Relation between virulence of Vibrio anguillarum strains and response to the host factors mucin, bile salts and cholesterol.

AIMS: In this study, we investigated the responsiveness of 15 Vibrio anguillarum strains to three host factors (mucin, bile salts and cholesterol). METHODS AND RESULTS: Three virulence-related phenotypes were investigated in this respect, i.e. motility, biofilm formation and exopolysaccharide production. Almost all V. anguillarum strains showed a significantly increased motility in the presence of either of the three host factors. Only five of the strains showed increased biofilm formation in the presence of host factors and only three strains showed increased exopolysaccharide production in the presence of the host factors. CONCLUSIONS: There were no significant correlations between the three putatively virulence-linked phenotypes (in the absence of host factors) and virulence to sea bass larvae. There was no correlation between responsiveness to the host factors (percentage increase in motility, biofilm formation or exopolysaccharide production in the presence of the three host factors) and virulence to sea bass larvae. However, the responses of these virulence-related phenotypes upon the addition of either of the three host factors were significantly correlated with each other. This result suggests that the mechanisms by which V. anguillarum responds to these three host factors is linked. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the mechanism by which V. anguillarum responds to the host factors mucin, bile salts and cholesterol seems to be linked, there is no correlation between host factor responsiveness and virulence towards sea bass larvae. This emphasizes that one should be careful when extrapolating results obtained for one particular strain to reach general conclusions on a species of pathogenic bacteria.

Expression of virulence genes in luminescent and nonluminescent isogenic vibrios and virulence towards gnotobiotic brine shrimp (Artemia franciscana).

AIMS: This study aimed to evaluate the expression levels of virulence gene regulators (luxR and toxR) and virulence factors (serine protease, metalloprotease and haemolysin) in luminescent and nonluminescent isogenic Vibrio harveyi and Vibrio campbellii. METHOD AND RESULTS: Nonluminescent variants have been reported before to become dominant in cultures of luminescent vibrios when grown under static conditions in the dark. Wild-type V. harveyi BB120, V. campbellii LMG 21363, quorum sensing mutants of V. harveyi BB120 and their previously reported nonluminescent isogenic counterparts were used in this study. The expression level of the virulence genes srp serine protease, vhp metalloprotease and vhh haemolysin, the quorum sensing master regulator gene luxR and the virulence regulator gene toxR in isogenic luminescent and nonluminescent strains were quantified using reverse transcriptase real-time PCR. These experiments revealed that the nonluminescent strains produced lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene (which is known to be regulated by quorum sensing). Finally, challenge tests with gnotobiotic brine shrimp (Artemia franciscana) larvae revealed that the nonluminescent strains are less virulent than their luminescent isogenic counterparts. CONCLUSION: Nonluminescent variants of V. harveyi and V. campbellii strains produce lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene and are less virulent to brine shrimp than their isogenic luminescent counterparts. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that adaptation of luminescent vibrios to specific growth conditions that result in a dominant nonluminescent phenotype is accompanied by a decreased adaptation to a host environment because of altered virulence gene regulation.

The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi.

AIMS: To study the potential biocontrol activity of bioflocs technology. METHODS AND RESULTS: Glycerol-grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth-inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing-regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. CONCLUSIONS: Bioflocs grown on glycerol as carbon source inhibit quorum sensing-regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond.

Quorum quenching bacteria protect Macrobrachium rosenbergii larvae from Vibrio harveyi infection.

AIMS: In this study, we investigated the effect of N-acyl homoserine lactone-degrading bacterial enrichment cultures (ECs) on larviculture of the giant freshwater prawn Macrobrachium rosenbergii. METHODS AND RESULTS: The larval performance in terms of larval growth, larval survival, larval quality, duration of the larval rearing process and microflora levels in the rearing water as well as inside the prawn gut was investigated. The application of the EC bacteria was performed in two ways: by adding them directly into the larval rearing water and via enriched Artemia nauplii used for larval feeding. The results of the study demonstrated that both ECs that were tested had a similar positive effect on larval survival and larval quality, whereas they did not affect larval growth or the duration of the larval rearing process. CONCLUSIONS: Under normal hatchery conditions, the optimal EC densities were found to be 10(6) CFU ml(-1) for adding into the rearing water and 5 × 10(8) CFU ml(-1) for enrichment of Artemia nauplii used for feeding of the larvae. In the hatchery, the ECs can be grown on waste streams of Artemia hatching. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of this kind of ECs could lead to a more sustainable aquaculture production, by replacing the use of antibiotics to control diseases.

Presence of typical and atypical virulence genes in vibrio isolates belonging to the Harveyi clade.

AIMS: The study was aimed at investigating the presence of typical and atypical virulence genes in isolates belonging to the Harveyi clade (Vibrio harveyi and Vibrio campbellii). METHODS AND RESULTS: Forty-eight vibrio isolates belonging to the Harveyi clade were screened for the presence of virulence genes that are typical for these bacteria and those found in human pathogenic vibrios such as Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus and aquatic pathogenic Vibrio anguillarum. The virulence genes were amplified by PCR with specific primers, and the presence further confirmed by dot blot hybridization. The virulence genes vhh, chiA, vhpA, toxR(Vh), luxR and serine protease, typical of Harveyi clade were detected in all the isolates. The haemolysin gene hlyA and the virulence regulator gene toxR(Vc) specific to V. cholerae and the V. anguillarum-specific flagellum gene (flaC) were present in some of the isolates. Challenge tests with gnotobiotic Artemia nauplii did not show any correlation between the presence of the virulence genes and virulence of the isolates. CONCLUSION: From our results, there appears a remote possibility that vibrios belonging to the Harveyi clade might acquire virulence genes from other vibrios in the aquatic environment through horizontal gene transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrios belonging to the Harveyi clade may be an important reservoir of virulence genes of other (human pathogenic) Vibrio species in the aquatic environment. The acquisition of virulence genes by horizontal transfer might increase the ability of Harveyi clade vibrios to infect aquatic organisms by increasing their virulence to a specific host by broadening their host range. The detection of such genes may forewarn the hatchery operators about a potentially virulent pathogen and thus help to develop management measures to handle the problem of vibriosis.

Virulence of luminescent and non-luminescent isogenic vibrios towards gnotobiotic Artemia franciscana larvae and specific pathogen-free Litopenaeus vannamei shrimp.

AIMS: This study was conducted to test the virulence of luminescent (L) and non-luminescent (NL) isogenic strains of Vibrio campbellii LMG21363, Vibrio harveyi BB120 (wild type) and quorum-sensing mutant strains derived from the wild type such as Vibrio harveyi BB152, BB170, MM30 and BB886. METHODS AND RESULTS: The NL strains could be obtained by culturing rifampicin-resistant luminescent strains in the dark under static condition. The virulence of the L and NL strains was tested in gnotobiotic Artemia franciscana larvae challenged with 10(4) CFU ml(-1) of bacteria. All luminescent isogenic tested strains showed higher virulence compared to the NL strains. The virulence of L and NL V. campbellii and V. harveyi BB120 was also tested in specific pathogen-free juvenile shrimp upon intramuscular injection with 10(6) CFU of bacteria. In contrast with Artemia, there was no significant difference in mortality between the groups challenged with L and NL strains (P > 0.05). The non-luminescent strains were not able to revert back to the luminescent state and quorum sensing did not influence this phenotypic shift. CONCLUSIONS: Luminescent Vibrio strains can switch to a non-luminescent state by culturing them in static conditions. The NL strains become less virulent as verified in Artemia. SIGNIFICANCE AND IMPACT OF THE STUDY: The luminescent state of Vibrio cells in a culture needs to be verified in order to assure maintenance of virulence.

Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates.

AIMS: To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios. METHODS AND RESULTS: Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free culture fluids of all strains significantly induced bioluminescence in the cholerae autoinducer 1, autoinducer 2 and harveyi autoinducer 1 reporter strains JAF375, JMH597 and JMH612, respectively. There was no relation between luminescence and signal production and virulence towards brine shrimp. CONCLUSIONS: There is a large difference between different strains of Vibrio campbellii and Vibrio harveyi with respect to bioluminescence. However, this is not reflected in signal production and virulence towards gnotobiotic brine shrimp. Moreover, there seems to be no relation between quorum sensing signal production and virulence towards brine shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here indicate that strains that are most brightly luminescent are not necessarily the most virulent ones and that the lower virulence of some of the strains is not due to a lack of autoinducer production.