Flagellar motility plays important role in Biofilm formation of Bacillus cereus and Yersinia enterocolitica.

Bacteria live either independently as planktonic cells or in organized surface associated colonies called as biofilms. Biofilms play an important role in increased pathogenesis of bacteria and it is assumed that motility is one of the contributing factors towards biofilm initiation. This study was planned to identify the role of flagella in biofilm formation by constructing flagellated (wild type) and physically disrupted variants (non-motile). Total 10 clinical bacterial strains were isolated and characterized. Morphological and biochemical study identified these strains as Enterobacter spp., Pseudomonas spp., Yersinia spp., Escherichia spp., Salmonella spp., Proteus spp., Staphylococcus spp., Streptococcus spp., Lactobacillus spp. and Bacillus spp. Among all strains, two strains including Yersinia spp and Bacillus spp. showed higher antibiotic resistance, hence studied at molecular and physiological level. Biofilm formation capacity of strains was analyzed using three methods including Congo red assay, Test tube assay and Liquid-interface coverslip assay. Afterwards, flagellar disintegration was induced by blending and centrifugation for 5, 10 and 15 minutes. 16S rRNA sequencing showed two strains as Bacillus cereus and Yersinia enterocolitica. Both strains produced significant biofilm by all three above mentioned methods. A motility test of these blended variants showed partial/diminished motility with increased blending time. The significant loss in biofilm formation after 15 minutes blending confirmed the important flagellar contribution to the initiation of biofilm formation. This biofilm defect observed in flagella paralysed/minus variants presumably may be due to defects in attachments to surface at early stages. This study indicated that flagellar motility is crucial initially for surface attachment and subsequently for biofilm formation.

Evaluation of a natural methionine source on broiler growth performance.

BACKGROUND: Methionine, a sulfur-containing amino acid, is essential for the health and growth of broilers, so its optimum level should be provided in broiler diets. Synthetic methionine sources used by poultry nutritionists may cause health hazards in broilers as, during conversion of synthetic methionine to the active form, homocysteine is produced which may be injurious to body tissues when there is a lack of coordination between methyl group donors and acceptors. Thus the present study evaluates the efficacy of a natural methionine source. RESULTS: The comparative growth performance of broilers fed synthetic and/or natural methionine was observed. Results revealed that the basal diet has a lower growth performance (P < 0.05) than all other diets. However, replacement of synthetic methionine with a naturally sourced methionine has no significant effect (P > 0.05) on weight gain, feed consumption or feed conversion ratio. Serum biochemistry values and slaughter data also indicated no effect due to two different methionine sources (P > 0.05). CONCLUSION: The results of the present study show that synthetic methionine may effectively be replaced with a naturally sourced methionine without affecting the health and growth performance of broilers. However, additional research is needed to further explore other natural sources of methionine.

Functional Studies of Cytogenotoxic Potential of Laticifer Proteins of Calotropis procera against Viral Disease.

Plant products are widely used for health and disease management. However, besides their therapeutic effects, some plants also have potential toxic activity. Calotropis procera is a well-known laticifer plant having pharmacologically active proteins playing a therapeutically significant role in curing diseases like inflammatory disorders, respiratory diseases, infectious diseases, and cancers. The present study was aimed to investigate the antiviral activity and toxicity profile of the soluble laticifer proteins (SLPs) obtained from C. procera. Different doses of rubber free latex (RFL) and soluble laticifer protein (ranging from 0.019 to 10 mg/mL) were tested. RFL and SLPs were found to be active in a dose-dependent manner against NDV (Newcastle disease virus) in chicken embryos. Embryotoxicity, cytotoxicity, genotoxicity, and mutagenicity of RFL and SLP were examined on chicken embryos, BHK-21 cell lines, human lymphocytes, and Salmonella typhimurium, respectively. It was revealed that RFL and SLP possess embryotoxic, cytotoxic, genotoxic, and mutagenic activity at higher doses (i.e., 1.25-10 mg/mL), while low doses were found to be safe. It was also observed that SLP showed a rather safer profile as compared to RFL. This might be due to the filtration of some small molecular weight compounds at the time of purification of SLPs through a dialyzing membrane. We suggest that SLPs could be used therapeutically against viral disorders but the dose should be critically monitored.

In vitro Antimicrobial Activity of Biogenically Synthesized Nickel and Zinc Nanoparticles against Selected Pathogenic Bacterial Strains.

Bacterial resistance to already present antibiotics demands for new approaches in field of medicine. Scientists prefer nanoparticles (NPs) due to their promising potential in many applications. Two bacterial strains, Escherichia coli and Bacillus subtilis were used for biogenic synthesis of NPs. Characterization of prepared NPs was accomplished using UV-vis spectroscopy and fourier transform infrared spectroscopy (FTIR). The prepared NPs were confirmed by the color change from pale yellow to having white deposition for Zn NPs while from dark green to light green for Ni NPs. UV-vis spectroscopy of E. coli and B. subtilis based ZnNPs showed highest peak at 354nm and 362nm, respectively. Likewise, E. coli and B. subtilis NiNPs showed peaks at 246 nm and 238 nm, respectively. Antibacterial activity of B. subtilis based ZnNPs showed significant (p ≤ 0.05) zone of inhibition (ZOI; 27.3±0.6) against B. subtilis and 26.66±0.67 against E. coli at 100 mg/mL. Antibacterial activity of E. coli based ZnNPs showed 8.3±0.3 ZOI against B. subtilis and 6.6±0.3 ZOI against E. coli while NiNPs showed (25.0±0.0 mm) (ZOI) against B. subtilis and (25.0 ± 0.3 mm) against E. coli. Minimum inhibitory concentration (MIC) of E. coli ZnNPs showed values of 6.7±0.3 µg/mL for E. coli and 4.7±0.3 µg/mL for B. subtilis. MIC of B. subtilis ZnNPs showed 5.3±0.3 µg/mL for E. coli and 6.6±0.3 µg/mL for B. subtilis while NiNPs showed 33.0±1.0 µg/mL against E. coli and 24.0±1.0 µg/mL against B. subtilis as effective inhibitory concentrations. Minimum bactericidal concentration (MBC) of E. coli ZnNPs showed 7.3±0.3 µg/mL for E. coli and 8.3±0.3 µg/mL for B. subtilis. MBC of B. subtilis ZnNPs showed 7.6±0.3 µg/mL for E. coli and 8.6±0.3 µg/mL for B. subtilis while NiNPs showed 45.7±1.3 µg/mL against E. coli and 33.0±1.0 µg/mL against B. subtilis as effective inhibitory concentrations. It was concluded from the current study that biogenically synthesized ZnNPs and NiNPs are effective as promising antibacterial agents and have potential applications in biomedical fields.

Honey Potential as Antibiofilm, Antiquorum Sensing and Dispersal Agent against Multispecies Bacterial Biofilm.

This study is first to test Pakistani honey bees, Apis dorsata and A. cerana honey samples as anti biofilm, anti quorum sensing (QS) and biofilm dispersal agents honey against multispecies biofilm of bacteria (obtained from obese patients). Briefly, five previously identified isolates Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Morganella morganii and Klebsiella pneumoniae (MT448672-MT448676) were selected. Antibiogram study of all five isolates was tested against three antibiotics viz., erythromycin (20 µg/mL), lincomycin (100 µg/mL) and rifampicin (100 µg/mL). In order to form multispecies biofilm, identified bacteria were grown in batch culture by mixing equal volumes (OD590nm = 0.1) of 2, 3 and 5 bacterial isolates. In total 11 groups (g1-g11) were made. Crystal violet (CV) staining method was used to evaluate the antibiofilm potential and biofilm dispersal potential of both honey samples. QS inhibition in P. aeruginosa was measured following culture supernatant method. Antibiogram study showed significant (p < 0.05) resistance by P. aeruginosa against tested antibiotics. E. coli, M. morganii and K. pneumoniae were significantly susceptible to erythromycin and S. aureus to lincomycin. Both honey samples at 2% and 5% concentrations showed significant (p < 0.05) inhibition potential of multispecies biofilm by all test groups (g1-g11). Though A. dorsata honey significantly inhibited biofilm formation at 2 and 5% against all groups but 2% concentration was highly significant against g2-g4 groups. Regarding A. cerana honey, 2% concentration was significantly effective against g1, g4-g7 and g9-g11 groups. Both honey samples significantly inhibited QS at 2 and 5%. The 5% concentration of A. dorsata honey significantly dispersed biofilm by all groups compared to 2% which showed dispersal potential only by g2 and g3 groups. Accordingly, honey samples showed significant antibiofilm, anti-QS and biofilm dispersal potentials thus can be considered as good alternative to antibiotics.

Biogenic Synthesis, Characterization and Antibacterial Properties of Silver Nanoparticles against Human Pathogens.

Biogenic synthesis of silver nanoparticles (AgNPs) is more eco-friendly and cost-effective approach as compared to the conventional chemical synthesis. Biologically synthesized AgNPs have been proved as therapeutically effective and valuable compounds. In this study, the four bacterial strains Escherichia coli (MT448673), Pseudomonas aeruginosa (MN900691), Bacillus subtilis (MN900684) and Bacillus licheniformis (MN900686) were used for the biogenic synthesis of AgNPs. Agar well diffusion assay revealed to determine the antibacterial activity of all biogenically synthesized AGNPs showed that P. aeruginosa AgNPs possessed significantly high (p < 0.05) antibacterial potential against all tested isolates. The one-way ANOVA test showed that that P. aeruginosa AgNPs showed significantly (p < 0.05) larger zones of inhibition (ZOI: 19 to 22 mm) compared to the positive control (rifampicin: 50 µg/mL) while no ZOI was observed against negative control (Dimethyl sulfoxide: DMSO). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) concentration against four test strains also showed that among all biogenically synthesized NPs, P. aeruginosa AgNPs showed effective MIC (3.3-3.6 µg/mL) and MBC (4.3-4.6 µg/mL). Hence, P. aeruginosa AGNPs were characterized using visual UV vis-spectroscopy, X-ray diffractometer (XRD), fourier transform infrared (FTIR) and scanning electron microscopy (SEM). The formation of peak around 430 nm indicated the formation of AgNPs while the FTIR confirmed the involvement of biological molecules in the formation of nanoparticles (NPs). SEM revealed that the NPs were of approximately 40 nm. Overall, this study suggested that the biogenically synthesized nanoparticles could be utilized as effective antimicrobial agents for effective disease control.

Cloning and over Expression Studies of Ovine Somatotropin cDNA of Kajli (sheep breed) in a Prokaryotic System.

Genetic studies including the quest, cloning and expression of genes encoding proteins responsible for various vital physiological processes and beneficial characteristics of economic perspective have made the biotechnology research progressively auspicious. Due to its great zootechnical and industrial importance somatotropin gene have been cloned from various animal species. Current study was designed to clone mature ovine growth hormone complementary DNA (oGH cDNA) of a sheep breed, Kajli and carry out over expression studies of cloned GH cDNA in a suitable prokaryotic expression system. Sheep GH cDNA was cloned in T/A (thymine / adenine) vector with signal peptide and confirmed by nested polymerase chain reaction (PCR) and restriction digestion. The gene was then ligated in pLEX expression vector and restricted plasmids showed a fragment insert of ~ 600 bps. Restriction analysis confirmed positive clones, were induced for protein expression analysis. The pET vectors (plasmid for expression by T7 RNA polymerase) have an isopropylthio-ß-galactoside (IPTG) inducible strong T7 promoter and Escherichia coli expression strain of BL21 (DE3) pLysS contains DNA fragment from T7 phage which harbors RNA polymerase. Therefore, for expressing recombinant proteins, cells were induced with various IPTG concentrations to optimize expression levels. Cells were induced with different IPTG concentrations (0.1 to 0.8 mM) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated maximum expression level of oGH at 5 hrs after induction of cells with 0.3 mM IPTG concentration with a molecular weight of 22 kDa. As for as cellular localization of protein is concerned accumulation of expressed oGH is observed in inclusion bodies. The successful expression of the cloned GH cDNA of sheep confirmed the functional viability of the clone. The above mentioned technique of genetic engineering has provided to boost the dairy industry by the production of large quantities of recombinant bovine somatotropin (rbST).