Endospore Staining Research Articles

Isolation, Characterization, and Selection of Bacillus sp. from Shallot Rhizosphere that Inhibits Fusarium oxysporum Growth

Bacillus sp. is a Plant Growth Promoting Rhizobacteria (PGPR) species that lives in the rhizosphere. This bacterium can produce antifungal compounds that suppress pathogenic fungi, such as Fusarium oxysporum. The aim of this research was isolate, characterize, and obtain Bacillus sp. that can inhibit F. oxysporum which causes twisted disease in shallots. Isolation was done by sampling 10 g of soil from shallot roots, placing it in 9 mL of sterile water, and carrying out a series of dilutions. The first dilution was incubated at 80 °C for 20 minutes to obtain Bacillus sp. on the selective medium HiChrome. Colony morphology, shape, cell color, Gram staining, catalase and endospore staining were observed from obtained isolates. An antagonist test was done to determine the inhibitory effects of isolates against the pathogenic fungus F. oxysporum. Four isolates of Bacillus sp. were obtained with irregular colony shape, dull white color without muccus, edges varying in shape between lobate and undulate, and raised elevation. The results of the antagonist test showed that the four isolates had the ability to inhibit the growth of the pathogenic fungus F. oxysporum with the greatest inhibition shown by isolate BM1 at 30.12%, with an antibiosis mechanism and hyphae swelling.

Nematocidal activity and biocontrol efficacy of endophytic Bacillus velezensis Pt-RP9 from Pinus tabuliformis against pine wilt disease caused by Bursaphelenchus xylophilus

Pine wilt disease (PWD) is a globally significant quarantine forest disease caused by Bursaphelenchus xylophilus (PWN), resulting in substantial ecological and economic losses. Traditional nematode management practices are neither cost-effective nor environmentally friendly, prompting the exploration of biocontrol as a promising alternative for managing this devastating forest disease. Obtaining novel and specific biocontrol agents is extremely crucial for the effective and precise control of PWD. In the present study, a total of 136 endophytic isolates were obtained from the roots, stems and needles of Pinus tabuliformis in the Qinling Mountains of China, which were then subjected to nematocidal activity assay against PWN in vitro. Nine endophytic bacterial isolates exhibited exceptionally strong nematocidal capacity, with a corrected mortality rate exceeding 90 %, which were then identified as the genus of Bacillus through morphological features, endospore staining, and 16S rDNA sequencing, with one strain as B. mycoides, two as B. cereus, and six as B. velezensis. Additionally, the inhibition effects of the three Bacillus species on the reproduction of PWN in vitro was assessed using an original detection model, with B. velezensis Pt-RP9 identified as the most promising strain. Subsequently, the biocontrol efficacy of B. velezensis Pt-RP9 against PWD was evaluated in greenhouse experiments. Pt-RP9 demonstrated significant biocontrol effectiveness against PWD, with control efficiencies ranging from 31.25 % to 68.89 % across all treatments, particularly showing improved efficacy when pine seedlings were pre-treated with Pt-RP9 before PWN inoculation. Furthermore, pine seedlings treated with Pt-RP9 exhibited significantly reduced PWN density and lipid peroxidation levels in cell membranes compared to the control groups, along with increased activities of peroxidase, catalase, and polyphenol oxidase. To our knowledge, this study is the first to showcase the nematocidal activity of endophytes from P. tabuliformis against PWN and their biocontrol efficacy against PWD, marking a significant advancement in the field. The findings highlight the potential of B. velezensis Pt-RP9 as a crucial biological control agent against PWD, presenting a novel and sustainable disease management approach for pine forests.

Isolation and Characterization of Indigenous Lactic Acid Bacteria from Pakatikng Rape, Dayak’s Traditional Fermented Food

West Kalimantan is one of the provinces with a variety of traditional foods. One of them is pakatikng rape, a traditional Dayak fermented food made from kelampai fruit. The fermentation process involves lactic acid bacteria which act as food preservatives because they have inhibitory substances known as bacteriocins. Exploration of lactic acid bacteria in the local food fermentation process, especially in West Kalimantan, has not been widely carried out. Therefore, the purpose of this research is to obtain LAB from kelampai fruit fermentation. The research methods included fermentation of kelampai fruit, isolation of LAB, screening for acid-producing LAB, characterization of colony morphology, gram staining, endospore staining, motility test, catalase test, and antibiotic sensitivity test. The results of the study obtained nine isolates that are capable of producing acid and have general characteristics of lactic acid bacteria, namely round cell shape, gram-positive, non-endospores, non-motile and catalase negative and four isolates that are resistant to antibiotics, namely KL-10 resistant to erythromycin, KL-15 and KL-19 resistant to Ofloxacin, and KL-20 resistant to amoxicillin.

Isolation and Characterization of Phyllosphere Microflora of Maize

Maize is renowned as the "Queen of Cereals" and is one of the world's most significant cereal crops. Several foliar and stalk rot diseases affect maize crops. The illness affected the majority of the cultivars that were issued. The influence of overuse of chemical fungicides on the environment and food safety has become a serious problem with the rise of ecological agriculture. Epiphytes are phyllosphere residents who can include a wide range of bacteria and filamentous fungi. Microbial interactions in the phyllosphere repress and promote plant pathogen colonisation and infection of tissues, increasing disease resistance and agricultural crop productivity, implying that phyllosphere microorganisms can play a key role in growth promotion and disease suppression. Bacteria from individual colonies were studied. Individual colony bacteria was examined for shape, size, colour, Gram staining, endospore staining, elevation and texture for morphological studies. Different biochemical tests viz., Catalase, Oxidase, Voges Prausker’s, Indole, Methyl red, Gelatin liquefaction were done for phyllosphere bacteria.

The 16S rRNA Analysis and Enzyme Screening of Bacillus from Rhizosphere Soil of Lombok Island

Bacillus are commonly found in nature, especially in soil and food. It has the ability to produce bioactive compounds as well as the enzyme. This study was aimed to isolate, identify, and screen their enzyme activities. Four soil samples from Mandalika, Lombok Island, West Nusa Tenggara (NTB), were used for isolation. Bacillus was isolated using the heat-shock method and characterized through Gram staining, endospore staining, and morphological phenotype. Bacillus identification was conducted based on 16S rRNA gene sequence. The hydrolytic enzyme activities were checked qualitatively using selective media, and the enzyme tested including amylase, galactosidase, lipase, protease, and cellulase. As many as twenty-two bacteria isolates were obtained from four soil samples and represented 15 distinct species. The member of bacteria genera successfully identified, consisted of Bacillus sp., Brevibacillus sp., and Fictibacillus sp. Bacillus sp. was the most isolated. Some of the isolated bacteria have the ability to produce lipase, protease, and cellulase that potential to be used in biotechnology processes.
 
 Keywords: Bacillus, hydrolytic enzymes, identification, screening

ISOLATION AND CHARACTERIZATION OF IAA-PRODUCING RIZOSPHERE BACTERIA FROM RICE PLANTS (ORYZA SATIVA L.) IN KEDUNGPANI URBAN VILLAGE, SEMARANG CITY

Rhizosphere bacteria live in the soil around plant roots. Various rhizosphere bacteria are able to produce the hormone Indole-3-Acetic Acid (IAA) which stimulates plant growth. This study aims to isolate and characterize IAA-producing rhizosphere bacteria in rice plants in the rice fields of Kedungpani urban village, Semarang city. Bacterial enumeration was carried out using the total plate count (TPC) method. The isolates obtained were characterized by observation of colony and cell morphology, gram staining, endospore staining, catalase test, and sugar fermentation test. The ability to produce the IAA hormone was tested using Salkowski's reagent. The enumeration results showed that the total bacteria in the sample was 2.9 x 105 CFU / g. A total of 10 isolates were obtained and were able to produce the IAA hormone with various concentrations. Isolates KP1, KP2, KP3, KP4, KP5, KP6, KP9, KP13, KP14, and KP15 produced IAA with concentrations of 3.389, 5.111, 3.000, 1.667, 1.944, 5.056, 5.444, 4.500, 5.056, and 10.833 ppm, respectively. The results showed that there were 5 isolates with the highest IAA concentrations, namely KP2, KP6, KP9, KP14, and KP15 which had the potential to be used as biostimulant agents for plants.

Identification and Characterization of Antagonistic Bacteria from Coffee Plant Litter

Nowadays, the use of antagonistic bacteria is widely applied in agriculture and used as biological control. A previous study which is yet to be identified based on physiological and biochemical characterizations discovered these organisms present in coffee plant litter could control fusarium wilt disease. Therefore, this study is a follow-up analysis that aims to identify coffee litter antagonistic bacteria with the ability to control fusarium wilt disease. The investigation techniques used were biochemical tests comprising of endospore staining, yellow colony growth test on media, bacterial purification and screening, macroscopic and microscopic characterization, Gram, the solubility of 3% KOH, fermentative oxidative, fluorescent pigment production, catalase, hypersensitivity, nitrogen-fixing, and phosphate solvent test. The results showed that antagonistic bacteria, including <em>Bacillus mycoides, Clostridium, Pseudomonas, Erwinia, </em>and <em>Alcaligenes</em> sp. were identified based on their character traits. This study represents new information on the bacteria types from coffee litter to control pathogenic fungi as an environmentally friendly biological control agent.

In-Vitro Antagonistic Effect of Bacillus thuringiensis on Ralstonia solanacearum, the Causal Agent of Bacterial Wilt Disease of Tomato (Lycopersicon esculentum Mill).

Tomato (Lycopersicon esculentum, Mill) is a rich source of vitamins, minerals and lycopene, which has many health benefits. However, its production is hampered by bacterial wilt caused by Ralstonia solanacearum resulting in significant yield losses. Use of chemicals in the control of plant pathogens has detrimental effects on humans and the environment in terms of leaving residues in soil which later find their way into underground waters. Therefore, it is desirable to find an alternative to chemical control of this bacterial pathogen. This study investigates the potential of native Bacillus thuringiensis (Bt) for biological control of Ralstonia solanacearum (Rs) under laboratory conditions. B. thuringiensis was isolated from cultivated soil, non- cultivated soil, stagnant water, sawdust, horse dung, grain dust, dead leaves and poultry manure. R. solanacearum was isolated from stem exudates of bacterial wilt infected plants and its pathogenicity assay was carried out using 2-week-old seedlings of Beske tomato variety. The Bt and R. solanacearum isolates were then characterized phenotypically. Bt isolates were further identified using endospore and parasporal staining techniques. All the Bt isolates were tested for in-vitro antagonistic activity on R. solanacearum using agar well diffusion method. Isolates Bt2, Bt16, Bt17, Bt32 and Bt34 were confirmed as Bacillus thuringiensis while isolate Rs was confirmed as R. solanacearum. Beske showed wilting symptoms from the fourth day of inoculation and eventual death of seedlings. The zone of inhibition exhibited ranged from 0.0 mm to 20.0 mm.
 Keywords: Bacillus thuringiensis, In-vitro, Bacterial wilt, Ralstonia solanacearum, Tomato

English

Four soil samples and water effluents each within the mining environment were tested in this study. Twenty six bacteria were isolated using nutrient agar and acidified nutrient agar. Gram staining and endospore staining were carried out to determine the Bacillus species used for bioremediation. Speciation of the microorganisms using 16S rRNA sequencing showed the organisms to be Bacillus cereus NK1, Lysinibacillus species TAI-282, Lysinibacillus fusifomis, Bacillus aryabhattai PM1 and Bacillus megaterium from the samples. The temperature of the water effluents ranged from 29.00 to 34.70°C. Sample C from beneficiation area had the highest temperature of 34.70°C. The pH ranged from 6.70 to 9.77 with effluent from primary crushing area two (PC2) having the highest of 9.77. Bioremediation of the water samples were carried out for 6 days using the identified Bacillus species from the mine site. For all the effluents treated, there was an increase in the concentration of magnesium with effluent from PC2 treated with L. fusifomis increasing to 7.453±0.004 from 4.278±0.003 and these values were significantly different at p≤0.05. Concentration of calcium in effluent from iron ore storage area increased from 1.350 ±0.002 to 15.450±0.004 after treatment with B. aryabhattai PM1, and the values were significantly at p≤0.05. Bacillus spp. from PC2 reduced the concentration of iron from 179.738 to 0.091 ppm on effluent from PC1, the concentration was significantly different at p≤0.05. The indigenous microorganisms from within the iron ore mining site had bioremediation potentials reducing the concentrations of most of the heavy metals present.   Key words: Iron ore, molecular analysis, bioremediation, Bacillus species.

Production of Extracellular Protease from Bacterial Co-cultures using Solid State Fermentation

Proteases (also known as peptidases or proteinases) are hydrolytic enzymes that cleave proteins into amino acids. They comprise 60% of the total industrial usage of enzymes worldwide and can be obtained from many sources. The current study aims to isolate and screen protease-producing bacterial strains from the soil and to produce protease from the bacterial co-cultures using solid-state fermentation (SSF). Primary screening of the protease-producing bacterial strains was carried out on skim milk agar and they were sub-cultured and preserved on the nutrient agar for further testing. Thirty-two compatibility tests of twenty-seven bacterial isolates were performed and SSF was carried out. Afterward, absorbance was taken at 660 nm against tyrosine as standard. According to the results, the bacterial co-culture 19 showed the highest absorbance with an enzyme activity of 10.2 U/ml. The bacterial strains of the co-culture 19 were identified through morphological and biochemical tests. Bacterial strain 1 was observed as cocci and irregular, while bacterial strain 2 was bacillus and rod-shaped. Both strains were positive for gram staining, catalase test, casein hydrolysis test and methyl red test. As for endospore staining, bacterial strain 1 was spore forming while bacterial strain 2 was a non-spore former. It was concluded that the bacterial co-culture 19 can act as a potent co-culture for protease production. Compatibility test was carried out to enhance the production of protease by utilizing cheap and readily available agro-waste products, which benefit the industry by being cost effective and the environment by being eco-friendly.

Production of Extracellular Protease from Bacterial Co-cultures using Solid State Fermentation

Proteases (also known as peptidases or proteinases) are hydrolytic enzymes that cleave proteins into amino acids. They comprise 60% of the total industrial usage of enzymes worldwide and can be obtained from many sources. The current study aims to isolate and screen protease-producing bacterial strains from the soil and to produce protease from the bacterial co-cultures using solid-state fermentation (SSF). Primary screening of the protease-producing bacterial strains was carried out on skim milk agar and they were sub-cultured and preserved on the nutrient agar for further testing. Thirty-two compatibility tests of twenty-seven bacterial isolates were performed and SSF was carried out. Afterward, absorbance was taken at 660 nm against tyrosine as standard. According to the results, the bacterial co-culture 19 showed the highest absorbance with an enzyme activity of 10.2 U/ml. The bacterial strains of the co-culture 19 were identified through morphological and biochemical tests. Bacterial strain 1 was observed as cocci and irregular, while bacterial strain 2 was bacillus and rod-shaped. Both strains were positive for gram staining, catalase test, casein hydrolysis test and methyl red test. As for endospore staining, bacterial strain 1 was spore forming while bacterial strain 2 was a non-spore former. It was concluded that the bacterial co-culture 19 can act as a potent co-culture for protease production. Compatibility test was carried out to enhance the production of protease by utilizing cheap and readily available agro-waste products, which benefit the industry by being cost effective and the environment by being eco-friendly.

Colony, morphological and biochemical characteristics of cotton phyllosphere bacteria and its antagonistic activity against the Alternaria leaf spot of cotton

Cotton crop is known to suffer from number of fungal, bacterial and viral diseases. Among them, Alternaria leaf spot caused by Alternaria spp. is predominant in causing economic losses to the cotton crop in the country. Phyllosphere inhabitants termed as epiphytes may consists of a variety of bacteria and filamentous fungi. They also play a key role in carbon and nitrogen cycling and help in important environmental processes such as methanol degradation and nitrification. So, bacterial phyllosphere cultures (P1 to P8) were isolated from the Bt and non Bt cotton by leaf imprint method and dilution method. The colony characters of isolates of bacteria pertaining to their shape, size, elevation, margin, texture, appearance and pigmentation were recorded. Gram’s staining and endospore staining revealed that P1, P2, P3, P5, P8 were Gram positive, P1, P2, P5 were showing endospore staining and P1, P4, P6, P7 were rod shaped. Biochemical tests revealed that the eight isolates Bt and non Bt cotton were differed in each test and showed positive for the catalase and oxidase test. The isolates P1, P5 and P6 showed positive results to Voges proskauer test. Isolates P2, P4 and P6 revealed positive results of phyllosphere bacteria to Indole test. Whereas, isolates P1, P2 and P8 showed negative reaction to methyl red test. The phyllosphere bacterial isolates P3, P5 and P7 recorded negative reaction to gelatin liquefaction. Screening of phyllosphere microflora against Alternaria macrospora was conducted following dual culture technique for all the isolates. Phyllosphere bacterial isolate P5 recorded maximum growth inhibition with 57.41 per cent followed by P4 isolate with 53.52 per cent growth inhibition over the test fungus Alternaria macrospora. Whereas least per cent growth inhibition was reported in isolate P6 with 45.63 per cent.

Isolation of alkali-thermotolerant cellulolytic bacteria from spent mushroom substrate

In this study, the isolation of new alkali-thermophilic bacteria with enhanced cellulase producing ability from the spent mushroom substrate was conducted. Repeated streaking was performed on the Microcrystalline-cellulose (MCC) to obtain a pure culture. Two thermophiles bacteria isolates were used for subsequent experiment. Morphological characteristic by gram staining and endospore staining also genetic identification of 16S rDNA was performed and the growth profile had been generated using Polymath Software. Filter paper assay (determination of enzyme activity), Bradford protein concentration were used in plotting the growth associated product curve. Based on the 16S rDNA sequencing results, the bacterium strains of Anoxybacillus geothermalis, incubated at 50˚C, pH 7 and Aeribacillus pallidus, incubated at 60˚C, pH 8 was successfully isolated. Both strains are gram-positive and endospore-forming bacteria. Based on the growth curve, the doubling time for Anoxybacillus geothermalis was calculated to be 49.5 minutes. Whereas, doubling time of Aeribacillus pallidus was 56.7 minutes. Both strains represent the growth assosiated product curve in which, when product formation is growth associated the specific rate of the product formation increases with specific growth rate and vice versa.

Isolation, Characterization and Identification of Contaminant Bacteria from Sugarcane (Saccharum officinarum L.) in vitro Culture in Tigray Biotechnology Center, Mekelle, Ethiopia

Plant tissue cultures can be contaminated by extensive diversity of bacteria and the contaminations are species specific. The contamination can reduce growth rate of shoot and root, multiplication factor, and even cause plant death. The study was conducted at Mekelle University, College of Veterinary Medicine and Tigray Biotechnology center and contaminated in vitro sugarcane culture obtained from laboratory of plant tissue culture. The objective of the study was to isolate, characterize, and identify bacteria from contaminated in vitro Sugarcane culture and to test the sensitivity of the isolates to the most commonly used antibiotics. Morphological, Gram stain, Endospore stain and Biochemical test method were used to isolate and identify the contaminates and disk diffusion method was used for the drug susceptibility test. Commonly available antibiotics were used for susceptibility testing. In the present study Escherichia, Bacillus and Micrococcus were isolated and identified as the major contaminant bacteria from in vitro sugarcane cultures. In antibacterial susceptibility test the isolates of Bacillus and Micrococcus were susceptible to Gentamicin, Chloramphenicol, Ciprofloxaciline, Tetracycline, Vancomycine, Streptomycin, Penicillin G and Kanamycine demonstrating the efficacy of these antimicrobials for the treatment of sugarcane in vitro culture contamination by incorporating in to the sugarcane media formulation while, the third isolate Escherichia were resistance to all antibiotic agents.

Identification of Bacteria on Seblak Food around University of Jember Based on Microbiological Criteria of Processed Food

The quality of food that is good in bacteriological, chemical and physical must always be maintained in order to avoid diseases or health problems. Healthy and safe food is an important factor to improve the standard of public health. Seblak is a ready-to-eat Indonesian food which until now has never been carried out research about what kinds of bacteria in it. Seblak is a food made from raw crackers which is then deliberately soaked using hot water to have a chewy texture. This study aims to determine what types of bacteria are contained in seblak, through the process of isolation and identification in the macroscopic, microscopic and biochemical way. This study used 5 samples, which was repeated 5 times for each sample. Bacteria were isolated from the sample using spread plate techniques and observed by growing colonies on the plate. Each different colonies was observed microscopically through gram staining and endospore staining. To strengthen the data, biochemical tests were also carried out, biochemical tests that have been done in this study were the oxidase test, catalase test, and indole test. The results of the study showed that the bacteria that were found from the samples are in the genus of Bacillus sp. because they show the morphological characteristics of the colonies that form concentric circles, meanwhile, microscopic observations show morphological characteristics of cells in the form of gram-negative bacilli and have the endospores.

The production of bioinsecticide using Bacillus thuringiensis

This study aims at determining the ability of Bacillus thuringiensis to induce a biocidal effect on mosquito larvae. The B. thuringiensis used in this work was isolated from soil samples obtained from the root of plantain trees in a garden at Umuoma community in Uli Town. Soil samples were subjected to high temperature of about 50oC in order to exclude other microbes that may contaminate the culture, B. thuringiensis was able to survive the heat condition because they are spore formers. To further enhance purity of the culture, the organism was cultured in Luria Bertani medium which is selective for Bacillus spp. Before the toxicity tests were carried out, various morphological tests such as endospore stain, Gram stain, macroscopic examination and biochemical tests such as starch hydrolysis, oxidase test, motility test, citrate test, methyl red test, indole test, sugar fermentation test and catalase test were carried out to identify the organism. The results showed the isolates to be Bacillus thuringiensis. To ensure the ability of the isolate to produce spores needed for toxicity testing, the isolate was subcultured in nutrient agar and incubated at 37oC for 7 days after which there was formation of spores in the growth medium. The isolate was then inoculated in sterile water containing mosquito larvae in other to test for its toxicity against the mosquito larvae. At intervals, observations were made to determine the rate at which the Isolates induce the lethal effects on the larvae by using the sterile loop to check for viable larva remaining at each set up. The viability is deduced from the ability of the viable larva to retain their motility when agitated with the sterile wire loop. The result for toxicity testing showed that set-up Pr1 had the highest number of dead larvae at 1 – 4 hours respectively, while Gr1 had the lowest number of dead larvae at 1. Statistical representation of the results showed that there was significant difference of p > 0.05 between time and the parasporal crystals of Bacillus thuringiensis against mosquito larva. Two- factor ANOVA without replication also showed that there was a significant difference between the set-­ups Pr1, Pr2, Gr1 and Db2, and the positive control used gave significant difference of p < 0.05. This study successfully demonstrated the toxicity of the parasporal crystals of Bacillus thuringiensis against mosquito larvae.

Isolation and Characterization of Amylase-Producing Amylolytic Bacteria from Rice Soil Samples

Amylase is an enzyme that plays a role in hydrolyzing starch to simple sugars. This protein is very important for various industrial processes. Microorganisms are considered as the best source of amylase production. This study was aimed at isolating and dentifying amylolytic bacteria from rice soil samples that have the potential to produce amylase enzymes. Rice soil samples were collected from two locations, namely the Percut Sei Tuan area and Labu Beach. Samples was isolated, then macroscopically identified with microscopy, and microscopically characterized with catalase test, endospore staining and gram staining. The identification results showed that 14 bacterial isolates from the two samples potential to produce amylase enzymes. The isolates which were characterized with catalase test showed positive results for all isolates. The characterization results with endospora staining showed 7 positive isolates and 7 negative isolates. Likewise with the results of gram staining characterization. Thus, from the two sample locations obtained amylolytic bacteria that potentially produce amylase enzymes.

Diversity of Bacillus thuringiensis cry genes in soils of Andhra Pradesh, India

A total of 61 bacterial strains was isolated in soils from Chittoor and Kadapa districts of Andhra Pradesh, India using sodium acetate selection method and preliminarily identified as Bacillus thuringiensis based on Gram's staining, endospore staining and crystal protein stud. These strains were tested against the insect, Spodoptera litura under laboratory conditions and found 18 strains with ≥ 50% insect mortality. Further, these eighteen strains were analyzed for the insecticidal crystal protein composition with fourteen cry specific primers. The results indicated that, among all 14 cry genes, cry1I was the observed in 11 strains, followed by cry1Aa in 10 strains. At the same time a strain, F493 collected from Talakona area identified as effective against S. litura with 100 per cent larval death and which was also identified with 8 different crystal protein encoding genes (cry1Aa, cry1Ac, cry1Fa1, cry1I, cry2, cry2A(a)1, cry8, cry9Ca1).

Effect of Bacillus velezensis on Aeromonas veronii-Induced Intestinal Mucosal Barrier Function Damage and Inflammation in Crucian Carp (Carassius auratus).

Aeromonas veronii is an emerging aquatic pathogen causing hemorrhagic septicemia in humans and animals. Probiotic is an effective strategy for controlling enteric infections through reducing intestinal colonization by pathogens. Here we report that the consumption of Bacillus velezensis regulated the intestinal innate immune response and decreased the degree of intestinal inflammation damage caused by the A. veronii in Crucian carp. In this study, we isolated four strains of B. velezensis, named C-11, S-22, L-17 and S-14 from apparently healthy Crucian carp, which exerted a broad-spectrum antimicrobial activity inhibiting both Gram-positive and Gram-negative bacteria especially the fish pathogens. B. velezensis isolates showed typical Bacillus characteristics by endospore staining, physiological and biochemical test, enzyme activity analysis (amylase, protease, and lipase), and molecular identification. Here, Bacillus-containing dietary was orally administrated to Crucian carp for 8 weeks before A. veronii challenge. Immunological parameters and the expression of immune-related genes were measured at 2, 4, 6, 8, and 10 weeks post-administration. The results showed that B. velezensis was found to promote the increase in the phagocytic activities of peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs), as well as the increase in interleukin 1β (IL-1β), IL-10 and tumor necrosis factor α (TNF-α) concentration of serum. Lysozyme levels (113.76 U/mL), ACP activity (25.32 U/mL), AKP activity (130.08 U/mL), and SOD activity (240.63 U/mL) were maximum (P < 0.05) in the B. velezensis C-11 treated group at 8 week. Our results showed that Crucian carp fed with the diet containing B. velezensis C-11 and S-22 developed a strong immune response with significantly higher (P < 0.05) levels of IgM in samples of serum, mucus of skin and intestine compared to B. velezensis L-17 and S-14 groups. Moreover, B. velezensis spores appeared to show no toxicity and damage in fish, which could inhabit the gut of Crucian carp. B. velezensis restrained up-regulation of pro-inflammation cytokines (IL-1β, IFN-γ, and TNF-α) mRNA levels in the intestine and head kidney at final stage of administration, and the expression of IL-10 was increased throughout the 10-week trial. A. veronii infection increased the population of inflammatory cells in the intestinal villi in the controls. In contrast, numerous goblet cells and few inflammatory cells infiltrated the mucosa in the B. velezensis groups after challenge with A. veronii. Compared with A. veronii group, B. velezensis could safeguard the integrity of intestinal villi. The highest post-challenge survival rate (75.0%) was recorded in B. velezensis C-11 group. The present data suggest that probiotic B. velezensis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection.IMPORTANCEIn this work, four Bacillus velezensis strains isolated from apparently healthy Crucian carp, which exhibited a broad-spectrum antibacterial activity especially the fish pathogens. Administration of B. velezensis induced the enhancement of the intestinal innate immune response through reducing intestinal colonization by pathogens. The isolation and characterization would help better understand probiotic can be recognized as an alternative of antimicrobial drugs protecting human and animal health.

Identificación de Bacillus toyonensis en heces de ganado cebú en el Departamento de Sucre, Colombia

Se evaluaron las características probióticas de cepas nativas aisladas de ganado cebú y se realizó identificación molecular de una de estas cepas, para considerar su posterior aplicación como aditivos microbianos en la alimentación bovina. Se usaron muestras de estiércol de terneros de levante, se aislaron bacterias y levaduras, determinándose la capacidad probiótica de estas cepas mediante pruebas como; resistencia a sales biliares (0.05, 0.1, 0.15 y 0.3 %), resistencia a pH ácido (pH 3, 4, 5.6, 7), tolerancia a NaCl (2, 4, 6, 8,10 %), actividad antagónica (Salmonella spp., y Escherichia coli), producción de gas a partir de la glucosa, y crecimiento a diferentes temperaturas (37 y 40 °C). La identificación preliminar de las cepas se realizó mediante: tinción de Gram, tinción de endosporas (método de Wirtz), prueba de la catalasa, prueba de oxidasa. Se seleccionó una de las cepas que superó las pruebas probióticas para su identificación mediante métodos moleculares y se realizó el análisis filogenético de la misma utilizando la base de datos NCBI Reference Sequence (RefSeq). Los ensayos se organizaron en un diseño completamente al azar, los resultados obtenidos por triplicado se sistematizaron en el software Statgraphics Centurion XVI y el efecto de los diferentes tratamientos se analizó estadísticamente mediante análisis de varianza. Se aislaron 10 cepas nativas, identificándose molecularmente a Bacillus toyonensis. Esta investigación representa el primer reporte molecular de B. toyonensis en estiércol de terneros de levante cebú en el Departamento de Sucre, el cual ha sido ampliamente utilizado como probiótico en la nutrición animal.