Detection and quantification of pathogenic Vibrio parahaemolyticus in shellfish by using multiplex PCR and loop-mediated isothermal amplification assay

Vibrio parahaemolyticus is a halophilic bacterium that commonly inhabits the marine and estuarine environments. This organism is also one of the leading causative pathogen of gastroenteritis often related to consumption of raw or undercooked seafood. In this study, molluscan shellfish (bloody clams and surf clams) and crustaceans (shrimps) were monitored in wet markets and hypermarkets. Two molecular methods were employed and compared to detect total and pathogenic V. parahaemolyticus in MPN enrichments: multiplex PCR and LAMP assay. The multiplex PCR was optimized to detect the total (toxR+), tdh+ and trh+ V. parahaemolyticus. On the other hand, the LAMP assay was employed to target the pathogenic strains only, the tdh+ and trh+, respectively. Out of 232 samples examined, 229 (98.7%) were positive for V. parahaemolyticus with counts ranging from 30 to >110, 000 MPN/g. Positive samples for tdh+ V. parahaemolyticus were obtained in 77 out of 232 (33.1%) samples ranging from 30 to >110, 000 MPN/g. Meanwhile, positive samples for trh+ were identified in 16 out of 232 (6.9%) samples examined ranging from 30 to 9600 MPN/g. Detection of samples with presence of tdh+ genes did not vary between methods, but a significant difference was observed when the LAMP assay was compared to PCR to detect trh+ V. parahaemolyticus. Therefore, on occasions where the density of the targeted genes is low, the LAMP assay serves as a better alternative. Nonetheless, this study constitutes an assessment of presence of total and potentially pathogenic V. parahaemolyticus in shellfishes for domestic consumption revealing the potential risk of contracting vibriosis if precautions and safety measures are not properly managed.     

Distribution,prevalence, molecular typing,and virulence of Vibrio parahaemolyticus isolated from different sources in coastal province Jiangsu,China

Vibrio parahaemolyticus is one of the most important foodborne pathogens in China, Japan and other countries. In this study we reported the distribution, prevalence, molecular characterization and virulence of V. parahaemolyticus from aquatic products, cooked food and clinical patients. In aquatic products, the prevalence of V. parahaemolyticus was 47.2% and the prevalence of tdh⁺ and trh⁺ strains was 8.5% and 1.5%, respectively. The prevalence of V. parahaemolyticus in clinical samples was 24.9% and 96.1% of the clinical strains were tdh⁺ and 62.7% of the clinical strains were orf8⁺. A total of 341 strains were classified into six molecular types and 23 subtype by random amplified polymorphic DNA (RAPD). Subtypes A₁ and subtype F₈ were the two major subtypes and strains of these two subtypes accounted for 45.5% and 25.8%, respectively. tdh⁺ strains mainly belonged to subtype A₁ (78.6%) and subtype F₈ (15.0%) and all the orf8⁺ strains belonged to subtype A₁. V. parahaemolyticus could be transmitted not only through seafood but also through freshwater products in food chains. The tdh⁺ strains especially orf8⁺ strains were the main toxigenic pathogen that caused foodborne diseases in Jiangsu, China.     

Seasonal prevalence of Vibrio species in retail shrimps with an emphasis on Vibrio parahaemolyticus

Seasonal prevalence of Vibrio species in shrimp samples from retail outlets in the South-western part of Iran was studied. A total of 300 samples were analyzed (75 samples in each season). Special attention was paid to the prevalence of total and pathogenic Vibrio parahaemolyticus. All the TCBS isolates were first identified to the genus level with PCR and then identified to the species level using a battery of biochemical reactions and tests. To investigate the pathogenicity of the isolated V. parahaemolyticus, multiplex PCR (tl, tdh and trh genes) was performed. Vibrios were detected during the whole investigation period, depending on the sampling season. They were detected in 18.6% of the winter samples, 64% of the spring samples, 70.6% of the summer samples and 41.3% of the autumn samples. Vibrio calviensis and Vibrio alginolyticus were dominant in samples of different seasons, with the average prevalence of 18.6% and 17.6%, respectively. V. parahaemolyticus was found in 4.0% of the winter samples, 13.3% of the spring samples, 18.6% of the summer samples and 8% of the autumn samples. During the period of this study, two tdh-positive strains were isolated, while no trh-positive V. parahaemolyticus strain was detected in samples of different seasons.     

Molecular characterizations of pathogenic Vibrio parahaemolyticus isolated from Southern Taiwan oyster-growing environment

Ninety-four virulent strains of Vibrio parahaemolyticus containing tdh and/or trh genes isolated from seawater, sediment, and oysters collected in Penghu, Tainan, and Changhua Lukang coastal water were characterized by analyses of O-group antigens, pulsed-field gel electrophoresis (PFGE) after digestion with SfiI enzyme, and randomly amplified polymorphic DNAs (RAPD). Analysis of virulence markers (tdh and trh) revealed that 60.6% of the isolates possessed only tdh, 13.8% of them possessed only trh, and 25.5% of them possessed both tdh and trh. Ten O-group serotypes (O1, O2, O3, O4, O5, O6, O7, O8, O10, and O11) were identified among the isolates with O1 group (28 isolates) being the most prevalent, followed by the O3 group (16 isolates) and O5 group (15 isolates). PFGE typing of the isolates revealed 94 patterns while RAPD analyses categorized the isolates into five major groups. Both PFGE and RAPD analyses showed high index of discrimination (DI) values. This study identified diverse serotypes and genotypes of virulent V. parahaemolyticus distributed in the oyster-growing environments, which can be used for risk assessment of V. parahaemolyticus infection associated with oyster consumption in Taiwan.     

Distribution,serological and molecular characterization of Vibrio parahaemolyticus from shellfish in the eastern coast of China

To investigate the prevalence of Vibrio parahaemolyticus in shellfish, a total of 288 samples from retail markets in four coastal provinces of eastern China were collected and analyzed monthly from December 2008 to November 2009. Altogether 172 isolates were isolated and identified, of which 2 isolates were tdh⁺ and 5 were trh⁺. The levels of V. parahaemolyticus were estimated by most probable number procedure and results suggested that the distribution of V. parahaemolyticus was disparity in season. Serotyping was performed among all isolates, 157 isolates were grouped into 9 O-groups and 42 isolates were determined by specific K-typing. Two tdh⁺ isolates were identified as O3:K6 and O4:K68 serovars. Random amplified polymorphic DNA (RAPD) was performed to assess the genetic diversity of all isolates. The results showed that there were 73 different patterns, which were clustered into 18 groups except 6 miscellaneous patterns. The two tdh⁺ isolates and two clinical isolates were grouped into the same cluster. This study demonstrated that V. parahaemolyticus from shellfishes were of high antigenic and genetic diversity. Comparison with serological method, RAPD might be a more efficient vehicle for epidemiology and risk assessment of V. parahaemolyticus.     

Comparison of toxR and tlh based PCR assays for Vibrio parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative bacterium found in marine and estuarine environments and is globally the leading cause of bacterial seafood-related illness. A real-time PCR assay for V. parahaemolyticus was developed for the marker toxR, with a large-scale and direct comparison of its applicability as a species-specific marker compared to the tlh gene carried out. Assays for tlh and toxR were used for 255 presumptive V. parahaemolyticus strains from our strain library, utilising both real-time (toxR) and conventional PCR assays (tlh). Of the 255 strains test, 254 results were in concordance; 255 strains were identified as being toxR positive (100%) and 254 strains were tlh positive (99.6%). The single discordant strain (isolate V12/023) was of interest, because it represented a presumptive V. parahaemolyticus strain, isolated from a clinical case. Whole genome sequence analysis and multi locus sequence typing of this single discordant strain was carried out, which unambiguously identified that the isolate was indeed V. parahaemolyticus. Genome analysis identified mismatches in the primer binding sites for the established tlh assay is likely responsible for the assay failing on this particular strain. The identification of false-negative results in strains that are implicated in human infections using the tlh assay and clearly highlights the relevance of the comparison with a toxR assay which showed 100% identification for the V. parahaemolyticus strains tested.     

Production and characterization of a monoclonal antibody against recombinant thermolabile hemolysin and its application to screen for Vibrio parahaemolyticus contamination in raw seafood

Thermolabile hemolysin gene (tlh) is regarded as a species-specific marker for Vibrio parahaemolyticus. To assess the utility of the tlh gene product, thermolabile hemolysin (TLH), as a marker to screen for V. parahaemolyticus-contamination in raw seafood, we generated a monoclonal antibody (MAb) against recombinant TLH and established with an enzyme-linked immunosorbent assay using the MAb (TLH-ELISA). TLH-ELISA testing of broth cultures for 78 V. parahaemolyticus strains showed positive results all around. In contrast, most broth cultures of 53 non-V. parahaemolyticus species tested yielded negative results.We devised a screening method using TLH-ELISA to check for low-level contamination of V. parahaemolyticus in raw seafood within 24 h and evaluated its ability. In testing of V. parahaemolyticus-spiked raw seafood, results suggested that our screening method can detect 100 most-probable-number (MPN) of V. parahaemolyticus/g. Further, on testing 119 commercial raw seafood samples with our screening method, 117 samples were determined to contain less than 100 MPN of V. parahaemolyticus/g. All of the 117 samples were also estimated by the MPN method to contain less than 100 MPN of V. parahaemolyticus/g. Taken together, these results suggest that our screening method using TLH-ELISA is useful to check for low-level (<100 MPN/g) of V. parahaemolyticus in raw seafood.     

The efficacy of grape seed extract,citric acid and lactic acid on the inactivation of Vibrio parahaemolyticus in shucked oysters

The efficacy of grape seed extract (GE), citric acid (CA) or lactic acid (LA) on the inactivation of Vibrio parahaemolyticus in shucked oysters was studied. The minimum inhibitory concentration (MIC) of GE, CA or LA against V. parahaemolyticus in TSB-1% NaCl was also determined. The shucked oysters were artificially inoculated with V. parahaemolyticus, the inoculated shucked oysters (25 g) were then dipped in solution of GE (0.0, 10.0, 20.0, 50.0, 100, 200, 300 and 500 mg mL⁻¹), CA (0.0, 5.0, 10.0, 15.0, 20.0, 50.0, 100, 200 and 300 mg mL⁻¹) or LA (0.0, 1.0, 5.0, 10.0, 15.0, 20.0, 50.0, 100 and 150 mg mL⁻¹) for 10 min. The population of V. parahaemolyticus in shucked oysters was determined. The MICs of GE, CA or LA against V. parahaemolyticus were 10.0, 5.0 or 1.0 mg mL⁻¹, respectively. A 500, 300 or 150 mg mL⁻¹ GE, CA or LA solutions were needed to reduce the population of V. parahaemolyticus to below the detection level (1.0 log g⁻¹) in shucked oysters.     

Validation of real-time PCR for detection of six major pathogens in seafood products

Seafood can pose a public health concern to consumers. It is often consumed raw and may be contaminated with several foodborne pathogens. In order to guarantee the safety of seafood, real-time polymerase chain reaction (PCR) protocols may be used as these enable results to be provided within 24 h.The first goal of our work was to develop real-time PCR protocols enabling the detection of six foodborne pathogens that may be present in seafood products (Campylobacter jejuni, Campylobacter coli, enterohemorrhagic Escherichia coli, Salmonella spp., Vibrio parahaemolyticus, and Vibrio vulnificus). The corresponding gene targets were: 50S/VS1, rfbE, ttr, tlh, and vvp. A multiplex PCR was also developed to detect the virulence genes of V. parahaemolyticus: tdh and trh. A total of 420 bacterial strains belonging to four different genera/strains were used in this study. Sensitivity and specificity were always 100%, except in the case of Salmonella spp., where three strains were not detected by our PCR protocols.The second objective of our work was to assess the detection limit of our real-time PCR protocols on artificially contaminated seafood products (raw shrimps, cooked shrimps, and raw mussels), purchased in public stores. Six different levels of contamination were assayed in four replicates for each matrix. The real-time PCR protocols enabled a better level of detection than the ISO methods, except for Salmonella in raw shrimps and for V. vulnificus in shrimps (raw and cooked). The estimated level of detection was between 1 and 47 cfu/25 g sample for the ISO norms and between 1 and 315 cfu/25 g sample for the real-time PCR protocols tailored in our work.The real-time PCRs developed in our work allowed for good selectivity, sensitivity, and specificity. The sensitivity on seafood products was estimated at a level of 100%, except for Salmonella (97%). In the spiking assays, the levels of detection were lower with the real-time PCR protocol than those obtained with the ISO method. This was not the case for V. vulnificus in raw and cooked shrimps and for Salmonella in raw shrimps.These real-time PCR protocols appear to be good alternative methods for surveillance of seafood products to ensure the absence of foodborne pathogens.One additional conclusion is that laboratories have to use enrichment media that are compatible with those recommended by ISO standards. This may facilitate the isolation of the pathogen if the real-time PCR protocol gives a suspect positive signal during the first step of the seafood analysis.     

Prevalence,pathogenicity, and serotypes of Vibrio parahaemolyticus in shrimp from Chinese retail markets

Vibrio parahaemolyticus is one of the most common foodborne pathogens in Asian countries. V. parahaemolyticus contamination of retail shrimp in China has been reported previously, and such contaminated shrimp have been linked to outbreaks of foodborne diseases. However, to date, the prevalence of V. parahaemolyticus in retail shrimp in China has not been determined. This study aimed at identifying the prevalence of V. parahaemolyticus in shrimps in Chinese retail market. From May 2012 to April 2013, a total of 273 shrimp samples was obtained from retail markets in 16 provinces (19 cities) of China. V. parahaemolyticus was detected in 103 (37.7%) of 273 samples by the most probable number method. Bacterial densities were less than 100 MPN/g in 95.1% (98/103) of the samples. Five trh-positive isolates were identified from 247 isolates, and none of the isolates were tdh-positive. In multiplex PCR-based O-antigen serotyping of these 247 pathogenic isolates, all O-antigen serotypes, except O9, were detected, and serotype O2 was found to be the most prevalent (detected in 82 isolates). This is the first report on V. parahaemolyticus prevalence in retail shrimp in China, and the findings of this study can be used for microbiological risk assessment of shrimp in China.     

Comparison of Vibrio parahaemolyticus isolates from aquatic products and clinical by antibiotic susceptibility,virulence, and molecular characterisation

Vibrio parahaemolyticus is a common foodborne pathogen found in aquatic products and represents a major threat to human health worldwide. Though not all this bacteria were harmful to human beings, the pathogenic V. parahaemolyticus always harbors either tdh (the thermostable direct hemolysin) or trh (TDH-related hemolysin) gene, or both. Additionally, the extensive use of antibiotics has been shown to be a contributing factor to the increasing incidence of antimicrobial-resistant strains. In this study, thirty-one clinical isolates were examined and compared with 95 (38.0%) aquatic product isolates (fishes, n = 28; shrimps, n = 67) collected from 250 samples in Guangdong, China. All isolates were studied by antibiotic susceptibility analysis, tdh and trh genes detection, serotyping and molecular typing (ERIC-PCR). The antimicrobial resistance patterns of these aquatic product isolates to 12 antimicrobial agents revealed that most of the isolates were resistant to streptomycin (90.53%). The isolates were also resistant to follow by ampicillin (33.68%) and cephalothin (30.53%). For clinical isolates, they were resistant to streptomycin (93.55%), ampicillin (87.10%), and cefazolin (64.52%). All isolates showed no resistance to azitromycin, chloramphenicol, ciprofloxacin, or nalidixic acid. The clinical isolates were positive for tdh (100%) and trh gene (77.42%), with ratios of only 2.11% and 28.42%, respectively in the aquatic product isolates. Serotyping detected shown that the isolates contained O1, O2, O3, O4, and O11, with the O3 serotype being the most common among the clinical isolates (48.39%), while the O2 (41.05%) makes the maximum proportion on aquatic product isolates. ERIC-PCR results demonstrated the isolates (n = 126) were classified into eight clusters, revealing genetic variation and relatedness between clinical and aquatic product isolates. This study provided a foundation for understanding the distinction between aquatic product and clinical isolates and yielded basic information for achieving food safety through control of V. parahaemolyticus contamination.     

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shellfish in Shanghai

Vibrio parahaemolyticus is a marine and estuarine bacterium that poses the greatest threat to human health worldwide. It has been the leading bacterial cause of seafood-borne illness. This study investigated the prevalence and drug resistance of V. parahaemolyticus isolated from retail shellfish in Shanghai. A total of 140 shellfish samples were collected from February 2014 to February 2015. The occurrence of V. parahaemolyticus in shellfish was 34.3%, which has increased compared to previous reports. In addition, discernible differences of total presumptive V. parahaemolyticus counts (TPVPC) were also observed in shellfish between market A and B. The results from PCR assays indicated that thermostable direct hemolysin (tdh) gene was positive in two isolates (2.1%), and the thermostable direct hemolysin-related hemolysin (trh) gene was not detected in all isolates. Antibiotic resistance profiles of those isolates were as follows: ampicillin (87.5%), cephazolin (31.3%), cephalothin (6.3%), amoxicillin/clavulanic acid (6.3%), piperacillin (6.3%), and amikacin (3.2%). Thirty-three out of 96 isolates were resistant to two or more antimicrobial agents. It is suggested that V. parahaemolyticus in retail shellfish could be a potential risk to consumers in Shanghai.     

PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state

Vibrio parahaemolyticus is a crucial foodborne pathogen. The viable but non-culturable (VBNC) state of V. parahaemolyticus cannot be detected by traditional culture methods. The objective of this study was to develop and evaluate a method that combines propidium monoazide (PMA) treatment with real-time fluorescent loop-mediated isothermal amplification (RF-LAMP) to detect and quantify VBNC cells of V. parahaemolyticus. Different states of cells were treated with PMA in dark for 5 min and subsequently exposed to a 650 W halogen lamp for 5 min. The cells were collected and DNA was amplified by RF-LAMP. The primers which targeted six distinct regions in the tlh gene of V. parahaemolyticus were used for the PMA-RF-LAMP assay. The results indicated that the treatment with 4 μg/mL of PMA and a further exposure to light for 5 min was suitable for PMA-RF-LAMP to distinguish viable cells from dead cells of V. parahaemolyticus. The developed assay exhibited remarkably high specificity, sensitivity and rapidity, without any cross reaction with the tested non-V. parahaemolyticus strains. There was good linear correlation between Ct values and log copy/mL in the range of 6.8–6.8 × 10⁷ copy/mL, with the reaction endpoints less than 30 cycles (30 min). It could detect as low as 14 copy/g of V. parahaemolyticus in spiked seafood samples (pomfret, shrimp, scallop, oyster and salted fish) without any interference from food matrices, dead cells and other bacteria.     

Application of grape seed extract in depuration for decontaminating Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas)

This study investigated the potential application of grape seed extract (GSE) in depuration to increase its efficacy in reducing Vibrio parahaemolyticus populations in Pacific oysters (Crassostrea gigas). Pacific oysters were inoculated with five clinical strains of V. parahaemolyticus to 10⁴⁻⁵ MPN/g and depurated in UV-irradiated artificial seawater (ASW) containing GSE at a level of 1.0% (1.8 mg/mL total phenolic contents as gallic acid equivalents) or 1.5% (3.1 mg/mL total phenolic contents as gallic acid equivalents) at 12.5 °C for up to 5 days. Changes of V. parahaemolyticus populations in oysters during depuration were analyzed every 24 h using the three-tube most probable number (MPN) method. The populations of V. parahaemolyticus in inoculated oysters decreased by 3.06 and 3.71 log MPN/g, respectively, after 4 and 5 days of depuration in ASW at 12.5 °C. However, populations of V. parahaemolyticus in oysters were reduced by 3.01 and 4.18 log MPN/g after 2 days of depuration at 12.5 °C in ASW containing 1.0 and 1.5% GSE, respectively. Further studies confirmed that depuration using ASW containing 1.5% GSE with 3.1 mg/mL total phenolic contents as gallic acid equivalents at 12.5 °C for two days was capable of achieving >3.52 log MPN/g reductions of V. parahaemolyticus in Pacific oysters.     

Pomegranate peel (Punica granatum L) extract and Chinese gall (Galla chinensis) extract inhibit Vibrio parahaemolyticus and Listeria monocytogenes on cooked shrimp and raw tuna

Vibrio parahaemolyticus and Listeria monocytogenes are bacterial pathogens associated with raw or ready-to-eat seafood products. Many compounds extracted from plant material have shown promise for inhibiting bacterial pathogens when applied to some foods. In this study, aqueous methanol extracts from pomegranate peel (Punica granatum L.) and Chinese gallnut (Galla chinensis) were tested against V. parahaemolyticus and L. monocytogenes on cooked shrimp and raw homogenized tuna. The extracts were applied to the shrimp by soaking for 2 min (5 mg/ml). The extracts (1.7 mg ml) were added to homogenized tuna and stirred. The antimicrobial assay on V. parahaemolyticus was conducted at 12 °C, and the assay on tuna was conducted at both 4° and 12 °C. Both Chinese gall and pomegranate peel extracts significantly inhibited the growth of V. parahaemolyticus in both shrimp and tuna. Only Chinese gall extract significantly inhibited growth of L. monocytogenes. Overall V. parahaemolyticus was more sensitive to both plant extracts compared with L. monocytogenes. Both plant extracts had stronger antimicrobial activity on shrimp compared with the tuna. Neither extract completely inhibited the growth of V. parahaemolyticus or L. monocytogenes.     

Effect of temperature on pathogenic and non-pathogenic Vibrio parahaemolyticus biofilm formation

Biofilm formation is crucial for the environmental survival and transmission of Vibrio parahaemolyticus, an important food-borne pathogen in seafood. The biofilm developmental process of pathogenic (n = 22) and non-pathogenic (n = 17) V. parahaemolyticus strains on polystyrene microtiter plates under 15 °C, 25 °C and 37 °C was investigated using crystal violet staining, and validated by confocal laser scanning microscopy. The results indicated that biofilm developmental process at 15 °C and 25 °C were divergent, biofilm formation increased continuously at 15 °C, while at 25 °C biofilm formation increased gradually and peaked at 12 h. Also the biofilm formation was dramatically elevated at 25 °C in comparison with that at 15 °C and 37 °C. Additionally, pathogenic strains, on average, formed more biofilm than non-pathogenic strains at all temperatures measured. Moreover, extensive strain variability was observed during biofilm formation and indexed using the coefficient of variation (CV). This index increased with increasing temperature and this index, at all temperatures, peaked after 12 h. The results of this study provide insight into the developmental process of biofilm, which allow us to further optimize strategies to control V. parahaemolyticus biofilm in food industry.     

Thermal inactivation and growth of Listeria monocytogenes during production and storage of caramel apples

During the fall of 2014, commercially produced pre-packaged caramel apples were linked to 35 cases of listeriosis in 12 states. In response, this study aimed to assess 1) the reduction of different outbreak and non-outbreak strains of Listeria monocytogenes during caramel dipping of apples, and 2) subsequent growth of the apple outbreak strains within caramel apples during storage at 22 and 4 °C. In aim 1, three unwaxed Jonathan apples were dip-inoculated with three different 4-strain L. monocytogenes cocktails (apple outbreak, unrelated outbreak or unrelated environmental) at ∼8 log CFU/apple, dried for 1 h, dipped for 5 s in caramel at 82, 88, 93 or 99 °C, cooled for 1 h at room temperature and assessed for survivors. In aim 2, Jonathan apples were spot-inoculated with the apple outbreak cocktail (∼3 log CFU/apple) at the stem juncture, dried for 1 h, pushed onto wooden sticks, and dipped in caramel at 82 °C. During storage at 4 and 22 °C for 28 and 14 days, respectively, four different apple sections (top, middle, bottom and core) were cut from three apples, homogenized and plated for Listeria. After dipping apples in caramel at 82 and 99 °C, the apple outbreak, unrelated outbreak and environmental Listeria strains decreased 2.0 ± 0.6 and 2.7 ± 0.1, 1.8 ± 0.3 and 2.6 + 0.1, and 1.7 ± 0.1 and 2.9 ± 0.2 logs, respectively, with the environmental cocktail significantly less heat resistant (P < 0.05) at 99 °C compared to the other two cocktails. After 14 days of storage at 22 °C, Listeria populations were significantly higher (P < 0.05) in the core (7.4 ± 0.6 log CFU/g) compared to the other three sections (4.9–5.4 log CFU/g). The same trend was seen for the core (7.7 ± 0.6 log CFU/g) and the other three sections (5.0–5.4 log CFU/g) after 28 days of storage at 4 °C. Since dipping in hot caramel cannot ensure pathogen elimination, producers of caramel apples should implement good agricultural practices, post-harvest preventive controls and refrigeration of the final product to minimize the risks from Listeria.     

Simultaneous detection of Vibrio cholerae,Vibrio alginolyticus,Vibrio parahaemolyticus and Vibrio vulnificus in seafood using dual priming oligonucleotide (DPO) system-based multiplex PCR assay

In this study, a rapid and reliable multiplex PCR assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus in seafood was developed using the dual priming oligonucleotide (DPO) system. Species-specific DPO primers were designed targeting the mdh, vvhA, colH and toxR genes for the discrimination of V. cholerae, V. vulnificus, V. alginolyticus and V. parahaemolyticus, respectively. Compared to conventional PCR assay, the DPO system-based multiplex PCR assay allowed a wider annealing temperature at 48 °C–68 °C to effectively amplify target genes followed an analytical detection limit of <1.5 × 10² CFU/mL (or g) for each Vibrio species in pure cultures or artificially contaminated food matrix. A total of 396 bacterial strains including 209 targets and 187 other bacterial strains were used to test the specificity of the DPO system-based multiplex PCR assay, and results showed that specific PCR product was only observed in target bacterial strains without nonspecific priming. Practical application of the assay indicated that target Vibrio species in seafood, clinical samples and foodborne outbreaks can be accurately detected. This DPO system-based multiplex PCR assay developed in this study would be a powerful tool for the rapid and reliable detection of the target Vibrio species.     

Effects of green tea extract on reducing Vibrio parahaemolyticus and increasing shelf life of oyster meats

This study investigated effects of tea extract on growth of pathogenic Vibrio parahaemolyticus and potential utilization in post-harvest treatment to extend shelf life of Pacific oysters (Crassostrea gigas). Longjing Tea, which exhibited strong bactericidal activity against V. parahaemolyticus, was selected to use in this study. Tea extract containing equal or higher than 4.6 g/l total phenolic contents (TPC) as gallic acid equivalents (GAE) determined by Folin-Ciocalteau method could reduce a mixture of five clinical V. parahaemolyticus strains in tryptic soy broth plus 1.5% NaCl from 4.5 log CFU/ml to non-detectable level (<1 log CFU/ml) within 8 h. A treatment of shucked oysters with tea extract containing 9.1 g/l TPC as GAE for 2 h at 23 ± 1 °C with oyster/tea extract ratio of approximate 0.9 g/ml resulted in greater (p < 0.05) V. parahaemolyticus reductions (0.8 log MPN/g) compared to controls (0.2 log MPN/g). The following shelf life study indicated that green tea treatment at oyster/tea extract ratio of approximate 0.7 g/ml could enhance reducing V. parahaemolyticus while retarding the growth of total bacteria in oysters during 5 ± 1 °C storage. Therefore, green tea might be utilized as a natural antimicrobial agent to inactivate V. parahaemolyticus in oysters and extend the shelf life during refrigeration storage.     

Effect of detergents as antibacterial agents on biofilm of antibiotics-resistant Vibrio parahaemolyticus isolates

Vibrio parahaemolyticus (V. parahaemolyticus) is a halophilic, Gram-negative human pathogen known as a leading cause of seafood-derived food poisoning. Due to high contamination rate of seafood in Asian countries, V. parahaemolyticus is considered as a food safety concern. V. parahaemolyticus is able to produce biofilm which is more resistant toward disinfectants and antibodies than its planktonic form. Thirty six V. parahaemolyticus isolates from seafood were tested for their susceptibility using 18 different antibiotics. Two V. parahaemolyticus isolates were resistant to bacitracin, chloramphenicol, rifampin, ampicillin, vancomycin, nalidixic acid, penicillin and spectinomycin. Fourteen V. parahaemolyticus isolates were found to be resistant to bacitracin, tetracycline, rifampin, ampicillin, vancomycin, penicillin and spectinomycin. The remaining two isolates were resistant to more than 2 antibiotics. Majority of the V. parahaemolyticus isolates (97.2%) showed MAR index > 0.2, indicating that these isolates were originated from high risk sources. To investigate effect of three common detergents on antibacterial-resistant V. parahaemolyticus, 16 V. parahaemolyticus isolates resistant to more than 7 antibiotics were selected. V. parahaemolyticus (ATCC 17802) was used as reference strain. Detergents were tested for their minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) and time–kill curves were constructed to assess the concentration between MIC and bactericidal activity of detergents. Detergents D1 (Linear alkyl benzene based) was found to be the most effective with MIC and MBC ranged between 97.656 and 1562.5 μg/ml and 781.25–3125 μg/ml, respectively. The time–kill curves demonstrated that the bactericidal endpoint for resistant V. parahaemolyticus isolates reached after 30 min incubation with D1 at concentration 8 × MIC. The isolate VP003 was killed at 8 × MIC within 0.5 h and the reduction in CFU/ml was 3 log units (99.9%). V. parahaemolyticus biofilms were formed in 96 wells microtiter plates at 37 °C and 24 h-old biofilm were used to test antibacterial activity of detergents. Results showed that biofilm-producing ability of antibacterial-resistant V. parahaemolyticus isolates were inhibited at 1562.5–6250 μg/ml of D1 and eradicated at 3125 – ≥50,000 μg/ml of D1. Detergents showed potential antimicrobial activity against V. parahaemolyticus