Abstract
A total of 449 samples including 385 seafood and 64 water samples in the Mekong Delta of Vietnam collected in 2015 and 2016 were examined. Of 385 seafood samples, 332 (86.2%) samples were contaminated with Vibrio parahaemolyticus and 25 (6.5%) samples were pathogenic V. parahaemolyticus carrying tdh and/or trh genes. The tdh gene positive V. parahaemolyticus strains were detected in 22 (5.7%) samples and trh gene positive V. parahaemolyticus strains were found in 5 (1.3%) samples. Of 25 pathogenic V. parahaemolyticus strains, two strains harbored both tdh and trh genes and the other 23 strains carried either tdh or trh gene. Of 64 water samples at aquaculture farms, 50 (78.1%) samples were contaminated with V. parahaemolyticus. No tdh gene positive V. parahaemolyticus strains were detected; meanwhile, trh gene positive V. parahaemolyticus strain was detected in 1 (1.6%) sample. Twenty-six pathogenic V. parahaemolyticus strains isolated were classified into 6 types of O antigen, in which the serotype O3:K6 was detected in 4 strains. All pathogenic strains were group-specific PCR negative except for 4 O3:K6 strains. The result of antimicrobial susceptibility test indicated that pathogenic strains showed high resistance rates to streptomycin (84.6%), ampicillin (57.7%) and sulfisoxazole (57.7%). These findings can be used for understanding microbiological risk of seafood in the Mekong Delta, Vietnam.
Keywords: antimicrobial susceptibility, serotype, tdh, trh, Vibrio parahaemolyticus
Vibrio parahaemolyticus, a gram-negative halophilic bacterium, is a vital agent of food poisoning in human [6]. The infection of this pathogen in human has been reported worldwide [16] and most reported cases link to seafood consumption [7, 18]. Thermostable direct hemolysin (TDH) encoded tdh gene and TDH-related hemolysin encoded trh gene are considered to be the most important virulent factors of human pathogenic V. parahaemolyticus [18]. In Vietnam, the infection of V. parahaemolyticus in human has been reported since 1983 [12]. The outbreak of V. parahaemolyticus with the predominance of pandemic O3:K6 strain from 1997 to 1999 was reported in Nha Trang in the middle region of Vietnam [4, 24]. Tai et al. [22] reported that V. parahaemolyticus was isolated at 8.3% from acute diarrheal patients in the South of Vietnam in 2010 and tdh or trh gene carrying strains dominated 41.7% of these V. parahaemolyticus infections. A few information on human pathogenic V. parahaemolyticus in environment has been published [22, 23] although human V. parahaemolyticus infection has been reported in Vietnam. In this century, a large volume of seafood and seafood products are produced in the Mekong Delta, the South of Vietnam. However, the information on prevalence of V. parahaemolyticus in this area has been not fully understood. In an effort to understand the risk of V. parahaemolyticus infection in Vietnam, particularly in the Mekong Delta, our objectives were to investigate prevalence of V. parahaemolyticus in seafood and water environment in the Mekong Delta and clarify some characteristics of this bacterium such as harboring virulent genes and serotype, harboring pandemic trait and antimicrobial resistance of virulent strains.
MATERIALS AND METHODS
Sample collection
A total of 449 samples including 385 seafood samples and 64 water samples were collected. Of 385 seafood samples, 330 retail samples including 298 shellfish samples [white hard clam (Meretrix lyrata), blood cockle (Anadara granosa), mud clam (Geloina coaxans), hakf-crenate ark (Anadara subcrenata) and antique ark (Anadara antiquata)] and 32 shrimp samples [banana shrimp (Penaeus merguiensis) and greasyback shrimp (Metapenaeus ensis)] were purchased from wet markets in Can Tho city and Tra Vinh province in the Mekong Delta in 2015 and 2016 and 55 farming samples including 16 clam samples [white hard clam (Meretrix lyrata)] and 39 shrimp samples [white leg shrimp (Litopenaeus vannamei) and black tiger shrimp (Penaeus monodon)] were collected at 2 clam farms and 39 shrimp ponds, respectively, in Tra Vinh province in 2016. Of 64 water samples, 22 and 42 samples were collected at 2 clam farms and 42 shrimp ponds, respectively, in Tra Vinh province in 2016. All samples were kept separately in sterile plastic bags placed in polystyrene foam boxes with ice and analyzed immediately as arrival at laboratory.
Enrichment culture and V. parahaemolyticus isolation
For seafood samples, a 25 g portion of seafood was mixed with 225 ml of Alkaline Peptone Water (APW, Nissui Co., Ltd., Tokyo, Japan) in sterile stomacher bag to form homogenate solution. For water samples, a 100 ml volume of water was mixed well with 100 ml of 2 times high concentrate APW. The mixture was incubated at 37°C for 18 hr. After that, a loopful of enrichment culture was inoculated on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Nissui) and CHROMagar Vibrio (CV) agar (CHROMagar Microbiology, Paris, France) and incubated at 37°C for 18 hr. After incubation, green colonies on TCBS agar and violet colonies on CV agar were picked up and subjected to biochemical tests. The strains showing glucose fermentation without gas production, lysine-positive, mobility-positive, indole-positive, oxidase-positive, VP- negative, no growth without NaCl and growth from 3 to 8% NaCl were identified as V. parahaemolyticus. The V. parahaemolyticus identified was confirmed by species-specific toxR gene detection using PCR method following protocol described by Kim et al. [13].
DNA extraction
DNA of V. parahaemolyticus strains was extracted using the boiling method as described previously [10].
Pathogenic gene detection
tdh gene: Loop-mediated isothermal amplification assay was applied to detect tdh gene using a Loopamp DNA amplification kit (Eiken Chemical Co., Ltd., Tokyo, Japan) as described by Yamazaki et al. [25]. The incubation was carried out in a Loop realtime tubidimeter (Realoop-30, Eiken Chemical) at 65°C for 60 min, following by 80°C for 2 min. A reaction was considered positive as the turbidity reached 0.1 within 60 min.
trh gene: PCR assay for trh gene examination was performed using the protocol described previously [2]. PCR amplified products were checked in 1.5% agarose gels by electrophoresis. After that, the gel was stained with ethidium bromide (0.5 mg/ml) and photographed under a UV transilluminator.
Serotyping
V. parahaemolyticus strains harboring tdh and/or trh genes isolated in this study were serotyped using commercial antisera test kit (Denka Seiken, Tokyo, Japan) following manufacturer’s instructions.
Pandemic trait detection
Group-specific PCR (GS-PCR) was carried out to find pandemic strains using protocol described by Matsumoto et al. [15].
Antimicrobial resistance examination
The antimicrobial susceptibility test was done using the disk diffusion method following the guideline of Clinical and Laboratory Standard Institute (CLSI) [5]. Nine antimicrobial agents were used in this study including ampicillin (10 µg), chloramphenicol (30 µg), gentamycin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), ofloxacin (10 µg), oxytetracycline (30 µg), streptomycin (10 µg) and sulfisoxazole (250 µg). The density of bacteria was adjusted to reach the turbidity of 0.5 McFarland standard. After that, bacteria were lawn on Muller Hilton agar (Benton, Dickinson and Co., Sparks, MD, U.S.A.) supplemented 3% NaCl plates. Antimicrobial disks (Benton, Dickinson and Co.) were then placed on agar plates and these agar plates were incubated at 37°C for 18 hr.
RESULTS
Prevalence of V. parahaemolyticus in seafood and water samples in the Mekong Delta
Seafood samples: The prevalence of V. parahaemolyticus in seafood samples obtained from wet markets and clam and shrimp farms was summarized in Table 1. V. parahaemolyticus strains were isolated in 288 of 330 (87.3%) samples from retail shops and in 44 of 55 (80.0%) samples from farms. Of 330 retail seafood samples, tdh and/or trh gene positive V. parahaemolyticus strains were detected in 24 (7.3%) samples. The tdh gene positive V. parahaemolyticus strains were detected in 22 (6.7%) samples and trh gene positive V. parahaemolyticus strains were found in 4 (1.2%) samples. Of 24 pathogenic V. parahaemolyticus strains, two strains harbored both tdh and trh genes and the other 22 strains carried either tdh or trh gene. Regarding to farming seafood samples, none of tdh gene positive V. parahaemolyticus strains was isolated; meanwhile, trh gene positive V. parahaemolyticus was found in 1 (1.8%) sample. Of farming seafood samples examined, the trh gene positive strain was detected only in the clam sample, dominating 6.3% clam samples collected. No pathogenic V. parahaemolyticus strains were detected in shrimp samples including in retail shops and in shrimp ponds. All tdh and/or trh gene carrying strains isolated was toxR gene positive (Table 3).
Table 1. Prevalence of V. parahaemolyticus in seafood samples in the Mekong Delta, Vietnam.
| Origin | Samples | No. of samples | No. of V. parahaemolyticus positive samples (%) | No. of human pathogenic V. parahaemolyticus positive samples (%) |
|||
|---|---|---|---|---|---|---|---|
| tdh gene | trh gene | Total | |||||
| Retail shops | Molluscan shellfish | ||||||
| White hard clam | 87 | 79 (90.8) | 10 (11.5) | 2 (2.3) | 11 (12.6)a) | ||
| Blood cockle | 85 | 80 (94.1) | 5 (5.9) | 1 (1.2) | 5 (5.9)a) | ||
| Mud clam | 60 | 51 (85.0) | 4 (6.7) | 1 (1.7) | 5 (8.3) | ||
| Antique ark | 40 | 32 (80.0) | 1 (2.5) | 0 (0.0) | 1 (2.5) | ||
| Hakf-crenate ark | 26 | 18 (69.2) | 2 (7.7) | 0 (0.0) | 2 (7.7) | ||
| Subtotal | 298 | 260 (87.2) | 22 (7.4) | 4 (1.3) | 24 (8.0) | ||
| Shrimp | |||||||
| Banana shrimp | 28 | 25 (89.3) | 0 (0.0) | 0 (0.0) | 0 (0.0) | ||
| Greasyback shrimp | 4 | 3 (75.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | ||
| Subtotal | 32 | 28 (87.5) | 0 (0.0) | 0 (0.0) | 0 (0.0) | ||
| Subtotal | 330 | 288 (87.3) | 22 (6.7) | 4 (1.2) | 24 (7.3) | ||
| Farms | Molluscan shellfish | ||||||
| White hard clam | 16 | 16 (100) | 0 (0.0) | 1 (6.3) | 1 (6.3) | ||
| Shrimp | |||||||
| White leg shrimp | 35 | 25 (71.4) | 0 (0.0) | 0 (0.0) | 0 (0.0) | ||
| Black tiger shrimp | 4 | 3 (75.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | ||
| Subtotal | 39 | 28 (71.8) | 0 (0.0) | 0 (0.0) | 0 (0.0) | ||
| Subtotal | 55 | 44 (80.0) | 0 (0.0) | 1 (1.8) | 1 (1.8) | ||
| Total | 385 | 332 (86.2) | 22 (5.7) | 5 (1.3) | 25 (6.5) | ||
a) One V. parahaemolyticus strain harbored both tdh and trh genes.
Table 3. Characteristics of human pathogenic V. parahaemolyticus strains isolated from seafood and water samples in the Mekong Delta, Vietnam.
| Serotype | Origin | tdh gene | trh gene | toxR gene | GS-PCR |
|---|---|---|---|---|---|
| O1:K1 | Retail shellfish | - | + | + | - |
| O1:K32 | Retail shellfish | + | - | + | - |
| O1:KUTa) | Retail shellfish | + | + | + | - |
| O1:KUT | Retail shellfish | + | + | + | - |
| O1:KUT | Retail shellfish | + | - | + | - |
| O2:KUT | Retail shellfish | + | - | + | - |
| O2:KUT | Retail shellfish | + | - | + | - |
| O2:KUT | Retail shellfish | + | - | + | - |
| O3:K6 | Retail shellfish | + | - | + | + |
| O3:K6 | Retail shellfish | + | - | + | + |
| O3:K6 | Retail shellfish | + | - | + | + |
| O3:K6 | Retail shellfish | + | - | + | + |
| O3:K7 | Retail shellfish | + | - | + | - |
| O3:K7 | Retail shellfish | + | - | + | - |
| O3:KUT | Retail shellfish | + | - | + | - |
| O3:KUT | Clam at clam farm | - | + | + | - |
| O4:K29 | Retail shellfish | + | - | + | - |
| O4:K34 | Retail shellfish | + | - | + | - |
| O4:K42 | Retail shellfish | + | - | + | - |
| O4:K42 | Retail shellfish | + | - | + | - |
| O4:K42 | Retail shellfish | + | - | + | - |
| O4:KUT | Retail shellfish | + | - | + | - |
| O4:KUT | Retail shellfish | + | - | + | - |
| O4:KUT | Retail shellfish | + | - | + | - |
| O5:K47 | Retail shellfish | - | + | + | - |
| O8:KUT | Water at clam farm | - | + | + | - |
a) Untypeable.
Water samples: Of 64 water samples collected at clam farms and shrimp ponds, 50 (78.1%) samples were V. parahaemolyticus positive (Table 2). No tdh gene positive V. parahaemolyticus was isolated from all samples although only 1 (1.6%) sample harbored trh gene positive V. parahaemolyticus (Table 2). This trh gene positive strain was isolated from clam farming environment, accounting for 4.5% samples collected at this area. The trh gene positive strain was also toxR gene positive (Table 3).
Table 2. Prevalence of V. parahaemolyticus in water samples in the Mekong Delta, Vietnam.
| Water samples | No. of samples | No. of V. parahaemolyticus positive samples (%) | No. of human pathogenic V. parahaemolyticus positive samples (%) |
||
|---|---|---|---|---|---|
| tdh gene | trh gene | ||||
| Molluscan shellfish farms | |||||
| White hard clam | 22 | 17 (77.3) | 0 (0.0) | 1 (4.5) | |
| Shrimp ponds | |||||
| White leg shrimp | 38 | 30 (78.9) | 0 (0.0) | 0 (0.0) | |
| Black tiger shrimp | 4 | 3 (75.0) | 0 (0.0) | 0 (0.0) | |
| Subtotal | 42 | 33 (78.6) | 0 (0.0) | 0 (0.0) | |
| Total | 64 | 50 (78.1) | 0 (0.0) | 1 (1.6) | |
Serotypes and pandemic trait
The serotypes and pandemic trait of pathogenic V. parahaemolyticus strains obtained in this study were shown in Table 3. The pathogenic strains were classified into 6 O serogroups and generated 13 serotypes. Notably, all of 4 O3:K6 strains harboring tdh gene from retail shellfish samples showed GS-PCR positive. The pandemic trait was only found in O3:K6 strains, but not in the others.
Antimicrobial resistance
Most of the pathogenic V. parahaemolyticus strains isolated in this study showed resistance to streptomycin (84.6%), ampicillin (57.7%) and sulfisoxazole (57.7%) (Table 4). Of 26 pathogenic strains, 5 (19.2%) and 20 (76.9%) strains showed resistance against one and more than one antimicrobial agents, respectively; while only 1 strain showed no resistance to antimicrobial agents (Table 5).
Table 4. Antimicrobial resistance of human pathogenic V. parahaemolyticus (n=26).
| Antimicrobial agents | No. of resistant strains (%) |
|---|---|
| Streptomycin | 22 (84.6) |
| Ampicillin | 15 (57.7) |
| Sulfisoxazole | 15 (57.7) |
| Kanamycin | 2 (7.7) |
| Chloramphenicol | 0 (0.0) |
| Gentamycin | 0 (0.0) |
| Nalidixic acid | 0 (0.0) |
| Oxfloxacin | 0 (0.0) |
| Oxytetracycline | 0 (0.0) |
Table 5. Resistant patterns of human pathogenic V. parahaemolyticus strains against 9 antimicrobial agents.
| Resistant patterns | No. of strains |
|---|---|
| KM-AMP-SFX-STMa) | 1 |
| KM-AMP-STM | 1 |
| AMP-SFX-STM | 6 |
| SFX-STM | 6 |
| AMP- STM | 4 |
| AMP-SFX | 2 |
| STM | 4 |
| AMP | 1 |
| No resistance | 1 |
| Total | 26 |
a) KM: Kanamycin; AMP: Ampicillin; SFX: Sulfisoxazole; STM: Streptomycin.
DISCUSSION
The pathogenicity of V. parahaemolyticus causing foodborne illness in human is usually associated with tdh and trh genes [18]. The contamination of this pathogen has been reported in seafood samples in some Southeast Asian countries such as Thailand [3, 17], Malaysia [2, 17, 19] and Indonesia [17]. However, few information on the prevalence of this pathogen in environment in the Mekong Delta has been reported. In this study, tdh and trh gene positive V. parahaemolyticus was isolated from retail shellfish and shellfish farms. This is the first report on the detection of human pathogenic V. parahaemolyticus in food in the Mekong Delta, Vietnam.
The human pathogenic V. parahaemolyticus is usually detected relatively at a high rate in molluscan shellfish samples. The tdh gene positive V. parahaemolyticus was detected from molluscan shelfish at 12% in Thailand [3], 11.1% in Malaysia and 9.1% in Indonesia [17]. It is known that molluscan shellfish are filter feeders and can accumulate pathogenic V. parahaemolyticus in their guts, providing high prevalence of this pathogenic bacterium compared to other species such as shrimp and fish [17, 20]. Therefore, the prevalence rate of pathogenic V. parahaemolyticus in shellfish samples seems to be higher than that in shrimp in this study. Moreover, many of retail shops in this study were located near the coast in the Mekong Delta. Shellfish from those shops were usually sold immediately after they were harvested at the coast. Therefore, prevalence of pathogenic V. parahaemolyticus in retail shellfish seems to reflect that in the environment in this coastal area in this study although tdh gene positive V. parahaemolyticus was not detected from farming shellfish and environment samples.
Serotype O3:K6 is known to be the predominant serotype isolated from V. parahaemolyticus infection in human [16]. The GS-PCR positive O3:K6 strain is indicated as the pandemic strain [15]. In this study, all of 4 O3:K6 strains detected in retail shellfish samples showed GS-PCR positive. These results indicate that the pandemic V. parahaemolyticus strain is prevalent in the environment in the Mekong Delta. Therefore, we should pay more attention on human V. parahaemolyticus infection in this region.
Pathogenic V. parahaemolyticus strains isolated in this study showed high resistance rates to streptomycin, ampicillin and sulfisoxazole. Similar observations were reported elsewhere in Southeast Asian countries [1, 9, 14, 21]. However, Marlina et al. [14] reported human pathogenic V. parahaemolyticus originated from Indonesia showed high resistance rates to tetracycline and gentamycin, although our isolates showed no resistance to those antibiotics. Almost all pathogenic V. parahaemolyticus strains examined in this study demonstrated multiple antimicrobial resistances. Other studies in some Southeast Asian countries also recorded similar results [1, 14]. In fact, several antimicrobial agents such as streptomycin, ampicillin and sulfisoxazone have been frequently used in agriculture and aquaculture for the last some decades; therefore, the resistance of bacteria to those antimicrobial agents could be facilitated by selective pressure [8, 11].
In conclusion, human pathogenic V. parahaemolyticus is prevalent in seafood relatively at high rate in the Mekong Delta and the pandemic serotype O3:K6 distributed in this area. Furthermore, pathogenic V. parahaemolyticus isolated in this area showed resistance to several antimicrobial agents and performed multidrug resistance. These findings can be used for monitoring microbiological risk of seafood in the Mekong Delta.
CONFLICT OF INTEREST
No conflicts were declared.
Acknowledgments
The authors thank Dr. Ryoichi KUBO in Kanto Chemical Co., Inc. for supporting our research.
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