Int. J. Aquat. Biol. (2019) 7(3): 166-174 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2019 Iranian Society of Ichthyology Original Article Study on probiotic potential of Bacillus species isolated from the intestine of farmed rainbow trout, Oncorhynchus mykiss Mina Seifzadeh, Mohammad Rabbani Khorasgani *,1Rasoul Shafiei Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran. s Article history: Received 22 April 2019 Accepted 25 June 2019 Available online 2 5 June 2019 Keywords: Bacillus Farmed rainbow trout Gastrointestinal tract Probiotic Abstract: The gastrointestinal tract of fishes is a complex ecosystem occupied by a large number of microorganisms, some of them could have potentially-valuable features. This research was conducted to study Bacillus species in the intestine of farmed rainbow trout to examine their probiotic properties, and to provide a new source of probiotics. A total of 23 farmed rainbow trout were sampled and their intestine samples were cultured. Following the morphological assay and biochemical analysis, isolated Bacilli were amplified by polymerase chain reaction and universal primers 27f and 1492r. Bacillus subtilis and B. amyloliquefaciens were isolated from 5 and 3 samples, respectively. Bacillus tequilensis, B. cereus and B. licheniformis were isolated from 1 sample. Probiotic properties of B. subtilis strain MSM 24, B. amyloliquefaciens strain TMM 25 and B. licheniformis strain MR 78 were confirmed. Since probiotic bacteria cause no foodborne diseases, their existence in farmed trout intestines, and their penetration into the fish tissues do not pose any risk to consumers’ health. Introduction The intestinal microbiota is an integral part of the gastrointestinal tract of fishes. Numerous internal and external factors regulate the microbial specifications of environment, thereby are affecting the fish intestinal microbiota (Earl et al., 2008). Bacteria are the main constituents of the intestinal microbiota of fish. The fish intestinal microbiota is characterized by high density and diversity of organisms with complex interactions (Nithya and Halami, 2013). Diverse microbial populations exist in the intestinal contents. The numbers of bacteria are higher in the intestinal contents than in the surrounding water. This shows that the intestines provide favorable environments for microorganisms. This microbial population can prevent infection by interfering with pathogens and/or limiting occupying surface for them (Huber et al., 2004). Studies have indicated that Bacillus species improve food consumption in fishes (Kavitha et al., 2018). Various studies reported that the intestinal microbiota of freshwater fishes encompasses of *Correspondence: Mohammad Rabbani DOI: https://doi.org/10.22034/ijab.v7i3.584 E-mail: m.rabbani@biol.ui.ac.ir different genera such as Acinetobacter, Aeromonas, Bacillus, Flavobacterium, Pseudomonas, Entero- bacteriaceae, and obligate anaerobic bacteria of the genera Bacteroides, Fusobacterium, and Clostridium (Hovda et al., 2007; Huber et al., 2004). Bacilli bacteria are present in water and soil and they can easily reach the trout farms. Given that rainbow trout are among high-quality and marketable fishes, its farming is considered as the largest aquaculture industry in Iran (Ghorbanzadeh and Nazari, 2015). Rainbow trout are sold as non-live form in many regions of Iran, which may lead to Bacilli bacteria penetrating to the fish flesh; as a result, it is necessary to determine the properties of these bacteria. Since probiotics have health benefits, they may provide an alternative way to reduce the use of drugs in aquaculture and simultaneously may avoid the development of antibiotic-resistant bacteria (Chen et al., 2016). Bacilli are being explored for the production and preservation of food for many centuries. The inherent https://www.ncbi.nlm.nih.gov/pubmed/?term=Earl%20AM%5BAuthor%5D&cauthor=true&cauthor_uid=18467096 https://www.sciencedirect.com/science/article/pii/S2352513418300176#! 167 Int. J. Aquat. Biol. (2019) 7(3): 166-174 ability of production of the proteins, enzymes, antimicrobial compounds, vitamins, carotenoids and other specifications are importance of study of bacilli in food industry. Additionally, Bacillus spp. are gaining interest in human health related functional foods researches coupled with their tolerance and survivability under hostile environment of gastrointestinal tract. Besides, Bacilli are more stable during manufacturing process, food storage and pharmaceutical industry that making them more suitable candidate for health promoting (Elshaghabee et al., 2017). Therefore, providing a new source of probiotic Bacilli is necessary. Hence, this research was conducted to study Bacillus species in the intestines of farmed rainbow trout, Oncorhynchus mykiss, by examining their probiotic properties, and providing a new source of probiotics. Materials and Methods Initial separation: A total of 23 farmed rainbow trout, 130-2230 g in weight, were collected from fish- farming ponds. The fish were sampled after euthanasia. First, the fish skin was washed using 70% alcohol. Then, their intestine was removed and washed by sterile distilled water to remove fecal matter. Next, the intestine was cut and rewashed by sterile distilled water to remove the remaining fecal matter. They were placed in sterile Petri dishes. The intestinal wall was scraped with a plastic spatula. All steps of sampling were done on ice. Undiluted mucus was used to explore the type of bacteria. Mucus was cultured on the nutrient agar (Cohen and Laux, 1995). Plates were incubated under aerobic conditions at 37°C for 5 days. After incubation, white colonies of gram-positive bacteria with 2-3 mm diameter were sampled. These bacteria were sub-cultured on nutrient agar under same conditions. Bacterial identification was carried out by morphological assay, chemical tests, PCR and probiotic tests. Morphological assay: Gram staining and fluorescent microscopy were used for morphological study of the bacteria. Bacterial identification by chemical methods: The bacteria were identified according to Bergey Manual of Systemic Bacteriology (Holt et al., 1994). Hence, they were identified by the mobility and catalase activity, followed by fermentation a set of carbohydrates, including arabinose, glucose, starch, mannose, mannitol, salicin, xylose, turanose and melibiose. The other biochemical tests were tween 20, o-nitrophenyl-β-d-galactopyranoside (ONPG), pH, concentration of NaCl range from 2-10%, temperature (5-65oC), citrate, VP and nitrate reduction. The ability to ferment lactose was utilized to differentiate Bacillus subtilis from B. amyloliquefaciens. The sugars were sterilized by filtration (Millipore filter, 0.45 μm pore size). Bacterial identification by PCR: In order to identify the bacteria using the PCR (Bioline, United Kingdom), they were cultured on nutrient agar. The culture medium was incubated at 37°C for 18 hours. Boiling method was used to extract DNA from bacteria (Dashti et al., 2009). The concentration and integrity of the extracted DNA was determined by ethidium bromide stained 0.8% agarose gel electrophoresis and compared against known concentrations of lambda DNA (Chandrasekara Bhagya et al., 2013). All chemicals used were molecular grade from Sigma Company. 16S ribosomal RNA gene was used. The PCR primers were 27f (AGAGTTT GATCCTG GCTCAG) and 1492r (TACGGYTACCTTGTTA CG ACTT). Master-mix of PCR was diluted to 10.30 μl reaction mixture contained 15 µl master, 1 µl each PCR primer and 8 µl sterile injectable water. The PCR program was as follow: initial denaturation at 94°C for 2 min followed by denaturation at 94°C for 1 min, annealing at 55°C for 1.5 min, 30 cycles of extension at 72°C for 1 min, and the final extension at 72°C for 3 min. The PCR product was visualized on a 1% agarose gel under UV light after ethidium bromide staining (Silva et al., 2013). Gel electrophoresis was carried out at 80 V. Amplicons were purified from agarose gel using the GFX PCR DNA and Gel Band Purification kit (GE Healthcare, United Kingdom). The PCR products were sequenced using the Sanger DNA sequencing (Teng et al., 2004). Sequences were identified using BLAST. The results were analyzed 168 Seifzadeh and Rabbani / Probiotic potential of Bacillus species isolated from rainbow trout using the BioEdit sequence editor. FASTA sequences aligned using Clustal X. PCR results aligned against bacterial DNA database (NCBI) (http://blast.ncbi. nlm.nih.gov). A homology greater than 97% was acceptable for identifying the genus. These bacteria were registered in the NCBI and DGBI database. Probiotic assay of the isolates: The isolates were screened for hemolytic activity, lecithinase, tolerance to acidic conditions, gastric juice and bile salt, and antibiotic resistance transferability. Then, based on the results, the selected bacteria were examined for adhesiveness and invasiveness. Microbial suspension was prepared for study of theses specifications (Iranian National Standard No. 19459. 2014). Microbial suspension preparation: The identified bacteria were cultured in nutrient broth at 37ºC for 24 hours. Culture media were centrifuged at 5000 rpm for 10 min. The supernatant was removed and pellets were washed in a phosphate-buffered saline (3 times). Then, the pellets were dissolved in the same buffer. These suspensions were adjusted to a turbidity equivalent to the 0.5 McFarland standard (Iranian National Standard No. 19459. 2014). Acidic tolerance: In order to evaluate acidic tolerance, pH of MRS broth (10 ml) adjusted to 2.5 and 4 using hydrochloric acid. 100 µl of microbial suspensions were inoculated into MRS broth. They were incubated at 37ºC for 3 and 4 hours. After that, 1 ml of these media were inoculated into nutrient agar, followed by incubation at 37ºC for 24 hours to determine colony forming unit (Iranian National Standard No. 19459. 2014). Gastric juice tolerance (pepsin and trypsin): In order to prepare the pepsin and trypsin media, 2 g of sodium chloride and 2.3 g of pepsin or trypsin were dissolved in 1 L of distilled water. The pH of these media were adjusted to 2-2.3. In order to evaluate gastric juice tolerance, the microbial suspension (2%) was inoculated into the pepsin and trypsin media, then, incubated at 37ºC for 24 hours. Thereafter, a 103 dilution was prepared from the incubated media using saline solution supplanted by 0.1% peptone water. One ml of these dilutions was cultured in nutrient agar using pour-plate method. Plates were incubated at 37ºC for 24 hours to determine colony forming unit (Iranian National Standard No. 19459. 2014). Bile salt resistance: In order to assess bile salt resistance, 100 µl of the microbial suspension were inoculated into nutrient broth containing 0.3% of fish bile, followed by incubation at 37ºC for 8 hours. Before and after the incubation period, absorbance of the suspension was measured at a wavelength of 600 nm using UV-VIS spectrophotometer (Eppendorf, Germany). A medium without bile was used as a control sample for each bacterium (Iranian National Standard No. 19459. 2014). Microbial adhesion and invasion: The adhesion assay was conducted according to Nithya and Halami (2013). The invasion assay was carried out as described by Rowan et al. (2001). The HepG-2 cell line was used. The cell monolayers were seeded at 5×104 cells per well (in RPMI 1640 medium plus 10% fetal calf serum) in 96-well plate. The cells were infected with filter-sterilized (0.2 μm) supernatants from overnight cultures (BHI, 30°C) of bacteria. 100 μl of supernatant were added to the cultured cells immediately after heat treatment (95°C, 10 min). Monolayers containing the bacterial supernatants were incubated overnight at 37°C in a 5% CO2 atmosphere. After overnight incubation, the suspension from each well was discarded and 25 μl of fresh complete medium (RPMI+10% fetal calf serum) containing 0.004 g/mL of MTT reagent (Sigma) was added. Samples were incubated for 3 hours at 37°C in 5% CO2 and the formazan product was solubilized by the addition of 100 ml of dimethyl sulfoxide. Optical densities of the suspensions were measured at 540 nm using an ELISA reader (Biotek, USA) and cyto- toxicity was calculated (Mohkam et al. 2016). The results of microbial adhesion and invasion tests were analyzed by SPSS Software. The samples were compared to each other by One Way ANOVA. T-test was used to compare the control with the test samples. Antibiotic susceptibility examination: The susceptibility of the Bacillus isolates to erythromycin (15 µg), penicillin (10 µg), gentamicin (10 µg), amoxi- cillin (25 µg), azithromycin (15 µg), chloramphenicol (30 µg), cefalotin (30 µg), clindamycin (2 µg), 169 Int. J. Aquat. Biol. (2019) 7(3): 166-174 streptomycin (10 µ g) and tetracycline (30 µg) were determined using Muller-Hinton agar. After incubation, the formation of clear zone around the discs monitored (Iranian National Standard No. 19459. 2014). Hemolytic activity test: This test was performed on all isolates. A loopful of overnight bacteria was cultured to spot form on blood agar plate. The plates were incubated for 24 hours at 37ºC and observed for clear zones around the colonies (Iranian National Standard No. 19459. 2014). Lecithinase activity: This test was performed using Mannitol-egg yolk-Polymyxin agar. A loopful of overnight bacteria was cultured to spot form on this medium. After incubation for 24 hr at 37ºC, the plates were checked for clear zone around the bacterial colonies (Iranian National Standard No. 19459. 2014). Abbreviations used: GIT, Gastrointestinal tract; ONPG, O-nitrophenyl-β-d-galactopyranoside; PCR, Polymerase chain reaction. Results Morphological characteristics: The detected bacteria appeared to form of short or long chains of large gram positive Bacilli. Their spores had situations of ellipsoidal, central and sub terminal. Chemical properties: Eleven Bacillus species were obtained from 23 examined fish. They showed catalase activity and could grow under both aerobic and anaerobic conditions. Bacillus cereus was arabinose, glucose, mannose, mannitol, salicin, xylose, and starch negative. However, it was citrate, VP, and nitrate reduction positive. This bacterium was unable to grow in the presence of salt (2-10%). Glucose, mannose, mannitol, salicin, xylose, arabinose, citrate and starch were used by B. subtilis, B. amyloliquefaciens, B. tequilensis and B. licheniformis. These bacteria were VP and nitrate reduction positive. These isolates were able to grow at the media containing 2-8% NaCl. Turanose and tween 20 were used by B. subtilis and B. amyloliquefaciens. Bacillus amyloliquefaciens was melibiose and lactose positive; however, it was ONPG negative. Bacillus subtilis was lactose negative; however, it was tween 80 positive. According to Table 1, 11 Bacilli species were identified in rainbow trout. Bacillus subtilis and B. amyloliquefaciens were isolated from 5 and 3 samples, respectively. Bacillus tequilensis, B. cereus and B. licheniformis were isolated from one sample. All the isolated Bacilli were motile. These bacteria were able to growth at pH 7; however, not at pH 10. Bacillus cereus MR11 was not able to grow at pH at 8-9. Bacillus cereus MR11 and B. licheniformis strain MR 78, but not the other Bacilli, were not able to grow at temperatures 10, 50 and 60°C. The isolated Bacilli were able to growth at temperatures of 20, 30 and 40°C, but not 5 and 65°C. Bacillus amyloliquefaciens was found in the small and large fishes. Bacillus subtilis was found in the small and medium fishes. Bacillus licheni formis and B. cereus were observed in the medium fish. Bacillus tequilensis was observed in the fish with 700 g weight. Molecular characteristics: The isolated bacteria were Table 1. The chemical identification results of Bacillus species isolated from the intestines of farmed rainbow trout (N= 11). Index Bacteria Weight Motility pH Capability at different temperatures 7 8 9 10 5 10 20 30 40 50 60 65 B. tequilensis strain MS21 700 + + - - - - + + + + + + - B. cereus MR11 355 + + - - - - - + + + - - - B. subtilis strain MT 13 480 + + + + - - + + + + + + - B. amyloliquefaciens MA 11 2040 + + + + - - + + + + + + - B. subtilis strain MSM 14 135 + + + + - - + + + + + + - B. amyloliquefaciens strain TR 15 215 + + + + - - + + + + + + - B. subtilis strain MR 20 247 + + + + - - + + + + + + - B. subtilis strain TAM 21 360 + + + + - - + + + + + + - B. amyloliquefaciens strain TMM 25 2230 + + + + - - + + + + + + - B. subtilis strain MSM 24 247 + + + + - - + + + + + + - B. licheniformis strain MR 78 350 + + + + - - - + + + - - - https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_948513421 170 Seifzadeh and Rabbani / Probiotic potential of Bacillus species isolated from rainbow trout 99% homologous to B. tequilensis, B. cereus, B. amyloliquefaciens and B. subtilis. Probiotic potentials: From all the Bacilli isolates, only B. licheniformis strain MR 78, B. amyloliquefaciens strain TMM 25, and B. subtilis strain MSM 24 were able to grow in the presence of bile salts and gastric juice, and different acidity levels. However, these bacteria had no hemolytic capability and lecithinase activity. Therefore, the other bacteria removed from cell toxicity and antibiotic resistance tests. As Table 4 shows, there was no significant difference in adhesion among the samples (P<0.05), but, there was significant difference between test and positive probiotic control in this factor (P>0.05). Adhesions of the test samples were significantly lower than the adhesion of the pathogenic control (L. monocytogenes) (P<0.05). No invasion was observed in B. subtilis strain MSM 24 and B. amyloliquefaciens strain TMM 25, but these bacteria had no significant difference in invasion compared to negative control and the other Bacilli. The highest invasion was related to the pathogenic control, which were significantly different compared to the other groups (P<0.05). As the Table 5 shows, B. subtilis strain MSM 24, B. amyloliquefaciens strain TMM 25 and B. licheniformis strain MR 78 have no antibiotic resistance. According to the Tables 3, 4 and 5, B. licheniformis strain MR 78, Table 2. Accession numbers and similarity of Bacillus species isolated from the intestine of farmed rainbow trout (N=11). Bacteria NCBI DGBI Similarity (%) B. tequilensis strain MS21 - LC458434 99 B. cereus strain MR11 MK395545.1 - B. subtilis strain MT 13 - LC458433 99 B. amyloliquefaciens strain MA 11 MK392154.1 - 99 B. subtilis strain MSM 14 MK400693.1 - 99 B. amyloliquefaciens strain TR 15 MK393391.1 - 99 B. subtilis strain MR 20 MK397791.1 - 99 B. subtilis strain TAM 21 MK397798.1 - 99 B. amyloliquefaciens strain TMM 25 MK394994.1 - 99 B. subtilis strain MSM 24 MK393445.1 - 99 B. licheniformis strain MR 78 MK395274.1 - 99 Table 3. The culture results of Bacillus species isolated from the intestine of the farmed rainbow trout in different conditions (N=11). Growth at Index Bacteria pH 4 pH 2.5 Bile 0.3% Gastric juice tolerance Lecithinase Activity Hemolytic activity 3h 4h 3h 4h Pepsin Trypsin B. tequilensis strain MS21 1×108±4.67 1×108±2.35 1×108±3.89 1×108±2.67 0.48±0.11 1×105±4.74 1×104±1.14 + + B. cereus strain MR11 1×108±4.13 1×108±3.56 1×108±2.87 1×108±3.78 0.56±0.16 1×104±4.71 1×106±3.91 + + B. subtilis strain MT 13 1×108±4.56 1×108±3.76 1×108±2.67 1×108±2.98 0.49±0.13 1×105±4.22 1×103±4.27 + + B. amyloliquefaciens strain MA 11 1×108±3.78 1×108±3.16 1×108±3.64 1×108±3.15 0.52±0.17 1×105±3.97 1×104±4.11 + + B. subtilis strain MSM 14 1×108±3.21 1×108±3.13 1×108±3.13 1×108±3.24 0.57±0.19 1×103±2.89 1×104±3.91 + + B. amyloliquefaciens strain TR 15 1×108±3.45 1×108±2.58 1×108±4.43 1×108±3.74 0.61±0.18 1×103±2.95 1×105±3.47 + + B. subtilis strain MR 20 1×104±2.19 1×103±4.15 1×103±3.17 1×102±4.45 0.91±0.15 1×105±2.31 1×104±3.31 + + B. subtilis strain TAM 21 1×108±4.15 1×108±3.18 1×108±3.78 1×108±2.54 0.06±0.02 1×108±4.17 1×108±3.42± _ _ B. amyloliquefaciens strain TMM 25 1×108±4.98 1×108±4.24 1×108±3.97 1×108±3.19 0.02±0.01 1×108±3.16 1×108±4.11 _ _ B. subtilis strain MSM 24 1×108±2.78 1×108±4.19 1×108±3.65 1×108±4.21 0.4±0.14 1×108±2.23 1×108±4.61 _ _ B. licheniformis strain MR 78 1×108±4.19 1×108±4.25 1×108±2.12 1×107±4.18 0.4±0.12 1×108±2.59 1×108±3.90 _ _ http://getentry.ddbj.nig.ac.jp/getentry/na/LC458434/?filetype=html http://getentry.ddbj.nig.ac.jp/getentry/na/LC458433/?filetype=html 171 Int. J. Aquat. Biol. (2019) 7(3): 166-174 B. amyloliquefaciens strain TMM 25, and B. subtilis strain MSM 24 had probiotic potentiality. Discussions Bacilli are diverse bacterial species, found ubiquitously in nature. They are highly adaptable to different environmental conditions and capable to grow in various environments, including the gastrointestinal tract of animals, soil and plants. Different strains of B. subtilis were isolated form different fishes, as probiotic for foods with aquatic origin viz. B. subtilis AB1 in rainbow trout (Newaj- Fyzul et al., 2007), B. subtilis in ornamental fish (Lalloo et al., 2017), B. subtilis in Labeo calbasu (Kavitha et al., 2018), B. subtilis strain VITNJ1 isolated from the gastrointestinal tract of Oreochromis niloticus (Efendi and Usra, 2014) and B. subtilis strain G024 in Paralichthys lethostigma (Chen et al., 2016). They recommended using the Bacillus isolates in the aquaculture industry for their probiotic potentiality. To the best of our knowledge, the present research is the first attempt to explore the isolation of B. subtilis from rainbow trout in Iran. Bacillus subtilis is found in the upper layers (1-3 cm) of various soils (Mongkolthanaruk, 2012), therefore, this bacterium enters into trout body through water. Hence, this species was observed in more fishes compared to the other Bacillus species. The present study reported the isolation of probiotic B. amyloliquefaciens from trout. It has been isolated from aquatic animals and other sources in different countries. Krishnan et al. (2014) isolated probiotic Bacillus species from the gastrointestinal tract of Anabas testudineus and Labeo rohita. Chen et al. (2016) isolated B. amyloliquefaciens strain N004 from the gastrointestinal tract of southern flounder and sediments of farming ponds of sea cucumber in China and recommended that it could be used as a probiotic strain. Kavitha et al. (2018) isolated probiotic B. amyloliquefaciens from the gastrointestinal tract of L. calbasu. Cao et al. (2010) isolated B. amyloliquefaciens from sediments in Shanghai. Sugita et al. (1998) isolated Bacillus species strain NM 12 from the gastrointestinal tract of Japanese coastal fishes. Nevertheless, there is no report about isolation of this bacterium from trout in Iran. Some members of B. amyloliquefaciens are found in plants and their roots and provide benefits for plant growth. Table 4. Adhesion and invasion percentages of Bacillus subtilis strain MSM 24, B. amyloliquefaciens strain TMM 25 and B. licheniformis strain MR 78 in cell culture medium (N=3). Invasion (%) Adhesion (%) Index Experiment 0a 0a Phosphate-buffered saline (as negative control) 0a 0.74±0.02b Bacillus subtilis strain MSM 24 0a 0.79±0.012b Bacillus amyloliquefaciens strain TMM 25 0.01±0.0001a 0.59±0.014b Bacillus licheniformis strain MR 78 0.09±0.003a 1.1±0.09b Bacillus coagulans (IBRC-M 10807) (as positive probiotic control) 35.1±1.2b 2.3±0.11c L. monocytogenes (IBRC-M 10671) ( as pathogen control) The different letters in the same column indicate significant differences (P<0.05). Table 5. The growth inhibition halo diameters of Bacillus licheniformis, B. amyloliquefaciens and B. subtilis (mm). Bacteria Antibiotic disk B. subtilis strain MSM 24 B. amyloliquefaciens strain TMM 25 B. licheniformis strain MR 78 Erythromycin (15 µg) 32±3.19 1.14±29 32±2.28 Penicillin (10 µg) 31±2.13 28±2.25 35±3.35 Amoxicillin (25 µg) 33±3.21 32±3.23 33±2.43 Gentamycin (10 µ g) 22±2.16 31±2.11 31±1.56 Azithromycin (15 µg) 32±3.24 34±3.18 33±2.53 Cefalotin (30 µg) 29±1.17 33±1.21 30±3.51 Clindamycin (2 µg) 31±2.35 35±3.39 34±3.41 Streptomycin (10 µ g) 29±3.41 30±3.59 32±2.65 Tetracycline (30 µg) 34 ±2.31 29±2.42 34±2.54 Chloramphenicol (30 µg) 33±2.56 33±2.34 30±1.52 172 Seifzadeh and Rabbani / Probiotic potential of Bacillus species isolated from rainbow trout Bacillus amyloliquefaciens strains are widely used in various commercial formulations to boost plant growth. These bacteria are readily transmitted to aquatic animals from water flowing and through plants that harbor the bacteria (Chen et al., 2007). Bacillus tequilensis spp. was isolated from the intestines of the farmed rainbow trout. Its isolation of aquatic animals has not been reported yet. There are a few reports of the presence of this bacterium in water. This indicates the effect of the physiochemical conditions of water or soil and/or antagonistic activity of other bacteria on the survival of this bacterium. Bacillus licheniformis strain MR 78, B. amyloliquefaciens strain TMM 25, and B. subtilis strain MSM 24 B. cereus spp. was isolated from the intestine of the farmed rainbow trout. Bacillus cereus was isolated from other fishes by Rasool et al. (2017) and Lalloo et al. (2007). Kavitha et al. (2018) also isolated B. cereus from the gastrointestinal tract of L. calbasu. Nevertheless, its isolation of farmed rainbow trout has not been reported from Iran. Bacillus cereus might be as vegetative cells or spores, which can adapt to the environmental changes, thus, it resists in environment. Then, penetrates to the rivers and trout farms (Rasool et al., 2017). Bacillus licheniformis spp. was isolated from the intestine of the farmed rainbow trout. Lalloo et al. (2007) reported the isolation of B. licheniformis from common carp. Pasmik et al. (2008) carried out a study on the isolation of B. licheniformis from Atlantic menhaden (Brevoortia tyrannus). Bacillus licheniformis may be a common flora of some fish species. It is also a part of fish feed. In addition, B. licheniformis is ubiquitous in the environment and used in treatment plants often enter into rivers and trout farms (Pasnik et al., 2008). Based on the results, B. licheniformis strain MR 78, B. amyloliquefaciens strain TMM 25, and B. subtilis strain MSM 24 have probiotic potentiality because they were resistant to bile salt, acid and gastric juice. Furthermore, they had no antibiotic resistance, lecithinase and hemolytic activity and toxicity in cell culture. Although, Lactobacilli constitute the main population of probiotics; however, other bacteria such as B. coagulans and B. subtilis and so on are also among probiotics or are regarded as probiotic candidates for the development of probiotic products (Elshaghabee et al., 2017). Conclusion The present study demonstrated that, of the Bacilli isolated from the trout intestine, B. licheniformis strain MR 78, B. amyloliquefaciens strain TMM 25, and B. subtilis strain MSM 24 have probiotic properties. Further studies are needed to determine the bacteria anti-micrbial properties and resistance to enable one for choosing the most suitable bacteria for use in food industry as probiotic. Acknowledgements We are sincerely grateful to the experts at Isfahan University and Inland Waters Aquaculture Research Center for their unwavering support. References Cao H., He S., Wei R., Diong M., Lu L. (2011). 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