Monitoring of vibrio species in oysters crassostrea virginica and seawater of Delaware bay and molecular characterization of vibrio parahaemolyticus
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ABSTRACT
Delaware Bay is the prime oystering ground in Delaware. Oysters naturally inhabit marine environments and, as filter-feeding mollusks, they tend to accumulate different microorganisms from seawater during their filtration process. Most of the illnesses caused by the consumption of raw and undercooked oysters are strongly associated with the oysters contaminated with Vibrio spp. Vibrio spp. are Gram-negative pathogenic bacteria from the family Vibrionaceae that negatively impact aquatic systems and human health. This study aimed to identify and differentiate Vibrio spp. in oyster and seawater samples from the Delaware Bay using CHROMagarᵀᴹ Vibrio medium which provides the means for easy differentiation of Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, and Vibrio alginolyticus based on the colony color. Pathogenic Vibrio parahaemolyticus was characterized using conventional PCR which was applied for the identification of total and pathogenic V. parahaemolyticus by screening the species-specific tlh gene, and the virulent tdh, trh, toxR, and vpm genes.
Vibrio spp. were differentiated and isolated, and total and pathogenic V. parahaemolyticus from oyster and seawater samples were identified. Oysters and seawater samples were collected once a month from June through October 2016, from Bowers Beach, and Lewes, Broadkill in Delaware Bay. A third site, Slaughter Beach, was added to this study from August through October 2016. Physico-chemical water quality parameters recoded on-site during samples collection include water and air temperature, salinity, pH, dissolved oxygen, turbidity, chlorophyll a, depth, conductivity, weather, wind direction, wind speed, and tide.
Oyster and seawater samples were serially diluted up to (10⁻⁶), and 100 µL of each dilution from each sample were spread plated on CHROMagarᵀᴹ Vibrio medium. V. parahaemolyticus, V. vulnificus/V. cholerae, and V. alginolyticus colonies were identified based on color development (mauve, green/blue, and colorless, respectively). Average Vibrio spp. in oyster ranged from <10 CFU g-1 (non-detectable) to 2.8×104 CFU g-1, while the average of Vibrio spp. in seawater samples ranged from 1.7×10 CFU mL-1 to 4.47×103 CFU mL-1. More Vibrio colony forming units were observed during the warmer months (June through September). As anticipated, the total colony forming units of Vibrio spp. were proportional in both oyster and seawater samples. The multiple regression model indicated a significant positive relationship between Vibrio spp. and V. parahaemolyticus Log10 CFUs and water temperature, (p < 0.05). The one-way ANOVA analysis showed no statistical significant association between Vibrio spp. Log10 CFUs and the sample type, oysters and seawater (p > 0.05).
A total of 165 bacterial isolates of Vibrio parahaemolyticus (mauve colonies) from oyster and seawater samples were examined for the presence of tlh, tdh, trh, toxR, and vpm genetic markers. Only 19% of the samples tested were positive for tdh and 24% were positive for trh while 83%, 65.5%, and 67% of the samples were positive for tlh, toxR, and vpm, respectively. This confirmed the specificity of tlh gene for V. parahaemolyticus species. Screening for tdh and trh genes is not sufficient for the surveillance of pathogenic or potential pathogenic V. parahaemolyticus but the reliability of toxR and vpm as gene markers is notably higher.