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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

In vitro Profiling of Probiotic Bacillus Species Isolated from the Gut Microbiota of Brackish Water Fish, Etroplus Suratensis (Pearlspot)

E
Ethirajan Manimozhi1
A
Arumugam Uma2,*
N
Nathan Felix3
A
Antony Cheryl3
S
Sethu Selvaraj4
S
S. Arun Sudhagar5
H
Harikrishan Lisha1
1Department of Aquatic Animal Health Management, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Dr. M.G.R. Fisheries College and Research Institute, Ponneri-601 204, Tamil Nadu, India.
2Directorate of Incubation and Vocational Training in Aquaculture, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, ECR-Muttukadu Chennai-600 001, Tamil Nadu, India.
3Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Vettar River View Campus, Nagapattinam-609 307, Tamil Nadu, India.
4Department of Aquaculture, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Dr. M.G.R. Fisheries College and Research Institute, Ponneri-601 204, Tamil Nadu, India.
5Fish Health, Peninsular and Marine Fish Genetic Resources Centre, ICAR – National Bureau of Fish Genetic Resources, Kochi-682 001, Kerala, India.

ABSTRACT

Background: In aquaculture, antibiotic misuse has accelerated antimicrobial resistance (AMR), endangering fish health, ecosystems and public safety. Probiotics offer a sustainable alternative by enhancing immunity, growth and pathogen resistance without promoting AMR. The pearlspot (Etroplus suratensis), a valuable brackish water fish in South Asia, remains vulnerable to bacterial diseases, yet research on autochthonous probiotics for its health management is limited.

Methods: The present investigation aimed to assess the probiotic potential of two bacterial isolates, Bacillus subtilis (BLCET3/SRLAAH/2023) and B. velezensis (BLCET4/SRLAAH/2023) isolated from the gut of a brackish water fish, E. suratensis. The probiotic potential of the isolates was analyzed by in vitro assays, molecular analysis of probiotic marker genes and in vivo biosafety assay.

Result: Among the two isolates, BLCET3 exhibited stronger antimicrobial activity against Streptococcus agalactiae, Aeromonas veronii and Enterococcus sp. Both BLCET3 and BLCET4 tolerated a pH range of 2-9 and bile salt concentrations up to 10%, produced extracellular enzymes and formed biofilms. BLCET3 showing significantly higher (P<0.05) auto-aggregation, co-aggregation and cell surface hydrophobicity. Neither isolate displayed antibiotic resistance in ABST assays and in vivo biosafety tests confirmed them as non-pathogenic. PCR analysis revealed the presence of key probiotic marker genes. These findings confirm the probiotic potential of both isolates for aquaculture applications.

INTRODUCTION

Disease is a major constraint to aquaculture production, causing an estimated 21% loss of global aquaculture output, equivalent to approximately US$ 10 billion annually (Peeler et al., 2023). In India, disease-related losses in aquaculture are estimated at US$ 2.48 billion, accounting for 14.95% of the annual production value (Patil et al., 2025).
       
In aquaculture, antibiotics play a vital role in preventing and treating bacterial infections. While antibiotics are widely used to manage these infections, their overuse has led to antibiotic resistance, raising global health concerns (Mukhtar et al., 2025). Probiotics have emerged as sustainable alternatives, offering health benefits such as improved digestion, pathogen inhibition and enhanced immunity in fish (Rathod et al., 2025). They prevent pathogen colonization via competitive exclusion, organic acid production and antimicrobial metabolites like bacteriocins (Tabassum et al., 2021).
       
Important probiotic selection criteria include their  acid and bile  tolerance for gut survival, adherence to epithelial surfaces and biofilm formation for colonisation (Hyronimus et al., 2000). Genetic markers enable precise probiotic strain identification, complementing microbiological techniques (Papadimitriou et al., 2015). Autochthonous probiotics from the fish gut are better adapted to aquatic environments than commercial probiotics sourced from terrestrial systems (Lalitha et al., 2022; Hoseinifar et al., 2024). Bacterial genera viz., Lactobacillus, Bifidobacterium, Streptococcus, Bacillus and Enterococcus exhibit probiotic potential (Nayak et al., 2010), with Bacillus species gaining prominence due to their resilience and effectiveness in aquaculture (Cheng et al., 2017). In brackish water aquaculture, Bacillus sp. enhanced growth, water quality, stress tolerance and disease resistance in finfish and shellfish species viz., Oreochromis niloticus, Labeo rohita, Penaeus vannamei and Scylla tranquebarica (Hortillosa et al., 2022; Jamil et al., 2025; Gunarto et al., 2023; Monier et al., 2023).
       
E. suratensis (Pearlspot), native to South Asian brackish water habitats, is commercially significant and suitable for polyculture systems. Despite its aquaculture importance, bacterial diseases remain a challenge in their culture activities (Swaminathan et al., 2022). Studies on autochthonous probiotics characterization from E. suratensis remain largely unexplored. To address this, two Bacillus species were isolated and characterized from the gut of E. suratensis and their probiotic potential were evaluated.

MATERIALS AND METHODS

Sample collection and isolated of gut-associated Bacillus species
 
Seventy-five healthy specimens of E. suratensis (46±2.4 g; 11.4±1.2 cm) were collected from Pulicat lagoon (13.4177o N, 80.3185oE) and transported alive to the State Referral Laboratory for Aquatic Animal Health, Chennai. After 48 h of starvation, the guts were aseptically dissected, homogenized and serially diluted in sterile saline (10-1 to 10-6). Samples were spread plated on Hichrome Bacillus agar and incubated at 37oC for 24-48 h. Morphologically distinct colonies were sub-cultured to obtain pure isolates, yielding 37 bacterial strains from the gut of E. suratensis.
 
Screening for antibacterial activity
 
Fish bacterial pathogens viz., Streptococcus agalactiae (NCBI Accession no. PP580699), Aeromonas veronii (Accession no. OR487464), Enterococcus sp. (Accession no.  PP600907)  obtained from the microbial archive in the State Referral Laboratory of Aquatic Animal Health - Madhavaram, Chennai was used. The antibacterial potential for the isolates was screened using disc diffusion method (Lapenda et al., 2015). The zones of inhibition (mm) against the bacterial pathogens were measures and the effective isolates., BLCET3 and BLCET4 were further characterised biochemically and molecular confirmated by PCR and sequencing.
 
Biochemical characterization
 
Morphological and biochemical characterization of the bacterial isolates BLCET3 and BLCET4 were carried out by gram-staining, motility test, oxidase, catalase, methyl red, voges-proskauer test, growth at 5% NaCl, indole, citrate utilization, carbohydrate fermentation and amino acid utilization tests (Berkeley et al., 1984).
 
Molecular confirmation of bacterial isolates
 
Total DNA was extracted from bacterial isolates BLCET3 and BLCET4 using commercial DNA extraction Kit (Qiagen, Germany). 16S rRNA gene of the bacteria was amplified following Weisburg et al. (1991). Amplified products (1484 bp) were visualized on a 1.5% ethidium bromide-stained agarose gel and documented (Bio-Rad). Sequenced products (Eurofins, India) were analyzed using NCBI BLAST, submitted to GenBank for accession numbers and a phylogenetic tree was constructed via Maximum Likelihood in MEGA 11.
 
In vitro screening for probiotic potential
 
The probiotic potential of Bacillus isolates was assessed through multiple in vitro assays. pH and bile tolerance were evaluated following Khan et al. (2021), with bacterial cultures adjusted to OD600 0.25 (10w CFU/mL). For pH tolerance, cultures were incubated in TSB (pH 2-9) at 37oC for 24 hours. For bile tolerance, isolates were exposed to bile salts (0%, 2.5%, 5% and 10% w/v) in PBS (pH 7.4) at 30oC for 1 hour and growth was measured at 600 nm (Shimadzu, Japan). Salt and phenol tolerance were tested in MRS broth with varying concentrations of NaCl (0-5% w/v) and phenol (0-5% v/v), incubated at 30oC for 24 hours and measured at 600 nm (Aswathy et al., 2008). Extracellular enzymatic activity was evaluated using nutrient agar supplemented with specific substrate: starch for amylolytic activity, carboxymethyl cellulose for cellulase activity, gelatin for gelatinase activity and skimmed milk for proteolytic activity. The bacterial isolates, BLCET3 and BLCET4 were streaked onto respective media and incubated at 30oC for 24 h. Amylase activity was detected by flooding with 1% iodine solution, gelatinase with 15% mercuric chloride and cellulase with 1% Congo red followed by washing with 1 M NaOH (Balan et al., 2012; Kasana et al., 2008). Auto-aggregation and co-aggregation assays followed Rastogi et al. (2020) and Handley et al. (1987). Cell hydrophobicity was tested using xylene, chloroform and ethyl acetate following the method of Patel et al. (2009). Biofilm formation was screened using congo red agar method (Ramesh et al., 2015). The isolates, BLCET3 and BLCET4 were streaked on Muller Hinton agar (Himedia, India) containing 0.8 g/l of Congo red dye and incubated for 48h at 37oC.
 
Detection of probiotic marker genes
 
The probiotic properties of BLCET3 and BLCET4 were confirmed by PCR amplification of marker genes, including 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (2,3-BIP), arginine/ornithine antiporter (ArcD), choloylglycine hydrolase (CGH), LuxS and the E1 β-subunit of the pyruvate dehydrogenase complex (E1-β) (Khan et al., 2021). DNA quality and concentration were checked spectrophotometrically (OD260/280 ~1.8-2.0) prior to amplification. PCR reactions included standard positive and negative controls to ensure specificity and reliability. Amplicons were resolved on 1.5% agarose gels and documented using a gel documentation system (Bio-Rad, Germany).
 
Safety evaluation of the probiotic isolates
 
The antibiotic susceptibility of BLCET3 and BLCET4 was evaluated using the Kirby-Bauer disk diffusion test on Mueller-hinton Agar (MHA) with sterile antibiotic discs (Himedia, India) following CLSI standards (Bauer et al., 1996). Zones of inhibition (ZOI in mm) were measured after 24 - 48 hrs of incubation at 37oC. For in vivo biosafety assessment, groups of 10 E. suratensis (23.5±2 g) in triplicates were intraperitoneally injected with 0.1 mL bacterial suspensions (109 CFU/mL) of each isolate. A control group received an equivalent volume of sterile PBS (pH 7.2). Fish were monitored daily for 10 days for signs of disease and mortality.
 
Statistical analysis
 
The results of in vitro tests were analyzed using the IBM SPSS Statistic 26 software. All the data were analyzed using one-way ANOVA employing Tukey’s post hoc test (p=0.05) to identify the significant differences among the groups. The results were represented as the mean ± standard error, with statistical significance at P<0.05.

RESULTS AND DISCUSSION

Bacillus spp. are promising probiotics in aquaculture due to their spore-forming nature, ease of large-scale production, enzyme synthesis, beneficial metabolites and immunost-imulatory properties (Nayak et al., 2010; Li et al., 2023). The healthy fish gut provides host-adapted autochthonous probiotics that enhance fish and ecosystem health while reducing the environmental risks linked to non–host-specific strains (Li et al., 2024; Fadel et al., 2025). A total of 37 bacterial isolates from the gut of 75 healthy E. suratensis specimens were screened for their antibacterial efficacy. Among them, two isolates BLCET3 and BLCET4, exhibited significant antibacterial activity against fish pathogens, including S. agalactiae, A. veronii and Enterococcus sp. The zones of inhibition (ZOI) were measured and are presented in Table 1. The antibacterial activity observed is likely attributed to the production of bacteriocins, enzymes and other antimicrobial metabolites (Naeem et al., 2018) which enhance the ability of these isolates to inhibit pathogenic bacterial growth effectively.

Table 1: Antibacterial activity of the bacterial isolates against targeted fish pathogens.


       
Biochemical characterization revealed metabolic variations between BLCET3 and BLCET4, particularly in their ability to ferment carbohydrates and utilize amino acids (Table 2). These findings suggest that both isolates possess a versatile metabolic profile, contributing to their survivability and functionality in the gut environment (Kuebutornye et al., 2020). Molecular characterization through 16S rRNA sequencing identified BLCET3 as B. subtilis (Accession No. PP851370) and BLCET4 as B. velezensis (Accession No. PP657343) (Fig 1).

Table 2: Biochemical characterization of the bacterial isolates.



Fig 1: Phylogram constructed using the neighbour-joining tree for Bacillus species.


       
The ability to withstand harsh gastrointestinal conditions is fundamental probiotic trait (Ghosh et al., 2017). Both BLCET3 and BLCET4 exhibited pH tolerance across a wide range (2-9), with optimal growth observed at pH 7-8 (Fig 2a). Notably, BLCET3 displayed slightly greater resilience at extreme pH levels compared to BLCET4. Similarly, both isolates tolerated bile concentrations up to 10% (w/v), although growth gradually decreased with increasing bile concentrations (Fig 2b). These findings are consistent with earlier report of Nayak et al. (2024), which highlighted the importance of pH and bile tolerance in probiotic bacteria.

Fig 2: In vitro assays for probiotic screening of bacterial isolate.


       
Both isolates demonstrated salt tolerance up to 5% NaCl and phenol tolerance up to 0.6%, with growth declining beyond these thresholds (Fig 2c, 2d). The ability to withstand salt stress aligns with observations by Emam and Dunlap (2020) who reported the role of osmolyte regulation in maintaining cellular balance under hypertonic conditions. Phenol tolerance, an indicator of resilience in the gastrointestinal environment, further validates the robustness of these isolates (Yadav et al., 2016).
       
Extracellular enzymes production viz., amylase, cellulase, gelatinase and protease were observed in both isolates, indicated by clear halo zones around colonies on substrate- specific agar plates. These enzymatic activities facilitate the breakdown of complex dietary components, improving nutrient absorption in the host (Banerjee et al., 2013). The enzymatic profile observed in BLCET3 and BLCET4 suggests their potential to enhance the digestive efficiency of E. suratensis.
       
Auto-aggregation and co-aggregation assays revealed strong aggregation abilities in both isolates. BLCET3 exhibited auto-aggregation of 78.34% and co-aggregation of 45.73%, while BLCET4 showed slightly lower values (71.92% and 39.47% respectively) (Fig 2e). These findings suggest that both strains can adhere to intestinal surfaces and competitively exclude pathogens, consistent with the results of Thankappan et al. (2015). Additionally, cell hydrophobicity assays demonstrated strong affinities for xylene, ethyl acetate and chloroform with BLCET3 showing higher hydrophobicity than BLCET4 (Fig 2f). Hydrophobicity is critical for bacterial adhesion to host intestinal mucosa, which supports colonization and persistence in the gut (Patel et al., 2009). Biofilm formation enhances bacterial survival, resistance to stress and pathogen exclusion (Guo et al., 2016) and the ability of probiotic strains to form biofilms enables them to outcompete pathogens for nutrients and colonization sites, thereby conferring protection to the host (Puvanasundram et al., 2022). Both the isolates produced black crystalline colonies on Congo red agar indicating their ability to form biofilm.
       
The functional probiotic attributes of BLCET3 and BLCET4 were validated through PCR amplification of key marker genes (Fig 3). These included 2,3-Bisphosphog-lycerate-independent phosphoglycerate mutase (gpmM) for acid stress tolerance (Kapse et al., 2019), Choloylglycine hydrolase (bsh) for bile salt deconjugation (Khan et al., 2021), Arginine/ornithine antiporter (ArcD) for acid and bile salt resistance (Mazhar et al., 2023), LuxS for quorum sensing and colonization efficiency (Jiang et al., 2021) and E1 β-subunit of pyruvate dehydrogenase complex (fbpA) for adhesion to intestinal epithelial cells (Oliveira et al., 2017).   

Fig 3: Agarose gel electrophoresis of PCR products of probiotic markers genes of bacterial isolates.

              

The presence of these marker genes in the bacterial isolates further affirms their probiotic nature in molecular level and similar findings was reported by Khan et al. (2021).
       
Antibiotic susceptibility is considered an important attribute of probiotics (Yu et al., 2023). Both the isolates demonstrated susceptibility to the majority of tested antibiotics (Table 3). The antibiotic susceptibility of the Bacillus isolates indicates a lower risk of AMR transfer, further supporting their potential as safe probiotic candidates. Our biosafety evaluation revealed that neither B. subtilis (BLCET3) nor B. velezensis (BLCET4) induced any signs of disease or mortality in the injected fish, suggesting their safety for in vivo applications. Our in vitro study will serve as a strongbase line for future in vivo trials assessing the immunomodulatory effects, while optimized downstream processing, formulation, encapsulation and delivery are critical for successful large-scale application of these probiotics in aquaculture (Ordanel et al., 2025).

Table 3: Antibiotic susceptibility of the bacterial isolated from the gut of E. suratensis.

CONCLUSION

The isolates B. subtilis (BLCET3) and B. velezensis (BLCET4) exhibited strong antimicrobial activity, enzymatic capabilities, pH and bile tolerance, biofilm formation, adhesion properties and absence of AMR. Probiotic marker genes and safety assessments further confirmed their potential as probiotics. Future studies are needed to evaluate their large-scale in vivo efficacy for fish health management.

ACKNOWLEDGEMENT

The authors acknowledge the research facilities extended by Tamil Nadu Dr. J. Jayalalithaa Fisheries University for this study. and National Agriculture Development Programme (NADP) titled “Integrated Disease Surveillance, Monitoring System and Seed Quality Assessment to Enhance Aquaculture Production in Tamil Nadu” for supporting this research work.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

REFERENCES


  1. Aswathy, R.G., Ismail, B., John, R.P. and Nampoothiri, K.M. (2008). Evaluation of the probiotic characteristics of newly isolated lactic acid bacteria. Applied Biochemistry and Biotechnology. 151: 244-255. doi: 10.1007/s12010-008-8183-6.

  2. Balan, S.S., Nethaji, R., Sankar, S. and Jayalakshmi, S. (2012). Production of gelatinase enzyme from Bacillus spp isolated from the sediment sample of Porto Novo Coastal sites. Asian Pacific Journal of Tropical Biomedicine. 2(3): S1811- S1816. doi:  10.1016/S2221-1691(12)60500-0.

  3. Banerjee, G., Ray, A.K., Askarian, F. and Ringø, E. (2013). Characteri- sation and identification of enzyme-producing autochthonous bacteria from the gastrointestinal tract of two Indian air- breathing fish. Beneficial Microbes. 4(3): 277-284.  doi:10. 3920/bm2012.0051.

  4. Bauer, A.W. (1996). Antibiotic susceptibility testing by a standardized single disc method. Am. J. of Clinc. Path. 45: 149-158. doi: https://cir.nii.ac.jp/crid/1571980075385493376.

  5. Berkeley, R.C.W., Logan, N.A., Shute, L.A. and Capey, A.G. (1984). Identification of Bacillus Species. In Methods in Microbiology. 16: 291-328. Academic press. doi:10. 1016/S0580-9517(08)70397-0.

  6. Cheng, A.C., Lin, H.L., Shiu, Y.L., Tyan, Y.C. and Liu, C.H., (2017). Isolation and characterization of antimicrobial peptides derived from Bacillus subtilis E20-fermented soybean meal and its use for preventing Vibrio infection in shrimp aquaculture. Fish and Shellfish Immunology.  67: 270- 279. doi:10.1016/j.fsi.2017.06.006.

  7. Emam, A.M. and Dunlap, C.A. (2020). Genomic and phenotypic characterization of Bacillus velezensis AMB-y1; a potential probiotic to control pathogens in aquaculture. Antonie Van Leeuwenhoek. 113(12): 2041-2052. doi:10.1007/s10482- 020-01476-5.

  8. Fadel, A., Mahmoud, M.A., Abdelsalam, M., Eissa, E.S.H. and Sherif, A.H. (2025). Aeromonas veronii infection in cultured Oreochromis niloticus: prevalence, molecular and histopat- hological characterization correlated to water physicoch- emical characteristics, with the protective autochthonous probiotic. Aquaculture International. 33(4): 298. doi: 10.1007/ s10499-025-01960-7.

  9. Ghosh, K., Banerjee, S., Moon, U.M., Khan, H.A. and Dutta, D. (2017). Evaluation of gut associated extracellular enzyme-producing  and pathogen inhibitory microbial community as potential probiotics in Nile tilapia, Oreochromis niloticus. International Journal of Aquaculture. 7. doi:10.5376/ija.2017.07.0023.

  10. Gunarto, H., Sulaeman, M. and Tampangalo, B.R. (2023). The effectivity of probiotic containing Bacillus spp. applicated in the rearing of mud crab Scylla tranquebarica larvae to produce crablet. In IOP Conference Series: Earth and Environmental Science. 1137(1): 012045). IOP Publishing.  doi: 10.1088/1755-1315/1137/1/012045.

  11. Guo, X., Chen, D.D., Peng, K.S., Cui, Z.W., Zhang, X.J., Li, S. and Zhang, Y.A. (2016). Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed. Fish and Shellfish Immunology. 52: 74-84. doi: 10.1016/j.fsi.2016.03.017.

  12. Handley, P.S., Harty, D.W., Wyatt, J.E., Brown, C.R., Doran, J.P. and Gibbs, A.C. (1987). A comparison of the adhesion, coaggre- gation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius. Journal of General Microbiology. 133(11): 3207-3217. doi: 10.1099/ 00221287-133-11-3207.

  13. Hortillosa, E.M., Amar, M.J.A., Nuñal, S.N., Pedroso, F.L. and Ferriols, V.M.E.N. (2022). Effects of putative dietary probiotics from the gut of milkfish (Chanos chanos) on the growth performance and intestinal enzymatic activities of juvenile Nile tilapia (Oreochromis niloticus). Aquaculture Research.  53(1): 98-108. doi:10.1111/are.15556.

  14. Hoseinifar, S.H., Hoseini, S.M., Taheri Mirghaed, A., Ghelichpour, M., Shirzad-Aski, H., Van Doan, H., El-Haroun, E., Safari, R. and Khanzadeh, M. (2024). Comparison of the effects of host- associated (autochthonous) and commercial probiotics on immune responses, growth parameters and intestinal microbiota of Caspian whitefish (Rutilus frisii kutum) fry. Frontiers in Marine Science. 11: 1446927. doi: 10. 3389/fmars.2024.1446927.

  15. Hyronimus, B., Le Marrec, C., Sassi, A.H. and Deschamps, A. (2000). Acid and bile tolerance of spore-forming lactic acid bacteria.  International Journal of Food Microbiology. 61(2-3): 193-197. doi: 10.1016/S0168-1605(00)00366-4.

  16. Jamil, H., Ghaffar, A., Afzal, F., Ahmad, H., Abbas, G., Fouad, D., Ataya, F.S. and Li, K. (2025). Attenuation of salinity-induced stress and improvement of brackish water aquaculture of Labeo rohita through dietary interventions of multi-species probiotics. Applied Water Science. 15(6): 119. doi: 10. 1007/s13201-025-02447-9.

  17. Jiang, J., Wu, C., Zhang, C., Zhang, Q., Yu, L., Zhao, J., Zhang, H., Narbad, A., Chen, W. and Zhai, Q. (2021). Strain-specific effects of Bifidobacterium longum on hypercholesterolemic rats and potential mechanisms. International Journal of Molecular Sciences. 22(3): 1305. doi: 10.3390/ijms22031305.

  18. Kapse, N.G., Engineer, A.S., Gowdaman, V., Wagh, S. and Dhakephalkar, P.K. (2019). Functional annotation of the genome unravels probiotic potential of Bacillus coagulans HS243. Genomics. 111(4): 921-929. doi: 10.1016/j.ygeno.2018.05.022.

  19. Kasana, R.C., Salwan, R., Dhar, H., Dutt, S. and Gulati, A. (2008). A rapid and easy method for the detection of microbial cellulases on agar plates using Gram’s iodine. Current Microbiology.  57: 503-507. doi: 10.1007/s00284-008-9276-8. 

  20. Khan, M.I.R., Kamilya, D., Choudhury, T.G., Tripathy, P.S. and Rathore, G. (2021). Deciphering the probiotic potential of Bacillus amyloliquefaciens COFCAU_P1 isolated from the intestine of Labeo rohita through in vitro and genetic assessment. Probiotics and Antimicrobial Proteins. 13(6): 1572-1584. doi: 10.1007/s12602-021-09788-2.

  21. Kuebutornye, F.K., Lu, Y., Abarike, E.D., Wang, Z., Li, Y. and Sakyi, M.E. (2020). In vitro assessment of the probiotic characteristics of three Bacillus species from the gut of Nile tilapia, Oreochromis niloticus. Probiotics and Antimicrobial Proteins. 12: 412-424.  doi: 10.1007/s12602-019-09562-5.

  22. Lalitha, N., Ronald, B.S.M., Chitra, M.A., Hemalatha, S., Senthilkumar, T.M.A. and Muralidhar, M. (2022). Characterization of lactic acid bacteria from the gut of Penaeus vannamei as potential probiotic. Indian Journal of Animal Research. 56(12): 1499-1505. doi: 10.18805/IJAR.B-4983.

  23. Lapenda, J.C., Silva, P.A., Vicalvi, M.C., Sena, K.X.F.R. and Nascimento, S.C. (2015). Antimicrobial activity of prodigiosin isolated from Serratia marcescens UFPEDA 398. World Journal of Microbiology and Biotechnology. 31: 399-406. doi: 10. 1007/s11274-014-1793-y.

  24. Li, R., Liu, J., Liu, Y., Cao, L., Qiu, W. and Qin, M. (2023). Probiotic effects of Bacillus subtilis on growth performance and intestinal microecological balance of growing to finishing pigs. Journal of Food Biochemistry. 2023(1): 7150917. doi: 10.1155/2023/7150917.

  25. Li, X., Lin, S., Li, X., Lu, K., Ma, R., Song, K., Lin, Y., Wang, L. and Zhang, C. (2024). Autochthonous potential probiotics improve the Tianchongyou feed utilization in spotted seabass (Lateolabrax maculatus). Aquaculture Reports. 39: 102491.  doi: 10.1016/j.aqrep.2024.102491.

  26. Mazhar, S., Khokhlova, E., Colom, J., Simon, A., Deaton, J. and Rea, K. (2023). In vitro and in silico assessment of probiotic and functional properties of Bacillus subtilis DE111®. Frontiers  in Microbiology. 13: 1101144. doi: 10.3389/fmicb.2022. 1101144.

  27. Monier, M.N., Kabary, H., Elfeky, A., Saadony, S., El-Hamed, N.N.A., Eissa, M.E. and Eissa, E.S.H. (2023). The effects of Bacillus species probiotics (Bacillus subtilis and B. licheniformis) on the water quality, immune responses and resistance of whiteleg shrimp (Litopenaeus vannamei) against Fusarium solani infection. Aquaculture International. 31(6): 3437-3455. doi:  10.1007/s10499-023-01136-1.

  28. Mukhtar, S., Hussain, S.M., Ali, S., Yilmaz, E., Shahzad, M.M., Munir, M., Nazish, N., Alshehri, M.A., Naeem, A., Amjad, M. and Yousaf, Z. (2025). Promoting sustainable aquaculture: Exploring antibiotic alternatives to fight against pathogens. International  Aquatic Research. 17(1): 1-15. doi: 10.22034/iar.2025. 2008914.1732.

  29. Naeem, M., Ahmed, I., Ahmed, S., Ahmed, Z., Riaz, M.N. and Ghazanfar, S. (2018). Screening of cattle gut associated Bacillus strains for their potential use as animal probiotic. Indian J. Anim. Res. 10. doi: 10.18805/ijar.B-948.

  30. Nayak, A., Harshitha, M., Dubey, S., Munang’andu, H.M., Chakraborty, A., Karunasagar, I. and Maiti, B. (2024). Evaluation of probiotic efficacy of Bacillus subtilis RODK28110C3 against pathogenic Aeromonas hydrophila and Edwardsiella tarda using in vitro studies and in vivo gnotobiotic zebrafish gut model system. Probiotics and Antimicrobial Proteins. 16(5): 1623-1637. doi: 10.1007/s12602-023-10127-w.

  31. Nayak, S.K. (2010). Probiotics and immunity: A fish perspective. Fish and Shellfish Immunology. 29(1): 2-14. doi: 10.1016/j.fsi.2010. 02.017.

  32. Oliveira, L.F., Salvador, S.L., Silva, P.H., Furlaneto, F.A., Figueiredo, L., Casarin, R. et al. (2017). Benefits of Bifidobacterium animalis subsp. lactis probiotic in experimental periodontitis. Journal of Periodontology.  88(2): 197-208. doi:  10.1902/ jop.2016.160217.

  33. Ordanel, A.M., Nuevaespaña, J.A.A., Ramos, I.I.F., Almarza, P.J.D. and Caipang, C.M.A. (2025). Recent advances and challenges in the industrial-scale production of probiotics in aquaculture. International Journal of Aquatic Biology. 13(2): 118-139. doi: 10.22034/ijab.v13i2.2434.

  34. Papadimitriou, K., Zoumpopoulou, G., Foligné, B., Alexandraki, V., Kazou, M., Pot, B. and Tsakalidou, E. (2015). Discovering probiotic microorganisms: In vitro, in vivo, genetic and omics approaches. Frontiers in Microbiology. 6: 58. doi: 10.3389/fmicb.2015.00058. 

  35. Patel, A.K., Ahire, J.J., Pawar, S.P., Chaudhari, B.L. and Chincholkar, S.B. (2009). Comparative accounts of probiotic characteristics of Bacillus spp. isolated from food wastes. Food Research International. 42(4): 505-510. doi: 10. 1016/j.foodres.2009.01.013.

  36. Patil, P.K., Geetha, R., Mishra, S.S., Abraham, T.J., Solanki, H.G., Krupesha Sharma, S.R., Pradhan, P.K., Manna, S.K., Avunje, S., Abhinaya, D. and Felix, K.T. (2025). Unveiling the economic burden of diseases in aquatic animal food production in India. Frontiers in Sustainable Food Systems.  9: 1480094. doi: 10.3389/fsufs.2025.1480094.

  37. Peeler, E.J., Caballero-Celli, R., Davila, C.E.S., Gomez, A.C., Gilbert, W., Gómez-Sánchez, M., Phan, V.T., Rushton, J., Schrijver, R.S. and Kennerley, A. (2023). Farm level bio-economic modelling of aquatic animal disease and health interventions.  Preventive Veterinary Medicine.  221: 106055.  doi: 10. 1016/j.prevetmed.2023.106055.

  38. Puvanasundram, P., Chong, C.M., Sabri, S., Yusoff, M.S.M., Lim, K.C. and Karim, M. (2022). Efficacy of single and multistrain probiotics on in vitro strain compatibility, pathogen inhibition, biofilm formation capability and stress tolerance. Biology. 11(11): 1644. doi:  10.3390/biology11111644.

  39. Ramesh, D., Vinothkanna, A., Rai, A.K. and Vignesh, V.S. (2015). Isolation of potential probiotic Bacillus spp. and assessment of their subcellular components to induce immune responses in Labeo rohita against Aeromonas hydrophila. Fish and Shellfish Immunology. 45(2): 268-276.  doi:  10.1016/j.fsi.2015. 04.018. 

  40. Rastogi, S., Mittal, V. and Singh, A. (2020). In vitro evaluation of probiotic potential and safety assessment of Lactobacillus mucosae strains isolated from donkey’s lactation.  Probiotics and Antimicrobial Proteins. 12: 1045-1056. doi: 10.1007/s12602-019-09610-0. 

  41. Rathod, A.C., Khedkar, C.D., Sarode, A.R., Kalyankar, S.D., Deosarkar, S.S., Lule, V.K. and Patil, P.S. (2025). Commercially available probiotic products for wellbeing of mankind, livestock and fishery: A Review. Asian Journal of Dairy and Food Research. 44(1): 01-07. doi: 10.18805/ajdfr.DR-1803.

  42. Swaminathan, T.R., Johny, T.K., Nithianantham, S.R., Sudhagar, A., Pradhan, P.K., Sulumane Ramachandra, K.S., Nair, R.R. and Sood, N. (2022). A natural outbreak of infectious spleen and kidney necrosis virus threatens wild pearlspot, Etroplus suratensis in Peechi Dam in the Western Ghats biodiversity hotspot, India. Transboundary and Emerging Diseases. 69(5): e1595-e1605. doi: 10.1111/tbed.14494.

  43. Tabassum, T., Mahamud, A.S.U., Acharjee, T.K., Hassan, R., Snigdha, T.A., Islam, T., Alam,R., Khoiam, M.U., Akter, F., Azad, M.R. and Al Mahamud, M.A. (2021). Probiotic supplementations improve growth, water quality, hematology, gut microbiota and intestinal morphology of Nile tilapia. Aquaculture Reports. 21: 100972. doi:  10.1016/j.aqrep.2021.100972.

  44. Thankappan, B., Ramesh, D., Ramkumar, S., Natarajaseenivasan, K. and Anbarasu, K. (2015). Characterization of Bacillus spp. from the gastrointestinal tract of Labeo rohita-towards to identify novel probiotics against fish pathogens.  Applied Biochemistry and Biotechnology. 175: 340-353. 175:340-53. doi: 10.1007/s12010-014-1270-y.

  45. Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology. 173(2): 697-703. doi: 10.1128/jb. 173.2.697-703.1991.

  46. Yadav, R., Puniya, A.K. and Shukla, P. (2016). Probiotic properties of Lactobacillus plantarum RYPR1 from an indigenous fermented beverage Raabadi. Frontiers in Microbiology. 7: 1683.  doi: 10.3389/fmicb.2016.01683.

  47. Yu, Y., Zhang, Y., Wang, Y., Liao, M., Li, B., Rong, X., Wang, C., Ge, J., Wang, J. and Zhang, Z. (2023). The genetic and phenotypic diversity of Bacillus spp. from the mariculture system in China and their potential function against pathogenic Vibrio. Marine Drugs. 21(4): 228.  doi: 10.3390/md21040228.
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    Full Research Article

    In vitro Profiling of Probiotic Bacillus Species Isolated from the Gut Microbiota of Brackish Water Fish, Etroplus Suratensis (Pearlspot)

    E
    Ethirajan Manimozhi1
    A
    Arumugam Uma2,*
    N
    Nathan Felix3
    A
    Antony Cheryl3
    S
    Sethu Selvaraj4
    S
    S. Arun Sudhagar5
    H
    Harikrishan Lisha1
    1Department of Aquatic Animal Health Management, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Dr. M.G.R. Fisheries College and Research Institute, Ponneri-601 204, Tamil Nadu, India.
    2Directorate of Incubation and Vocational Training in Aquaculture, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, ECR-Muttukadu Chennai-600 001, Tamil Nadu, India.
    3Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Vettar River View Campus, Nagapattinam-609 307, Tamil Nadu, India.
    4Department of Aquaculture, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Dr. M.G.R. Fisheries College and Research Institute, Ponneri-601 204, Tamil Nadu, India.
    5Fish Health, Peninsular and Marine Fish Genetic Resources Centre, ICAR – National Bureau of Fish Genetic Resources, Kochi-682 001, Kerala, India.

    ABSTRACT

    Background: In aquaculture, antibiotic misuse has accelerated antimicrobial resistance (AMR), endangering fish health, ecosystems and public safety. Probiotics offer a sustainable alternative by enhancing immunity, growth and pathogen resistance without promoting AMR. The pearlspot (Etroplus suratensis), a valuable brackish water fish in South Asia, remains vulnerable to bacterial diseases, yet research on autochthonous probiotics for its health management is limited.

    Methods: The present investigation aimed to assess the probiotic potential of two bacterial isolates, Bacillus subtilis (BLCET3/SRLAAH/2023) and B. velezensis (BLCET4/SRLAAH/2023) isolated from the gut of a brackish water fish, E. suratensis. The probiotic potential of the isolates was analyzed by in vitro assays, molecular analysis of probiotic marker genes and in vivo biosafety assay.

    Result: Among the two isolates, BLCET3 exhibited stronger antimicrobial activity against Streptococcus agalactiae, Aeromonas veronii and Enterococcus sp. Both BLCET3 and BLCET4 tolerated a pH range of 2-9 and bile salt concentrations up to 10%, produced extracellular enzymes and formed biofilms. BLCET3 showing significantly higher (P<0.05) auto-aggregation, co-aggregation and cell surface hydrophobicity. Neither isolate displayed antibiotic resistance in ABST assays and in vivo biosafety tests confirmed them as non-pathogenic. PCR analysis revealed the presence of key probiotic marker genes. These findings confirm the probiotic potential of both isolates for aquaculture applications.

    INTRODUCTION

    Disease is a major constraint to aquaculture production, causing an estimated 21% loss of global aquaculture output, equivalent to approximately US$ 10 billion annually (Peeler et al., 2023). In India, disease-related losses in aquaculture are estimated at US$ 2.48 billion, accounting for 14.95% of the annual production value (Patil et al., 2025).
           
    In aquaculture, antibiotics play a vital role in preventing and treating bacterial infections. While antibiotics are widely used to manage these infections, their overuse has led to antibiotic resistance, raising global health concerns (Mukhtar et al., 2025). Probiotics have emerged as sustainable alternatives, offering health benefits such as improved digestion, pathogen inhibition and enhanced immunity in fish (Rathod et al., 2025). They prevent pathogen colonization via competitive exclusion, organic acid production and antimicrobial metabolites like bacteriocins (Tabassum et al., 2021).
           
    Important probiotic selection criteria include their  acid and bile  tolerance for gut survival, adherence to epithelial surfaces and biofilm formation for colonisation (Hyronimus et al., 2000). Genetic markers enable precise probiotic strain identification, complementing microbiological techniques (Papadimitriou et al., 2015). Autochthonous probiotics from the fish gut are better adapted to aquatic environments than commercial probiotics sourced from terrestrial systems (Lalitha et al., 2022; Hoseinifar et al., 2024). Bacterial genera viz., Lactobacillus, Bifidobacterium, Streptococcus, Bacillus and Enterococcus exhibit probiotic potential (Nayak et al., 2010), with Bacillus species gaining prominence due to their resilience and effectiveness in aquaculture (Cheng et al., 2017). In brackish water aquaculture, Bacillus sp. enhanced growth, water quality, stress tolerance and disease resistance in finfish and shellfish species viz., Oreochromis niloticus, Labeo rohita, Penaeus vannamei and Scylla tranquebarica (Hortillosa et al., 2022; Jamil et al., 2025; Gunarto et al., 2023; Monier et al., 2023).
           
    E. suratensis     (Pearlspot), native to South Asian brackish water habitats, is commercially significant and suitable for polyculture systems. Despite its aquaculture importance, bacterial diseases remain a challenge in their culture activities (Swaminathan et al., 2022). Studies on autochthonous probiotics characterization from E. suratensis remain largely unexplored. To address this, two Bacillus species were isolated and characterized from the gut of E. suratensis and their probiotic potential were evaluated.

    MATERIALS AND METHODS

    Sample collection and isolated of gut-associated Bacillus species
     
    Seventy-five healthy specimens of E. suratensis (46±2.4 g; 11.4±1.2 cm) were collected from Pulicat lagoon (13.4177o N, 80.3185oE) and transported alive to the State Referral Laboratory for Aquatic Animal Health, Chennai. After 48 h of starvation, the guts were aseptically dissected, homogenized and serially diluted in sterile saline (10-1 to 10-6). Samples were spread plated on Hichrome Bacillus agar and incubated at 37oC for 24-48 h. Morphologically distinct colonies were sub-cultured to obtain pure isolates, yielding 37 bacterial strains from the gut of E. suratensis.
     
    Screening for antibacterial activity
     
    Fish bacterial pathogens viz., Streptococcus agalactiae (NCBI Accession no. PP580699), Aeromonas veronii (Accession no. OR487464), Enterococcus sp. (Accession no.  PP600907)  obtained from the microbial archive in the State Referral Laboratory of Aquatic Animal Health - Madhavaram, Chennai was used. The antibacterial potential for the isolates was screened using disc diffusion method (Lapenda et al., 2015). The zones of inhibition (mm) against the bacterial pathogens were measures and the effective isolates., BLCET3 and BLCET4 were further characterised biochemically and molecular confirmated by PCR and sequencing.
     
    Biochemical characterization
     
    Morphological and biochemical characterization of the bacterial isolates BLCET3 and BLCET4 were carried out by gram-staining, motility test, oxidase, catalase, methyl red, voges-proskauer test, growth at 5% NaCl, indole, citrate utilization, carbohydrate fermentation and amino acid utilization tests (Berkeley et al., 1984).
     
    Molecular confirmation of bacterial isolates
     
    Total DNA was extracted from bacterial isolates BLCET3 and BLCET4 using commercial DNA extraction Kit (Qiagen, Germany). 16S rRNA gene of the bacteria was amplified following Weisburg et al. (1991). Amplified products (1484 bp) were visualized on a 1.5% ethidium bromide-stained agarose gel and documented (Bio-Rad). Sequenced products (Eurofins, India) were analyzed using NCBI BLAST, submitted to GenBank for accession numbers and a phylogenetic tree was constructed via Maximum Likelihood in MEGA 11.
     
    In vitro screening for probiotic potential
     
    The probiotic potential of Bacillus isolates was assessed through multiple in vitro assays. pH and bile tolerance were evaluated following Khan et al. (2021), with bacterial cultures adjusted to OD600 0.25 (10w CFU/mL). For pH tolerance, cultures were incubated in TSB (pH 2-9) at 37oC for 24 hours. For bile tolerance, isolates were exposed to bile salts (0%, 2.5%, 5% and 10% w/v) in PBS (pH 7.4) at 30oC for 1 hour and growth was measured at 600 nm (Shimadzu, Japan). Salt and phenol tolerance were tested in MRS broth with varying concentrations of NaCl (0-5% w/v) and phenol (0-5% v/v), incubated at 30oC for 24 hours and measured at 600 nm (Aswathy et al., 2008). Extracellular enzymatic activity was evaluated using nutrient agar supplemented with specific substrate: starch for amylolytic activity, carboxymethyl cellulose for cellulase activity, gelatin for gelatinase activity and skimmed milk for proteolytic activity. The bacterial isolates, BLCET3 and BLCET4 were streaked onto respective media and incubated at 30oC for 24 h. Amylase activity was detected by flooding with 1% iodine solution, gelatinase with 15% mercuric chloride and cellulase with 1% Congo red followed by washing with 1 M NaOH (Balan et al., 2012; Kasana et al., 2008). Auto-aggregation and co-aggregation assays followed Rastogi et al. (2020) and Handley et al. (1987). Cell hydrophobicity was tested using xylene, chloroform and ethyl acetate following the method of Patel et al. (2009). Biofilm formation was screened using congo red agar method (Ramesh et al., 2015). The isolates, BLCET3 and BLCET4 were streaked on Muller Hinton agar (Himedia, India) containing 0.8 g/l of Congo red dye and incubated for 48h at 37oC.
     
    Detection of probiotic marker genes
     
    The probiotic properties of BLCET3 and BLCET4 were confirmed by PCR amplification of marker genes, including 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (2,3-BIP), arginine/ornithine antiporter (ArcD), choloylglycine hydrolase (CGH), LuxS and the E1 β-subunit of the pyruvate dehydrogenase complex (E1-β) (Khan et al., 2021). DNA quality and concentration were checked spectrophotometrically (OD260/280 ~1.8-2.0) prior to amplification. PCR reactions included standard positive and negative controls to ensure specificity and reliability. Amplicons were resolved on 1.5% agarose gels and documented using a gel documentation system (Bio-Rad, Germany).
     
    Safety evaluation of the probiotic isolates
     
    The antibiotic susceptibility of BLCET3 and BLCET4 was evaluated using the Kirby-Bauer disk diffusion test on Mueller-hinton Agar (MHA) with sterile antibiotic discs (Himedia, India) following CLSI standards (Bauer et al., 1996). Zones of inhibition (ZOI in mm) were measured after 24 - 48 hrs of incubation at 37oC. For in vivo biosafety assessment, groups of 10 E. suratensis (23.5±2 g) in triplicates were intraperitoneally injected with 0.1 mL bacterial suspensions (109 CFU/mL) of each isolate. A control group received an equivalent volume of sterile PBS (pH 7.2). Fish were monitored daily for 10 days for signs of disease and mortality.
     
    Statistical analysis
     
    The results of in vitro tests were analyzed using the IBM SPSS Statistic 26 software. All the data were analyzed using one-way ANOVA employing Tukey’s post hoc test (p=0.05) to identify the significant differences among the groups. The results were represented as the mean ± standard error, with statistical significance at P<0.05.

    RESULTS AND DISCUSSION

    Bacillus spp. are promising probiotics in aquaculture due to their spore-forming nature, ease of large-scale production, enzyme synthesis, beneficial metabolites and immunost-imulatory properties (Nayak et al., 2010; Li et al., 2023). The healthy fish gut provides host-adapted autochthonous probiotics that enhance fish and ecosystem health while reducing the environmental risks linked to non–host-specific strains (Li et al., 2024; Fadel et al., 2025). A total of 37 bacterial isolates from the gut of 75 healthy E. suratensis specimens were screened for their antibacterial efficacy. Among them, two isolates BLCET3 and BLCET4, exhibited significant antibacterial activity against fish pathogens, including S. agalactiae, A. veronii and Enterococcus sp. The zones of inhibition (ZOI) were measured and are presented in Table 1. The antibacterial activity observed is likely attributed to the production of bacteriocins, enzymes and other antimicrobial metabolites (Naeem et al., 2018) which enhance the ability of these isolates to inhibit pathogenic bacterial growth effectively.

    Table 1: Antibacterial activity of the bacterial isolates against targeted fish pathogens.


           
    Biochemical characterization revealed metabolic variations between BLCET3 and BLCET4, particularly in their ability to ferment carbohydrates and utilize amino acids (Table 2). These findings suggest that both isolates possess a versatile metabolic profile, contributing to their survivability and functionality in the gut environment (Kuebutornye et al., 2020). Molecular characterization through 16S rRNA sequencing identified BLCET3 as B. subtilis (Accession No. PP851370) and BLCET4 as B. velezensis (Accession No. PP657343) (Fig 1).

    Table 2: Biochemical characterization of the bacterial isolates.



    Fig 1: Phylogram constructed using the neighbour-joining tree for Bacillus species.


           
    The ability to withstand harsh gastrointestinal conditions is fundamental probiotic trait (Ghosh et al., 2017). Both BLCET3 and BLCET4 exhibited pH tolerance across a wide range (2-9), with optimal growth observed at pH 7-8 (Fig 2a). Notably, BLCET3 displayed slightly greater resilience at extreme pH levels compared to BLCET4. Similarly, both isolates tolerated bile concentrations up to 10% (w/v), although growth gradually decreased with increasing bile concentrations (Fig 2b). These findings are consistent with earlier report of Nayak et al. (2024), which highlighted the importance of pH and bile tolerance in probiotic bacteria.

    Fig 2: In vitro assays for probiotic screening of bacterial isolate.


           
    Both isolates demonstrated salt tolerance up to 5% NaCl and phenol tolerance up to 0.6%, with growth declining beyond these thresholds (Fig 2c, 2d). The ability to withstand salt stress aligns with observations by Emam and Dunlap (2020) who reported the role of osmolyte regulation in maintaining cellular balance under hypertonic conditions. Phenol tolerance, an indicator of resilience in the gastrointestinal environment, further validates the robustness of these isolates (Yadav et al., 2016).
           
    Extracellular enzymes production viz., amylase, cellulase, gelatinase and protease were observed in both isolates, indicated by clear halo zones around colonies on substrate- specific agar plates. These enzymatic activities facilitate the breakdown of complex dietary components, improving nutrient absorption in the host (Banerjee et al., 2013). The enzymatic profile observed in BLCET3 and BLCET4 suggests their potential to enhance the digestive efficiency of E. suratensis.
           
    Auto-aggregation and co-aggregation assays revealed strong aggregation abilities in both isolates. BLCET3 exhibited auto-aggregation of 78.34% and co-aggregation of 45.73%, while BLCET4 showed slightly lower values (71.92% and 39.47% respectively) (Fig 2e). These findings suggest that both strains can adhere to intestinal surfaces and competitively exclude pathogens, consistent with the results of Thankappan et al. (2015). Additionally, cell hydrophobicity assays demonstrated strong affinities for xylene, ethyl acetate and chloroform with BLCET3 showing higher hydrophobicity than BLCET4 (Fig 2f). Hydrophobicity is critical for bacterial adhesion to host intestinal mucosa, which supports colonization and persistence in the gut (Patel et al., 2009). Biofilm formation enhances bacterial survival, resistance to stress and pathogen exclusion (Guo et al., 2016) and the ability of probiotic strains to form biofilms enables them to outcompete pathogens for nutrients and colonization sites, thereby conferring protection to the host (Puvanasundram et al., 2022). Both the isolates produced black crystalline colonies on Congo red agar indicating their ability to form biofilm.
           
    The functional probiotic attributes of BLCET3 and BLCET4 were validated through PCR amplification of key marker genes (Fig 3). These included 2,3-Bisphosphog-lycerate-independent phosphoglycerate mutase (gpmM) for acid stress tolerance (Kapse et al., 2019), Choloylglycine hydrolase (bsh) for bile salt deconjugation (Khan et al., 2021), Arginine/ornithine antiporter (ArcD) for acid and bile salt resistance (Mazhar et al., 2023), LuxS for quorum sensing and colonization efficiency (Jiang et al., 2021) and E1 β-subunit of pyruvate dehydrogenase complex (fbpA) for adhesion to intestinal epithelial cells (Oliveira et al., 2017).   

    Fig 3: Agarose gel electrophoresis of PCR products of probiotic markers genes of bacterial isolates.

                  

    The presence of these marker genes in the bacterial isolates further affirms their probiotic nature in molecular level and similar findings was reported by Khan et al. (2021).
           
    Antibiotic susceptibility is considered an important attribute of probiotics (Yu et al., 2023). Both the isolates demonstrated susceptibility to the majority of tested antibiotics (Table 3). The antibiotic susceptibility of the Bacillus isolates indicates a lower risk of AMR transfer, further supporting their potential as safe probiotic candidates. Our biosafety evaluation revealed that neither B. subtilis (BLCET3) nor B. velezensis (BLCET4) induced any signs of disease or mortality in the injected fish, suggesting their safety for in vivo applications. Our in vitro study will serve as a strongbase line for future in vivo trials assessing the immunomodulatory effects, while optimized downstream processing, formulation, encapsulation and delivery are critical for successful large-scale application of these probiotics in aquaculture (Ordanel et al., 2025).

    Table 3: Antibiotic susceptibility of the bacterial isolated from the gut of E. suratensis.

    CONCLUSION

    The isolates B. subtilis (BLCET3) and B. velezensis (BLCET4) exhibited strong antimicrobial activity, enzymatic capabilities, pH and bile tolerance, biofilm formation, adhesion properties and absence of AMR. Probiotic marker genes and safety assessments further confirmed their potential as probiotics. Future studies are needed to evaluate their large-scale in vivo efficacy for fish health management.

    ACKNOWLEDGEMENT

    The authors acknowledge the research facilities extended by Tamil Nadu Dr. J. Jayalalithaa Fisheries University for this study. and National Agriculture Development Programme (NADP) titled “Integrated Disease Surveillance, Monitoring System and Seed Quality Assessment to Enhance Aquaculture Production in Tamil Nadu” for supporting this research work.

    CONFLICT OF INTEREST

    The authors declare that there is no conflict of interest.

    REFERENCES


    1. Aswathy, R.G., Ismail, B., John, R.P. and Nampoothiri, K.M. (2008). Evaluation of the probiotic characteristics of newly isolated lactic acid bacteria. Applied Biochemistry and Biotechnology. 151: 244-255. doi: 10.1007/s12010-008-8183-6.

    2. Balan, S.S., Nethaji, R., Sankar, S. and Jayalakshmi, S. (2012). Production of gelatinase enzyme from Bacillus spp isolated from the sediment sample of Porto Novo Coastal sites. Asian Pacific Journal of Tropical Biomedicine. 2(3): S1811- S1816. doi:  10.1016/S2221-1691(12)60500-0.

    3. Banerjee, G., Ray, A.K., Askarian, F. and Ringø, E. (2013). Characteri- sation and identification of enzyme-producing autochthonous bacteria from the gastrointestinal tract of two Indian air- breathing fish. Beneficial Microbes. 4(3): 277-284.  doi:10. 3920/bm2012.0051.

    4. Bauer, A.W. (1996). Antibiotic susceptibility testing by a standardized single disc method. Am. J. of Clinc. Path. 45: 149-158. doi: https://cir.nii.ac.jp/crid/1571980075385493376.

    5. Berkeley, R.C.W., Logan, N.A., Shute, L.A. and Capey, A.G. (1984). Identification of Bacillus Species. In Methods in Microbiology. 16: 291-328. Academic press. doi:10. 1016/S0580-9517(08)70397-0.

    6. Cheng, A.C., Lin, H.L., Shiu, Y.L., Tyan, Y.C. and Liu, C.H., (2017). Isolation and characterization of antimicrobial peptides derived from Bacillus subtilis E20-fermented soybean meal and its use for preventing Vibrio infection in shrimp aquaculture. Fish and Shellfish Immunology.  67: 270- 279. doi:10.1016/j.fsi.2017.06.006.

    7. Emam, A.M. and Dunlap, C.A. (2020). Genomic and phenotypic characterization of Bacillus velezensis AMB-y1; a potential probiotic to control pathogens in aquaculture. Antonie Van Leeuwenhoek. 113(12): 2041-2052. doi:10.1007/s10482- 020-01476-5.

    8. Fadel, A., Mahmoud, M.A., Abdelsalam, M., Eissa, E.S.H. and Sherif, A.H. (2025). Aeromonas veronii infection in cultured Oreochromis niloticus: prevalence, molecular and histopat- hological characterization correlated to water physicoch- emical characteristics, with the protective autochthonous probiotic. Aquaculture International. 33(4): 298. doi: 10.1007/ s10499-025-01960-7.

    9. Ghosh, K., Banerjee, S., Moon, U.M., Khan, H.A. and Dutta, D. (2017). Evaluation of gut associated extracellular enzyme-producing  and pathogen inhibitory microbial community as potential probiotics in Nile tilapia, Oreochromis niloticus. International Journal of Aquaculture. 7. doi:10.5376/ija.2017.07.0023.

    10. Gunarto, H., Sulaeman, M. and Tampangalo, B.R. (2023). The effectivity of probiotic containing Bacillus spp. applicated in the rearing of mud crab Scylla tranquebarica larvae to produce crablet. In IOP Conference Series: Earth and Environmental Science. 1137(1): 012045). IOP Publishing.  doi: 10.1088/1755-1315/1137/1/012045.

    11. Guo, X., Chen, D.D., Peng, K.S., Cui, Z.W., Zhang, X.J., Li, S. and Zhang, Y.A. (2016). Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed. Fish and Shellfish Immunology. 52: 74-84. doi: 10.1016/j.fsi.2016.03.017.

    12. Handley, P.S., Harty, D.W., Wyatt, J.E., Brown, C.R., Doran, J.P. and Gibbs, A.C. (1987). A comparison of the adhesion, coaggre- gation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius. Journal of General Microbiology. 133(11): 3207-3217. doi: 10.1099/ 00221287-133-11-3207.

    13. Hortillosa, E.M., Amar, M.J.A., Nuñal, S.N., Pedroso, F.L. and Ferriols, V.M.E.N. (2022). Effects of putative dietary probiotics from the gut of milkfish (Chanos chanos) on the growth performance and intestinal enzymatic activities of juvenile Nile tilapia (Oreochromis niloticus). Aquaculture Research.  53(1): 98-108. doi:10.1111/are.15556.

    14. Hoseinifar, S.H., Hoseini, S.M., Taheri Mirghaed, A., Ghelichpour, M., Shirzad-Aski, H., Van Doan, H., El-Haroun, E., Safari, R. and Khanzadeh, M. (2024). Comparison of the effects of host- associated (autochthonous) and commercial probiotics on immune responses, growth parameters and intestinal microbiota of Caspian whitefish (Rutilus frisii kutum) fry. Frontiers in Marine Science. 11: 1446927. doi: 10. 3389/fmars.2024.1446927.

    15. Hyronimus, B., Le Marrec, C., Sassi, A.H. and Deschamps, A. (2000). Acid and bile tolerance of spore-forming lactic acid bacteria.  International Journal of Food Microbiology. 61(2-3): 193-197. doi: 10.1016/S0168-1605(00)00366-4.

    16. Jamil, H., Ghaffar, A., Afzal, F., Ahmad, H., Abbas, G., Fouad, D., Ataya, F.S. and Li, K. (2025). Attenuation of salinity-induced stress and improvement of brackish water aquaculture of Labeo rohita through dietary interventions of multi-species probiotics. Applied Water Science. 15(6): 119. doi: 10. 1007/s13201-025-02447-9.

    17. Jiang, J., Wu, C., Zhang, C., Zhang, Q., Yu, L., Zhao, J., Zhang, H., Narbad, A., Chen, W. and Zhai, Q. (2021). Strain-specific effects of Bifidobacterium longum on hypercholesterolemic rats and potential mechanisms. International Journal of Molecular Sciences. 22(3): 1305. doi: 10.3390/ijms22031305.

    18. Kapse, N.G., Engineer, A.S., Gowdaman, V., Wagh, S. and Dhakephalkar, P.K. (2019). Functional annotation of the genome unravels probiotic potential of Bacillus coagulans HS243. Genomics. 111(4): 921-929. doi: 10.1016/j.ygeno.2018.05.022.

    19. Kasana, R.C., Salwan, R., Dhar, H., Dutt, S. and Gulati, A. (2008). A rapid and easy method for the detection of microbial cellulases on agar plates using Gram’s iodine. Current Microbiology.  57: 503-507. doi: 10.1007/s00284-008-9276-8. 

    20. Khan, M.I.R., Kamilya, D., Choudhury, T.G., Tripathy, P.S. and Rathore, G. (2021). Deciphering the probiotic potential of Bacillus amyloliquefaciens COFCAU_P1 isolated from the intestine of Labeo rohita through in vitro and genetic assessment. Probiotics and Antimicrobial Proteins. 13(6): 1572-1584. doi: 10.1007/s12602-021-09788-2.

    21. Kuebutornye, F.K., Lu, Y., Abarike, E.D., Wang, Z., Li, Y. and Sakyi, M.E. (2020). In vitro assessment of the probiotic characteristics of three Bacillus species from the gut of Nile tilapia, Oreochromis niloticus. Probiotics and Antimicrobial Proteins. 12: 412-424.  doi: 10.1007/s12602-019-09562-5.

    22. Lalitha, N., Ronald, B.S.M., Chitra, M.A., Hemalatha, S., Senthilkumar, T.M.A. and Muralidhar, M. (2022). Characterization of lactic acid bacteria from the gut of Penaeus vannamei as potential probiotic. Indian Journal of Animal Research. 56(12): 1499-1505. doi: 10.18805/IJAR.B-4983.

    23. Lapenda, J.C., Silva, P.A., Vicalvi, M.C., Sena, K.X.F.R. and Nascimento, S.C. (2015). Antimicrobial activity of prodigiosin isolated from Serratia marcescens UFPEDA 398. World Journal of Microbiology and Biotechnology. 31: 399-406. doi: 10. 1007/s11274-014-1793-y.

    24. Li, R., Liu, J., Liu, Y., Cao, L., Qiu, W. and Qin, M. (2023). Probiotic effects of Bacillus subtilis on growth performance and intestinal microecological balance of growing to finishing pigs. Journal of Food Biochemistry. 2023(1): 7150917. doi: 10.1155/2023/7150917.

    25. Li, X., Lin, S., Li, X., Lu, K., Ma, R., Song, K., Lin, Y., Wang, L. and Zhang, C. (2024). Autochthonous potential probiotics improve the Tianchongyou feed utilization in spotted seabass (Lateolabrax maculatus). Aquaculture Reports. 39: 102491.  doi: 10.1016/j.aqrep.2024.102491.

    26. Mazhar, S., Khokhlova, E., Colom, J., Simon, A., Deaton, J. and Rea, K. (2023). In vitro and in silico assessment of probiotic and functional properties of Bacillus subtilis DE111®. Frontiers  in Microbiology. 13: 1101144. doi: 10.3389/fmicb.2022. 1101144.

    27. Monier, M.N., Kabary, H., Elfeky, A., Saadony, S., El-Hamed, N.N.A., Eissa, M.E. and Eissa, E.S.H. (2023). The effects of Bacillus species probiotics (Bacillus subtilis and B. licheniformis) on the water quality, immune responses and resistance of whiteleg shrimp (Litopenaeus vannamei) against Fusarium solani infection. Aquaculture International. 31(6): 3437-3455. doi:  10.1007/s10499-023-01136-1.

    28. Mukhtar, S., Hussain, S.M., Ali, S., Yilmaz, E., Shahzad, M.M., Munir, M., Nazish, N., Alshehri, M.A., Naeem, A., Amjad, M. and Yousaf, Z. (2025). Promoting sustainable aquaculture: Exploring antibiotic alternatives to fight against pathogens. International  Aquatic Research. 17(1): 1-15. doi: 10.22034/iar.2025. 2008914.1732.

    29. Naeem, M., Ahmed, I., Ahmed, S., Ahmed, Z., Riaz, M.N. and Ghazanfar, S. (2018). Screening of cattle gut associated Bacillus strains for their potential use as animal probiotic. Indian J. Anim. Res. 10. doi: 10.18805/ijar.B-948.

    30. Nayak, A., Harshitha, M., Dubey, S., Munang’andu, H.M., Chakraborty, A., Karunasagar, I. and Maiti, B. (2024). Evaluation of probiotic efficacy of Bacillus subtilis RODK28110C3 against pathogenic Aeromonas hydrophila and Edwardsiella tarda using in vitro studies and in vivo gnotobiotic zebrafish gut model system. Probiotics and Antimicrobial Proteins. 16(5): 1623-1637. doi: 10.1007/s12602-023-10127-w.

    31. Nayak, S.K. (2010). Probiotics and immunity: A fish perspective. Fish and Shellfish Immunology. 29(1): 2-14. doi: 10.1016/j.fsi.2010. 02.017.

    32. Oliveira, L.F., Salvador, S.L., Silva, P.H., Furlaneto, F.A., Figueiredo, L., Casarin, R. et al. (2017). Benefits of Bifidobacterium animalis subsp. lactis probiotic in experimental periodontitis. Journal of Periodontology.  88(2): 197-208. doi:  10.1902/ jop.2016.160217.

    33. Ordanel, A.M., Nuevaespaña, J.A.A., Ramos, I.I.F., Almarza, P.J.D. and Caipang, C.M.A. (2025). Recent advances and challenges in the industrial-scale production of probiotics in aquaculture. International Journal of Aquatic Biology. 13(2): 118-139. doi: 10.22034/ijab.v13i2.2434.

    34. Papadimitriou, K., Zoumpopoulou, G., Foligné, B., Alexandraki, V., Kazou, M., Pot, B. and Tsakalidou, E. (2015). Discovering probiotic microorganisms: In vitro, in vivo, genetic and omics approaches. Frontiers in Microbiology. 6: 58. doi: 10.3389/fmicb.2015.00058. 

    35. Patel, A.K., Ahire, J.J., Pawar, S.P., Chaudhari, B.L. and Chincholkar, S.B. (2009). Comparative accounts of probiotic characteristics of Bacillus spp. isolated from food wastes. Food Research International. 42(4): 505-510. doi: 10. 1016/j.foodres.2009.01.013.

    36. Patil, P.K., Geetha, R., Mishra, S.S., Abraham, T.J., Solanki, H.G., Krupesha Sharma, S.R., Pradhan, P.K., Manna, S.K., Avunje, S., Abhinaya, D. and Felix, K.T. (2025). Unveiling the economic burden of diseases in aquatic animal food production in India. Frontiers in Sustainable Food Systems.  9: 1480094. doi: 10.3389/fsufs.2025.1480094.

    37. Peeler, E.J., Caballero-Celli, R., Davila, C.E.S., Gomez, A.C., Gilbert, W., Gómez-Sánchez, M., Phan, V.T., Rushton, J., Schrijver, R.S. and Kennerley, A. (2023). Farm level bio-economic modelling of aquatic animal disease and health interventions.  Preventive Veterinary Medicine.  221: 106055.  doi: 10. 1016/j.prevetmed.2023.106055.

    38. Puvanasundram, P., Chong, C.M., Sabri, S., Yusoff, M.S.M., Lim, K.C. and Karim, M. (2022). Efficacy of single and multistrain probiotics on in vitro strain compatibility, pathogen inhibition, biofilm formation capability and stress tolerance. Biology. 11(11): 1644. doi:  10.3390/biology11111644.

    39. Ramesh, D., Vinothkanna, A., Rai, A.K. and Vignesh, V.S. (2015). Isolation of potential probiotic Bacillus spp. and assessment of their subcellular components to induce immune responses in Labeo rohita against Aeromonas hydrophila. Fish and Shellfish Immunology. 45(2): 268-276.  doi:  10.1016/j.fsi.2015. 04.018. 

    40. Rastogi, S., Mittal, V. and Singh, A. (2020). In vitro evaluation of probiotic potential and safety assessment of Lactobacillus mucosae strains isolated from donkey’s lactation.  Probiotics and Antimicrobial Proteins. 12: 1045-1056. doi: 10.1007/s12602-019-09610-0. 

    41. Rathod, A.C., Khedkar, C.D., Sarode, A.R., Kalyankar, S.D., Deosarkar, S.S., Lule, V.K. and Patil, P.S. (2025). Commercially available probiotic products for wellbeing of mankind, livestock and fishery: A Review. Asian Journal of Dairy and Food Research. 44(1): 01-07. doi: 10.18805/ajdfr.DR-1803.

    42. Swaminathan, T.R., Johny, T.K., Nithianantham, S.R., Sudhagar, A., Pradhan, P.K., Sulumane Ramachandra, K.S., Nair, R.R. and Sood, N. (2022). A natural outbreak of infectious spleen and kidney necrosis virus threatens wild pearlspot, Etroplus suratensis in Peechi Dam in the Western Ghats biodiversity hotspot, India. Transboundary and Emerging Diseases. 69(5): e1595-e1605. doi: 10.1111/tbed.14494.

    43. Tabassum, T., Mahamud, A.S.U., Acharjee, T.K., Hassan, R., Snigdha, T.A., Islam, T., Alam,R., Khoiam, M.U., Akter, F., Azad, M.R. and Al Mahamud, M.A. (2021). Probiotic supplementations improve growth, water quality, hematology, gut microbiota and intestinal morphology of Nile tilapia. Aquaculture Reports. 21: 100972. doi:  10.1016/j.aqrep.2021.100972.

    44. Thankappan, B., Ramesh, D., Ramkumar, S., Natarajaseenivasan, K. and Anbarasu, K. (2015). Characterization of Bacillus spp. from the gastrointestinal tract of Labeo rohita-towards to identify novel probiotics against fish pathogens.  Applied Biochemistry and Biotechnology. 175: 340-353. 175:340-53. doi: 10.1007/s12010-014-1270-y.

    45. Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology. 173(2): 697-703. doi: 10.1128/jb. 173.2.697-703.1991.

    46. Yadav, R., Puniya, A.K. and Shukla, P. (2016). Probiotic properties of Lactobacillus plantarum RYPR1 from an indigenous fermented beverage Raabadi. Frontiers in Microbiology. 7: 1683.  doi: 10.3389/fmicb.2016.01683.

    47. Yu, Y., Zhang, Y., Wang, Y., Liao, M., Li, B., Rong, X., Wang, C., Ge, J., Wang, J. and Zhang, Z. (2023). The genetic and phenotypic diversity of Bacillus spp. from the mariculture system in China and their potential function against pathogenic Vibrio. Marine Drugs. 21(4): 228.  doi: 10.3390/md21040228.
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