Total Vibrio Parahaemolyticus Research Articles

PREVALENCE OF HEAT-RESISTANT VIBRIO PARAHAEMOLYTICUS IN RETAIL SEAFOOD

Previous work using a modified two-step enrichment method recovered total Vibrio parahaemolyticus, including the regular and heat/pasteurization-resistant strains. These strains would remain undetectable by exclusive cultivation protocol, attributed to their viable-but-nonculturable (VBNC) property, from environmental shellfish seafood. Hence, this suggests the need to validate retail seafood safety from this human pathogen and reform the inspection examination and cooking protocols mandated by the regulators to mitigate vibriosis recurrence. This modified method was further explored with raw, frozen shellfish seafood products from retail stores nationwide in the US for heat/pasteurization-resistant V. parahaemolyticus using nourishment only (one-step enrichment) or nourishment and heating selection (two-step enrichment) combined, followed by selective cultivation on thiosulphate-citrate-bile-salt agar (TCBS) media. Both presumptive regular and heat-resistant V. parahaemolyticus were recovered from processed shrimps (deveined/non-deveined, shell, and tail on), and non-processed oysters and crabs. Of 29 isolates recovered, nine (31%) were confirmed as heat-resistant (two from oyster; 7%) and regular (seven, two from shrimp; five from oyster; 24%) V. parahaemolyticus using 16S rDNA bacterial identification. However, none of them were resistant to pasteurization conditions. Virulence phenotype and genotype examinations indicated the presence of hemolysis- and urease-positive V. parahaemolyticus and that their unparallel phenotype/genotype correlation could be attributed to gene manipulation. For the first time, the present study reveals the prevalence of total V. parahaemolyticus, both regular and heat-resistant pathogenic strains, in retail shellfish products using the modified two-step enrichment technique and acclaims the need to reform the investigative and cooking standard protocol of USDA and other food safety regulatory entities.

The correlation between ectoparasite infestation and total Vibrio parahaemolyticus bacteria in Pacific white shrimp (Litopenaeus vannamei) in Super Intensive Ponds

This study aimed to determine the type, intensity, degree of ectoparasite infestation and its correlation to the total V. parahaemolyticus bacteria in super-intensive pacific white shrimp culture. In this study, sampling was carried out in 3 super-intensive pacific white shrimp pond areas spread out in East Java, namely Bangil, Tuban, and Lamongan with 50 shrimps (PL30-PL 40). The obtained data underwent regression and correlation analysis. Based on the results, there were three types of ectoparasites, namely Zoothamnium sp., Epistylis sp. and Vorticella sp. High ectoparasite intensity was found in pacific white shrimp from Lamongan and Tuban ponds, namely 76 and 55 individuals/shrimp, respectively, showing the heavy infestation. High total V. parahaemolyticus bacteria was found in Tuban (1.16 × 105 CFU/gr) and Lamongan (1.16 × 105 CFU / gr) ponds. Based on the results, the coefficient value was R = 0.807 showing positive correlation of V. parahaemolyticus with the increasing parasite intensity and low oxygen levels.

Bacteriophages Against Pathogenic Vibrios in Delaware Bay Oysters (Crassostrea virginica) During a Period of High Levels of Pathogenic Vibrio parahaemolyticus

Eastern oysters (Crassostrea virginica) from three locations along the Delaware Bay were surveyed monthly from May to October 2017 for levels of total Vibrio parahaemolyticus, pathogenic strains of V. parahaemolyticus and Vibrio vulnificus, and for strain-specific bacteriophages against vibrios (vibriophages). The objectives were to determine (a) whether vibriophages against known strains or serotypes of clinical and environmental vibrios were detectable in oysters from the Delaware Bay and (b) whether vibriophage presence or absence corresponded with Vibrio abundances in oysters. Host cells for phage assays included pathogenic V. parahaemolyticus serotypes O3:K6, O1:KUT (untypable) and O1:K1, as well as clinical and environmental strains of V. vulnificus. Vibriophages against some, but not all, pathogenic V. parahaemolyticus serotypes were readily detected in Delaware Bay oysters. In July, abundances of total and pathogenic V. parahaemolyticus at one site spiked to levels exceeding regulatory guidelines. Phages against three V. parahaemolyticus host serotypes were detected in these same oysters, but also in oysters with low V. parahaemolyticus levels. Serotype-specific vibriophage presence or absence did not correspond with abundances of total or pathogenic V. parahaemolyticus. Vibriophages were not detected against three V. vulnificus host strains, even though V. vulnificus were readily detectable in oyster tissues. Selected phage isolates against V. parahaemolyticus showed high host specificity. Transmission electron micrographs revealed that most isolates were ~ 60-nm diameter, non-tailed phages. In conclusion, vibriophages were detected against pandemic V. parahaemolyticus O3:K6 and O1:KUT, suggesting that phage monitoring in specific host cells may be a useful technique to assess public health risks from oyster consumption.

Evaluation of Ice Slurries as a Control for Postharvest Growth of Vibrio spp. in Oysters and Potential for Filth Contamination

Raw oyster consumption is the most common route of exposure for Vibrio spp. infections in humans. Vibriosis has been increasing steadily in the United States despite efforts to reduce the incidence of the disease. Research has demonstrated that ice is effective in reducing postharvest Vibrio spp. growth in oysters but has raised concerns of possible contamination of oyster meat by filth (as indicated by the presence of fecal coliform bacteria or Clostridium perfringens). This study examined the use of ice slurries (<4.5°C) to reduce Vibrio growth. Ice slurries showed rapid internal cooling of oysters, from 23.9°C (75°F) to 10°C (50°F) within 12 min. The initial bacterial loads in the ice slurry waters were near the limits of detection. Following repeated dipping of oysters into ice slurries, water samples exhibited significant (P < 0.05) increases in median levels of fecal coliforms (9.5 most probable number [MPN]/100 ml), C. perfringens (280 MPN/100 ml), Vibrio vulnificus (11,250 MPN/ml), and total Vibrio parahaemolyticus (3,900 MPN/ml). The microbial load in oyster meat, however, was unchanged after 15 min of submergence, with no significant differences (P < 0.05) in levels of filth indicator (range, 250 to 720 MPN/100 g) or Vibrio spp. (range, 9,000 to 20,000 MPN/g) bacteria. These results support the use of ice slurries as a postharvest application for rapid cooling of oysters to minimize Vibrio growth.

Detection of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae with respect to seasonal fluctuations in temperature and plankton abundance

Over a 1-year period, bi-monthly estuarine surface water and plankton samples (63-200 and > 200 μm fractions) were assayed by polymerase chain reaction for the prevalence of total Vibrio parahaemolyticus, V. vulnificus and V. cholerae and select genes associated with clinical strains found in each species. Neither temperature nor plankton abundance was a significant correlate of total V. parahaemolyticus; however, the prevalence of genes commonly associated with clinical strains (trh, tdh, ORF8) increased with temperature and copepod abundance (P < 0.05). The prevalence of total V. vulnificus and the siderophore-related viuB gene also increased with temperature and copepod and decapod abundance (P < 0.001). Temperature and copepod abundance also covaried with the prevalence of V. cholerae (P < 0.05), but there was no significant relationship with ctxA or other genes commonly found in clinical strains. Results show that genes commonly associated with clinical Vibrio strains were more frequently detected in association with chitinous plankton. We conclude that V. parahaemolyticus, V. vulnificus, V. cholerae and subpopulations that harbour genes common to clinical strains respond distinctly to seasonal changes in temperature as well as shifts in the taxonomic composition of discrete plankton fractions.

The relationship of Vibrio parahaemolyticus densities and water quality factor during oyster culture

Vibrio parahaemolyticus(VP)is a pathogen of bacterial food poisoning associated with shellfish.VP,which is detected both in human and fish,has even higher concentrations in oysters and other filter-feeding shellfish.In order to study the relationship of VP densities versus water quality factor including water salinity,temperature,pH and dissolved oxygen in the course of oyster culture,we detected total VP densities in the oyster and water quality factor at the oyster farm in Yangxi,Guangdong from March to November in 2009.The results showed that the detection ratio of total VP in oysters was 94.38%.The water temperature was correlated positively with total VP in oysters,but the salinity was correlated negatively with total VP in oysters.The dissolved oxygen and pH didn't show correlation with total VP in oysters.Based on the regression analysis,a quadratic relationship for VP densities versus water temperature and salinity was found.The regression equation of VP densities versus water temperature was:LgVP=0.093T-0.685(R2=0.336);The regression equation of VP densities versus salinity was:LgVP=1.199+0.116S-0.004S2(R2=0.217);The best fit regression equation of VP densities versus water temperature and salinity was LgVP=-1.941+0.116T+0.058S-0.001S2(R2=0.414).The results of this paper may provide some reference data for the risk analysis of VP during oyster culture.

Real-Time PCR Quantification of Vibrio parahaemolyticus in Oysters Using an Alternative Matrix

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26°C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r =−0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus–specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.