Antagonism against fish pathogens by cellular components and verification of probiotic properties in autochthonous bacteria isolated from the gut of an Indian major carp,Catla catla (Hamilton)

Probiotic potential of the autochthonous bacteria in catla, Catla catla has been evaluated through determination of antagonistic activity (in vitro) of the cellular components of gut bacteria against seven fish pathogens. Altogether 208 strains were isolated, inhibitory activity of the isolates was evaluated through cross‐streaking and 16 primarily selected antagonistic strains were confirmed using the double‐layer method. Four bacteria that showed antagonism against ≥4 pathogens were selected as putative probiotics. The intracellular, extracellular, whole‐cell and heat‐killed cell components exhibited bactericidal activity against the pathogens. In addition, the selected strains were capable of producing different extracellular enzymes, competent to grow in intestinal mucus and could tolerate diluted bile juice. Analysis of 16SrRNA partial gene sequence revealed that both the strains CC1FG2 and CC1FG4 were Bacillus methylotrophicus (KF559344 and KF559345), while the isolates CC1HG5 and CC2HG7 were Bacillus subtilis subsp. spizizenii (KF559346) and Enterobacter hormaechei (KF559347) respectively. Bio‐safety evaluation through intra‐peritoneal injection of the isolates did not induce any pathological signs or mortalities in C. catla. The study confirmed probiotic properties of autochthonous gut bacteria in C. catla and demonstrated potential for using them as bio‐control agents. However, in vivo studies are essential to explore their efficacy in the commercial aquaculture.     

Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel (Anguilla japonica) for its use in aquaculture

In the present study, a potential Lactobacilli probiotics were isolated from Japanese eels (Anguilla japonica) and characterized and evaluated for their possible use in eel farming. Sixteen Lactobacilli were isolated from intestines of Japanese eels, using selective media. The lactobacilli strains (represented as PL1 to PL16) were screened by their ability to produce digestive enzyme. Among these, three strains (PL11, PL13 and PL16) producing four digestive enzymes (amylase, cellulase, protease and phytase) simultaneously were characterized further using API ZYM kit. From these, PL11 (Lactobacillu (L.) pentosus) was identified as potential probiotics candidate producing 15 enzymes among 20 tested. Further examination of biological activities of PL11 revealed tolerance against pH, artificial bile juice and antibacterial activity against several fish pathogenic bacteria. The in vitro competitive exclusion assay also revealed 88.4% reduction in adhesion of fish pathogen (Edwardsiella tarda) by PL11 to host intestinal mucus. In vitro incubation of Japanese eel foregut with Baclight‐labelled PL11 showed colonization of the enterocyte surface by confocal and scanning electron microscopy. In summary, PL11 isolated from eels could serve as a potential probiotics with acid and bile tolerance, production of digestive enzymes, antibacterial activity and inhibition of fish pathogen adhesion to intestinal mucus.     

In vitro oocyte maturation and blood‐circulating steroid hormones in Sterlet (Acipenser ruthenus) and Siberian sturgeon (Acipenser baeri)

The oocyte of Sterlet (Acipenser ruthenus) and Siberian sturgeon (Acipenser baeri) was evaluated after incubation with 17α, 20β‐Dihydroxyprogesterone (DHP) in artificial media. Concentration of testosterone (T), progesterone (P4) and 17‐β estradiol (E2) in blood and their relation to fertilization rate were measured during experimental period. The oocytes at the same developmental stages were incubated in SIS (based on sturgeon blood ionic composition), RM2 (Ringer solution modified for sturgeons) and L‐15 (Leibovitz medium) artificial mediums in the presence of 1 μg/ml of 17α, 20β‐Dihydroxyprogesterone for 12, 18 and 24 hr. The result of this study demonstrated that fertilization rate decreased with the increasing duration of incubation in all mediums. In the Siberian and Sterlet sturgeon, the highest fertilization rate was observed in the oocytes incubated in RM2 (0.81 ± 0.4) and SIS (0.44 ± 0.8) mediums for 12 hr respectively. In the Siberian sturgeon, fertilization rate decreased significantly (p < .05) with incubation time in all three mediums. In Starlet, fertilization rate decreased significantly (p < .05) with time and reached a minimum after 24 hr (0.16 ± 0.1) in L‐15 medium. No significant (p > .05) differences were observed between blood plasma hormones and fertilization rate. The results of this study indicated that 17α, 20β‐Dihydroxyprogesterone is essential for induction of in vitro oocyte maturation in these species.     

Quantitative dietary copper requirement of juvenile Siberian sturgeon,Acipenser baerii,and effects on muscle composition and some enzymatic activities

An 8‐week feeding trial was conducted to quantify dietary copper (Cu) requirement of juvenile Siberian sturgeon, Acipenser baerii. Five isonitrogenous diets were formulated to provide actual dietary copper values of 1.8, 5.7, 10.1, 15.9 and 28.3 mg Cu per kg diet. Experimental diets were fed to the Siberian sturgeon (27.57 ± 0.24 g) in triplicate to apparent satiation for 8 weeks. At the end of experiment, weight gain (WG), specific growth rate (SGR) and protein efficiency ratio (PER) were significantly increased with increasing dietary Cu level up to 10.1 mg/kg and then decreased with further increases in dietary Cu level (p < .05). The Cu concentration in the liver and cartilage was positively correlated with the respective concentrations in the diet (p < .05), while muscle and serum Cu concentrations remained significantly unchanged (p > .05). Superoxide dismutase and glutathione peroxidase had the highest activities in serum of fish fed with 15.9 and 28.3 mg Cu per kg diet, respectively. Analysis by the broken‐line regression of SGR, crude protein content and superoxide activity demonstrated that the optimum dietary Cu requirements in juvenile Siberian sturgeon were 9.51, 9.58 and 16.10 mg/kg diet, respectively.     

Evaluating the probiotic potential and adhesion characteristics of Bacillus spp. isolated from the intestine of Rhynchocypris lagowskii Dybowski

Probiotics have been widely used in aquaculture worldwide especially in China as antibiotics have been banned in aquaculture. In the present study, 27 potential strains were isolated from Rhynchocypris lagowskii Dybowski intestinal tract towards the assessment of their probiotic potential. Two potential probiotics were finally screened from the 27 candidate strains according to the results of enzyme-producing ability, in vitro against pathogens ability and antibiotic sensitivity test. The 2 potential probiotics were identified and confirmed on the basis of their colony morphology, biochemical characteristics, and 16S rRNA sequencing. The probiotic strains, LSG2-3–2 and LSG3-6, were identified to be Bacillus methylotrophicus and B. tequilensis, respectively. Further studies showed that LSG2-3–2 and LSG3-6 had excellent tolerance to high temperature (80℃), low pH (3–5), bile salts (0.3%), intestinal juice (10%), and gastric juice (0.5%). The adhesion rates of LSG2-3–2 and LSG3-6 in the in vitro intestinal mucosal adhesion model were 17.74% and 24.04%, respectively. Analysis of their bacteria surface adhesive proteins revealed that the lectins on the bacterial surface of LSG2-3–2 and LSG3-6 were mainly protein and glycoprotein properties, respectively. The adhesion receptor components in the mucus proteins of the two strains were all protein properties. The results of the inhibitory adhesion test indicated that LSG3-6 had a higher inhibitory effect on Aeromonas hydrophila and LSG2-3–2 had a better inhibitory effect on A. veronii. The biosafety assay confirmed that the isolates were not pathogenic to the host fish. Based on the presently observed characteristic features, it can be concluded that LSG2-3–2 (B. methylotrophicus) and LSG3-6 (B. tequilensis) retrieved from the digestive tract of R. lagowskii can be used for the healthy breeding and disease prevention of R. lagowskii.

     

Revealing the compositions of the intestinal microbiota of three Anguillid eel species using 16S rDNA sequencing

Anguilla japonica, A. marmorata and A. bicolor pacifica are important eel species for aquaculture in Taiwan. In the past, seldom studies focused on the intestinal microbiome of them or the probiotics used in their cultivation. Probiotics are microbes that can improve the health of organisms, and the microbes isolated from the intestines of organisms might be the potential probiotics. Therefore, the aim of this study was to collect the intestinal mucus samples of these three eel species for investigating the autochthonous microbiome using 16S rDNA sequencing. The diversity, abundance of the intestinal microbiome and compositions of all the libraries were identified. Moreover, the composition of intestinal microbiome of eel was suggested that it would be affected by the waters and the characteristics of different eel species. Furthermore, most of the identified predominant genera of the intestinal microbiome of these three eel species, such as Cetobacterium, Clostridium, Shewanella, Acinetobacter and Bacteroides, seemed to have great potential as the probiotics. In the future, more studies shall be conducted to promote the practical application of these candidate probiotics for improving the techniques of eel farming.     

Effects of dietary zinc level on performance,zinc status,tissue composition and enzyme activities of juvenile Siberian sturgeon,Acipenser baerii (Brandt 1869)

This study conducted to evaluate the effect of dietary zinc (Zn) levels on feed utilization, tissue Zn composition and serum enzyme activities of juvenile Siberian sturgeon, Acipenser baerii. Five isoenergetic and isonitrogenous semi‐purified diets were formulated with increasing Zn sulphate (ZnSO₄.5H₂O) level to provide the actual Zn values of 14.7 (control), 20.8, 27.3, 37.7 and 46.4 mg/kg diet. Each diet was assigned to three groups of 20 experimental Siberian sturgeons with uniform size (initial weight of 26.52 ± 0.94 g) for a period of 8‐week feeding trial. Results showed that growth performance and muscle protein content were significantly increased with increasing dietary Zn level up to 27.3 mg/kg (p < .05), beyond which they remained significantly unchanged (p > .05). Muscle lipid content significantly declined with increasing dietary Zn level. While muscle and serum Zn contents were not significantly changed among treatments (p > .05), liver Zn content tended to rise with increasing dietary Zn supplementation. Alkaline phosphatase, superoxide dismutase and glutathione peroxidase activities were also raised with increasing dietary Zn level. The adequate amount of dietary Zn requirements for the Siberian sturgeon was estimated to be 28.24 mg/kg based on the relative growth rate and 34.60 mg/kg based on the liver Zn content.     

Isolation and characterization of an atypical Siberian sturgeon herpesvirus strain in Russia: novel North American Acipenserid herpesvirus 2 strain in Europe?

Siberian sturgeon herpesvirus (SbSHV) was isolated in Russia for the first time in 2006. Nine SbSHV isolates were recovered from different fish hatcheries producing the same cytopathic effect in cell cultures, the same clinical signs and mortality kinetics in virus‐infected fish and the same virus neutralization pattern and shared identical nucleotide sequences. In 2011, a new isolate was recovered from juvenile sturgeon, which caused completely different cytopathic effect. That isolate was not readily neutralized by Siberian sturgeon hyperimmune antisera, and its DNA was not recognized by the routine PCR developed for SbSHV detection. Molecular study of the novel isolate revealed that it was more closely related to North American Acipenserid herpesvirus 2 (AciHV‐2) isolates from white sturgeon, while the genome sequences of the former SbSHV isolates showed high similarity to the AciHV‐2 isolated from shortnose sturgeon. While clinical signs and mortality caused by the novel isolate in infected Siberian sturgeon were similar to those of the formerly described SbSHV isolates, the incubation period and mean time to death produced by the novel isolate were twice as long. The differences between the former isolates and the recent one suggest that a novel SbSHV strain emerged in Europe and the molecular findings imply its North American origin.     

Bacterial Change in the Gut and Hepatopancreas of Tiger Shrimp Reared in Bioaugmented System

The study was conducted in nine 150‐L capacity glass aquaria for 192 h to determine the changes in the microflora in the gut and hepatopancreas of tiger shrimp, Penaeus monodon, after bioaugmenting (water treatment and probiotics). Results showed that luminous bacterial counts were significantly lower in bioaugmented than in nonbioaugmented systems (range 0–5.9 × 10³ cfu/g and 0–3.2 × 10³ cfu/g in gut and hepatopancreas, respectively). Biochemical tests of isolates showed that the bioaugmented systems were dominated by Gram‐positive Bacillus and Streptococcus and the Gram‐negative Vibrio dominated the control. In water‐treated and probiotics‐fed bioaugmented system, the change in bacterial dominance to Bacillus became evident 2 h in the gut and 24 h in the hepatopancreas. Bacterial dominance shifted to Vibrio species after 120 h. Shrimp in the control were observed to become lethargic after 72 h as Vibrio species significantly increased in number. Dissolved oxygen and unionized ammonia levels were significantly lower in bioaugmented system compared with the control.     

Inhibition of Vibrio spp. by 2‐Hydroxyethyl‐11‐hydroxyhexadec‐9‐enoate of Marine Cyanobacterium Leptolyngbya sp. LT19

Seven marine cyanobacteria were isolated from two regions of the Gulf of Thailand and evaluated by the agar diffusion method for antibacterial activity. Inhibitory compound was purified from the crude methanol extract and its structure was elucidated based on extensive spectroscopic analysis, including IR, 1D and 2D NMR spectra as well as mass spectrometry. A novel antimicrobial compound produced by the marine cyanobacterium Leptolyngbya sp. LT19 was identified to be a 2‐hydroxyethyl‐11‐hydroxyhexadec‐9‐enoate which has so far never been reported in microorganisms. Biological assays revealed that this novel compound exhibited antibacterial activities against the Gram‐negative, persistent shrimp pathogens, Vibrio harveyi and V. parahaemolyticus with minimal inhibitory concentration of 250–1000 and 350–1000 μg mL^?1 respectively.     

Mixed mycobacterial infections in farmed sturgeons

Based on microbiological and histopathological examinations and DNA sequencing, several outbreaks of mycobacteriosis in the reared sturgeons, including Chinese sturgeon (Acipenser sinensis Gray) and Amur sturgeon (Acipenser schrencki), were identified during 2009 to 2010. Forty‐nine isolates of non‐tuberculous mycobacteria(NTM)were isolated from 19 diseased sturgeons. In total, seven species of Mycobacterium were identified, namely, Mycobacterium chelonae, Mycobacterium marinum, Mycobacterium gordonae, Mycobacterium fortuitum, Mycobacterium szulgai, Mycobacterium arupense and Mycobacterium porcinum. Among them, M. marinum was found to be more prevalent (89.5%) compared with the other mycobacterial species. When two molecular biological methods, PCR‐DGGE (denaturing gradient gel electrophoresis) analysis and rpoB gene library sequencing, were used to analyse the mycobacterial DNAs extracted from the diseased fish tissues, mixed infections of two or three mycobacterial species were found being the predominant infection form (94.7%) in sturgeon mycobacteriosis. M. marinum was the only one species that caused sturgeon mycobacteriosis alone. Virulence assay showed that M. marinum possessed stronger pathogenicity to zebrafish killing 100% of fish in 28 days at 10³ cfu/fish than the other species. These results suggested that M. marinum is the major pathogenic bacteria in sturgeon mycobacteriosis. To the best of our knowledge, this study is the first report on mycobacteriosis in farmed Chinese and Amur sturgeons as well as the first isolation of M. porcinum and M. arupense from fish.     

Improvement of growth performance,water quality and disease resistance against Vibrio harveyi of postlarval whiteleg shrimp (Litopenaeus vannamei) by administration of mixed microencapsulated Bacillus probiotics

The objective of this study was to investigate the effects of mixed Bacillus on growth, water quality and disease resistance against Vibrio harveyi in whiteleg shrimp (Litopenaeus vannamei). Postlarval shrimp (PL₃₀) were fed with (a) a basal diet (the control), (b) a diet containing mixed freeze‐dried Bacillus probiotics (FB) and (c) addition of mixed microencapsulated Bacillus probiotics (MB) in culture water. Addition of FB and MB probiotics improved (p < .05) growth, feed efficiency, survival and culture water quality (ammonia and nitrite) compared to the control group although there was no difference (p > .05) between the two treated groups. Bacillus numbers in gastrointestinal tracts and culture water of FB‐ and MB‐administrated shrimp were higher (p < .05) than in the control. After a 30‐day culture, shrimp were infected with V. harveyi and monitored for 10 days. A significant reduction (p < .05) in cumulative mortality was observed in FB‐ and MB‐supplemented shrimp (43.24% and 45.05%, respectively), compared to the control (63.06%). This finding demonstrated that administration of microencapsulated probiotics was as effective as freeze‐dried probiotics for improving growth, feed efficiency, survival, Bacillus in gastrointestinal tracts, water quality (ammonia and nitrite) and conferring disease resistance to V. harveyi.     

Adhesion to sole, Solea senegalensis Kaup, mucus of microorganisms isolated from farmed fish, and their interaction with Photobacterium damselae subsp. piscicida

Abstract Most studies carried out to select microorganisms as candidate probiotics have focused on in vitro antagonism tests, such as the production of inhibitory compounds against pathogenic microorganisms. However, attachment to mucous surfaces could be another criterion to be considered when selecting potential probiotics for aquaculture. Nineteen isolates obtained from farmed Senegalese sole, Solea senegalensis Kaup, and gilthead sea bream, Sparus aurata L., have been evaluated for their capacity to adhere to skin and intestinal mucus of Senegalese sole, and their antagonistic effect against Photobacterium damselae subsp. piscicida, an important pathogen for farmed sole. The isolates from gilthead sea bream showed the highest percentage of adhesion to sole mucus, whilst the pathogenic microorganisms assayed and the isolates from sole showed, in general, a lower ability to adhere to sole mucus. The results suggest that the adhesion to fish mucus was more dependent on the isolate tested than on the host mucus. The isolates from gilthead sea bream also showed a higher antagonistic activity against P. damselae subsp. piscicida than those from Senegalese sole. Four isolates were selected, on the basis of their adhesive ability and antagonistic effect on P. damselae subsp. piscicida, to study their interactions with the pathogen in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions. The results obtained show the ability of three isolates to reduce the adhesion of P. damselae subsp. piscicida to sole mucus under displacement and competition conditions. The adhesion of the pathogen to sole intestinal mucus was also significantly reduced when three isolates were assayed under exclusion conditions.     

Selection of lactic acid bacteria as candidate probiotics for Vibrio parahaemolyticus depuration in pacific oysters (Crassostrea gigas)

The application of probiotics in food is now widespread and is widely accepted by consumers. Lactic Acid Bacteria (LAB) were isolated from traditional salted fish and then characterized by its ability to inhibit Vibrio parahaemolyticus growth using minimal inhibitory concentration tests. Five out of these strains were identified as Lactobacillus delbrueckii, Lactobacillus casei and three Lactobacillus gasseri by PCR using 16S and 23S rRNA genes. Antibiofilm activity of Lactobacillus spp. extracts were also tested in 96 polystyrene plates. A potential antibiofilm effect was demonstrated as most LAB. Although most LAB extract were able to eradicate pre‐formed biofilm, results demonstrated that five Lactobacillus spp. exhibited a stronger inhibitory effect against V. parahaemolyticus in infected oysters. Vibrio parahaemolyticus viable cells number declined from 10⁶ UFC to 10⁴ UFC after 3 days of incubation with Lactobacillus spp. Probiotic applications, in biological control of seafood associated pathogens can be an alternative solution, providing consumer with a product of good quality owing to the use of non‐toxic compounds. Based on our results, LAB could be used as a bioprotective culture in oyster's depuration to prevent V. parahaemolyticus growth.     

Isolation and evaluation of putative probiotic strains from different teleost to prevent Pseudomonas aeruginosa infection in Cyprinus carpio

Probiotics renowned as valuable microbes serve as a potential alternative to control diseases in aquaculture and are considered as an efficient and environment‐friendly approach to reduce the use of antibiotics. The present study aims at the isolation of putative probiotic bacteria from the intestinal tract of different fish species from the Doaba region of Punjab, India. In this study, isolated bacterial strains were characterized based on their morphological, biochemical and molecular characterization by 16S rRNA gene sequencing, followed by in vitro evaluation of different selection parameters described in FAO/WHO guidelines. A total of 169 different bacterial strains were isolated from the gastrointestinal tract of 52 different fish species. After in vitro evaluation, out of 169 bacterial strains only five bacteria (S3, S7, BDK2', BDK7 and BDK9) identified as Enterococcus and Bacillus species showed antagonistic activity against the fish pathogen Pseudomonas aeruginosa (MTCC 4 673). These isolates were screened based on their response towards bile tolerance, pH tolerance, adhesion and drug susceptibility to different antibiotic discs. And, the in vivo results indicated improved growth and survival against the infection (P. aeruginosa) after oral administration of the probiotics. The observations of in vitro and in vivo evaluation indicate that these isolated probiotic strains serve as effective probiotics and can be used as a novel and safe treatment to cure current issues in aquaculture.     

Pathogenic threats and probiotic use in larviculture of the scallop,Pecten maximus

During a series of experiments, bacteriological elements in scallop larval rearing were investigated: larvae susceptibility to pathogens as a function of their age, and the use of probiotic bacteria during larviculture. Younger larvae (d5 PF) were highly more susceptible to pathogenic‐challenge than their older siblings, which were challenged at an older age (d15 PF). A challenge with 10⁴ CFU mL^?1 of V. pectenicida killed 100% of d5 PF larvae 7 days following challenge, yet killed only 9% of d15 PF larvae 9 days following challenge. Use of the probiotics Phaeobacter gallaeciensis, Alteromonas macleodii 0444 and Neptunomonas sp. 0536, provided for larger larvae, a high yield of competent larvae and, perhaps more importantly, protection against pathogen‐challenge similar to levels achieved from antibiotic use. When challenged with V. pectenicida, d29 survivals were 20.3%, 85.1% and 75.0% respectively for control (no probiotic), antibiotic treated, and ‘probiotic mix’ administered larvae. Use of potential probiotic Pseudoalteromonas sp. D41 appeared to hinder scallop larvae. Future use of probiotics in scallop larval rearing would benefit from combined use of P. gallaeciensis, A. macleodii 0444 and Neptunomonas sp. 0536.     

PCR‐DGGE analysis of the autochthonous gut microbiota of grouper Epinephelus coioides following probiotic Bacillus clausii administration

The autochthonous microbiota in the foregut, midgut and hindgut of juvenile grouper Epinephelus coioides following the dietary administration of probiotic Bacillus clausii for 60 days were assessed using polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). A complex and generally similar bacterial composition along the digestive tract of E. coioides was detected in the DGGE profiles, while several bacteria showed regional specialization. Similarity dendrogram revealed that the bacterial composition of the foregut was more similar to the midgut than the hindgut. Samples collected from the probiotic group and the control group showed generally similar DGGE patterns, while no significant difference in the total number of bands and Shannon index were observed between the probiotic group and the control group, suggested that probiotic B. clausii exerted no significant effect on the gut microbiota of E. coioides. However, various potentially beneficial bacteria, such as Enterococcus sp.‐like and Bacillus pumilus‐like bacterium were selectively stimulated by probiotic B. clausii, while some potential harmful species, like Staphylococcus sp.‐like and Vibrio ponticus‐like bacterium were depressed. These indicated that the gut microbiota was modified to some degree by probiotic B. clausii. Sequences analysis showed that the autochthonous gut bacteria of E. coioides could be classified into four groups, i.e. Proteobacteria, Firmicutes, Actinobacteria and unclassified bacteria.     

Luminal uptake of Vibrio (Listonella) anguillarum by shed enterocytes – a novel early defence strategy in larval fish

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A‐gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod‐like formations. The engulfed structures were positive for protein A‐colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread‐like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.     

Integrated application of two different screening strategies to select potential probiotics from the gut of channel catfish Ictalurus punctatus

In this study, 342 bacterial strains were isolated from the gut of channel catfish Ictalurus punctatus. Two different screening strategies, including the production of antagonistic effects against pathogens and the capacity of secreting exoenzyme, were used to select potential probiotics from the bacterial strains. Among them, 6 isolates showed antagonism against the pathogens, 15 isolates could produce high amylase and protease, and 3 isolates (BHI344, BH0142 and BH03124) had both the two characteristics . Then, BHI344, BH0142, and BH03124 were tested for survival rate in channel catfish bile and at low pH, antimicrobial susceptibilities, and pathogenicity in channel catfish. The results of the obtained assays showed that BHI344 possessed the broadest inhibitory potential, a strong ability of secreting extra-cellular amylase and protease, high viability in simulated gastrointestinal conditions, and sensitivity to most of the used antibiotics and non-pathogenic to channel catfish. Therefore, it could be selected as the most promising probiotics candidate. BHI344 was identified as Bacillus subtilis based on the phenotypic characterization and 16S rRNA gene sequencing.