Isolation and characterization of a novel antimicrobial oxatetracyclo ketone from Bacillus stercoris MBTDCMFRI Ba37 isolated from the tropical estuarine habitats of Cochin.

Antimicrobial compounds from the safest source have gained greater relevance because of their wide spectrum of possible applications, especially in aquaculture industry, where pathogenic threat and antibacterial resistance are serious concerns. Bacillus stercoris MBTDCMFRI Ba37 isolated from mangrove environment of tropical estuarine habitats of Cochin exhibited a wide spectrum of antibacterial activity against major aquaculture pathogens belonging to genus Vibrio and Aeromonas. The structural characterization of the antibacterial compound from this strain may help in identifying their role as a biocontrol agent in aquaculture and allied sectors. The highest antibacterial activity was detected in 3rd day culture, grown in a modified Bacillus medium containing 1% of glycerol and 0.5% of glutamic acid at 30 °C, pH 8.0 and 15 ppt saline conditions. The inhibitory activity of the cell free supernatant was evident even at 20% v/v dilution. Preliminary studies on the nature of antibacterial action indicated that the bioactive principle is stable at temperatures up to 70 °C, between pH 6-9 and instable to lyzozyme and proteolytic enzymes. Bioassay guided purification followed by spectroscopic characterization of active fractions of B. stercoris MBTDCMFRI Ba37 revealed that the compound 1-(1-Hydroxyethyl)-1,7,10,12,13,15,17 heptamethyl-16-oxatetracyclo[8.7.0.02,3.012,13]heptadecan-5-one, is responsible for its major antibacterial activity. This is the first report on isolation and characterization of an antibacterial compound from the species B. stercoris. The results of this study indicated that B. stercoris MBTDCMFRI Ba37 has beneficial antibacterial properties which could be useful in developing novel antimicrobial therapeutics against a variety of aquaculture and other pathogens.

Evaluation of in vitro and in vivo potential of Bacillus subtilis MBTDCMFRI Ba37 as a candidate probiont in fish health management.

Bacillus subtilis MBTDCMFRI Ba37 (B. subtilis Ba37), an antibacterial strain isolated from the tropical estuarine habitats of Cochin, was evaluated for in vitro and in vivo potential, and its application as a candidate probiont in fish health management. B. subtilis Ba37 was characterized using their morphological and biochemical properties. It exhibited exoenzymatic activities, tolerance to various physiological conditions and a wide spectrum of antibacterial activity against aquaculture pathogens such as Vibrio and Aeromonas. In co-culture assay, B. subtilis Ba37 inhibited Vibrio anguillarum O1 (V. anguillarum O1) even with the initial cell count of 104 CFUmL-1. Cytotoxicity assay performed using the cell free supernatant (CFS) of B. subtilis Ba37 revealed its non toxic nature. A twenty one days of feeding trial was conducted in juveniles of Etroplus suratensis (E.suratensis) by administrating B. subtilis Ba37 to evaluate its efficacy on growth, immune parameters and antioxidant enzyme activities. Overall the supplementation of B. subtilis Ba37 enhanced significantly (P < 0.05) the survival rate, weight gain, specific growth (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and feed efficiency (FE) of the fed animals as compared with the control. The immune parameters and antioxidant activities such as total protein, alkaline phosphatase (ALP), superoxide dismutase (SOD) and catalase were also improved significantly (P < 0.05) while serum alanine aminotransferase (SGOT) and serum aspartate aminotransferase (SGPT) activities were decreased slightly than the control. After fifteen days of challenge test, the fish fed with B. subtilis Ba37 showed higher relative percentage survival (RPS) than the control. Thus the study indicated the advantages of B. subtilis Ba37 to be used as a candidate probiont, which could be effectively utilized in managing diseases in aquaculture systems and to improve the health of the host.

Antibacterial assay guided isolation of a novel hydroxy-substituted pentacyclo ketonic compound from Pseudomonas aeruginosa MBTDCMFRI Ps04.

Antimicrobial compounds from the natural source have gained greater relevance because of their wide spectrum of possible applications, especially in the aquaculture industry where pathogenic threat and antibacterial resistance are serious concerns. In this regard, Pseudomonas aeruginosa MBTDCMFRI Ps04 (P. aeruginosa Ps04) strain isolated from the tropical estuarine habitats of Cochin was evaluated for its antibacterial potential against major aquaculture pathogens. The physiological conditions for the maximum production of the active metabolite were also optimized. An activity-guided approach was employed further to isolate and characterize the secondary metabolite responsible for the inhibitory potential. It was found that the cell free supernatant (CFS) of P. aeruginosa Ps04 exhibited strong antibacterial activity against major aquaculture pathogens belonging to the genus Vibrio and Aeromonas and retained its potential even at 30% (v/v) dilution. The highest antibacterial activity was detected from 3rd day culture, grown in glycerol alanine media (1% each) as carbon and nitrogen source, respectively, at 30 °C, pH 7.0 and at a salinity of 20 parts per thousand (ppt). The activity of the antagonistic principle was found to be stable against variations in pH (pH 2-pH 12), temperature (up to 120 °C) and enzymatic treatments. Bioassay-guided purification followed by spectroscopic characterization of active fractions of P. aeruginosa Ps04 revealed that the compound 4-Hydroxy-11-methylpentacyclo [11.8.0.02,3.011, 12.016,17]henicosa-1,3,5,8(9),17-penten-14-one is responsible for its major antibacterial activity. The results of this study indicated that P. aeruginosa Ps04 has beneficial antibacterial properties which could be used in developing novel antimicrobial therapeutics against a variety of aquaculture pathogens.

A comparative study of coastal and clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium having a versatile metabolic potential and great ecological and clinical significance. The geographical distribution of P. aeruginosahas revealed the existence of an unbiased genetic arrangement in terrestrial isolates. In contrast, there are very few reports about P. aeruginosa strains from marine environments. The present work was aimed at studying the distribution of P. aeruginosa in coastal waters along the Indian Peninsula and understanding the environmental influence on genotypic, metabolic and phenotypic characteristics by comparing marine and clinical isolates. Of the 785 marine isolates obtained on selective media, only 32 (~4.1%) were identified as P. aeruginosa, based on their fatty acid methyl ester profiles. A low Euclidian distance value (< 2.5) obtained from chemotaxonomic analysis suggested that all the environmental (coastal and marine) isolates originated from a single species. While UPGMA analyses of AP-PCR and phenotypic profiles separated the environmental and clinical isolates, fatty acid biotyping showed overlapping between most clinical and environmental isolates. Our study revealed the genetic diversity among different environmental isolates of P. aeruginosa. While biogeographical separation was not evident based solely on phenotypic and metabolic typing, genomic and metatranscriptomic studies are more likely to show differences between these isolates. Thus, newer and more insightful methods are required to understand the ecological distribution of this complex group of bacteria.

A comparative study of coastal and clinical isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium having a versatile metabolic potential and great ecological and clinical significance. The geographical distribution of P. aeruginosahas revealed the existence of an unbiased genetic arrangement in terrestrial isolates. In contrast, there are very few reports about P. aeruginosa strains from marine environments. The present work was aimed at studying the distribution of P. aeruginosa in coastal waters along the Indian Peninsula and understanding the environmental influence on genotypic, metabolic and phenotypic characteristics by comparing marine and clinical isolates. Of the 785 marine isolates obtained on selective media, only 32 (~4.1%) were identified as P. aeruginosa, based on their fatty acid methyl ester profiles. A low Euclidian distance value (< 2.5) obtained from chemotaxonomic analysis suggested that all the environmental (coastal and marine) isolates originated from a single species. While UPGMA analyses of AP-PCR and phenotypic profiles separated the environmental and clinical isolates, fatty acid biotyping showed overlapping between most clinical and environmental isolates. Our study revealed the genetic diversity among different environmental isolates of P. aeruginosa. While biogeographical separation was not evident based solely on phenotypic and metabolic typing, genomic and metatranscriptomic studies are more likely to show differences between these isolates. Thus, newer and more insightful methods are required to understand the ecological distribution of this complex group of bacteria.

Molecular approach for the rapid detection of Bacillus and Pseudomonas genera--dominant antagonistic groups--from diverse ecological niches using colony multiplex PCR.

Bacillus and Pseudomonas are the dominant groups of bacteria known for their antagonistic potential against many plant and animal pathogens. Presently, exploration of these genera with antagonistic property for disease management of aquaculture system is gaining more importance to overcome the use of antibiotics and related resistance issues. Rapid screening and identification of these genera from diverse bacterial populations by conventional methods is laborious, cost-intensive, and time-consuming. To overcome these limiting factors, in the present study, a colony multiplex PCR (cmPCR) method was developed and evaluated for the rapid detection of Bacillus and Pseudomonas. The technique amplifies the partial 16S rRNA gene of Bacillus and Pseudomonas with a product size of ~1,100 and ~375 bp, respectively, using single forward (BSF2) and two reverse primers (PAGSR and BK1R). Reliability of the cmPCR method was confirmed by screening 472 isolates obtained from ten different eco-stations, of which 133 isolates belonged to Bacillus and 32 to Pseudomonas. The cmPCR method also helped to identify six different Pseudomonas spp. and 14 different Bacillus spp. from environmental samples. Of the total 472 isolates studied, 46 showed antagonistic activity, among which 63 % were Bacillus and 17.4 % were Pseudomonas. Thus, the newly developed molecular approach provides a quick, sensitive, and potential screening tool to detect novel, antagonistically important Bacillus and Pseudomonas genera for their use in aquaculture. Further, it can also act as a taxonomic tool to understand the distribution of these genera from wide ecological niches and their exploitation for diverse biotechnological applications.

Diversity and characterization of antagonistic bacteria from tropical estuarine habitats of Cochin, India for fish health management.

Mortalities due to pathogenic bacteria are a major problem in aquaculture, especially in larval rearing systems. Use of antibiotics to overcome this problem is not an option any more due to the increasing antibiotic resistance among pathogens. The present study aims to understand the diversity of bacteria with antagonistic properties in the tropical estuarine habitats of Cochin, located along the southwest coast of India, and to use them as an alternative to antibiotics in aquaculture. Among the 4,870 isolates screened, approximately 1 % showed significant antibacterial activity against six common aquaculture pathogens belonging to the genera Aeromonas and Vibrio. The antagonistic bacteria were identified as Bacillus (81 %) and Pseudomonas (19 %) using biochemical and 16S rRNA gene sequence homology. The isolates showing stable and higher levels of antibacterial activity were subjected to enzymatic expression profile, antibiotic resistance pattern and abiotic stress tolerance assays. As a result, five Pseudomonas spp. and four Bacillus spp., were identified as promising antagonistic isolates that could be exploited as probionts or microbial products (MP's), to control bacterial diseases in aquaculture rearing systems.