Search Results (419)

There have been reports of an association between the gut microbiota and the development of chronic liver disease, fibrosis, and carcinogenesis; however, it is not yet possible to reach a definite conclusion. In this cross-sectional study, we examined the association between the presence or absence of hepatocellular carcinoma (HCC) and the gut microbiota in patients with chronic hepatitis B virus (HBV) infection. The study subjects consisted of 62 consecutive HBV patients admitted to our hospital who provided informed consent to participate in the study. We performed 16S rRNA analysis using DNA extracted from fecal pellets. The sequencing depth per sample was 80,000 to 90,000 reads. We calculated the proportion of each bacterial genus so that the total for each sample added up to 100%. The male-to-female ratio was 49/13, the median age was 67 years, and 46 of the patients had HCC. Twenty microbial phyla spanning 41 classes, 79 orders, 163 families, and 431 genera were identified. Receiver operating characteristic (ROC) analysis was performed on the identified bacterial taxa, from the level of phylum down to genus, to assess their ability to distinguish between patients with and without HCC. Several bacteria with an area under the curve (AUC) > 0.65 were identified as follows: TM7 phylum TM7-3 class (AUC = 0.700); Firmicutes phylum Clostridiales class Lachnobacterium genus, Dialister genus, Ruminococcus genus, and Roseburia genus (AUC = 0.670, 0.668, 0.667, and 0.660, respectively); and Firmicutes phylum Erysipelotrichi class (AUC = 0.656). Combining three of these taxa resulted in high discriminative power (p = 0.000585) with a sensitivity and specificity of 0.761 and 0.750, respectively. A similar trend was observed in the subgroup analysis based on liver reserve capacity. Even after adjusting for factors related to liver reserve capacity in the multivariate analysis, an association between these bacterial genera and HCC was confirmed. Our results suggest that gut microbiota may be associated with the prevalence of HCC in HBV patients. Full article

Background/Objectives: Bitter taste receptors (T2Rs), specifically T2R38, are present in the respiratory epithelium and react with bacterial quorum-sensing molecules to induce an innate immunity response. Although TAS2R38 polymorphisms have been correlated with susceptibility to chronic rhinosinusitis (CRS), they have not yet been explored in odontogenic rhinosinusitis (ORS), a distinct form of CRS with particular microbial and inflammatory features. We aim to establish a proof-of-concept methodology for investigating TAS2R38 genetic variants in ORS using direct maxillary sinus tissue analysis and demonstrate the feasibility of this translational approach. Methods: We conducted a prospective pilot case–control study of 36 ORS patients and 37 controls undergoing septoplasty without sinonasal disease. Maxillary sinus mucosal biopsies were obtained intraoperatively with informed consent. Genomic DNA was extracted using the PureLink Genomic DNA Mini Kit and quantified via NanoDrop spectrophotometry. TAS2R38 haplotypes were determined and classified as taster (PAV/PAV), non-taster (AVI/AVI), or intermediate (PAV/AVI) phenotype. Results: Among fully classifiable canonical TAS2R38 phenotypes (32 ORS patients, 28 controls), distributions were: tasters 12.5% vs. 25.0%, non-tasters 31.3% vs. 25.0%, and intermediate 56.3% vs. 50.0%. AVI/AVI non-taster status was not significantly associated with ORS susceptibility (OR = 1.36, 95% CI: 0.44–4.25; Fisher’s exact p = 0.775). Conclusions: This proof-of-concept study demonstrates that genotyping-grade genomic DNA can be recovered from acutely inflamed maxillary sinus mucosa, validating this substrate for future tissue-based expression, functional, and microbiome analyses not obtainable from peripheral samples; germline genotyping itself does not require sinus tissue. The observed difference in non-taster prevalence (31.3% vs. 25.0%) did not reach statistical significance and is reported descriptively. This directional trend is hypothesis-generating only and, given the limited statistical power, does not constitute evidence for an association. The demonstrated feasibility, together with the established biological rationale, supports an adequately powered confirmatory study and lays the foundation for future investigation of taste receptor genetics in ORS pathogenesis, and potentially personalized therapeutic strategies. Full article

Background: Cervical cancer is primarily caused by the human papillomavirus (HPV), with persistent infections progressing to low- (LGSIL) and high-grade (HGSIL) lesions. Emerging evidence indicates that the cervicovaginal microbiota influences HPV persistence and disease progression, although the underlying metabolic mechanisms remain unclear. Therefore, we assessed the relationship between the cervicovaginal microbiota and the metabolic milieu in women with cervical dysplasia and HPV infections. Methods: We recruited 36 non-menopausal, non-pregnant women who were classified as negative, LGSIL, or HGSIL based on pathology and HPV results. Cervical swabs were collected for genomic DNA extraction to characterize bacterial communities using 16S rRNA sequencing and to perform HPV genotyping. Cervical lavages were collected for untargeted metabolomic profiling using Gas Chromatography-Mass Spectrometry. Integrative multiomic analysis was performed using the MIMOSA2 pipeline. Results: Although bacterial community structure was not different between groups, women with HGSIL had higher richness and exhibited a higher abundance of Prevotella bivia, Prevotella buccalis, and Lachnospiraceae G-9 oral taxon 924. Lesion-positive samples had shifts in tyramine and putrescine, biogenic amines linked to cancer development. Specifically, Pseudomonas was identified as a potential contributor to tyramine oxidation. Conclusions: Cervical lesions and HPV risk are associated with shifts in the cervicovaginal microbial metabolic milieu, highlighting the role of low-abundant anaerobic bacteria. Despite the small sample size, biogenic amines were associated with anaerobic taxa and microbial dysbiosis. These findings warrant further assessment of microbial-derived metabolites and their potential to promote tumor progression by driving a pro-inflammatory, metabolically altered microenvironment. Full article

Background: Pulmonary tuberculosis (TB) is a significant trigger of acute exacerbations of chronic obstructive pulmonary disease (AECOPD), so its timely and accurate diagnosis is essential. Also, the risk factors for TB occurrence in this population remain unclear. This study aimed to evaluate the performance of metagenomic next-generation sequencing (mNGS) for TB diagnosis in AECOPD patients, as well as to identify the associated risk factors. Methods: A retrospective observational cohort of 659 AECOPD patients with suspected pulmonary infection was enrolled. The microbial cell-free nucleic acids in bronchoalveolar lavage fluid samples were extracted and subjected to mNGS detection. The clinical data for each patient were collected from the hospital information system. The statistical analyses were performed with SPSS version 25.0. Results: A total of 170 cases, included for final analyses, were categorized into TB (n = 41), bacterial infection (n = 73), and non-infective control (n = 56) groups. Among these groups, the TB group had the highest intensive care unit (ICU) admission rate (46.34%) and longest median hospital stay (19.50 days) (p < 0.01). For TB diagnosis, mNGS demonstrated a greater sensitivity (86.00%), a lower specificity (93.30%), and a higher area under the curve (AUC, 0.877) than TB-DNA detection (70.21%, 100%, 0.848, respectively) and Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay (63.83%, 100.00%, 0.870, respectively). Notably, mNGS identified the bacterial or viral co-infections in 18.00% of TB cases. Furthermore, the stringently mapped read number determined by mNGS showed a positive correlation with ICU admission rate (r = 0.76) and in-hospital mortality (r = 0.77). The lower body mass index (BMI) and reduced natural killer (NK) cell count were identified as the independent risk factors in the TB group (both p < 0.05). Conclusions: For the diagnosis of pulmonary TB in AECOPD patients, mNGS demonstrated comparable performance to TB-DNA detection and Xpert MTB/RIF assay, and also mNGS identified co-infections. In addition, a lower BMI and reduced NK cell count were identified as the independent risk factors for TB occurrence in this cohort. Full article

Background/Objectives: Age-related macular degeneration (AMD) is a multifactorial retinal disease involving inflammatory, metabolic, and genetic factors. Increasing evidence suggests that the gut microbiome may contribute to systemic pathways involved in retinal homeostasis. This exploratory pilot study investigated gut microbiome alterations in AMD patients and controls using long-read whole-genome sequencing. Methods: Bacterial DNA was extracted from fecal samples and analyzed using Oxford Nanopore sequencing, followed by taxonomic profiling, alpha and beta diversity analyses, and differential abundance testing. Results: AMD patients showed significantly reduced microbial diversity, reflected by lower richness, Shannon and Simpson indices. Species-level beta diversity analyses revealed significant differences in microbial community composition, particularly with Bray-Curtis metrics, alongside increased inter-individual microbial heterogeneity in AMD samples. Differential abundance analyses identified the depletion of several potentially beneficial commensal taxa, including Faecalibacterium prausnitzii and Parabacteriodes distasonis, whereas Staphylococcus aureus was enriched in AMD patients. Comparisons between wet and dry subtypes showed no significant differences in alpha or beta diversity. Conclusions: Overall, the findings support the presence of gut microbial dysbiosis in AMD characterized by reduced diversity, abundance-driven community shifts, and increased microbiome heterogeneity. Given the small cohort size, cross-sectional design and lack of functional analysis, these results should be considered preliminary and hypothesis-generating. Full article

Background/Objectives: Pregnancy is characterized by complex physiological, hormonal, and immunological changes that influence the oral environment and the microbial composition of the oral cavity. Emerging evidence suggests that maternal oral dysbiosis may be associated with systemic inflammatory responses and may potentially influence pregnancy outcomes. This systematic review aimed to evaluate the current clinical evidence regarding the association between maternal oral dysbiosis and adverse pregnancy outcomes, including preterm birth, low birth weight, and gestational complications. Methods: A systematic search of PubMed, Scopus, Web of Science, and Cochrane Library was conducted for studies published between January 2013 and September 2025. Observational studies and clinical trials examining the relationship between maternal oral dysbiosis or periodontal pathogens and pregnancy outcomes in pregnant women were included. Study selection was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines, and the review was prospectively registered in PROSPERO (CRD420261383855). Data were extracted on the study design, population characteristics, microbiological assessment methods, and reported pregnancy outcomes. Ten studies met the inclusion criteria of this review. Results: Seven of the ten included studies reported significant associations between the increased prevalence of periodontal pathogens, including Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia, and adverse pregnancy outcomes, particularly preterm birth and low birth weight (LBW). Several studies have identified oral bacterial DNA in placental tissues, supporting the potential hematogenous microbial translocation pathways. However, heterogeneity in microbiological assessment techniques and study designs limits the comparability of the findings. Conclusions: Current evidence suggests that maternal oral dysbiosis may be associated with the inflammatory pathways linked to adverse pregnancy outcomes. Further prospective studies and standardized microbiome analyses are required to clarify the role of the oral microbiome in maternal and fetal health. Integrating oral health assessments into prenatal care may be an important strategy for improving maternal and neonatal outcomes. Full article

Background: Human hospitals and veterinary centres are hotspots for resistant microbes and plasmids, and metagenomic sequencing offers an agnostic insight into microbiomes, resistomes, and mobilomes, informing strategies for reducing AMR spread. Methods: Environmental samples, including wastewater and surface swabs, were taken from a tertiary human hospital ward (36 samples) and a companion animal veterinary hospital (48 samples) in London. Whole DNA was extracted and metagenomic sequencing undertaken using Oxford Nanopore Technologies’ MinION. Data were analyzed for microbiomes, resistomes and mobilomes and compared. Results: Microbial diversity analyses highlight higher richness across human hospital (HH) environmental samples, but more evenness in veterinary hospital (VH) environmental samples. Diversity showed distinct microbial communities in the HH and VH samples. There were significantly more total antimicrobial resistance gene (ARG) types (p < 0.0001) in the environmental HH samples compared with the environmental VH samples. There was a significantly higher mean number of Enterobacteriales plasmid types (p ≤ 0.0001) in the HH samples. There were significantly more total Gram-Positive plasmid types (p ≤ 0.0001) in the VH samples. Discussion: This research highlights the presence of human and animal pathogens, ARGs and mobile genetic elements in clinical environments, underscoring the importance of multisectoral surveillance. Integrating taxonomic, resistome, and mobilome analyses provides a better understanding of the potential for AMR dissemination at the human–animal–environment interface. This provides insights relevant for the development of targeted surveillance and mitigation strategies within a OH framework. Full article

Antimicrobial resistance genes threaten the effective treatment of infectious diseases, underscoring the importance of their control in line with the EU One Health policy. Wastewater treatment plants are recognized hotspots for antimicrobial resistance. We assessed whether multi-channel mixed-matrix membranes (MCMMMs)—polyethersulfone (PES)/polyvinylpyrrolidone (PVP) ultrafiltration membranes with embedded activated carbon—can concurrently reduce microorganisms and extracellular DNA in wastewater effluent, building on prior reports of micropollutant removal. We evaluated the performance of MCMMMs in removing Escherichia coli and Saccharomyces cerevisiae as model organisms, as well as colony-forming units (CFUs) from wastewater effluent at a transmembrane pressure of 1 bar with a filtration area of 66 cm² over 1 h. DNA was extracted from wastewater effluent following filtration and analyzed to assess changes in microbial community composition. MCMMMs achieved log₁₀ reductions of 5.47 ± 0.42 (Escherichia coli), 5.99 ± 0.46 (Saccharomyces cerevisiae), and 2.79 ± 0.31 (wastewater CFU); reductions by pure PES/PVP membranes were comparable: higher for Escherichia coli and wastewater CFUs, lower for Saccharomyces cerevisiae. Amplicon sequencing showed altered relative abundances in wastewater effluent. Collectively, these findings demonstrate the potential of MCMMMs to simultaneously remove microorganisms, extracellular DNA, and micropollutants, highlighting their suitability for water treatment applications within the One Health framework. Full article

Background: Oral dysbiosis during pregnancy has been associated with adverse outcomes, including preterm birth, premature rupture of membranes (PROM), and low birth weight, yet oral health remains an underappreciated component of routine prenatal care. Dental caries and gingival bleeding are frequently reported during pregnancy and may remain clinically relevant in the immediate postpartum period, but their relationship with specific oral pathogens in postpartum women has been insufficiently characterised, particularly in Eastern European populations. Methods: This exploratory cross-sectional, single-centre study included 60 postpartum women recruited consecutively at “Cuza-Vodă” Clinical Hospital of Obstetrics and Gynecology, Iași, Romania, between December 2025 and February 2026. All participants completed a structured questionnaire covering obstetric history, demographic characteristics, and oral hygiene behaviours and underwent a standardised clinical oral examination by two calibrated examiners. Before study initiation, the two examiners underwent a joint calibration session based on the predefined visual oral assessment criteria used in this study and agreed on uniform recording procedures for visible dental caries, self-reported gingival bleeding during brushing, tooth mobility, and overall oral status. Saliva samples were collected after delivery. Genomic DNA was extracted using a magnetic-bead protocol and analysed by Real-Time PCR using TaqMan-based assays to detect four oral pathogens: Porphyromonas gingivalis, Streptococcus mutans, Mycoplasma salivarium, and Fusobacterium nucleatum. Results: Most participants were primiparous (55.0%) and delivered at term (≥37 weeks of gestation; 78.3%). The prevalence of pathogen detection was: P. gingivalis 38.3% (23/60), S. mutans 70.0% (42/60), M. salivarium 71.7% (43/60), and F. nucleatum 100% (60/60). Poly-microbial carriage was common: 15.0% of participants carried all three variable pathogens simultaneously (S. mutans, M. salivarium, and P. gingivalis), and the most frequent two-pathogen combination was S. mutans + M. salivarium (30.0%). No statistically significant associations were identified between pathogen detection and clinical or obstetric variables, consistent with limited statistical power in this small convenience sample. Conclusions: This exploratory study provides the first salivary prevalence estimates for these four oral pathogens in postpartum women in Northeast Romania. The high prevalence of poly-microbial carriage, including the novel quantitative estimate for M. salivarium, provides an empirical foundation for power calculations and future confirmatory research integrating standardised periodontal assessment with pregnancy outcome data. Full article

Background/Objectives: Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. Emerging evidence highlights the role of the gut microbiota in the development and progression of CRC. Microbial dysbiosis is hypothesized to contribute to chronic inflammation through a variety of mechanisms, such as the production of free radicals, which induce mutagenesis and immune dysregulation in the host, ultimately leading to diseases such as cancer. Methods: Tumor tissue samples or healthy mucosa tissue were collected for bacterial DNA extraction. The V3–V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Bioinformatics analysis was performed with QIIME2, including quality control, DADA2 denoising, alpha and beta diversity calculation, and taxonomic classification using the SILVA database. Results: Differences in microbial composition were observed between groups. The healthy controls exhibited high relative abundances of beneficial genera such as Faecalibacterium, Bacteroides, and Asteroleplasma, whereas the patients with CRC showed enrichment of atypical genera including Novosphingobium, Bradyrhizobium, and Undibacterium. Alpha diversity was lower in the CRC group, and clear clustering by group was observed in the beta diversity analysis. LEfSe analysis identified potential bacterial biomarkers associated with CRC at both the species and genus levels. Conclusions: The findings of this study support the hypothesis that colorectal cancer is associated with distinct alterations in gut microbiota composition, such as an increase in the Novosphingobium genus and a decrease in the Bacteroides genus. An exploratory description of these microbial profiles may aid in the development of microbiome-based diagnostic and therapeutic strategies and contribute to current knowledge of the role of the gut microbiota in CRC in southern Peru. Full article

Background/Objectives: Medication-related osteonecrosis of the jaw (MRONJ) is a rare but severe complication of antiresorptive therapy for osteoporosis. This study investigated bacterial adhesion and microbial composition on two suture materials and their potential impact on early wound healing following tooth extraction in patients receiving antiresorptive therapy. Methods: In this prospective exploratory clinical study with partially randomized allocation, female patients undergoing antiresorptive therapy were evaluated for clinical parameters, including the Mombelli Plaque Index (MPI), Mombelli Bleeding Index (MBI), oral smear analysis, and Early Wound Healing Score (EHS). Suture samples (Vicryl and Monocryl, Ethicon, Germany) were removed after 10 days, measured, and weighed. Bacterial DNA was isolated and quantified by qPCR targeting the albumin and 16S rRNA genes. In addition, 16S rRNA gene amplicon sequencing was performed to assess the microbial community composition. Statistical and bioinformatic analyses were conducted to compare materials and evaluate the clustering patterns. Results: Fifty-two suture samples were analyzed. Vicryl exhibited significantly higher 16S rRNA gene copy numbers than Monocryl, indicating increased bacterial colonization, whereas albumin gene copy numbers were significantly higher in Monocryl. The suture weight correlated primarily with albumin gene copy numbers. Amplicon sequencing revealed no material-dependent differences in the microbial composition; instead, samples clustered predominantly by patient, particularly in split-mouth cases. The wound healing outcomes based on the EHS were comparable between materials. Conclusions: Although Vicryl and Monocryl differ in bacterial load and host material deposition, the microbial community composition is primarily patient-specific and the clinical healing outcomes are similar. Surgical management and patient-related factors appear more critical than suture material selection. Full article

Growing Angus steers (n = 20) were blocked by weight and randomly assigned to one of two treatment groups: Control group (CON, n = 10) fed a feedlot ration ad libitum, or a ruminally protected hydrogenated fat-embedded calcium gluconate (HFCG) treatment group (HFCG, n = 10), which was fed the control ration top-dressed at 16 g/head/day for 55 days. During the slaughter process, digesta samples were collected from the cecum, colon, and rectum. Acetate concentrations were greater in the cecal and rectal digesta of steers (p ≤ 0.05) in the HFCG treatment group. Propionate concentrations were greater in the cecal, colonic, and rectal (p ≤ 0.05) digesta of steers in the HFCG treatment group. Butyrate concentrations were greater (p = 0.098) in the colon digesta of steers in the HFCG treatment group; however, they were not different in the cecal and rectal digesta. To determine the microbial composition within each section of the hindgut, DNA was extracted, and 16S rRNA gene sequencing was performed. Data were analyzed using a General Linear Model with dietary treatment as the main effect. Species richness in the cecum, colon, and rectum was not different between treatments. Erysipelotrichaceae, Peptostreptococcaceae, and Atopobiaceae abundances were increased (p ≤ 0.05) in the cecal bacterial community of steers in the HFCG group, while a significant decrease (p ≤ 0.05) in Rikenellaceae and Muribaculaceae abundances was recorded within the same bacterial community. In the colon bacterial community of steers in the HFCG group, Ruminococcaceae and Muribaculaceae abundances were elevated (p ≤ 0.05), while there was a reduction (p ≤ 0.05) in Lachnospiraceae, Erysipelotrichaceae, Atopobiaceae, and Peptostreptococcaceae abundances. Paeniclostridium, Romboutsia, and Turicibacter abundances were increased (p ≤ 0.05) in the cecal bacterial community of steers in the HFCG group, while there was a decrease (p ≤ 0.05) in Rikenellaceae_RC9 _gut_group abundance within the same bacterial community. In the colon microbiota of steers in the HFCG group, Turicibacter abundance was decreased (p ≤ 0.05). Supplementing growing steers with HFCG impacted some members of the bacterial populations, which have important roles in gut homeostasis and health, along with the formation of beneficial end-products in the gastrointestinal tract. Full article

Excessive lipid accumulation, a hallmark characteristic of high-fat diet (HFD)-induced obesity, has become a worldwide challenge, necessitating the exploration of secure and efficacious natural products for its intervention. In the present work, a polysaccharide (MCP) was extracted and purified from Mesembryanthemum crystallinum L., a novel halophyte, and its physicochemical properties, in vitro fermentation characteristics, lipid-lowering activity, and underlying mechanisms were systematically investigated. Physicochemical analysis revealed that MCP is an acidic polysaccharide, with galacturonic acid as the predominant monosaccharide component, broad molecular weight distribution, and a porous structural morphology. In vitro fermentation experiments demonstrated that MCP could be effectively utilized by human fecal microbiota, significantly promoting the yield of short-chain fatty acids (SCFAs), particularly butyrate at high concentrations, which outperformed inulin. 16S rDNA sequencing uncovered that MCP optimized microbiota composition by enriching SCFA-producing beneficial bacteria (Prevotella_9, Faecalibacterium) while suppressing opportunistic pathogens (Megamonas, Escherichia-Shigella). Metabolomic analysis of fermentation broth revealed that MCP significantly affected microbial glycerophospholipid metabolic pathways. Experiments in Caenorhabditis elegans (C. elegans) confirmed that MCP inhibited HFD-induced lipogenesis, which was linked to the regulation of the nhr-49/sbp-1-mediated lipogenesis pathway. For the first time, using an antibiotic-induced microbiota depletion model in C. elegans, the lipid-lowering effect of MCP was observed to disappear, suggesting a potential role of the gut microbiota in mediating this effect. This investigation establishes a scientific basis for MCP as a novel prebiotic or dietary supplement for managing obesity-related lipid accumulation. Full article

Fermentation is a type of biological process conducted domestically or commercially to preserve foods and beverages, produce alcohol, add nutritional value and improve aroma and flavor. The natural fermentation of flour in water to obtain a leaven for baking, lately scrutinized in the laboratory with the application of metagenomic methods, has been ubiquitous since the dawn of civilization. Commercially, single culture or defined mixtures of microorganisms are used for their predictability, but regularly fed two-domain microorganism cultures are favored in less industrialized and domestic operations. Fungi principally produce the carbon dioxide responsible for leavening. The bacteria produce acid in the bread commonly known as sourdough for its aroma and flavor. A leaven made by fermentation using flour and water can be stored while it is dormant. We studied a mature culture that is fed twenty-fold with water and flour by incubating it for 24 h, sampling it regularly for pH measurements, and plating it. The colonies were suspended for micrography and DNA extraction for PCR and Sanger sequencing. The metagenomic DNAs were analyzed for bacterial and fungal composition. The proportions of the plant and microbial DNA endogenous to the flour decline rapidly, and the predominant bacteria and fungi in mature leaven propagate, without overlap between the respective microbiomes. Full article

While essential oils are gaining momentum as a strategy to modulate rumen function and potentially reduce enteric methane in cattle, little is known about how their bioactive components, terpenes, affect rumen microbes. Our objective was to evaluate how in vivo doses of thymol affect the structure and function of the rumen microbial community via whole genome shotgun sequencing (WGS). Four beef steers were used in a 4 × 4 Latin square with four 28 d periods. Steers consumed ad libitum forage and received one of four thymol doses (0 [CON], 120 [120-T], 240 [240-T], and 480 [480-T] mg/kg forage intake). Rumen contents were separated into liquid and solid fractions, DNA was extracted, analyzed via WGS, and assessed with orthogonal contrasts. After FDR correction, no taxa were affected by thymol; however, raw p-values demonstrated responses to thymol supplementation for solid-associated uncultured Lachnospiraceae bacterium (p = 0.04), uncultured Methanobrevibacter (p = 0.05), and uncultured Coriobacteriaceae bacterium (p = 0.02). Liquid-associated uncultured Prevotellaceae bacterium (p = 0.03), Prevotella sp. (p = 0.04), and Bacteroides sp. (p = 0.02) also responded to thymol, with the highest abundances observed at various thymol doses. Genes involved in energy production and amino acid metabolism transport were observed at the highest abundances at 240-T, while genes associated with cell cycle control, cell division, and chromosome partitioning were present in the highest abundances at 120-T. The findings suggest that thymol exerts dose-dependent effects on rumen microbial abundances and functional pathways, with 240 mg/kg forage intake appearing to be the most effective dose to downregulate methanogenic enzymes while also enhancing the enzymes associated with metabolism without negatively impacting microbial diversity. Full article