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Background: Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays. Methodology/Principal Findings: We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog's Phenotype MicroArray (TM) technology and some virulence factor assays. Conclusions/Significance: Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum.

The effect of heat-treatment on the internal temperature of raw cockles (Anadara granosa) and survival of their intrinsic flora of Vibrio spp. as well as of inoculated V. cholerae 0139 was examined. The cockles were purchased from markets in Malaysia and had an average weight including shells of 8.90±2.45 g. In one experiment heatpenetration of individual cockles was examined. Cockles weighing <8 g (including shell) exhibited maximum internal temperatures of between 50 and 75°C when heated in water at 99°C for 10 s and 71–93°C when heated for 30 s. Cockles weighing >12 g exhibited maximum internal temperatures between 42 and 58°C when heated in water at 99°C for 10 s and 56–69°C when heated for 30 s. In another experiment, heat-treatment of 10 cockles treated as a group at 99°C for 10 or 30 s resulted in reduction of levels of intrinsic Vibrio spp. (enumerated directly on thiosulphate–citrate–bile salt sucrose agar; TCBS) from 5.73 to 3.15 log cfu g−1 or below 1 log cfu g−1, respectively. The levels of Vibrio spp. after heat-treatment decreased with an increase in numbers of cockles grouped together during treatment. In a third experiment V. cholerae 0139 was inoculated into cockles and subjected to heat-treatment at 99°C for 0, 10, 15, 20, 25 or 30 s. The levels of Vibrio spp. in uninoculated, non-heat-treated cockles was 4.89 log cfu g−1 on TCBS, and the predominant species were V. parahaemolyticus and V. alginolyticus. V. cholerae 0139 inoculated into cockles with an average weight of 13.5±1.90 g (including shell) decreased for samples examined immediately after heat-treatment from 6 log cfu g−1 initially to 3.5 log cfu g−1 after 25 s and <1 log cfu g−1 (TCBS) after 30 s of heat-treatment. The most probable number method by enrichment in alkaline peptone water gave in general within 1 log unit higher counts than TCBS direct enumeration. TCBS direct enumeration and MPN counts were up to 2.38 or 1.30 log units higher, respectively, for samples heat-treated for 20 s

The predominant microbial flora of a specific Malaysian food ingredient. chili bo (containing 9% ground dried chilies. 0.6% acetic acid, and 5 to 10% cornstarch, wt/vol) stored for up to 25 days at 28°C without added benzoic acid (product A) and with 7,000 ppm of added benzoic acid (product B) was examined. Aerobic plate counts for both products were initially 6.2 to 6.5 log CFU/g increasing to 8.5 log CFU/g for product A after 4 days. Aerobic plate counts for product B did not increase during storage. Lactic acid bacteria (LAB) counts increased in product A from 4.8 log CFU/g to 8.3 log CFU/g and in product B from 2.1 log CFU/g to 7.6 log CFU/g after 17 days. Growth of yeast occurred in product A. Both products exhibited spoilage after 1 to 2 days of storage at 28°C indicated as accumulation of gas bubbles. In addition surface growth of molds (product A) or whitish discoloration (product B) was observed later in storage. For product A the predominant isolates were LAB. Bacillus pumilus, Bacillus subtilis, Staphylococcus spp., and yeasts. B. pumilus and B. subtilis predominated initially whereas the other types of microorganisms predominated after 25 days of storage. B. pumilus and B. subtilis were also predominant in product B, but after 25 days of storage a homofermentative LAB was found in higher numbers (7.6 log CFU/g). Isolates of heterofermentative LAB but not homofermentative LAB or B. pumilus or B. subtilis were able to produce gas during growth in chili be sterilized by autoclaving at 121°C for 15 min. Growth of heterofermentative LAB, B. pumilus, and B. subtilis was inhibited by acidifying agents, a nisin-containing supernatant, or incubation at low temperatures.