ABSTRACT
Faecal contamination of bivalve molluscan shellfish (BMS) can lead to infections from enteric pathogens if consumed. Across Europe, the faecal indicator bacteria Escherichia coli, is used to determine contamination of BMS harvesting areas. The reference most probable number (MPN) method for E. coli in BMS takes around 48 h from sample receipt to result. In this study, an alternative method was developed in which the final, E. coli confirmation step in the MPN method (usually carried out on chromogenic TBX agar) was replaced by presence/absence real-time PCR (qPCR). This qPCR-MPN method was directly compared with the reference TBX-MPN method using 194 BMS samples consisting of mussels (Mytilus spp.), Pacific oysters (Crassostrea gigas) and common cockles (Cerastoderma edule). The qPCR-MPN method correlated positively with the TBX-MPN method (Kendall's tau coefficient = 0.812). However, the strength of this correlation varied between BMS species, with mussels having the poorest correlation (0.677) followed by Pacific oysters (0.795) and common cockles (0.890). There were some samples for which the difference between the two methods was higher than might be expected by statistical probability alone. Variations in the way in which the two confirmation methods work may account for much of this variation. This method may serve as an ad hoc, rapid assessment method that is complementary to the official reference method and could be easily implemented in many official control laboratories.
ABSTRACT
The recent emergence of Vibrio infections at high latitudes represents a clear human health risk attributable to climate change. Here, we investigate the population dynamics of three Vibrio species: Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae within a British coastal estuarine site, with contrasting salinity and temperature regimes during an intense heatwave event. Water samples were collected weekly through the summer of 2018 and 2019 and filtered using membrane filtration and subsequently grown on selective media. Suspected vibrios were confirmed using a conventional species-specific PCR assay and further analysed for potential pathogenic markers. Results showed that Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae were present at high concentrations throughout both years, with their populations at substantially greater abundances corresponding to conditions of higher water temperatures during the heatwave of 2018 and at lower salinity sites, which is comparable to the results of previous studies. A subset of strains isolated during the extreme heatwave event in 2018 (46 Vibrio parahaemolyticus, 11 Vibrio cholerae and 4 Vibrio vulnificus) were genomically sequenced. Analysis of these 63 sequenced strains revealed a broad phenotypic and genomic diversity of strains circulating in the environment. An analysis of pathogenicity attributes identified a broad array of virulence genes across all three species, including a variety of genes associated with human disease. This study highlights the importance of the need for an increased Vibrio spp. surveillance system in temperate regions and the potential impact warming events such as heatwaves may have on the abundance of potentially pathogenic bacteria in the environment.
