ABSTRACT
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.
Subject(s)
Artemia/microbiology , Bacterial Proteins/metabolism , Vibrio/pathogenicity , Virulence Factors/metabolism , Animals , Artemia/growth & development , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression , Genes, Regulator , Germ-Free Life , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Larva/microbiology , Luminescence , Metalloproteases/genetics , Metalloproteases/metabolism , Quorum Sensing , Repressor Proteins/genetics , Repressor Proteins/metabolism , Serine Proteases/genetics , Serine Proteases/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Vibrio/genetics , Vibrio/metabolism , Virulence/genetics , Virulence Factors/geneticsABSTRACT
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.
Subject(s)
Vibrio/pathogenicity , Virulence Factors/genetics , Animals , Artemia/microbiology , Genes, Bacterial , Polymerase Chain Reaction/methods , Vibrio/classification , Vibrio/genetics , Vibrio/isolation & purification , Virulence/geneticsABSTRACT
AIMS: Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR. METHODS AND RESULTS: Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0.95, while ERIC-PCR showed a DI of 0.94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. CONCLUSIONS: The use of protein profiling in combination with other established typing methods such as RAPD and ERIC-PCR generates useful information in the case of V. parahaemolyticus associated with seafood. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the usefulness of nucleic acid and protein-based studies in understanding the relationship between various isolates from seafood.
