Comparative analysis of commercially available kits for optimal DNA extraction from bovine fecal samples.

In the field of metagenomic research, the choice of DNA extraction methods plays a pivotal yet often underestimated role in shaping the reliability and interpretability of microbial community data. This study delves into the impact of five commercially available DNA extraction kits on the analysis of bovine fecal microbiota. Recognizing the importance of accurate DNA extraction in elucidating microbial community dynamics, we systematically assessed DNA yield, quality, and microbial composition across these kits using 16S rRNA gene sequencing. Notably, the FastDNA spin soil kit yielded the highest DNA concentration, while significant variations in quality were observed across kits. Furthermore, differential abundance analysis revealed kit-specific biases that impacted taxa representation. Microbial richness and diversity were significantly influenced by the choice of extraction kit, with QIAamp DNA stool minikit, QIAamp Power Pro, and DNeasy PowerSoil outperforming the Stool DNA Kit. Principal-coordinate analysis revealed distinct clustering based on DNA isolation procedures, particularly highlighting the unique microbial community composition derived from the Stool DNA Kit. This study also addressed practical implications, demonstrating how kit selection influences the concentration of Gram-positive and Gram-negative bacterial taxa in samples. This research highlights the need for consideration of DNA extraction kits in metagenomic studies, offering valuable insights for researchers striving to advance the precision and depth of microbiota analyses in ruminants.

An antimicrobial metabolite n- hexadecenoic acid from marine sponge-associated bacteria Bacillus subtilis effectively inhibited biofilm forming multidrug-resistant P. aeruginosa.

Effective drug candidates to obstruct the emergence of multidrug-resistant pathogens have become a major concern. A potent antimicrobial producer was isolated from a marine sponge designated as MSI38 and was identified as Bacillus subtilis by 16SrDNA sequencing. The active antimicrobial fraction was purified, and the metabolite was identified as n-hexadecanoic acid by spectroscopic analysis. The fish-borne pathogen Pseudomonas aeruginosa FP012 was found to be multidrug-resistant and poses a risk of disease to food handlers and consumers in general. The compound showed a potent bactericidal effect against P. aeruginosa FP012 with a MIC of 31.33 ± 5.67 mg L-1 and MBC of 36.66 ± 5.17 mg L-1. The time-based biofilm inhibitory potential of MSI38 and ciprofloxacin was analyzed by confocal laser scanning microscopy. A synergistic effect of MSI38 and ciprofloxacin on biofilm showed 85% inhibition.

Effective utilization of wheat gluten by lipopeptide biosurfactant in the formulation of cookies.

BACKGROUND: Gluten-free food products are in demand due to the gluten sensitivity of individuals around the globe. Lipopeptide biosurfactants are widely used in food formulations for wetting, foaming, emulsion stabilization, anti-adhesive and anti-microbial properties. Lipopeptide biosurfactants can be used for gluten utilization and the formulation of food products. RESULTS: The strain Bacillus licheniformis MS48 was isolated from the marine sponge associated bacteria and found to be the ideal biosurfactant producer with an emulsification activity of 70%. Biosurfactant production was optimized using lactose, yeast extract, and sodium chloride (NaCl). The 1,1-diphenyl- 2-picryl hydrazyl (DPPH) radical scavenging activity was found to increase with an increase in the concentration of biosurfactant. The conformation changes in the gluten  due to the treatment of lipopeptide biosurfactant was identified by thiol quantification and scanning electron microscopy. With the addition of lipopeptide MS48 to cookie dough, cookies with softer and smooth textures were developed. The conformation changes in the gluten   in the lipopeptide incorporated  cookie dough was visualized by scanning electron microscopy. The lipopeptide biosurfactant incorporation in cookie dough causes appropriate changes in the structural network of gluten. The lipopeptide-incorporated cookies had improved the spread factor and texture. CONCLUSION: Lipopeptide biosurfactant with effective radical scavenging activity aided in the utilization of gluten. Incorporating lipopeptide in cookie dough assisted in the production of cookies with improved textural and sensory properties. This study is a novel approach to the application of lipopeptide biosurfactant in gluten utilization for the development of cookies. © 2023 Society of Chemical Industry.

Insights into the role of vaginal microbiome in women's health.

The vaginal microbiome is a complex and dynamic microecosystem that fluctuates continually throughout a woman's life. Lactobacillus, a bacterium that possesses antibacterial properties dominates a healthy vaginal microbiome. Bacterial vaginosis is the most common vaginal disorder that has been linked with the dysbiosis of normal vaginal microbiota. Despite the importance of vaginal microbiome, little is known about functions it performs especially, how it helps in protecting the female reproductive tract. This knowledge gap is a significant impediment to the development of effective and feasible clinical treatments that might be required to improve women's health. Thus, a deeper understanding of the functional aspects and not just the composition of vaginal microbiome may aid in improving the diagnostics and treatment strategies. Recent advancement in molecular methods and computational biology have allowed researchers to acquire more knowledge about the vaginal microbiome. The use of metagenomics (culture-independent high-throughput technology) and bioinformatics tools have improved our understanding of the vaginal microbiome. In this review, we have attempted to explore the factors that may alter normal vaginal microbiota homeostasis such as age, sexual behavior, ethnicity, and hygiene, and so forth. We also discuss the role of probiotics in restoring healthy vaginal microbiome.

Degradation intermediates of polyhydroxy butyrate inhibits phenotypic expression of virulence factors and biofilm formation in luminescent Vibrio sp. PUGSK8.

Luminescent vibrios are ubiquitous in the marine environment and are the causative agents of vibriosis and mass mortality in many aquatic animals. In aquatic environments, treatments cannot be limited to the diseased population alone, therefore treatment of the entire aquatic system is the only possible approach. Thus, the use of antibiotics to treat part of the infected animals requires a dose based on the entire biomass, which results in the treatment of uninfected animals as well as non-target normal microbial flora. A treatment method based on anti-virulence or quorum quenching has recently been proposed as an effective treatment strategy for aquatic animals. Polyhydroxy butyrates (PHB) are bacterial storage molecules, which accumulate in cells under nutritional stress. The degradation of PHB releases short-chain ß-hydroxy butyric acid, which may act as anti-infective molecule. To date, there is very limited information on the potential anti-infective and anti-virulence mechanisms involving PHB. In this study, we aim to examine the effect of PHB on inhibition of the virulence cascade of Vibrio such as biofilm formation, luminescence, motility behaviour, haemolysin and quorum sensing. A luminescent Vibrio PUGSK8, tentatively identified as Vibrio campbellii PUGSK8 was tested in vitro for production of extracellular virulence factors and then established as a potential shrimp pathogen based on in vivo challenge experiments. The ability of Vibrio PUGSK8 to form biofilms and the effect of PHB on biofilm formation was tested in a 96-well microtitre-plate assay system. The motility behaviour of Vibrio PUGSK8 was evaluated using twitching, swimming and swarming plate assays. Reporter strains such as Chromobacterium violaceum CV026 and Agrobacterium tumefaciens were used to detect quorum-sensing molecules. Gas chromatography-mass spectrometry spectral analysis was performed to elucidate the fragmentation pattern and structure of N-hexanoyl homoserine lactone. PHB depolymerase activity in Vibrio PUGSK8 was quantified as the amount of the enzyme solution to hydrolyse 1 µg of PHB per min. An in vivo challenge experiment was performed using a gnotobiotic Artemia assay. Of the 27 isolates tested, the Vibrio PUGSK8 strain was selected for target-specific assays based on the high intensity of luminescence and production of virulence factors. The virulence cascade detected in Vibrio PUGSK8 include luminescence, motility behaviour, biofilm formation, quorum sensing and haemolysin production. Thus inhibition/degradation of the virulence cascade would be an effective approach to contain Vibrio infections in aquatic animals. In this report, we demonstrate that the degradation intermediate of PHB effectively inhibits biofilm formation, luminescence, motility behaviour, haemolysin production and the N-acyl-homoserine lactone (AHL)-mediated quorum-sensing pathway in PUGSK8. Interestingly, the growth of Vibrio PUGSK8 remains unaffected in the presence of PHB, with PHB degradation being detected in the media. PHB depolymerase activity in Vibrio PUGSK8 results in the release of degradation intermediates include a short-chain ß-hydroxy butyric acid, which inhibits the virulence cascade in Vibrio PUGSK8. Thus, a molecule that targets quorum sensing and the virulence cascade and which is species/strain-specific could prove to be an effective alternative to antimicrobial agents to control the pathogenesis of Vibrio, and thereby help to contain Vibrio outbreaks in aquatic systems.

A halotolerant thermostable lipase from the marine bacterium Oceanobacillus sp. PUMB02 with an ability to disrupt bacterial biofilms.

A halotolerant thermostable lipase was purified and characterized from the marine bacterium Oceanobacillus sp. PUMB02. This lipase displayed a high degree of stability over a wide range of conditions including pH, salinity, and temperature. It was optimally active at 30 °C and pH 8.0 respectively and was stable at higher temperatures (50-70 °C) and alkaline pH. The molecular mass of the lipase was approximately 31 kDa based on SDS-PAGE and MALDI-ToF fingerprint analysis. Conditions for enhanced production of lipase by Oceanobacillus sp. PUMB02 were attained in response surface method-guided optimization with factors such as olive oil, sucrose, potassium chromate, and NaCl being evaluated, resulting in levels of 58.84 U/ml being achieved. The biofilm disruption potential of the PUMB02 lipase was evaluated and compared with a marine sponge metagenome derived halotolerant lipase Lpc53E1. Good biofilm disruption activity was observed with both lipases against potential food pathogens such as Bacillus cereus MTCC1272, Listeria sp. MTCC1143, Serratia sp. MTCC4822, Escherichia coli MTCC443, Pseudomonas fluorescens MTCC1748, and Vibrio parahemolyticus MTCC459. Phase contrast microscopy, scanning electron microscopy, and confocal laser scanning microscopy showed very effective disruption of pathogenic biofilms. This study reveals that marine derived hydrolytic enzymes such as lipases may have potential utility in inhibiting biofilm formation in a food processing environment and is the first report of the potential application of lipases from the genus Oceanobacillus in biofilm disruption strategies.