ORIGINAL ARTICLE Quorum-Quenching Activity of the AHL-Lactonase from Bacillus licheniformis DAHB1 Inhibits Vibrio Biofilm Formation In Vitro and Reduces Shrimp Intestinal Colonisation and Mortality G. Vinoj & B. Vaseeharan & S. Thomas & A. J. Spiers & S. Shanthi Received: 28 October 2013 /Accepted: 2 July 2014 /Published online: 25 July 2014 # Springer Science+Business Media New York 2014 Abstract Vibrio parahaemolyticus is a significant cause of gastroenteritis resulting from the consumption of undercooked sea foods and often cause significant infections in shrimp aquaculture. Vibrio virulence is associated with biofilm for- mation and is regulated by N-acylated homoserine lactone (AHL)-mediated quorum sensing. In an attempt to reduce vibrio colonisation of shrimps and mortality, we screened native intestinal bacilli from Indian white shrimps (Fenneropenaeus indicus) for an isolate which showed biofilm-inhibitory activity (quorum quenching) against the pathogen V. parahaemolyticus DAHP1. The AHL-lactonase (AiiA) expressed by one of these, Bacillus licheniformis DAHB1, was characterised as having a broad-spectrum AHL substrate specificity and intrinsic resistance to the acid conditions of the shrimp intestine. Purified recombinant AiiA inhibited vibrio biofilm development in a cover slip assay and significantly attenuated infection and mortality in shrimps reared in a recirculation aquaculture system. Investigation of intestinal samples also showed that AiiA treatment also re- duced vibrio viable counts and biofilm development as determined by confocal laser scanning microscopy (CLSM) imaging. These findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture. Keywords AHL-lactonase . AiiA . Bacillus . Biofilm . Quorum quenching . Vibrio parahaemolyticus Introduction Vibrio parahaemolyticus is a major cause of gastroenteritis world-wide, and contaminates fresh and seawater produce, including fish, molluscs, shrimps, and prawns, and can be found in lakes, rivers, and coastal seas (Sarkar et al. 1985; Thompson et al. 2004; Yeung and Boor 2004; Su and Liu 2007). Vibrio virulence involves arrange of factors (Johnson 2013), including biofilm formation, which is regulated by quorum signalling (QS) using N-acylated homoserine lactones (AHLs) including C6-HSL (N-hexanoyl-L-homoserine lac- tone, HHL) (Fuqua and Parsek 2001; Ng and Basslern 2009; Kamruzzaman et al. 2010; Defoirdt et al. 2011; Fast and Tipton 2012). Disruption of QS by quorum quenching has been suggested as an anti-infective strategy to control pathogenic bacteria through the interference of the col- onisation processes, including biofilm formation and the invasion of host tissues (Rasmussen and Givskov 2006; Defoirdt et al. 2008; Czajkowski and Jafra 2009). This might be achieved by treatments with exogenous AHL- lactonases that degrade AHLs, though these would need to have broad substrate specificity, as AHLs vary in chain length (4–16 carbons) and oxo or hydroxyl sub- stitutions. Some bacteria, especially Bacillus spp., may use AHL-lactonases in quorum quenching to boost com- petitive strength in soil, and a number of AHL- Electronic supplementary material The online version of this article (doi:10.1007/s10126-014-9585-9) contains supplementary material, which is available to authorized users. G. Vinoj : B. Vaseeharan (*) : S. Shanthi Crustacean Molecular Biology and Genomics Laboratory, Department of Animal Health and Management, Alagappa University, Science Block, 4th Floor, Burma Colony, Karaikudi 630 004, Tamil Nadu, India e-mail: vaseeharanb@gmail.com S. Thomas Cholera and Environmental Microbiology Laboratory, Department of Molecular Microbiology, Rajiv Gandhi Centre for Biotechnology, Trivandrum, Kerala 695014, India A. J. Spiers SIMBIOS Centre, Abertay University, Dundee DD4 1HG, United Kingdom Mar Biotechnol (2014) 16:707–715 DOI 10.1007/s10126-014-9585-9