Indian Journal of Geo Marine Sciences Vol. 46 (06), June 2017, pp. 1116-1127 Diversity and enzymatic profile of bacterial flora in the gut of an estuarine fish, Mugil jerdoni Ankita A. Datta, Amit K. Sharma, Rahul Kundu & Satya P. Singh* UGC-CAS Department of Biosciences, Saurashtra University, Rajkot 360 005, India *[E-mail: [email protected]] Received 12 August 2015 ; revised 07 December 2015 In order to examine the bacterial diversity and enzymatic potential, the isolates were screened for the amylolytic, cellulolytic, lipolytic and proteolytic activities using selective media. Significant proportion of the isolates (44%) exhibited lipase activity, while only few (11%) had protease activity. The 16S rRNA gene sequence analysis revealed that most of the isolates related to the genera Bacillus, Acinetobacter, Staphylococcus, Aeromonas, Psychrobacter, Dietzia and Isoptericola. Examined bacteria displayed significant tolerance against varying salt concentrations. Most of the isolates displayed antagonism against 10 selected target organisms. Bacteria were also assessed for their resistance and sensitivity against different antibiotics. Staphylococcus epidermis MJMG8.1 and Dietzia sp. MJMG8.2 grew significantly in the presence of different organic solvents. [Key Words: Microbial enzymes, antibiotic resistance, 16S rRNA sequencing, phylogeny, microbial diversity, fish-gut microflora] Introduction The culturable bacterial flora in the gut of Staphylococcus, unidentified anaerobes and yeast are freshwater and marine water fishes has been explored reported to produce exogenous enzymes. Amylases, in limited sense 1. Bacterial population in the fish gut proteases, lipases, cellulases, chitinases and few is governed by various environmental and intrinsic others have been produced by the gut flora 5, 7. factors such as species, developmental stage, feeding Studies have revealed that the gut microflora prevents strategy, structure of the digestive system and other the establishment of opportunistic pathogens in the physiological factors 2. Gastrointestinal tract of fish is gastrointestinal tract 8, 9. Few bacterial genera have rich in the nutritional additives and provides a unique been suggested as the probiotics since they have niche for the survival of the selected, but a diverse potentiality to inhibit the colonization of pathogens in group of the microorganisms. The gut bacterial flora the gastrointestinal tract by producing various can be categorized into two groups as autochthonous antibacterial compounds 10. (indigenous), which colonize the gut to become Mugil jerdoni (greenback mullet) is an estuarine resident flora, and allochthonous (incidental visitors), fish belonging to the order Mugiliformes and family appearing transiently without colonization 3, 4. Mugilidae and can tolerate a range of the salinity. It The gut bacterial flora represents the significant feeds on small algae, diatoms and benthic detrital and diversified enzymatic potential, which might play material taken along the sand and mud 11. The fish is a major role in the fermentative digestive metabolism one of the important estuarine aquaculture species in of fish 5, 6. Various enzyme-producing microbes are India. It is a popular edible fish with high market isolated and identified from the gastrointestinal tract value. The fish used in the present study was collected of various fishes. Bacillus, Acinetobacter, Aeromonas, from Jodiya, Gulf of Kutch, Gujarat, India (lat Psychrobacter, Photobacterium, Pseudomonas, 22.70000N, long 70.30000E) and used for the Vibrio, Microbacterium, Enterobacter, Micrococcus, commercial fishery business. To the best of our INDIAN J. MAR. SCI., VOL. 46, NO. 06, JUNE 2017 1117 understanding, there is no information on the diversity Isolated bacteria were characterized on the basis of of the gut microflora of this fish. the morphological, physiological and biochemical Present study focused on the isolation and features. For morphological characterization, cultural enumeration of the aerobic and facultative anaerobes characteristics, colony morphology, cell size, shape, in the digestive tract, assessment of the diversity and margin, elevation, consistency, texture, opacity, enzymatic profile of the gut bacterial flora from the pigmentation and Gram-staining characteristics were midgut of an estuarine fish, Mugil jerdoni. investigated. Effect of temperature, pH and NaCl (0.5 Amylolytic, proteolytic, lipolytic and cellulolytic %, 2 %, 5 %) and 7% (w/v) was assessed on the activities were estimated, and the antibiotic growth of the isolated bacteria. Bacterial isolates sensitivity, antimicrobial activities, the biochemical were further diversified on the basis of the characteristics, salt tolerance and organic solvent antibiogram and biochemical examinations, such as tolerance were examined to evaluate the bacterial MR (Methyl Red), VP (Voges Prausker), urea, diversity. Further, the identification and classification utilization, Simmon Citrate, nitrate reduction, of the bacteria was carried out by 16S rRNA gene hydrolysis of starch, gelatin, H2S production and the analysis. ability to utilize different carbohydrates. Biochemical tests were performed as per the procedures described Materials and Methods in Bergey’s Manual of Systematic Bacteriology 13. During August-September 2013, the estuarine fish Gut bacteria were examined for the extracellular Mugil jerdoni was obtained from Jodiya, Gulf of protease, amylase, cellulase, and lipase activities. Kutch (22.70000N, 70.30000E), Gujarat, India. Bacteria were spread onto the surface of the gelatin, Average weight and length of all the four fishes taken starch, CMC and tributyrin agar for protease, for study were 155 ±2.5g and 17±0.25 cm, amylase, cellulase, and lipase activities, respectively. respectively. Fish was sacrificed and washed Inoculated plates were incubated at 37 °C for 48 h and properly with sterile distilled water followed by the after sufficient growth; the plates were flooded with surface sterilization with 70 % ethanol. Apparently different solutions for the zone of clearance. asymptomatic fish was dissected aseptically in a Qualitative estimation of the extracellular enzymes laminar air flow chamber and the gut was carefully was based on the zone of clearance around the colony. removed from the abdominal cavity. The midgut was The ratio of the zone of clearance to the colony then washed with chilled sterile N-saline solution diameter was used to compare the relative enzyme (0.89 % w/v NaCl) to remove the non-adherent secretion by the bacteria isolates. bacteria. One gram (wet weight) of the midgut was Antibiotic sensitivity/resistance of the bacterial squeezed with a pinch of sterile sand (Sigma) in a isolates against various antibiotics was determined glass mortar pestle for 5mins and then homogenized using the dodeca universal discs (Hi-Media). The 30 with 10 ml sterile N-saline solution. Homogenized antibiotics discs and concentration range were: gut sample was further centrifuged at 4000 rpm for 10 Cefpodoxime (CPD 10 μg), Chloramphenicol (C, 30 min at 4 ºC in a cooling centrifuge. μg), Vancomycin (VA, 30 μg), Streptomycin (S, 10 The homogenate of the gut segments were serially μg), Rifampicin (RIF, 5 μg), Levofloxacin (LE, 5 μg), 12 diluted from 10-1 to 10-10 with sterilized N-saline . Ceftraiaxone (CTR, 30 μg), Clindamycin (CD, 2 μg), Samples from each dilution stage were thoroughly Augmentin (AMC, 30 μg), Amikacin (AK, 30 μg), mixed by shaking and 0.1 mL of the homogenate was Cefixime (CFM, 5 μg), Tetracycline (TE, 30 μg), Co- spread on the growth medium. Supernatants from Trimoxazole (COT, 25 μg), Colistin (CL, 10 μg), serial dilutions of the gut homogenous were poured Netillin (NET, 30 μg), Norfloxacin (NX, 10 μg), aseptically over triplicate plates of nutrient agar (pH Ciprofloxacin (CIP, 5 μg), Cephotaxime (CTX, 30 7.0; NaCl 0.5 % w/v) and complex medium agar (pH μg), Gentamicin (GEN, 10 μg), Furazolidone (FR, 50 8.0; NaCl 2.0 % w/v), respectively for the μg), Amoxycillin (AMX, 10 μg), Ampicillin (AMP, enumeration and enrichment of bacteria. The 10 μg), Cefuroxime (CXM, 30 μg), Cefradroxil (CFR, inoculated plates were incubated at 37 ºC for 24 h and 30 μg), Penicillin (P, 10 units), Cefaclor (CF, 30 μg), examined for the bacterial growth. The well isolated Azithromycin (AZM, 15 μg), Erythromycin (E, 15 colonies with different morphological features were μg), Cefaperazone (CPZ, 75 μg) and Clarithromycin further purified by streaking and re-streaking on fresh (CLR, 5 μg). Bacterial inoculums were spread onto o media. Pure cultures were preserved at -20 C in 15 % the nutrient agar plates, impregnated with the (v/v) glycerol as cryo-preservant. antibiotic disc and the zones of the inhibition were 1118 DATTA et al.: DIVERSITY AND ENZYMATIC PROFILE OF BACTERIAL FLORA IN THE GUT OF MUGIL JERDONI observed after incubating the plates at 37 ºC for 24-48 step-4, extension at 72 °C for 60 s. Subsequently, the h. Inhibition of the growth of the test organisms was steps 2, 3 and 4 were repeated for 35 cycles, followed taken as the sensitivity of the organisms, while the by a final elongation step at 72 ºC for 2 min. Besides, resistance against an antibiotic was reflected by the the reaction included a positive control (E.coli growth of the test organism and absence of the genomic DNA) and, two negative controls, one inhibition zone. without template DNA and another without any Antimicrobial susceptibility tests were carried out primer. Similarity search was carried