Abstract
Vibrio parahaemolyticus is an environmental organism normally found in subtropical estuarine environments which can cause seafood-related human infections. Clinical disease is associated with diagnostic presence of tdh and/or trh virulence genes and identification of these genes in our preliminary isolates from retail shellfish prompted a year-long surveillance of isolates from a temperate estuary in the north of England. The microbial and environmental analysis of 117 samples of mussels, seawater or sediment showed the presence of V. parahaemolyticus from mussels (100%) at all time-points throughout the year including the colder months although they were only recovered from 94.9% of seawater and 92.3% of sediment samples. Throughout the surveillance, 96 isolates were subjected to specific PCR for virulence genes and none tested positive for either. The common understanding that consuming poorly cooked mussels only represents a risk of infection during summer vacations therefore is challenged. Further investigations with V. parahaemolyticus using RAPD-PCR cluster analysis showed a genetically diverse population. There was no distinct clustering for “environmental” or “clinical” reference strains although a wide variability and heterogeneity agreed with other reports. Continued surveillance of isolates to allay public health risks are justified since geographical distribution and composition of V. parahaemolyticus varies with Future Ocean warming and the potential of environmental strains to acquire virulence genes from pathogenic isolates. The prospects for intervention by phage-mediated biocontrol to reduce or eradicate V. parahaemolyticus in mussels was also investigated. Bacteriophages isolated from enriched samples collected from the river Humber were assessed for their ability to inhibit the growth of V. parahaemolyticus strains in-vitro and in-vivo (with live mussels). V. parahaemolyticus were significantly reduced in-vitro, by an average of 1 log−2 log units and in-vivo, significant reduction of the organisms in mussels occurred in three replicate experimental tank set ups with a “phage cocktail” containing 12 different phages. Our perspective biocontrol study suggests that a cocktail of specific phages targeted against strains of V. parahaemolyticus provides good evidence in an experimental setting of the valuable potential of phage as a decontamination agent in natural or industrial mussel processing (343w).
Highlights
The use of chromogenic agar was more effective in detecting V. parahaemolyticus isolates than Thiosulphate Citrate Bile Salt Sucrose (TCBS)
It was observed that more presumptive colonies of V. parahaemolyticus were detected on chromogenic agar than on TCBS, when the same amount of sample was plated onto the different selective agar
The violet color of V. parahaemolyticus remained on Chromogenic vibrio (CV) agar even when they were physically covered by other colored colonies produced by different vibrio species or other bacteria whereas V. parahaemolyticus colonies on TCBS agar were occasionally hidden by the yellow color produced by sucrose-fermenting bacteria such as V. alginolyticus
Summary
V. parahaemolyticus is a Gram-negative bacterium commonly found in marine and estuarine coastal environments (Feldhusen, 2000; Ceccarelli et al, 2013; Letchumanan et al, 2014; Malcolm et al, 2015; Raghunath, 2015) and can cause shellfish-related gastroenteritis (Hazen et al, 2015; Raghunath, 2015), wound infection and septicaemia in sub-tropical environments (Daniels et al, 2000; Zhang and Orth, 2013). Two well-described haemolysins, thermostable direct haemolysin genes (tdh) and tdh—related haemolysin (trh) are present (Honda and Iida, 1993; Nishibuchi and Kaper, 1995; Bej et al, 1999; Harth et al, 2009; Johnson et al, 2012; Zhang and Orth, 2013; Letchumanan et al, 2014; Raghunath, 2015) The detection of these gene sequences by PCR amplification targeted by specific oligonucleotide primers are the most important predictive measure of the pathogenicity of V. parahaemolyticus strains for human illness (Johnson et al, 2012). The method generates “fingerprints” that can be used to compare bacteria both at the inter-species and intra-species level with high discriminating power and (a) does not require previous knowledge of sequences in the DNA of the isolate under study (b) produces a DNA pattern that allows comparison of many loci simultaneously, (c) simple and relatively low cost and requires only nanogram amounts of template DNA
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