16S rRNA Gene Research Articles

Environmental microbiota from substrate may interfere with microbiome-based identification of forensically relevant body fluids: A pilot study

The microbiome is a promising tool for identifying body fluids which can be deposited on various substrates at a crime scene. Body fluids collected from crime scenes are not entirely free from substrate microbes whose effects on the microbiome-based identification of body fluids are not well understood. In this study, five body fluids (peripheral blood, menstrual blood, nasal secretions, saliva, and semen) were deposited on sterile swabs, bedspreads, and floors under indoor exposure conditions for 7 days. The microbial communities in the samples were characterized using amplicon sequencing targeted V4 region of 16S rRNA gene. The results showed that the microbial communities of fresh samples deposited on sterile swabs clustered together according to the type of body fluid. The microbial composition of the body fluids deposited on the bedspread and floor is significant different from those deposited on sterile swabs. The microbial communities of mock body fluids were a mixture of microbes from pure body fluids and environmental microbes. FEAST analysis showed that the microbes of mock saliva samples were mainly from pure body fluids (51.53 % and 63.04 % on the bedspread and floor, respectively), but not from substrates (25.70 % and 18.92 % on the bedspread and floor, respectively). Contrary results were observed in peripheral blood, mock nasal secretion, and semen samples. All samples were mainly clustered based on the substrate, but not on the type of body fluid in the PCoA visualization. PERMANOVA results showed that the substrate accounted for more of the variance (R2 = 0.211, P < 0.001) than the type of body fluid (R2 = 0.152, P < 0.001). MicroDecon was used to remove contamination by microbes from the substrate of mock body fluid samples. PCoA and PERMANOVA were performed using decontaminated data. The results showed that samples were no longer clustered based on the substrate, and the type of body fluid (R2 = 0.240, P < 0.001) accounted for more of the variance in the microbial communities of samples than the substrate (R2 = 0.108, P < 0.001). Our results suggest that environmental microbiota from substrates may interfere with the microbiome-based identification of forensically relevant body fluids. To some extent, decontamination could decrease the effects of the substrate on the microbial communities of the samples and enhance the ability to distinguish between the types of body fluids. This pilot study will be valuable in promoting the application of microbiome-based stain analysis in forensics.

ISOLATION AND MOLECULAR IDENTIFICATION OF INDIGENOUS BACTERIOCIN-PRODUCING WEISSELLA CIBARIA

Globally, over 6.22 million deaths are associated with antibiotic resistance. Bacteriocins, a set of antimicrobial peptides synthesized on the ribosomes, are widely viewed as a potential answer to this issue. This is due to their pore-forming ability and antimicrobial activity against antibiotic-resistant pathogens. The aim of this study is to isolate bacteriocin-producing Weissella cibaria and evaluate its antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, Streptococcus sp., Candida sp. and Rhizopus stolonifer. Weissella cibaria man1 was isolated by inoculating deMan Rogosa Sharpe (MRS) broth with small pieces of ripe Mangifera indica (mango), 24-hour incubation at 370C, 10-fold serial dilution and plating on MRS agar. Molecular identification was achieved by DNA extraction, amplification of the 16S rRNA gene through polymerase chain reaction (PCR), agarose gel electrophoresis, gene sequencing, and BLASTN homology searches in the National Center for Biotechnology Information (NCBI). Antimicrobial activity of the bacteriocin was determined by agar well diffusion assay. Mangifera indica (mango) was found to harbor bacteriocin-producing Weissella cibaria man1. The bacteriocin (weissellicin man1) exhibited a broad spectrum of antimicrobial activity. Weissellicin man1 suppressed the growth of several target pathogens (Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, Candida sp. and Rhizopus stolonifer) but had no inhibitory action against Escherichia coli, Streptococcus sp., Staphylococcus aureus. In conclusion, weissellicin man1 from Weissella cibaria man1 has a broad-spectrum of antimicrobial action. These findings will facilitate further evaluation of the antimicrobial potency of weissellicin man1.

Exploring Fresh Lettuce (Lactuca sativa) as a Dairy-Free Probiotic Source

Probiotics are living microorganisms that, when given in appropriate amounts, have optimistic effects on body. This study explores the potential of fresh lettuce (Lactuca sativa) leaves as a probiotic source for individuals with dairy allergies. Gram staining and the catalase test were used to identify the bacteria that were recovered from lettuce leaves as gram-positive, catalase-negative strains. The resistance to antibiotics and their capacity to tolerate low pH and bile salt concentrations both essential for surviving the digestive tract were evaluated. The bacteria showed a high tolerance to bile salts and low pH, but they were susceptible to ampicillin, streptomycin, gentamycin, chloramphenicol, and neomycin. Tests for gas production in the presence of glucose revealed no gas production. Molecular techniques such as PCR and 16S rRNA gene sequencing revealed the presence of Enterococcus lactis, Enterococcus durans, Lactobacillus paracasei, and Lactobacillus casei

Comparative analysis of the intestinal microbiota diversity in wild and farmed Gryllus bimaculatus

Abstract This study aims to investigate the structure and diversity of the intestinal microbiome of Gryllus bimaculatus from wild and farmed environments. Additionally, we sought to identify the core microbes to get information on the microbial diversity of the gut microbiome to the farm condition. We analyzed the microbial community diversity in intestinal samples of both wild and farmed G. bimaculatus using high-throughput sequencing of 16S rRNA gene and ITS rRNA amplicons. Using the PICRUSt2 software, we predicted the functional capabilities of bacterial communities. The analysis indicated significant differences in the diversity of the microbial communities (bacteria) within the gut of crickets under farmed and wild conditions, with notably higher bacterial diversity observed in the gut of farmed crickets compared to those in the wild. The gut bacterial communities exhibited significant similarity across the two conditions, and the variations in diversity are primarily influenced by species with low abundance. Genus-level analysis revealed that the abundance of pathogens, including genera Listeria, Staphylococcus, and Candida, was significantly higher in the gut microbiota of farmed crickets than in the wild ones. By identifying core genera (present in over 80% of the samples), we discovered 19 bacterial genera and 1 fungal genus. The core bacterial genera constituted 60.35% and 87.10% of the cricket’s gut bacterial abundance in farmed and wild environments, respectively. The functional prediction analysis performed using PICRUSt2 identified a significant increase of pathways about “Terpenoids and polyketides metabolism”, and “Infectious disease: bacterial” in the farmed group; as well as a significant decrease in pathways about “Environmental adaptation”, “Development and regeneration”, and the “Nervous system”, compared to the wild group. In conclusion our results showed that the gut microbial community of the G. bimaculatus could be influenced by its environment and diet. However, this influence is primarily manifested in the variation of low-abundance genera, with the gut microbial community remains dominated by a few microbial groups. Therefore, it is speculated that the combination of core and edge species in the gut microbial community of the G. bimaculatus may constitute an effective strategy that not only maintains the core functions of the community but also adapts to environmental changes through the diversity of edge species. The high population density and excessive feeding in the farmed condition could have allowed for the creation of a more diverse gut microbiota for G. bimaculatus, including potential pathogens. Therefore, when breeding G. bimaculatus, the environmental hygiene management should be strengthened to reduce the carrying and transmission of pathogens.

Amycolatopsis melonis sp. nov., a novel protease-producing and cellulose-degrading actinobacterium isolated from soil.

A novel protease-producing and cellulose-degrading actinobacterium, designated strain NEAU-NG30T, was isolated from a melon rhizosphere soil sample collected in Harbin, Heilongjiang Province, China, and established its status using a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NEAU-NG30T was closely related to Amycolatopsis bullii DSM 45802T (98.7%) and Amycolatopsis vancoresmycina DSM 44592T (98.3%). The phospholipid profile contained diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylinositol. The diagnostic sugars in cell hydrolysates were determined to be galactose and arabinose. Cell walls contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinone was MK-9(H4). The major fatty acids were iso-C15:0 and iso-C16:0. Meanwhile, genome analysis of sequences revealed a genome size of 9 338 250 bp and a DNA G+C content of 71.6%. In addition, the average nucleotide identity values and the level of digital DNA-DNA hybridization between strain NEAU-NG30T and its reference strains fall below the thresholds typically used for delineating prokaryote species. According to phenotypic, chemotaxonomic and genotypic studies, it is indicated that strain NEAU-NG30T is considered to be a novel species of the genus Amycolatopsis, for which the name Amycolatopsis melonis sp. nov. is proposed, with NEAU-NG30T (=MCCC 1K08677T=JCM 35654T) as the type strain.

Sustainable Abiotic-Biotic Dechlorination of Perchloroethene with Sulfidated Nanoscale Zero-Valent Iron as Electron Donor Source.

Combining organohalide-respiring bacteria with nanoscale zero-valent iron (nZVI) represents a promising approach for remediating chloroethene-contaminated aquifers. However, limited information is available regarding their synergistic dechlorinating ability for chloroethenes when nZVI is sulfidated (S-nZVI) under the organic electron donor-limited conditions typically found in deep aquifers. Herein, we developed a combined system utilizing a mixed culture containing Dehalococcoides (Dhc) and S-nZVI particles, which achieved sustainable dechlorination with repeated rounds of spiking with 110 μM perchloroethene (PCE). The relative abundance of Dhc considerably increased from 5.2 to 91.5% after five rounds of spiking with PCE, as evidenced by 16S rRNA gene amplicon sequencing. S-nZVI corrosion generated hydrogen as an electron donor for Dhc and other volatile fatty acid (VFA)-producing bacteria. Electron balance analysis indicated that 68.1% of electrons from Fe0 consumed in S-nZVI were involved in dechlorination, and 6.2, 1.1, and 3.2% were stored in formate, acetate, and other VFAs, respectively. The produced acetate possibly served as a carbon source for Dhc. Metagenomic analysis revealed that Desulfovibrio, Syntrophomonas, Clostridium, and Mesotoga were likely involved in VFA production. These findings provide valuable insights into the synergistic mechanisms of biotic and abiotic dechlorination, with important implications for sustainable remediation of electron donor-limited aquifers contaminated by chloroethenes.

Roseobacter sinensis sp. nov., a marine bacterium capable to synthesize arachidonic acid.

Strain WL0113T was isolated from surface seawater of the coast of Lianyungang, Jiangsu province, PR China. Strain WL0113T shared highest 16S rRNA gene sequence similarity with Roseobacter insulae YSTF-M11T (98.8%), followed by R. cerasinus AI77T (98.8%), R. ponti MM-7T (98.0%). Strain WL0113T was Gram-stain-negative, cream, aerobic, non-motile and coccoid- to oval-shaped, and able to grow at pH 6.5-9.0 (optimum, pH 7.0-8.0), at 10-37°C (optimum, 28°C) and in the presence of 1-5% (w/v; optimum, 2.5%) NaCl. Ubiquinone-10 was detected as dominant. The main fatty acids (> 5%) of the strain WL0113T were C16:0, iso-C17:0 3OH, C20:4ω6,9,12,15c (arachidonic acid), and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The major polar lipids include phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, glycophospholipid, unknown aminolipid, unknown phospholipid, and two unknown polar lipids. The ANI and dDDH values between strain WL0113T and Roseobacter cerasinus were 80.4% and 23.0%, respectively. The genomic DNA G + C content of strain WL0113T was 63.1%. Based on these data, it is proposed that strain WL0113T represent novel species of the genus Roseobacter, for which the name Roseobacter sinensis sp. nov. is proposed. The type strain is WL0113T (= GDMCC 1.3082T = JCM 35567T).

A Molecular Approach to Understand the Involvement of Mycoplasma in Suppurative Otitis Cases of Cattle in Chittoor District of Andhra Pradesh

In bovines the economic losses mainly due to the outbreak of diseases, leads to mortality, morbidity, treatment cost and reduced production. Recently in August month of 2024, received 13 bovine suppurativeotitis samples from outbreak in KuppamMandal of Chittoor district. The history having the purulent discharges with foul smell from ear infections of cattle that to crossbreed cattle of all age groups are affected. After 5 days of appearing aural discharges the animals were also shown the nervous signs, head tilting and other clinical findings includes unilateral or bilateral ear droop, epiphora, ptosis, abnormal nystagmus, strabismus, regurgitation, stiff neck, opisthotonus, facial hyperesthesia, purulent aural discharge, nasopharyngeal collapse, recumbency and finally death were noticed in severely affected animals. All the samples were processed for cultural tests by inoculating in PPLO selective broth and selective media PPLO agar then incubated anaerobically at 370C and observed the color development in broth pink to yellow color after 3- 5 days of incubation indicative of positive growth and fried egg micro colonies on selective PPLO agar media after 8-14 days of incubation observed under low power and 40X. The DNA was isolated from all the samples and screened for presence of Mycoplasma by targeting 16s rRNA gene and found that out of 13 samples 10 were positive for genus Mycoplasma and produced the predicted 280bp size product in all positive samples. The suppurativeotitis in cattle usually is caused by Actinomyces spp., Corynebacterium pseudotuberculosis, E. coli, Heamophilus, P.multocida, Pseudomonas spp, Streptococcus spp., and Mycoplasma bovis. This study was targeted only the emerging pathogen i.e the cellwall deficient bacteria Mycoplasma because of infections is hampered by a lack of effective vaccines and specific treatments, leads to increasing trends in antimicrobial resistance. Concluded that isolation and identification of the pathogenic ........

Oral supplementation of heat-killed Enterococcus faecalis strain EC-12 relieves gastrointestinal discomfort and alters the gut microecology in academically stressed students.

Stress significantly affects gastrointestinal and mental health, and the gut microbiota plays a pivotal role in this process. Enterococcus faecalis strain EC-12 (EC-12) is a lactic acid bacterium that has several health benefits. To investigate the impact of oral supplementation with heat-killed EC-12 on the discomfort caused by stress, a randomised, double-blind, placebo-controlled trial was conducted with students under academic stress taking EC-12 (n= 14) or a placebo (n= 13) daily for one week. Improvement in the students' symptoms was assessed using the visual analogue scale. Faecal microbiota was characterised by next-generation sequencing of 16S rRNA genes, and faecal metabolites and short-chain fatty acids were analysed using a GC-MS metabolomics approach. Significant improvements in abdominal pain and rumbling of the stomach were found in the EC-12 group compared to the placebo group, but no changes were observed in mental symptoms or salivary cortisol levels. The relative abundance of E. faecalis significantly increased in the EC-12 group after the trial; however, the composition and diversity of the gut microbiota did not change significantly. Functional analysis of the gut microbiota suggested that EC-12 intake alters specific metabolic pathways. Although the levels of faecal short-chain fatty acids did not change between the groups before and after the trial, EC-12 intake altered the composition of faecal metabolites, with a significant increase in tryptamine levels. The ratio of students with improved symptoms to those with increased tryptamine levels was calculated based on the number of students with elevated faecal tryptamine levels who showed symptomatic improvements. The ratio of improved rumbling stomach was higher than that of other types of digestive discomfort. These results suggest that oral supplementation with EC-12 has a potentially beneficial effect on stress-induced gastrointestinal discomfort, which may occur through alterations in gut microbiota composition and metabolism. This study was registered at the University Hospital Medical Information Network Center (UMIN) under the UMIN ID: UMIN000048184.

DNA Barcoding Using 18S rRNA Gene Fragments for Identification of Tick-Borne Protists in Ticks in the Republic of Korea

The objective of this study was to evaluate the diversity and prevalence of tick-borne protists in the Republic of Korea via DNA barcoding using 18S rRNA gene fragments and PCR. Between 2021 and 2022, questing ticks were collected using the flagging method, with a total of 13,375 ticks collected and pooled into 1003 samples. Of these, 50 tick pools were selected for DNA barcoding targeting the V4 and V9 regions of 18S rRNA using the MiSeq platform. A taxonomic analysis of the amplicon sequence variants identified three genera of protozoa, namely Hepatozoon canis, Theileria luwenshuni, and Gregarine sp. However, the number and abundance of protists detected were different depending on the primer sets, and T. gondii was not identified in DNA barcoding. Furthermore, conventional PCR confirmed the presence of H. canis, Toxoplasma gondii, T. luwenshuni, and Theileria sp. in the collected ticks. This study identified H. canis and T. gondii in Ixodes nipponensis for the first time. It demonstrated that the results of DNA barcoding using 18S rRNA gene fragments can vary depending on the primer sets and further optimization is required for library construction to identify tick-borne protists in ticks. Despite these limitations, the findings highlight the potential of DNA barcoding using 18S rRNA gene fragments for screening the diversity of tick-borne protists in ticks.

Fermentation Profile, Bacterial Community Structure, Co-Occurrence Networks, and Their Predicted Functionality and Pathogenic Risk in High-Moisture Italian Ryegrass Silage

This study aimed to assess the fermentation characteristics, bacterial community structure, co-occurrence networks, and their predicted functionality and pathogenic risk in high-moisture Italian ryegrass (IR; Lolium multiflorum Lam.) silage. The IR harvested at heading stage (208 g dry matter (DM)/kg fresh weight) was spontaneously ensiled in plastic silos (10 L scale). Triplicated silos were opened after 1, 3, 7, 15, 30, and 60 days of fermentation, respectively. The bacterial community structure on days 3 and 60 were investigated using high-throughput sequencing technology, and 16S rRNA-gene predicted functionality and phenotypes were determined by PICRUSt2 and BugBase tools, respectively. After 60 days, the IR silage exhibited good ensiling characteristics indicated by large amounts of acetic acid (~58.7 g/kg DM) and lactic acid (~91.5 g/kg DM), relatively low pH (~4.20), acceptable levels of ammonia nitrogen (~87.0 g/kg total nitrogen), and trace amounts of butyric acid (~1.59 g/kg DM). Psychrobacter was prevalent in fresh IR, and Lactobacillus became the most predominant genus after 3 and 60 days. The ensilage process reduced the complexity of the bacterial community networks in IR silage. The bacterial functional pathways in fresh and ensilaged IR are primarily characterized by the metabolism of carbohydrate and amino acid. The pyruvate kinase and 1-phosphofructokinase were critical in promoting lactic acid fermentation. A greater (p &lt; 0.01) abundance of the “potentially pathogenic” label was noticed in the bacterial communities of ensiled IR than fresh IR. Altogether, the findings indicated that the high-moisture IR silage exhibited good ensiling characteristics, but the potential for microbial contamination and pathogens still remained after ensiling.

Microbial community dynamics in blood, faeces and oral secretions of neotropical bats in Casanare, Colombia

Bats are known reservoirs for a wide range of pathogenic microorganisms, including viruses, bacteria, fungi, helminths, and protozoa, which can be transmitted and infect other zoonotic organisms. Various studies have utilised next-generation sequencing (NGS) to describe the pathogens associated with bats. Although most have characterised microbial communities in specific body fluids, few have analysed the composition and diversity of these microbial communities across different body fluids at the individual level. In this study, we employed two next-generation sequencing techniques: amplicon-based sequencing of the V4 hypervariable region of the 16S- and 18S-rRNA genes and viral metagenomics, to describe the prokaryotic, eukaryotic, and viral communities present in blood, faeces, and oral swab samples collected from two genera of bats (Carollia and Phyllostomus) in the department of Casanare, eastern Colombia. A total of 60 samples corresponding to the three bodily fluids were processed and analysed. The results indicated that the microbial communities across the body fluids were mainly composed of bacteria, fungi, protozoa, and various DNA and RNA viruses, showing a variability of microbial genera and species. The abundances, diversity metrics, and correlations of these microorganisms displayed patterns associated with bat genus and body fluids, suggesting that the ecological characteristics of these microbial communities may be influenced by the ecological and physiological traits of the bats. Additionally, we found similar community compositions of bacteria, some fungal genera, and viruses in the three body fluids, indicating a possible circulation of these microbes within the same bat. This could be due to microbial movement from the gut microbiota to other physiological systems or transmission via blood-feeding vectors. Furthermore, our results revealed the presence of various microbes of public health concern, including Bartonella spp., Mannheimia haemolytica, Rhodotorula spp., Piroplasmida spp., Toxoplasma gondii, Alphacoronavirus spp., and Bat circovirus. The abundance of these pathogenic microbial species across the three bodily fluids suggests potential transmission routes from bats to other organisms, which may contribute to the emergence of zoonotic disease outbreaks. These findings highlight the variability of microorganisms present within the same bat and the different pathogen-host interactions that may regulate the presence and transmission of these zoonotic microbes. Further research is required to elucidate the genomic features, ecological interactions, and biological activities of these microbial communities in bats.

Associations between Gut Microbiota and Microbial Metabolites in Adjuvant- induced Arthritis Rats with Moist Heat Arthralgia Spasm Syndrome.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease. According to Traditional Chinese Medicine (TCM) syndromes theory, moist heat arthralgia spasm syndrome is the most prevalent syndrome of RA patients in the active period. However, the mechanism of alteration of gut microbiota in RA with moist heat arthralgia spasm syndrome has not been reported until now. This study focused on the alteration of gut microbiota in adjuvant-induced arthritis rats with moist heat arthralgia spasm syndrome, elaborated its regulation mechanism, and analyzed the associations between gut microbiota and microbial metabolites. The disease-syndrome combination rat model of RA with moist heat arthralgia spasm syndrome was constructed with Adjuvant-Induced Arthritis (AIA) under damp-heat stimulating. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measure serum biochemical indicators. Damages of ankle joints were observed using hematoxylin and eosin (H&E). 16 small ribosomal subunit RNA (16S rRNA) gene sequencing was conducted to assess the gut microbiota composition and function on feces from rats. Alterations in fecal metabolites profiling were evaluated by fecal metabolomics through Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS). Pearson correlation analysis was performed to explore the associations of altered gut microbiota and microbial metabolites in Model rats. The imbalance of gut microbiota in Model rats was accompanied by metabolic disorders. Lactobacillus, Prevotellaceae_NK3B31_group, Allobaculum, Prevotellaceae_UCG_001, Alloprevotella, and Dubosiella were found to be dominant genera in Model rats. In total, 357 metabolites were significantly altered in Model rats and predominantly enriched into fatty acid degradation and glycerophospholipid metabolism. Pearson correlation analysis showed that TNF-α and IL-1β were associated with Prevotellaceae_Ga6A1_group and 3R-hydroxy-docosan-5S-olide, alpha-N-(3-hydroxy-14-methyl-pentadecanoyl)-ornithine, 17-methyl-trans-4,5- methylenenona-decanoic acid, Semiplenamide F. The key differential microbiota genera and differential microbial metabolites may become important targets for the treatment of RA and provide the theoretical basis for exploring the pathogenesis of RA.

Comparative genomic analysis of two putative novel Idiomarina species from hypersaline miocene deposits

BackgroundHypersaline habitats, as extreme environments, are a great source of well-adapted organisms with unique properties as they have evolved various strategies to cope with these extreme conditions. Bioinformatics and genomic mining may shed light on evolutionary relationships among them. Therefore, the aim of this study was to assess the biodiversity and especially the strategies evolved within the Idiomarina genus, with the primary focus on the taxonomy and genomic adaptations of two novel strains affiliated with Idiomarina genus isolated from unique environment – brines of two Early Miocene salt deposits.ResultsBoth analyzed species belonged to the Idiomarina loihiensis cluster with similarity levels of 16S rRNA gene sequences as high as 99.5% and showed a significant genome size reduction, known characteristic of Idiomarina genomes, though within the genome of Sol25 strain the lowest extent of the carbohydrate utilization genes reduction was observed t among the Idiomarina species. Moreover, the comparative genome analyses indicated that despite both strains being isolated from geographically and geologically similar environments (brines from at least 12 Ma), the species showed higher relatedness to other Idiomarina species than to each other.ConclusionThe present findings highlighted the importance of genomic data in resolving taxonomic uncertainties and understanding of adaptation strategies of extremophiles. Geographic isolation likely contributed to population divergence of the Idiomarina genus, and the recent study offered insights into biogeographic patterns and allopatric speciation of this bacterial group.

Isolation of Heavy Metal-Tolerant and Anti-Phytopathogenic Plant Growth-Promoting Bacteria from Soils.

In this study, by isolating multifunctional soil bacteria that can promote plant development, resist heavy metals, exhibit anti-phytopathogenic action against plant diseases, and produce extracellular enzymes, we hope to improve the effectiveness of phytoremediation techniques. To isolate multifunctional soil bacteria, we used soils with diverse characteristics as isolation sources. To look into the diversity and structural traits of the bacterial communities, We conducted amplicon sequencing of the 16S rRNA gene on five types of soils and predicted functional genes using Tax4Fun2. The isolated bacteria were evaluated for their multifunctional capabilities, including heavy metal tolerance, plant growth promotion, anti-phytopathogenic activity, and extracellular enzyme activity. The genes related to plant growth promotion and anti-phytopathogenic activity were most abundant in forest and paddy soils. Burkholderia sp. FZ3 and FZ5 demonstrated excellent heavy metal resistance (≤ 1 mM Cd and ≤ 10 mM Zn), Pantoea sp. FC24 exhibited the highest protease activity (24.90 μmol tyrosine·g-DCW-1·h-1), and Enterobacter sp. PC20 showed superior plant growth promotion, especially in siderophore production. The multifunctional bacteria isolated using traditional methods included three strains (FC24, FZ3, and FZ5) from the forest and one strain (PC20) from paddy field soil. These results indicate that, for the isolation of beneficial soil microorganisms, utilizing target gene information obtained from isolation sources and subsequently exploring target microorganisms is a valuable strategy.

Co-frequency or contrary? The effects of Qiwei Baizhu Powder and its bioactive compounds on mucosa-associated microbiota of mice with antibiotic-associated diarrhea

Qiwei Baizhu Powder (QWBZP) has been proven effective in treating antibiotic-associated diarrhea (AAD), and the mechanism is associated with regulating the gut microbiota. However, the role of the bioactive compounds of QWBZP in regulating the gut microbiota is still unclear. In this study, 24 mice were divided into a normal control group (N), a model group (R), a QWBZP decoction group (TW), and a QWBZP-TG group (TG). AAD mouse models were established by mixed antibiotic administration. After modeling, mice in the TW group and TG group were treated with QWBZP decoction and QWBZP-TG, respectively. Mice in the N group and R group were gavaged with sterile water. 16S rRNA gene sequencing was used to investigate the changes of mucosa-associated microbiota (MAM) in the small intestine of mice. Moreover, the levels of diamine oxidase (DAO), D-Lactate, secretory immunoglobulin A (sIgA), interleukin 6 (IL-6), IL-10, and tumor necrosis factor-α (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA) kits. The results showed that QWBZP-TG significantly altered the diversity, structure, and abundance of MAM in the AAD mice. QWBZP-TG exerted a stronger suppression effect on Escherichia and Clostridium compared with QWBZP decoction. Meanwhile, QWBZP-TG downregulated the abundance of Lactobacillus, which elicited an opposite effect to QWBZP decoction. Prevotella was the signature bacteria that responded to the QWBZP-TG intervention. Furthermore, both QWBZP decoction and QWBZP-TG decreased the levels of DAO, D-Lactate, sIgA, IL-6, and TNF-α in the AAD mice. The role of glycosides is to help QWBZP ameliorate diarrhea symptoms by inhibiting the proliferation of diarrhea-associated bacteria, reducing inflammation and regulating immunity.

Shen-Bai-Jie-Du decoction suppresses the progression of colorectal adenoma to carcinoma through regulating gut microbiota and short-chain fatty acids

BackgroundShen-Bai-Jie-Du decoction (SBJDD), a traditional Chinese herb formula developed based on evidence-based medicine, is efficacy to reduce the recurrence and carcinogenesis of colorectal adenoma. However, the mechanism of SBJDD to treat colorectal adenoma remains unclear. The present study aims to investigate the efficacy and mechanism of SBJDD on colorectal adenoma carcinogenesis from the aspects of regulating gut microbiota and short-chain fatty acids (SCFAs).MethodsTwenty-one patients diagnosed with colorectal adenoma were recruited in the study and required to take SBJDD for four consecutive weeks. Analysis of gut microbiota was conducted using 16S rRNA gene amplicon sequencing, while levels of SCFAs in fecal and serum samples were determined through HPLC–MS/MS. Additionally, twenty-four Apcmin/+ mice were randomly assigned to normal diet (ND), high-fat diet (HFD), and SBJDD groups. The pharmacological effects and mechanism of SBJDD on colorectal adenoma carcinogenesis were assessed using RT-qPCR, HE staining, IHC staining, Western blot, IF staining, and Flow cytometry assays.ResultsOur clinical study has shown that SBJDD can regulate the gut microbiota composition and enhance SCFAs production in patients with colorectal adenoma. SBJDD alleviated colorectal adenoma formation and carcinogenesis, as well as protected the integrity of the intestinal barrier in the Apcmin/+ mice model compared to the HFD group. Additionally, SBJDD was found to regulate gut microbiota capable of producing SCFAs. G protein-coupled receptors GPR43, GPR41, and GPR109a were effectively activated in the SBJDD group, while HDAC1 and HDAC3 were inhibited. Furthermore, decreased expression levels of interleukin 1 beta (IL-1β) and interleukin 6 (IL-6), along with elevated expression level of interleukin 10 (IL-10), were observed in the colorectal tissue of the SBJDD group. Finally, SBJDD exhibited the ability to reduce the proportion of M1-type macrophages while increasing the proportion of M2-type macrophages.ConclusionsOur study objectively demonstrated the pharmacological effects of SBJDD in inhibiting the progression of colorectal adenoma and investigated its mechanisms in terms of regulating gut microbiota, increasing SCFAs, and reducing colorectal inflammation.

Aspirin inhibits atherosclerosis through macrophage pyroptosis by interacting with Lactobacillus johnsonii and its metabolite butyrate

Abstract Introduction Atherosclerotic cardiovascular disease (ASCVD) is a chronic progressive disease closely related to metabolism and inflammation. Aspirin inhibits cyclooxygenase (COX) and has antiplatelet aggregation effects, considered as the cornerstone of ASCVD treatment. Numerous studies have shown that gut microbiota (GM) and its metabolites affects host metabolism. Studies have also shown that people taking aspirin exhibit specific changes in the microbiota profile. However, whether the interaction of aspirin with GM and its metabolites affects the therapeutic efficacy of aspirin in treating atherosclerosis (AS) have not been reported. Purpose This study aimed to explore the interaction of aspirin with the GM and its metabolites in the treatment of AS, to reveal the specific mechanism of the GM-metabolite axis and provide potential new targets for the combined treatment of AS. Methods To establish the mouse model of atherosclerosis by high-fat diet, and the atherosclerotic plaque burden and composition were analyzed. The role of GM in aspirin anti-atherosclerosis was explored by antibiotics and fecal microbiota transplantation (FMT). The third generation 16S RNA sequencing was used to explore the effects of aspirin on species diversity and abundance of GM, and to screen differential microorganisms for intervention of AS mice. UHPLC-MS and GC-MS were used to explore the changes of intestinal metabolites induced by aspirin. The correlation between gut microbiota and metabolites was also investigated. Finally, in vitro and in vivo experiments were performed to confirm the effects and mechanisms of metabolites on macrophage pyroptosis and atherosclerosis. Results Aspirin has anti-atherosclerosis effect. After removing gut microbiota, the therapeutic effect of aspirin on atherosclerosis was decreased. Three generations of 16S rRNA gene sequencing results showed that aspirin supplementation alters the diversity of gut microbiota, and increased the relative abundance of Lactobacillus johnsonii(L.johnsonii) and Ligilactobacillus murinus(L.murinus). After gavage of L.johnsonii and L.murinus, the plaque burden and macrophage pyroptosis were significantly reduced in AS mice. Colonization with L.johnsonii and L.murinus significantly increased the abundance of probiotics and butyrate level in the feces of AS mice and L.johnsonii and L.murinus were positively correlated with butyrate. Supplementation of butyrate inhibited macrophage pyroptosis and atherosclerosis in vivo. In vitro, butyrate could inhibit the expression of NLRP3/Caspase-1/IL-1β and pyroptosome production, and this process mainly by activating butyrate receptors GPR41, GPR43 and GPR109A. Conclusions The gut microbiota may mediate the anti-AS effect of aspirin by promoting an increase in the abundance of L.johnsonii and L.murinus. Butyrate produced by Lactobacillus may mediate the anti-AS effect of aspirin, and inhibiting macrophage pyroptosis and the progression of AS.

Characteristics of the Gut Microbiota Composition of the Arctic Zone Residents in the Far Eastern Region

Background. In many studies over the past decade, scientists have made a connection between the composition of gut microbiota and human health. A number of publications have shown that gut bacteria are involved in many metabolic and physiological processes of the organism. The composition of the gut microbiome is unique for each person and is formed under the influence of various factors associated with both the individual characteristics of the body and the characteristics of the environment. Different regional characteristics make it necessary for the body to adapt to certain conditions, including temperature fluctuations. Living in areas with low temperatures, such as the Arctic zone, dictates the need for increased energy consumption, which affects the composition of the gut microbiome. Methods. In our study, an extensive questionnaire was conducted among the participants, where many questions were included about the dietary preferences of the study participants, which allowed them us to further divide them into groups according to their diets. Stool samples were collected from participants from 3 groups: Arctic native, Arctic newcomer and the control group. The next step was the isolation of bacterial DNA and sequencing the 16S rRNA gene. The analysis of the results of the diversity of the intestinal microbiota was carried out both with and without taking into account the dietary preferences of the participants. Results. As a result of comparing the intestinal microbiota obtained from residents of the Arctic zone with the gut microbiota of residents of other regions with a milder climate, significant differences are found. These differences may be related to limited food resources and a reduction in the variety of food products characteristic of this Arctic region. t was also found that representatives of the bacterial families Christensenellaceae and Muribaculaceae dominated the control group, both with traditional nutrition and with a dairy-free diet in comparison with the Arctic groups. The control group was dominated by representatives of the Prevotellaceae, Enterobacteriaceae and Comamonadaceae families compared to the Arctic group (with a traditional diet). The results also show that the number of representatives of the families Desulfovibrionaceae (with traditional diet) and Enterobacteriaceae (with milk-free diet) is growing in the Arctic group. Conclusions. In the course of this work, bacterial families characteristic of people living in the Arc-tic zone of the Far Eastern region of the Russian Federation were identified. Poor diet, difficult climatic conditions, and problems with logistics and medical care can have a strong impact on the health of this population. The main type of diet for the inhabitants of the Arctic is the traditional type of diet. They consume a large number of low-cost products, obtainget animal protein from poultry and canned food, and also eat a small number of fresh vegetables and fruits. Such a diet is due to the social status of the study participants and the climatic and geographical features of the region (difficulties in agriculture). With such a diet, we observe a decrease in representatives of the Christensenellaceae, Muribaculaceae, Eubacteriaceae, and Prevotellaceae families and an increase in representatives of the Enterobacteriaceae and Desulfovibrionaceae families among Arctic residents. This imbalance in the futuremay cause, this population may to develop various diseases in the future, including chronic diseases such as obesity, intestinal dysbiosis, inflammatory bowel diseases, and type 2 diabetes.

Soil Properties and Rhizosphere Microbes Community Structure Reveal Nitrogen Uptake Preferences and Nitrogen Use Efficiency of Two Ecotypes of Paphiopedilum micranthum

Paphiopedilum micranthum, an IUCN Red List species, is discontinuously distributed in the karst limestone mountain of southwest China and exhibits ecological specialization, typically through lithophytic and terrestrial ecotypes. Whether the distribution of rhizosphere bacteria and fungi in these different habitats is random or reflects soil preferences requires further investigation. A total of 73 samples from the core distribution areas in China, representing all habitats in two sites, were analyzed for soil differences by comparing edaphic properties and microbial community structure based on high-throughput sequencing of bacterial 16S rRNA genes and fungal ITS region sequences, alongside soil physiochemical data. The results showed no significant differences in microbial community richness and diversity across the heterogeneous habitats. However, significant differences in taxa were observed across various habitats. Dominant bacterial phyla included Actinobacteriota, Proteobacteria and Acidobacteriota, with dominant genera such as Crossiella, Pseudonocardia, 67-14, Mycobacterium and RB41. The primary fungal phyla were Basidiomycota and Ascomycota, featuring prominent genera such as Phlegmacium, Archaeorhizomyces, Trechispora, and Lepiota. There were 16 bacterial genera and 13 fungal genera associated with nitrogen transformation and fixation. Alkali-hydrolyzed nitrogen (AN) was identified as a main driver of soil bacterial and fungal community variation. Based on an analysis of soil physicochemical properties, ammonium nitrogen content was consistently higher than nitrate nitrogen across different habitats. Furthermore, across all heterogeneous habitats, P. micranthum showed no significant differences in nitrate nitrogen, ammonium nitrogen, or their ratio. The nitrogen-use efficiency of P. micranthum ranged from 7.73% to 9.87%, with the highest efficiency observed in the terrestrial habitat of Shedu. These results suggest that P. micranthum prefers habitats rich in organic matter and nitrogen, showing a preference for ammonium nitrogen uptake in natural conditions. Heterogeneous habitats affect plant nitrogen-use efficiency as well as changes in microbial community composition.