Insights into the methodological perspectives for screening polyunsaturated fatty acids-containing bacteria.

Polyunsaturated fatty acids (PUFA) are vital molecules in the pharmaceutical, medical, and nutritional industries. Exploration of bacterial strains capable of producing significant amounts of PUFAs offers a promising avenue for biotechnological applications and industrial-scale production. However, an extensive screening of several samples from diverse sources is highly needed to identify a potential strain. The present study provides the results of the evaluation of 15 different screening methodologies (including changes in existing protocols in terms of reagent concentration, incubation temperature and time) for identifying PUFA-producing bacteria in comparison to the gold standard method (Gas chromatography-mass spectrometry), for the first time. The results determined the most effective techniques for each critical PUFA, leading to an optimized screening process that saves time and resources. The H2O2 plate assay using 0.5% or 1% H2O2 for 72 & 96 h of incubation at 15 °C consistently outperformed others for finding bacteria containing total nutritionally important long chain-PUFA (LC-PUFA), linoleic acid, and arachidonic acid. Whereas the 2,3,5-triphenyl tetrazolium chloride broth assay at 10-15 °C was the most effective and semiquantitative screening methodology for eicosapentaenoic acid (EPA) and alpha-linolenic acid-containing bacteria. Apart from the methodological perspectives, the study also revealed certain potential strains to be targeted in the ongoing research on PUFA-containing bacteria. Further, the manuscript forms the first report on the presence of docosahexaenoic acid (DHA) in Shewanella decolorationis, EPA in Psychrobacter maritimus and Micrococcus aloeverae, and both EPA and DHA in Arthrobacter rhombi. Altogether, the paper generates several thought-provoking insights on the methodological perspectives and identifies potential PUFA-containing bacteria with practical applications in future bacteria-based PUFA research.

In vitro efficacy of aquaculture antimicrobials and genetic determinants of resistance in bacterial isolates from tropical aquaculture disease outbreaks.

Understanding the efficacy of antimicrobials against pathogens from clinical samples is critical for their responsible use. The manuscript presents in vitro efficacy and antimicrobial resistance (AMR) genes in seven species of fish pathogens from the disease outbreaks of Indian aquaculture against oxytetracycline, florfenicol, oxolinic acid, and enrofloxacin. In vitro efficacy was evaluated by minimum inhibitory concentration and minimum bactericidal concentration. The gene-specific PCR screened AMR genes against quinolones (qnrA, qnrB, and qnrS) and tetracyclines (tetM, tetS, tetA, tetC, tetB, tetD, tetE, tetH, tetJ, tetG, and tetY). The results showed that Aeromonas veronii (45%) showed the maximum resistance phenotype, followed by Streptococcus agalactiae (40%), Photobacterium damselae (15%), Vibrio parahaemolyticus (10%), and Vibrio vulnificus (5%). There was no resistance among Vibrio harveyi and Vibrio alginolyticus against the tested antimicrobials. The positive association between tetA, tetB, tetC, tetM, or a combination of these genes to oxytetracycline resistance and qnrS to quinolone resistance indicated their potential in surveillance studies. The prevalence of resistance phenotypes (16.43%) and evaluated AMR genes (2.65%) against aquaculture antimicrobials was low. The resistance phenotype pattern abundance was 0.143. All the isolates showed susceptibility to florfenicol. The results help with the appropriate drug selection against each species in aquaculture practices.

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.

Analysis of nonribosomal peptide synthetase genes in haemolymph microbes of marine crabs.

Nonribosomal peptide synthetases (NRPS) are multi-domain enzymes that have innumerably beneficial health applications. Realizing the significance of marine microorganisms in search for NRPS sequences, study was conducted for analysis of NRPS gene sequences of marine crab haemolymph bacteria for the first time. Strains belonging to five different species were found to have NRPS genes. The study generated NRPS sequences from four bacterial species, for which NRPS gene information was not available earlier. Two new putative adenylation domain signatures were identified from phylum Firmicutes. In silico analysis of amino acid sequences from four species showed less identity (42-50%) to the characterized NRPS compounds that integrate serine residue in active site, suggesting the novelty or uncharacterized nature. Altogether, the study warrants future research exploiting marine crab haemolymph bacteria, an unexplored niche of microbial genetic wealth to discover microbial novel NRPS genes and natural products using emerging tools and technologies.

Curcumin Enriched VCO Protects against 7,12-Dimethyl Benz[a] Anthracene-Induced Skin Papilloma in Mice.

Virgin coconut oil (VCO) and turmeric are traditionally being used in Indian cuisine systems; VCO is a natural combination of medium-chain triglyceride and polyphenols with established pharmacological potential. Curcumin isolated from turmeric is renowned for its anticancer properties, however, with limited clinical success due to poor bioavailability. Considering the lipophilic nature of VCO, curcumin added to VCO is expected to have synergistic/additive actions. In this study, the chemopreventive potential of curcumin enriched VCO (VCr) (4 and 8 mL/Kg orally) was analyzed in 7,12-dimethyl benz[a]anthracene (DMBA;470 nmoles/200 µL/week for two weeks topical)/croton oil (3% v/v in 200 µL acetone twice a week for 6 weeks topical) induced skin papilloma. In DMBA control animals, an average incidence of 13 papilloma/mice (latency period of 11.6 ± 1.5 weeks) was recorded. Pretreatment with VCrH (8 mL/kg) had a 60% inhibition of tumor index, and an increased latency period (12.5 ± 0.9 weeks). Additionally, DMBA/croton oil-induced reduction in glutathione levels and concomitant increase in thiobarbituric acid reactive substance (TBARS) in the skin microenvironment were restored by VCr. The study thus suggests that the VCr promotes antioxidant status in vivo and imparts an improved anticarcinogenic potential. However, further studies are necessary to ascertain the improvement in bioavailability of curcumin .

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.

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.

Degradation of marine crustacean shell wastes through single-stage co-fermentation using proteolytic and chitinolytic bacteria.

Management of crustacean shell waste (SW) through an eco-friendly technique is an environmental obligation to control pollution. The present study showed a novel approach through the simultaneous application of proteolytic and chitinolytic bacteria to effectively degrade unprocessed crustacean SW. For this, the bacteria with concurrent chitinolytic and proteolytic activity (Bacillus subtilis, Priestia megaterium, or Bacillus amyloliquefaciens) were applied either alone or in combination with one proteolytic strain (Paenibacillus alvei) in the unprocessed lobster, crab, and shrimp SW. The method degraded the shells with high deproteinization (> 90%) and demineralization efficiency (> 90%). The degradation was confirmed through scanning electron microscopy. The highest weight loss achieved with shrimp, crab, and lobster shells was 93.67%, 82.60%, and 83.33%, respectively. B. amyloliquefaciens + P. alvei combination produced the highest weight loss in crab and lobster SW, whereas all combinations produced statistically similar weight loss in shrimp SW. There was a concurrent production of N-acetyl glucosamine (up to 532.89, 627.87, and 498.95 mg/g of shrimp, lobster, and crab shell, respectively, with P. megaterium + P. alvei and B. amyloliquefaciens + P. alvei in all SW) and amino acids (4553.8, 648.89, 957.27 µg/g of shrimp, lobster, and crab shells, respectively with B. subtilis + P. alvei in shrimp and B. amyloliquefaciens + P. alvei in crab and lobster). Therefore, it is concluded that, for the first time, efficient degradation of crustacean shell waste was observed using chitinolytic and proteolytic bacterial fermentation with the obtention of byproducts, providing a basis for further application in SW management.

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.

A flow cytometric journey into cell cycle analysis.

Cell cycle involves a series of changes that lead to cell growth and division. Cell cycle analysis is crucial to understand cellular responses to changing environmental conditions. Since its inception, flow cytometry has been particularly useful for cell cycle analysis at single cell level due to its speed and precision. Previously, flow cytometric cell cycle analysis relied solely on the measurement of cellular DNA content. Later, methods were developed for multiparametric analysis. This review explains the journey of flow cytometry to understand different molecular and cellular events underlying cell cycle using various protocols. Recent advances in the field that overcome the shortcomings of traditional flow cytometry and expand its scope for cell cycle studies are also discussed.

Flow cytometry: principles, applications and recent advances.

Flow cytometry (FCM) is a sophisticated technique that works on the principle of light scattering and fluorescence emission by the specific fluorescent probe-labeled cells as they pass through a laser beam. It offers several unique advantages as it allows fast, relatively quantitative, multiparametric analysis of cell populations at the single cell level. In addition, it also enables physical sorting of the cells to separate the subpopulations based on different parameters. In this constantly evolving field, innovative technologies such as imaging FCM, mass cytometry and Raman FCM are being developed in order to address limitations of traditional FCM. This review explains the general principles, main applications and recent advances in the field of FCM.

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.

Heat Shock Proteins (HSPs): A Novel Target for Cancer Metastasis Prevention.

BACKGROUND: Heat shock proteins (HSPs) are predominant molecular chaperones which are actively involved in the protein folding; which is essential in protecting the structure and functioning of proteins during various stress conditions. Though HSPs have important physiological roles, they have been well known for their roles in various pathogenic conditions such as carcinogenesis; however, limited literature has consolidated its potential as an anti-metastatic drug target. OBJECTIVES: The present review outlines the role of different HSPs on cancer progression and metastasis; possible role of HSP inhibitors as anti-neoplastic agents is also discussed. METHODS: The data were collected from PubMed/Medline and other reputed journal databases. The literature that was too old and had no significant role to the review was then omitted. RESULTS: Despite their strong physiological functions, HSPs are considered as good markers for cancer prognosis and diagnosis. They have control over survival, proliferation and progression events of cancer including drug resistance, metastasis, and angiogenesis. Since, neoplastic cells are more dependent on HSPs for survival and proliferation, the selectivity and specificity of HSP-targeted cancer drugs remain high. This has made various HSPs potential clinical and experimental targets for cancer prevention. An array of HSP inhibitors has been in trials and many others are in experimental conditions as anticancer and anti-metastatic agents. Several natural products are also being investigated for their efficacy for anticancer and anti-metastatic agents by modulating HSPs. CONCLUSION: Apart from their role as an anticancer drug target, HSPs have shown to be promising targets for the prevention of cancer progression. Extensive studies are required for the use of these molecules as anti-metastatic agents. Further studies in this line may yield specific and effective antimetastatic agents.

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..(AU)

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.