Isolation of bacterial antagonistic strains of the genus Bacillus, with potential for the control of fish pathogenic microbiota

Quantitative and qualitative analyses of bacterial flora associated with pond water, gills, and intestine of polycultured healthy common carp (Cyprinus carpio) and African catfish (Clarias gariepinus) were carried out and identified to species level where possible. Total viable bacterial counts in the pond water ranged from 9.2 ± 5.5 × 103 to 6.6 ± 5.1 × 104 colony-forming units (cfu)/mL; in the gill filaments of carp and catfish, 3.3 ± 3.8 × 106 to 7.9 ± 5.6 × 106 and 1.1 ± 4.6 × 105 to 2.3 ± 5.2 × 106 cfu/g, respectively; and in the intestine of carp and catfish, 1.4 ± 2.9 × 1010 to 1.7 ± 6.0 × 1011 and 2.7 ± 3.4 × 1010 to 1.0 ± 4.5 × 1011 cfu/g, respectively. Gram-negative rod-shaped bacteria dominated the populations: 90% in carp, 89% in catfish, 80% in water, and 86% in the total populations. Altogether, 14 bacterial species of 10 genera were identified in total populations. Pond water bacteria had a reflection on the bacterial composition of the gills and intestine of carp and catfish. Aeromonas hydrophila, Shewanella putrefaciens, Vibrio cholerae, Staphylococcus sp., and Vibrio vulnificus appeared as the common bacteria in the populations, where the first three were highly significantly abundant (P < 0.0001). Moreover, A. hydrophila was the most significantly dominant bacteria (32%; P < 0.005) among the total populations. Pantoea sp. and Pasteurella pneumotropica were present only in carp and catfish, respectively, but Corynebacterium urealyticum and Micrococcus sp. were present only in pond water.

The present study focused on the use of extra-label drugs in commercial aquaculture. Data was collected through questionnaire interview with 30 drug retailers and 30 commercial aqua farmers in MymensinghSadar and Trishalupazila of Mymensingh district. All together 94 extra-label drugs of different groups were identified which included antibiotics, disinfectants, nutritional supplements, probiotics, gas remover and saline. Six groups of antibiotics having 10 different active compounds with 46 trade names were found in the drug retailer shops.These drugs were primarily prepared either for the use in veterinary or poultry but were found using indiscriminately in aquaculture. All these drugs were marketed by 18 companies in the study areas. It was observed that 83% drugs were not labeled for aquaculture purpose. Majority (77%) of the commercial aqua farmers used extra-label drugs in their farms and 73% of them never received any prescription from qualified personnel before use. Most of the farmers were unable to calculate appropriate doses and had no idea about risk, safety issue and toxicity reaction of using extra-label drugs. Farmers generally got suggestion from the drug retailer regarding application of drugs. The results also revealed that extra-label use of veterinary and poultry drugs in aquaculture is a common practice by commercial aqua farmers. Thus, the use of drugs in aquaculture should have a sufficient regulatory system in place. It is important to produce and use appropriate labeled drugs under a sufficient regulatory system for safe fish production in the aquaculture of Bangladesh [J Bangladesh Agril Univ 2021; 19(1.000): 160-167]

Motor units from the cat tibialis posterior muscle were examined for an association between physiological and biochemical properties. Functionally isolated motor units were categorized on the basis of their physiological properties. This was followed by quantitative microbiochemical analysis of single muscle fibers from each unit, identified in cross sections using the glycogen-depletion method. The activities of malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase distinguished between fatigable (type FF) and fatigue-resistant (types FR and S) units. The activities of both lactate dehydrogenase and adenylokinase were higher in fast- than in slow-contracting units. Cluster analyses, based on both physiological and biochemical properties or on biochemical properties alone, produced groupings identical to types FF, FR, and S. The association between physiological and biochemical properties substantiates the idea that biochemically distinct groups of motor units correspond to physiologically identifiable groups.

Efforts to increase microbial populations in fish intestines tract can be done by utilizing microbes in the fish's digestive tract as probiotics. The application of Bacillus subtilis in fish feed mixtures can provide better growth performance in modifying microbes in the intestines of fish. The aim of this research was to analyze the bacterial community in the intestines of Common Carp(Cyprinus carpio) that have been given probiotics from the bacteria Bacillus subtilis with metagenome sequencing using 16S rRNA through Next Generation Sequencing (NGS) technology. Common carp samples were collected from maintenance conducted with different treatments, namely control fish without probiotic administration and fish treatment with mixed feed probiotic Bacillus subtilis. This research was conducted in the Laboratory of Fisheries Biotechnology, Faculty of Fisheries and Marine Science, Padjajaran University. Then molecular identification was carried out using Illumina's NGS in Novogene, Singapore. The results of the identification of using probiotic bacteria Bacillus subtilis in Common carp(Cyprinus carpio) feed affect the bacterial community in the fish intestine. In the intestines of carp that were given probiotics, the highest abundance was Proteobacteria, Fusobacteriota, and firmicutes.The highest abundance at the genus level in both samples was domiciled by the genus Cetobacterium followed by the Sphingomonas and Aeromonas.

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

Quantitative and qualitative estimation of bacterial flora present in pond water, sediments, gills, and intestine of healthy common carp Cyprinus carpio cultured in Saudi Arabia were performed and identified to species level where possible. Mean total viable bacterial counts in pond water ranged from 1.2 ± 2.9 × 104 to 2.5 ± 3.5 × 105 cfu/mL; in sediments, 9.3 ± 2.1 × 107 to 2.7 ± 3.5 × 109 cfu/g; in gills filaments, 4.3 ± 2.9 × 106 to 1.6 ± 3.9 × 107 cfu/g; and in intestine, 8.7 ± 4.1 × 109 to 5.4 ± 3.2 × 1010 cfu/g. Gram-negative rod-shaped bacteria dominated (76%) the populations. In total, 12 bacterial genera and 15 species were identified. Pond water and sediment bacteria had the reflection on bacterial composition of gills and intestine of carp. Intestinal bacteria showed more diversification in contrast to gill bacteria. Aeromonas hydrophila, Shewanella putrefaciens, Vibrio cholerae, Corynebacterium urealyticum, Vibrio vulnificus, Vibrio sp., and Staphylococcus sp. were the common bacteria in all the populations. In pond water and carp intestine, A. hydrophila, S. putrefaciens, V. cholerae, and C. urealyticum were the most dominant bacteria (prevalence ≥ 10%) where pond sediments and the carp gills experienced with more one dominant bacterium V. vulnificus. Only the A. hydrophila covered one fourth (25%) of the total bacterial populations.

The feed carbon‑nitrogen (C:N) ratio and carbohydrate type (CT – starch vs. non-starch polysaccharides (NSP)) are important due to their potential to improve pond's production and mineralization capacity. This experiment investigates the impact of feed C:N ratio and CT on feed intake of fish, their growth parameters, body composition, and the natural food web in a carp-tilapia pond polyculture. The polyculture consists of catla ( Catla catla ), rohu ( Labeo rohita ), Silver carp ( Hypophthalmichthys molitrix) , and mono-sex Nile tilapia ( Oreochromis niloticus ). The study employs a three-by-two factorial design, including three feed C:N ratios (low, medium, and high) and two feed carbohydrate types (CT – starch-diet (rich in starch but also contains NSP), and NSP-diet (rich in NSP but also contains starch)). Results suggest that increasing the feed C:N ratio does not decrease fish productivity at the pond level. The effect of feed C:N ratio on each of the four species in the polyculture revealed that the growth of rohu and catla did not decrease with a high feed C:N, whereas the growth of silver carp improved and of tilapia decreased. The feed carbohydrate type notably influenced the overall pond fish production, by 27 % when using NSP-diets compared to starch-diets ( P < 0.05). The effect of feed CT on each species showed that the biomass gain and growth of rohu and catla were not affected by NSP-diets, while growth of silver carp and tilapia were improved by NSP-diets. Since silver carp mostly feeds on natural food in pond systems, their enhanced growth when fed high carbohydrate diets (high C:N ratio) – especially those rich in non-starch polysaccharides (NSP-diet) – suggests that these diets improved natural food production in the pond. The production and protein content of tilapia went down when fed a high C:N feed. This confirms that tilapia could not fully compensate for low protein in their diet through the consumption of natural food. This limitation is likely due to the intense grazing pressure exerted by four species on the available natural food, which also obscures the effects of the feed C:N ratio and carbohydrate type on the natural food web parameters. Apart from improved fish production, other observed positive effects of the NSP-diet in this experiment were low TDS in pond water and stable phytoplankton diversity as compared with the starch-diet. Considering that the total fish and carp production remained unaffected by the feed C:N ratio ( P > 0.05), as did the overall fish protein content ( P > 0.05), we can conclude that protein-rich diet (low C:N) is less necessary for pond culture system because a carbohydrate-rich diet (High C:N) can yield similar production. Furthermore, regardless of the feed C:N ratio, the NSP-diet consistently enhanced total pond fish production. This allows feed formulators greater flexibility in choosing plant-based NSP-rich feed ingredients when making pond feed. • Increasing feed C:N ratio does not decrease fish production in pond. • An NSP-rich diet improves fish production in ponds. • An NSP-rich diet may enhance natural food production in ponds. • In pond polyculture, silver carp and tilapia benefit from an NSP-rich diet. • Pond feeds can accommodate higher levels of plant-based by-products.

The purpose of this study was to isolate soy curd forming bacterial strains. Soy curd forming bacteria were isolated from Kimchi, a traditional Korean vegetable food that is fermented using lactic acid bacteria. Among 196 bacterial strains, ten isolates (strain No. 2-2-2, 2-15-2, 2-18-1, 2-19-2, 3-4-1, 3-4-2, 3-8-1, 3-8-3, 3-17-1, 4-39-5) formed firm soy curd. The isolated bacterial strains were identified by molecular biological and biochemical analyses. The genomic DNAs extracted from the isolated bacterial strains were used as a template for PCR amplification of 16S rDNA region. By comparing the results of the 16s rDNA sequences with GenBank data, the isolated strains were identified as Leuconostoc mesenteroides group and Lactobacillus sakei group. The phylogenetic position of soy curd forming strains and their related taxa were investigated using neighbor-joining method. L. mesenteroides group was further identified as L. mesenteroides subsp. dextranicum based on biochemical properties. L. sakei group was named Lactobacillus sp., because it showed a variety of biochemical properties.

Bacteria of the genus Bacillus make up a significant (8–12%) part of the soil microbiome. Manifestation of their biological activity, in particular, the antagonistic and lytic activity against other microorganisms directly depends on their exometabolites. According to the literature, such properties of soil bacteria of the genus Bacillus thus can be related to their various lytic exoenzymes. Aim. To evaluate the role of lytic exoenzymes of the studied soil bacteria strains of the genus Bacillus in the manifestation of their biological (antagonistic, lysing) activity. Methods. The antagonistic activity of bacteria strains of the genus Bacillus against phytopathogenic micromycetes was determined by the method of double culture in Petri dishes on potato-glucose agar. For qualitative analysis of the presence of extracellular enzymes, strains of bacteria of the genus Bacillus were plated on Petri dishes with solid mineral-salt medium and a suitable substrate inducer. The ratio of the diameter of substrate hydrolysis zone to the diameter of the colony was taken as the relative enzymatic activity of the culture. Bacteriolytic activity of the studied strains was determined by the change in optical density of living cells of phytopathogenic bacteria suspension at 540 nm. Results. Six strains of bacteria of the genus Bacillus were selected by the results of preliminary screening, with at least five types of lytic activity, namely proteolytic, chitinase, amylolytic, cellulase, and xylanase of different levels (low, average, high). Analysis of the antagonistic activity of the selected strains of bacteria of the genus Bacillus to the main groups of phytopathogenic bacteria (six test cultures) singled out the strain Bacillus sp. 41 for a careful study of the nature and spectrum of its antagonism. Analysis of the level of antagonistic activity of the selected Bacillus strains against the phytopathogenic micromycetes showed that the minimum decrease of antagonism (the decrease of growth inhibition zones) during the observation period (at the 3rd and 7th days) was in Bacillus sp. 41 strain. Therefore, only this strain showed a stable and relatively wide range of antagonistic activity against phytopathogens of bacterial and fungal etiology. The nature of this antagonism is probably complex and conditioned by the participation of various biochemical mechanisms, in particular, the synthesis of a complex of lytic exoenzymes. To assess the lysing activity of Bacillus strains, three strains with the highest proteolytic and cellulolytic activity of exoenzymes were taken from the six previously chosen. Only Bacillus sp.1913 strain showed high (70%) lytic activity against gram-negative polyphagous phytopathogen Pseudomonas syringae pv. syringae UCM B-1027T. Such activity of the strain did not manifest against the rest of the phytopathogenic test cultures. The high lytic activity of Bacillus sp. 1913 strain may be associated with high activity of exogenous proteases and cellulases of the lytic complex, which is quite consistent with the literature data on the lytic activity of bacteria of the genus Bacillus. Conclusions. The spectrum and activity of lytic exoenzymes of strains of the studied soil bacteria of the genus Bacillus indicate the indirect participation of these enzymes in the manifestation of biological activity (antagonistic and lytic).

Biocontrol potential of endophytic Bacillus spp. associated to wild Solanaceous species and their secondary metabolites against various potato (Solanum tuberosum L.) storage diseases

Aquaculture is currently one of the most rapidly growing food production industries in the world. The increasing global importance for this industry stems primarily from the fact that it is reducing the gap between the supply and demand for fish products. Commercial aquaculture contributes significantly to the economies of many countries since high-value fish species are a major source of foreign exchange. This review looks at the aquaculture industry, the issues raised by the production of fish through aquaculture for food security, the sustainability of the practice to agriculture, what the future holds for the industry in the next 10-20 years, and why there is a need to have available analytical procedures to regulate the safe use of chemicals and veterinary drugs in aquaculture.

Laboratory experiments were conducted to study the efficacy of bacterial isolates and strains on Phelipanche ramosa haustorium initiation at the Bio-pesticides and Bio-fertilizers Department, Environment, Natural Resources and Desertification Research Institute (ENDRI), NCR, Khartoum, Sudan. Fifteen soil borne bacteria isolates (9 organic nitrogen users and 6 mineral nitrogen users) and 3 bacterial strains (Bacillus circulans, B. megatherium var. phosphaticum and Azospirillum brasiliense) were tested. All tested bacterial isolates and strains caused a significant (P≤ 0.5) reduction in haustorium initiation as compared to control in response to DMBQ (2, 6 dimethoxybenzequinone). Organic nitrogen using bacterial isolates ISO5M and ISO22M reduced haustoria by 32 to 54% as compared to the corresponding control. While the mineral nitrogen using bacterial isolate ISO1S and Bacillus circulans strain reduced haustorium by 37 to 45% compared to medium control. The most efficient isolates were identified as Serratia odorifera (ISO22M) with probability 95% and Rhizobium radiobacter (ISO5M) with probability 99%.

Utonga-kupsu, Hentak and Ngari are traditional fermented fish products produced by the Manipuri people living in the North-Eastern part of India. The present study was designed with the aim to isolate, identify and characterize the microorganisms present in these fermented foods. Bacterial pure cultures were isolated using serially diluted samples and were further identified by conventional biochemical tests and Sanger sequencing of 16s rRNA gene. Results show that the number of bacterial count in Nutrient agar and Starch casein agar was 14-20 and 10-16CFU/g, respectively. A total of 46 morphologically different bacterial strains were identified and assigned under the phylum Firmicutes. Identified bacterial strains belonged to the genus Bacillus and Staphylococcus and majority of the isolates were Bacillus subtilis and Staphylococcus nepalensis. Bacterial isolate HNS60 isolated from Hentak and identified as Bacillus subtilis was shown to possess high antimicrobial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Most of the identified bacteria were shown to possess DPPH radical scavenging as well as phosphatase, amylase, protease and cellulose activities. The isolate HNS60 contain high antimicrobial, enzymatic and probiotics activity which might responsible for the possible health benefits of the fermented foods. Utonga-kupsu, Hentak and Ngari thus can be further exploited as a potential source of probiotics and natural antioxidants.

A protein-based electrospun nanofiber from cold water fish gelatin (FG) including bovine lactoferrin (L) as an antimicrobial substance for food packaging applications was developed. Various amounts of L (0, 5, 10, 15, and 20%) were incorporated into FG electrospun nanofibers in order to test antimicrobial activity by disc diffusion method against Pseudomonas fluorescens, Acinetobacter johnsonii, Aeromonas hydrophila, Flavobacterium psychrophilum, Shewanella putrefaciens, and Escherichia coli commonly cause problems in food safety especially in fish products. It was obviously seen that 15% and 20% wt L incorporated FG electrospun nanofibers had significant inhibition zone against all bacterial strains while 5% and 10% L-FG had lower antimicrobial effects. In order to recommend fish gelatin as a food packaging material, mechanical properties should be enhanced to be competitive with synthetic polymers. It was revealed that mechanical strength of gelatin electrospun nanofibers depended on both fiber morphology and bioactive substance content. Neat FG (N-FG ) bead-free electrospun mats had higher fiber diameter (815 ±40 nm) while 15% and 20% L-FG had relatively lower diameter with beaded morphology, i.e., 348 ±32 nm, 229 ± 44 nm respectively. The tensile strength of 20% L-FG mats was significantly lower than the N-FG mats due to beady and thinner morphology. It can be concluded that L-FG electrospun nanofibers with high antimicrobial activity and improvable tensile strength is promising for active packaging applications.Keywords: Electrospinning, Pseudomonas spp., Escherichia coli, Shewanella spp., biodegradable, active packaging

The age and quality of pond bottom soil affect water quality and production of Pangasius hypophthalmus in the tropical environment