Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro In vitro and in vivo effect of 2,6-Di-tert-butyl-4-methylphenol as an antibiofilm agent against quorum sensing mediated biofilm formation of Vibrio spp. Sivasubramanian Santhakumari a , Rengarajan Jayakumar b , Ravichandran Logalakshmi a , Narayanan Marimuthu Prabhu c , Abdul Kuthus Abdul Nazar b , Shunmugiah Karutha Pandian a , Arumugam Veera Ravi a, ⁎ a Department of Biotechnology, Science Campus, Alagappa University, Karaikudi 630 003, Tamil Nadu, India b Mandapam Regional Centre, Central Marine Fisheries Research Institute, Mandapam, Tamil Nadu, India c Department of Animal Health and Management, Alagappa University, Karaikudi 630 003, Tamil Nadu, India ARTICLE INFO Keywords: 2,6-Di-tert-butyl-4-methylphenol Seafood Quorum sensing Biofilm biomass Litopenaeus vannamei ABSTRACT This study unveils the in vitro and in vivo antibiofilm potential of 2,6-Di-tert-butyl-4-methylphenol (DTBMP) from Chroococcus turgidus against Vibrio spp. In the preliminary study, cell free culture supernatant (CFCS) of C. turgidus inhibited the violacein production in biomarker strain Chromobacterium violaceum and its mutant strain CV026 in a dose dependent manner. The effective biofilm inhibitory concentration (BIC) of pure compound DTBMP from C. turgidus was identified as 250 μg/ml concentration in tested Vibrio species. Furthermore, DTBMP proved to effectively inhibit the bioluminescence production in V. harveyi and other biofilm related virulence traits such as exopolysaccharides (EPS) production, hydrophobicity index, swimming and swarming motility at its BIC concentration in three major pathogenic vibrios: V. harveyi, V. parahaemolyticus and V. vulnificus. The antibiofilm potential of DTBMP was validated through light, confocal laser scanning and scanning electron microscopic analyses. In addition, the non-bactericidal effect of DTBMP was determined through growth curve and 2,3-bis (2-methyloxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Real-time PCR studies revealed the down-regulation of master quorum sensing (QS) regulator genes of V. harveyi such as luxR, luxS, luxP, luxQ and luxO on treatment with DTBMP. In vivo results confirmed that DTBMP augmented the survival rate of Litopenaeus vannamei larvae up to 75, 88 and 66% upon infection with V. harveyi, V. para- haemolyticus and V. vulnificus, respectively. The results of this study ascertain the promising effects of DTBMP as an antibiofilm agent, which could be positively explored to treat biofilm-associated vibrios infections in aqua- culture. 1. Introduction Vibrios are halophilic Gram-negative rod or curve-shaped bacteria widely distributed in marine environments. They are documented as one of the leading causes of foodborne disease outbreaks associated with seafood consumption around the world (Soto-Rodriguez et al., 2003). Vibriosis is caused by several pathogenic Vibrio spp. (Austin and Zhang, 2006; Garcia et al., 2009) and are frequently isolated from a variety of seafood organisms including codfish, flounder, clam, octopus, shrimp, crab, lobster, crawfish, scallop and oysters (Baffone et al., 2000; Liston, 1990). Recently, consumption of seafood especially shrimp, has greatly increased worldwide (Norhana et al., 2012). Shrimp is found to be most vulnerable to contamination with a variety of bacterial pathogens, especially by members of the Vibrio family (Sharma et al., 2010). The prevalence of vibrios in shrimp has resulted in severe eco- nomic losses and total mortality in many countries, particularly in Asia, including China, India, Thailand, Vietnam and Iran (Gopal et al., 2005; Vaseeharan and Ramasamy, 2003; Zarei et al., 2012). V. harveyi is a key bioluminescent bacterium known to infect a wide range of seafood organisms especially black tiger prawn (Penaeus monodon), and causes up to 100% mortality in many Asian countries (Farmer et al., 2005). Similarly, V. parahaemolyticus and V. vulnificus are recognized as im- portant seafood-borne pathogens and as the primary cause of human gastroenteritis, wound infections and septicemia due to consumption of raw or undercooked shrimp posing as a potential risk to public health (Drake et al., 2007; Kaysner and DePaola, 2001; Su and Liu, 2007). V. https://doi.org/10.1016/j.ijfoodmicro.2018.05.024 Received 22 December 2017; Received in revised form 17 May 2018; Accepted 22 May 2018 ⁎ Corresponding author. E-mail address: aveeraravi@rediffmail.com (A. Veera Ravi). International Journal of Food Microbiology 281 (2018) 60–71 Available online 25 May 2018 0168-1605/ © 2018 Elsevier B.V. All rights reserved. T