16S rRNA Gene Sequence Analysis Research Articles

Comparative Analysis of Intestinal Morphology and Gut Microbiota of Spinibarbus sinensis Under Different Aquaculture Systems

Fish gut health is influenced by various factors, with the environment being a significant one. S. sinensis is a key aquaculture species in China, yet research on the impact of different aquaculture systems on its intestinal health remains limited. This study aims to explore the changes in intestinal morphology and gut microbiota of S. sinensis under two aquaculture systems. The juveniles of S. sinensis were divided into two groups and cultured in traditional ponds (CT) and an in-pond tank culture system (JY), with equal amounts of feed provided daily over a 72-day experimental period. The results showed no significant differences in growth performance metrics, including the specific growth rate, weight gain rate, hepatosomatic index, and viscerosomatic index between the two groups. In terms of intestinal morphology, the JY group villus width was significantly wider than the CT group, and the number of goblet cells in the CT group was significantly higher than that of the JY group (p < 0.05), which suggested that the fish in the JY group may have better intestinal nutrient absorption capacity, while the water quality in the CT group may be worse. The 16S rRNA gene sequencing analysis of the gut microbiota showed that the JY group had a significantly higher Shannon index compared to the CT group (p < 0.05), indicating greater species richness and evenness. Principal Coordinates Analysis (PCoA) revealed a distinct clustering of gut microbiota between the two groups. At the phylum level, the relative abundance of Fusobacteriota was significantly higher in the CT group, whereas Bacteroidota and Proteobacteria were significantly higher in the JY group (p < 0.05). Furthermore, KEGG pathway predictions indicated differences in the potential metabolic capabilities of the gut microbiota between the two groups (p < 0.05). Overall, this study is the first to conduct a comparative analysis of the growth performance, intestinal tissue morphology, and gut microbiota of S. sinensis under two different aquaculture systems, which has valuable implications for the further optimization of aquaculture practices.

Pooled analysis of oral microbiome profiles defines robust signatures associated with periodontitis.

Oral microbial dysbiosis has been associated with periodontitis in studies using 16S rRNA gene sequencing analysis. However, this technology is not sufficient to consistently separate the bacterial species to species level, and reproducible oral microbiome signatures are scarce. Here, using ultra-deep metagenomic sequencing and machine learning tools, we defined a simple two-factor decision tree, based on Tannerella forsythia and Fretibacterium fastidiosum, that was highly associated with periodontitis. Altogether, we defined robust oral microbiome signatures relevant to the pathophysiology of periodontitis that can help define promising targets for microbiome therapeutic modulation when caring for patients with periodontitis.

Biofilm Formation on Excavation Damaged Zone Fractures in Deep Neogene Sedimentary Rock.

Deep underground galleries are used to access the deep biosphere in addition to mining and other engineering applications, such as geological disposal of radioactive waste. Fracture networks developed in the excavation damaged zone (EDZ) are concerned with accelerating mass transport, where microbial colonization might be possible due to the availability of space and nutrients. In this study, microbial biofilms at EDZ fractures were investigated by drilling from a 350-m-deep gallery and subsequent borehole logging at the Horonobe Underground Research Laboratory (URL). By using microscopic and spectroscopic techniques, the dense colonization of microbial cells was demonstrated at the surfaces of the EDZ fractures with high hydraulic conductivity. 16S rRNA gene sequence analysis revealed the dominance of gammaproteobacterial lineages, the cultivated members of which are aerobic methanotrophs. The near-complete genomes from Horonobe groundwater, affiliated with the methanotrophic lineages, were fully equipped with genes involved in aerobic methanotrophy. Although the mediation of aerobic methanotrophy remains to be demonstrated, microbial O2 production was supported by the presence of genes in the near-complete genomes, such as catalase and superoxide dismutase that produce O2 from reactive oxygen species and a nitric oxide reductase gene with the substitutions of amino acids in motifs. It is concluded that the EDZ fractures provide energetically favorable subsurface habitats for microorganisms.

The choice of 16S rRNA gene sequence analysis impacted characterization of highly variable surface microbiota in dairy processing environments.

Accurate knowledge of the microbiota collected from surfaces in food processing environments is important for food quality and safety. This study assessed discrepancies in taxonomic composition and alpha and beta diversity values generated from eight different bioinformatic workflows for the analysis of 16S rRNA gene sequences extracted from the microbiota collected from surfaces in dairy processing environments. We found that the microbiota collected from environmental surfaces varied widely in density (0-9.09 log10 CFU/cm2) and Shannon alpha diversity (0.01-3.40). Consequently, depending on the sequence analysis method used, characterization of low-abundance genera (i.e., below 1% relative abundance) and the number of genera identified (114-173 genera) varied considerably. Some low-abundance genera, including Listeria, varied between the amplicon sequence variant (ASV) and operational taxonomic unit (OTU) methods. Centered log-ratio transformation inflated alpha and beta diversity values compared to rarefaction. Furthermore, the ASV method also inflated alpha and beta diversity values compared to the OTU method (P < 0.05). Therefore, for sparse, uneven, low-density data sets, the OTU method and rarefaction are better for taxonomic and ecological characterization of surface microbiota.IMPORTANCECulture-dependent environmental monitoring programs are used by the food industry to identify foodborne pathogens and spoilage biota on surfaces in food processing environments. The use of culture-independent 16S rRNA amplicon sequencing to characterize this surface microbiota has been proposed as a tool to enhance environmental monitoring. However, there is no consensus on the most suitable bioinformatic analyses to accurately capture the diverse levels and types of bacteria on surfaces in food processing environments. Here, we quantify the impact of different bioinformatic analyses on the results and interpretation of 16S rRNA amplicon sequences collected from three cultured dairy facilities in New York State. This study provides guidance for the selection of appropriate 16S rRNA analysis procedures for studying environmental microbiota in dairy processing environments.

Sorlinia euscelidii gen. nov., sp. nov., a novel acetic acid bacterium isolated from the leafhopper Euscelidius variegatus (Hemiptera: Cicadellidae).

Acetic acid bacteria - belonging to the Acetobacteraceae family - are found in the gut of many sugar-feeding insects. In this study, six strains have been isolated from the hemipteran leafhopper Euscelidius variegatus. While they exhibit high 16S rRNA gene sequence similarities to uncultured members of the Acetobacteraceae family, they could not be unequivocally assigned to any particular type species. Considering the clonality of the six isolates, the EV16PT strain was used as a representative of this group of isolates. The genome sequence of EV16PT is composed of a 2.388 Mbp chromosome, with a DNA G+C content of 57 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence and whole-genome multilocus sequence analysis indicate that EV16PT forms a monophyletic clade with the uncultivated endosymbiont of Diaphorina citri, the Candidatus Kirkpatrickella diaphorinae. Such a phylogenetic clade is positioned between those of Asaia-Swaminathania and Kozakia. The genomic distance metrics based on gene and protein sequences support the proposal that EV16PT is a new species belonging to a yet-undescribed genus. It is a rod-shaped Gram-stain-negative bacterium, strictly aerobic, non-motile, non-spore-forming, showing optimal growth without salt (NaCl) at 30 °C and pH of 6-7. The major quinone is Q10, and the dominant cellular fatty acids (>10%) are C18:l ω7c, C19 : 0 cyclo ω6c, C16 : 0 and C19 : 1 2OH. The polar lipid profile comprises diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine, along with unidentified aminophospholipids, glycophospholipids, aminolipids and lipids. Based on a polyphasic approach, including phylogenetic, phylogenomic, genome relatedness, phenotypic and chemotaxonomic characterisations, EV16PT (= KCTC 8296T, = DSM 117028T) is proposed as a representative of a novel species in a novel genus with the proposed name Sorlinia euscelidii gen. nov., sp. nov., in honour of Prof. Claudia Sorlini, an Italian environmental microbiologist at the University of Milan who inspired the research on microbial diversity, including symbiosis in plants and animals.

A New Bacterial Strain Producing Both of the Surfactin and Fengycin Lipopeptide Biosurfactant with Strong Emulsifications on Crude Oil.

A new lipopeptide-producing strain Cytobacillus sp. R3-1 was isolated from the production water of the Daqing oilfield in China and identified based on 16S rRNA gene sequence analyses. The strain R3-1 is capable of simultaneously producing both of the surfactin and fengycin, the two major families of the lipopeptide biosurfactant. The chemical structures of the surfactin and fengycin were confirmed by a combination of the ESI-MS, FT-IR, and amino acid analyses, and the impact of various temperatures, pH, and NaCl concentrations on the emulsifying activity (E24) was investigated. The lipopeptide biosurfactant produced by the strain R3-1 exhibited strong emulsifying activity with E24 value over 60% on crude oil and different hydrocarbons, including the cyclohexane, hexadecane, benzene, toluene, kerosene, diesel oil, and liquid paraffin. Meanwhile, it showed excellent emulsifying activity across a broad range of conditions of the temperature up to 60 °C, NaCl tolerance up to 100 g/L, and pH values between 5 and 9, which suggests that the strain R3-1 is a valuable microbial candidate for the simultaneous production of the surfactin and fengycin lipopeptide biosurfactant with strong emulsifying properties and stability under diverse environmental conditions and is a potential application in environmental bioremediation and enhanced oil recovery.

The influence of perilipin 5 deficiency on gut microbiome profiles in murine metabolic dysfunction-associated fatty liver disease (MAFLD) and MAFLD-hepatocellular carcinoma.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has emerged as the leading cause of hepatocellular carcinoma (HCC) worldwide. Over the years, Perilipin 5 (PLIN5) has been recognized as a key regulator of both MAFLD and HCC development. In our previous studies we demonstrated that deficiency in Plin5 reduces the severity of MAFLD and HCC in mice. Interestingly, it has been established that patients with MAFLD and HCC exhibit various changes in their gut microbiome profiles. The gut microbiome itself has been shown to play a role in modulating carcinogenesis and the immune response against cancer. Therefore, we conducted a study to investigate the alterations in fecal microbiome composition in wild type (WT) and Plin5-deficient (Plin5 -/-) mice models of MAFLD and MAFLD-induced HCC (MAFLD-HCC). We utilized 16S rRNA gene sequencing analysis to profile the composition of gut bacteria in fecal samples. Notably, we discovered that the absence of Plin5 alone is already associated with changes in gut microbiota composition. Moreover, feeding the mice a Western diet (WD) resulted in additional microbial alterations. Interestingly, Plin5 -/- animals exhibited an enrichment of the beneficial taxa Lactobacillus in both animal models. Our findings identify Plin5 as a major regulator of gut microbiota during the development of MAFLD and MAFLD-HCC.

Identification and characteristics of Plesiomonas shigelloides isolated from Mastacembelus armatus

Plesiomonas shigelloides (P. shigelloides), a dominant pathogenic strain identified as YY001, was successfully isolated and characterized from the hepatopancreas of diseased Mastacembelus armatus (M. armatus) for the first time. The strain was identified through a comprehensive approach that included morphological, physiological, and biochemical characterization, as well as 16S rRNA, 23S rRNA, and gyrB gene sequence analysis. Strain YY001 is classified as a Gram-negative bacterium, with optimal growth conditions observed at 28°C and pH 7.0. Results from the bacterium hemolysis test indicated the absence of hemolytic activity in YY001. However, the strain exhibited amplification of nine virulence genes: nanE, waaA, clpP, ssb, actP, aphA, astA, astD, and astB. Drug-resistance testing showed that YY001 displayed resistance only to penicillin G, oxacillin, norfloxacin, and tetracycline, while remaining sensitive to the majority of other antibiotics. Furthermore, nine drug-resistance genes, including emrD, catB, macB, ksgA, tolC, bacA, MdfA, sul1, and DegP, were detected through amplification. Experimental infection demonstrated that YY001 induced symptoms such as abdominal swelling, visceral edema, and hemorrhage, with hepatopancreatic manifestations being particularly severe. The median lethal dosage of strain YY001 in M. armatus was 9.7×108 colony-forming units (CFU)/mL within 48 h, and 4.9×106 CFU/mL within 192 h under the conditions of intraperitoneal injection. The hepatopancreas, gill, and intestine of infected fish showed obvious pathological symptoms, including tissue loosening, hemorrhage, and cell necrosis. The results of this study provided evidence for a more comprehensive understanding of P. shigelloides strain YY001 infection in M. armatus.

Chronic Exposure to Petroleum-Derived Hydrocarbons Alters Human Skin Microbiome and Metabolome Profiles: A Pilot Study.

Petroleum-derived substances, like industrial oils and grease, are ubiquitous in our daily lives. Comprised of petroleum hydrocarbons (PH), these substances can come into contact with our skin, potentially causing molecular disruptions and contributing to the development of chronic disease. In this pilot study, we employed mass spectrometry-based untargeted metabolomics and 16S rRNA gene sequencing analyses to explore these effects. Superficial skin samples were collected from subjects with and without chronic dermal exposure to PH at two anatomical sites: the fingers (referred to as the hand) and arms (serving as an intersubject variability control). Exposed hands exhibited higher bacterial diversity (Shannon and Simpson indices) and an enrichment of oil-degrading bacteria (ODB), including Dietzia, Paracoccus, and Kocuria. Functional prediction suggested enriched pathways associated with PH degradation in exposed hands vs non-exposed hands, while no differences were observed when comparing the arms. Furthermore, carboxylic acids, glycerophospholipids, organooxygen compounds, phenol ethers, among others, were found to be more abundant in exposed hands. We observed positive correlations among multiple ODB and xenobiotics, suggesting a chemical remodeling of the skin favorable for ODB thriving. Overall, our study offers insights into the complex dysregulation of bacterial communities and the chemical milieu induced by chronic dermal exposure to PH.

Fimbriimonadales performed dissimilatory nitrate reduction to ammonium (DNRA) in an anammox reactor

Bacteria belonging to the order Fimbriimonadales are frequently detected in anammox reactors. However, the principal functions of these bacteria and their potential contribution to nitrogen removal remain unclear. In this study, we aimed to systematically validate the roles of Fimbriimonadales in an anammox reactor fed with synthetic wastewater. High-throughput 16S rRNA gene sequencing analysis revealed that heterotrophic denitrifying bacteria (HDB) were the most abundant bacterial group at the initial stage of reactor operation and the abundance of Fimbriimonadales members gradually increased to reach 38.8 % (day 196). At the end of reactor operation, Fimbriimonadales decreased to 0.9 % with an increase in anammox bacteria. Correlation analysis demonstrated nitrate competition between Fimbriimonadales and HDB during reactor operation. Based on the phylogenetic analysis, the Fimbriimonadales sequences acquired from the reactor were clustered into three distinct groups, which included the sequences obtained from other anammox reactors. Metagenome-assembled genome analysis of Fimbriimonadales allowed the identification of the genes narGHI and nrfAH, responsible for dissimilatory nitrate reduction to ammonium (DNRA), and nrt and nasA, responsible for nitrate and nitrite transport. In a simulation based on mass balance equations and quantified bacterial groups, the total nitrogen concentrations in the effluent were best predicted when Fimbriimonadales was assumed to perform DNRA (R2 = 0.70 and RMSE = 18.9). Moreover, mass balance analysis demonstrated the potential contribution of DNRA in enriching anammox bacteria and promoting nitrogen removal. These results prove that Fimbriimonadales compete with HDB for nitrate utilization through DNRA in the anammox reactor under non-exogenous carbon supply conditions. Overall, our findings suggest that the DNRA pathway in Fimbriimonadales could enhance anammox enrichment and nitrogen removal by providing substrates (nitrite and/or ammonium) for anammox bacteria.

Streptomyces albidocamelliae sp. nov., an endophytic actinomycete isolated from the leaves of Camellia oleifera.

A novel actinobacterium strain, HUAS 14-6T, was isolated from the healthy leaves of Camellia oleifera collected from Changde City, Hunan Province, China. Strain HUAS 14-6T produced tight spiral spore chains consisting of oval or spherical spores with a smooth surface. 16S rRNA gene sequence analysis revealed that strain HUAS 14-6T belonged to the genus Streptomyces and shared highest similarity to Streptomyces bungoensis DSM 41781T (99.72%). Phylogenetic analysis based on 16S rRNA gene sequences indicated strain HUAS 14-6T was in a clade with S. bungoensis DSM 41781T. However, the ANIm and dDDH between strain HUAS 14-6T and S. bungoensis DSM 41781T were 88.16% and 31.2%, respectively, far less than the species-level thresholds. Phylogenetic trees based on the five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) showed that strain HUAS 14-6T formed a separate branch, indicating that this strain could belong to a potential new species. Pairwise MLSA distances between strain HUAS 14-6T and all type strains exhibiting 16S rRNA gene sequence similarities of ≥98.7% to it were much higher than the maximum range of 0.014 recommended for delineating a new Streptomyces species. Based on polyphasic taxonomic study, HUAS 14-6T represents a novel species within the genus Streptomyces for which the name Streptomyces albidocamelliae sp. nov. is proposed. The type strain is HUAS 14-6T (=MCCC 1K08365T=JCM 35920T).

Pathogenomic profiling: Decoding the pathogenic potential of virulent Bacillus cereus associated with mortality of farmed Labeo rohita (Hamilton, 1822), in India

Bacillus cereus is widely used as probiotics in aquaculture. However, present research work identified B. cereus from diseased fish (Labeo rohita) associated with mass mortality in Hooghly, West Bengal, India. The bacterium was identified based on morphological, biochemical characterization and molecular identification, which comprise 16S rRNA gene and housekeeping gene sequencing analysis. Furthermore, the pathogenic potential of the isolates was confirmed by the presence of different toxin-encoding genes viz. hemolytic enterotoxin (hblA, hblC, hblD), non-hemolytic enterotoxin (nheA, nheB, nheC), cytotoxin K (cytK) and enterotoxin FM (entFM), Diarrheal enterotoxin (bceT) and Cereolysin AB. All these toxin-encoding genes were regulated by a master virulence regulating transcription factor plcr, which is also present in this isolate. Additionally, this bacterium also secretes caseinase and lecithinase, which helps the bacteria to invade the host tissue. The intraperitoneal injection of B. cereus at a dose of 3.6× 106 CFU ml−1 resulted in 100 % mortality of L. rohita. The histopathological changes revealed that the bacterium induces severe damage and necrosis in the liver and kidney of the infected fish. To understand the innate immune response once bacteria invade the host, a quantitative gene expression study was carried out using different immune-related genes and stress-related genes viz. Complement factor 3a (C3a), Toll-like receptor (TLR), Myeloid differentiation primary response 88 (Mydd88), Interleukin 6 (IL6), Nucleotide oligomerization domain (NOD)-like receptor, Catalase (CAT), Glutathione peroxidase (GPx) and Superoxide dismutase (SOD) in the hepatic and renal tissue. The over-expression of TLR, NOD and Mydd88 shared a common downstream inflammatory signaling pathway and the upregulation of antioxidant enzymes SOD, CAT and GPx was evident for combating strategies as well as safeguarding the host's cells from oxidative damage. Overall, this study showed that an isolated strain of B. cereus exhibits high pathogenicity, which may be attributed to climate change, urbanization or horizontal gene transfer. This presents a potential threat to the aquaculture industry and raises concerns about the possibility of cross-contamination within the human population.

Unique gel-like colony forming bacterium Novosphingobium pituita sp. nov., isolated from a membrane bioreactor (MBR) treating sewage

A novel, gelatinous, colony-forming, rod-shaped bacterial strain, designated IK01T was isolated from biofilms formed on the membrane surface of a sewage-treating membrane bioreactor (MBR). Strain IK01T produced gelatinous and almost transparent colonies at lower medium concentrations. Fourier transform infrared analysis of the gelatinous colony matrix showed that the matrix could be a biofilm substance. This suggests that strain IK01T is a fouling-causing bacteria in the MBR. Furthermore, 16S rRNA gene sequence analysis showed that strain IK01T was phylogenetically placed in the genus Novosphingobium. The average nucleotide identity values for IK01T and the other 50 species of the genus Novosphingobium ranged from 78.5 to 83.9 %. Correspondingly, the estimated digital DNA-DNA hybridization values ranged from 20.8 to 24.4 %. The genomic DNA G + C content was 66.0 %. The predominant fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c), summed feature 3 (C16:1ω7c and/or C16:1ω6c), and C14:0 2-OH. A polar lipid profile revealed phosphatidylethanolamine, two unidentified phospholipids, and three aminoglycophospholipids as major compounds. The major respiratory quinone was ubiquinone Q-10. Genotypic, chemotaxonomic, and phenotypic analyses characterized the newly identified strain IK01T, as a novel species of the genus Novosphingobium, for which we propose the name Novosphingobium pituita sp. nov. The type strain is IK01T (NBRC 116408T = DSM 116658T).

5-aminosalicylic acid alleviates colitis and protects intestinal barrier function by modulating gut microbiota in mice.

5-aminosalicylic acid (5-ASA) is widely used in the treatment of ulcerative colitis (UC), but its anti-inflammatory mechanism is complex and has not been fully understood. DSS model was used to test the effect of 5-ASA. Tight junction and Ki-67 were detected by western blot, immunofluorescence, and immunohistochemistry or qPCR. 16S rRNA gene sequencing of gut microbiota and subsequent bioinformatics and statistical analysis were performed to identify the specific bacteria which were associated with the treatment effect of 5-ASA. GC-MS was performed to test short-chain fatty acids (SCFAs). Antibiotic-treated mice were used to demonstrate the key role of endogenous gut microbiota. Here, we found that 5-ASA alleviated dextran sulfate sodium (DSS)-induced colitis in mice. Moreover, 5-ASA significantly repaired the intestinal barrier. At the molecular level, 5-ASA markedly raised the expression of tight junction proteins including JAM-A and occludin and cell proliferation marker Ki-67 in mice. In addition, bacterial 16S rRNA gene sequencing and bioinformatics analysis showed that 5-ASA significantly modulated the DSS-induced gut bacterial dysbiosis. In detail, it stimulated the growth of protective bacteria belonging to Faecalibaculum and Dubosiella, which were negatively correlated with colitis parameters, and blocked the expansion of pro-inflammatory bacteria such as Escherichia-Shigella and Oscillibacter, which were positively correlated with colitis in mice. Meanwhile, 5-ASA increased the cecal acetate level. Most notably, 5-ASA was no longer able to treat colitis and reverse gut barrier dysfunction in antibiotic-treated mice that lacked endogenous gut microbiota. Our data suggested that the anti-inflammatory activity of 5-ASA required the inherent intestinal flora, and the gut microbiota was a potential and effective target for the treatment of ulcerative colitis.

Biscuits for the gut: A symphony of FODMAPs and dietary fibre in gut microbiome for irritable bowel syndrome (IBS) management

Emerging evidence suggests gut microbiome alterations significantly influence irritable bowel syndrome (IBS) symptoms with fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) implicated in triggering symptoms.This study investigated the impact of low and high FODMAP biscuit models on gut microbiota including: Biscuit Flour Control (BiFC), Low-FODMAP Control (LFC), Low-FODMAP High-Fibre Prototype (LFP), and Wholemeal Flour Control (WMC). Biscuits were subjected to in vitro digestion which was followed by in vitro colonic fermentation. 16S rRNA gene sequencing and fatty acid analyses were performed at the end of fermentation.Alpha diversity analysis revealed samples fermented with LFP induced higher diversity compared to samples fermented with BiFC and LFC. The relative abundance of Escherichia-Shigella was significantly lower in LFP fermentates than BiFC fermentates. LEfSe analysis showed LFC fermentates were characterized by higher levels of Citrobacter and Ligilactobacillus. Significantly reduced acetate and butyrate levels were found in LFP fermentates compared to LFC fermentates. WMC fermentates had higher acetate and propionate levels compared to other biscuit fermentates.In conclusion, incorporating low-FODMAP, high-fibre food prototypes may enhance gut microbial diversity and reduce potentially pathogenic bacteria such as Escherichia-Shigella. These findings highlight the potential of personalized dietary interventions, such as the manipulation of biscuit formulations, in modulating gut microbiota and managing IBS symptoms. Industrial relevanceThe low FODMAP diet has been developed as an effective method to moderate the symptoms of IBS patients by reducing bloating and gas in the colon. Our results revealed that the low-FODMAP, high-fibre biscuit model proved more effective than the low-FODMAP biscuit for generating a gut microbiota that could contribute to easing IBS symptoms. Future research should focus on improving this formulation and adapting it to treat the different forms of IBS. This study also demonstrates the efficacy of using colon fermentation models to devise food formulations for gut microbiome-associated diseases.

Isolation and Preliminary Characterization of Probiotic Isolates from Healthy Thai Adult Feces

Probiotics are known for health-promoting microorganisms, for examples, assisting gastrointestinal digestion and short chain fatty acid producers. Several current oral human probiotics were derived from human samples; yet, of various ethnic (genetics) and dietary patterns. As probiotics from similar ethnic (genetics) and dietary pattern might confer certain advantages (e.g., more compatibility with hosts), this study isolated and characterized probiotics from healthy Thai adults. To screen for potential probiotic isolates from healthy Thai adult feces, and preliminary characterize these probiotic isolates by biochemical tests and 16S rRNA gene sequencing, following the established Thailand’s Food and Drug Administration (FDA) and National Center for Genetic Engineering and Biotechnology (BIOTEC) guidelines. Twenty fecal samples with clinically verified healthy Thai males and females aged 22-42 years were cultivated on MRS (or M17) media along the broad probiotic screenings (pH 4.0 and 0.1% oxgall) for bacterial probiotic propagation. All derived colonies of different morphologies were continued colony PCR using universal probiotic primers (specific for genera Bifidobacterium, Enterococcus, Lactobacillus, Pediococcus and Streptococcus thermophilus), and only PCR-positive colonies from any of these probiotic primer pair were further cultivation for isolates. Then, these isolates were screened and characterized general probiotic properties that included acid tolerance (pH 2.0), bile salt tolerance (0.3% bile salt), hydrophobicity, antibiotic (ampicillin, ciprofloxacin, erythromycin and vancomycin) susceptibility, no hemolysis activity, catalase, and potassium hydroxide (KOH). The final passing isolates represented potential probiotic isolates that could perhaps maintain in gastrointestinal tract with safety, and were full-length 16S rRNA gene sequenced to identify the genus and species by BLASTN against non-redundant GenBank database. A total of 295 bacterial isolates were obtained from the cultivated on MRS (or M17) media along the broad probiotic screenings from 20 fecal samples. Then, following the general probiotic property characterizations and the full-length 16S rRNA gene sequence analysis, 7 potential isolates were finally derived: 2 Bifidobacterium animalis subsp. lactis, 1 Lactobacillus (new genus name as Limosilactobacillus) reuteri, 1 L. reuteri or L. vaginalis, and 3 Pediococcus pentosaceus. These isolates exhibited strong acid and bile salt tolerances, and relatively high cell surface hydrophobicity. These isolates were susceptible to ampicillin and erythromycin, similar to a reference probiotic control Lactobacillus casei strain Shirota; and the CU13-450 and CU13-451 were also susceptible to vancomycin, similar to a reference control Bifidobacterium breve. Catalase-negative consistent with these bacteria in intestinal tract as anaerobes or facultative anaerobes, and KOH-negative result inferred the Gram-positive bacteria consistent with the full-length 16S rRNA gene sequence identification (Bifidobacterium, Lactobacillus, and Pediococcus are Gram-positive bacteria). No hemolysis activity was observed for these isolates. These 7 probiotic isolates had general probiotic properties and with confirmed the 16S rRNA gene sequences, that follow the Thailand’s FDA and BIOTEC guidelines. However, additional beneficial function investigations, such as in vitro epithelial cell adhesion and in vivo animal model, and whole genome sequencing, should be performed to better validate their possible human health benefits and safety.

Molecular detection and phylogenetic tree assay of Pseudomonus aeruginosa isolated from otitis cases of cats and humans, Iraq

The aim of this study was toward isolation and identification of P. aeruginosa from cats with otitis externa in Basra city Using culture media, Biochemical tests and Polymerase chain reaction. Pseudomonas aeruginosa was isolated from 46 isolates that involved 32 cases out of 75 of feline otitis externa (21.3%) and in 14 (9.3%) from 75 human samples .the clinical signs of otitis externa were head shaking, scratching, excessive ear wax, malodor, pain during palpation, alopecia and pus and/or blood. Culture was done on Pseudomonas chrome agar revealed positive samples with production of greenish-blue pigmentation by the bacteria .polymerase chain reaction revealed that isolates were Pseudomonas aeruginosa using specific primers for P. aeruginosa identification 16S rRNA and the result was positive for the 15 isolates. a phylogenetic tree analysis of P. aeruginosa was performed using the 16S rRNA gene sequence analysis which illustrated the phylogenetic relationship between P. aeruginosa strains isolated from cats and other related genera. In conclusion, this study confirmed the higher prevalence of P. aeruginosa in otitis externa infection of cats when compared to human. The using of molecular assay and 16S rRNA gene contributed effectively in detection of P. aeruginosa. Therefore, this study suggests the application of molecular techniques in identification of different microorganisms. Additionally, moreover studies are necessary to detect the prevalence of P. aeruginosa in different infections in animals as well as human.

Isolation, Molecular Characterization and Determination of Antagonistic Properties of Alkalitolerant Streptomyces Members from Van Lake-Çarpanak Island Soil

Çarpanak Island is one of the islands in Lake Van, the easternmost and largest lake in Türkiye. In this study, soil samples taken from three different locations of Çarpanak island, Lake Van, were inoculated into 4 different selective isolation media using the dilution plate technique, and 42 actinomycete isolates were stocked. PCR amplification of the 16S rRNA gene of 13 isolates selected according to their morphological differences was performed with 27F and 1525R primers. Phylogenetic trees were constructed with the neighbor-joining algorithm in the MEGA 7.0 software. According to 16S rRNA gene sequence analysis, it was determined that 13 isolates belonged to the genus Streptomyces. The antimicrobial activities of 13 isolates against 8 pathogens and the ability of these isolates to produce lipase, amylase, protease and pectinase were determined. Except for CA43 and CA62 isolates, other Streptomyces isolates have the ability to produce lipase. Other Streptomyces isolates except CA59 and CA94 can synthesize amylase. In addition, 3 of the 13 Streptomyces isolates, CA40, CA61 and CA94, do not have the ability to synthesize protease. No isolate is capable of producing pectinase enzyme. As a result of this study, it was observed that most of the Streptomyces isolates had higher lipase enzyme production abilities than other enzyme groups.

Effect of Isoflavone on Muscle Atrophy in Ovariectomized Mice.

Sarcopenia, characterized by muscle mass decline due to aging or other causes, is exacerbated by decreased estrogen levels after menopause in women. Isoflavones, a class of flavonoids acting on estrogen receptors, may have beneficial effects on metabolic disorders. We examined these effects in ovariectomized mice fed a high-fat, high-sucrose diet (HFHSD). At 7 weeks old, female C57BL6/J mice (18-20 g, n = 12) underwent bilateral ovariectomy (OVX), and were then fed a high-fat, high-sucrose diet starting at 8 weeks of age. Half of the mice received isoflavone water (0.1%). Metabolic analyses, including glucose and insulin tolerance tests, were conducted. Muscle analysis involved grip strength assays, next-generation sequencing, quantitative RT-PCR, and western blotting of skeletal muscle after euthanizing the mice at 14 weeks old. Additionally, 16S rRNA gene sequence analysis of the gut microbiota was performed. The results demonstrated that isoflavone administration did not affect body weight, glucose tolerance, or lipid metabolism. In contrast, isoflavone-treated mice had higher grip strength. Gene expression analysis of the soleus muscle revealed decreased Trim63 expression, and western blotting showed inactivation of muscle-specific RING finger protein 1 in isoflavone-treated mice. Gut microbiota analysis indicated higher Bacteroidetes and lower Firmicutes abundance in the isoflavone group, along with increased microbiota diversity. Gene sets related to TNF-α signaling via NF-κB and unfolded protein response were negatively associated with isoflavones. Isoflavone intake alters gut microbiota and increases muscle strength, suggesting a potential role in improving sarcopenia in menopausal women.

Distinct Communities of Bacteria and Unicellular Eukaryotes in the Different Water Masses of Cretan Passage Water Column (Eastern Mediterranean Sea).

Elucidating marine microbiota diversity and dynamics holds significant importance due to their role in maintaining vital ecosystem functions and services including climate regulation. This work aims to contribute in the understanding of microbial ecology and networking in one of the world's most understudied marine regions, the Eastern Mediterranean Sea. High-throughput 16S and 18S rRNA gene sequencing analysis was applied to study the diversity of bacteria and unicellular eukaryotes in the different water masses of the Cretan Passage during two seasonally-different sampling expeditions. We assumed that microbial associations differ between the surface and deepwater masses and created co-occurrence networks to evaluate this hypothesis. Our results unveiled vertical variations in both bacterial and unicellular eukaryotic diversity with species fluctuations indicative of seasonality being recorded in the surface water mass. Heterotrophic taxa and grazers related to organic matter degradation and nutrient cycling were enriched in the deepest water layers. Moreover, surface waters presented a higher number of microbial associations indicating abundant ecological niches compared to the deepest layer, possibly related to the lack of bottom-up resources in the oligotrophic deep ocean. Overall, our data provide insight in a heavily stressed, yet underexplored, marine area that requires further research to unravel the ecological roles of marine microbes. To our knowledge, this is the first study that combines molecular biology tools to provide data on both planktic prokaryotes and unicellular eukaryotes across the different water masses in this marine region of the Eastern Mediterranean basin.