Behind the Paper - Aquatic bacteria: Interfering with biofilm formation in pathogenic Vibrio

Discover the story behind our paper, "Degradation intermediates of polyhydroxy butyrate inhibits phenotypic expression of virulence factors and biofilm formation in luminescent Vibrio sp. PUGSK8", which was published in npj Biofilms and Microbiomes

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Our article entitled "Degradation intermediates of polyhydroxy butyrate inhibits phenotypic expression of virulence factors and biofilm formation in luminescent Vibrio sp. PUGSK8" was published in the journal npj Biofilms and Microbiomes. The Nature Research team had a few questions for us about our article, which we have answered below.

What was the main aim of your research and why did you decide to investigate this?

Our main aim of this research work was to develop an effective treatment method to contain vibriosis in aquatic systems. In aquatic environment, treatment of individual animals is not possible, then treatment of the system is the only possible approach. Then use of antibiotics to treat part of the infected animals requires a dose based on entire biomass would carry treatment to uninfected animals as well as to the non-target normal microbial flora. This type of existing treatment methods using antibiotics could increase the level of antibiotics resistance and residual accumulation in harvested fish/shrimp. Indeed antibiotics are becoming increasingly ineffective in the control of pathogenic vibrios and this coupled with the fact that the use of antibiotics in animal husbandry is banned in many countries; has resulted in an increased interest in the use of alternate treatment methods to treat vibrio outbreaks in shrimp aquaculture. Therefore safer, eco-friendly approaches are important to control and prevent pathogenic outbreaks of Vibrios.

A treatment-method based on anti-virulence or quorum quenching was proposed in this study 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 molecules. 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 in Vibrio include biofilm, luminescence, motility behavior, hemolysin and quorum sensing.

How did you go about designing your study?

This study was designed based on already existing preliminary data in our Laboratory include,

1. Antiadhesive effect of poly-hydroxy butyrate from a marine Brevibacterium casei MSI04 against shrimp pathogenic vibrios (Kiran et al. 2014).

2. 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.

In an in vitro treatment experiments (effect PHB against biofilms of vibrios), we observed PHB depolymerase activity in Vibrio PUGSK8 results in the release degradation intermediates which inhibits luminescence in Vibrio PUGSK8. Therefore we designed this study to test the effect of degradation intermediates of PHB, released as a result of PHB depolymerase activity in Vibrio PUGSK8, on inhibition of the virulence cascade of Vibrio PUGSK8 such as biofilm, luminescence, motility behavior, hemolysin and quorum sensing.

What challenges did you face?

We find difficulty in assigning a taxonomic position to Vibrio PUGSK8 under Vibrio clade. For instance luminescent V. campbellii belonging to the V. harveyi clade are known to use the quorum sensing molecule N-hexanoylhomoserine lactone. The quorum sensing molecules identified in luminescent vibrios include C6 homoserine lactone in V. fischeri and C4 homoserine lactone in V. harveyi. But there is little conclusive information on quorum sensing molecules produced in various other species in the V. harveyi clade.

What were the key findings from your research?

Of the twenty seven 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 behavior, biofilm formation, quorum sensing and hemolysin production. In this report, we demonstrate that the degradation intermediate of PHB effectively inhibits biofilm formation, luminescence, motility behavior, hemolysin production and the N-acyl-homoserine lactone (AHL)-mediated quorum sensing pathway in PUGSK8. The growth of Vibrio PUGSK8 remains unaffected in the presence of PHB, with PHB degradation being detected in the media. Interestingly, a pathogen degrades PHB for its nutrient utilization results in the release of degradation intermediates which inhibits phenotypic expression of virulence factors. This phenomenon makes a pathogen become a normal microflora in the aquatic system. Disruption of the quorum sensing systems in Vibrio would an effective and environmentally friendlier alternate to antimicrobial agents to control the pathogen. Thus PHB treatments could be an eco-friendly non-invasive effectively strategy to contain Vibrio infection in shrimp aquaculture. It is well established that the treatment of individual infected aquatic animals is neither feasible nor possible, with entire system treatment approaches involving antibiotics culminating in resistant and residual impacts. The PHB based treatment would be an effective anti-infective strategy to achieve aquatic system treatment approaches to contain Vibrio outbreaks in shrimp aquaculture.

What next? What further research is needed in this area?

Further research is planned and research proposal is being generated on elucidation of PHB degradation fragment pattern, docking of targets with AHL and other virulence factors and extended spectrum of activity on other luminescent Vibrio clades.

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