Prospects for Biocontrol of Vibrio parahaemolyticus Contamination in Blue Mussels (Mytilus edulus)-A Year-Long Study

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).

Keywords: Mytilus edulis; RAPD-PCR; Vibrio parahaemolyticus; bacteriophage; chromogenic agar; genetic diversity; temperate estuary.

Figure 1

(A) Map of the East Coast Region of England showing sampling site (Cleethorpes) along the River Humber. (B) Representative agar plate showing lytic activity of bacteriophage on lawn of host V. parahaemolyticus.

Figure 2

(A) Number of Vibrio parahaemolyticus isolated from seawater samples in relation to seawater temperature and salinity. R² = 0.068; P = 0.262 (seawater) and R² = 0.024; P = 0.153 (salinity). R² = 0.066; P = 0.256 (temperature) and R² = 0.006; P = 0.074 (salinity) were observed for counts of V. parahaemolyticus in sediment. And R² = 0.014; P = −0.120 (temperature) and R² = 0.094; P = −0.307 (salinity) were observed for counts of V. parahaemolyticus. (B) Number of Vibrio parahaemolyticus isolated from sediment samples in relation to sediment temperature and salinity. R² = 0.068; P = 0.262 (seawater) and R² = 0.024; P = 0.153 (salinity). R² = 0.066; P = 0.256 (temperature) and R² = 0.006; P = 0.074 (salinity) were observed for counts of V. parahaemolyticus in sediment. And R² = 0.014; P = - 0.120 (temperature) and R² = 0.094; P = −0.307 (salinity) were observed for counts of V. parahaemolyticus. (C) Number of Vibrio parahaemolyticus isolated from mussels in relation to seawater temperature and salinity. R² = 0.068; P = 0.262 (seawater) and R² = 0.024; P = 0.153 (salinity). R² = 0.066; P = 0.256 (temperature) and R² = 0.006; P = 0.074 (salinity) were observed for counts of V. parahaemolyticus in sediment. And R² = 0.014; P = −0.120 (temperature) and R² = 0.094; P = −0.307 (salinity) were observed for counts of V. parahaemolyticus.

Figure 3

(A) Agarose (1.5%) gel electrophoresis of RAPD-PCR products for primer GEN1-50-08 of V. parahaemolyticus isolates. Lanes Lane 1: BD1; Lane 2: BD2; Lane 3: BD3; Lane 4: BD4; Lane 5: BD6; Lane 6: BD7; Lane 7: BD35; Lane 8: BD9; Lane 9: Q-4 DNA ladder; Lane 10: BD14; Lane 11: BD37; Lane 13: BD33; Lane 14: BD54; Lane 15: BD34; Lane 16: BD39. (B) Agarose (1.5%) gel electrophoresis of RAPD-PCR products for primer OPD16 of V. parahaemolyticus isolates. Lane 1: CHL16; Lane 2: CHL1; Lane 3: CHL14; Lane 4: BD7; Lane 5: BD84; Lane 6: BD1; Lane 7: BD3; Lane 8: BD5; Lane 9: Q-4 DNA ladder; Lane 10: BD34; Lane 11: BD30; Lane 12: BD29; Lane 13: BD77; Lane 14: BD71; Lane 15: BD47; Lane 16: BD75.

Figure 4

(A) Simplified dendrogram based on UPGMA generated by Gel compare software showing genetic similarity from RAPD profiles of V. parahaemolyticus isolates (Gen1-50-08). (B) Simplified dendrogram based on UPGMA generated by Gel compare software showing genetic similarity from RAPD profiles of V. parahaemolyticus isolates (OPD16).

Figure 5

Effect of bacteriophage treatment on Vibrio parahaemolyticus SPA2 (A) or SPA3 (B) in Tryptone Soya Broth incubated at 30, 37, and 40°C. Each bar represents the mean V. parahaemolyticus counts of duplicate samples. Error bars denote the standard error of the means of triplicate experiments.

Figure 6

Effect of bacteriophage treatment on V. parahaemolyticus in seawater, sediment or mussels after 72 h. Each line represents the mean of duplicate bacteria counts. Error bars denote the standard error of the mean duplicate bacterial counts.

Figure 7

Representative electrophoresis gel (1.0%) of bacteriophage DNA.