Degradation intermediates of polyhydroxy butyrate inhibits phenotypic expression of virulence factors and biofilm formation in luminescent Vibrio sp. PUGSK8

George Seghal Kiran, Sethu Priyadharshini, Alan D W Dobson, Elumalai Gnanamani, Joseph Selvin

Luminescent vibrios are ubiquitous in the marine environment and are the causative agents of vibriosis and mass mortality in many aquatic animals. In aquatic environments, treatments cannot be limited to the diseased population alone, therefore treatment of the entire aquatic system is the only possible approach. Thus, the use of antibiotics to treat part of the infected animals requires a dose based on the entire biomass, which results in the treatment of uninfected animals as well as non-target normal microbial flora. A treatment method based on anti-virulence or quorum quenching has recently been proposed as an effective treatment strategy for aquatic animals. Polyhydroxy butyrates (PHB) are bacterial storage molecules, which accumulate in cells under nutritional stress. The degradation of PHB releases short-chain β-hydroxy butyric acid, which may act as anti-infective molecule. To date, there is very limited information on the potential anti-infective and anti-virulence mechanisms involving PHB. In this study, we aim to examine the effect of PHB on inhibition of the virulence cascade of Vibrio such as biofilm formation, luminescence, motility behaviour, haemolysin and quorum sensing. A luminescent Vibrio PUGSK8, tentatively identified as Vibrio campbellii PUGSK8 was tested in vitro for production of extracellular virulence factors and then established as a potential shrimp pathogen based on in vivo challenge experiments. The ability of Vibrio PUGSK8 to form biofilms and the effect of PHB on biofilm formation was tested in a 96-well microtitre-plate assay system. The motility behaviour of Vibrio PUGSK8 was evaluated using twitching, swimming and swarming plate assays. Reporter strains such as Chromobacterium violaceum CV026 and Agrobacterium tumefaciens were used to detect quorum-sensing molecules. Gas chromatography-mass spectrometry spectral analysis was performed to elucidate the fragmentation pattern and structure of N-hexanoyl homoserine lactone. PHB depolymerase activity in Vibrio PUGSK8 was quantified as the amount of the enzyme solution to hydrolyse 1 μg of PHB per min. An in vivo challenge experiment was performed using a gnotobiotic Artemia assay. Of the 27 isolates tested, the Vibrio PUGSK8 strain was selected for target-specific assays based on the high intensity of luminescence and production of virulence factors. The virulence cascade detected in Vibrio PUGSK8 include luminescence, motility behaviour, biofilm formation, quorum sensing and haemolysin production. Thus inhibition/degradation of the virulence cascade would be an effective approach to contain Vibrio infections in aquatic animals. In this report, we demonstrate that the degradation intermediate of PHB effectively inhibits biofilm formation, luminescence, motility behaviour, haemolysin production and the N-acyl-homoserine lactone (AHL)-mediated quorum-sensing pathway in PUGSK8. Interestingly, the growth of Vibrio PUGSK8 remains unaffected in the presence of PHB, with PHB degradation being detected in the media. PHB depolymerase activity in Vibrio PUGSK8 results in the release of degradation intermediates include a short-chain β-hydroxy butyric acid, which inhibits the virulence cascade in Vibrio PUGSK8. Thus, a molecule that targets quorum sensing and the virulence cascade and which is species/strain-specific could prove to be an effective alternative to antimicrobial agents to control the pathogenesis of Vibrio, and thereby help to contain Vibrio outbreaks in aquatic systems.