Diverse Profiles of N‐acyl Homoserine l‐Lactones, Biofilm, Virulence Genes and Integrons in Food‐Borne Aeromonas Isolates

Abstract

Aeromonas are regarded as opportunistic as well as primary pathogens of humans and fish, and are associated with gastroenteritis and septicemia in humans. Production of N-acyl-homoserine lactone (AHL) signal molecules and biofilm was determined in 22 Aeromonas isolates, from different food products in India, using thin-layer chromatography (TLC) analysis and microtiter-plate assay, respectively. Overall, highly heterogeneous patterns of AHL production were observed, with the production of N-butanoyl homoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL) by the majority (81.8%) of Aeromonas food isolates. Moreover, putative N-pentanoyl homoserine lactone (C5-HSL), N-heptanoyl homoserine lactone (C7-HSL), and N-octanoyl homoserine lactone (C8-HSL) were produced by 72.7%, 27.3%, and 9.1% of isolates, respectively. This is the 1st report of production of C7-HSL by Aeromonas species. Aeromonas food isolates were highly variable in their biofilm forming abilities with majority of them as weak biofilm producers in 2 different media, TSB and M9 minimal medium supplemented with 0.4% glucose. The genes encoding for putative virulence factors, glycerophospholipid cholesterol acyltransferase (gcat), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), serine protease (ser), polar flagella (fla), and lateral flagella (lafA) were present in 95.5%, 59.1%, 22.7%, 81.8%, 77.3%, and 22.7% of the strains, respectively. Class 1 integrons (100 to 3000 bp) were found in 68.2% of food isolates; whereas, 50% isolates contained class 2 integrons (150 to 1600 bp). This study provides a baseline data on the diversity of AHLs, biofilm forming ability and presence of virulence genes and integrons in Aeromonas food isolates from India.

Biofilm formation by Aeromonas strains under food-related environmental stress conditions

“…veronii CECT 4257 T ) only in M9 minimal medium, whereas none of the strains could form moderate‐to‐strong biofilm in TSB medium (Figure ). Our results are in consonance with earlier studies where the majority of Aeromonas isolates from food and water formed weak‐to‐moderate biofilm in TSB (Igbinosa, Chigor, Igbinosa, Obi, & Okoh, ; Nagar et al, ). The influence of growth medium on the biofilm formation by Aeromonas strains is unclear, as insignificant difference in the biofilm formation by 50% of Aeromonas strains in TSB and M9 minimal medium was observed (Figure ).…”

“…Aeromonas strains obtained from similar class of source (food, environment, and clinical) did not have comparable biofilm‐forming abilities. Similar observations were made by other researchers: Aeromonas (Nagar et al, ), Salmonella (Stepanovic, Cirkovic, Ranin, & Svabic‐Vlahovic, ), and L. monocytogenes (Nilsson et al, ). Biofilm formation is an intricate and incompletely understood process regulated by diverse factors including the growth medium (Harvey, Keenan, & Gilmour, ).…”

“…CECT 839 T , CECT 4257 T , CECT 4246, CECT 7112, and AH1N were kindly provided by Dr. Valérie Leclère, University Lille, France; MTCC 2654, MTCC 1608, and MTCC 3249 were obtained from Microbial Type Culture Collection (MTCC, Chandigarh, India), whereas A331 and A85 were isolated and identified in our lab (Nagar, Shashidhar, & Bandekar, ). The cultures were revived, sub‐cultured, grown, and inoculated in 96‐well flat‐bottomed microtiter plates (Falcon; BD Biosciences, NJ) according to detailed method described by Nagar, Sinha, and Bandekar (). The effect of different stress conditions on biofilm formation was studied by inoculating 20 µl of overnight grown culture (30°C, 150 rpm, 16 h), standardized to an optical density of 0.02 (approximately 2.0 × 10 7 CFU/ml) in individual well containing 180 µl of test medium (adjusted to defined pH or salt concentration, with/without food preservatives).…”

“…The present study indicates that diverse Aeromonas strains respond differently to the different media composition. Various researchers have reported considerable strain variability in the biofilm production for food-borne pathogens: Aeromonas in TSB and M9 minimal media supplemented with 0.4% glucose (Nagar et al, 2015), Listeria monocytogenes in brain-heart infusion (BHI) broth (Nilsson, Ross, & Bowman, 2011), Salmonella enterica in TSB (Lianou & Koutsoumanis, 2012), and Escherichia coli in M63 minimal media supplemented with glucose and casamino acids, BHI, and diluted TSB (Skyberg, Siek, Doetkott, & Nolan, 2007).…”

Characterization of virulence, cell surface characteristics and biofilm‐forming ability of Aeromonas spp. isolates from fish and sea water

“…). Aeromonas biofilms may serve as continuous source of cross‐contamination during fish/food processing (Nagar, Sinha & Bandekar ) and are potential threats in the food industry and to public health via direct contact with infected animals as well as consumption of contaminated food and water (Tomás ; Chen et al . ).…”