Symbiosis DOI 10.1007/s13199-017-0474-7
Culturable autochthonous gut bacteria in rohu, Labeo rohita. In vitro growth inhibition against pathogenic Aeromonas spp., stability in gut, bio-safety and identification by 16S rRNA gene sequencing Anjan Mukherjee 1 & Dipanjan Dutta 1 & Sudeshna Banerjee 1 & Einar Ringø 2 & Eva Marie Breines 3 & Ellinor Hareide 3 & Goutam Chandra 4 & Koushik Ghosh 1
Received: 20 September 2016 / Accepted: 27 January 2017 # Springer Science+Business Media Dordrecht 2017
Abstract Autochthonous endosymbiotic gut bacteria antagonistic against pathogenic Aeromonas spp. have been evaluated in rohu, Labeo rohita for characterization of putative probiotics. Four promising pathogen inhibitory bacteria (23 strains out of 225 isolates showed antagonism) were selected by double layer assay, following which inhibition pattern was examined through in vitro growth curve and statistical analyses. Cell free supernatant (CFS) of the selected gut bacteria significantly inhibited the growth of pathogenic aeromonads. While, CFS of strain LR3FG26 was the most efficient among them. Selected strains were γ haemolytic and susceptible to most of the common antibiotics that demonstrated their likely non-pathogenic and eco-friendly nature. Additionally, selected bacteria produced different exo-enzymes (digestive and antinutritional factors degrading), could grow better in skin mucus than intestinal mucus (exceptionally, LR3FG26 grew better in skin mucus) and tolerated diluted bile juice (2–20%). 16S rRNA partial gene sequence analyses and Blast search in NCBI GenBank unveiled that the strains LR1FG1,
* Koushik Ghosh
[email protected];
[email protected] 1
Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Golapbag, Burdwan, West Bengal 713104, India
2
Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, UiT The Arctic University of Norway, -9037 Tromsø, NO, Norway
3
Department of Arctic and Marine Biology, Faculty of Bioscience, Fisheries and Economics, UiT The Arctic University of Norway, -9037 Breivika, NO, Norway
4
Parasitology Laboratory, Department of Zoology, The University of Burdwan, Burdwan, West Bengal 713104, India
LR2HG13, LR3FG26 and LR3HG4 were similar with the type strains of Bacillus methylotrophicus (NR116240), Bacillus amyloliquefaciens (NR117946), Pseudomonas fluorescens (NR113647) and Bacillus licheniformis (NR118996), respectively. Application of these symbiotic pathogen-inhibitory bacteria in pathogen challenge studies is required to appraise their probiotic effects in vivo. Keywords Carp . Fish pathogen . Cell free supernatant . Inhibition efficiency . Bacillus . Probiotics . Hormetic-responses
1 Introduction Extension, intensification and diversification of aquaculture have increased the incidences of disease outbreaks in the past few decades. The motile Aeromonad septicaemia by far the most common among the bacterial diseases of freshwater fish that has been associated with several species of the genus Aeromonas, viz., A. hydrophila, A. caviae, A. veronii, A. schuberti, A. salmonicida, A. sobria etc. Amongst these, A. hydrophila (Wahli et al. 2005) and A. sobria (Majtán et al. 2012), were documented as causative agents for large scale mortality in fish. Oral administration of antibacterial drugs as feed supplement is the most common prophylactic treatment for bacterial diseases in fish (Ran et al. 2012). However, indiscriminate usage of antibiotics has often been criticized for bringing about environmental hazards and unpredictable longterm effects on public health through the spread of drugresistant pathogenic strains. In addition, application of antibiotics as a protective measure may alter the gut microenvironment affecting the autochthonous symbiotic microbial population that has been demonstrated to contribute in
Mukherjee A. et al.
nutrient utilization and disease prevention in host fish (Irianto and Austin 2002; Ray et al. 2012; Dutta et al. 2015). Therefore, numerous countries have imposed restrictions on antibiotic use in aquaculture (Kesarcodi-Watson et al. 2008). This has encouraged seeking alternatives to the antibiotics for aquaculture application. Expanded interest on symbiotic gut bacteria in fish as the probiotic bio-control agents has come up with the fact that gut bacteria often produce bacteriocin like compounds that could antagonize pathogenic bacteria (Merrifield et al. 2010; Askarian et al. 2011). Probiotics are gaining increasing scientific and commercial interest in aquaculture practice. Gut symbionts usually maintain a close association with the host and put forth their advantageous effect on the host for better physiological function and immunity (Banerjee and Ray 2016). Therefore, endosymbionts selected as probiotics encompass a sustainable and environment-friendly approach to produce benefit to the host that might involve improvement in feed utilization, modulation of protective gastrointestinal microflora, enhancement of immune responses and inhibition of pathogens (Ran et al. 2012). It has been suggested that a probiotic strain is likely to be much effective in the host species from where it was originated, i.e., autochthonous and symbiotic (Verschuere et al. 2000). During the last few decades, selection of probiotics in consequence of likely improvement of nutrient availability due to exogenous enzymes secreted by the autochthonous gut microflora has been widely emphasized in the freshwater teleosts including the Indian major carps (IMC) (Ray et al. 2012). In contrast, antagonism towards the pathogens has been less accentuated for screening of putative probiotics from the IMCs (Dutta et al. 2015; Mukherjee and Ghosh 2016). The IMCs are highly nutritious and constitute nearly 70% of the Indian aquaculture production, wherein rohu (Labeo rohita) is the most preferred species comprising about 35% of the IMC production (FAO 2000). India contributed nearly 1 million tons out of the global production of 1.2 million tons of L. rohita in 2005 (Giri et al. 2012). Therefore, development of suitable probiotic strains for L. rohita holds promise to sustain aquaculture production of this tropical freshwater fish. Although isolation of pathogen inhibitory bacteria is a common practice in the global scenario (Ringø et al. 2005; Askarian et al. 2012; Mukherjee and Ghosh 2016), the specific pattern of inhibition for a pathogen in presence of any antimicrobial-producing bacteria has never been considered to select suitable probiotics. Apart from the positive functional attributes (viz., exogenous enzymes, antagonism towards pathogens etc.); it is also important to consider that the putative probiotics should be non-pathogenic, non-haemolytic and kept alive in gastrointestinal condition (Verschuere et al. 2000; Balcázar et al. 2006). Moreover, selection of antibiotic resistant strains should be avoided to reduce environmental risks associated with the drug-resistant strains (Robredo et al. 2000; Irianto and Austin 2002). To date, the screening of probiotics
has been reasonably benefit oriented, lacking selection criteria to exclude adverse side effects. Therefore, prior to in vivo application of a putative probiotic organism, screening and selection of the candidate probiotics through various in vitro selective criteria and in vivo bio-safety assay are to be considered with prime importance. Thus, the presently reported study addressed evaluation of culturable autochthonous symbiotic gut microbiota in rohu to recognize promising probiotics through multistep screening and validation. An extensive collection of autochthonous bacteria were tested for antagonism against reference pathogenic Aeromonas strains. In this regard, the present study proposes application of suitable statistical treatment to evaluate the trend of inhibition of a specific pathogen by a putative probiont based on their in vitro growth inhibition pattern. Further, effective antagonistic strains were evaluated for different exoenzyme production, growth in fish mucus and tolerance to the bile juice. The safety of selected strains was also assessed in terms of haemolytic activity, antibiotic susceptibility and in vivo bio-safety evaluation through intra-peritoneal injection to see whether the target fish may develop any pathological symptoms.
2 Materials and methods 2.1 Sample collection and isolation of autochthonous gut bacteria Healthy and disease free rohu, Labeo rohita (Average weight: 275 ± 8.9 g; length 32.5 ± 2.73 cm) were collected from three different polyculture ponds (5 from each, 15 altogether) of Burdwan (23°14″N, 87°39″E), West Bengal, India. The specimens were starved for 48 h and anaesthetized with MS-222 (tricaine methanesulfonate; Sigma-Aldrich Corp., USA). GI tracts were dissected out, divided into PI (proximal intestine) and DI (distal intestine) segments and gut samples were processed following the methods described by Mandal and Ghosh (2013) and Mukherjee and Ghosh (2016) for isolation of autochthonous gut microorganisms. Pooled sample of each segment from the specimens collected from each pond was considered as a replicate, and therefore, there were three replicates in the study. Homogenates of the gut segments were serially diluted and spread on soybean casein digest medium (tryptone soya agar, TSA; HiMedia). Following incubation (48 h, 30 °C), distinct colonies were randomly isolated, cultured on TSA plates and pure cultures were preserved (4 °C) for further study. 2.2 Antimicrobial activity assay Antibacterial activity of the gut bacteria was examined against four pathogenic Aeromonas spp. by the ‘double-layer’ method (Dopazo et al. 1988). The pathogenic strains, Aeromonas
Culturable autochthonous gut bacteria in rohu, Labeo rohita
hydrophila MTCC-1739 (AH), A. salmonicida MTCC-1945 (AS) and A. sobria MTCC 3613 (ASo) were obtained from the Microbial Type Culture Collection, Chandigarh, India, whereas, A. veronii KT737240 (AV) was isolated from a diseased fish. Antagonism against the pathogenic strains in ‘double-layer’ assay was determined through measurement of the zone of inhibition (halo, diameter in mm) and presented as scores. Promising antagonistic bacteria were selected on the basis of cumulative scores. Further, the strong pathogen inhibitory gut bacteria were confirmed through ‘micro-titer plate assay’ as described elsewhere (Ringø 2008; Ringø et al. 2005; Salma et al. 2011; Askarian et al. 2012). Briefly, log phase culture of the promising gut bacteria was subjected to centrifugation (10,000 rpm, 15 mins) to obtain the cell free supernatant (CFS). CFS from each bacterium was filter sterilized and evaluated for likely inhibition of the tested fish pathogens. Filter sterilized CFS (50 μL) was added to of fresh neutral sterile medium (150 μL) in microtitre wells, and was inoculated with the cultures of pathogenic bacteria (10 μL, 107 CFU mL−1). The control set containing 200 μL of neutral sterile medium was also inoculated with the pathogenic strains. Experiments were conducted in triplicates. The data on the growth measured in terms of O.D. (600 nm) were used to measure the inhibition efficiency (IE) using the following formulae: Inhibition Eff iciency ðIEÞ ¼
OD in presence of CFS of probiotics OD of pathogen only
Where, IE>1 indicated growth promotion, IE = 1 indicated no inhibition (NI) and IE
