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

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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 out in-silico using five Gram positive and five Gram negative using BLAST of NCBI. In the present study, the fish human pathogens by cross streak method 14. The gut bacterial isolates, exhibiting different profiles with human pathogens used for the study were: Shigella sp. respect to the conventional microbiological features, clinical, Micrococcus clinical, Salmonella typhi were further identified by the 16S rRNA gene MTCC 98, Bacillus megaterium, Bacillus cereus sequencing. Culture dependent diversity and MTCC 430, Escherichia coli MTCC 1697, phylogeny of the fish gut bacteria were assessed using 17 Enterobacter aerogens MTCC 111, Bacillus subtilis polyphasic approach . The nucleotide sequence data MTCC 441, Staphylococcus aureus MTCC 1144 and reported in this study was submitted to GenBank Pseudomonas aeruginosa MTCC 2587.The bacteria under the accession numbers from KJ623582 to examined for displaying the inhibitory action were KJ623590. A phylogenetic tree was constructed to termed as the ‘test strains’, whereas those used as a compare the evolutionary distances among the target were termed as the ‘target strains’. The test bacterial isolates. Phylogenetic and evolutionary 18 strains were streaked across one-third of an agar plate analyses were carried out using MEGA version 6 . and incubated at 37 ºC. After the visible growth, the Bootstrap analysis for 100 replicates was performed target strains were streaked perpendicular to the initial to estimate the confidence of tree topologies. streaking line and the plates were further incubated at The patterns and phenotypic characters of each 37ºC. The antagonistic effect was indicated by the isolates were compared and scored. A matrix based on failure of the target strain to grow in the confluence the presence and absence of the characters was area. constructed, and the data was analyzed using the 19 The bacterial isolates were cultured in minimal cluster analysis by the PAST program. Jaccard 20 Davis broth supplemented with 10 different organic similarity coefficient SJ was calculated to determine solvents at 30 %, (v/v), without any additional carbon the relationships between the phenotypic characters of and nitrogen sources. Cultures were incubated at 37 the isolated gut bacteria. Similarity matrix was °C for 48-72 h and the microbial growths were transformed into a phenogram by the unweighted pair determined by measuring the dry cell weight (DCW) group method with arithmetic averages (UPGMA). 15, 16 . Six organic solvents of high log Pow value; n- Further, the cluster analysis was carried out and hexane (3.9), isooctane (4.5), heptane (4.6), phenogram constructed as recently described by 17 cyclohexane (3.4), chlorobenzene (3.61) and toluene Kikani et al. . (2.8) and four organic solvents with low log Pow value; methanol (0.82), propanol (0.25), butanol (0.9) Results and 1, 4-dioxan (0.03) were selected for the present The relative length of the midgut used for the study. Organic solvents with low log Pow are isolation of the bacteria was 5.5 cm. Average number considered highly toxic to the microorganisms. of the aerobic and facultative anaerobic colony- 3 The selected nine bacterial isolates from the fish forming units (cfu) was 2.3 x 10 per gram of the gut displaying high enzyme-production and midgut. Altogether, a total of 18 bacterial isolates antimicrobial activities were further investigated by with different morphological characteristics were 16S rRNA gene sequencing. Genomic DNA from identified and obtained. Majority of the isolates each bacterial isolate was amplified and sequenced, displayed yellow and orange color pigmentation. using the 16S universal bacterial (27F& 1492R and The bacterial isolates were characterized on the 518F & 800R) sets of primers. 1 µl of template DNA basis of their biochemical characteristics. Among the added in 20 µl of standard PCR reaction solution. isolates, 33 % isolates displayed oxidase activity. Amplification protocol consisted of step-1, the initial Nitrate reduction, citrate utilization and urea denaturation at 94 ºC for 1 min; step-2, denaturation hydrolysis were represented by 50, 28 and 22 % at 94 ºC for 45 s; step-3, annealing at 55 ºC for 60 s; isolates, respectively, as highlighted in Table 1.

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Table 1- Biochemical characterization of the bacterial isolates

Biochemical Tests Isolates 1 2 3 4 5 6 7 8 9 10 11 7.1 + - - - - - + + - - - 7.2 + ------+ 7.3 + + ------+ + + 7.4 ------+ + - 7.5 ------+ - - + 7.6 - - - + - - - + - - - 7.7 - - - + - - + + - - - 7.8 + + - - - - - + - - + 7.9 - - - + - - - - - + - 7.10 - - - + + - - + - + + 7.11 - - - + + - + + - + + 7.12 - + - + + + - + - - - 7.13 - + - - - + + + - - + 7.14 - - - + - - + + - - + 8.1 - - - + - + - + - - - 8.2 - - - + - + - + - - - 8.3 - + - - - - + + - - - 8.4 + + - - - - - + - - -

*Table Abbreviations: 1- Citrate Utilization Majority of the isolates fermented all the sugars, Test, 2- Methyl Red Test, 3- VogesProskauer Test, 4- except trehalose, and interestingly, none of our Nitrate Reduction Test, 5- Indole Production Test, 6- isolates displayed production of gas or acid. Patterns Urea Hydrolysis Test, 7- Oxidase Test, 8- Growth in of the sugar utilization profile are documented in the 2%lysozyme, 9- Gelatin Hydrolysis Test, 10- Starch Table 2. Majority of the isolates utilized lactose and Hydrolysis Test, 11- Lipid Hydrolysis Test xylose, which emerged as the most favored carbon ‘+’ indicates positive test; ‘-‘indicates negative test source, whereas sucrose and fructose were much less preferred carbon source for the isolates. Significant difference was evident in the ability to utilize sugar by Interestingly, about 78 % of the total isolates the isolates, as reflected by the fact that lactose (72 exhibited growth in the presence of 2% lysozyme. %), xylose (72 %), rhamnose (61 %), inositol (56 %), Based on the Triple Iron Sugar (TSI) test, 50% of the arabinose (50 %), mannitol (50 %), maltose (39 %), isolates were identified as obligate aerobes, while the cellobiose (39 %), raffinose (33 %), fructose (28 %), rest were facultative anaerobes. and sucrose (22 %) were variably utilized by the The organisms grew over a salt concentration range studied bacterial isolates (Fig.1). of 0.5-2 % (w/v) and about 90 % of them displayed The extracellular enzyme production by the significant decrease in growth at 7 % (w/v) NaCl. bacterial isolates was qualitatively estimated. Optimal growth for the majority of the isolates was at Significantly high proportion of the bacteria isolates 0.5% (w/v) salt concentration. Two Actinobacteria, (44 %) exhibited lipase production, while amylase Dietzia sp. MJMG8.2 and Isoptericola variabilis and cellulase production was evident in 28 % and MJMG8.4 displayed maximum growth at 0.5 % (w/v) 11% isolates. Some isolates produced more than one NaCl concentration; while Acinetobacter sp. enzyme. Aeromonas punctata MJMG7.11 exhibited MJMG7.2 and Staphylococcus epidermis MJMG8.1 both cellulase and amylase activities and grew optimally at 7 % (w/v) NaCl. Ability of the Staphylococcus hemolyticus MJMG7.10 had amylase, isolates to ferment various sugars was investigated lipase and cellulase activities. Bacillus sp. MJMG7.3, under aerobic conditions. The extent of the sugar interestingly, had capability to produce all the four utilization was highly variable among the isolates. enzymes.

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Table 2- Sugar fermentation ability of the bacterial isolates MJMG8.4, were sensitive against all the drugs examined. Largest number of bacteria displaying Isolates 1 2 3 4 5 6 7 8 9 10 11 12 resistance were detected for AMP (39 %); AMC, CLR, E (22 %); CXM, CFR, CTX, CIP (17 %) and 7.1 + + + + + + + - - + + + CTR, AK, NET, NX, P, AZM, CPZ (11 %). Only 7.2 + + - + - + + - - + + + 5.5% of the isolates had resistance against CF, COT, 7.3 + + - + - + + - - + - + CL, C, VA, RIF, CFM and TE. About 78 % of the isolates were resistant against at least one of the 7.4 - - + - + + + - + - - - drugs, and 44 % of them displayed multi-resistance 7.5 - - - + - + + - - + + + characteristics. Staphylococcus pasteuri MJMG7.13 7.6 - - - + ------+ was resistant against five antibiotics; Ak, Cot, Net, Ctx and E, while, Bacillus sp. MJMG7.3 and 7.7 - - + + + + + - + + + + Psychrobacter sp. MJMG7.7 confirmed resistance 7.8 - + - + + + + - - - + + against four antibiotics; Amc, Ak, E, Clr and Cf, 7.9 - - - - + + + - + - - - Azm, E, Clr respectively.

7.10 - - - + + - + - - - - - Antibacterial activities of the isolates were examined 7.11 - - - + - - + - + - + + against various Gram positive and Gram negative test 7.12 + - + + + + + - - - - + human pathogens, by measuring the zone of inhibition. In the current study, the antibacterial 7.13 - - + + + - - - + + + + activities were evident against the Gram-negative 7.14 - + - - + - - - + - + + pathogens such as: Shigella, Salmonella typhi, 8.1 ------Escherichia coli, Enterobacter aerogens and Pseudomonas; and Gram-positive bacteria such as: 8.2 - - - + - - - - - + + + Micrococcus, Bacillus megaterium, Bacillus cereus, 8.3 ------Bacillus subtilis and Staphylococcus aureus. Overall, 8.4 - - + + + ------+ good antibacterial activity was observed against Micrococcus, Escherichia coli and Salmonella typhi. Acinetobacter sp. MJMG7.2 and Dietzia sp. *Table Abbreviations: 1- Sucrose, 2- Fructose, 3- MJMG8.2 showed antibacterial activity against five Cellobiose, 4- Xylose, 5-Inositol, 6- Arabinose, 7- human pathogens with a large zone of inhibition, Rhamnose, 8- Trehalose, 9- Raffinose, 10- Malotose, which can be considered as the most promising gut 11- Lactose bacteria with respect to the antibacterial action. ‘+’ indicates utilization of the sugar by the isolate; Bacillus sp. MJMG7.3, isolate MJMG7.1 and ‘-‘indicates no utilization of the sugar by the isolate MJMG7.5 displayed antibacterial activity against five human pathogens, while Isoptericola variabilis MJMG8.4 inhibited Gram negative bacteria, The isolates were examined for their sensitivity and Pseudomonas. resistance against 30 different antibiotics. Isolates varied in their sensitivity against these antibiotics. The growths of the bacterial isolates were studied Antibiotic resistance of the gut bacterial flora in the presence of 10 different organic solvents (data represented in Fig.2 highlighted that all the bacterial not shown). As previously mentioned, the solvents strains were resistant against the Gram positive- with a low log Pow are highly toxic to the organisms. specific antibiotics. Majority of the isolates were Interestingly, some of the bacterial isolates did exhibit sensitive against many antibiotics, whereas few growth in the presence of the low log Pow solvents. isolates exhibited zone of inhibition of more than 40 About 94 % of the isolates displayed growth in 1,4- mm in diameter against levofloxacin and tetracycline. Dioxan, and two gut bacterial isolates; Isoptericola Four bacterial isolates, Acinetobacter sp. MJMG7.2, variabilis MJMG8.4 and isolate MJMG7.4 had Aeromonas caviae MJMG7.11, Staphylococcus highest growth. epidermidis MJMG8.1 and Isoptericola variabilis

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Table 3- Antibacterial susceptibility against 10 human pathogens

Isolate 1 2 3 4 5 6 7 8 9 10

7.1 + + - - - + + - - + 7.2 + + + + - + - - - - 7.3 + + + - - + - + - - 7.4 + - + + - + + - - - 7.5 + + + - - + - - - + 7.6 + + - - - + - - - + 7.7 - + - - - + - - - - 7.8 - + + ------7.9 - - - + - + - - - + 7.10 - + + ------7.11 - + + - - - - - + - 7.12 - - - - - + + - + - 7.13 ------7.14 - + ------8.1 ------+ + - - 8.2 - + - + + - + + - - 8.3 + ------+ 8.4 - - - + - - - + - +

*Table Abbreviations: 1- Shigella, 2- Micrococcus, The phenogram (Fig. 4) is a diagrammatic 3- Salmonella typhi, 4- Bacillus megaterium, 5- representation of the taxonomic relationships among Bacillus cereus, 6- Escherichia coli, 7- Enterobacter the bacteria, based on the overall similarity of many aerogens,8- Bacillus subtilis, 9- Staphylococcus characteristics without regards to any evolutionary aureus, 10- Pseudomonas history. Four clusters, each containing two different ‘+’ indicates growth of the human pathogen; isolates, were formed at or above the 96 % similarity ‘-‘indicates no growth of the human pathogen level. Trends suggested that the phenotypic and phylogenetic data did not considerably coincide. The Sj/UPGMA phenogram showed some differences Staphylococcus epidermis MJMG8.1, Dietzia sp. from the dendrogram. Bacterial community of the MJMG8.2 and isolate MJMG8.3 represented growth present study could be grouped into 3 clusters on the in the presence of all the 10 different solvents, basis of phylogenetic tree highlighted in Fig.3; whereas Bacillus sp. MJMG7.3 and Psychrobacter sp. Staphylococcus sp. (cluster 1), Dietzia/Isoptericola MJMG7.7 were able to grow in all the solvents except sp. (cluster 2), and Aeromonas/Acinetobacter sp. methanol. Acinetobacter sp. MJMG7.2 and (cluster 3). However, from the phenogram/UPGMA Staphylococcus hemolyticus MJMG7.10 were able to cluster analysis, 4 clusters each containing more than grow in the presence of 4 separately added solvents. two different isolates were identified. These clusters The bacterial community from gut flora could be are labeled as A to D. Cluster-A consisted of grouped into 3 major clusters (Fig. 3).Cluster-1 Psychrobacter sp. and Staphylococcus sp., cluster-B comprised of Staphylococcus genera and includes included Staphylococcus sp., Dietzia and Isoptericola isolates MJMG7.13, MJMG8.1 and MJMG7.10; sp., while cluster-C consisted of Staphylococcus sp. whereas the cluster-2 included genera Dietzia sp. and cluster-D included Bacillus sp. and Acinetobacter strain MJMG8.2 and Isoptericola variabilis strain sp. Significantly, the phenotypic data may be useful to MJMG8.4. The cluster-3 included Acinetobacter sp. explore the biotechnological potentials of the studied MJMG 7.2 and Psychrobacter sp. MJMG 7.7. gut bacteria, using their biochemical properties.

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Fig. 1- Sugar fermentation ability of the isolates

Fig. 2c- Universal 3 (Hi media) disc

Fig. 2a- Percentage of the isolates displaying antibiotic resistance in Universal 1 (Hi media) disc

Fig. 2b -Universal 2 (Hi media) disc Fig. 3- Phylogenetic tree exhibiting the phylogenetic relationship between the isolates with their nearest homologs

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Discussion Bacteria are widely found in the habitats where tract of the fish. Enzyme producing Aeromonas fishes reside and taken up by the fish along with their species have been detected in the gut of fishes such diet. The digestive tract of fishes is inhabited by dense as, grass carp 30 and gray mullet 31. Presence of the bacterial population and the bacterial counts vary Staphylococcus species in the gastrointestinal tract of among the different types of fishes 21, 22.Differences in fishes has been reported in recent years. In the present the diversity and population size have been reported study, three Staphylococcus strains, namely in different regions of the digestive tract. Total viable Staphylococcus hemolyticus, Staphylococcus pasteuri count was 2.3 x 103 cfu g-1 in the midgut region which and Staphylococcus epidermis were identified, the is comparable with a bacterial count of 1.8 x 103 to finding which is quite similar to that for the Atlantic 1.8 x 105 bacteria g-1 reported for a commercial fish of salmon 7. Isoptericola variabilis, an Actinobacteria, Japan 1. This value is significantly lower than those has been isolated from a river soil sample 32. It has reported for humans and terrestrial animals been reported that the gut of mature Mugil contains (approx.1011cfu g-1). This possibly reflects that the sand, ingested along with the food from the bottom number of anaerobes (1010-1011cfu g-1) in the intestine substrates of estuaries and this might be the possible of endotherms is much higher when compared to that reason for the presence of Actinomycetes groups, of fishes and hence, the anaerobic microbial Dietzia and Isoptericola, in the gut of fish. communities of fish intestines are rarely considered 23, 24. The bacteria present in the gastrointestinal tract of fishes are related to their aquatic habitats and vary with the salinity of the habitat. Treatment of the gastrointestinal tract with N-saline solution prior to isolation ensured that the microbes isolated belong to the autochthonous gut flora. In the present study, 16S rDNA sequencing was used to differentiate nine potent and phenotypically distinct bacterial isolates. It was demonstrated that the Firmicutes and Proteobacteria were predominantly isolated from the midgut of the fish 7, which is also in line of some previous study with various fishes such as yellow catfish, rainbow trout and Atlantic salmon 4, 7, 25. Interestingly, some genera identified in the present Fig. 4- The phenogram, exhibiting the phenotypic relatedness study, i.e. Dietzia and Isoptericola have not been among the bacteria dwelling in the midgut of Mugil jerdoni previously reported from the gut of the mullets. Bacillus species have previously been detected in the Mugil jerdoni is frequently found in the estuarine gut of a wide variety of fishes, such as Atlantic environment which is a zone of active mixing of the salmon 7, 26, 27. The importance of the genera Bacillus 6 fresh and saline waters. Adult fishes are found in in aquaculture has been earlier discussed . Earlier water of 0-7.5 % (w/v) salinity, while juveniles can studies indicated the presence of Acinetobacter in the tolerate a wider salinity range. Bacteria are ingested digestive tract of fish 28 and an increase in its number 7 along with water at the larval stage and they colonize when the fish was fed a chitin supplemented diet . A the gastrointestinal tract to become autochthonous Gram-negative strain MJMG7.7 showing 99 % flora in juvenile fish. The results on the salt tolerance similarity with Psychrobacter sp., belonging to the 29 in present study indicated that, the gut bacterial family Moraxellaceae, was isolated. Yang et al. isolates were able to tolerate variations in salt demonstrated that the autochthonous microbial concentration. diversity was enhanced when the fishes were fed with diet containing probiotic Psychrobacter. As per a Mugil jerdoni is a diurnal feeder, consuming previous study, Aeromonas, a fish pathogen, dominant mainly small algae, zooplankton, dead plant matter among the cultivable bacteria in the intestine of fish, and detritus. The occurrence of diversified enzyme is known to cause various infections in the alimentary

1124 DATTA et al.: DIVERSITY AND ENZYMATIC PROFILE OF BACTERIAL FLORA IN THE GUT OF MUGIL JERDONI

producing indigenous intestinal bacteria indicates activity which was in agreement with the previous their possible role in the digestive process, by studies from a variety of fishes; Sea bass larvae, producing extracellular enzymes. Bacteria isolated Atlantic salmon and Artic charr 7, 38, 39. from the midgut of Mugil jerdoni were assessed The presence of enzyme producing bacterial flora qualitatively for amylase, cellulase, lipase and in the gastrointestinal tract of fishes can be correlated protease activities to ascertain their role in the with their feeding habitats. Kar and Ghosh 26 reported exogenous production of digestive enzymes. higher occurrence of the proteolytic bacterial Yokoi and Yasumasu 33 believed that fish do not populations in a carnivorous fish, Channa punctatus possess any cellulase, but Shcherbina and compared to that in herbivorous fish, Labeo rohita. Kazlauskiene 34 reported the presence of microbial Being omnivorous in nature, the occurrence of cellulase in the gastrointestinal tract of the common proteolytic bacteria was very limited in the digestive carp and more importantly cellulose activity resulted tract of Mugil jerdoni. from a ‘stable’ microflora in the digestive tract, The enzyme producing bacteria would certainly irrespective of the feeding habit. Presence of the play an important role in the digestibility of the food abundant cellulolytic bacteria in the gut of the and may assist in feed formulation for the commercial omnivorous and herbivorous well documented production of mullets in estuarine aquaculture. compared to the carnivorous fishes. Cellulase activity Due to the increased usage of antibiotics in the in the gut may be due to the cellulose procured from aquaculture, multiple antibiotic resistances have been the ingestion of plant detritus. Prejs and Blaszczyk 35 reported in the fish pathogens and bacteria 40, 41. Mugil established a positive correlation between the jerdoni is an edible fish and is usually consumed in cellulase activity and the amount of the dead plant cooked form in India, and therefore, the fish might be materials in the gut of cyprinid and salmonid species. a low risk food, even if it is contaminated with enteric Present study indicated the presence of five bacteria species. The antibiotic susceptibility profile cellulolytic bacteria in the gastrointestinal tract of the showed that few bacterial isolates were resistant to as omnivorous estuarine fish. Bacillus sp. MJMG7.3 and many as 8 antibiotics out of the 30 antibiotics Aeromonas punctata MJMG7.11 displayed a cellulase examined in the present study. The presence of activity which is in consistence with the report by resistance in Aeromonas, isolated from a retail fish Askarian et al. 7 and Jiang et al.30. and the high incidence of the multiple antimicrobial The presence of the exogenous amylase activity is resistance has been reported 42. Interestingly, about more prominent in the digestive tract of the 22% isolates were sensitive against all the antibiotics omnivorous and herbivorous as compared to the suggesting that these strains might be nutritionally carnivorous fishes. Sugita et al. 36 detected amylase beneficial to the host organism and hence of low risk producers at high densities in the digestive tract of to humans. four omnivorous fishes and the absence of aerobic The gastrointestinal flora is known to produce amylase producers in the gut of carnivorous fish. Das various bioactive substances such as different classes et al. 37 reported amylase producing Brevibacillus of bacteriocins, biotin 43 and organic acids to exert parabrevis and Bacillus licheniformis from the their antimicrobial property. In the present study, the gastrointestinal tracts of brackish water teleosts. bacterial isolates effectively inhibited the Gram Significant amylase activity was exhibited by Bacillus positive pathogens as compared to the Gram negative sp. MJMG7.3, Staphylococcus hemolyticus pathogens. Similar studies have been carried out with MJMG7.10 and Aeromonas punctata MJMG7.11 in the Lactobacillus sp. isolated from Mugil cephalus 44. the present study. There is no report on the presence In a recent study, the antibacterial activities of several of amylase producing Staphylococcus hemolyticus lactic acid bacteria were tested against several human species in the gut of estuarine fish. pathogens in which, Acinetobacter sp. and Bacillus Lipase producing bacteria in the digestive tract of sp. did not exhibit antibacterial activity when tested fish aid in the digestion of triglycerides. Extracellular against four fish pathogens: A. salmonicida, V. lipase activity was presented by eight isolates in anguillarum, M. viscosa and C. maltaromaticum, which maximum activity was shown by the isolate respectively 45. In the present study our two isolates, MJMG7.14. Acinetobacter sp. MJMG7.2, Bacillus sp. Acinetobacter sp. MJMG7.2 and Bacillus sp. MJMG7.3, Staphylococcus pasteuri MJMG7.13 and MJMG7.3 displayed antibacterial activities against Aeromonas punctata MJMG7.11 did exhibit a lipase five human pathogens.

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The toxicity of an organic solvent is closely related sp. MJMG7.3 and Aeromonas punctata MJMG7.11 to its hydrophobicity, as expressed by the log Pow (representatives of cluster C and D) did exhibit lipase values. In general, the Gram-negative bacteria show and cellulase activities which was also corroborated relatively higher solvent tolerances than the Gram- by previously reported variety of fishes; Sea bass positive bacteria 46, which may be due to the larvae, Atlantic salmon and Artic charr 7, 38. Members difference in the composition of the cell envelope. of the clusters B and C were sensitive to Ampicillin. Previous studies have reported that the most important The presence of resistance pattern, in our studies in hydrocarbon-degrading bacteria both in marine and case of Aeromonas sp. was similar to an earlier report soil environments are Achromobacter, Acinetobacter, by Radu et al. 42.While, the members of the clusters A Alcaligenes, Arthrobacter, Bacillus, Flavobacterium, and D were sensitive to the Amikacin, Co- Nocardia, Pseudomonas and the coryneforms 47. Oil Trimoxazole, Erythromycin and Clarithromycin. hydrocarbon-degrading bacteria belonging to the Interestingly, no member of the cluster D was able to genera Aeromonas, Pseudomonas, Flavobacterium grow in the presence of 2.0 % (w/v) lysozyme. and Micrococcus were isolated from the digestive Similar analysis has recently been carried out using tracts of fish and among which the species Aeromonas this approach in our laboratory for the analysis of the eucrenophila and Aeromonas media dominated 48. In diversity of the haloalkaliphilic bacteria from the the present study, Aeromonas punctata MJMG7.11 saline desert Bhatt and Singh 50. showed increased cell biomass in the presence of organic solvents; methanol, butanol, toluene, 1,4- Conclusion dioxan, cyclohexane and chlorobenzene. Dietzia sp. The bacterial community was assessed using both MJMG8.2 grew in the presence of all the organic the phylogenetic and the phenotypic relationships in solvents which correspond with a previous study in the present study. Further, the diversity was judged on which Dietzia was able to grow on n-alkanes of C12 the basis of the biocatalytic potential, sugar to C38 and branched alkanes 49. The enzymes of fermentation capability, antibiotic bacteria present in the digestive tract partly degrade sensitivity/resistivity, antimicrobial activities against the organic solvents consumed by the fish along with human pathogens and organic solvent tolerance of the the food or water. isolates. This study would be of practical significance In the present study, the bacterial isolates with respect to the feed formulation and disease exhibiting different profiles of the conventional prevention in commercial aquaculture 29, 45. microbiological features were further identified by the sequencing of 16S rRNA gene. The phylogenetic tree Acknowledgement was constructed to compare the evolutionary Authors greatly acknowledge the financial and distances between the isolated bacteria. Overall, the infrastructural support of the University Grant bacterial community of the present study could be Commission (UGC), New Delhi under various grouped into 3 clusters, as Staphylococcus sp., programmes including CAS and Saurashtra Dietzia/Isoptericola sp. and Aeromonas/Acinetobacter University, Rajkot. AKS gratefully acknowledges the sp. Research Fellowship in Sciences for Meritorious From the phenogram/UPGMA analysis, four Students by UGC, New Delhi, India. clusters each containing more than two different isolates were formed. These clusters are labeled in References alphabetic sequence, A to D. In addition, there were 1 Austin, B., The bacterial microflora of fish, Sci. World J., three two-member clusters and one three-member 6(2006), 931–945. 2 Cahill, M.M., Bacterial flora of fishes: a review, Microbial cluster.The clusters reveal different carbon source Ecol., 19(1990), 21–41. utilization patterns. Members of the clusters A and B 3 Ringø, E. & Birkbeck, T.H., Intestinal microflora of fish were able to grow well in a wide range of solvents, larvae and fry, Aquacult. Res., 30(1999), 73–93. while the members of the clusters C and D were less 4 Kim, D., Brunt, J. & Austin, B., Microbial diversity of versatile, displaying low growth percentage in intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss), J. Appl. Microbiol., 102(2007), solvents in Minimal Davis broth. Members of the 1654–1664. clusters C and D in general had good proteolytic 5 Bairagi, A., Ghosh, K.S., Sen, S.K. & Ray, A.K., Enzyme activity and were potent source of extracellular producing bacterial flora isolated from fish digestive tracts, amylases, proteases, cellulases and lipases. Bacillus Aquacult. Int., 10(2002), 109–121.

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