growth with a shorter generation time and a faster growth rate, leading to a higher density of
V. parahaemolyticus in samples [
26].
It is well known that not all
V. parahaemolyticus strains cause diseases in aquatic animals and humans, but strains harboring the
tdh and
trh genes produce hemolysin factors that induce inflammatory gastroenteritis, and these genes are considered as virulent indicators of pathogenic strains [
5]. Therefore, detecting these genes in isolates is crucial for mitigating potential risks to human health. In this study, we identified a significantly higher prevalence of the
trh gene (21.6%) than the
tdh gene (8.1%) among 37
V. parahaemolyticus isolates. This pattern aligns with previous research, which found the
trh and
tdh genes in 15.9% and 6.1% of seawater isolates from Korea [
25] and in 19.8% and 9.9% of isolates from seafood in China [
27], respectively. In contrast, the
tdh gene was detected at a higher level (48%) than the
trh gene (8.3%) in an estuarine system in South Carolina, the United States in a study by Gutierrez et al. [
28] in 2013. Similarly,
tdh-positive
V. parahaemolyticus was also more prevalent than
trh-positive bacteria in oyster environments in Taiwan [
29] and in coastal water in Saudi Arabia [
30]. The differences in the distribution of
tdh+/
trh+ V. parahaemolyticus strains are possibly due to the sampling techniques, the geographical origin, seasonal effects and the method of their detection [
12]. For example, Parveen et al. [
31] reported that real-time PCR could help increase the detection of
tdh- and
trh-positive
V. parahaemolyticus to 13% and 40%, respectively, for water samples compared to the conventional techniques.
The antimicrobial resistance of
Vibrio species has emerged as one of the most significant threats to fish farming, food safety, and public health [
32]. Therefore, monitoring the antimicrobial susceptibility of
V. parahaemolyticus is very important for evaluating its potential effects on environmental and human health. Our results indicated that more than 72% of
V. parahaemolyticus isolates from mariculture were resistant to ampicillin, erythromycin, and tetracycline. These findings broadly support the work of other studies, in which 86% to 100% of
V. parahaemolyticus strains isolated from marine environments [
33,
34] were resistant to ampicillin and tetracycline, and 42% to 48.3% of isolates from shrimp mariculture were resistant to erythromycin [
12,
35]. This high frequency of resistance confirms that these antimicrobials are widely used and becoming less effective against
V. parahaemolyticus, likely due to the extensive use of antibiotics in the areas studied. In Vietnam, 64% of fish farms reported using at least 1 antibiotic for disease treatment and prevention, with 10% to 21% of farms utilizing tetracycline [
36]. Additionally, significant levels of antibiotic residues, including ampicillin, erythromycin, and tetracycline, have been detected in aquaculture water in Vietnam, which may contribute to the increasing resistance rates to these antimicrobials [
37]. Conversely, other studies have shown that ampicillin, tetracycline, and erythromycin were effective against
V. parahaemolyticus strains, with over 90% of isolates being inhibited [
10,
38–
40]. The variability in resistance of
Vibrio to antibiotics may be attributed to geographical differences or variations in testing methodologies [
5].
Significantly, all tested
V. parahaemolyticus isolates were found to be susceptible to third-generation cephalosporins (ceftazidime and cefotaxime) and chloramphenicol. However, 2 isolates showed intermediate resistance to cefotaxime, and 3 isolates exhibited intermediate resistance to chloramphenicol. These findings are consistent with those of previous studies [
12,
13,
39,
41], which reported that over 90% of
V. parahaemolyticus strains were susceptible to these antibiotics. Therefore, these drugs are considered the most effective antimicrobials for treating
V. parahaemolyticus infections in the areas studied.
Ciprofloxacin, a fluoroquinolone antibiotic, has been broadly employed as an alternative treatment for tetracycline-resistant bacteria [
33], while aminoglycosides (kanamycin and gentamicin) antibiotics are commonly used in aquaculture production and show substantial effectiveness against a broad spectrum of bacteria [
42]. However, the use of these antibiotics for the prevention and treatment of
V. parahaemolyticus may not be effective, as the rate of antibiotic resistance among 37
V. parahaemolyticus isolates in the current study ranged from 13.5% to 27.0%. Comparable resistance rates have been observed in previous studies, with 9.5% to 39.8% of
V. parahaemolyticus strains showing resistance to kanamycin, ciprofloxacin, and gentamicin [
24,
43]. The increasing resistance of
V. parahaemolyticus to fluoroquinolones and aminoglycosides could be attributed to their extensive use in human medicine and aquaculture production, which may lead to significant public health concerns [
44].
MAR indexing has been recognized as an efficient and cost-effective method for tracking the sources of bacterial contamination. Letchumanan et al. [
5] suggest that MAR index values greater than 0.2 indicate high-risk sources of antibiotic contamination, posing a potential threat to human health. Regrettably, our study revealed that 67.57% of
V. parahaemolyticus isolates had a MAR index exceeding 0.2, indicating a multidrug resistance phenotype. Our findings suggest that these
V. parahaemolyticus strains were recovered from sources where antibiotics are frequently used, which could be hazardous. This is in contrast to the findings of Mok et al. [
13], who reported that only 1.9% of
V. parahaemolyticus strains isolated from the Korean coast were resistant to 3 antibiotics. Nonetheless, our results are in general agreement with previous observations [
6,
24,
45], which showed that more than half of the environmental
V. parahaemolyticus isolates exhibited multidrug resistance. Furthermore, the MAR index values in this study ranged from 0.11 to 0.55, which are significantly lower than those reported by Ahmed et al. [
46], who found all
V. parahaemolyticus isolates were resistant to at least 7 antimicrobial agents. The variation in the MAR index values may be attributed to differences in sample sources, geographic distribution, the number and types of antibiotics tested, and the methodologies employed [
47].
The presence of ARGs is the basis for bacterial resistance, and a high detection rate of ARGs indicates an elevated risk of ARG transmission and a significant potential for bacteria to develop resistance [
48]. In the present study, the
qnrA gene was detected in all 10 ciprofloxacin-resistant isolates, demonstrating a strong correlation between phenotypic resistance and genotype (the presence of
qnrA). This result aligns with previous research [
44,
45], in which all fluoroquinolone-resistant isolates carried the
qnrA and
qnrS genes. Conversely, Jeamsripong et al. [
49] showed that 77.8% of 594
V. parahaemolyticus isolates harbored the
qnr gene, but all isolates were susceptible to ciprofloxacin. This difference is possibly due to the evolution, mutation, and silencing of resistance genes [
50] or unknown mechanisms that remain to be elucidated.
Similarly, a strong relationship between the tetracycline resistance phenotypes of
V. parahaemolyticus isolates and the presence of ARG was also determined, as
tetA was detected in 23 of 27 isolates that exhibited resistance to tetracycline. The overall level was found to be much higher than reported in other studies [
45,
51] in which the detection rate of
tetA was 28% to 30% of tetracycline-resistant isolates. Meanwhile,
tetA was undetected in all
V. parahaemolyticus strains that exhibited resistance to tetracycline [
5,
52]. A possible explanation for this difference might be that
V. parahaemolyticus strains used other ARGs (
tetM and
tetS) against tetracycline [
53].
Interestingly, a high percentage of isolates showed resistance to ampicillin and/or erythromycin; however, none of the 32 or 28 isolates resistant to ampicillin/erythromycin carried the genes for macrolide or β-lactam antibiotic resistance. This discrepancy between the resistance phenotypes and genotypes for ampicillin and/or erythromycin has been noted in previous studies [
5,
12,
45]. This may occur because resistance phenotypes can be governed by various ARGs and mechanisms [
12]. Another potential explanation is that the resistance phenotypes are regulated by the efflux systems of the bacterial cells [
54].
The
aac(3)-IV gene was detected in susceptible and intermediate-resistance isolates, but not in gentamicin-resistant strains. Similarly, Beshiru and Igbinosa [
45] only found the
aac(3)-IV gene in 2 intermediate gentamicin-resistant isolates. However, the findings of the current study do not support the previous research [
12], in which positive results were found for the
aac(3)-IV gene in 12 of 61 isolates that exhibited resistance to gentamicin. Nevertheless, susceptible isolates harbored the
aac(3)-IV gene, posing a potential risk for the preservation and transmission of ARGs [
12].
Biofilm formation is a significant virulence factor for pathogenic bacteria during infection. Several
Vibrio species, including
V. parahaemolyticus, can produce biofilms, enabling the bacteria to establish infections and enhance their resistance to hostile environments, such as antibiotics and the host immune response [
55]. This study found that more than 83% of
V. parahaemolyticus isolates were biofilm producers (
Figure 2). The rate observed in this investigation was 16% lower than that of previous studies, in which 100% of
V. parahaemolyticus strains isolated from aquatic animals and marine environments [
47,
56,
57] were able to produce biofilms. The difference in the capacity to form biofilm may be due to the physical conditions (temperature, pH, etc.), and the surfaces where cells attach [
58]. Another important finding is that isolates with multidrug resistance (64.9%, 24/37) were more likely to produce biofilms than non-multidrug-resistant isolates (18.9%, 7/37). This supports the possibility that biofilm formation is associated with increased antimicrobial resistance, as suggested in the literature [
59–
61]. Moreover, the
trh gene was detected in 6 biofilm-producing strains that were resistant to 4 or 5 antibiotics. This outcome is consistent with Letchumanan’s findings [
62], which indicated that 9 out of 13
trh-positive strains exhibited resistance to 4 or more antimicrobials. Interestingly, a correlation between biofilm phenotype and associated genes was also identified in this study, as 3 genes (
VP0950,
VP0952, and
VP0962) that mediate biofilm formation were present in
V. parahaemolyticus isolates that were identified as biofilm producers. Similar results were also reported in other studies [
47,
56].
The paper presents intriguing findings on the prevalence of ARGs and the virulence genes of
V. parahaemolyticus strains in aquaculture environments and their implications for public health. However, this study did not assess the prevalence of
V. parahaemolyticus in fish, which could pose a direct threat to human health. Nevertheless, it is widely recognized that
V. parahaemolyticus is an adept swimmer capable of attaching to aquatic animals [
63]. Thus, a contaminated water body may potentially infect all its fish inhabitants [
64]. Alarmingly, some
V. parahaemolyticus isolates identified in the current study carry ARGs and virulence genes, which could pose a significant risk to human health. Aquatic ecosystems, particularly, are acknowledged as hotspots for the environmental spread of pathogenic microorganisms, ARGs, and antimicrobials [
1]. Furthermore, the intricate environmental conditions of tropical aquatic habitats may prompt genetic differentiation and adaptive variation in organisms. These changes can enhance their pathogenicity or lead to the emergence of new strains with distinct virulence factors in aquatic hosts [
32]. Therefore, to better understand the genotypic variation of
V. parahaemolyticus isolates, to determine the relatedness between clinical and environmental isolates, and to clarify the relationships of
V. parahaemolyticus virulence genes across different marine environmental sources, further research is required.
