{"paper_id":"27a03c9b-b4e7-4f6a-a044-bbc521b3a846","body_text":"Genomic and Functional Insights into a Novel Lactococcus sp. AK05 from Cirrhinus mrigala with Potent Antagonism Against Aeromonas hydrophila | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genomic and Functional Insights into a Novel Lactococcus sp. AK05 from Cirrhinus mrigala with Potent Antagonism Against Aeromonas hydrophila Anasuya Karjee, Soumya Chatterjee, Ranadhir Chakraborty This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7873003/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Feb, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 12 You are reading this latest preprint version Abstract Resistance in pathogens has increased as a result of the extensive usage of antibiotics in aquaculture. This emphasizes how urgently safe and efficient probiotic substitutes are needed. In our study, we isolated and characterized a new strain of Lactococcus sp., called AK05, from the gut of Cirrhinus mrigala . The phenotypic tests showed that it can survive harsh conditions, including a pH of 4, 0.8% bile salts, and even up to 8% NaCl. It also showed strong autoaggregation at 95.65%, co-aggregation with Aeromonas hydrophila subsp. hydrophila MTCC 1739 at 82.2%, and antimicrobial activity, with a 15.66 mm inhibition zone. A safety evaluation revealed γ-hemolysis, no gelatinase or DNase activity, and vulnerability to the majority of antibiotics, with the exception of trimethoprim. With 97.61% ANI and 78.9% dDDH, whole-genome sequencing showed that AK05 and Lactococcus lactis subsp. hordniae are closely related, yet only have 21% phenotypic similarity. This supports its classification as a unique strain. Our genomic analysis found genes that produce bacteriocins, like lactococcin, as well as genes that provide acid and bile tolerance, adhesion, and carbohydrate use. These traits, along with its ability to contain A. hydrophila , make AK05 a promising option for managing diseases without antibiotics and improving gut health in aquaculture. Lactococcus sp. AK05 genome analysis probiotic traits bacteriocin genes fish gut microbiome aquaculture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction The term probiotics was originally referred to as “organisms and substances which contribute to intestinal microbial balance” ( 1 ). In the year 1989, Fuller revised the definition into “live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance” ( 2 ). In the present time, probiotics are defined as “live microorganisms which when administered in adequate amount, confer health benefits on the host” ( 3 ), as stated by the World Health Organization (WHO) and Food and Agriculture Organization of the United Nations (FAO). Numerous types of microorganisms have been considered as probiotics, such as bacteria including various genera like Lactococcus , Lactobacillus , Bifidobacterium , Streptococcus , Shewanella ( 4 ), etc., yeasts ( Saccharomyces cerevisae and Sachharomyces boulardii ) ( 5 ) and filamentous fungi such as Aspergillus oryzae, A. cristatus ( 6 ) and Candida pintolopesii ( 7 ). To be regarded as a probiotic, the live feed must be capable of persisting in the stomach’s acidic conditions, and in the small intestine it must endure the existence of bile, also, it must have antimicrobial properties against pathogens, should be able to adhere to the gut lining of the intestine, must be able to utilize a range of sugar derivatives for their growth to act in full potential and after fulfilling all the above-mentioned criteria, they need to be non-pathogenic to the host and must not be resistant to antibiotics. Owing to the tremendous increase in world population, there has been an increase in demand in the food supply sector globally. It is estimated that by the year 2050, the requirement of meat for human consumption is going to be 500 Mt (Megatonnes) and to meet this humongous demand for food, there has been a vast expansion in the aquaculture sector ( 8 ). Due to the sudden boost in the cultivation of aquatic animals, there have been outbreaks of bacterial, viral, and fungal diseases ( 9 ), which have resulted in losses of more than 50% of aquatic organisms. To combat such diseases, various methods have been implemented like the use of antibiotics, vaccination of the aquatic animals, etc. Since the discovery of antibiotics, there has been an unchecked use of them for humans as well as other organisms. Such vast and uncontrolled use of antibiotics has been causing subsequent contamination in the environment by giving rise to antibiotic-resistant bacteria. In aquaculture, probiotics such as Bacillus subtilis and Lactobacillus plantarum are used against fish pathogen Vibrio anguillarum , in Japanese eel ( 10 ), B. subtilis and processed yeast against Edwardsiella tarda through mucosal and systemic immunity and they also release antimicrobials such as lysozymes, bacteriocins or hydrogen peroxide, in host Olive Flounder ( 11 ), Lactococcus lactis and Lactobacillus rhamnosus against Streptococcus agalactiae through an increase in immunity in host Nile tilapia ( 12 ), Enterococcus casselifavus against Aeromonas hydrophila , in the host Cyprinus carpio showed improved growth rate, better hematological parameters and increase in immunity ( 13 ). However, Lactococcus is not well-studied in fish farming because of a number of reasons. For example, Lactococcus garvieae has become a major fish pathogen, and more research is needed on its antimicrobial susceptibility ( 14 ). Also, other probiotic candidates like Bacillus subtilis are getting more attention. Lactococcus lactis is acknowledged for its probiotic capabilities ( 15 ). Nevertheless, the potential of other Lactococcus species and strains, along with the mechanisms of action of lactic acid bacteria in fish, necessitate additional research ( 16 ) Few studies combine whole-genome evidence with phenotypic evaluation to identify new strains of lactic acid bacteria (LAB), even though there is growing interest in LAB as probiotics for aquaculture. Correct taxonomic placement, the identification of functional genes related to probiotic traits, and biosafety evaluation all rely on this kind of integration. In order to describe the antimicrobial potential, stress tolerance, adhesion ability, and safety profile of the LAB strain that we isolated from the gut of C. mrigala , we used both experimental and bioinformatic methods. Its phylogenetic position, genomic novelty, and the existence of genes underlying observed probiotic phenotypes were all ascertained through whole-genome sequencing. According to the results, Lactococcus sp. AK05 is a unique candidate for microbiome modification in aquaculture systems both genomically and functionally. Materials and methods Standard assays were used to assess probiotic traits: Sample collection and lactic acid bacteria (LAB) isolation Healthy live fish specimens reared in the local hatchery, belonging to the group of Indian major carp C. mrigala of average weight 139.14 ± 45.18 g, were procured from Shivmandir (26°42'25.4\"N 88°21'41.3\"E), West Bengal, India. Samples were transported alive in plastic containers to the OMICs Laboratory, Department of Biotechnology, University of North Bengal, for dissection purposes following the approved guidelines of the Committee for the Purpose of Control & Supervision of experiments on animals (CPCSEA). Prior permission was sought from the CPCSEA, Registration No: 840/GO/Re/S/04/CPCSEA, the University of North Bengal, for performing the experiments. Fishes were anesthetized using clove oil (eugenol) with a dose volume ≥ 100 mg/L ( 17 ) followed by aseptic removal of gastrointestinal tracts (GITs). Aseptically removed GIT, weighing around 2.52 ± 1.29 g, was homogenized and serially diluted in PBS (pH 7.4). Morphologically distinct colonies were clonally purified from serially diluted samples spread out onto a de Man Rogosa Sharpe Agar plate (MRSA, Himedia) and cultured anaerobically for 48 h at 37 o C. Tests were conducted on purified strains to determine their suitability as probiotics. Probiotic Characterization Test for determining antagonistic activity From the overnight grown cultures of the eight bacterial isolates, the supernatant was obtained by centrifuging the cultures for 15 min at 1252 g , and subsequent analysis was done. Agar well-diffusion assay was done in Mueller Hinton (Himedia) agar plate (MHA) ( 18 ). Overnight grown culture of the pathogenic bacterium Aeromonas hydrophila subsp. hydrophila MTCC 1739 (100 µl) ( 19 ) was spread evenly over the MHA plate surface. Wells with 6 mm diameter were punched aseptically into the agar plate and 50 µl of the supernatant from each isolated bacterial strain was added to respective wells. Subsequent incubation of the MHA plates was done for 24 h at 30°C to observe distinct inhibition zones to observe the antibacterial activity of the metabolites secreted by the test isolates ( 20 ). Test for acid and bile salt tolerance The bacterial isolates were evaluated for their acid-tolerant properties in MRS broth. Individual calibration of the MRS broth to pH 2 or 3 or 4 or 5 or 6 or 7 with 6 N HCl was done. Each MRS broth having a different pH was inoculated with 1% of overnight-grown bacterial cultures and 37°C was taken as the incubation temperature. At sampling times 0, 3, 6, and 24 h, 1 ml culture was taken, dilution was done serially in phosphate-buffered saline (PBS, pH 7), and incubated at 37°C, spread onto Tryptone Soya Agar (TSA) plates, and observed for countable colony forming units (CFUs) after 24 h. Additionally, MRS broth was supplemented with different percentages of conjugated bile salt solution collected from the fish gall bladder, i.e., 0.3%, 0.5%, and 0.8% (v/v) respectively. MRS broth medium supplemented with fish gall bladder extract was inoculated with 1% of overnight-grown bacterial isolates. Bacterial growth was monitored at 2 h intervals till 8 h by measuring absorbance with a UV-Vis spectrophotometer at 600 nm( 21 ). Test for NaCl tolerance The capability of the bacterial strains to grow in high salt concentration was assessed by inoculating 1% overnight grown bacterial cells in MRS broth enriched with different concentrations of NaCl (1% to 8% respectively). The absorbance was calculated with a UV- Vis spectrophotometer at 600 nm at intervals of 0, 2.5, 5, and 24 h respectively ( 22 ). Test for autoaggregation Bacterial cell auto-aggregation was evaluated for their ability to endure and grow in the GIT and confer advantageous outcomes. Overnight grown cell cultures using MRS broth were harvested at 1252 g for 15 min and washed two times in PBS solution (pH 7.4). Incubation of the cells was done by suspending them in PBS under static conditions at 37°C. The upper layer of the suspension was measured for its absorbance at 600 nm at an interval of 0, 2, 5, and 24 h respectively ( 23 ). The auto-aggregation percentage was calculated using the formula Autoaggregation % = [(A1 - A2)/ (A1) x 100] Where A1 is the initial optical density (O.D.) of the cell suspension in PBS, and A2 is the O.D. of the cell suspension in PBS after static incubation for the defined period. Test for co-aggregation : Co-aggregation of the bacterial isolates was done against two pathogens, Staphylococcus aureus and A. hydrophila subsp. hydrophila MTCC 1739. The bacterial isolates and the pathogens were cultured overnight in MRS broth and TSB, respectively, and washed two times in PBS solution (pH 7.4). The washed cells of the test isolates and the pathogens were finally resuspended separately in PBS (pH 7.4), and then each of the PBS-suspended test bacterial strains and the pathogen was mixed in equal volume (1:1 ratio) and incubated at 37°C. The O.D. of the suspensions was calculated at an interval of 0, 2, 5, and 24 h respectively at 600 nm. The co-aggregation percentage was measured by the following formula: [A Isolate + A pathogen ] − [A mix (A Isolate + A pathogen)])/ 2 × 100, where A Isolate , A pathogen , and A mix represent the absorbance of test bacterial isolate, pathogen, and the mixture of test bacterial isolate and pathogen, respectively ( 24 ). Determination of cell surface hydrophobicity of the test bacterial isolates : The hydrophobicity of the test bacterial cells was determined by measuring their affinity towards hydrophobic substances like hexane and ethyl acetate ( 25 ). Bacterial cells were grown overnight and harvested by centrifugation at 1252 g for 15 min, washed two times in sterile PBS (pH 7.4), and again suspended in the same. Each of the finally resuspended bacterial cultures in PBS and the hydrocarbon (hexane or ethyl acetate) were mixed in a ratio of 1:3, vortexed vigorously, and left for 30 min in static condition. The absorbances of the aqueous phase just after mixing (initial) and after incubation (final) were taken at 600 nm in a UV-Vis spectrophotometer. Cell surface hydrophobicity (CSH) was measured using the following formula: CSH % = \\(\\:\\frac{Initial\\:OD-Final\\:OD}{Initial\\:OD}\\:\\times\\:100\\) Carbohydrate utilization The carbohydrate fermentation test was performed using the KB009 HiCarbo kit (Hi-Media, India). 50 µl of overnight-grown bacterial cultures were inoculated into each well and grown at 37°C for a duration of 24–48 h. Color changes due to a change in pH were observed ( 26 ). Pathogenicity and safety assessment tests Hemolysis test This test was done to assess the hemolytic properties of the bacteria. The isolates were grown on TSA plates containing 5% defibrinated sheep blood. The plates were evaluated for clear zones around the bacterial growth after a period of 24 h of incubation at 37°C ( 27 ). DNase agar test DNase (deoxyribonuclease) production by the bacteria was examined in this study. To DNase agar (Himedia) plates, a loopful of overnight–grown bacterial culture was streaked and incubated at 37°C for 24 h. A clear zone was assessed around the colonies, which hydrolysed DNA ( 28 ). Gelatin liquefaction test The freshly grown bacteria was stabbed on a slant made from Gelatin agar (Hi-Media) with the help of a sterile inoculating loop and grown for 24 h at 37°C. After the growth of the bacterial culture, stabbed slants were kept for 3 h under refrigeration to confirm the liquefaction activity caused by microbial processes and not because of the temperature used for incubation ( 29 ). Antibiotic susceptibility test A modified standard Kirby-Bauer procedure was followed to test the antibiotic susceptibility profile ( 30 ). The isolates were evaluated for their antibiotic susceptibility against different classes of antibiotics, viz. Penicillin (Ampicillin - Amp10), Cephalosporins (Cefotaxime - CTX30), Carbapenems (Imipenem - IMP10), Fluoroquinolones (Levofloxacin - LVX5), Glycopeptides and lipoglycopeptides (Vancomycin - VA30), Marcrolides (Erythromycin - E15), Tetracyclines (Tigecycline - TGC15), Oxazolidinones (Linezolid - LZ10) and other Miscellaneous Group (Nitrofurantoin - NIT30, Trimethoprim - TR10) antibiotic discs (Himedia Ltd., India). The inhibition zones around the discs were measured with the help of an antibiotic zone scale (Himedia) in millimeters (mm). Genomic Characterization Whole genome Extraction and Sequencing Overnight grown cells of the most potent probiotic strain were taken and extraction of the genomic DNA was carried out by using the isolation kit by Purelink genomic DNA (Thermo Fisher Scientific, USA). The whole genome sequencing was done using Illumina (Novaseq 6000), 150PE platform, with a prepared DNA library. Default parameters were used to assemble high-quality reads into contigs that are > 200 bp using Unicycler v0.5.0 ( 31 ). The submission of the raw sequence reads was done to the NCBI Sequence Read Archive, and the entire genome shotgun projects were deposited to DDBJ/ENA/GenBank (SRA). The draft genome sequence annotations were done using the NCBI GenBank file. Bioinformatics analysis The whole genome tree of the strain AK05 was created using Type (Strain) Genome Server (TYGS) ( https://tygs.dsmz.de/ ) ( 32 ). Using the pipeline described earlier by Dongwook Kim et. al., 2021 ( 33 ). EzAAI was used to produce a matrix of average amino acid identity (AAIm) values. It supported the large-scale calculations of AAI matrices and, thus, using UPGMA (unweighted pair-group method with arithmetic mean), a dendrogram was constructed using all the related taxa belonging to the genus Lactococcus and the strain AK05. To find out the similarities between species in the Lactococcus group, digital DNA-DNA hybridization (dDDH) using Genome to Genome Distance Calculation (GGDC) version3.0 ( https://ggdc.dsmz.de/phylogeny-service.php ) ( 34 ) and the average nucleotide identity (ANI) ( https://www.ezbiocloud.net/tools/ani ) values using EZbiocloud were calculated, respectively ( 35 ). Corroboration of phylogenetic inference with the Jaccard similarity coefficient was scored between AK05 and the nearest phylogenetic relative, Lactococcus lactis subsp. hordniae CCUG32210. Jaccard similarity coefficient was determined by the following formula: \\(\\:\\text{J}\\left(\\text{A},\\text{B}\\right)=\\:\\frac{\\left|\\text{A}\\text{n}\\text{B}\\right|}{\\left|\\text{A}?\\text{B}\\right|}\\) , where A ∩ B stands for the number of phenotypic tests recorded as positive as well as negative in both the sets corresponding to the strains, A and B; A U B stands for the total number of tests conducted in both sets, A and B. The ratio multiplied by 100 gives the % similarity coefficient, ( 36 – 38 ) Table S1 . To detect the genes accountable for the production of antimicrobial substances like bacteriocin and other secondary metabolites, bioinformatics tools BAGEL v.4 ( 39 ) and antiSMASH version 7.1 ( 40 ) were used respectively ( 41 ). Genes coding for enzymes responsible for the production, modification, and complex carbohydrate breakdown present in AK05 were mined using Carbohydrate-Active Enzyme Annotation (dbCAN-3) database ( https://bcb.unl.edu/dbCAN2/blast.php ) ( 42 ). Genes related to pathogenicity and virulence in the genome of AK05 were identified using Comprehensive Antibiotic Resistance Database (CARD) v3.1.1 ( 43 ) PathogenFinder 1.1 ( https://cge.food.dtu.dk/services/PathogenFinder/ ) ( 44 ) and VirulenceFinder 2.0 ( https://cge.food.dtu.dk/services/VirulenceFinder/ ) ( 45 ) from Centre for Genomic Epidemiology (CGE). Functional protein-protein interaction through STRING database: STRING (v12.0) software was used to predict the interactions between different proteins of interest. Either the protein sequence or the protein identification can be used to access the STRING database. The functional partners of a given protein are ranked according to their degree of confidence ( 46 ). Genetic data, the transmission of relationships or interactions between species, gene co-expression analysis, enhanced data collections, database analysis, and literature research are the sources of the forecasts. Each source is offered as proof. In STRING, every single score for a related piece of data or interaction is highlighted independently and shown as a colored line. A probabilistic confidence score is provided for each of these relationships. The degree of interaction among nodes connected by a multitude of evidence is indicated by confidence ratings. The highest level of assurance is indicated by a high aggregate score, which usually exceeds the individual score. STRING employs a unique scoring mechanism based on standards of various kinds of connections in comparison to a common reference set. In STRING, confidence scores are divided into four categories: low (0.1), medium (0.4), high (0.7) and highest (0.9). For this investigation, we used EnoA, Ecf, MsmK, ZitP, ZitQ, EC6.4.1.1, LmrA, LmrC, LmrD, SrtA, SrtC, Llmg1152, Llmg2465, PS102, PS103, PS501, LysP, PtPl, Phage lysin (Table S2) as our protein queries and numerous proteins as our input format. As the model organism, Lactococcus lactis was selected for this study. The functional protein networking was created by setting the network type to \"full\" STRING network, the \"meaning of network edges\" to evidence, allowing all active sources of interactions, setting the minimum required interaction score to the default value of 0.4, and, for display purposes, limiting the number of interactions to 10 in the first shell and 20 in the second shell under the basic settings. Among the clustering alternatives, k-means clustering was selected, with four clusters and solid lines for the edges between the clusters. Ultimately, the produced STRING network was exported in PNG format for analysis and visual interpretation. RESULTS Isolation of lactic acid bacteria (LAB) Three healthy specimens of C. mrigala (average weight 139.14 ± 45.18 g) were collected from a local hatchery in West Bengal, India (26°42'25.4\"N 88°21'41.3\"E). Fish were anesthetized with clove oil (≥ 100 mg/L) and dissected under aseptic conditions. Gastrointestinal tracts were homogenized, serially diluted, and plated on de Man Rogosa Sharpe Agar (MRSA; HiMedia). Nine morphologically distinct colonies were clonally purified from serially diluted samples spread out onto a de Man Rogosa Sharpe Agar plate (MRSA, Himedia) and cultured anaerobically for 48 h at 37 o C. With purified strains, tests for suitability as probiotics were determined. Probiotic Characterization Antagonism : Agar well diffusion assay of the isolated strains was tested against bacteria A. hydrophila subsp. hydrophila MTCC 1739. The highest diameter of inhibition zones was shown by AK16 and AK05, the zones measured were 15.33 and 15.66 mm in diameter, and then AK01 and AK06 showed inhibition zones of 10.33 and 11 mm in diameter. The remaining isolates showed inhibition zones less than 10 mm in diameter (Table 1 ). Hence, the three strains with the highest zone diameters AK06, AK16, and AK05 were selected for further screening processes. Table 1 Inhibition zone diameter of the Antagonistic assay of the isolated strains against the pathogen A. hydrophila subsp. hydrophila MTCC 1739 using agar well diffusion test Strains Inhibition zone diameter (mm) AK01 10.33 ± 0.57 AK06 11 ± 1 AK09 9.66 ± 0.57 AK11 7.66 ± 1.52 AK16 15.33 ± 1.52 AK17 8.66 ± 0.57 AK18 7.33 ± 0.57 AK19 7.66 ± 0.57 AK05 15.66 ± 1.52 Stress tolerance : Acid and bile salt tolerance: Three strains, AK06, AK16, and AK05, were further selected on the basis of maximum inhibition zone obtained from antagonistic assay. It was observed that the tolerance of the strains to different pH was not uniform. AK06 and AK05 were able to tolerate pH 4 till the 24th h of incubation while AK16 failed to survive beyond 6 h of incubation at pH 4 (Fig. 1 ). All three strains showed growth in different percentages of fish bile extract i.e., 0.3%, 0.5%, and 0.8%. The strains AK05 and AK16 had entered the mid-log phase by the 4th and 6th h of incubation for all three percentages of the bile acid respectively. But the strain AK06 entered the mid-log phase by the 4th, 6th, and 8th h of intervals for 0.3%, 0.5%, and 0.8% respectively. Thus, all the strains showed growth in 0.3%, 0.5%, and 0.8% of the bile acid in different intervals (Fig. 1 ). NaCl tolerance: All three strains reached the log phase by the 24th h at the highest concentration of 5% (Fig. 1 ). Autoaggregation test The highest autoaggregation property was shown by the strain AK05, 95.65%, and then AK06, 95.07% followed by AK16 which is 94.90% at 24 h (Fig. 2 ). Co-aggregation The co-aggregation percentage of AK05 against the pathogens Staphylococcus aureus and A. hydrophila subsp. hydrophila MTCC 1739 was 75.25% and 82.20%, respectively, while AK06 and AK16 demonstrated 68.64% and 83.80% and 42.45% and 74.78%, respectively (Fig. 3 ). Cell surface hydrophobicity test The strain AK05 had greater affinity for the non-hydrophobic hydrocarbon hexane (92.91%) than ethyl acetate (26.66%), according to the cell surface hydrophobicity test (CSH%). According to Fig. 4 , the other strains, AK06 and AK16, demonstrated 33.53% and 29.61% affinity for hexane and 59.75% and 54.64% affinity for ethyl acetate, respectively. Carbohydrate utilization test One of the probiotic screening procedures was the carbohydrate fermentation test, where the bacterial isolates AK05, AK06, and AK16 were able to ferment the majority of the carbohydrates, which is regarded as a positive probiotic characteristic (Table 2 ). In the context of the carbohydrate utilization test, the results showed no differences between the three strains. Table 2 Carbohydrate fermentation test by using KB009 HiCarbo kit (Hi-Media, India) S.No Carbohydrates AK05 AK06 AK16 1 Lactose + + + 2 Xylose + + + 3 Maltose + + + 4 Fructose + + + 5 Dextrose + + + 6 Raffinose + + + 7 Trehalose + + + 8 Melibiose + + + 9 Sucrose + + + 10 L-Arabinose + + + 11 Mannose + + + 12 Inulin + + + 13 Sodium gluconate + + + 14 Glycerol + + + 15 Salicin + + + 16 Dulcitol + + + 17 Inositol + + + 18 Sorbitol + + + 19 Mannitol + + + 20 Adinitol + + + 21 Arabitol + + + 22 Erythritol + + + 23 Alpha-Methyl-D-glucoside + + + 24 Rhamnose + + + 25 Cellobiose + + + 26 Melezitose + + + 27 Alpha-Methyl-D-mannoside + + + 28 Xylitol + + + 29 ONPG + + + 30 Esculin + + + 31 D-Arabinose + + + 32 Citrate - - - 33 Malonate - - - 34 Sorbose + + + ‘+’ denotes utilization of carbohydrate; ‘−’ denotes no utilization of carbohydrate Pathogenicity and Safety assessment tests Hemolytic test, gelatinase liquefaction test, and DNase agar tests were done; γ-hemolysis, negative gelatinase activity, and negative DNase activity were demonstrated by AK05, AK06, and AK16. According to these findings, all three strains showed no detrimental activity and are therefore acceptable for use in the food and dairy industries. Antibiotic susceptibility test Table 3 Antibiotic susceptibility profiling and interpretation using the latest EUCAST guidelines. Classes of Antibiotics Antibiotics Bacterial strains AK05 (mm) AK06 (mm) AK16 (mm) Penicillin Ampicillin (10µg) 21.66 ± 1.52 (S) 13.33 ± 0.57 (R) 11.66 ± 0.57 (R) Cephalosporins Cefotaxime (30µg) 23.66 ± 0.57 (S) 25.66 ± 1.15 (S) 32 ± 1(S) Carbapenems Imipenem (10µg) 28.33 ± 0.57 (S) 26.66 ± 0.57 (S) 34.66 ± 0.57 (S) Fluoroquinolones Levofloxacin (5µg) 21.66 ± 1.15 (I) 19.66 ± 0.57 (I) 23.66 ± 0.57 (I) Glycopeptides and lipoglycopeptides Vancomycin (30µg) 18.66 ± 0.57 (S) 14.66 ± 1.15 (S) 14.66 ± 0.57 (S) Marcrolides, Erythromycin (15µg) 21.33 ± 0.57 (S) 24.66 ± 1.15 (S) 23 ± 1(S) Tetracyclines Tigecycline (30µg) 20.66 ± 0.57 (S) 14.66 ± 0.57 (R) 17.33 ± 0.57 (R) Oxazolidinones Linezolid (10µg) 22.66 ± 1.15 (S) 20.66 ± 1.15 (S) 17.66 ± 0.57 (R) Miscellaneous Group Nitrofurantoin(30µg) 15.66 ± 1.52 (S) 18.33 ± 0.57 (S) 15.66 ± 0.57 (S) Trimethoprim(10µg) (R) 9.33 ± 0.57 (R) 9.66 ± 0.57 (R) ‘S’ denotes susceptible; ‘R’ denotes resistant The strain AK05 was demonstrated to be the most suitable probiotic candidate in terms of antibiotics-susceptibility profile, being resistant to only trimethoprim among seven classes of antibiotics tested. The other two strains, AK06 and AK16 were multiple-antibiotic resistant; in addition to trimethoprim, they were resistant to both ampicillin and tigecycline. Furthermore, AK16 has also resisted linezolid (Table 3 ). All three isolates were initially identified using 16S rRNA phylogeny ( 47 ). The three strains were all members of the Lactococcus genus. While AK05 split off to stand out among other species of the genus Lactococcus , AK06 and AK16 were revealed to be closely linked to L. garvieae (Fig. S1 , S2, S3). Thus, the two strains, AK16 and AK06, were not chosen for further research because the literature indicated that Lactococcosis caused by L. garvieae is one of the main causative organisms for fish disorders ( 48 ). Therefore, the strain AK05 being the most potent probiotic candidate underwent whole genome sequencing and sequence analysis. Phylogenetic placement of the strain AK05 and its whole genome sequence analysis The whole genome phylogenetic analyses (Fig. 5 ) have placed the strain AK05 on a discrete branch sharing a common ancestor with Lactococcus lactis subsp. hordniae CCUG 32210. To further clarify the taxonomic position of strain AK05, a phylogenomic dendrogram was compiled on the basis of amino acid identity between representative Lactococcus species (Fig. 6 ). AK05 was located in the Lactococcus lactis clade and clustered closely with L. lactis subsp. hordniae CCUG 32210 and L. lactis subsp. cremoris strains KW2 and ATCC 19257. The short branch lengths that separate AK05 from these subspecies reflect very high amino acid identity, validating that AK05 is a member of the L. lactis species complex. This positioning differentiates AK05 from other Lactococcus species like L. garvieae , L. raffinolactis , and L. plantarum , justifying its status as a new isolate within the L. lactis lineage. Between AK05 and L. lactis subsp. hordniae CCUG 32210, the dDDH and orthoANI estimates yielded high genomic values of 78.90% dDDH and 97.61% orthoANI, respectively, beyond species and subspecies demarcation levels (Table S3). In agreement, a phenotypic comparative analysis using the Jaccard similarity coefficient indicated limited phenotypic similarity of 21% for 23 phenotypic tests (this study for AK05 and reported findings for L. lactis subsp. hordniae CCUG 32210) (Table S1 ). AK05 can thus be considered a strain of the taxon that is both genetically and phenotypically different. More genomic insights : The comprehensive genome characteristics of AK05 have been presented in Table 4 . Table 4 Details of the genome of Lactococcus sp. AK05 Characteristics AK05 Bioproject accession no. PRJNA1141375 SRA accession no. SRR30829574 BioSample accession no. SAMN42887626 GenBank accession no. JBFTEI000000000 No.of contigs 184 Genome size (bp) 2,555,466 Genome coverage (bp) 121 GC content (%) 35.64 N50 (bp) 201128 AntiSMASH (version 7.1.0 web server) revealed that Ribosomally synthesized and post-translationally modified Peptides (RiPP-like), RiPPs modified by radical S-adenosylmethionine enzymes (Ras-RiPP), Type III polyketide synthase (T3PKS), terpene-like precursor, and Betalactone (biosynthetic gene cluster) protein-coding genes were present in the genome of AK05. BAGEL v4 web server confirmed the presence of Enterolysin A protein coding gene in the AK05’s genome (Fig. 7 ). Study of antibiotic resistance gene observed via CARD v3.1.1 The draft genome of AK05 was analyzed via CARD database of antibiotic resistance gene and strict hit was observed against the antibiotic, vancomycin Y and vancomycin W belonging to the class of Glycopeptide antibiotic (Fig. 8 ). Safety assessment through sequence analysis For genotypic corroboration, pathogenicity and virulence assessments were done using PathogenFinder version 1.1 and VirulenceFinder 2.0 where no hits were found in both the cases, thus, it can be stated that Lactococcus sp. AK05 is non-pathogenic and non-virulent. Protein-protein interaction via STRING (v12.0) A protein interacting network was created using the STRING database, forming different clusters of proteins belonging to different functional traits. The proteins EnoA, Ecf, MsmK, ZitP, ZitQ, EC6.4.1.1, BirA1, and BirA2, were related to stress (acid and salt stresses) represented by the red color cluster. The yellow cluster including LmrA, LmrC and LmrD belonged to proteins related to bile acid tolerance. The cell adhesion, autoaggregation, and co-aggregation proteins included SrtA, SrtC, Llmg1152, and Llmg2465 (cluster green). The blue cluster included proteins related to bacteriocin biosynthesis i.e., PS102 and PS103. Also, several bacteriocin biosynthesis and exopolysaccharide proteins were found in a scattered manner in the genome which included PS501, LysP, PtPl and Phage lysin (Fig. 9 ). Discussion Isolating probiotics from host-specific organisms is more effective than from other sources, as these strains demonstrate better colonization properties and re-establish a balanced and healthy homeostatic state ( 20 ). In the pursuit of enhancing aquaculture sustainability and fish health, the exploration of probiotics derived from the fish gut microbiota has attracted significant interest. Commensal bacteria, naturally residing within the fish gut, have shown to be more effective as probiotics due to their established relationship with the host. Commensal microorganisms act best in similar environments ( 49 ) showing improved growth, better immunological responses, and defense against diseases in aquatic organisms ( 50 ). The LAB isolates exhibited antimicrobial activities against a common fish pathogen A. hydrophila subsp. hydrophila MTCC 1739. Studies have found that A. hydrophila is the most common bacteria causing diseases in freshwater fishes and responsible for huge economic losses in the aquaculture sector worldwide ( 51 ). Morphologically distinct LAB strains exhibiting antimicrobial activities were primarily selected, from which the best ones were chosen based on other probiotic traits. A key characteristic of a probiotic is to be able to endure adverse environments of the fish’s gastrointestinal tract i.e., high tolerance to acid and bile ( 52 , 53 ). In the acid tolerance test, AK05 showed more than 4-fold increase in growth at pH 5 in 24 h from the time of inoculation, and similar gradual growth was observed in pH 6 and pH 7, while AK16 and AK06 showed more than a 3-fold increase in growth at pH 5 and gradual growth till pH 7 in 24 h from the time of inoculation (Fig. 1 ). The bile acid tolerance test showed that AK05 reached a mid-log phase in the 4th h of growth in presence of 0.3%, 0.5%, and 0.8% bile acid, while the other two strains showed gradual growth in lower percentages of bile acid, suggesting that they can survive in the harsh environment of the intestine (Fig. 1 ). The intestinal fluid of C. mrigala has a pH range of 6.2 to 7.1 and digestion takes place mainly due to bile and pancreatic juices due to the absence of gastric glands in the intestinal bulb. Along with the acid and bile tolerance the probiotic must be able to tolerate stresses like salt tolerance, all three strains showed prominent growth of up to 5% of NaCl in the medium (Fig. 1 ), thus, indicating that it can grow in the stressed environment without getting affected in its osmolarity. Bacteria having a high tolerance to NaCl stress can be used in food products and aquaculture. To effectively survive, colonize, and provide advantages to the host’s health, the probiotic must possess some key characteristics like auto-aggregation, co-aggregation, and cell surface hydrophobicity. Probiotic strains including the genus Lactobacillu s and Lactococcus exhibited properties inhibiting the growth of harmful bacteria to the lining of the gastrointestinal tract (GIT), this inhibition can take place due to several reasons like, competition for adhesion sites due to the presence of specific adhesin and receptors, production of antimicrobial substances, immune response modulation, alteration of gut pH, and biofilm formation leading to the prevention of the pathogen adhesion ( 52 , 54 – 56 ). The strain AK05 showed auto-aggregation properties which rose from 19.03% to 95.65% during 5 to 24 h of incubation. The co-aggregation percentage shown by AK05 against pathogens, S. aureus , and A. hydrophila subsp. hydrophila MTCC 1739 increased from 19.1–75.3% and 60- 82.2% respectively during 5 to 24 h of incubation. The cell surface hydrophobicity test showed that the CSH percentage of AK05 was more towards the non-polar substance, hexane, indicating better adherence to the gut epithelial cells. To survive in the gut environment of the host, the probiotic must be able to import and metabolize a range of carbohydrates. Carbohydrate utilization was tested by Hi-Carbo Kit, and it was found that all three strains were able to oxidize/ferment almost all carbohydrates used except citrate and malonate; making them desirable for being used as probiotics. Besides the carbohydrate-utilizing trait, all three strains were found to be non-hemolytic, non-gelatinolytic, and DNase-negative, and can be considered safe for consumption. Another important trait of a probiotic strain is its susceptibility to antibiotics. Among the three strains, AK05 was regarded as the most suitable probiotic candidate as per the antibiotic-susceptibility profile, being sensitive to all antibiotics tested except trimethoprim. AK05 was identified to belong to the genus Lactococcus , inspite of high genomic similarities indicated by dDDH value of 79.9% and orthoANI value of 97.61%, the Jaccard similarity index showed only 21% of the phenotypic characteristics (Table S1 , S3). Genomic data confirms AK05 as a new strain in Lactococcus that is highly genetically similar to L. lactis subsp. hordniae yet phenotypically different. Having unique functional genes also supports its uniqueness. Genomic assessment of the strain AK05 for probiotic traits corroborated with the phenotypic expression establishing it as a good probiotic candidate. Bacteriocin production by a probiotic is one of the most important antibacterial features to act against pathogens. The presence of bacteriocins, lactococcin 972, and bacteriocin immunity proteins-producing genes corroborated with the antimicrobial phenotype against the pathogen A. hydrophila subsp. hydrophila MTCC 1739. Detailed information regarding the location of genes related to bacteriocin and bacteriocin-related protein production is given in Table S4. Lactococcin 972 was reported to be produced by Lactococcus lactis subsp. lactis IPLA972 ( 57 ). It inhibits cell elongation, unable to form septa, resulting in cell death of closely related bacteria like Lactococcus lactis M1614 ( 58 ). It also inhibits cell wall biosynthesis, as it bears a lipid II binding motif that binds to lipid II essential for peptidoglycan synthesis of the target bacteria ( 59 ). As these properties impart antibacterial activity to the strain Lactococcus sp. AK05, the strain itself should possess a certain mechanism via which it gets protection against its bacteriocin. Bacteriocin-producing bacteria protect themselves by several mechanisms; the most important one is the production of bacteriocin immunity proteins. The genome of AK05 possessed genes encoding proteins providing immunity against bacteriocin. Genes coding for such immunity proteins are in the same cluster and often co-regulated ( 60 ). Another mechanism by which bacteria protects itself from its bacteriocin is when Lactococcin A binds with man-PTS (mannose phosphotransferase protein system) receptor proteins IIC and IID on the target bacteria. Concurrently, the immunity protein LciA (lactococcin immunity protein A) binds to this lactococcin A-receptor complex preventing the Lactococcin A from causing death of the producer cell by membrane permeabilization ( 61 ). Also, blockage of the pore formed by the bacteriocin can take place by the bacteriocin immunity protein, thus maintaining cellular integrity ( 62 ). Competitive binding of bacteriocin immunity protein and bacteriocin with the receptor of the target bacteria also might take place, thus, the immunity protein occupies the binding site for bacteria leading to the target cell’s protection ( 63 ). Web servers, antiSMASH version 7.1.0, and BAGEL v4 revealed the presence of antimicrobial proteins like Ribosomally synthesized and post-translationally modified peptides (RiPP), and Ras-RiPP, Type III Polyketide synthases, betalactone, and Enterolysin A (Fig. 7 ). RiPP and Ras-RiPP proteins belong to a group of antimicrobial peptides (AMP) and the post-translational modifications impart functional diversity to them ( 64 ). RiPPs and Ras-RiPPs secondary metabolites are found to be produced by certain genera of LAB-like, Lactobacillus , Streptococcus , and Lactococcus having antibacterial properties ( 65 ). Also, Type III polyketide synthases (T3PKS) are a class of enzymes taking part in the biosynthesis of polyketides, which are a group of secondary metabolites. For example, T3PKS 2, 4- diacetylphloroglucinol synthesized by Pseudomonas has antimicrobial activity against soil-borne fungal plant pathogens ( 66 ). T3PKS like ArsB and ArsC produced by Azotobacter vinelandii inhibits the growth of certain microorganisms, and Germicidin derivatives control germination of spore produced by Streptomyces sp., thus, managing their population ( 67 , 68 ). Enterolysin A, class III heat-labile bacteriocin, disrupts cell membranes and compromises cellular integrity, leading to cell death; its regulated expression in L. lactis has shown a bactericidal effect, highlighting its potential use as a preservative in food applications ( 69 ). Beta-lactones are natural products, produced by some bacterial genera such as Pseudomonas , Streptomyces , Kitasatospora , etc. targeting various classes of enzymes such as hydrolases, transferases, ligases, and oxidoreductases have bioactivity against bacteria, fungi, or human cancer cell lines ( 70 ). Furthermore, beta-lactones are inhibitors of homoserine transacetylase (HTA) which can inactivate through an irreversible acylation of ser143, the transacetylase activity of homoserine in the enzyme’s active site, thus, intervening in the biosynthesis of methionine and threonine essential for cellular functions in microorganisms ( 71 ) (Table S5). Acid tolerance phenotype of the strain Lactococcus sp. AK05 corroborates with the presence of a high copy number gene coding for amino acid permease, which is an integral membrane protein responsible for the absorption of extracellular amino acids into the cell for elevating the intracellular pH level. This is beneficial for the bacteria for survival in stressful environments like low nutrient availability, low pH environments, etc. Here, the amino acids are decarboxylated to produce amines and in this process, protons are consumed raising the pH level ( 72 ). The F0F1 ATP synthase helps maintain the pH homeostasis by extruding the protons, helping the bacteria survive in low acidic environments. The genome of AK05 revealed the presence of all the genes responsible for the synthesis of F0F1 ATP synthase subunits. The main role of F0F1 ATP synthase in acid tolerance has been reported in Listeria monocytogenes , Lactobacillus casei , Streptococcus mutans , Lactococcus lactis ( 73 – 77 ) (Table S6). As the bacteria were tolerant to high percentages of bile acids (Fig. 1 ), this was substantiated by the presence of genes like lmrCD , which is induced by the presence of cholate and related bile acids in Lactococcus lactis . Proteins coded by lmrCD function as efflux pumps, removing bile acids from the cell. During the bile acid stress, the cholate is taken up as sodium salt, thus, there is upregulation of Na + /H + antiporter which counteracts Na + toxicity ( 78 ). Hence, the presence of several copy numbers of Na +/ H + antiporter genes in the genome of AK05 proved that under salt stress environment these antiporters help in extruding excess Na + from the cell in exchange for H + , thus, maintaining osmotic balance inside the cell ( 79 ) (Table S7). To survive and proliferate the bacteria must be able to aggregate and adhere to the host’s intestinal tract. The presence of several copy numbers of mucin binding proteins and cell wall binding proteins suggested (Table S8 and S9) that the bacteria have better potential to adhere to the mucosal layer found in Lactococcus lactis , Bifidobacterium bifidum, Lactobacillus reuteri ( 80 , 81 ) and aggregate together which might result in biofilm formation. The formation of biofilm further inhibits the adherence of pathogenic bacteria and protects from the harsh environment of the gut, also influenced by exopolysaccharide production, contributing to the stability and functionality of the gut environment ( 81 ). The mucin-binding protein further influences the immune response by acting as an epitope that interacts with the host immune cells by promoting broad-spectrum antibody reaction also facilitating retention in the host’s intestinal layer ( 82 – 84 ). The gene srtA encodes for SrtA, present in the cell membrane, anchors a typical motif LPxTG, by cleaving the protein at ‘x’ position facilitating a covalent attachment of the protein to the peptidoglycan layer of the cell wall which helps in adhesion and aggregation function of the bacteria ( 85 ). Another kind of Sortase which is encoded by the gene srtC , is important for polymerization of pili subunit which further facilitates autoaggregation and biofilm formation reported in Lactoccus lactis ( 86 ). The carbohydrate fermentation test (Table 2 ) showed that the strain AK05 could utilize a wide range of carbohydrates. This suggests that the strain consists of a diverse range of enzymes capable of breaking down various carbohydrates; this property makes the strain survive in different ecological niches with varied nutrient availability. DBCAN 3 web server was used to annotate the carbohydrate-active enzymes, showing the presence of 34 glycoside hydrolase (GHs) family enzymes, 11 glycoside transferase (GTs) enzymes, five carbohydrate-binding modules (CBMs), four carbohydrate esterases (CE), and seven multidomains (Table S10). The presence of glycoside hydrolase family proteins and carbohydrate esterases suggests that the complex carbohydrates are acted upon by these enzymes and broken down into simple carbohydrates enabling L. lactis to utilize substrates for growth ( 87 ). GH2, GH13, and GH32 present in Lactobacillus paracasei were responsible for the breakdown of oligosaccharides ( 88 ). Thus, it can be said that the strain AK05 has the potential to degrade various types of carbohydrates in dietary fibers, which when degraded results in the formation of short-chain fatty acids (SCFAs) like butyrate, propionate, and acetate which have anti-inflammatory activity improving the gut barrier function and immune response by preventing against infectious diseases ( 89 ). The STRING (v12.0) database was used to elucidate the complex network of protein-protein interactions related to genes responsible for the functional properties of a probiotic, thus, interactions were performed using key proteins (Table S2). A vital property of a probiotic is to survive in a gastric acidic environment. Thus, the phenotypic study showed that the strain AK05 can survive at pH 4 up to 24 h, which corroborated with the genotypic study showing various genes that are responsible for producing proteins imparting the property of acid tolerance to the strain (Table S6). Thus, the literature study brought out the key proteins, EnoA, EcfA2, MsmK, ZitP, ZitQ, and EC6.4.1.1, responsible for stress tolerance (Table S2). EnoA is a key enzyme for energy production involved in the glycolytic pathway, the ecfA2 gene encodes an energy coupling factor transporter ATPase (EcfA2), and overexpression of this gene significantly enhances acid tolerance in L. lactis . Similarly, overexpression of ZitP and ZitQ, metal ABC transporters proteins have been found to impart improved acid tolerance to L. lactis ( 90 ). The overexpression of Msmk protein under acid stress leads to higher ATP concentration. Providing the cells with the necessary energy to combat the acid stress condition, Msmk is also involved in the utilization of carbohydrates, which is essential for microbial cells to adjust their metabolism in maintaining optimum cellular functions during stress; also, this protein helps in up-regulation of genes like coaD and arg involved in energy generation pathways associated with ATP production ( 91 ). EC6.4.1.1 (pyruvate carboxylase) catalyzes the conversion of pyruvate to oxaloacetate (OAA), which is an essential step in gluconeogenesis and lipogenesis. During stress conditions, upregulation of pyruvate carboxylase enhances the metabolic flux towards the production of OAA which is further utilized in the production of amino acids, nucleotides, etc. A separate yellow cluster was formed involving proteins like LmrA, LmrC, and LmrD which are related to bile acid tolerance. It was also found that the strain AK05 could tolerate and grow in a high percentage of bile acid (0.8%). The presence of ABC-Type multidrug resistance transporter, LmrCD, plays a crucial role in extruding bile acids from the cell functioning as a pump, thereby, avoiding the accumulation of bile acids intracellularly and increasing its survivability in bile acid-rich environments ( 78 ). Another important trait of being a high-quality probiotic is adherence and aggregation properties; the phenotypic tests revealed that AK05 has a 95.65% autoaggregation percentage and co-aggregation percentages of 74.1% and 82.32%, against S. aureus and A. hydrophila subsp. hydrophila MTCC 1739. The analysis of the genome showed the presence of genes responsible for these properties, srtA and srtC (coding for sortase), llmg_2465 (coding for YmcF), llmg_1152 (coding for YwcG) (Table S2), formed a separate green cluster. Sortases A and C, both play crucial roles in adhesion purposes, here, proteins with an LPXTG- motif are predominantly anchored by SrtA to the cell wall, whereas SrtC is involved in biofilm formation enhancing adherence to the host tissues ( 85 ). Both llmg_1152 and llmg_2465 proteins mediate adhesion to the mucosal surfaces, thus, helping in colonization in the gastrointestinal tract due to the presence of mucus-binding domain and LPXTG motifs ( 84 ). One of the most important aspects of a probiotic is to possess the property of antimicrobial activity, apart from the phenotypic test, the genome mining also showed some important proteins responsible for imparting antimicrobial properties. The protein-protein interaction was performed in the STRING database, where the blue-colored cluster (ps102 and ps103) was obtained along with separate units in white, including ps501, lysP, ps461 (phage lysin). This blue-colored cluster and the white units involved proteins related to antimicrobial properties. LysL and LysP are phage lysins that cause the cell wall lysis of other closely related bacteria and are highly specific due to the presence of a cell wall binding domain (CBD) exhibiting a narrow range ( 92 ) (Fig. 9 ). Lactococcus sp. AK05 has arisen to be a potent probiotic strain, for use in aquaculture and food industries. AK05 exhibited antimicrobial activity against the pathogenic strain A. hydrophila subsp. hydrophila MTCC 1739 which is known to cause considerable economic losses in freshwater aquaculture. Various characteristics like the ability to tolerate low pH, and elevated salt and bile acid concentrations along with the properties like cell surface hydrophobicity, auto-aggregation, and co-aggregation with harmful bacteria make it well suited to thrive and colonize within the harsh environment of the gastrointestinal tract and provide a beneficial impact on the host. Also, genomic analysis has confirmed the presence of genes associated with the above-mentioned functions. To support a healthy gut microbiome and to maintain stability in the host’s intestinal environment, these kinds of traits of probiotics are very crucial. AK05 was also able to produce bacteriocin ( viz . lactococcin 972) that effectively inhibits pathogenic bacterial growth. The genomic profile also revealed the presence of genes encoding bacteriocin immunity proteins that safeguard AK05 from its antimicrobial compounds. Antibiotic resistance pattern showed AK05’s resistance towards trimethoprim only, while it was susceptible to all the rest of the broad-spectrum antibiotics. These genomic and phenomic features reinforced the claim that AK05 is an effective probiotic strain that can enhance the gut health of aquatic species and can be used as a viable, natural solution for pathogen control. Statements and Declarations Conflict of Interest There are no academic or financial conflicts of interest for the authors regarding the publication of this work. Data availability statement Data is available in the NCBI Database under the GenBank Accession id : JBFTEI000000000; BioSample Accession id: SAMN42887626; BioProject Accession id: PRJNA1141375; SRA Accession id: SRR30829574 Acknowledgement Authors, Anasuya Karjee and Soumya Chatterjee acknowledge the Council of Scientific and Industrial Research (CSIR- NEW DELHI, GOVT. OF INDIA) as they received research grant in the form of Senior Research Fellowship during this work (CSIR-SRF; AWARD NO.- 09/285(0093)/2019-EMR-I, 09/1151(0006)/2019-EMR-I respectively). Authors’ contributions Conceptualization: Ranadhir Chakraborty; Methodology: Anasuya Karjee; Formal analysis and investigation: Anasuya Karjee, Soumya Chatterjee, and Ranadhir Chakraborty; Writing - original draft preparation: Anasuya Karjee; Writing - review and editing: Ranadhir Chakraborty, Anasuya Karjee, and Soumya Chatterjee; Funding acquisition: Anasuya Karjee; Resources: Ranadhir Chakraborty; Supervision: Ranadhir Chakraborty. References Parker RB (1974) Probiotics, the other half of the antibiotics story. Anim Nutr Health 29:4–8 Fuller R (1989) Probiotics in man and animals. J Appl Bacteriol 66(5):365–378 Sánchez B, Delgado S, Blanco-Míguez A, Lourenço A, Gueimonde M, Margolles A (2017) Probiotics, gut microbiota, and their influence on host health and disease. 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Front Microbiol. ;14 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.doc Fig. S1Evolutionary analysis of Lactococcus sp. AK06 by Maximum Likelihood method Fig. S2Evolutionary analysis of Lactococcus sp. AK16 by Maximum Likelihood method Fig. S3 Evolutionary analysis of strain Lactococcus sp. AK05 by Maximum Likelihood method Cite Share Download PDF Status: Published Journal Publication published 13 Feb, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Revision requested 16 Nov, 2025 Reviews received at journal 07 Nov, 2025 Reviews received at journal 07 Nov, 2025 Reviews received at journal 01 Nov, 2025 Reviewers agreed at journal 25 Oct, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers agreed at journal 20 Oct, 2025 Reviewers agreed at journal 19 Oct, 2025 Reviewers invited by journal 19 Oct, 2025 Editor assigned by journal 17 Oct, 2025 Submission checks completed at journal 17 Oct, 2025 First submitted to journal 15 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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13:59:09\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":115765,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGraphical representation of the tolerance of strains AK05, AK06, and AK16 under different stress conditions, a) acid tolerance at pH 4, 5, 6, and 7, b) bile acid tolerance at concentrations 0.3%, 0.5%, and 0.8%, and c) NaCl tolerance at concentrations 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% at regular time intervals i) Ak05, ii) AK06, iii) AK16\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/ac7fddf345d1b021480a904e.png\"},{\"id\":96918499,\"identity\":\"25a0c30b-c23f-44ac-9c1c-982b6a55824b\",\"added_by\":\"auto\",\"created_at\":\"2025-11-27 14:12:02\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":31238,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAutoaggregation % of AK05, AK06, and, AK16 at different time intervals\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/844d8fb05fdf3a0742d44dc2.png\"},{\"id\":96918662,\"identity\":\"5288cde1-46d6-41fe-a3df-a84888fc68ca\",\"added_by\":\"auto\",\"created_at\":\"2025-11-27 14:12:17\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":65698,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCo-aggregation % of AK05, AK06, and, AK16 against pathogens \\u003cem\\u003eS. aureus \\u003c/em\\u003eand \\u003cem\\u003eA. hydrophila\\u003c/em\\u003esubsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC1739\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/e84b9751c7394bbae3091840.png\"},{\"id\":96831823,\"identity\":\"a19b5cbf-d117-4caa-9882-7eb41edaf99b\",\"added_by\":\"auto\",\"created_at\":\"2025-11-26 13:59:09\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":32416,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGraphical representation of CSH % of the strains AK05, AK06, and AK16 towards hydrocarbons hexane and ethyl acetate\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/7a98424e47ba33d10a2ad55a.png\"},{\"id\":96918024,\"identity\":\"ae643706-bee7-44ff-b596-f4ee66d89de3\",\"added_by\":\"auto\",\"created_at\":\"2025-11-27 14:11:01\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":81741,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eWhole genome tree of the strain AK05 using TYGS\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/30cf39742a0f24b07c1a9e0f.png\"},{\"id\":96831829,\"identity\":\"28f9d9a6-32fe-4240-837c-191a26fa251b\",\"added_by\":\"auto\",\"created_at\":\"2025-11-26 13:59:09\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":163272,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA dendrogram was constructed based on the amino acid identity (AAI) of strain \\u003cem\\u003eLactococcus\\u003c/em\\u003esp. AK05 and related taxa, using the EZAAI pipeline\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/cb43ec02b94485f2e699d50f.png\"},{\"id\":96831834,\"identity\":\"48e5e1b3-8976-4017-85b6-094447f3b7c1\",\"added_by\":\"auto\",\"created_at\":\"2025-11-26 13:59:09\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":301790,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePresence of putative gene clusters responsible for biosynthetic genes responsible for producing secondary metabolites, like a) Ripp-like, and b) Ras-Ripp proteins, and c) T3PKS, along with the biosynthesis of antibacterial substances like d) Betalactone and e) Enterolysin A, are predicted by the software like antiSMASH version 7.1.0and BAGEL v4\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/59040b3646697a440518d776.png\"},{\"id\":96831832,\"identity\":\"52888664-8139-4958-a7d7-ecae9c3d187e\",\"added_by\":\"auto\",\"created_at\":\"2025-11-26 13:59:09\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":55581,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAntibiotic resistance gene in \\u003cem\\u003eLactococcus\\u003c/em\\u003e sp. AK05, observed in CARD v3.1.1 database\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/4f3b34041933f9af3e4f8971.png\"},{\"id\":96919028,\"identity\":\"c902b28d-d8a7-41ca-a742-26c990114f92\",\"added_by\":\"auto\",\"created_at\":\"2025-11-27 14:13:02\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":229141,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eProtein-protein interaction observed of proteins belonging to different functional traits of probiotics forming different clusters using STRING (v12.0)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/8a4667c486b2c9a6d23a9af2.png\"},{\"id\":102785586,\"identity\":\"a3caba1a-7e93-4280-8b3d-293ef00b68d5\",\"added_by\":\"auto\",\"created_at\":\"2026-02-16 16:08:26\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2626647,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/add085f4-b1b2-4048-a940-0ca1e90a0694.pdf\"},{\"id\":96918810,\"identity\":\"7fc61817-aacd-48b7-a18d-e0b3d5553d8f\",\"added_by\":\"auto\",\"created_at\":\"2025-11-27 14:12:38\",\"extension\":\"doc\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":888320,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFig. S1\\u003c/strong\\u003eEvolutionary analysis of \\u003cem\\u003eLactococcus\\u003c/em\\u003esp. AK06 by Maximum Likelihood method\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFig. S2\\u003c/strong\\u003eEvolutionary analysis of \\u003cem\\u003eLactococcus\\u003c/em\\u003esp. AK16 by Maximum Likelihood method\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFig. S3\\u003c/strong\\u003e Evolutionary analysis of strain \\u003cem\\u003eLactococcus\\u003c/em\\u003e sp. AK05 by Maximum Likelihood method\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SupplementaryFile.doc\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7873003/v1/ab89736cf9ca24bdfc1c7888.doc\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Genomic and Functional Insights into a Novel Lactococcus sp. AK05 from Cirrhinus mrigala with Potent Antagonism Against Aeromonas hydrophila\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe term probiotics was originally referred to as \\u0026ldquo;organisms and substances which contribute to intestinal microbial balance\\u0026rdquo; (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e). In the year 1989, Fuller revised the definition into \\u0026ldquo;live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance\\u0026rdquo; (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). In the present time, probiotics are defined as \\u0026ldquo;live microorganisms which when administered in adequate amount, confer health benefits on the host\\u0026rdquo; (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e), as stated by the World Health Organization (WHO) and Food and Agriculture Organization of the United Nations (FAO). Numerous types of microorganisms have been considered as probiotics, such as bacteria including various genera like \\u003cem\\u003eLactococcus\\u003c/em\\u003e, \\u003cem\\u003eLactobacillus\\u003c/em\\u003e, \\u003cem\\u003eBifidobacterium\\u003c/em\\u003e, \\u003cem\\u003eStreptococcus\\u003c/em\\u003e, \\u003cem\\u003eShewanella\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e), etc., yeasts (\\u003cem\\u003eSaccharomyces cerevisae\\u003c/em\\u003e and \\u003cem\\u003eSachharomyces boulardii\\u003c/em\\u003e) (\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e) and filamentous fungi such as \\u003cem\\u003eAspergillus oryzae, A. cristatus\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e) and \\u003cem\\u003eCandida pintolopesii\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e). To be regarded as a probiotic, the live feed must be capable of persisting in the stomach\\u0026rsquo;s acidic conditions, and in the small intestine it must endure the existence of bile, also, it must have antimicrobial properties against pathogens, should be able to adhere to the gut lining of the intestine, must be able to utilize a range of sugar derivatives for their growth to act in full potential and after fulfilling all the above-mentioned criteria, they need to be non-pathogenic to the host and must not be resistant to antibiotics.\\u003c/p\\u003e\\u003cp\\u003eOwing to the tremendous increase in world population, there has been an increase in demand in the food supply sector globally. It is estimated that by the year 2050, the requirement of meat for human consumption is going to be 500 Mt (Megatonnes) and to meet this humongous demand for food, there has been a vast expansion in the aquaculture sector (\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e). Due to the sudden boost in the cultivation of aquatic animals, there have been outbreaks of bacterial, viral, and fungal diseases (\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e), which have resulted in losses of more than 50% of aquatic organisms. To combat such diseases, various methods have been implemented like the use of antibiotics, vaccination of the aquatic animals, etc. Since the discovery of antibiotics, there has been an unchecked use of them for humans as well as other organisms. Such vast and uncontrolled use of antibiotics has been causing subsequent contamination in the environment by giving rise to antibiotic-resistant bacteria. In aquaculture, probiotics such as \\u003cem\\u003eBacillus subtilis\\u003c/em\\u003e and \\u003cem\\u003eLactobacillus plantarum\\u003c/em\\u003e are used against fish pathogen \\u003cem\\u003eVibrio anguillarum\\u003c/em\\u003e, in Japanese eel (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e), \\u003cem\\u003eB. subtilis\\u003c/em\\u003e and processed yeast against \\u003cem\\u003eEdwardsiella tarda\\u003c/em\\u003e through mucosal and systemic immunity and they also release antimicrobials such as lysozymes, bacteriocins or hydrogen peroxide, in host Olive Flounder (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e), \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e and \\u003cem\\u003eLactobacillus rhamnosus\\u003c/em\\u003e against \\u003cem\\u003eStreptococcus agalactiae\\u003c/em\\u003e through an increase in immunity in host Nile tilapia (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e), \\u003cem\\u003eEnterococcus casselifavus\\u003c/em\\u003e against \\u003cem\\u003eAeromonas hydrophila\\u003c/em\\u003e, in the host \\u003cem\\u003eCyprinus carpio\\u003c/em\\u003e showed improved growth rate, better hematological parameters and increase in immunity (\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e). However, \\u003cem\\u003eLactococcus\\u003c/em\\u003e is not well-studied in fish farming because of a number of reasons. For example, \\u003cem\\u003eLactococcus garvieae\\u003c/em\\u003e has become a major fish pathogen, and more research is needed on its antimicrobial susceptibility (\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eAlso, other probiotic candidates like \\u003cem\\u003eBacillus subtilis\\u003c/em\\u003e are getting more attention. \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e is acknowledged for its probiotic capabilities (\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e). Nevertheless, the potential of other \\u003cem\\u003eLactococcus\\u003c/em\\u003e species and strains, along with the mechanisms of action of lactic acid bacteria in fish, necessitate additional research (\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e)\\u003c/p\\u003e\\u003cp\\u003eFew studies combine whole-genome evidence with phenotypic evaluation to identify new strains of lactic acid bacteria (LAB), even though there is growing interest in LAB as probiotics for aquaculture. Correct taxonomic placement, the identification of functional genes related to probiotic traits, and biosafety evaluation all rely on this kind of integration. In order to describe the antimicrobial potential, stress tolerance, adhesion ability, and safety profile of the LAB strain that we isolated from the gut of \\u003cem\\u003eC. mrigala\\u003c/em\\u003e, we used both experimental and bioinformatic methods. Its phylogenetic position, genomic novelty, and the existence of genes underlying observed probiotic phenotypes were all ascertained through whole-genome sequencing. According to the results, \\u003cem\\u003eLactococcus\\u003c/em\\u003e sp. AK05 is a unique candidate for microbiome modification in aquaculture systems both genomically and functionally.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cp\\u003eStandard assays were used to assess probiotic traits:\\u003c/p\\u003e\\n\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003eSample collection and lactic acid bacteria (LAB) isolation\\u003c/h2\\u003e\\n \\u003cp\\u003eHealthy live fish specimens reared in the local hatchery, belonging to the group of Indian major carp \\u003cem\\u003eC. mrigala\\u003c/em\\u003e of average weight 139.14\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;45.18 g, were procured from Shivmandir (26\\u0026deg;42\\u0026apos;25.4\\u0026quot;N 88\\u0026deg;21\\u0026apos;41.3\\u0026quot;E), West Bengal, India. Samples were transported alive in plastic containers to the OMICs Laboratory, Department of Biotechnology, University of North Bengal, for dissection purposes following the approved guidelines of the Committee for the Purpose of Control \\u0026amp; Supervision of experiments on animals (CPCSEA). Prior permission was sought from the CPCSEA, Registration No: 840/GO/Re/S/04/CPCSEA, the University of North Bengal, for performing the experiments. Fishes were anesthetized using clove oil (eugenol) with a dose volume\\u0026thinsp;\\u0026ge;\\u0026thinsp;100 mg/L (\\u003cspan class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e) followed by aseptic removal of gastrointestinal tracts (GITs). Aseptically removed GIT, weighing around 2.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.29 g, was homogenized and serially diluted in PBS (pH 7.4). Morphologically distinct colonies were clonally purified from serially diluted samples spread out onto a de Man Rogosa Sharpe Agar plate (MRSA, Himedia) and cultured anaerobically for 48 h at 37 \\u003csup\\u003eo\\u003c/sup\\u003eC. Tests were conducted on purified strains to determine their suitability as probiotics.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003eProbiotic Characterization\\u003c/h3\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTest for determining antagonistic activity\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFrom the overnight grown cultures of the eight bacterial isolates, the supernatant was obtained by centrifuging the cultures for 15 min at 1252 \\u003cem\\u003eg\\u003c/em\\u003e, and subsequent analysis was done. Agar well-diffusion assay was done in Mueller Hinton (Himedia) agar plate (MHA) (\\u003cspan class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e). Overnight grown culture of the pathogenic bacterium \\u003cem\\u003eAeromonas hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739 (100 \\u0026micro;l) (\\u003cspan class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e) was spread evenly over the MHA plate surface. Wells with 6 mm diameter were punched aseptically into the agar plate and 50 \\u0026micro;l of the supernatant from each isolated bacterial strain was added to respective wells. Subsequent incubation of the MHA plates was done for 24 h at 30\\u0026deg;C to observe distinct inhibition zones to observe the antibacterial activity of the metabolites secreted by the test isolates (\\u003cspan class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTest for acid and bile salt tolerance\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe bacterial isolates were evaluated for their acid-tolerant properties in MRS broth. Individual calibration of the MRS broth to pH 2 or 3 or 4 or 5 or 6 or 7 with 6 N HCl was done. Each MRS broth having a different pH was inoculated with 1% of overnight-grown bacterial cultures and 37\\u0026deg;C was taken as the incubation temperature. At sampling times 0, 3, 6, and 24 h, 1 ml culture was taken, dilution was done serially in phosphate-buffered saline (PBS, pH 7), and incubated at 37\\u0026deg;C, spread onto Tryptone Soya Agar (TSA) plates, and observed for countable colony forming units (CFUs) after 24 h. Additionally, MRS broth was supplemented with different percentages of conjugated bile salt solution collected from the fish gall bladder, i.e., 0.3%, 0.5%, and 0.8% (v/v) respectively. MRS broth medium supplemented with fish gall bladder extract was inoculated with 1% of overnight-grown bacterial isolates. Bacterial growth was monitored at 2 h intervals till 8 h by measuring absorbance with a UV-Vis spectrophotometer at 600 nm(\\u003cspan class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTest for NaCl tolerance\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe capability of the bacterial strains to grow in high salt concentration was assessed by inoculating 1% overnight grown bacterial cells in MRS broth enriched with different concentrations of NaCl (1% to 8% respectively). The absorbance was calculated with a UV- Vis spectrophotometer at 600 nm at intervals of 0, 2.5, 5, and 24 h respectively (\\u003cspan class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTest for autoaggregation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBacterial cell auto-aggregation was evaluated for their ability to endure and grow in the GIT and confer advantageous outcomes. Overnight grown cell cultures using MRS broth were harvested at 1252 \\u003cem\\u003eg\\u003c/em\\u003e for 15 min and washed two times in PBS solution (pH 7.4). Incubation of the cells was done by suspending them in PBS under static conditions at 37\\u0026deg;C. The upper layer of the suspension was measured for its absorbance at 600 nm at an interval of 0, 2, 5, and 24 h respectively (\\u003cspan class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e). The auto-aggregation percentage was calculated using the formula\\u003c/p\\u003e\\n\\u003cp\\u003eAutoaggregation % = [(A1 - A2)/ (A1) x 100]\\u003c/p\\u003e\\n\\u003cp\\u003eWhere A1 is the initial optical density (O.D.) of the cell suspension in PBS, and A2 is the O.D. of the cell suspension in PBS after static incubation for the defined period.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTest for co-aggregation\\u003c/strong\\u003e: Co-aggregation of the bacterial isolates was done against two pathogens, \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e and \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739. The bacterial isolates and the pathogens were cultured overnight in MRS broth and TSB, respectively, and washed two times in PBS solution (pH 7.4). The washed cells of the test isolates and the pathogens were finally resuspended separately in PBS (pH 7.4), and then each of the PBS-suspended test bacterial strains and the pathogen was mixed in equal volume (1:1 ratio) and incubated at 37\\u0026deg;C. The O.D. of the suspensions was calculated at an interval of 0, 2, 5, and 24 h respectively at 600 nm. The co-aggregation percentage was measured by the following formula: [A\\u003csub\\u003eIsolate\\u003c/sub\\u003e + A\\u003csub\\u003epathogen\\u003c/sub\\u003e] \\u0026minus; [A\\u003csub\\u003emix\\u003c/sub\\u003e (A\\u003csub\\u003eIsolate\\u003c/sub\\u003e + A\\u003csub\\u003epathogen)])/\\u003c/sub\\u003e2 \\u0026times; 100, where A\\u003csub\\u003eIsolate\\u003c/sub\\u003e, A\\u003csub\\u003epathogen\\u003c/sub\\u003e, and A\\u003csub\\u003emix\\u003c/sub\\u003e represent the absorbance of test bacterial isolate, pathogen, and the mixture of test bacterial isolate and pathogen, respectively (\\u003cspan class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDetermination of cell surface hydrophobicity of the test bacterial isolates\\u003c/strong\\u003e: The hydrophobicity of the test bacterial cells was determined by measuring their affinity towards hydrophobic substances like hexane and ethyl acetate (\\u003cspan class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e). Bacterial cells were grown overnight and harvested by centrifugation at 1252 \\u003cem\\u003eg\\u003c/em\\u003e for 15 min, washed two times in sterile PBS (pH 7.4), and again suspended in the same. Each of the finally resuspended bacterial cultures in PBS and the hydrocarbon (hexane or ethyl acetate) were mixed in a ratio of 1:3, vortexed vigorously, and left for 30 min in static condition. The absorbances of the aqueous phase just after mixing (initial) and after incubation (final) were taken at 600 nm in a UV-Vis spectrophotometer. Cell surface hydrophobicity (CSH) was measured using the following formula:\\u003c/p\\u003e\\n\\u003cdiv class=\\\"Heading\\\"\\u003eCSH % =\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\frac{Initial\\\\:OD-Final\\\\:OD}{Initial\\\\:OD}\\\\:\\\\times\\\\:100\\\\)\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/div\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCarbohydrate utilization\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe carbohydrate fermentation test was performed using the KB009 HiCarbo kit (Hi-Media, India). 50 \\u0026micro;l of overnight-grown bacterial cultures were inoculated into each well and grown at 37\\u0026deg;C for a duration of 24\\u0026ndash;48 h. Color changes due to a change in pH were observed (\\u003cspan class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003ch3\\u003ePathogenicity and safety assessment tests\\u003c/h3\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eHemolysis test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis test was done to assess the hemolytic properties of the bacteria. The isolates were grown on TSA plates containing 5% defibrinated sheep blood. The plates were evaluated for clear zones around the bacterial growth after a period of 24 h of incubation at 37\\u0026deg;C (\\u003cspan class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDNase agar test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDNase (deoxyribonuclease) production by the bacteria was examined in this study. To DNase agar (Himedia) plates, a loopful of overnight\\u0026ndash;grown bacterial culture was streaked and incubated at 37\\u0026deg;C for 24 h. A clear zone was assessed around the colonies, which hydrolysed DNA (\\u003cspan class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGelatin liquefaction test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe freshly grown bacteria was stabbed on a slant made from Gelatin agar (Hi-Media) with the help of a sterile inoculating loop and grown for 24 h at 37\\u0026deg;C. After the growth of the bacterial culture, stabbed slants were kept for 3 h under refrigeration to confirm the liquefaction activity caused by microbial processes and not because of the temperature used for incubation (\\u003cspan class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAntibiotic susceptibility test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA modified standard Kirby-Bauer procedure was followed to test the antibiotic susceptibility profile (\\u003cspan class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e). The isolates were evaluated for their antibiotic susceptibility against different classes of antibiotics, \\u003cem\\u003eviz.\\u003c/em\\u003e Penicillin (Ampicillin - Amp10), Cephalosporins (Cefotaxime - CTX30), Carbapenems (Imipenem - IMP10), Fluoroquinolones (Levofloxacin - LVX5), Glycopeptides and lipoglycopeptides (Vancomycin - VA30), Marcrolides (Erythromycin - E15), Tetracyclines (Tigecycline - TGC15), Oxazolidinones (Linezolid - LZ10) and other Miscellaneous Group (Nitrofurantoin - NIT30, Trimethoprim - TR10) antibiotic discs (Himedia Ltd., India). The inhibition zones around the discs were measured with the help of an antibiotic zone scale (Himedia) in millimeters (mm).\\u003c/p\\u003e\\n\\u003ch3\\u003eGenomic Characterization\\u003c/h3\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eWhole genome Extraction and Sequencing\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOvernight grown cells of the most potent probiotic strain were taken and extraction of the genomic DNA was carried out by using the isolation kit by Purelink genomic DNA (Thermo Fisher Scientific, USA). The whole genome sequencing was done using Illumina (Novaseq 6000), 150PE platform, with a prepared DNA library. Default parameters were used to assemble high-quality reads into contigs that are \\u0026gt;\\u0026thinsp;200 bp using Unicycler v0.5.0 (\\u003cspan class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e). The submission of the raw sequence reads was done to the NCBI Sequence Read Archive, and the entire genome shotgun projects were deposited to DDBJ/ENA/GenBank (SRA). The draft genome sequence annotations were done using the NCBI GenBank file.\\u003c/p\\u003e\\n\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003eBioinformatics analysis\\u003c/h2\\u003e\\n \\u003cp\\u003eThe whole genome tree of the strain AK05 was created using Type (Strain) Genome Server (TYGS) (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://tygs.dsmz.de/\\u003c/span\\u003e\\u003c/span\\u003e) (\\u003cspan class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e). Using the pipeline described earlier by Dongwook Kim et. al., 2021 (\\u003cspan class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e). EzAAI was used to produce a matrix of average amino acid identity (AAIm) values. It supported the large-scale calculations of AAI matrices and, thus, using UPGMA (unweighted pair-group method with arithmetic mean), a dendrogram was constructed using all the related taxa belonging to the genus \\u003cem\\u003eLactococcus\\u003c/em\\u003e and the strain AK05. To find out the similarities between species in the \\u003cem\\u003eLactococcus\\u003c/em\\u003e group, digital DNA-DNA hybridization (dDDH) using Genome to Genome Distance Calculation (GGDC) version3.0 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://ggdc.dsmz.de/phylogeny-service.php\\u003c/span\\u003e\\u003c/span\\u003e) (\\u003cspan class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e) and the average nucleotide identity (ANI) (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.ezbiocloud.net/tools/ani\\u003c/span\\u003e\\u003c/span\\u003e) values using EZbiocloud were calculated, respectively (\\u003cspan class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e).\\u003c/p\\u003e\\n \\u003cp\\u003eCorroboration of phylogenetic inference with the Jaccard similarity coefficient was scored between AK05 and the nearest phylogenetic relative, \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e subsp. \\u003cem\\u003ehordniae\\u003c/em\\u003e CCUG32210.\\u003c/p\\u003e\\n \\u003cp\\u003eJaccard similarity coefficient was determined by the following formula: \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\text{J}\\\\left(\\\\text{A},\\\\text{B}\\\\right)=\\\\:\\\\frac{\\\\left|\\\\text{A}\\\\text{n}\\\\text{B}\\\\right|}{\\\\left|\\\\text{A}?\\\\text{B}\\\\right|}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e, where A \\u0026cap; B stands for the number of phenotypic tests recorded as positive as well as negative in both the sets corresponding to the strains, A and B; A U B stands for the total number of tests conducted in both sets, A and B. The ratio multiplied by 100 gives the % similarity coefficient, (\\u003cspan class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u0026ndash;\\u003cspan class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e) Table \\u003cspan class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e.\\u003c/p\\u003e\\n \\u003cp\\u003eTo detect the genes accountable for the production of antimicrobial substances like bacteriocin and other secondary metabolites, bioinformatics tools BAGEL v.4 (\\u003cspan class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e) and antiSMASH version 7.1 (\\u003cspan class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e) were used respectively (\\u003cspan class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e). Genes coding for enzymes responsible for the production, modification, and complex carbohydrate breakdown present in AK05 were mined using Carbohydrate-Active Enzyme Annotation (dbCAN-3) database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://bcb.unl.edu/dbCAN2/blast.php\\u003c/span\\u003e\\u003c/span\\u003e) (\\u003cspan class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e). Genes related to pathogenicity and virulence in the genome of AK05 were identified using Comprehensive Antibiotic Resistance Database (CARD) v3.1.1 (\\u003cspan class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e) PathogenFinder 1.1 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://cge.food.dtu.dk/services/PathogenFinder/\\u003c/span\\u003e\\u003c/span\\u003e) (\\u003cspan class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e) and VirulenceFinder 2.0 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://cge.food.dtu.dk/services/VirulenceFinder/\\u003c/span\\u003e\\u003c/span\\u003e) (\\u003cspan class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e) from Centre for Genomic Epidemiology (CGE).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003eFunctional protein-protein interaction through STRING database:\\u003c/h3\\u003e\\n\\u003cp\\u003eSTRING (v12.0) software was used to predict the interactions between different proteins of interest. Either the protein sequence or the protein identification can be used to access the STRING database. The functional partners of a given protein are ranked according to their degree of confidence (\\u003cspan class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e). Genetic data, the transmission of relationships or interactions between species, gene co-expression analysis, enhanced data collections, database analysis, and literature research are the sources of the forecasts. Each source is offered as proof. In STRING, every single score for a related piece of data or interaction is highlighted independently and shown as a colored line. A probabilistic confidence score is provided for each of these relationships. The degree of interaction among nodes connected by a multitude of evidence is indicated by confidence ratings. The highest level of assurance is indicated by a high aggregate score, which usually exceeds the individual score. STRING employs a unique scoring mechanism based on standards of various kinds of connections in comparison to a common reference set. In STRING, confidence scores are divided into four categories: low (0.1), medium (0.4), high (0.7) and highest (0.9).\\u003c/p\\u003e\\n\\u003cp\\u003eFor this investigation, we used EnoA, Ecf, MsmK, ZitP, ZitQ, EC6.4.1.1, LmrA, LmrC, LmrD, SrtA, SrtC, Llmg1152, Llmg2465, PS102, PS103, PS501, LysP, PtPl, Phage lysin (Table S2) as our protein queries and numerous proteins as our input format. As the model organism, \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e was selected for this study. The functional protein networking was created by setting the network type to \\u0026quot;full\\u0026quot; STRING network, the \\u0026quot;meaning of network edges\\u0026quot; to evidence, allowing all active sources of interactions, setting the minimum required interaction score to the default value of 0.4, and, for display purposes, limiting the number of interactions to 10 in the first shell and 20 in the second shell under the basic settings.\\u003c/p\\u003e\\n\\u003cp\\u003eAmong the clustering alternatives, k-means clustering was selected, with four clusters and solid lines for the edges between the clusters. Ultimately, the produced STRING network was exported in PNG format for analysis and visual interpretation.\\u003c/p\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eIsolation of lactic acid bacteria (LAB)\\u003c/h2\\u003e\\u003cp\\u003eThree healthy specimens of \\u003cem\\u003eC. mrigala\\u003c/em\\u003e (average weight 139.14\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;45.18 g) were collected from a local hatchery in West Bengal, India (26\\u0026deg;42'25.4\\\"N 88\\u0026deg;21'41.3\\\"E). Fish were anesthetized with clove oil (\\u0026ge;\\u0026thinsp;100 mg/L) and dissected under aseptic conditions. Gastrointestinal tracts were homogenized, serially diluted, and plated on de Man Rogosa Sharpe Agar (MRSA; HiMedia). Nine morphologically distinct colonies were clonally purified from serially diluted samples spread out onto a de Man Rogosa Sharpe Agar plate (MRSA, Himedia) and cultured anaerobically for 48 h at 37 \\u003csup\\u003eo\\u003c/sup\\u003eC. With purified strains, tests for suitability as probiotics were determined.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eProbiotic Characterization\\u003c/h2\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section3\\\"\\u003e\\u003ch2\\u003e\\u003cem\\u003eAntagonism\\u003c/em\\u003e:\\u003c/h2\\u003e\\u003cp\\u003eAgar well diffusion assay of the isolated strains was tested against bacteria \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739. The highest diameter of inhibition zones was shown by AK16 and AK05, the zones measured were 15.33 and 15.66 mm in diameter, and then AK01 and AK06 showed inhibition zones of 10.33 and 11 mm in diameter. The remaining isolates showed inhibition zones less than 10 mm in diameter (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Hence, the three strains with the highest zone diameters AK06, AK16, and AK05 were selected for further screening processes.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eInhibition zone diameter of the Antagonistic assay of the isolated strains against the pathogen \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739 using agar well diffusion test\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"2\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eStrains\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eInhibition zone diameter (mm)\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAK01\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e10.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAK06\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e11\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAK09\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e9.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAK11\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e7.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.52\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAK16\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e15.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.52\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAK17\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e8.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAK18\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e7.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAK19\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e7.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAK05\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e15.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.52\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e\\u003cem\\u003eStress tolerance\\u003c/em\\u003e:\\u003c/h2\\u003e\\u003cp\\u003eAcid and bile salt tolerance: Three strains, AK06, AK16, and AK05, were further selected on the basis of maximum inhibition zone obtained from antagonistic assay. It was observed that the tolerance of the strains to different pH was not uniform. AK06 and AK05 were able to tolerate pH 4 till the 24th h of incubation while AK16 failed to survive beyond 6 h of incubation at pH 4 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eAll three strains showed growth in different percentages of fish bile extract i.e., 0.3%, 0.5%, and 0.8%. The strains AK05 and AK16 had entered the mid-log phase by the 4th and 6th h of incubation for all three percentages of the bile acid respectively. But the strain AK06 entered the mid-log phase by the 4th, 6th, and 8th h of intervals for 0.3%, 0.5%, and 0.8% respectively. Thus, all the strains showed growth in 0.3%, 0.5%, and 0.8% of the bile acid in different intervals (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eNaCl tolerance: All three strains reached the log phase by the 24th h at the highest concentration of 5% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eAutoaggregation test\\u003c/h2\\u003e\\u003cp\\u003eThe highest autoaggregation property was shown by the strain AK05, 95.65%, and then AK06, 95.07% followed by AK16 which is 94.90% at 24 h (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCo-aggregation\\u003c/h2\\u003e\\u003cp\\u003eThe co-aggregation percentage of AK05 against the pathogens \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e and \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739 was 75.25% and 82.20%, respectively, while AK06 and AK16 demonstrated 68.64% and 83.80% and 42.45% and 74.78%, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCell surface hydrophobicity test\\u003c/h2\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003eThe strain AK05 had greater affinity for the non-hydrophobic hydrocarbon hexane (92.91%) than ethyl acetate (26.66%), according to the cell surface hydrophobicity test (CSH%). According to Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, the other strains, AK06 and AK16, demonstrated 33.53% and 29.61% affinity for hexane and 59.75% and 54.64% affinity for ethyl acetate, respectively.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCarbohydrate utilization test\\u003c/h2\\u003e\\u003cp\\u003eOne of the probiotic screening procedures was the carbohydrate fermentation test, where the bacterial isolates AK05, AK06, and AK16 were able to ferment the majority of the carbohydrates, which is regarded as a positive probiotic characteristic (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). In the context of the carbohydrate utilization test, the results showed no differences between the three strains.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eCarbohydrate fermentation test by using KB009 HiCarbo kit (Hi-Media, India)\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"5\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eS.No\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eCarbohydrates\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eAK05\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eAK06\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eAK16\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e1\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eLactose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e2\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eXylose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e3\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eMaltose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e4\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eFructose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e5\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDextrose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e6\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eRaffinose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e7\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eTrehalose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e8\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eMelibiose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e9\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSucrose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e10\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eL-Arabinose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e11\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eMannose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e12\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eInulin\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e13\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSodium gluconate\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e14\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eGlycerol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e15\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSalicin\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e16\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDulcitol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e17\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eInositol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e18\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSorbitol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e19\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eMannitol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e20\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAdinitol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e21\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eArabitol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e22\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eErythritol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e23\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAlpha-Methyl-D-glucoside\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e24\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eRhamnose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e25\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eCellobiose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e26\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eMelezitose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e27\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAlpha-Methyl-D-mannoside\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e28\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eXylitol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e29\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eONPG\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e30\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eEsculin\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e31\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eD-Arabinose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e32\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eCitrate\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e-\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e-\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e-\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e33\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eMalonate\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e-\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e-\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e-\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e34\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSorbose\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e+\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003ctfoot\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"5\\\"\\u003e\\u003csup\\u003e\\u0026lsquo;+\\u0026rsquo; denotes utilization of carbohydrate; \\u0026lsquo;\\u0026minus;\\u0026rsquo; denotes no utilization of carbohydrate\\u003c/sup\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tfoot\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003ePathogenicity and Safety assessment tests\\u003c/h2\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003eHemolytic test, gelatinase liquefaction test, and DNase agar tests were done; γ-hemolysis, negative gelatinase activity, and negative DNase activity were demonstrated by AK05, AK06, and AK16. According to these findings, all three strains showed no detrimental activity and are therefore acceptable for use in the food and dairy industries.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eAntibiotic susceptibility test\\u003c/h2\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eAntibiotic susceptibility profiling and interpretation using the latest EUCAST guidelines.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"5\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eClasses of Antibiotics\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eAntibiotics\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c5\\\" namest=\\\"c3\\\"\\u003e\\u003cp\\u003eBacterial strains\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eAK05 (mm)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eAK06 (mm)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eAK16 (mm)\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePenicillin\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eAmpicillin (10\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e21.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.52 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e13.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (R)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e11.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (R)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCephalosporins\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eCefotaxime (30\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e23.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e25.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.15 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e32\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1(S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCarbapenems\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eImipenem (10\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e28.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e26.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e34.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eFluoroquinolones\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eLevofloxacin (5\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e21.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.15 (I)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e19.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (I)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e23.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (I)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eGlycopeptides and lipoglycopeptides\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eVancomycin (30\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e18.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e14.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.15 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e14.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMarcrolides,\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eErythromycin (15\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e21.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e24.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.15 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e23\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1(S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eTetracyclines\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eTigecycline (30\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e20.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e14.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (R)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e17.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (R)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eOxazolidinones\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eLinezolid (10\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e22.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.15 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e20.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.15 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e17.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (R)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eMiscellaneous Group\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eNitrofurantoin(30\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e15.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.52 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e18.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e15.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (S)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eTrimethoprim(10\\u0026micro;g)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e(R)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e9.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (R)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003e9.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57 (R)\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003e\\u0026lsquo;S\\u0026rsquo; denotes susceptible; \\u0026lsquo;R\\u0026rsquo; denotes resistant\\u003c/p\\u003e\\u003cp\\u003eThe strain AK05 was demonstrated to be the most suitable probiotic candidate in terms of antibiotics-susceptibility profile, being resistant to only trimethoprim among seven classes of antibiotics tested. The other two strains, AK06 and AK16 were multiple-antibiotic resistant; in addition to trimethoprim, they were resistant to both ampicillin and tigecycline. Furthermore, AK16 has also resisted linezolid (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eAll three isolates were initially identified using 16S rRNA phylogeny (\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e). The three strains were all members of the \\u003cem\\u003eLactococcus\\u003c/em\\u003e genus. While AK05 split off to stand out among other species of the genus \\u003cem\\u003eLactococcus\\u003c/em\\u003e, AK06 and AK16 were revealed to be closely linked to \\u003cem\\u003eL. garvieae\\u003c/em\\u003e (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e, S2, S3). Thus, the two strains, AK16 and AK06, were not chosen for further research because the literature indicated that \\u003cem\\u003eLactococcosis\\u003c/em\\u003e caused by \\u003cem\\u003eL. garvieae\\u003c/em\\u003e is one of the main causative organisms for fish disorders (\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e). Therefore, the strain AK05 being the most potent probiotic candidate underwent whole genome sequencing and sequence analysis.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003ePhylogenetic placement of the strain AK05 and its whole genome sequence analysis\\u003c/h2\\u003e\\u003cp\\u003eThe whole genome phylogenetic analyses (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e) have placed the strain AK05 on a discrete branch sharing a common ancestor with \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e subsp. \\u003cem\\u003ehordniae\\u003c/em\\u003e CCUG 32210. To further clarify the taxonomic position of strain AK05, a phylogenomic dendrogram was compiled on the basis of amino acid identity between representative \\u003cem\\u003eLactococcus\\u003c/em\\u003e species (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). AK05 was located in the \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e clade and clustered closely with \\u003cem\\u003eL. lactis\\u003c/em\\u003e subsp. \\u003cem\\u003ehordniae\\u003c/em\\u003e CCUG 32210 and \\u003cem\\u003eL. lactis\\u003c/em\\u003e subsp. \\u003cem\\u003ecremoris\\u003c/em\\u003e strains KW2 and ATCC 19257. The short branch lengths that separate AK05 from these subspecies reflect very high amino acid identity, validating that AK05 is a member of the \\u003cem\\u003eL. lactis\\u003c/em\\u003e species complex. This positioning differentiates AK05 from other \\u003cem\\u003eLactococcus\\u003c/em\\u003e species like \\u003cem\\u003eL. garvieae\\u003c/em\\u003e, \\u003cem\\u003eL. raffinolactis\\u003c/em\\u003e, and \\u003cem\\u003eL. plantarum\\u003c/em\\u003e, justifying its status as a new isolate within the \\u003cem\\u003eL. lactis\\u003c/em\\u003e lineage. Between AK05 and \\u003cem\\u003eL. lactis\\u003c/em\\u003e subsp. \\u003cem\\u003ehordniae\\u003c/em\\u003e CCUG 32210, the dDDH and orthoANI estimates yielded high genomic values of 78.90% dDDH and 97.61% orthoANI, respectively, beyond species and subspecies demarcation levels (Table S3). In agreement, a phenotypic comparative analysis using the Jaccard similarity coefficient indicated limited phenotypic similarity of 21% for 23 phenotypic tests (this study for AK05 and reported findings for \\u003cem\\u003eL. lactis\\u003c/em\\u003e subsp. \\u003cem\\u003ehordniae\\u003c/em\\u003e CCUG 32210) (Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). AK05 can thus be considered a strain of the taxon that is both genetically and phenotypically different.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e\\u003cb\\u003eMore genomic insights\\u003c/b\\u003e:\\u003c/h2\\u003e\\u003cp\\u003eThe comprehensive genome characteristics of AK05 have been presented in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eDetails of the genome of \\u003cem\\u003eLactococcus\\u003c/em\\u003e sp. AK05\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"2\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCharacteristics\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAK05\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eBioproject accession no.\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003ePRJNA1141375\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eSRA accession no.\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSRR30829574\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eBioSample accession no.\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSAMN42887626\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eGenBank accession no.\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eJBFTEI000000000\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eNo.of contigs\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e184\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eGenome size (bp)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e2,555,466\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eGenome coverage (bp)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e121\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eGC content (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e35.64\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eN50 (bp)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e201128\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eAntiSMASH (version 7.1.0 web server) revealed that Ribosomally synthesized and post-translationally modified Peptides (RiPP-like), RiPPs modified by radical S-adenosylmethionine enzymes (Ras-RiPP), Type III polyketide synthase (T3PKS), terpene-like precursor, and Betalactone (biosynthetic gene cluster) protein-coding genes were present in the genome of AK05. BAGEL v4 web server confirmed the presence of Enterolysin A protein coding gene in the AK05\\u0026rsquo;s genome (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e\\u003ch2\\u003eStudy of antibiotic resistance gene observed via CARD v3.1.1\\u003c/h2\\u003e\\u003cp\\u003eThe draft genome of AK05 was analyzed via CARD database of antibiotic resistance gene and strict hit was observed against the antibiotic, vancomycin Y and vancomycin W belonging to the class of Glycopeptide antibiotic (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eSafety assessment through sequence analysis\\u003c/h2\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003eFor genotypic corroboration, pathogenicity and virulence assessments were done using PathogenFinder version 1.1 and VirulenceFinder 2.0 where no hits were found in both the cases, thus, it can be stated that \\u003cem\\u003eLactococcus\\u003c/em\\u003e sp. AK05 is non-pathogenic and non-virulent.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cdiv id=\\\"Sec25\\\" class=\\\"Section3\\\"\\u003e\\u003ch2\\u003eProtein-protein interaction via STRING (v12.0)\\u003c/h2\\u003e\\u003cp\\u003eA protein interacting network was created using the STRING database, forming different clusters of proteins belonging to different functional traits. The proteins EnoA, Ecf, MsmK, ZitP, ZitQ, EC6.4.1.1, BirA1, and BirA2, were related to stress (acid and salt stresses) represented by the red color cluster. The yellow cluster including LmrA, LmrC and LmrD belonged to proteins related to bile acid tolerance. The cell adhesion, autoaggregation, and co-aggregation proteins included SrtA, SrtC, Llmg1152, and Llmg2465 (cluster green). The blue cluster included proteins related to bacteriocin biosynthesis i.e., PS102 and PS103. Also, several bacteriocin biosynthesis and exopolysaccharide proteins were found in a scattered manner in the genome which included PS501, LysP, PtPl and Phage lysin (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig12\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIsolating probiotics from host-specific organisms is more effective than from other sources, as these strains demonstrate better colonization properties and re-establish a balanced and healthy homeostatic state (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e). In the pursuit of enhancing aquaculture sustainability and fish health, the exploration of probiotics derived from the fish gut microbiota has attracted significant interest. Commensal bacteria, naturally residing within the fish gut, have shown to be more effective as probiotics due to their established relationship with the host. Commensal microorganisms act best in similar environments (\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e) showing improved growth, better immunological responses, and defense against diseases in aquatic organisms (\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eThe LAB isolates exhibited antimicrobial activities against a common fish pathogen \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739. Studies have found that \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e is the most common bacteria causing diseases in freshwater fishes and responsible for huge economic losses in the aquaculture sector worldwide (\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e). Morphologically distinct LAB strains exhibiting antimicrobial activities were primarily selected, from which the best ones were chosen based on other probiotic traits. A key characteristic of a probiotic is to be able to endure adverse environments of the fish\\u0026rsquo;s gastrointestinal tract i.e., high tolerance to acid and bile (\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e). In the acid tolerance test, AK05 showed more than 4-fold increase in growth at pH 5 in 24 h from the time of inoculation, and similar gradual growth was observed in pH 6 and pH 7, while AK16 and AK06 showed more than a 3-fold increase in growth at pH 5 and gradual growth till pH 7 in 24 h from the time of inoculation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The bile acid tolerance test showed that AK05 reached a mid-log phase in the 4th h of growth in presence of 0.3%, 0.5%, and 0.8% bile acid, while the other two strains showed gradual growth in lower percentages of bile acid, suggesting that they can survive in the harsh environment of the intestine (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The intestinal fluid of \\u003cem\\u003eC. mrigala\\u003c/em\\u003e has a pH range of 6.2 to 7.1 and digestion takes place mainly due to bile and pancreatic juices due to the absence of gastric glands in the intestinal bulb. Along with the acid and bile tolerance the probiotic must be able to tolerate stresses like salt tolerance, all three strains showed prominent growth of up to 5% of NaCl in the medium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), thus, indicating that it can grow in the stressed environment without getting affected in its osmolarity. Bacteria having a high tolerance to NaCl stress can be used in food products and aquaculture.\\u003c/p\\u003e\\u003cp\\u003eTo effectively survive, colonize, and provide advantages to the host\\u0026rsquo;s health, the probiotic must possess some key characteristics like auto-aggregation, co-aggregation, and cell surface hydrophobicity. Probiotic strains including the genus \\u003cem\\u003eLactobacillu\\u003c/em\\u003es and \\u003cem\\u003eLactococcus\\u003c/em\\u003e exhibited properties inhibiting the growth of harmful bacteria to the lining of the gastrointestinal tract (GIT), this inhibition can take place due to several reasons like, competition for adhesion sites due to the presence of specific adhesin and receptors, production of antimicrobial substances, immune response modulation, alteration of gut pH, and biofilm formation leading to the prevention of the pathogen adhesion (\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR55\\\" citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e). The strain AK05 showed auto-aggregation properties which rose from 19.03% to 95.65% during 5 to 24 h of incubation. The co-aggregation percentage shown by AK05 against pathogens, \\u003cem\\u003eS. aureus\\u003c/em\\u003e, and \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739 increased from 19.1\\u0026ndash;75.3% and 60- 82.2% respectively during 5 to 24 h of incubation. The cell surface hydrophobicity test showed that the CSH percentage of AK05 was more towards the non-polar substance, hexane, indicating better adherence to the gut epithelial cells. To survive in the gut environment of the host, the probiotic must be able to import and metabolize a range of carbohydrates. Carbohydrate utilization was tested by Hi-Carbo Kit, and it was found that all three strains were able to oxidize/ferment almost all carbohydrates used except citrate and malonate; making them desirable for being used as probiotics. Besides the carbohydrate-utilizing trait, all three strains were found to be non-hemolytic, non-gelatinolytic, and DNase-negative, and can be considered safe for consumption. Another important trait of a probiotic strain is its susceptibility to antibiotics. Among the three strains, AK05 was regarded as the most suitable probiotic candidate as per the antibiotic-susceptibility profile, being sensitive to all antibiotics tested except trimethoprim.\\u003c/p\\u003e\\u003cp\\u003eAK05 was identified to belong to the genus \\u003cem\\u003eLactococcus\\u003c/em\\u003e, inspite of high genomic similarities indicated by dDDH value of 79.9% and orthoANI value of 97.61%, the Jaccard similarity index showed only 21% of the phenotypic characteristics (Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e, S3). Genomic data confirms AK05 as a new strain in \\u003cem\\u003eLactococcus\\u003c/em\\u003e that is highly genetically similar to \\u003cem\\u003eL. lactis\\u003c/em\\u003e subsp. \\u003cem\\u003ehordniae\\u003c/em\\u003e yet phenotypically different. Having unique functional genes also supports its uniqueness.\\u003c/p\\u003e\\u003cp\\u003eGenomic assessment of the strain AK05 for probiotic traits corroborated with the phenotypic expression establishing it as a good probiotic candidate. Bacteriocin production by a probiotic is one of the most important antibacterial features to act against pathogens. The presence of bacteriocins, lactococcin 972, and bacteriocin immunity proteins-producing genes corroborated with the antimicrobial phenotype against the pathogen \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739. Detailed information regarding the location of genes related to bacteriocin and bacteriocin-related protein production is given in Table S4. Lactococcin 972 was reported to be produced by \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e subsp. \\u003cem\\u003elactis\\u003c/em\\u003e IPLA972 (\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e). It inhibits cell elongation, unable to form septa, resulting in cell death of closely related bacteria like \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e M1614 (\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e). It also inhibits cell wall biosynthesis, as it bears a lipid II binding motif that binds to lipid II essential for peptidoglycan synthesis of the target bacteria (\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e). As these properties impart antibacterial activity to the strain \\u003cem\\u003eLactococcus\\u003c/em\\u003e sp. AK05, the strain itself should possess a certain mechanism via which it gets protection against its bacteriocin. Bacteriocin-producing bacteria protect themselves by several mechanisms; the most important one is the production of bacteriocin immunity proteins. The genome of AK05 possessed genes encoding proteins providing immunity against bacteriocin. Genes coding for such immunity proteins are in the same cluster and often co-regulated (\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e). Another mechanism by which bacteria protects itself from its bacteriocin is when Lactococcin A binds with man-PTS (mannose phosphotransferase protein system) receptor proteins IIC and IID on the target bacteria. Concurrently, the immunity protein LciA (lactococcin immunity protein A) binds to this lactococcin A-receptor complex preventing the Lactococcin A from causing death of the producer cell by membrane permeabilization (\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e). Also, blockage of the pore formed by the bacteriocin can take place by the bacteriocin immunity protein, thus maintaining cellular integrity (\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e). Competitive binding of bacteriocin immunity protein and bacteriocin with the receptor of the target bacteria also might take place, thus, the immunity protein occupies the binding site for bacteria leading to the target cell\\u0026rsquo;s protection (\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e). Web servers, antiSMASH version 7.1.0, and BAGEL v4 revealed the presence of antimicrobial proteins like Ribosomally synthesized and post-translationally modified peptides (RiPP), and Ras-RiPP, Type III Polyketide synthases, betalactone, and Enterolysin A (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). RiPP and Ras-RiPP proteins belong to a group of antimicrobial peptides (AMP) and the post-translational modifications impart functional diversity to them (\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e). RiPPs and Ras-RiPPs secondary metabolites are found to be produced by certain genera of LAB-like, \\u003cem\\u003eLactobacillus\\u003c/em\\u003e, \\u003cem\\u003eStreptococcus\\u003c/em\\u003e, and \\u003cem\\u003eLactococcus\\u003c/em\\u003e having antibacterial properties (\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e). Also, Type III polyketide synthases (T3PKS) are a class of enzymes taking part in the biosynthesis of polyketides, which are a group of secondary metabolites. For example, T3PKS 2, 4- diacetylphloroglucinol synthesized by \\u003cem\\u003ePseudomonas\\u003c/em\\u003e has antimicrobial activity against soil-borne fungal plant pathogens (\\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e). T3PKS like ArsB and ArsC produced by \\u003cem\\u003eAzotobacter vinelandii\\u003c/em\\u003e inhibits the growth of certain microorganisms, and Germicidin derivatives control germination of spore produced by \\u003cem\\u003eStreptomyces\\u003c/em\\u003e sp., thus, managing their population (\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e68\\u003c/span\\u003e). Enterolysin A, class III heat-labile bacteriocin, disrupts cell membranes and compromises cellular integrity, leading to cell death; its regulated expression in \\u003cem\\u003eL. lactis\\u003c/em\\u003e has shown a bactericidal effect, highlighting its potential use as a preservative in food applications (\\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e69\\u003c/span\\u003e). Beta-lactones are natural products, produced by some bacterial genera such as \\u003cem\\u003ePseudomonas\\u003c/em\\u003e, \\u003cem\\u003eStreptomyces\\u003c/em\\u003e, \\u003cem\\u003eKitasatospora\\u003c/em\\u003e, etc. targeting various classes of enzymes such as hydrolases, transferases, ligases, and oxidoreductases have bioactivity against bacteria, fungi, or human cancer cell lines (\\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e70\\u003c/span\\u003e). Furthermore, beta-lactones are inhibitors of homoserine transacetylase (HTA) which can inactivate through an irreversible acylation of ser143, the transacetylase activity of homoserine in the enzyme\\u0026rsquo;s active site, thus, intervening in the biosynthesis of methionine and threonine essential for cellular functions in microorganisms (\\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e71\\u003c/span\\u003e) (Table S5). Acid tolerance phenotype of the strain \\u003cem\\u003eLactococcus\\u003c/em\\u003e sp. AK05 corroborates with the presence of a high copy number gene coding for amino acid permease, which is an integral membrane protein responsible for the absorption of extracellular amino acids into the cell for elevating the intracellular pH level. This is beneficial for the bacteria for survival in stressful environments like low nutrient availability, low pH environments, etc. Here, the amino acids are decarboxylated to produce amines and in this process, protons are consumed raising the pH level (\\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e72\\u003c/span\\u003e). The F0F1 ATP synthase helps maintain the pH homeostasis by extruding the protons, helping the bacteria survive in low acidic environments. The genome of AK05 revealed the presence of all the genes responsible for the synthesis of F0F1 ATP synthase subunits. The main role of F0F1 ATP synthase in acid tolerance has been reported in \\u003cem\\u003eListeria monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eLactobacillus casei\\u003c/em\\u003e, \\u003cem\\u003eStreptococcus mutans\\u003c/em\\u003e, \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e (\\u003cspan additionalcitationids=\\\"CR74 CR75 CR76\\\" citationid=\\\"CR73\\\" class=\\\"CitationRef\\\"\\u003e73\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR77\\\" class=\\\"CitationRef\\\"\\u003e77\\u003c/span\\u003e) (Table S6).\\u003c/p\\u003e\\u003cp\\u003eAs the bacteria were tolerant to high percentages of bile acids (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), this was substantiated by the presence of genes like \\u003cem\\u003elmrCD\\u003c/em\\u003e, which is induced by the presence of cholate and related bile acids in \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e. Proteins coded by \\u003cem\\u003elmrCD\\u003c/em\\u003e function as efflux pumps, removing bile acids from the cell. During the bile acid stress, the cholate is taken up as sodium salt, thus, there is upregulation of Na\\u003csup\\u003e+\\u003c/sup\\u003e/H\\u003csup\\u003e+\\u003c/sup\\u003e antiporter which counteracts Na\\u003csup\\u003e+\\u003c/sup\\u003e toxicity (\\u003cspan citationid=\\\"CR78\\\" class=\\\"CitationRef\\\"\\u003e78\\u003c/span\\u003e). Hence, the presence of several copy numbers of Na\\u003csup\\u003e+/\\u003c/sup\\u003eH\\u003csup\\u003e+\\u003c/sup\\u003e antiporter genes in the genome of AK05 proved that under salt stress environment these antiporters help in extruding excess Na\\u003csup\\u003e+\\u003c/sup\\u003e from the cell in exchange for H\\u003csup\\u003e+\\u003c/sup\\u003e, thus, maintaining osmotic balance inside the cell (\\u003cspan citationid=\\\"CR79\\\" class=\\\"CitationRef\\\"\\u003e79\\u003c/span\\u003e) (Table S7).\\u003c/p\\u003e\\u003cp\\u003eTo survive and proliferate the bacteria must be able to aggregate and adhere to the host\\u0026rsquo;s intestinal tract. The presence of several copy numbers of mucin binding proteins and cell wall binding proteins suggested (Table S8 and S9) that the bacteria have better potential to adhere to the mucosal layer found in \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e, \\u003cem\\u003eBifidobacterium bifidum, Lactobacillus reuteri\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR80\\\" class=\\\"CitationRef\\\"\\u003e80\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR81\\\" class=\\\"CitationRef\\\"\\u003e81\\u003c/span\\u003e) and aggregate together which might result in biofilm formation. The formation of biofilm further inhibits the adherence of pathogenic bacteria and protects from the harsh environment of the gut, also influenced by exopolysaccharide production, contributing to the stability and functionality of the gut environment (\\u003cspan citationid=\\\"CR81\\\" class=\\\"CitationRef\\\"\\u003e81\\u003c/span\\u003e). The mucin-binding protein further influences the immune response by acting as an epitope that interacts with the host immune cells by promoting broad-spectrum antibody reaction also facilitating retention in the host\\u0026rsquo;s intestinal layer (\\u003cspan additionalcitationids=\\\"CR83\\\" citationid=\\\"CR82\\\" class=\\\"CitationRef\\\"\\u003e82\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR84\\\" class=\\\"CitationRef\\\"\\u003e84\\u003c/span\\u003e). The gene \\u003cem\\u003esrtA\\u003c/em\\u003e encodes for SrtA, present in the cell membrane, anchors a typical motif LPxTG, by cleaving the protein at \\u0026lsquo;x\\u0026rsquo; position facilitating a covalent attachment of the protein to the peptidoglycan layer of the cell wall which helps in adhesion and aggregation function of the bacteria (\\u003cspan citationid=\\\"CR85\\\" class=\\\"CitationRef\\\"\\u003e85\\u003c/span\\u003e). Another kind of Sortase which is encoded by the gene \\u003cem\\u003esrtC\\u003c/em\\u003e, is important for polymerization of pili subunit which further facilitates autoaggregation and biofilm formation reported in \\u003cem\\u003eLactoccus lactis\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR86\\\" class=\\\"CitationRef\\\"\\u003e86\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eThe carbohydrate fermentation test (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e) showed that the strain AK05 could utilize a wide range of carbohydrates. This suggests that the strain consists of a diverse range of enzymes capable of breaking down various carbohydrates; this property makes the strain survive in different ecological niches with varied nutrient availability. DBCAN 3 web server was used to annotate the carbohydrate-active enzymes, showing the presence of 34 glycoside hydrolase (GHs) family enzymes, 11 glycoside transferase (GTs) enzymes, five carbohydrate-binding modules (CBMs), four carbohydrate esterases (CE), and seven multidomains (Table S10). The presence of glycoside hydrolase family proteins and carbohydrate esterases suggests that the complex carbohydrates are acted upon by these enzymes and broken down into simple carbohydrates enabling \\u003cem\\u003eL. lactis\\u003c/em\\u003e to utilize substrates for growth (\\u003cspan citationid=\\\"CR87\\\" class=\\\"CitationRef\\\"\\u003e87\\u003c/span\\u003e). GH2, GH13, and GH32 present in \\u003cem\\u003eLactobacillus paracasei\\u003c/em\\u003e were responsible for the breakdown of oligosaccharides (\\u003cspan citationid=\\\"CR88\\\" class=\\\"CitationRef\\\"\\u003e88\\u003c/span\\u003e). Thus, it can be said that the strain AK05 has the potential to degrade various types of carbohydrates in dietary fibers, which when degraded results in the formation of short-chain fatty acids (SCFAs) like butyrate, propionate, and acetate which have anti-inflammatory activity improving the gut barrier function and immune response by preventing against infectious diseases (\\u003cspan citationid=\\\"CR89\\\" class=\\\"CitationRef\\\"\\u003e89\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eThe STRING (v12.0) database was used to elucidate the complex network of protein-protein interactions related to genes responsible for the functional properties of a probiotic, thus, interactions were performed using key proteins (Table S2). A vital property of a probiotic is to survive in a gastric acidic environment. Thus, the phenotypic study showed that the strain AK05 can survive at pH 4 up to 24 h, which corroborated with the genotypic study showing various genes that are responsible for producing proteins imparting the property of acid tolerance to the strain (Table S6). Thus, the literature study brought out the key proteins, EnoA, EcfA2, MsmK, ZitP, ZitQ, and EC6.4.1.1, responsible for stress tolerance (Table S2). EnoA is a key enzyme for energy production involved in the glycolytic pathway, the \\u003cem\\u003eecfA2\\u003c/em\\u003e gene encodes an energy coupling factor transporter ATPase (EcfA2), and overexpression of this gene significantly enhances acid tolerance in \\u003cem\\u003eL. lactis\\u003c/em\\u003e. Similarly, overexpression of ZitP and ZitQ, metal ABC transporters proteins have been found to impart improved acid tolerance to \\u003cem\\u003eL. lactis\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR90\\\" class=\\\"CitationRef\\\"\\u003e90\\u003c/span\\u003e). The overexpression of Msmk protein under acid stress leads to higher ATP concentration. Providing the cells with the necessary energy to combat the acid stress condition, Msmk is also involved in the utilization of carbohydrates, which is essential for microbial cells to adjust their metabolism in maintaining optimum cellular functions during stress; also, this protein helps in up-regulation of genes like \\u003cem\\u003ecoaD\\u003c/em\\u003e and \\u003cem\\u003earg\\u003c/em\\u003e involved in energy generation pathways associated with ATP production (\\u003cspan citationid=\\\"CR91\\\" class=\\\"CitationRef\\\"\\u003e91\\u003c/span\\u003e). EC6.4.1.1 (pyruvate carboxylase) catalyzes the conversion of pyruvate to oxaloacetate (OAA), which is an essential step in gluconeogenesis and lipogenesis. During stress conditions, upregulation of pyruvate carboxylase enhances the metabolic flux towards the production of OAA which is further utilized in the production of amino acids, nucleotides, etc.\\u003c/p\\u003e\\u003cp\\u003eA separate yellow cluster was formed involving proteins like LmrA, LmrC, and LmrD which are related to bile acid tolerance. It was also found that the strain AK05 could tolerate and grow in a high percentage of bile acid (0.8%). The presence of ABC-Type multidrug resistance transporter, LmrCD, plays a crucial role in extruding bile acids from the cell functioning as a pump, thereby, avoiding the accumulation of bile acids intracellularly and increasing its survivability in bile acid-rich environments (\\u003cspan citationid=\\\"CR78\\\" class=\\\"CitationRef\\\"\\u003e78\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eAnother important trait of being a high-quality probiotic is adherence and aggregation properties; the phenotypic tests revealed that AK05 has a 95.65% autoaggregation percentage and co-aggregation percentages of 74.1% and 82.32%, against \\u003cem\\u003eS. aureus\\u003c/em\\u003e and \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739. The analysis of the genome showed the presence of genes responsible for these properties, \\u003cem\\u003esrtA\\u003c/em\\u003e and \\u003cem\\u003esrtC\\u003c/em\\u003e (coding for sortase), \\u003cem\\u003ellmg_2465\\u003c/em\\u003e (coding for YmcF), \\u003cem\\u003ellmg_1152\\u003c/em\\u003e (coding for YwcG) (Table S2), formed a separate green cluster. Sortases A and C, both play crucial roles in adhesion purposes, here, proteins with an LPXTG- motif are predominantly anchored by SrtA to the cell wall, whereas SrtC is involved in biofilm formation enhancing adherence to the host tissues (\\u003cspan citationid=\\\"CR85\\\" class=\\\"CitationRef\\\"\\u003e85\\u003c/span\\u003e). Both llmg_1152 and llmg_2465 proteins mediate adhesion to the mucosal surfaces, thus, helping in colonization in the gastrointestinal tract due to the presence of mucus-binding domain and LPXTG motifs (\\u003cspan citationid=\\\"CR84\\\" class=\\\"CitationRef\\\"\\u003e84\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eOne of the most important aspects of a probiotic is to possess the property of antimicrobial activity, apart from the phenotypic test, the genome mining also showed some important proteins responsible for imparting antimicrobial properties. The protein-protein interaction was performed in the STRING database, where the blue-colored cluster (ps102 and ps103) was obtained along with separate units in white, including ps501, lysP, ps461 (phage lysin). This blue-colored cluster and the white units involved proteins related to antimicrobial properties. LysL and LysP are phage lysins that cause the cell wall lysis of other closely related bacteria and are highly specific due to the presence of a cell wall binding domain (CBD) exhibiting a narrow range (\\u003cspan citationid=\\\"CR92\\\" class=\\\"CitationRef\\\"\\u003e92\\u003c/span\\u003e) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig12\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003cem\\u003eLactococcus\\u003c/em\\u003e sp. AK05 has arisen to be a potent probiotic strain, for use in aquaculture and food industries. AK05 exhibited antimicrobial activity against the pathogenic strain \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739 which is known to cause considerable economic losses in freshwater aquaculture. Various characteristics like the ability to tolerate low pH, and elevated salt and bile acid concentrations along with the properties like cell surface hydrophobicity, auto-aggregation, and co-aggregation with harmful bacteria make it well suited to thrive and colonize within the harsh environment of the gastrointestinal tract and provide a beneficial impact on the host. Also, genomic analysis has confirmed the presence of genes associated with the above-mentioned functions. To support a healthy gut microbiome and to maintain stability in the host\\u0026rsquo;s intestinal environment, these kinds of traits of probiotics are very crucial. AK05 was also able to produce bacteriocin (\\u003cem\\u003eviz\\u003c/em\\u003e. lactococcin 972) that effectively inhibits pathogenic bacterial growth. The genomic profile also revealed the presence of genes encoding bacteriocin immunity proteins that safeguard AK05 from its antimicrobial compounds. Antibiotic resistance pattern showed AK05\\u0026rsquo;s resistance towards trimethoprim only, while it was susceptible to all the rest of the broad-spectrum antibiotics. These genomic and phenomic features reinforced the claim that AK05 is an effective probiotic strain that can enhance the gut health of aquatic species and can be used as a viable, natural solution for pathogen control.\\u003c/p\\u003e\"},{\"header\":\"Statements and Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eConflict of Interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThere are no academic or financial conflicts of interest for the authors regarding the publication of this work.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData is available in the NCBI Database under the \\u003c/strong\\u003eGenBank Accession id\\u003cstrong\\u003e: \\u003c/strong\\u003eJBFTEI000000000; BioSample Accession id: SAMN42887626; BioProject Accession id: PRJNA1141375; SRA Accession id: SRR30829574\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAuthors, Anasuya Karjee and Soumya Chatterjee acknowledge the Council of Scientific and Industrial Research (CSIR- NEW DELHI, GOVT. OF INDIA) as they received research grant in the form of Senior Research Fellowship during this work (CSIR-SRF; AWARD NO.- 09/285(0093)/2019-EMR-I, 09/1151(0006)/2019-EMR-I respectively).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors’ contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization: Ranadhir Chakraborty; Methodology: Anasuya Karjee; Formal analysis and investigation: Anasuya Karjee, Soumya Chatterjee, and Ranadhir Chakraborty; Writing - original draft preparation: Anasuya Karjee; Writing - review and editing: Ranadhir Chakraborty, Anasuya Karjee, and Soumya Chatterjee; Funding acquisition: Anasuya Karjee; Resources: Ranadhir Chakraborty; Supervision: Ranadhir Chakraborty.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eParker RB (1974) Probiotics, the other half of the antibiotics story. 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Springer International Publishing, Cham, pp 1\\u0026ndash;14\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMart\\u0026iacute;nez Cruz P, Ib\\u0026aacute;\\u0026ntilde;ez AL, Monroy Hermosillo OA, Ram\\u0026iacute;rez Saad HC (2012) Use of Probiotics in Aquaculture. ISRN Microbiol 2012:1\\u0026ndash;13\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLee S, Katya K, Park Y, Won S, Seong M, hamidoghli A et al (2017) Comparative evaluation of dietary probiotics Bacillus subtilis WB60 and Lactobacillus plantarum KCTC3928 on the growth performance, immunological parameters, gut morphology and disease resistance in Japanese eel, Anguilla japonica. 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Front Microbiol. ;14\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"world-journal-of-microbiology-and-biotechnology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"wibi\",\"sideBox\":\"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)\",\"snPcode\":\"11274\",\"submissionUrl\":\"https://submission.nature.com/new-submission/11274/3\",\"title\":\"World Journal of Microbiology and Biotechnology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Lactococcus sp. AK05, genome analysis, probiotic traits, bacteriocin genes, fish gut microbiome, aquaculture\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7873003/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7873003/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eResistance in pathogens has increased as a result of the extensive usage of antibiotics in aquaculture. This emphasizes how urgently safe and efficient probiotic substitutes are needed. In our study, we isolated and characterized a new strain of \\u003cem\\u003eLactococcus\\u003c/em\\u003e sp., called AK05, from the gut of \\u003cem\\u003eCirrhinus mrigala\\u003c/em\\u003e. The phenotypic tests showed that it can survive harsh conditions, including a pH of 4, 0.8% bile salts, and even up to 8% NaCl. It also showed strong autoaggregation at 95.65%, co-aggregation with \\u003cem\\u003eAeromonas hydrophila\\u003c/em\\u003e subsp. \\u003cem\\u003ehydrophila\\u003c/em\\u003e MTCC 1739 at 82.2%, and antimicrobial activity, with a 15.66 mm inhibition zone. A safety evaluation revealed γ-hemolysis, no gelatinase or DNase activity, and vulnerability to the majority of antibiotics, with the exception of trimethoprim. With 97.61% ANI and 78.9% dDDH, whole-genome sequencing showed that AK05 and \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e subsp. \\u003cem\\u003ehordniae\\u003c/em\\u003e are closely related, yet only have 21% phenotypic similarity. This supports its classification as a unique strain. Our genomic analysis found genes that produce bacteriocins, like lactococcin, as well as genes that provide acid and bile tolerance, adhesion, and carbohydrate use. These traits, along with its ability to contain \\u003cem\\u003eA. hydrophila\\u003c/em\\u003e, make AK05 a promising option for managing diseases without antibiotics and improving gut health in aquaculture.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Genomic and Functional Insights into a Novel Lactococcus sp. 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