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, 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.
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References
Askarian F, Kousha A, Salma W, Ringø E (2011) The effect of lactic acid bacteria administration on growth, digestive enzymes activity and gut microbiota in Persian sturgeon (Acipenser persicus) and beluga (Huso huso ) fry. Aquac Nutr 17:488–497
Askarian F, Zhou Z, Olsen RE, Sperstad S, Ringø E (2012) Culturable autochthonous gut bacteria in Atlantic salmon (Salmo salar L.) fed diets with or without chitin. Characterization by 16S rRNA gene sequencing, ability to produce enzymes and in vitro growth inhibition of four fish pathogens. Aquaculture 326–329:1–8
Austin B, Stuckey LF, Robertson PAW, Effendi I, Griffith DRW (1995) A probiotic strain of Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalii. J Fish Dis 18:93–96
Bailey MJ, Biely P, Poutanen K (1992) Inter-laboratory testing of methods for assay of xylanase activity. J Biotechnol 23:257–270
Balcázar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Múzquiz JL (2006) The role of probiotics in aquaculture. Vet Microbiol 114:173–186
Balcázar JL, Vendrell D, de Blas I, Ruiz-Zarzuela I, Múzquiz JL, Girones O (2008) Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish. Aquaculture 278:188–191
Banerjee G, Ray AK (2016) Bacterial symbiosis in the fish gut and its role in health and metabolism. Symbiosis. doi:10.1007/s13199-016-0441-8
Bernfeld P (1955) Methods in enzymology. Academic Press, New York, pp 149–150
Bier M (1955) Methods in enzymology. Academic Press, New York, pp 627–642
Bøgwald J, Dalmo RA (2014) Aquaculture nutrition: gut health, probiotics and prebiotics. Wiley-Blackwell Publishing, Oxford, pp 53–74
Burbank DR, La-Patra SE, Fornshell G, Cain KD (2012) Isolation of bacterial probiotic candidates from the gastrointestinal tract of rainbow trout, Oncorhynchus mykiss (Walbaum), and screening for inhibitory activity against Flavobacterium psychrophilum. J Fish Dis 35:809–816
Cahill MM (1990) Bacterial flora of fishes: a review. Microb Ecol 19:21–41
Calabrese EJ, Baldwin LA (2003) HORMESIS: the dose-response revolution. Annu Rev Pharmacol Toxicol 43:175–197
Chythanya R, Karunasagar I, Karunasagar I (2002) Inhibition of shrimp pathogenic vibrios by a marine Pseudomonas I-2 strain. Aquaculture 208:1–10
De Smet I, Hoorde LV, Woestyne MV, Christiaens H, Verstraete W (1995) Significance of bile salt hydrolytic activities of lactobacilli. J Appl Bacteriol 79:292–301
Denison DA, Koehn RD (1977) Cellulase activity of Poronia oedipus. Mycologia 69:592–603
Dopazo C, Lemos M, Lodeiros C, Bolinches J, Barja J, Toranzo A (1988) Inhibitory activity of antibiotic producing marine bacteria against fish pathogens. J Appl Bacteriol 65:97–101
Dutta D, Ghosh K (2015) Screening of extracellular enzyme-producing and pathogen inhibitory gut bacteria as putative probiotics in mrigal, Cirrhinus mrigala (Hamilton, 1822). Int J Fish Aquat Stud 2:310–318
Dutta D, Banerjee S, Mukherjee A, Ghosh K (2015) Selection and probiotic characterization of exoenzyme-producing bacteria isolated from the gut of Catla catla (Actinopterygii: Cypriniformes: Cyprinidae). Acta Ichthyol Piscat 45:373–384
Eissa N, El-Gheit ESA, Shaheen AA (2014) Protective effect of Pseudomonas fluorescens as a probiotic in controlling fish pathogens. Am J BioSci 5:175–181
FAO (2000) The state of world fisheries and aquaculture. FAO, Rome
Fjellheim AJ, Klinkenberg G, Skjermo J, Aasen IA, Vadstein O (2010) Selection of candidate probionts by two different screening strategies from Atlantic cod (Gadus morhua L.) larvae. Vet Microbiol 144:153–159
Geraylou Z, Vanhove-Maarten PM, Souffreau C, Rurangwa E, Buyse J, Ollevier F (2014) In vitro selection and characterization of putative probiotics isolated from the gut of Acipenser baerii (Brandt, 1869). Aquac Res 45:341–352
Ghosh K, Sen SK, Ray AK (2002) Characterization of bacilli isolated from gut of rohu, Labeo rohita, fingerlings and its significance in digestion. J Appl Aquac 12:33–42
Ghosh S, Sinha A, Sahu C (2007) Isolation of putative probionts from the intestines of Indian major carps. Isr J Aquacult Bamidgeh 59:127–132
Ghosh K, Roy M, Kar N, Ringø E (2010) Gastrointestinal bacteria in rohu, Labeo rohita (Actinopterygii: Cypriniformes: Cyprinidae): scanning electron microscopy and bacteriological study. Acta Ichthyol Piscat 40:129–135
Giri SS, Sukumaran V, Sen SS, Vinumonia J, Nazeema-Banu B, Jena PK (2011) Antagonistic activity of cellular components of potential probiotic bacteria, isolated from the gut of Labeo rohita, against Aeromonas hydrophila. Probiotics Antimicrob Prot 3:214–222
Giri SS, Sen SS, Sukumaran V (2012) Effects of dietary supplementation of potential probiotic Pseudomonas aeruginosa VSG-2 on the innate immunity and disease resistance of tropical freshwater fish, Labeo rohita. Fish Shellfish Immunol 32:1135–1140
Gram LJ, Melchiorsen B, Spanggaard I, Huber T, Nielsen H (1999) Inhibition of Vibrio anguillarum by Pseudomonas fluorescens strain AH2 - a possible probiotic treatment of fish. Appl Environ Microbiol 65:969–973
Ibrahem MD (2015) Evolution of probiotics in aquatic world: potential effects, the current status in Egypt and recent prospective. J Adv Res 6:765–791
Irianto A, Austin B (2002) Use of probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 25:1–10
Kesarcodi-Watson A, Kaspar H, Lategan MJ, Gibson L (2008) Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 274:1–14
Kinnear PR, Gray CD (2009) SPSS 17 made simple. Psychology Press, East Sussex
Klare I, Konstabel C, Werner G, Huys G, Vankerckhoven V, Kahlmeter G, Hildebrandt B, Müller-Bertling S, Witte W, Goossens H (2007) Antimicrobial susceptibilities of Lactobacillus, Pediococcus and Lactococcus human isolates and cultures intended for probiotic or nutritional use. J Antimicrob Chemother 59:900–912
Krishnan R (2014) Probiotic potential of Bacillus species isolated from freshwater fish Anabas testudineus in Labeo rohita. Int J Multidiscip Res Dev 1(1):46–50
Lalloo R, Moonsamy G, Ramchuran S, Görgens J, Gardiner N (2010) Competitive exclusion as a mode of action of a novel Bacillus cereus aquaculture biological agent. Lett Appl Microbiol 50:563–570
Legendre P, Legendre L (1998) Numerical ecology. Elsevier, Amsterdam
Loh JY, Lim YY, Harmin SA, Ting ASY (2014) In vitro assessment on intestinal microflora from commonly farmed fishes for control of the fish pathogen Edwardsiella tarda. Turk J Vet Anim Sci 38:257–263
Lowry OH, Rosenbrough WJ, Fair HL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 195:265–275
Luo Z, Bai XH, Chen CF (2014) Integrated application of two different screening strategies to select potential probiotics from the gut of channel catfish Ictalurus punctatus. Fish Sci 80(6):1269–1275
Majtán J, Černy J, Ofúkaná A, Takáč P, Kozánek M (2012) Mortality of therapeutic fish Garra rufa caused by Aeromonas sobria. Asian Pac J Trop Biomed 2:85–87
Mandal S, Ghosh K (2013) Isolation of tannase-producing microbiota from the gastrointestinal tracts of some freshwater fish. J Appl Ichthyol 29:145–153
Maslow JN, Dawson D, Carlin EA, Holland SM (1999) Hemolysin as a virulence factor for systemic infection with isolates of Mycobacterium avium Complex. J Clin Microbiol 37:445–446
Mattson MP (2008) Hormesis defined. Ageing Res Rev 7:1–7
Merrifield DL, Dimitroglou A, Foey A, Davies SJ, Baker RR, Bøgwald J, Castex M, Ringø E (2010) The current status and future focus of probiotic and prebiotic applications for salmonids. Aquaculture 302:1–18
Mesalhy AS, Abd-El-Rahman AM, John G, Mohamed MF (2008) Characterization of some bacteria isolated from Oreochromis niloticus and their potential use as probiotics. Aquaculture 277:1–6
Moriarty DJW (1996) Microbial biotechnology, a key ingredient for suistainable aquaculture. INFO Fish Int 4:29–33
Mukherjee A, Ghosh K (2016) Antagonism against fish pathogens by cellular components and verification of probiotic properties in autochthonous bacteria isolated from the gut of an Indian major carp, Catla catla (Hamilton). Aquac Res 47:2243–2255
Mukherjee A, Dutta D, Banerjee S, Einar R, Breines EM, Hareide E, Chandra G, Ghosh K (2016) Potential probiotics from Indian major carp, Cirrhinus mrigala. Characterization, pathogen inhibitory activity, partial characterization of bacteriocin and production of exoenzymes. Res Vet Sci 108:76–84
Muñoz-Atienza E, Gómez-Sala B, Araújo C, Campanero C, del Campo R, Hernández PE, Herranz C, Cintas LM (2013) Antimicrobial activity, antibiotic susceptibility and virulence factors of lactic acid bacteria of aquatic origin intended for use as probiotics in aquaculture. BMC Microbiol 13:1–22
National Committee for Clinical Laboratory Standards (2012) Performance standards for antimicrobial disk susceptibility test; approved standard-ninth edition. Wayne: Clinical and Laboratory Standards Institute
Nikoskelainen S, Salminen S, Bylund G, Ouwehand AC (2001) Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish. Appl Environ Microbiol 67:2430–2435
Nurhidayu A, Ina-Salwany MY, Mohd-Daud H, Harmin SA (2012) Isolation, screening and characterization of potential probiotics from farmed tiger grouper (Epinephelus fuscoguttatus). Afr J Microbiol Res 6:1924–1933
Ouwehand AC, Salminen S, Isolauri E (2002) Probiotics: an overview of beneficial effects. Antonie Van Leeuwenhoek 82:279–289
Pychyński T, Malanowska T, Kozłowski M (1981) Tło bakteryjne branchionekrozy karpi (bacterial aetiology in branchionecrosis of carp). Med Weter 37(12):742–743
Ran C, Carrias A, Williams MA, Capps N, Dan BCT, Newton JC, Kloepper JW, Ooi EL, Browdy CL, Terhune JS, Liles MR (2012) Identification of Bacillus strains for biological control of catfish pathogens. PLoS One 7(9):e45793. doi:10.1371/journal.pone.0045793
Ray AK, Ghosh K, Ringø E (2012) Enzyme-producing bacteria isolated from fish gut: a review. Aquac Nutr 18:465–492
Ringø E (2008) The ability of carnobacteria isolated from fish intestine to inhibit growth of fish pathogenic bacteria: a screening study. Aquac Res 39:171–180
Ringø E, Strøm E, Tabachek JA (1995) Intestinal microflora of salmonids: a review. Aquac Res 26:773–789
Ringø E, Schillinger U, Holzapfel W (2005) Antibacterial abilities of lactic acid bacteria isolated from aquatic animals and the use of lactic acid bacteria in aquaculture. In: Holzapfel W, Naughton P (eds) Microbial ecology in growing animals. Elsevier, Edinburgh, pp 418–453
Ringø E, Sperstad S, Myklebust R, Mayhew TM, Olsen RE (2006) The effect of dietary inulin on bacteria associated with hindgut of Arctic charr (Salvelinus alpinus L.). Aquac Res 37:891–897
Robredo B, Singh KV, Baquero F, Murray BE, Torres C (2000) Vancomycin-resistant enterococci isolated from animals and food. Int J Food Microbiol 54:197–204
Ross NW, Firth KJ, Wang A, Burka JF, Johnson SC (2000) Changes in hydrolytic enzyme activities of naive Atlantic salmon Salmo salar skin mucus due to infection with the salmon louse Lepeophtheirus salmonis and cortisol implantation. Dis Aquat Org 41:43–51
Sakata T (1990) Microflora in the digestive tract of fish and shell-fish. In: Lesel R (ed) Microbiology in Poecilotherms. Elsevier, Amsterdam, pp 171–176
Salma W, Zhou Z, Wang W, Askarian F, Kousha A, Ebrahimi MT, Myklebust R, Ringø E (2011) Histological and bacteriological changes in intestine of beluga (Huso huso) following ex vivo exposure to bacterial strains. Aquaculture 314:24–33
Sánchez-Ortiz AC, Luna-González A, Campa-Córdova ÁI, Escamilla-Montes R, del Flores-Miranda MC, Mazón-Suástegui JM (2015) Isolation and characterization of potential probiotic bacteria from pustulose ark (Anadara tuberculosa) suitable for shrimp farming. Lat Am J Aquat Res 43:123–136
Shenderov BA (2011) Probiotic (symbiotic) bacterial languages. Anaerobe 17:490–495
Smith LS (1989) Digestive functions in teleost fishes. In: Halver JE (ed) Fish nutrition, Academic Press Inc., p 331–422
Talpur AD, Memon AJ, Khan MI, Ikhwanuddin M, Danish-Daniel MM, Abol-Munafi AB (2012) Isolation and screening of lactic acid bacteria from the gut of blue swimming crab, P pelagicus as in vitro inhibition assay and small scale in vivo model for validation of isolates as probiotics. J Fishery Aquat Sci 7:1–28
Vaughan EE, Heilig HG, Ben-Amor K, de Vos WM (2005) Diversity, vitality and activities of intestinal lactic acid bacteria and bifidobacteria assessed by molecular approaches. FEMS Microbiol Rev 29:477–490
Verschuere L, Rombaut G, Sorgeloos P, Verstraete W (2000) Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 64:655–671
Vine NG, Leukes WD, Kaiser H (2004) In vitro growth characteristics of five candidate aquaculture probiotics and two fish pathogens grown in fish intestinal mucus. FEMS Microbiol Lett 231:145–152
Wahli T, Burr SE, Pugovkin D, Mueller O, Frey J (2005) Aeromonas sobria, a causative agent of disease in farmed perch, Perca fluviatilis L. J Fish Dis 28:141–150
Walter HE (1984) Methods of enzymatic analysis. Verlag Chemie, Weinheim, pp 270–277
Yanke LJ, Selinger LB, Cheng KJ (1999) Phytase activity of Selenomonas ruminantium: a preliminary characterization. Lett Appl Microbiol 29:20–25
Zar JH (1999) Biostatistical analysis. Pearson Education Singapore Pte. Ltd., New Delhi (Indian Branch)
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The first author is obliged to the DST-INSPIRE programme for the research fellowship. The authors are grateful to Head of Department of Zoology, The University of Burdwan, West Bengal, India; The University Grants Commission (UGC-SAP-DRS programme), New Delhi, India and The Department of Science and Technology (FIST programme), New Delhi, India for research support. The authors are grateful to Dr. G. Aditya for rendering help in statistical analyses of data.
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Mukherjee, A., Dutta, D., Banerjee, S. et al. 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. Symbiosis 73, 165–177 (2017). https://doi.org/10.1007/s13199-017-0474-7
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DOI: https://doi.org/10.1007/s13199-017-0474-7