Current Perspective on the Membrane-Damaging Action of Thermostable Direct Hemolysin, an Atypical Bacterial Pore-forming Toxin

Thermostable direct hemolysin (TDH) is the major virulence determinant of the gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin (PFT). TDH shares remarkable structural similarity with the actinoporin family of eukaryotic PFTs produced by the sea anemones. Unlike most of the PFTs, it exists as tetramer in solution, and such assembly state is crucial for its functionality. Although the structure of the tetrameric assembly of TDH in solution is known, membrane pore structure is not available yet. Also, the specific membrane-interaction mechanisms of TDH, and the exact role of any receptor(s) in such process, still remain unclear. In this mini review, we discuss some of the unique structural and physicochemical properties of TDH, and their implications for the membrane-damaging action of the toxin. We also present our current understanding regarding the membrane pore-formation mechanism of this atypical bacterial PFT.

Prevalence and Characterization of Food-Borne Vibrio parahaemolyticus From African Salad in Southern Nigeria

The demand for minimally processed vegetables (African salad) has increased partly due to its inclusion in ready-to-eat foods. Nevertheless, the associated risk of the presence of emergent foodborne pathogens, such as Vibrio parahaemolyticus might be underestimated. The present study was designed to isolate and characterize foodborne V. parahaemolyticus from minimally processed vegetables using culture-based methods and molecular approach. A total of 300 samples were examined from retail outlets between November 2018 and August 2019 from Southern Nigeria. The prevalence of vibrios from the overall samples based on the colonial proliferation of yellow, blue-green and/or green colonies on thiosulfate citrate bile salts sucrose agar was 74/300 (24.6%). An average of two green or blue-green colonies from respective plates was screened for V. parahaemolyticus using analytical profile index (API) 20 NE. Polymerase chain reaction further confirmed the identity of positive V. parahaemolyticus. The counts of V. parahaemolyticus ranged from 1.5 to 1,000 MPN/g. A total of 63 recovered V. parahaemolyticus were characterized further. The resistance profile of the isolates include ampicillin 57/63 (90.5%), cefotaxime 41/63 (65.1%), ceftazidime 30/63 (47.6%), amikacin 32/63 (50.8%), kanamycin 15/63 (23.8%), and oxytetracycline 16/63 (25.4%). The multiple antibiotic index ranged from 0–0.81. The formation of biofilm by the isolates revealed the following: strong formation 15/63 (23.8%), moderate formation 31/63 (49.2%), weak formation 12/63 (19.1%), and no formation 5/63 (7.9%). A total of 63/63 (100%), 9/63 (14.3%), and 20/63 (31.8%) of the isolates harbored the tox R gene, TDH-related hemolysin (trh) and thermostable direct hemolysin (tdh) determinants respectively. The isolates with O2 serogroup were most prevalent via PCR. Isolates that were resistant to tetracycline, kanamycin, and chloramphenicol possessed resistant genes. The presence of multidrug-resistant vibrios in the minimally processed vegetables constitutes a public health risk and thus necessitates continued surveillance.

Characterization and Analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in Pandemic and Non-Pandemic Vibrio parahaemolyticus Isolates from Seafood Sources

Vibrio parahaemolyticus is one of the significant seafood-borne pathogens causing gastroenteritis in humans. Clustered regularly interspaced short palindromic repeats (CRISPR) are commonly detected in the genomes of V. parahaemolyticus and the polymorphism of CRISPR patterns has been applied as a genetic marker for tracking its evolution. In this work, a total of 15 pandemic and 36 non-pandemic V. parahaemolyticus isolates obtained from seafood between 2000 and 2012 were characterized based on hemolytic activity, antimicrobial susceptibility, and CRISPR elements. The results showed that 15/17 of the V. parahaemolyticus seafood isolates carrying the thermostable direct hemolysin gene (tdh+) were Kanagawa phenomenon (KP) positive. The Multiple Antibiotic Resistance (MAR) index ranged between 0.1 and 0.4, and 45% of the isolates have an MAR index ≥ 0.2. A total of 19 isolates were positive for CRISPR detection, including all tdh+ trh− isolates, two of tdh− trh+, and each of tdh+ trh+ and tdh− trh−. Four spacer types (Sp1 to Sp4) were identified, and CRISPR-positive isolates had at least one type of spacer homolog to the region of Vibrio alginolyticus megaplasmid. It is of interest that a specific CRISPR profile and spacer sequence type was observed with correlations to the hemolysin genotype (tdh/trh). Thus, these provide essential data on the exposure of foreign genetic elements and indicate shared ancestry within different genotypes of V. parahaemolyticus isolates.

Vibrio parahaemolyticus: a review on the pathogenicity, antibiotic resistance, foodborne outbreaks, and detection methods

Vibrio parahaemolyticus is a Gram-negative bacterium that is a natural inhabitant of the marine habitat. V. parahaemolyticus is a human foodborne pathogen linked to the consumption of contaminated raw and undercooked seafood. V. parahaemolyticus pathogenicity has been linked to the presence of two virulence gene that is thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). The emergence of antibiotic resistant strain of V. parahaemolyticus is a menace to public health. V. parahaemolyticus is linked to several foodborne diseases in Asian countries including Japan, China and Taiwan and has been acknowledged as the major cause of human gastroenteritis in the United States. The emergence of pathogenic Vibrio species in shellfish in Malaysia requires persistent monitoring and public enlightenment on food safety. Several detection methods based on its virulence factors are used in detecting V. parahaemolyticus. This review will provide an insight on V. parahaemolyticus, its pathogenicity, antibiotic resistance, foodborne outbreaks and detection methods.

Effect of food matrix type on growth characteristics and hemolysin production of Vibrio alginolyticus

The growth and hemolysin production of two V. alginolyticus strains (HY9901 and ATCC17749T) at 30 °C in briny tilapia, shrimp, scallop, oyster, pork, chicken, freshwater fish and egg fried rice were investigated. Bacterial counts were enumerated by plate counting. Hemolysin production was evaluated by blood agar and hemolytic titer tests. The two V. alginolyticus strains displayed similar growth and hemolysin production patterns in the foods. Based on the goodness of fit primary model statistics (R 2 , MSE, BF, AF), the modified Gompertz model was a better fit to V. alginolyticus growth in foods than the logistic model. Growth kinetic parameters of V. alginolyticus displayed a higher μ max and shorter λ in briny tilapia > shrimp > freshwater fish > egg fried rice > scallop > oyster > chicken > pork. It was notable that the V. alginolyticus counts were similar at the stationary phase, with no significant growth behavior difference between raw and cooked foods. Significantly higher (p < 0.05) thermostable direct hemolysin (TDH) activity was produced by V. alginolyticus in briny tilapia > freshwater fish > shrimp > chicken > egg fried rice > scallop > oyster > pork. But the hemolytic titer was not consistent with the TDH activity, being significantly higher (p < 0.05) in briny tilapia > egg fried rice > shrimp > freshwater fish > chicken > scallop > oyster > pork. Contrary to current belief, V. alginolyticus displayed a higher hemolysin production in some non-seafoods (freshwater fish, egg fried rice and chicken) than in scallop or oyster. This is the first report of growth and toxicity of V. alginolyticus in different food matrices and confirmation that some non-seafood contaminated with V. alginolyticus can be even more pathogenic. This study will enhance the awareness of non-seafood safety and improve the V. alginolyticus risk assessment accuracy.

Assessment of Vibrio parahaemolyticus levels in oysters (Crassostrea virginica) and seawater in Delaware Bay in relation to environmental conditions and the prevalence of molecular markers to identify pathogenic Vibrio parahaemolyticus strains

This study identified Vibrio parahaemolyticus in oyster and seawater samples collected from Delaware Bay from June through October of 2016. Environmental parameters including water temperature, salinity, dissolved oxygen, pH, and chlorophyll a were measured per sampling event. Oysters homogenate and seawater samples were 10-fold serially diluted and directly plated on CHROMagarᵀᴹ Vibrio medium. Presumptive V. parahaemolyticus colonies were counted and at least 20% of these colonies were selected for molecular chracterization. V. parahaemolyticus isolates (n = 165) were screened for the presence of the species-specific thermolabile hemolysin (tlh) gene, the pathogenic thermostable direct hemolysin (tdh)/ thermostable related hemolysin (trh) genes, the regulatory transmembrane DNA-binding gene (toxR), and V. parahaemolyticus metalloprotease (vpm) gene using a conventional PCR. The highest mean levels of the presumptive V. parahaemolyticus were 9.63×103 CFU/g and 1.85×103 CFU/mL in the oyster and seawater samples, respectively, during the month of July. V. parahaemolyticus levels in oyster and seawater samples were significantly positively correlated with water temperature. Of the 165 isolates, 137 (83%), 110 (66.7%), and 108 (65%) were tlh+, vpm+, and toxR+, respectively. Among the V. parahaemolyticus (tlh+) isolates, 7 (5.1%) and 15 (10.9%) were tdh+ and trh+, respectively, and 24 (17.5%), only oyster isolates, were positive for both genes. Potential pathogenic strains that possessed tdh and/or trh were notably higher in oyster (39%) than seawater (15.6%) isolates. The occurrence of total V. parahaemolyticus (tlh+) was not necessarily proportional to the potential pathogenic V. parahaemolyticus. Co-occurrence of the five genetic markers were observed only among oyster isolates. The co-occurrence of the gene markers showed a relatedness potential of tdh occurrence with vpm. We believe exploring the role of V. parahaemolyticus metalloprotease and whether it is involved in the toxic activity of the thermostable direct hemolysin (TDH) protein can be of significance. The outcomes of this study will provide some foundation for future studies regarding pathogenic Vibrio dynamics in relation to environmental quality.

Effect of food matrix type on growth characteristics and hemolysin production of Vibrio alginolyticus

Background: Vibrio alginolyticus is an important seafood-borne pathogen. There is increasing evidence that V.alginolyticus can also contaminate non-seafood by cross contamination and thereby cause food poisoning in humans. The growth and hemolysin production of V. alginolyticus at 30 °C in briny tilapia, shrimp, scallop, oyster, pork, chicken, freshwater fish and egg fried rice were investigated. Bacterial counts were enumerated by plate counting. Hemolysin production was evaluated by blood agar and hemolytic titer tests.Results: Based on the goodness of fit primary model statistics (R 2 , MSE, BF, AF), the modified Gompertz model was a better fit to V. alginolyticus growth in foods than the logistic model. Growth kinetic parameters of V. alginolyticus displayed a higher μ max and shorter λ in briny tilapia > shrimp > freshwater fish > egg fried rice > scallop > oyster > chicken > pork. It was notable that the V. alginolyticus counts were similar at the stationary phase, with no significant growth behavior difference between raw and cooked foods. However, higher thermostable direct hemolysin activity and hemolytic titer were observed in briny tilapia > egg fried rice > shrimp > freshwater fish > chicken > scallop > oyster > pork.Conclusion: V. alginolyticus growth was good in all food matrix types tested. Contrary to current belief, V. alginolyticus displayed a higher hemolytic activity in some non-seafoods (freshwater fish, egg fried rice and chicken) than in scallop or oyster. This is the first report of growth and toxicity of V. alginolyticus in different food matrices and confirmation that some non-seafood contaminated with V. alginolyticus can be even more pathogenic. This study will enhance the awareness of non-seafood safety and improve the V. alginolyticus risk assessment accuracy.

Effect of food matrix type on growth characteristics and hemolysin production of Vibrio alginolyticus

Background: The growth and hemolysin production of V. alginolyticus at 30 °C in briny Tilapia , shrimp, scallop, oyster, pork, chicken, freshwater fish and egg fried rice were investigated. Bacterial counts were enumerated by plate counting. Hemolysin production was evaluated by blood agar and hemolytic titer tests. Results: Based on the goodness of fit primary model statistics (R 2 , MSE, BF, AF), the modified Gompertz model was a better fit to V. alginolyticus growth in foods than the logistic model. Growth kinetic parameters of V. alginolyticus displayed a higher μ max and shorter λ in briny Tilapia > shrimp > freshwater fish > egg fried rice > scallop > oyster > chicken > pork. It was notable that the V. alginolyticus counts were similar at the stationary phase, with no significant growth behavior difference between raw and cooked foods. However, higher thermostable direct hemolysin activity and hemolytic titer were observed in briny Tilapia > egg fried rice > shrimp > freshwater fish > chicken > scallop > oyster > pork. Conclusion: V. alginolyticus growth was good in all food matrix types tested. Contrary to current belief, V. alginolyticus displayed a higher hemolytic activity in some non-seafoods (freshwater fish, egg fried rice and chicken) than in scallop or oyster. This is the first report of growth and toxicity of V. alginolyticus in non-seafood. This finding will significantly improve the accuracy of microbial risk assessment of V. alginolyticus in different food matrices especially during warmer climatic periods when it is most prevalent.