16S Ribosomal Ribonucleic Acid Research Articles

Use of FISH-FLOW as a Method for the Identification and Quantification of Bacterial Populations.

The gastrointestinal tract (GIT) harbors the largest group of microbiotas among the microbial communities of the human host. The resident organisms typical of a healthy gut are well adapted to the gastrointestinal environment while alteration of these populations can trigger disorders that may affect the health and well-being of the host. Various investigations have applied different tools to study bacterial communities in the gut and their correlation with gastrointestinal disorders such as inflammatory bowel disease (IBD), obesity, and diabetes. This study proposes fluorescent in situ hybridization, combined with flow cytometry (FISH-FLOW), as an alternative approach for phylum level identification of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria and quantification of target bacteria from the GIT based on analysis of fecal samples, where results are validated by quantitative polymerase chain reaction (qPCR) and 16S ribosomal ribonucleic acid (16s rRNA) sequencing. The results obtained via FISH-FLOW experimental approach show high specificity for the developed probes for hybridization with the target bacteria. The study, therefore, suggests the FISH-FLOW as a reliable method for studying bacterial communities in the gut with results correlating well with those of metagenomic investigations of the same fecal samples.

Effects of Shaofu Zhuyu decoction on intestinal flora and fibrosis in a mouse model of endometriosis

Shaofu Zhuyu decoction has been widely used to treat gynecological diseases; however, its mechanism of action in endometriosis remains unclear. We analyzed Shaofu Zhuyu decoction’s chemical composition using ultra-high performance liquid chromatography-mass spectrometry. In an endometriosis mouse model, ectopic lesions weight measurements and hematoxylin and eosin staining were used to assess the therapeutic efficacy of Shaofu Zhuyu decoction. Effects on intestinal microflora were analyzed using 16S ribosomal ribonucleic acid sequencing, and impacts on focal fibrosis were analyzed using Masson’s trichrome staining. Moreover, fibrosis- and metabolism-related proteins were assessed using immunohistochemistry and enzyme-linked immunosorbent assay. The study identified 157 chemical constituents within Shaofu Zhuyu decoction. Shaofu Zhuyu decoction treatment in mice with endometriosis resulted in a reduction in ectopic lesions weight (P<0.05) and delayed disease progression. Moreover, it improved the diversity and abundance of intestinal flora, and decreased the expression of Lachnospiraceae (P<0.05), Rikenellaceae (P<0.01), Ruminococcaceae (P<0.01), Lachnoclostridium (P<0.05), and unclassified_f__Ruminococcaceae (P<0.05). Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment in carbohydrate, amino acid, and lipid metabolism pathways. Masson’s trichrome staining revealed that compared to the untreated group, the Shaofu Zhuyu decoction group exhibited significantly reduced collagen deposition areas (P<0.001), lower TGF-β1 (P<0.001), COL1A1 (P<0.05), and α-SMA (P<0.01) expression in ectopic lesions, along with increased serum adiponectin (P<0.05), decreased serum leptin (P<0.05), TGF-β1 (P<0.001), and CTGF (P<0.05). Shaofu Zhuyu decoction regulates the intestinal flora of mice with endometriosis while also reducing fibrosis at the lesion site. These findings highlight novel mechanisms for its efficacy in alleviating endometriosis.

Therapeutic delivery of CCL2 modulates immune response and restores host-microbe homeostasis.

Many chronic inflammatory diseases are attributed to disturbances in host-microbe interactions, which drive immune-mediated tissue damage. Depending on the anatomic setting, a chronic inflammatory disease can exert unique local and systemic influences, which provide an exceptional opportunity for understanding disease mechanism and testing therapeutic interventions. The oral cavity is an easily accessible environment that allows for protective interventions aiming at modulating the immune response to control disease processes driven by a breakdown of host-microbe homeostasis. Periodontal disease (PD) is a prevalent condition in which quantitative and qualitative changes of the oral microbiota (dysbiosis) trigger nonresolving chronic inflammation, progressive bone loss, and ultimately tooth loss. Here, we demonstrate the therapeutic benefit of local sustained delivery of the myeloid-recruiting chemokine (C-C motif) ligand 2 (CCL2) in murine ligature-induced PD using clinically relevant models as a preventive, interventional, or reparative therapy. Local delivery of CCL2 into the periodontium inhibited bone loss and accelerated bone gain that could be ascribed to reduced osteoclasts numbers. CCL2 treatment up-regulated M2-macrophage and downregulated proinflammatory and pro-osteoclastic markers. Furthermore, single-cell ribonucleic acid (RNA) sequencing indicated that CCL2 therapy reversed disease-associated transcriptomic profiles of murine gingival macrophages via inhibiting the triggering receptor expressed on myeloid cells-1 (TREM-1) signaling in classically activated macrophages and inducing protein kinase A (PKA) signaling in infiltrating macrophages. Finally, 16S ribosomal ribonucleic acid (rRNA) sequencing showed mitigation of microbial dysbiosis in the periodontium that correlated with a reduction in microbial load in CCL2-treated mice. This study reveals a novel protective effect of CCL2 local delivery in PD as a model for chronic inflammatory diseases caused by a disturbance in host-microbe homeostasis.

Erythrocyte Very Long-Chain Saturated Fatty Acids, Gut Microbiota-Bile Acid Axis, and Incident Coronary Artery Disease in Adults: A Prospective Cohort Study

BackgroundPrior research has highlighted inverse associations between concentrations of circulating very long-chain saturated fatty acids (VLCSFAs) and coronary artery disease (CAD). However, the intricate links involving VLCSFAs, gut microbiota, and bile acids remain underexplored. ObjectivesThis study examined the association of erythrocyte VLCSFAs with CHD incidence, focusing on the mediating role of gut microbiota and fecal bile acids. MethodsThis 10-y prospective study included 2383 participants without CHD at baseline. Erythrocyte VLCSFAs [arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0)] were measured using gas chromatography at baseline, and 274 CHD incidents were documented in triennial follow-ups. Gut microbiota in 1744 participants and fecal bile acid metabolites in 945 participants were analyzed using 16S ribosomal ribonucleic acid sequencing and ultra-performance liquid chromatography-tandem mass spectrometry at middle-term. ResultsThe multivariable-adjusted hazard ratios (95% confidence interval) for CHD incidence in highest compared with lowest quartiles were 0.87 (0.61, 1.25) for C20:0, 0.63 (0.42, 0.96) for C22:0, 0.59 (0.41, 0.85) for C24:0, and 0.57 (0.39, 0.83) for total VLCSFAs. Participants with higher total VLCSFA concentrations exhibited increased abundances of Holdemanella, Coriobacteriales Incertae Sedis spp., Ruminococcaceae UCG-005 and UCG-010, and Lachnospiraceae ND3007 group. These 5 genera generated overlapping differential microbial scores (ODMSs) that accounted for 11.52% of the total VLCSFAs-CHD association (Pmediation = 0.018). Bile acids tauro_α_ and tauro_β_muricholic acid were inversely associated with ODMS and positively associated with incident CHD. Opposite associations were found for glycolithocholic acid and glycodeoxycholic acid. Mediation analyses indicated that glycolithocholic acid, glycodeoxycholic acid, and tauro_α_ and tauro_β_muricholic acid explained 56.40%, 35.19%, and 26.17% of the ODMS-CHD association, respectively (Pmediation = 0.002, 0.008, and 0.020). ConclusionsElevated erythrocyte VLCSFAs are inversely associated with CHD risk in the Chinese population, with gut microbiota and fecal bile acid profiles potentially mediating this association. The identified microbiota and bile acid metabolites may serve as potential intervention targets in future studies.This trial was registered at www.clinicaltrials.gov as NCT03179657.

Reduced Abundance of Phocaeicola in Mucosa-associated Microbiota Is Associated with Distal Colorectal Cancer Metastases Possibly through an Altered Local Immune Environment.

The aim of this study was to identify the microbiota whose decrease in tumor area was associated with the metastatic process of distal colorectal cancer (CRC). Twenty-eight consecutive patients with distal CRC undergoing surgical resection in our hospital were enrolled. Microbiota in 28 specimens from surgically resected colorectal cancers were analyzed using 16S ribosomal ribonucleic acid gene amplicon sequencing and the relative abundance (RA) of microbiota was evaluated. The densities of tumor-infiltrating lymphocytes (TIL) and tumor associated macrophages (TAM) in the colorectal cancers were immunohistochemically evaluated. Phocaeicola was the most abundant microbiota in normal mucosa. The RA of Phocaeicola in tumor tissues tended to be lower than that in normal mucosa although the difference was not significant (p=0.0732). The RA of Phocaeicola at tumor sites did not correlate either with depth of tumor invasion (pT-stage) or tumor size, however they were significantly reduced in patients with nodal metastases (p<0.05) and those with distant metastases (p<0.001). The RA of Phocaeicola at tumor sites showed positive correlation with the densities of CD3(+) or CD8(+) TIL. Since P. vulgatus was the most dominant species (47%) of the Phocaeicola, the RA of P. vulgatus and CRC metastasis and its association with TIL and TAM were also investigated. P. vulgatus showed a similar trend to genus Phocaeicola but was not statistically significant. A relative reduction of Phocaeicola attenuates the local anti-tumor immune response in distal CRC, which may facilitate metastatic spread.

Effects of humic acid application on bacterial diversity under maize cultivation

AbstractThe fundamental ecosystem processes in soil are regulated by microbial communities, and community diversity is implicated by soil environmental conditions. Humic acid (HA) improves soil quality and fertility, stimulating the microbial environment, but the detailed effects remain poorly understood. We investigated the effects of HA rates on soil bacterial diversity, particularly on species richness and community composition in the rhizosphere of corn (Zea mays). Inorganic fertilizer (T2), HA 0.5% (T3), HA 1.0% (T4), HA 1.5% (T5), and HA 2.0% (T6) were applied in soil. Initial soil (O1) and control after harvesting (T1) were included. A total of 3601 operational taxonomic units were captured from the overall sample, and analysis of 16S ribosomal ribonucleic acid amplicon sequencing data indicated that HA did not notably impact species richness. Intriguingly, HA induced changes in bacterial community composition, along with the relative prevalence of specific taxa. Certain associations between soil chemical properties and abundance distribution have been uncovered. Notably, exchangeable Mg2+, Ca2+, and available phosphorus were strongly related to the relative abundance of bacterial phyla. Furthermore, HA potentially shaped the specific bacterial taxa, as the application of HA at different rates had distinct effects on the member of bacterial abundance of each taxon. These findings enhance our understanding of communities potentially being increased or shifted by HA rate addition in short‐term corn cultivation.

Neglected role of iron redox cycle in direct interspecies electron transfer in anaerobic methanogenesis: Inspired from biogeochemical processes

Anaerobic digestion is an indispensable technical option towards green and low-carbon wastewater treatment, with interspecies electron transfer (IET) playing a key role in its efficiency and operational stability. The exogenous semiconductive iron oxides have been proven to effectively enhance IET, while the cognition of the physicochemical-biochemical coupling stimulatory mechanism was circumscribed and remains to be elucidated. In this study, semiconductive iron oxides, α-Fe2O3, γ-Fe2O3, α-FeOOH, and γ-FeOOH were found to significantly enhance syntrophic methanogenesis by 76.39, 72.40, 37.33, and 32.64% through redirecting the dominant IET pathway from classical interspecies hydrogen transfer to robust direct interspecies electron transfer (DIET). Their alternative roles as electron shuttles potentially substituting for c-type cytochromes were conjectured to establish an electron transport matrix associated with conductive pili. Distinguished from the conventional electron conductor mechanism of conductive Fe3O4, semiconductive iron oxides facilitated DIET intrinsically through the capacitive Fe(III/II) redox cycles coupled with secondary mineralization. The growth of Aminobacterium, Sedimentibacter, and Methanothrix was enriched and the gene copy numbers of Geobacteraceae 16S ribosomal ribonucleic acid were selectively flourished by 2.0-∼4.5- fold to establish a favorable microflora for DIET pathway. Metabolic pathways of syntrophic acetogenesis from propionate/butyrate and CO2 reduction methanogenesis were correspondingly promoted. The above findings provide new insights into the underlying mechanism of iron minerals enhancing the DIET-oriented pathway and offer paradigms for redox-mediated energy harvesting biological wastewater treatment.

Developing a microbial community structure index (MCSI) as an approach to evaluate and optimize bioremediation performance.

Much attention is placed on organohalide-respiring bacteria (OHRB), such as Dehalococcoides, during the design and performance monitoring of chlorinated solvent bioremediation systems. However, many OHRB cannot function effectively without the support of a diverse group of other microbial community members (MCMs), who play key roles fermenting organic matter into more readily useable electron donors, producing corrinoids such as vitamin B12, or facilitating other important metabolic processes or biochemical reactions. While it is known that certain MCMs support dechlorination, a metric considering their contribution to bioremediation performance has yet to be proposed. Advances in molecular biology tools offer an opportunity to better understand the presence and activity of specific microbes, and their relation to bioremediation performance. In this paper, we test the hypothesis that a specific microbial consortium identified within 16S ribosomal ribonucleic acid (rRNA) gene next generation sequencing (NGS) data can be predictive of contaminant degradation rates. Field-based data from multiple contaminated sites indicate that increasing relative abundance of specific MCMs correlates with increasing first-order degradation rates. Based on these results, we present a framework for computing a simplified metric using NGS data, the Microbial Community Structure Index, to evaluate the adequacy of the microbial ecosystem during assessment of bioremediation performance.

Comparative evaluation of 16S rRNA primer pairs in identifying nitrifying guilds in soils under long-term organic fertilization and water management.

Compared with 454 sequencing technology, short-read sequencing (e.g., Illumina) technology generates sequences of high accuracy, but limited length (<500 bp). Such a limitation can prove that studying a target gene using a large amplicon (>500 bp) is challenging. The ammonia monooxygenase subunit A (amoA) gene of ammonia-oxidizing archaea (AOA), which plays a crucial part in the nitrification process, is such a gene. By providing a full overview of the community of a functional microbial guild, 16S ribosomal ribonucleic acid (rRNA) gene sequencing could overcome this problem. However, it remains unclear how 16S rRNA primer selection influences the quantification of relative abundance and the identification of community composition of nitrifiers, especially AOA. In the present study, a comparison was made between the performance of primer pairs 338F-806R, 515F-806R, and 515F-907R to a shotgun metagenome approach. The structure of nitrifier communities subjected to different long-term organic matter amendment and water management protocols was assessed. Overall, we observed higher Chao1 richness diversity of soil total bacteria by using 515F-806R compared to 338F-806R and 515F-907R, while higher Pielou's evenness diversity was observed by using 515F-806R and 515F-907R compared to 338F-806R. The studied primer pairs revealed different performances on the relative abundance of Thaumarchaeota, AOB, and NOB. The Thaumarchaeota 16S rRNA sequence was rarely detected using 338F-806R, while the relative abundances of Thaumarchaeota detected using 515F-806R were higher than those detected by using 515F-907R. AOB showed higher proportions in the 338F-806R and 515F-907R data, than in 515F-806R data. Different primers pairs showed significant change in relative proportion of NOB. Nonetheless, we found consistent patterns of the phylotype distribution of nitrifiers in different treatments. Nitrosopumilales (NP) and Nitrososphaerales (NS) clades were the dominant members of the AOA community in soils subject to controlled irrigation, whereas Ca. Nitrosotaleales (NT) and NS clades dominated the AOA community in soils subject to flooding irrigation. Nitrospira lineage II was the dominant NOB phylotype in all samples. Overall, ideal 16S rRNA primer pairs were identified for the analysis of nitrifier communities. Moreover, NP and NT clades of AOA might have distinct environmental adaptation strategies under different irrigation treatments.

Relationship Between the Gut Microbiota and Neurological Deficits in Patients With Cerebral Ischemic Stroke

Objective The aim of the paper was to investigate the composition and structure of intestinal flora in patients with cerebral ischemic stroke (CIS), and to investigate the relationship between gut microbiota (GM) and different levels of stroke severity. Methods In this study, 47 CIS patients (16 mild, 21 moderate, and 10 severe) and 15 healthy controls were included. General information, clinical data, and behavioral scores of the enrolled subjects were collected. Deoxyribonucleic acid in fecal intestinal flora was extracted and detected using high-throughput Illumina 16S ribosomal ribonucleic acid sequencing technology. Finally, the correlation between the community composition of intestinal microbiota and National Institutes of Health Stroke Scale (NIHSS) score in CIS patients was analyzed. Results Compared with healthy controls, there was no statistically significant difference in Alpha diversity among CIS patients, but the principal coordinate analysis showed significant differences in the composition of the GM among stroke patients with different degrees of severity and controls. In CIS patients, Streptococcus was significantly enriched, and Eshibacter—Shigella, Bacteroides, and Agathobacter were significantly down-regulated (P < .05). In addition, the relative abundance of Blautia was negatively correlated with the NIHSS score. Conclusions Our results show that different degrees of CIS severity exert distinct effects on the intestinal microbiome. This study reveals the intestinal microecological changes after brain injury from the perspective of brain–gut axis. Intestinal microorganisms not only reveal the possible pathological process and indicate the severity of neurologic impairment, but also make targeted therapy possible for CIS patients.

The Cellular Microbiome of Visceral Organs: An Inherent Inhabitant of Parenchymal Cells.

The cell is the basic unit of life. It is composed of organelles and various organic and inorganic biomolecules. Recent 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing studies have revealed the presence of tissue bacteria in both tumor and normal tissues. Recently, we found that the liver microbiome resided in hepatocytes. Here, we further report on the cellular microbiome in the parenchymal cells of visceral organs as inherent inhabitants. We performed 16S rRNA gene sequencing on visceral organs of male adult Sprague Dawley (SD) rats, pregnant rats, newborn rats, and fetuses and placentas; then, we performed fluorescence in situ hybridization and immunofluorescence in visceral organs. Furthermore, we performed Western blotting on nuclear and cytoplasmic extractions of visceral organs of SD rats and cell lines HepG2, Huh-7, Hepa1-6, and HSC-T6. A high abundance of 16S rRNA gene was detected in the visceral organs of male adult, pregnant, newborn, and fetal rats as well as their placentas. The number of operational taxonomic units (OTUs) of visceral bacteria was higher than that of the feces and ileum bacteria. Bacterial 16S rRNA, lipopolysaccharide (LPS), and lipoteichoic acid (LTA) were found in the parenchymal cells of visceral organs, as well as in HepG2, Huh-7, HSC-T6, and Hepa1-6 cells. LPS consistently appeared in the nucleus of cells, while LTA was mainly found in the cytoplasm. In conclusion, the cellular microbiome is an intrinsic component of cells. Gram-negative bacteria are located in the nucleus, and Gram-positive bacteria are located in the cytoplasm. This differs from the gut microbiome and may be inherited.

Comparing Oral Microbiota Changes after Pulpotomy/Pulpectomy in Pediatric Patients.

Pediatric dental procedures like pulpotomy and pulpectomy are commonly performed to treat pulp-related dental issues in primary teeth. The choice of instrumentation, such as traditional rotary instruments or laser technology, can impact the oral microbiota. This study aims to compare the changes in oral microbiota following pulpotomy/pulpectomy in pediatric patients treated with traditional rotary instruments versus laser technology. A total of 60 pediatric patients, aged 4-10 years, requiring pulpotomy or pulpectomy were randomly assigned to two groups: group A (traditional) and group B (laser). In group A, traditional rotary instruments were used for pulp removal, while in group B, a dental laser was utilized. Saliva and plaque samples were collected before the procedure (baseline), immediately after the procedure, and at the one-month follow-up. Microbial deoxyribonucleic acid (DNA) was extracted and analyzed using 16S ribosomal ribonucleic acid (rRNA) sequencing to assess changes in the oral microbiota. At baseline, both groups exhibited similar microbial diversity and composition. However, immediate post-procedure results showed a significant decrease in microbial diversity in both groups, with group B (laser) demonstrating a more pronounced reduction. At the one-month follow-up, group A (traditional) showed a partial recovery of microbial diversity, while group B (laser) continued to exhibit lower microbial diversity compared to baseline. Notably, group B (laser) displayed a shift in microbial composition with a decrease in pathogenic species and an increase in beneficial bacteria compared to group A. This study highlights that both traditional rotary instruments and laser technology have a significant impact on the oral microbiota of pediatric patients following pulpotomy/pulpectomy. While both methods initially reduce microbial diversity, laser treatment appears to have a more sustained effect, favoring the growth of beneficial bacteria over time. Further research is needed to assess the long-term clinical implications of these microbiota changes for the oral health of pediatric patients.

Unveiling the gut microbiota and metabolite profiles in guinea pigs with form deprivation myopia through 16S rRNA gene sequencing and untargeted metabolomics

AimThe aim of this study was to confirm the presence of the form deprivation myopia (FDM) guinea pig eye-gut axis and investigate the relationship between serum vasoactive intestinal peptide (VIP), lipopolysaccharides (LPS), specific gut microbiota and their metabolites. Method20 specific-pathogen-free (SPF) guinea pigs were divided into the FDM and the control(Con) group. Following model induction, serum levels of VIP and LPS were quantified. A combination of 16S ribosomal ribosomal Ribonucleic Acid (rRNA) gene sequencing, non-targeted metabolomics and bioinformatics analysis were employed to identify disparities in gut microbiota and metabolites between the two groups of guinea pigs. ResultCompared to the control group, FDM guinea pigs exhibited a significant trend towards myopia, along with significantly elevated concentrations of LPS and VIP (p < 0.0001). Furthermore, Ruminococcus_albus emerged as the predominant bacterial community enriched in FDM (p < 0.05), and demonstrated positive correlations with 10 metabolites, including l-Glutamic acid, Additionally, Ruminococcus_albus exhibited positive correlations with VIP and LPS levels (p < 0.05). ConclusionThe findings suggest that the Ruminococcus_Albus and glutamate metabolic pathways play a significant role in myopia development, leading to concurrent alterations in serum VIP and LPS levels in FDM guinea pigs. This underscores the potential of specific gut microbiota and their metabolites as pivotal biomarkers involved in the pathogenesis of myopia.

Gut Microbiota in Patients with Prediabetes.

Prediabetes is characterized by abnormal glycemic levels below the type 2 diabetes threshold, and effective control of blood glucose may prevent the progression to type 2 diabetes. While the association between the gut microbiota, glucose metabolism, and insulin resistance in diabetic patients has been established in previous studies, there is a lack of research regarding these aspects in prediabetic patients in Asia. We aim to investigate the composition of the gut microbiota in prediabetic patients and their differences compared to healthy individuals. In total, 57 prediabetic patients and 60 healthy adult individuals aged 18 to 65 years old were included in this study. Biochemistry data, fecal samples, and 3 days of food records were collected. Deoxyribonucleic acid extraction and next-generation sequencing via 16S ribosomal ribonucleic acid metagenomic sequencing were conducted to analyze the relationship between the gut microbiota and dietary habits. Prediabetic patients showed a lower microbial diversity than healthy individuals, with 9 bacterial genera being less abundant and 14 others more abundant. Prediabetic patients who consumed a low-carbohydrate (LC) diet exhibited higher diversity in the gut microbiota than those who consumed a high-carbohydrate diet. A higher abundance of Coprococcus was observed in the prediabetic patients on an LC diet. Compared to healthy individuals, the gut microbiota of prediabetic patients was significantly different, and adopting an LC diet with high dietary fiber consumption may positively impact the gut microbiota. Future studies should aim to understand the relationship between the gut microbiota and glycemic control in the Asian population.

Features of the vagina microbiotope in patients with adenomyosis

The objective: to assess the composition of the vaginal microbiota in women of reproductive age with adenomyosis.Materials and methods. A comprehensive clinical-laboratory and instrumental prospective examination of 224 patients aged 30 to 50 years (average age – 42.0±1.8 years) with adenomyosis, who were included in the main study group, was conducted. The patients of the main group were divided into 2 subgroups depending on the form of adenomyosis – I group (diffuse form) included 106 women, II group (nodular form) – 118 persons. The control group included 84 healthy women of reproductive age with a regular ovulatory menstrual cycle and no history of gynecological diseases.Patients underwent microscopic examination of vaginal secretions, bacterioscopic examination of vaginal smears, stained according to Gram. Lactobacillus cultures were identified by polymerase chain reaction (PCR) and 16S ribosomal ribonucleic acid analysis.​Results. During the microscopic examination of the vaginal secretion, more than 15 leukocytes in the field of view were detected in 84 (37.5±3.2%) patients of both groups: in 40 (47.9±3.3%) patients of the I group and in 44 (52.1±3.3%) – II group. During the bacterioscopic examination of vaginal swabs, stained according to Gram, no gonococci and trichomonads were found in the main group. Candida fungi were detected microscopically in 158 patients (70.5±3.1%) of the main group: in 71 (66.9±4.6%) of the I group and in 87 (33.1±4.3%) of II group.Genotyping results showed that lactobacilli isolated from women with adenomyosis belonged to seven species: Lactobacillus fermentum, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus amnionii, Lactobacillus jensenii, Lactobacillus crispatus, and Lactobacillus delbrueskii. The majority of lactobacilli strains have high inhibitory activity against all test strains, except for Candida albicans (39 cases, 46.4%). High adhesion to human erythrocytes was determined by 54.7% (46 cases) of the studied cultures.All lactobacilli strains were resistant to metronidazole, 83.3% (70 cases) to kanamycin, 57.1% (48 cases) to vancomycin, all strains were sensitive to roxithromycin, amoxiclav, and ampicillin. Moderate sensitivity to gentamicin and cefazolin was found in 51.9% (43 cases) of cultures, peroxide-forming activity in 80.9% (68 cases).The use of quantitative diagnostic thresholds has led to significant improvements in diagnostic performance. PCR showed 88% sensitivity and 95% specificity for Gardnerella vaginalis and 96% sensitivity and 97% specificity for Atopobium vaginae.Conclusions. It has been established that bacterial vaginosis and microorganisms associated with bacterial vaginosis are independently associated with adenomyosis. The results of the study confirmed that vaginal microorganisms, which are usually diagnosed in women with adenomyosis, ascend to the endometrium from the vagina vertically, which leads to infection of the upper parts of the genital tract.

Short-Term Ingestion of Essential Amino Acid Based Nutritional Supplements or Whey Protein Improves the Physical Function of Older Adults Independently of Gut Microbiome.

Dietary proteins and essential amino acids (EAAs) are the major nutritional supplements that support the growth and activity of gut microbes contributing to the wellbeing of their host. This study hypothesizes that daily supplementation of the diet with either EAAs or whey protein for 12 weeks would improve the gut microbiome of older adults. The stool samples are processed and subjected to Illumina-based 16S ribosomal ribonucleic acid (rRNA)gene amplicon sequencing. In both groups, the most abundant families are found in order of relative abundance included: Bacteroidaceae, Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Rikenellaceae, Enterobacteriaceae, Oscillospiraceae, Tannerellaceae, and Akkermansiaceae, which indicate that these subjects are able to maintain a same healthy microbial diversity in their guts. A significant finding is a reduction of proinflammatory cytokine, interleukin-18 (IL-18) in the EAAs group. It also uses the standard 6-min walking test (6MWT) as a measure of cardiopulmonary fitness. At the end of the study, the subjects in the EAAs group perform significantly better in the 6MWT as compared to the whey group. It seems plausible that the improved physical performance and reduced proinflammatory cytokine, IL-18 seen in the EAAs group, are independent of changes in gut microbiota.

Temporal gut microbiota variability and association with dietary patterns: From the one-year observational Diet, Cancer, and Health - Next Generations MAX study

BackgroundKnowledge about the variability of gut microbiota within an individual over time is important to allow meaningful investigations of the gut microbiota in relation to diet and health outcomes in observational studies. Plant-based dietary patterns have been associated with a lower risk of morbidity and mortality and may alter gut microbiota in a favorable direction. ObjectivesTo assess the gut microbiota variability during one year and investigate the association between adherence to diet indexes and the gut microbiota in a Danish population. MethodsFour hundred forty-four participants were included in the Diet, Cancer, and Health - Next Generations MAX study (DCH-NG MAX). Stool samples collected up to three times during a year were analyzed by 16S ribosomal ribonucleic acid gene sequencing. Diet was obtained by 24-hour dietary recalls. Intraclass correlation coefficient (ICC) was calculated to assess temporal microbial variability based on 214 individuals. Diet indexes (Nordic, Mediterranean, and plant-based diets) and food groups thereof were associated with gut microbiota using linear regression analyses. ResultsWe found that 91 out of 234 genera had an ICC >0.5. We identified three subgroups dominated by Bacteroides, Prevotella 9, and Ruminococcaceae and adherence to diet indexes differed between subgroups. Higher adherence to diet indexes was associated with the relative abundance of 22 genera. Across diet indexes, higher intakes of fruit, vegetables, whole grains/cereals, and nuts were most frequently associated with these genera. ConclusionsIn the DCH-NG MAX study, 39% of the genera had an ICC >0.5 over one year, suggesting that these genera could be studied with health outcomes in prospective analyses with acceptable precision. Adherence to the Nordic, Mediterranean, and plant-based diets differed between bacterial subgroups and was associated with a higher abundance of genera with fiber-degrading properties. Fruits, vegetables, whole grains/cereals, and nuts were frequently associated with these genera.

P334 The longitudinal analysis of fecal microbiota in response to ustekinumab treatment in patients with Crohn’s disease

Abstract Background Emerging evidence supports that treatment of Crohn’s disease (CD) can alter the intestinal microbiota, which can play a significant role in the pathogenesis and therapeutic outcomes of CD. Ustekinumab (UST) has also been reported to modify the intestinal microbiota during a short-term treatment period. However, the long-term impact of UST treatment on the intestinal microbiota has rarely been studied. Methods We prospectively enrolled patients with CD who were planning to start UST treatment and collected fecal samples before (baseline) and after UST administration at week 20 and week 52. Fecal bacterial taxonomic diversity and composition were analyzed through 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing. The difference in the intestinal microbiota between clinical remitters and non-remitters defined by Crohn’s disease activity index (CDAI) was investigated, along with the longitudinal changes in the intestinal microbiota. Results A total of 124 fecal samples acquired from 43 patients were analyzed (median age 30 years, male 76.7%, median CDAI at baseline 183.2). Clinical remission was observed in 34.9% (n=15/43), 61.0% (n=25/41), and 60.0% (n=24/40) at baseline, week 20, and week 52, respectively. Shannon’s alpha diversity index increased significantly at week 20 and week 52 compared to baseline (both P&amp;lt;0.001). Compared to non-remitters, a significant increase of Shannon’s alpha diversity index was also observed among remitters (P=0.044) across all time points, while no significant difference was noted at each time point. The beta diversity analysis based on the Bray-Curtis distance revealed a significant difference among remitters at week 20 (P=0.027), however, there were no significant differences at other time points. Compared to the baseline, the abundance of the Firmicutes phylum was higher at week 20 (P=0.048) and 52 (P=0.019). The Lactobacillus genus (P=0.042), and Lactobacillus gasseri group (P=0.016) were significantly less abundant in remitters, while the Lachnospiraceae family (P=0.017) was more abundant in remitters. At week 20, Bifidobacterium genus (P=0.027) and Bifidobacterium pseudolongum group (P=0.040) were significantly more abundant in remitter while the Lactobacillus genus was less abundant (P=0.009) in remitter (Figure 1). Conclusion The alteration of fecal microbiota after UST treatment may have the potential to serve as a biomarker for predicting clinical remission. Financial Support This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1A2C2095096), the grant (2020IT0012) from the Asan Institute for Life Sciences, Seoul, Korea and the grant (CR 2017-4) from CELLTRION PHARM, Inc., Chungcheongbuk-do, Korea.

Effect of 15 days -6° head-down bed rest on microbial communities of supragingival plaque in young men.

The microgravity environment astronauts experience during spaceflight can lead to an increased risk of oral diseases and possible changes in oral microecology. In this study, we aimed to assess changes in the microbial community of supragingival plaques to explore the effects of spaceflight microgravity environment on oral microecology. Sixteen healthy male volunteers were recruited, and supragingival plaque samples were collected under -6° head-down bed rest (HDBR) at five-time points: day 1 before HDBR; days 5, 10, and 15 of HDBR; and day 6 of recovery. Bacterial genomic DNA was sequenced using gene sequencing technology with 16S ribosomal ribonucleic acid V3-V4 hypervariable region amplification and the obtained data were analyzed bioinformatically. Alpha diversity analysis showed a significant increase in species richness in supragingival plaque samples on day 15 of HDBR compared with that at pre-HDBR. Beta diversity analysis revealed that the community composition differed among the groups. Species distribution showed that, compared with those at pre-HDBR, the relative abundances of Corynebacterium and Aggregatibacter increased significantly during HDBR, while those of Veillonella, Streptococcus, and Lautropia decreased significantly. Moreover, compared with those at pre-HDBR, the relative abundance of Leptotrichia increased significantly on day 6 of recovery, whereas the relative abundances of Porphyromonas and Streptococcus decreased significantly. Network analysis showed that the interaction relationship between the dominant genera became simpler during HDBR, and the positive and negative correlations between them showed dynamic changes. Phylogenetic investigation of communities by reconstruction of unobserved states analysis showed that the amino acid metabolism function of plaque microorganisms was more enriched during HDBR. In summary, in a 15-day simulated microgravity environment, the diversity, species distribution, interaction relationship, and metabolic function of the supragingival plaque microbial community changed, which suggests that microgravity may affect the oral microecosystem by changing the balance of supragingival plaque microbial communities and further leading to the occurrence and development of oral diseases.

FEASIBILITY OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF METHICILLIN-SUSCEPTIBLE AND METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS IN TISSUE SAMPLES

To date, few studies have investigated the feasibility of the loop-mediated isothermal amplification (LAMP) assay for identifying pathogens in tissue samples. This study aimed to investigate the feasibility of LAMP for the rapid detection of methicillin-susceptible or methicillin-resistant Staphylococcus aureus (MSSA or MRSA) in tissue samples, using a bead-beating DNA extraction method. Twenty tissue samples infected with either MSSA (n = 10) or MRSA (n = 10) were obtained from patients who underwent orthopedic surgery for suspected musculoskeletal infection between December 2019 and September 2020. DNA was extracted from the infected tissue samples using the bead-beating method. A multiplex LAMP assay was conducted to identify MSSA and MRSA infections. To recognize the Staphylococcus genus, S. aureus, and methicillin resistance, 3 sets of 6 primers for the 16S ribosomal ribonucleic acid (rRNA) and the femA and mecA genes were used, respectively. The limit of detection and sensitivity (detection rate) of the LAMP assay for diagnosing MSSA and MRSA infection were analyzed. The results of this study suggest that the LAMP assay performed with tissue DNA samples can be a useful diagnostic method for the rapid detection of musculoskeletal infections caused by MSSA and MRSA.