Pectinase from a Fish Gut Bacterium, Aeromonas guangheii (SS6): Production, Cloning and Characterization.

During the present research, 11 gut bacteria were isolated from the freshwater fish, Systomus sarana (General name: olive barb) and upon screening, the strains produced extracellular pectinase enzyme. Among them, the SS6 strain was found to produce a high quantity of 208.731 U/ml pectinase and through molecular characterization the SS6 strain was identified as Aeromonas guangheii. During the culture of SS6 strain, a set of parameters were optimized through the response surface methodology with a Box-Behnken design, for the production of the enzyme. The optimal conditions were found to be 2.11% of maltose, 2.20% of yeast extract, 6.5 of pH, and a temperature of 27.3 °C at 32-h incubation. Under the above conditions, the activity of pectinase production was enhanced to 371 U/ml. The purified pectinase's molecular weight was determined to be ~ 50 kDa (by 10% 2-D PAGE). Totally, nine peptides were identified from the purified pectinase enzyme through the MALDI-TOF-MS analysis and MASCOT tool was used to get the mass spectrum of the peak at 2211 of peptide that indicated the reference pectinase protein. The referenced gene primer (pectate lyases) was PCR amplified and its nucleotide sequence was analyzed. The exo-pelA gene was cloned in pREST vector, which was found to be over expressed in Escherichia coli BL21. The ORF encoded for a mature protein comprising of 425 amino acids (1236 nucleotides) with a predicted molecular weight of ~ 48.7 kDa. The present findings underline the potential of the fish-gut microbes as a source of biotechnologically important enzymes.

Isolation of a bacterial strain from the gut of the fish, Systomus sarana, identification of the isolated strain, optimized production of its protease, the enzyme purification, and partial structural characterization.

BACKGROUND: The present study focuses on the isolation of Bacillus thuringiensis bacterium from the gut of fresh water fish, Systomus sarana, the innovative optimization of culture parameters to produce maximum protease enzyme, by the isolated bacterium, and the elucidation of peptide profile of the protease. And the experimental data and results were authenticated through the response surface method (RSM) and Box-Behnken design (BBD) model. RESULTS: During the RSM optimization, the interaction of the highest concentrations (%) of 2.2 maltose, 2.2 beef extract, and 7.0 pH, at 37 °C incubation, yielded a maximum protease enzyme of 245 U/ml by the fish gut-isolated, B. thuringiensis. The spectral analysis of the obtained enzyme revealed the presence of major functional groups at the range of 610-3852 cm-1 viz., alkynes (-C≡C-H: C-H stretch), misc (P-H phosphine sharp), α, ß-unsaturated aldehydes, and through PAGE analysis, its molecular weight was determined as 27 kDa. The enzyme's MALDI-TOF/MS analysis revealed the presence of 15 peptides from which the R.YHTVCDPR.L peptide has been found to be a major one. CONCLUSIONS: The fish gut-isolated bacterium, B. thuringiensis, SS4 exhibited the potential for high protease production under the innovatively optimized culture conditions, and the obtained result provides scope for applications in food and pharmaceutical industries.

Variability in in vitro biofilm production and antimicrobial sensitivity pattern among Pasteurella multocida strains.

Biofilm production, hitherto an uncharacterized feature among circulating Pasteurella multocida strains, was studied along with the antibiotic susceptibility pattern. On the basis of biofilm formation ability, all the strains were categorized into four groups under six different culture conditions: strong biofilm-forming (22%), moderate (19%), weak (51%), and non-adherent (7%). Strains from serogroups A and B formed significant biofilms in at least one culture condition whereas strains from serogroup D were unable to form biofilms. All strains were found to be susceptible to tetracycline. In addition, the correlation between diverse factors (host, capsule type, clinical condition and the tadD gene) as well as antimicrobial susceptibility in biofilm production were analyzed by Joint distribution models, and showed that enrofloxacin and azithromycin resistant strains were positively correlated with strong biofilm production.

Comparative genetic diversity analysis based on virulence and repetitive genes profiling of circulating Pasteurella multocida isolates from animal hosts.

Virulence associated and/or housekeeping/repetitive genes either in single or multiple copies are being extensively targeted for bacterial pathogen detection and differentiation in epidemiological studies. In the present study, isolation of Pasteurella multocida from different animals and their genetic profiling based on the capsular types, virulence and repetitive elements (ERIC/REP) were carried out. A total 345 clinical samples from apparently healthy and diseased (pneumonic, septicaemia) animals (sheep, goat, pig, cattle, buffalo and rabbits) from different geographical regions of Karnataka, Uttar Pradesh, Mizoram and Assam states of India were screened. A total of 32% of the samples were found positive, of which 41 P. multocida isolates recovered. Virulence profiling of isolates indicated that omp87, ompA, ptfA, sodA, sodC, nanB, fur and exbB were present in 100% of isolates. Whereas, prevalence of other genes were; nanH (90%), ompH (71%), pfhA (63%), plpB (80%), hsf-1 (12%), hsf-2 (37%), pmHAS (78%), toxA (73%), hgbA (37%), hgbB (81%), tbpA (78%) and fimA (98%), among isolates. There was no influence of host or place on prevalence of virulence genes when assessed by fitting a Hierarchial Bayesian ordinal regression model. There was correlation (positive and negative) between broad groups of virulence genes. Both repetitive gene profiles (ERIC and REP) generated multiple amplicons (~200 to ~4000 bp). Cluster analysis with ERIC profiles revealed 5 clusters and 3 non- typable isolates with higher discriminatory power (D = 0.7991) than the REP-PCR profiles (D = 00.734) which revealed 4 clusters and 6 non- typable isolates. The results showed that a considerable level of genetic diversity exists among circulating P. multocida isolates despite belonging to the same geographical origin. The genetic diversity or clustering based on either virulence or repetitive elements among isolates could be largely driven by multiple factors acting together which lead to manifestations of particular disease symptoms.

Comparative extraction of Salmonella bongori derived metabolites and their toxicity on bacterial pathogens, mosquito-larvae, zebrafish-embryo and brine-shrimp: A modified approach.

The present study pertains to two different (standard and adapted) extraction-procedures to extract bacterial extracellular metabolites from the cell-free supernatant (CFS) of S. bongori. Metabolites were extracted with the different polarity solvents using lyophilized-CFS mediated procedure, which revealed more number of compounds than standard procedure. The crude-extracts (CEs) were characterized using FTIR, HPLC and GC-MS analyses. The commonly presented compounds in standard (ME, EA & HE) and lyophilization-mediated extracts (LME, LEA & LHE) were identified through Heat-map analysis. Antibacterial assay: all CEs showed considerable activity on tested MTCC-strains, in which, LME and LEA were found preponderant. Larvicidal bioassay: LME resulted maximum mortality than other CEs on Culex-larvae. Zebrafish embryo-toxicity assay: except HE, all CEs exhibited toxicity at 100 ppm after 96 hpf. Brine shrimp-toxicity assay: ME, LME, EA and LEA have shown significant mortality after 24 h. With these observations, the adapted-extraction-procedure could form significance in the drug development process.

Isolation and growth inhibition potential of entomopathogenic nematodes against three public health important mosquito vectors.

The prevalence of mosquito vector borne diseases and the resistance of mosquitoes to conventional pesticides have been of important public concern to the mosquito endemic countries. Present study was conducted to identify the native bio-larvicidal potential of the entomopathogenic nematodes; Steinernema siamkayai (KPR-4) Heterohabditis indica (KPR-8), Steinernema glaseri and Steinernema abbasi. The isolated nematodes were subsequently cultured and evaluated their larvicidal potential against the larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Among the tested four different nematode species, the S. abassi exerted the highest mortality against A. aegypti (97.33%), the H. indica (KPR-8) against A. stephensi (97.33%) and the S. siamkayai (KPR-4) against C. quinquefasciatus (98.67%). The maximal mosquito-larvicidal property of EPNs was found with the LC50 and LC90 values (IJs/larvae): S. abbasi = 12.47 & 54.35 on A. aegypti; H. indica KPR-8 = 19.88 & 66.81 on A. stephensi and S. siamkayai KPR-4 = 16.69 & 58.97 on C. quinquefasciatus, respectively. The presently generated data on the molecular and larvicidal characteristics of the entomopathogenic nematodes form an important baseline data that upon further research would lead to the development of eco-friendly mosquito-control agent.

Bio-pesticidal effects of Trichoderma viride formulated titanium dioxide nanoparticle and their physiological and biochemical changes on Helicoverpa armigera (Hub.).

The control of agricultural pests through eco-friendly nanopesticides is a challenge of crucial environmental importance nowadays. The current study was aimed to discover a novel biopesticides through Trichoderma viride mediated synthesis of titanium dioxide nanoparticles (TDNPs). The main chemical and physical features of the TDNPs were assessed by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), energy dispersive X-ray (EDX) and size distribution and shape of the NPs studied through the scanning electron microscope (SEM), high-resolution transmission electron microscope (HR-TEM) and dynamic light scattering (DLS). The extracellular synthesized nanoparticles were evaluated for their larvicidal, antifeedant and pupicidal activities against Helicoverpa armigera. TDNPs exhibited highest mortality rate on first (100%), second (100%) and third (92.34%), instar larvae of H. armigera at 100 ppm. The detoxifying enzymes such as, ß-glucosidase and carboxylesterase were reduced whereas glutathione S-transferase increased during the treatment of TDNPs against H. armigera at 100 ppm. No toxic effects were found on Eudrilus eugeniae filter paper and artificial soil assays treated with TDNPs at 100 ppm. However, cypermethrin was toxic to earthworms after 72 h treatment. Therefore, TDNPs could act as significant inhibitors on the development of H. armigera, although, no adverse effect was found on earthworms.