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Vitamins mediate a web of cross-feeding interactions in the human gut. Many gram-positive gut microbes, in particular, are predicted to be vitamin auxotrophs. Previous studies of these microbes, however, have tended to use rich media, precluding controlled perturbations of low-abundance nutrients. We tested the ability of diverse Lachnospiraceae, the most common gram-positive bacteria in the gut, to grow on a chemically defined medium. Even though this medium contained riboflavin, we found that predicted riboflavin auxotrophs grew poorly, including the bile metabolizer Clostridium scindens. High-dose riboflavin supplementation enhanced growth, but also revealed that, surprisingly, C. scindens catabolizes riboflavin into lumichrome, making it the first reported anaerobe to do so. The only previously described catabolic pathway for riboflavin requires oxygen and has no homologs in C. scindens. In high-dose riboflavin, a single gene neighborhood with an aldolase, oxidoreductases, and a riboflavin kinase/adenylyltransferase was upregulated, suggesting an alternative anaerobic degradation or overflow pathway. Similar neighborhoods were detected in several other Lachnospiraceae, including Faecalicatena fissicatena, the only other anaerobe reported to degrade riboflavin. Reanalysis of published metabolomic data showed that, in vivo, both riboflavin and lumichrome were more abundant in colonized (vs germ-free) mouse ceca, and that, in vitro, Lachnospiraceae isolates depleted riboflavin while certain gram-negative isolates overproduced it. These results demonstrate that a member of the Lachnospiraceae can anaerobically convert an essential B vitamin into lumichrome, a molecule recently shown to have anti-inflammatory properties. Vitamin catabolism may both structure cross-feeding interactions in the gut and affect host health.IMPORTANCELachnospiraceae, the most prevalent human gut gram-positive bacteria, produce many health-relevant metabolites, but are genetically intractable and often grown in rich medium, complicating physiological studies. Unexpectedly, through comparative experiments in a chemically defined medium, we identify the first anaerobe that can catabolize riboflavin to lumichrome and show that it induces a specific gene neighborhood while doing so, suggesting a novel pathway. Variants of this neighborhood are conserved in a handful of Lachnospiraceae, including the only other anaerobe reported to degrade riboflavin (to hydroxyethylflavin). These results potentially explain decades-old observations implicating gut microbes in riboflavin catabolism. Furthermore, riboflavin catabolites have recently been shown to inhibit host mucosal-associated invariant T (MAIT) cell activation, suggesting an additional mechanism by which commensal Lachnospiraceae may dampen inflammation.

Urinary tract infections (UTIs) rank among the most common infections globally, with many linked to indwelling urinary catheters. Our prior culture-based longitudinal evaluation of long-term catheterized nursing home residents revealed persistent asymptomatic colonization by pathogens and demonstrated that CAUTI onset was not necessarily due to new pathogen acquisition. In this study, we optimized metagenomics methods to examine the ecological structure underlying persistent colonization and the transition to infection. We present a comprehensive longitudinal metagenomic analysis of catheterized urine specimens, revealing colonization dynamics of 69 microbial species across 198 samples from 9 individuals. Descriptive ecological metrics were combined with Bayesian mixed-effects models that accounted for repeated within-participant sampling to identify clusters of co-occurring species, determine the impact of perturbations such as antibiotic exposure and catheter changes on community structure, and identify taxa predictive of infection sign and symptom onset. Longitudinal specimens clustered into three main ecological phenotypes: 1) moderate diversity, unstable communities (3 participants); 2) high diversity, stable communities that resisted disruption even after multiple catheter changes (3 participants); and 3) low diversity, pathogen-dominated communities (3 participants). Catheter changes alone did not significantly disrupt community composition, while antibiotic exposures induced major shifts often followed by re-colonization with the same genera within subsequent weeks. Six clusters of species were identified for which relative abundances correlated across perturbations to the microbial community, including a mutually exclusive Enterobacterales cluster and fastidious-anaerobe group cluster. 24 species were found to correlate with onset of signs and symptoms of infection, 11 of which were missed by standard urine culture. The catheterized urinary tract represents a novel ecosystem that is resilient to disruption by catheter changes but susceptible to antibiotic perturbation. Antibiotic exposure did deplete all species associated with signs and symptoms but also depleted potentially benign microbes. Our findings have direct implications for catheter management protocols and antibiotic stewardship in long-term catheterized patients. Prospective evaluation using this framework in a larger cohort can help translate these ecological insights into clinical decision-making tools.

Small intestinal bacterial (SIBO) or fungal overgrowth (SIFO) involves excessive microbial growth in the small intestine. While jejunal aspirate is the gold standard for diagnosis, duodenal aspiration is easier to perform. This study determined and compared the diagnostic yield of tandemly performed duodenal and jejunal aspirates and cultures. Patients with gas and bloating symptoms and suspected SIBO/SIFO underwent enteroscopy, during which duodenal and jejunal aspirates were sequentially collected using a 2&#xa0;mm Liguory catheter under aseptic conditions. Cultures were performed for aerobic, anaerobic, and fungal organisms. The endoscopic time, diagnostic yield, and concordance rates were compared. Of 57 patients, 24 (42%) had positive cultures for SIBO and SIFO with diagnostic yield of 33% for both duodenal and jejunal aspirates. The overall concordance rate between duodenal and jejunal aspirates was 82% (47/57), with Cohen's kappa of 0.61. Among positive cases, the concordance was 58% (14/24). Using jejunal aspirates as the gold standard, duodenal aspirates had a sensitivity of 74%, specificity of 87%, positive predictive value of 74%, and negative predictive value of 87%. Aerobes predominated (79%), with 15% anaerobes and 6% fungi. Common aerobes included Streptococcus, Haemophilus, Klebsiella, Rothia, and Neisseria, and anaerobes included Clostridium and Bacteroides, with similar prevalence at both sites. Jejunal aspiration took significantly longer to perform than duodenal (p&#x2009;<&#x2009;0.001). Duodenal and jejunal aspirates have comparable diagnostic yields for detecting SIBO/SIFO. With similar microbial profiles but shorter procedural time, duodenal aspirates offer a practical and efficient alternative for routine evaluation although may miss diagnosis.

Periodontitis is a widespread chronic inflammatory disease driven by biofilm-forming oral bacteria. Porphyromonas gingivalis, a proteolytic anaerobe that secretes gingipains, plays a central role in disease progression by disrupting host defense mechanisms. Although mechanical debridement and antibiotic therapy are standard treatments, they are often ineffective against biofilm-associated bacteria and may contribute to antimicrobial resistance. Antimicrobial peptides (AMPs) have emerged as promising alternatives; however, their application in the oral cavity is frequently limited by proteolytic degradation. Here, we rationally designed short, symmetric AMPs with enhanced resistance to gingipain-mediated proteolysis and evaluated their antimicrobial and antibiofilm activities. Among these, APP3 demonstrated potent antimicrobial activity (MIC = 4-8 &#x3bc;M) against P. gingivalis, Fusobacterium nucleatum, and Streptococcus gordonii, while exhibiting minimal cytotoxicity and hemolysis. APP3 retained antimicrobial activity following exposure to gingipain-containing P. gingivalis culture supernatants. Furthermore, APP3 effectively inhibited mono- and dual-species biofilms involving P. gingivalis and F. nucleatum, accompanied by downregulation of genes associated with adhesion and biofilm maturation. Structural analyses revealed that APP3 adopts an &#x3b1;-helical conformation in membrane-mimicking environments and exerts antibacterial activity through membrane disruption. Collectively, these findings identify APP3 as a protease-resistant and selective AMP, demonstrating a structure-based strategy for targeting gingipain-associated pathogenic mechanisms relevant to periodontitis.

Oronasal fistula complicates 15-55% of primary cleft palate repairs, with recurrence rates approaching 43% after secondary closure, and global fistula rates have risen despite decades of iterative technical refinement, a trend that mechanical closure quality alone cannot explain. This narrative review synthesizes evidence from PubMed/MEDLINE, Scopus, and Web of Science from inception through April 2026 to argue that ONF is increasingly recognizable as a biologically mediated complication in which oral microbiome dysbiosis and innate immune dysregulation are primary, historically underrecognized determinants of palatal wound failure that act in synergy with, rather than independently of, mechanical and technical factors. Children with cleft lip and palate harbor a preoperative dysbiotic oral microbiome characterized by reduced alpha diversity, enrichment of Gram-negative anaerobes, and elevated proportions of pathobionts, including Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella spp., establishing an unfavorable immunological baseline before the first surgical incision. Perioperative broad-spectrum antibiotic prophylaxis compounds this trajectory by depleting commensal communities, while suture-associated polymicrobial biofilms sustain a persistent antigenic depot at the healing flap margin. Unremitting pathogen-associated molecular pattern exposure drives sustained TLR4-NF-&#x3ba;B signaling, NLRP3 inflammasome activation, macrophage M1 polarization arrest, neutrophil extracellular trap-mediated matrix degradation, and complement-coagulation amplification at the wound interface. Failure of the specialized pro-resolving mediator class switch leaves the wound frozen in a self-sustaining inflammatory state, precluding re-epithelialization and adequate collagen deposition. Direct human biopsy evidence for these pathways at palatoplasty wound margins remains limited; the causal temporal relationship between dysbiosis and wound breakdown remains unresolved; and all translational proposals require prospective validation in cleft-specific cohorts. Reducing ONF burden demands a conceptual shift from purely mechanical closure paradigms toward precision perioperative strategies that pair preoperative microbiome profiling, targeted immune modulation, and resolution-phase biomarker monitoring with sound surgical fundamentals.

Autoinducer-2 (AI-2) is a LuxS-dependent product of the activated methyl cycle (AMC) that functions as a quorum-sensing signal in diverse bacteria. Fusobacterium nucleatum is a genetically heterogeneous oral anaerobe comprising four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA). Previous studies have reported that FNN and FNP strains produce AI-2 and have proposed that AI-2-mediated quorum sensing contributes to biofilm formation and virulence. However, the distribution and functional relevance of AI-2 across all subspecies have not been systematically examined. Here, we show that AI-2 production is restricted to FNA strains. Genomic analysis revealed that FNN and FNV lack luxS, whereas FNP carries a disrupted luxS homolog. Consistent with these findings, AI-2 bioassays using the Vibrio harveyi BB170 reporter detected AI-2 exclusively in FNA strains. Deletion of luxS in FNA abolished AI-2 production, but resulted in minimal transcriptional changes, and exogenous AI-2 failed to elicit strong transcriptional responses in non-producing subspecies. These results demonstrate that AI-2 production in F. nucleatum is subspecies-specific and uncoupled from quorum sensing. Our findings revise current assumptions regarding AI-2-mediated communication in F. nucleatum and reveal previously unrecognized metabolic divergence within the species complex. Periodontitis affects nearly half of adults in the United States and remains a leading cause of tooth loss worldwide. Fusobacterium nucleatum is a central member of oral biofilms and has also been linked to adverse pregnancy outcomes and colorectal cancer. Although autoinducer-2 (AI-2)-mediated quorum sensing has been proposed to contribute to its biofilm formation and virulence, our study demonstrates that AI-2 production is confined to subsp. animalis and is absent in other subspecies. Moreover, AI-2 does not function as a conserved quorum-sensing regulator in this species. These findings fundamentally revise prevailing assumptions about AI-2 signaling in F. nucleatum and suggest that subspecies-specific metabolic traits, rather than universal quorum sensing, may underlie ecological adaptation and host association.

Sympathetic overactivity and gut microbiota dysbiosis drive chronic heart failure (CHF) progression. This study explored the associations of mesenteric denervation (MDN) with cardioprotection and potential involvement of the nerve-microbiota-heart axis in MI-induced CHF mice. C57BL/6 mice were randomized into control, CHF, and CHF_MDN groups. CHF was induced by LAD ligation, and MDN was performed via phenol ablation of the superior mesenteric artery. MDN was associated with suppressed mesenteric sympathetic activity, improved left ventricular ejection fraction, and reduced myocardial fibrosis and serum BNP. MDN correlated with restoration of colonic tight junction protein expression, reduced myocardial NLRP3 inflammasome activation, and decreased systemic inflammation (LPS, TNF-&#x3b1;, IL-6). 16S rRNA sequencing showed MDN reshaped gut microbiota (decreased F/B ratio, increased facultative anaerobes). Untargeted metabolomics identified 30 differential metabolites in CHF_MDN mice; MDN reduced pro-inflammatory metabolites (hippuric acid, deoxycholic acid) and increased metabolites linked to energy metabolic reprogramming (2-hydroxybutyric acid, ketoleucine, acetylglycine). Integrated analysis revealed that Campylobacterota and Halobacterota (altered by MDN) correlated with sarcosine and linoleic acid. MDN improves cardiac function in MI-induced CHF, alongside inhibition of mesenteric sympathetic activity, restoration of intestinal barrier function, reduction of inflammation, and modulation of gut microbiota and metabolic profiles. These findings suggest the nerve-microbiota-heart axis as a potential therapeutic target.

Brain abscesses demand prompt, accurate pathogen identification; however, identification using conventional culture is limited, especially for anaerobic and polymicrobial infections. We compared the diagnostic and clinical utility of metagenomic next-generation sequencing (mNGS) with that of conventional culture in patients with brain abscess. We retrospectively included 115 patients with confirmed brain abscess pathogens. Seventy-two patients underwent both mNGS and conventional culture, and 43 underwent culture alone. We evaluated diagnostic performance, pathogen profiles, adjustments to antimicrobial regimens, and clinical outcomes. mNGS detected pathogens in 86.1% of patients versus 44.4% for culture (Cohen's kappa test p=0.004; McNemar's test p=0.0001). It identified mixed infections in 53.2% of cases, whereas culture predominantly revealed single pathogens. mNGS produced substantially higher detection rates than culture for anaerobic bacteria (50.0% vs 16.7%) and oral-derived bacteria (77.6% vs 61.1%). Antimicrobial regimens were adjusted in 54.2% of patients based on mNGS results; 61.5% of these adjustments involved de-escalation, and vancomycin was discontinued in 77.8% of patients. mNGS use was associated with a lower surgical intervention rate (47.2% vs 65.1%, P = 0.002). There were no differences in length of hospital stay, fever duration, Glasgow Outcome Scale score, or hospitalization costs. In eight patients without reported dental history, mNGS revealed occult odontogenic foci, enabling source control and potentially reducing recurrence risk. mNGS outperformed conventional culture for detecting mixed infections, anaerobes, and pathogens of d origin. It may inform targeted antimicrobial therapy and assist in identifying the infection source. In this single&#x2011;center retrospective study, which is subject to potential selection bias, mNGS use was associated with a lower rate of surgical intervention; however, this finding should be interpreted as an association rather than causation, and prospective studies are needed to confirm this observation. These findings support the integration of mNGS into diagnostic algorithms for brain abscess.

Additive manufacturing (AM), particularly fused deposition modeling (FDM), holds promise for producing surgical instruments in austere settings. However, standard sterilization methods often degrade thermoplastics used in FDM. This study evaluates whether the high temperatures inherent to FDM can reduce bioburden on post-print materials. Polycarbonate filament was inoculated with E. coli and S. aureus and then 3D printed at 260&#xb0;C or 300&#xb0;C. Printed samples and preprint controls were cultured under aerobic, anaerobic, and fungal conditions. Sterility was assessed over a 14-day incubation period. Out of 30 printed samples, 5 (16.6%) showed microbial contamination, compared to 30/30 (100%) positive controls (p&#x226a;< 0.01). No significant difference was found between print temperatures or culture conditions. FDM significantly reduced microbial contamination under non-sterile operating conditions. This suggests further investigations should focus on a potential FDM single-stage, point-of-care sterilization method. Further work is needed to define critical parameters and validate efficacy against more resilient pathogens.

This work reports on the application of intermittent aeration (IA) in continuous-flow systems for biological nitrogen removal from low C/N wastewater (3.5 gCOD/gNH4-N). In similar scenarios, low organic matter concentrations hamper the denitrification step due to e-donor limitation of ordinary heterotrophic organisms (OHO) (e-donor need: 2.86/(1-Yobs) gCOD/gNO3-N). A 25&#x202f;L completely stirred bench-scale bioreactor equipped with on-line probes (pH, DO, ORP, NH4-N, NO3-N) was operated under IA at a moderate sludge retention time (SRT close to 25&#x202f;&#xb1;&#x202f;5 days) for 160 days of operation. The experimental period was structured in three main steps: continuous aeration (CA) (STEP 1); time-controlled IA (STEP 2); probe-controlled IA (STEP 3). The organic matter removal efficiency was not markedly affected by IA with removal efficiency increasing from 90% (STEP 1) to 98% (STEP 3) after a slight decrease down to 82% (STEP 2), likely due to a transitional phase during microbial adaptation. After the application of the IA process, a decrease in Yobs was observed from 0.43 to 0.26 gCODbiomass/gCODrem for STEP 1 and STEP 3, respectively. This resulted in about 40% less of sludge production, and disposal costs saving. Because a lower Yobs reduces the e-donor demand during the denitrification step, the biological nitrogen removal efficiency increased from 46.7% (STEP2,a) to 78.2% (STEP3,a). STEP 3 (probe-controlled IA) proved to be the best optimized operational condition compared to STEP 2 (time-controlled IA), due to the probe-controlled logic that avoids excessive aerated or undesirable anaerobic periods especially in cases of low influent organic and/or nitrogen loads. These remarkable results highlight that the IA process, operated at moderately high SRT and optimized using probe-controlled logic, is an attractive strategy for biological nitrogen removal from low C/N wastewater.

Acetamiprid is a frequently detected neonicotinoid insecticide that is widely present in water bodies and may disrupt the stability of the anaerobic ammonium oxidation (anammox) process. This study investigated the response threshold and mechanistic transition of the anammox system under long-term acetamiprid stress. The system remained stable at 0-2.5 mg/L acetamiprid, and nitrogen removal efficiency (NRE) did not change significantly. At 5-15 mg/L acetamiprid, NRE was maintained at approximately 80%, whereas the NO3--N/NH4+-N ratio increased to 0.40, and specific anammox activity (SAA) declined. This apparent maintenance of reactor performance was likely sustained by community-level functional compensation. At 50 mg/L acetamiprid, reactive oxygen species (ROS) levels increased by 74%, the protective effect of extracellular polymeric substances (EPS) weakened, and NRE decreased by 9.11%, indicating that the compensatory capacity of the microbial community had been exceeded and that the reactor had entered an unstable state. Overall, the reactor exhibited a stage-dependent transition from apparent stability to latent functional impairment and ultimately to overt instability. Community and metagenomic analyses further suggested that acetamiprid exposure reduced the ecological dominance and functional contributions of Candidatus Kuenenia and Candidatus Jettenia, while increasing the relative importance of Candidatus Brocadia and associated populations such as Ignavibacterium, and enhancing their stress response and xenobiotic-related functions. This transition indicates that the system shifted from a mode dominated by core anammox bacteria to a more distributed, multispecies compensatory state. These findings provide new insights into the stability boundaries and failure transitions of the anammox system under pesticide stress.

Full-length transcriptomic analysis indicates that alternative splicing, particularly intron retention, is a prominent component of the anaerobic stress response in Chlamydomonas reinhardtii and may contribute to metabolic adaptation through transcript isoform remodeling. Alternative splicing (AS) is a pivotal biological process that enhances transcriptomic plasticity in eukaryotes, especially under environmental stress. In this study, we integrated PacBio Iso-Seq and Illumina RNA-seq technologies to characterize the full-length transcriptome of Chlamydomonas reinhardtii under dark anaerobic conditions. Anaerobic treatment was associated with extensive remodeling of transcript structures, with intron retention emerging as the predominant splicing type. Transcript-structure changes were concentrated in genes related to carbon metabolism, pyruvate conversion, and fatty acid biosynthesis. Several key genes showed clear condition-dependent transcript isoform changes, including putative isoform switching events, which were further supported by qRT-PCR analysis. These findings support the view that AS-associated transcript-structure remodeling contributes to the anaerobic response of Chlamydomonas reinhardtii and provide a high-quality transcriptomic resource for future studies of stress adaptation and gene regulation in green algae.

Aspiration-related lung diseases comprise a broad spectrum of disorders resulting from the entry of oropharyngeal or gastric contents into the lower respiratory tract. Clinical manifestations range from acute aspiration pneumonitis and aspiration pneumonia to chronic airway and parenchymal injury, including bronchiolitis, bronchiectasis, fibrosis, and lipoid pneumonia. Aspiration pneumonitis is driven primarily by chemical injury, whereas aspiration pneumonia results from infection caused by aspirated microorganisms in susceptible hosts. Contemporary microbiologic data indicate that aspiration pneumonia more closely resembles community- or hospital-acquired pneumonia than the classic anaerobic infection paradigm. Imaging plays a central role in diagnosis, yet radiographic findings are highly variable and frequently contribute to diagnostic uncertainty. Chest radiography may be entirely normal or demonstrate only subtle gravity-dependent or perihilar opacities, while in other cases it reveals multifocal or diffuse air-space opacities that overlap substantially with other infectious and inflammatory lung diseases. Computed tomography is considerably more sensitive and depicts a broad spectrum of acute and chronic aspiration-related abnormalities, including ground-glass opacities, consolidation, tree-in-bud opacities, centrilobular nodules, aspiration bronchiolitis, bronchiectasis, fibrotic remodeling, and dendriform pulmonary ossification, while also demonstrating characteristic findings in selected aspiration-related conditions. Recognition of these imaging patterns, together with clinical history and swallowing evaluation, is critical for establishing the diagnosis and identifying the underlying mechanism of aspiration. Management requires integration of clinical history, imaging, swallowing assessment, and selective adjunctive testing, with treatment directed toward supportive care, appropriate antimicrobial therapy when infection is present, prevention of recurrent aspiration through correction of the underlying cause, and multidisciplinary management when indicated. Early diagnosis and intervention are essential to prevent recurrent lung injury and progressive pulmonary damage.

Uncultured Nap2-2B bacteria (order Desulfotomaculales; formerly family Peptococcaceae) are frequently detected in methanogenic hydrocarbon-degrading environments, yet their metabolic diversity remains poorly understood. Here, we analysed 17 GTDB r232 metagenome-assembled genomes (MAGs) from four genera within this clade. A bac120 phylogeny places Nap2-2B as a monophyletic family-level lineage within Desulfotomaculales. Glycyl radical enzyme phylogeny and operon context reveal strict substrate partitioning: SCADC1-2-3 encodes alkylsuccinate synthase for aliphatic hydrocarbon activation, 46-80 and UBA4053 encode benzylsuccinate synthase for aromatic activation, and JAIMBK01 lacks hydrocarbon activation genes but retains complete dissimilatory sulfate reduction pathway genes. Pangenome-level pathway reconstruction identifies complementary cofactor biosynthetic potential, notably in cobalamin and pantothenate biosynthesis, consistent with possible cofactor complementation. Genome-scale metabolic modeling suggests that the alkane-degrading SCADC1-2-3 lineage can support syntrophic hexane degradation, whereas the aromatic lineage cannot grow on the alkane FBA test because it lacks AssA and PFOR. A parallel aromatic-substrate FBA for 46-80 MAGs did not yield growth under minimal curation, reflecting the greater complexity of the downstream benzoyl-CoA pathway. Together, these data support a syntrophic guild structured by substrate partitioning, putative cofactor complementation, and distinct electron-disposal strategies that may shape methanogenic hydrocarbon attenuation in anoxic tailings environments.

Accurate detection of Clostridioides difficile (C. difficile) is essential for diagnosing and monitoring infection (CDI). Culture remains important for epidemiological investigations and strain characterization, but its success depends on effective stool pretreatment to enhance spore survival and suppress competing flora. This study aimed to compare the diagnostic yield, specifically culture isolation rates of C. difficile, between two stool pretreatment techniques: the Wadsworth Anaerobic Method (WAM) and the Bailey & Scott Method (BSM), using samples from clinically suspected CDI cases. A total of 337 stool samples from suspected CDI patients were processed. All samples were screened using glutamate dehydrogenase (GDH) and Toxin A/B immunoassays. Each specimen underwent both WAM and BSM pretreatment, followed by anaerobic culture for C. difficile isolation. Culture positivity rates were compared between methods and correlated with GDH/toxin results. Of the 337 samples, 34 (10.08%) were Toxin A/B &#xb1; GDH positive, while 19 (5.63%) were GDH positive but toxin negative. Overall, C. difficile was isolated from 21 samples (6.23%). Overall, under the study conditions, the C. difficile isolation rate was approximately 9.5-fold higher with WAM pretreatment than with BSM pretreatment (19 vs 2 isolates). Culture positivity was higher among GDH/toxin-positive samples than toxin-negative ones. Notably, five isolates were recovered from GDH- and toxin-negative samples, indicating that culture can detect organisms missed by immunoassays. Stool pretreatment significantly impacts C. difficile culture recovery. WAM showed superior performance over BSM, suggesting its utility for improving culture-based diagnosis and epidemiological studies. Higher isolation rates in GDH/toxin-positive samples indicate that toxin detection correlates with increased bacterial burden and improved likelihood of culture recovery.

Per- and polyfluoroalkyl substances, one of the most prevalent and persistent emerging contaminants in sludge, may drive the dissemination of antimicrobial resistance and pathogenicity during sludge treatment. However, the mechanisms underlying perfluorooctane sulfonate (PFOS)-mediated propagation of antibiotic resistance genes (ARGs) and virulence factors (VFs) remain poorly understood. This study investigated the effects of PFOS (1 and 10&#x202f;&#x3bc;g/g-dw) on ARGs dynamics and virulence risks. Quantitative PCR and metagenomic analysis revealed that PFOS stress led to the widespread enrichment of ARGs, the total abundance of mobile genetic elements (MGEs) and VFs also increased by 33.22-37.62% and 6.71-8.41%, respectively. Metagenomic binning results demonstrated that most metagenome-assembled genomes carrying ARGs or VFs simultaneously harbored MGEs. Mechanistically, excessive reactive oxygen species production and enhanced substrate-level phosphorylation for ATP generation may contribute to the increased horizontal transfer potential of ARGs under PFOS stress, which further facilitated the convergence of antimicrobial resistance and virulence traits within pathogens. Furthermore, PFOS may have hindered the negative regulation of the RhlI/RhlR quorum sensing system on the Type III secretion system, stimulating the secretion of VFs. This study elucidates the mechanisms by which PFOS promotes the dissemination of ARGs and pathogenicity during anaerobic digestion, highlighting the potentially overlooked environmental health risks of PFOS during sludge disposal.

Anaerobic sludge digestion is widely applied in municipal wastewater treatment plants for sludge stabilisation and renewable energy recovery through biogas production. However, digestion performance is governed by upstream biological treatment configuration, which determines sludge origin and biodegradability. This study presents a comparative assessment based on experimentally generated from a full-scale high-rate activated sludge (HRAS) system and literature-based datasets for conventional activated sludge (CAS) systems. Long-term anaerobic batch digestion tests were conducted under mesophilic conditions using plant-representative mixed sludge prepared from primary and secondary sludges collected from HRAS treatment line of a full-scale municipal wastewater treatment plant in T&#xfc;rkiye. Methane production and solids reduction were evaluated. The experimental results were further interpreted using plant-wide modelling implemented in the SUMO simulation platform. Methane yields normalised to volatile solids fed varied substantially with sludge origin and sludge age. The HRAS plant operated at the lowest sludge age exhibited the highest methane yield (530 L CH4/kg VSfed), whereas literature reported CAS systems yielded between 193 and 375 L CH4/kg VSfed. When normalised to treated wastewater, biogas production from CAS systems in T&#xfc;rkiye was significantly lower than that from HRAS configurations and reported CAS systems in Europe and North America, reflecting differences in carbon capture and upstream processes. Good agreement between experimental observations and plant-wide modelling confirms that anaerobic digestion performance cannot be optimised independently of upstream treatment design. Overall, the results highlight the importance of high-rate carbon capture strategies and unit wastewater-based performance indicators for improving plant-wide energy recovery in wastewater treatment systems.

Hydrogen sulfide, a typical malodorous pollutant generated during the anaerobic fermentation of manure, can cause severe harm to livestock health and lead to significant economic losses in the aquaculture industry when excessively accumulated. To address this issue, this work developed a highly sensitive ternary oxide sensor for detecting hydrogen sulfide in livestock farming environments. CuBi2O4 (CBO) microspheres assembled from nanoparticles were synthesized via coprecipitation, and iron oxide nanoparticles were subsequently loaded onto the copper bismuthate surface using a hydrothermal method, followed by systematic characterization of the composite material. The results revealed that, compared to pure copper bismuthate, the composite material exhibited significantly reduced response and recovery times of 42 and 37 s, accordingly, with an optimal operating temperature of 215 &#xb0;C. Due to the incorporation of iron oxide and its narrow bandgap properties, a heterojunction was formed at their interface. This not only resulted in a completely opposite resistance-temperature trend compared to intrinsic copper bismuthate but also effectively suppressed baseline drift and enhanced resistance to humidity. Furthermore, by integrating an optimized Gray Wolf Optimizer (GWO) with multiple models (RF, SVM, and LSTM), high-precision prediction of hydrogen sulfide concentration was achieved. This work proposes a novel strategy to enhance practicality of ternary oxide-based gas sensors in agricultural hydrogen sulfide detection through machine learning.

Fornicata is a group of eukaryotes adapted to anaerobic and microaerophilic environments. These organisms generate ATP anaerobically through substrate-level phosphorylation. ADP-forming acetyl-CoA synthetase (ACS) is one of the key enzymes of this process. While ACS characteristics in the parasite Giardia intestinalis have been studied, those in free-living species remain unknown. Here, we investigated the ATP-generating activities of recombinant ACS (rACS) of free-living fornicates Aduncisulcus paluster and Kipferlia bialata and compared them with Gasterophilus intestinalis. All rACSs exhibited the highest activity toward the substrate acetyl-CoA and the next toward n-propionyl-CoA. For acetyl-CoA, rApACS exhibited higher affinity (lower KM) and lower catalytic turnover (kcat) than the other two that displayed comparable kinetic profiles. For n-propionyl-CoA, rApACS also exhibited lower KM and kcat than the other two, while rGiACS exhibited far higher kcat than the other two, indicating that rGiACS could utilize n-propionyl-CoA effectively. For the substrate ADP, rKbACS exhibited the highest kcat among the three. These results suggest that ACSs from free-living fornicates are active ATP-generating enzymes, and that during the evolution of fornicates, the ATP-generating activity has been conserved, while the subtle changes in kinetic properties have occurred on their ACSs.