Seasonal Variation of Culturable Benthic Soil Prokaryotic Microbiota as Potential Fish Pathogens and Probiotics from an Aquaculture Farm in East Kolkata Wetlands, india

Rising demand in the aquaculture sector tends towards finding innovative ways to promote better yield and profitability. Benthic soil microbiota can provide an insight into the potent opportunistic fish pathogens as well as probiotics present in the aquaculture system. This study reports the seasonal diversity and abundance of fifteen culturable pathogenic bacterial strains belonging to the genera of Comamonas, Aeromonas, Providencia, Klebsiella, Escherichia, Acinetobacter, Serratia, Stenotrophomonas, Staphylococcus, and Enterobacter along with nine probiotic strains native to genera of Bacillus and Pseudomonas isolated from an aquaculture farm benthic soil, located in East Kolkata Wetlands, West Bengal, India. Strains are isolated using traditional microbial culture methods and tested for their antimicrobial susceptibility against commonly available antibiotics. 16S rDNA analysis was done for the identification of the strains and the establishment of their phylogenetic relationships. Among the isolates, B. pumilus strain S8 in the pre-monsoon sample, E. coli strain M2aR1 in the monsoon sample, and A. hydrophila strain P6dF1 in the post-monsoon sample were the most abundant having MPN counts of 275±21 x 106 CFU/gram dry soil, 278±18 x 106 CFU/gram dry soil, and 321±28 x 106 CFU/gram dry soil respectively. Data on the temporal diversity, abundance, and drugsusceptibility of prokaryotic fish-pathogens and probiotics can be used to formulate measures for sustainable aquaculture practices with reduced maintenance costs.

as natural sinks for waste recycling and attenuation of floods. Secondly, these water bodies are utilised for a thriving culture of several fish species 20 . The sewage fed aquaculture ponds, locally known as bheris, are unique in their operating procedure as they utilize municipal waste products as a fish feed with the occasional addition of inert feeds 21 . However, the seasonal dynamics of the physicochemical properties of water and benthic soil play crucial roles in the microbiome of the benthic soil of aquaculture farms 22,23 . Though several references are available on the description of the microbial communities of East Kolkata Wetlands, seasonal dynamics of the benthic soil prokaryotic microbiota emphasizing on the variation of probable fish pathogens and probiotics are scarce.

MATERIAl AND METhoDS Media and Chemicals
All chemicals and media were procured from Himedia (India) and Sigma Aldrich Chemical (USA). Tryptone Soya Agar (TSA) was used as enrichment media for the isolation of strains. Routine subculture and the permanent stocks were made in Nutrient agar (NA). Antibiotic Susceptibility test was done on Mueller Hinton Agar (MHA). Culture media were subjected to sterilization at 120°C temperature and 20 psi pressure for 15 minutes before inoculation.

Sample collection
Benthic soil samples were acquired aseptically from an aquaculture farm in East Kolkata Wetlands (Lat -22.5699° Long-88.4394°) from the top surface of the soil below 60cm of the water column. Samples of three seasons in the year 2019 viz. Pre-monsoon sample (April), Monsoon sample (August), and Post monsoon sample (December) were chosen for prokaryotic analyses in this study.

Isolation of bacterial strains
Isolation of culturable bacterial strains was done according to the method given by Vieira and Nahas 2005 24 , with some minor alterations. 5gms (wet weight) of each soil sample was first diluted and homogenized in sterile water (50 ml) with intermittent cooling in an ice bath for 30 minutes. The homogenate was then passed through a sterile 2mm mesh. Filtrates were serially diluted up to 10 8 fold, and 100 µl from the last four dilutions (10 5 to 10 8 ) of each filtrate were spread on TSA plates (in triplicates), which were then incubated at 37°C for 48 hours. A dry weight of soil was calculated by incubating 50 grams of benthic soil in 105°C for a period of 8 hours, and the final weight was used to calculate the wet weight to dry weight conversion factor 25 .

Most Probable Number Count
Colonies that appeared on Each TSA plate after 48 hours of incubation, were marked based on colony morphology (shape, size, colour, transparency, margin contour, and surface topology) and were analysed for the Most Probable Number (MPN) count was done according to the method given by Janssen et al. 2002 26 with some minor alterations. Conversion factors of wet weight to dry weight of each soil sample and dilution factor of the inoculum were considered in the MPN count. The count was taken as a mean along with standard deviation for each dilution (from the triplicate count) and results were expressed in CFU x 10 6 /gram dry soil. For the process of purification, colonies were transferred to NA plates and repeated subcultures were done until the strains were free from conglomeration. Each purified strain was stored permanently in NA stab cultures at 4°C and 80% glycerol stock at -20°C.

Biochemical characterization
A total of fourteen biochemical tests, including determination of Gram Character, Methyl Red (MR), tests for gelatinase, Triple Sugar Iron (TSI), degradation of starch, Voges-Proskauer (VP), indole production, citrate, oxidase, motility tests, etc. were performed with each purified strains and they were characterized following the methods of Bergey's Manual of Systematic Bacteriology 27 .

Molecular Characterization Isolation of Genomic DNA and 16S rRNA gene amplification
Isolation of Genomic DNA from the cultured bacterial strains was done using a bacterial genomic DNA isolation kit (GCC Biotech, India). Genomic DNA was electrophoresed through 0.8% agarose gel and was measured spectrophotometrically at 260 nm wavelength to estimate purity. The samples were subjected to 16S rRNA gene amplification using a Gradient Thermal Cycler (Biorad Laboratories, USA). Universal primer 27f (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3') encompassing V1-V9 hypervariable region of 16S rDNA were used for PCR amplification in conjunction with 2X PCR MasterMix (Thermo Fisher Scientific, India). The PCR amplification was carried out for 30 cycles and the amplicons (1.4 kbp approx.) were tested in 1.5% agarose gel using Ethidium Bromide stain. Purification of the amplicons was done using the Agarose Gel purification kit (NEB, USA) and sequencing was done from Xcelris Labs Ltd. (Ahmedabad, India).

in -silico analysis
Sequences were analysed in-silico using BLASTn (NCBI database), the hits were recorded for finding the nearest neighbour with the highest Max score. The aligned sequences were obtained in the FASTA format for downstream analyses in MEGA 7 31 . CLUSTALW algorithm was used for alignment analyses and the data thus obtained were considered for phylogenetic analyses using Neighbor-Joining method 32 . The evolutionary distance was calculated by Maximum Likelihood  Composite method 33 and the branch lengths were calculated based on base substitution per site. Bootstrap tests of 500 replicates were done to construct the consensus tree to represent the evolutionary history of the taxa analysed 34 . Nearest four neighbours of each isolate from the BLASTn hits were used to create the phylogenetic tree. The partial 16S rDNA sequences of all strains were deposited into the GENBANK database.

Statistical analysis
Experiments on the enumeration of bacterial MPN were performed in triplicate and mean values were indicated along with standard deviation (SD). SPSS 17.0 (SPSS Inc., Chicago, USA) was used for the Statistical analyses.

Isolation of bacterial strains
In total, two hundred and forty-eight culturable bacterial strains were isolated and purified from the aquaculture benthic soil samples collected from three seasons. Molecular phylogenetic analyses revealed that several bacterial strains were common in samples from more than one season. Amongst the isolates, only twenty-four strains were revealed to have either presumptive pathogenic or probiotic values to fish and are reported in this communication. In the pre-monsoon season sample, a total of six strains (Bacillus flexus strain S1a, Aeromonas punctata strain S2, Bacillus pumilus strain S8, Bacillus subtilis strain S8a1, Comamonas aquatica strain SAC1 and Bacillus cereus strain SWA6a) were isolated. Cumulatively, eight strains were found in the monsoon season sample (Bacillus thuringiensis strain M10, Aeromonas enteropelogenes strain M11, Escherichia coli strain M2aR1, Pseudomonas aeruginosa strain M2F1, Bacillus flexus strain M3, Acinetobacter junii strain M5fR1, Serratia marcescens strain M5hR1 and Stenotrophomonas maltophilia strain M6aR1). Finally, from the post-monsoon season sample, another ten strains were isolated (Escherichia coli strain P1bR1, Providencia vermicola strain P2fR1, Enterobacter cloacae strain P4cR1, Bacillus flexus strain P5a, Klebsiella pneumoniae strain P5aR1, Serratia marcescens strain P5bR1, Bacillus cereus strain P6b1, Aeromonas hydrophila strain P6dF1, Comamonas aquatica strain P7, and Staphylococcus aureus strain P8d3a1). Isolate B. flexus strain S1a, A. punctata strain S2, B. pumilus strain S8, B. subtilis strain S8a1 were found in the samples from both the pre-monsoon and monsoon season whereas, E. coli strain M2aR1, B. flexus strain M3 and S. marcescens strain M5hR1 strains were found to occur in the monsoon and post-monsoon samples.

MPN count
The MPN counts of bacterial strains isolated from all three seasons are depicted in Figure 1. Isolate B. pumilus strain S8 in the premonsoon sample, isolate E. coli strain M2aR1 in the monsoon sample, and A. hydrophila strain P6dF1 in the post-monsoon sample were the most abundant having MPN counts of 275±21 x 10 6 CFU/ gram dry soil, 278±18 x 10 6 CFU/gram dry soil, and 321±28 x 10 6 CFU/gram dry soil respectively. Strains were found to vary drastically in abundance in different seasons revealed by the difference in seasonal abundances of B. flexus strain S1a,

Biochemical Characterization
The biochemical characteristics of all the isolates are compiled in Table 1. Among the isolates, nine were Gram positives and rest were Gram-negative bacteria. Fermentation of sugar as analysed by TSI tests revealed a varying degree of fermentation potentiality amongst the isolates, in both aerobic and anaerobic conditions. All the strains tested were DNase negative and were unable to produce H 2 S in TSI media.

Antibiogram
The results of the antibiogram of the bacterial strains are given in

Molecular Characterization and phylogenetic analyses
BLASTn results revealed the identity of the isolates and taxonomic names were assigned based on the nearest neighbour of the NCBI database. Table 3 depicts the BLASTn analysis of the bacterial strains. Phylogenetic analysis of the fifteen pathogenic strains is given in Figure 2 and the optimal sum of the branch length was found to be 3.522. The phylogenetic relationship of the nine probiotic isolates is shown in Figure 3 with an optimal sum of branch length 1.478. Both the trees are drawn to scale and are shown as legend in the figure.
With the help of online literature databases, a review was done highlighting the pathogenic effect of the isolates on fish species cultivated in the farm and also to ascertain the efficacy of the species isolated as probiotics. Data obtained from the literature survey is summarised for the pathogens and probiotics in Table 4 and  Table 2. Antibiogram results of twenty-four isolates from the soil sample against thirteen different Antibiotics. The table is colour coded according to the legend given below Table 5 respectively. Notably, among the isolates, three species are belonging to genus Aeromonas were found that cause Aeromoniasis, which is by far the worst affecting disease, causing high fish morbidity in aquaculture fish cultivation scenario and is advised by several citations [35][36][37][38][39] . However, it is also appealing that among the probiotics isolated, several of them are reported to impart resistance against the pathogens isolated from the same source as well as boosting the immunity of the cultured fish specimens [40][41][42][43] .

DISCUSSIoN
This study reveals several pathogens and probiotic species present in the benthic soil of the aquaculture farm under study. The farm cultivates Indian and exotic major carps along with some catfishes and cichlid fishes and there is ample chance that any of these fish species may get affected by the pathogens revealed in this study. The presence of pathogens in a fish farm is never evaluated until or unless a disease breakout occurs with high fish morbidity 44 . Mass fish morbidity is reported caused by pathogens like A. hydrophila 10,35,45 , A punctata 36 , K. pneumonia 46,47 causing a huge loss in revenue. Bacterial isolates identified in this study revealed that the benthic soil sample collected from post-monsoon season harbors a huge number of opportunistic pathogens with A. hydrophila being the most abundant. It is also noteworthy that, in comparison with the other two seasons, diversity and abundance of the opportunistic pathogens are much higher in the post-monsoon season sample as the occurrence of new genera of pathogens viz. Klebsiella, Enterobacter and Providencia were noticed along with strains of Escherichia and Serratia. This high abundance of diverse opportunistic pathogens could be attributed to the mixture of urban sewage and rainwater runoff from adjacent areas during monsoon and post-monsoon seasons.

Journal of Pure and Applied Microbiology
Several antibiotic-resistant strains have been reported by scientists due to the indiscriminate antibiotic use in aquaculture farms 48,49 and also pathogens like E. coli can transmit the resistance to other pathogens possibly through horizontal gene transfer 44 . Since the study reveals the antibiotic susceptibility of the strains, indiscriminate use of antibiotics could be avoided and specific treatment measures can be implemented.
The prokaryotic probiotics present in soil and water also boost the immunity of culturable fishes 8,50-53 against several pathogens, and thus, steps could be formulated for the aid of the probiotics. These probiotic bacteria can also act as a dietary supplement for the fish specimens, mitigating the problem raised by inert fish feeds 43,[54][55][56] . Probiotic species also engage in the continuance of biogeochemical cycles by several enzymes that help in the biodegradation   of detritus materials in the benthos 18 . Probable probiotic species isolated in this study produces enzymes like cellulase 57 , lipase (unpublished data), and hence could be forerunners in the maintenance of the aquatic ecosystem. These probiotics are often consumed from the soil by bottom feeders thus circulating them along the food chain and could alter the gut microcosm of fish beneficially 58 . Further, it was revealed that the presence of probiotics is maximum is pre-monsoon season and declines rapidly from the monsoon season and thereafter. This is because the farm practices liming and the addition of organic fertilizers once annually in the pre-monsoon season leading to the amplification of probiotics. The monsoon rains bring a lot of agricultural and domestic runoffs as well as sewage overloads reducing the quality of water and soil, thus diminishing the number of probiotics and leading to the growth of pathogens. Coliforms are also found abundant in the soil during monsoon and post-monsoon season. This is probably the first report that provides a more in-depth insight into the culturable microcosm from an aquaculture farm of East Kolkata Wetlands, emphasizing their roles as fish pathogens and probiotics. This project could lead to early detection of pathogens and formulation of remedial measures even before the onset of the fish pathogenesis. This could be done by formulating a suitable bioaugmentation program to reinforce the growth of probiotics and eradication of the pathogens ensuring sustainable and profitable aquaculture.