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Candidatus (Ca.) Allocryptoplasma is a newly identified, genetically diverse genus of vector-borne bacteria closely related to established blood-borne zoonotic pathogens, including Anaplasma, Ehrlichia, and Neoehrlichia species. Ca. Allocryptoplasma is a globally distributed pathogen detected across taxonomically diverse tick species and varied ecological niches. It maintains a complex enzootic circulation involving multiple vertebrate classes, including birds, small mammals, primates, and particularly reptiles, which serve as reservoir hosts. Methods: To characterize this emerging agent, multi-locus genetic DNA sequence analyses using the maximum likelihood method were performed on 16S rRNA, sucA, and rpoB genes amplified from the blood and vectors of marine iguanas and lizard species sampled across multiple islands in the Galápagos archipelago. Results: The compiled genomic data demonstrate that the Ca. Allocryptoplasma detected in the Galápagos lizards and marine iguanas represents a distinct lineage and a novel Ca. Allocryptoplasma species. Conclusions: Given its broad host plasticity, widespread geographic footprint, and vector-mediated transmission, the potential of Ca. Allocryptoplasma as a hidden, emerging zoonotic blood pathogen warrants intensive further evaluation. Full article

Background: Crude oil refinery waste sludge (COWS) ranks among the most compositionally complex and ecotoxicologically hazardous industrial residues. Although bulk total petroleum hydrocarbon (TPH) and summed polycyclic aromatic hydrocarbon (PAH) removal are routinely reported, the metabolite-level biochemical fate of individual petrogenic compounds, spanning ring dihydroxylation, catechol cleavage, and entry into central carbon metabolism, remains largely unmapped under co-composting with diverse animal manures. Objectives: This study aimed to construct a metabolite-resolved, microbially anchored biochemical fate map of crude oil sludge during co-composting. Methods: Aerobic microcosms combining crude oil sludge, garden soil, and a wood-chip bulking agent were amended separately with poultry, horse, cow, or swine/pig manure alongside an unamended control and then incubated at 22 °C for 300 days. Analyses integrated untargeted gas chromatography-mass spectrometry (GC-MS) metabolomics, targeted PAH quantification (EPA Methods 3541/8270), 16S rRNA gene amplicon sequencing (Illumina MiSeq, V1–V3, paired-end 300 bp), physicochemical monitoring, and culture-dependent isolation, with National Institute of Standards and Technology (NIST) Mass Spectral library annotation. Results: GC-MS resolved 1169 metabolite features across 17 samples, comprising 538 annotated compounds within 11 chemical classes and 631 unknowns, of which 151 recurred in at least 10 samples. Petrogenic markers (n-alkanes C₁₄–C₃₆, hopanoids, steranes, and alkylated dibenzothiophenes) and ring-cleavage intermediates (2-hydroxyfluorene, 1,4-naphthoquinone, phenanthrene-methanol, benzenediols, butanedioic acid, fatty alcohols C₁₆–C₂₀) elucidated a four-stage degradation cascade consistent with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways map01220 and map00624. PAH mean-removal ranked swine/pig (88.0%) > horse (87.0%) > poultry (80.5%) > cow (79.1%) > control (68.2%). Sequencing recovered 2969 operational taxonomic units (OTUs) enriched in Pseudomonas, Achromobacter, Stutzerimonas, Dietzia, Gordonia, and Mycobacterium, with Pseudomonas dominating high-removal systems; respiration peaked at 18.7 mg CO₂-C g⁻¹ in poultry treatments. Conclusions: This work establishes a metabolite-resolved map linking hydrocarbonoclastic taxa to separate degradation steps. The co-occurrence of oxygenated PAH intermediates with decreasing parent PAH concentrations serves as an indicator of transformation processes and may assist in identifying potential residual-risk signals, thereby supporting remediation evaluation and process optimization. Full article

Coastal green tides may alter nearshore bioaerosols through coupled marine, atmospheric, and meteorological processes, yet their effects on airborne microbial communities remain poorly resolved. Atmospheric samples were collected in Aoshan Bay, Qingdao, China, during five phases of the Ulva prolifera green tide in 2019 (pre-bloom, 19 April; early bloom, 15 June; middle bloom, 15 July; late bloom, 6 August; post-bloom, 30 August); seawater samples were collected at one nearshore site on each of the five sampling dates, with microbial sequencing performed for the middle-bloom (15 July) and late-bloom (6 August) phases. Bacterial and fungal communities were characterized; although bioaerosols may also contain microalgae and viruses, this study profiled only the bacterial and fungal fractions, using bacterial 16S rRNA gene (V3-V4 region) and fungal internal transcribed spacer (ITS2) amplicon sequencing and evaluated together with meteorological variables, air-pollutant concentrations, and 72-h backward air-mass trajectories. Proteobacteria dominated the airborne bacterial assemblages (81.28–97.83%), with Sphingomonas as the most abundant genus (47.85–89.84%). Basidiomycota and Ascomycota dominated the fungal assemblages, whereas Cryptococcus and Alternaria were the major fungal genera. Community richness and composition varied across bloom phases. Chytridiomycota was undetected before the bloom (0%), appeared after bloom onset, and reached its highest relative abundance during the middle phase (8.19%). Spatial patterns indicated joint terrestrial and marine influences, although bacterial communities in seawater and air remained highly dissimilar. Temperature, relative humidity, particulate matter, ozone, and air-mass origin were associated with changes in microbial diversity and composition. These findings provide an observational baseline for coastal bioaerosol dynamics during a macroalgal green tide, extending the HAB–bioaerosol literature—which has focused predominantly on cyanobacterial blooms—to a large green macroalga. Bacteria and fungi showed contrasting environmental responses: bacterial richness increased with temperature, whereas fungal diversity declined. Greater compositional similarity between seawater and air for fungi than for bacteria suggests differential environmental filtering at the air–sea interface and implies that multiple source pathways—direct aerosolization, sea-surface release, and in-situ atmospheric production—may differentially shape the two domains. Given the single-date-per-phase sampling design, the absence of sequenced laboratory contamination controls, and the lack of absolute abundance data, these results should be regarded as preliminary and hypothesis-generating, underscoring the need for ASV-level source tracking, controlled chamber experiments, and replicated multi-year designs in future assessments of bloom–atmosphere interactions. Full article

Soil salinisation and periodic flooding jointly shape coastal wetland ecosystems, but how coexisting halophytes differ in their rhizosphere bacterial communities under these conditions remains insufficiently understood. Here, we investigated rhizosphere bacterial communities associated with Suaeda glauca Bunge and Tamarix chinensis Lour. in saline–alkaline habitats of the Yellow River Delta, China. Twenty quadrat-level rhizosphere samples were collected across four plant–habitat groups, and near-full-length 16S rRNA gene amplicons were sequenced using Pacific Biosciences single-molecule real-time sequencing. Our analysis revealed that hydrological habitat and plant identity together contributed to differences in rhizosphere bacterial community composition. Across the dataset, 2325 bacterial operational taxonomic units were identified. T. chinensis showed higher Shannon and Gini–Simpson diversity, whereas richness patterns depended on habitat and the metric examined. Meanwhile, exploratory genus-level association networks revealed host- and habitat-dependent differences in node number, network density and average degree. PICRUSt2-based functional prediction suggested contrasting predicted functional response patterns: the S. glauca rhizosphere showed 19 significantly altered predicted pathways between flooded and non-flooded habitats, whereas the T. chinensis rhizosphere showed no significant pathway shifts after multiple-testing correction. These findings suggest that coexisting halophytes are associated with divergent rhizosphere bacterial community patterns under saline–alkaline and flooding-associated habitat conditions. Full article

Gut microbiota plays an important role in host metabolism, immunity, and environmental adaptation, yet its response to contrasting wintering habitats in migratory waterbirds remains poorly understood. We compared the gut microbiota of whooper swans (Cygnus cygnus) wintering in a natural riverine habitat (Swan Bay, Hejing County) and an urban wetland habitat (Worker’s Park, Korla City) in Xinjiang, China. A total of 34 fresh fecal samples were analyzed using 16S rRNA gene sequencing. We identified 2207 operational taxonomic units (OTUs), of which 734 were shared between habitats, indicating the presence of a conserved core microbiota. The urban wetland group exhibited a significantly higher Simpson index, whereas beta diversity analysis revealed significant differences in community structure between habitats (p < 0.05). Both groups were dominated by Firmicutes, Fusobacteriota, Proteobacteria, Campylobacterota, and Bacteroidota, although their relative abundances differed markedly. LEfSe analysis identified distinct habitat-associated taxa, and PICRUSt2 prediction suggested greater enrichment of metabolism-related pathways in the natural riverine group. Overall, wintering habitat was associated with differences in gut microbial composition and predicted function, providing new insight into the ecological adaptation of migratory waterbirds under contrasting environmental conditions. Full article

The purpose of the present study was to investigate how periodontitis and diabetes mellitus (DM) are associated with Alzheimer’s disease (AD) through microRNA (miRNA) using AD model mice. The experimental period was 8 weeks. Twenty-four male knock-in mice (B6-AppNL-G-F/NL-G-F/J) were divided into four groups: control group fed a normal diet (C), DM group fed a high-fat/sucrose diet (DM), periodontitis (P) group, and DM + periodontitis (DM+P) group. Memory performance was compared using the Y-maze test. Next-generation sequencing was performed on brain samples, and fold changes in miRNA expression were calculated by comparing the DM+P and C groups. Integrated miRNA–mRNA analysis identified putative miRNA-targeted mRNAs, and protein expression of the top candidate gene was assessed. Memory function in the DM+P group was significantly lower than in the C group. Among the seven mRNAs identified by the integrated analysis, Neurod1 showed the greatest decrease in expression, and it was predicted to be regulated by miR-693-3p. Neurod1 protein expression in the hippocampus was significantly lower in the DM+P group than the C group. Our results suggest that the combined exposure to periodontitis and DM was associated with AD-like pathological changes and identified the miR-693-3p/Neurod1 pair as a candidate regulatory axis. Full article

The earliest gut and nasal bacterial community profiles after birth remain incompletely characterized, particularly when both sites are examined in parallel. We aimed to characterize these profiles according to delivery mode within the first 48 h of life in a Mexican cohort. Meconium and nasal wash samples from 60 neonates (30 vaginal and 30 cesarean deliveries) were analyzed by V3–V4 16S rRNA gene amplicon sequencing on an Illumina MiSeq platform, followed by QIIME 2 processing. All 60 meconium samples yielded analyzable 16S rRNA profiles. Nasal washes yielded analyzable profiles in 10/30 vaginally delivered and 12/30 cesarean-delivered neonates (p = 0.79). No statistically significant differences in alpha diversity were detected by delivery mode in gut samples (observed ASVs, p = 0.842; Shannon index, p = 0.183; Pielou’s evenness, p = 0.121; Faith’s phylogenetic diversity, p = 0.574) or nasal samples (p = 0.356, 0.947, 0.644, and 0.114, respectively). In unadjusted PERMANOVA comparisons, gut community structure differed by delivery mode for Bray–Curtis (p = 0.001), weighted UniFrac (p = 0.021), and unweighted UniFrac (p = 0.013) distances, whereas nasal comparisons were not statistically significant. Traditional ANCOM identified Staphylococcus as the feature with the highest W statistic in cesarean-delivered neonates (W = 65). Descriptive phylum-level profiles were examined across maternal urinary tract infection, premature rupture of membranes, maternal comorbidities, maternal BMI, hours since birth, and recorded antibiotic exposure; these comparisons were not multivariable and cannot exclude residual confounding. Delivery mode was associated with differences in early gut bacterial community structure in this cohort. Nasal findings were inconclusive because only 22 washes yielded analyzable 16S rRNA profiles and the workflow was not optimized to quantify ultra-low-biomass bacterial DNA. These cross-sectional data do not establish persistence, causality, or the timing at which nasal communities become stable. Full article

Endometrial senescence and the endometrial microbiota have both been implicated in the regulation of endometrial receptivity, yet their relationship remains poorly understood. The aim of this study was to investigate associations between p16-positive endometrial cells and microbiota composition during the implantation window and to determine whether these relationships differ according to implantation outcome. Endometrial senescence was assessed by p16 immunohistochemistry and digital image analysis, whereas microbial composition was characterized by 16S rRNA gene sequencing in endometrial biopsies collected from 68 women prior to undergoing transfer of a single euploid embryo, which was performed within six months of biopsy under the same hormonal preparation protocol. No significant differences in luminal epithelial or stromal p16 abundance were observed according to subsequent implantation outcome. Although senescence was not directly associated with implantation success, stromal p16 expression demonstrated multiple associations with the endometrial microbiota. Increased stromal p16-positivity was associated with lower relative abundance of Lactobacillus and higher abundance of Delftia. Notably, in exploratory subgroup analyses more pronounced associations were observed in women who achieved pregnancy, including a negative correlation between stromal p16 expression and Lactobacillus abundance (ρ = −0.48, p = 0.005) and positive correlation with Delftia abundance (ρ = 0.42, p = 0.016). Species-level analyses revealed implantation outcome-specific associations involving Lactobacillus iners and Limosilactobacillus vaginalis. In addition, stromal p16 expression was associated with differences in microbial co-occurrence networks, indicating broader effects on microbial community organization. Together, these findings suggest that stromal, but not luminal, senescence is closely linked to endometrial microbiota composition and microbial community organization during the window of implantation. The observation that the strongest senescence–microbiota associations occurred in women who subsequently achieved successful implantation supports the hypothesis that coordinated senescence–microbiota relationships may represent a previously underrecognized feature of the receptive endometrial microenvironment. Full article

In the context of the global shift toward sustainable energy systems and circular economy strategies, anaerobic digestion is a well-established biotechnology for the valorisation of organic residues, the production of biogas as a renewable energy carrier, and the generation of nutrient-rich digestate. This study aimed to evaluate the stability and methane yield of a two-stage co-digestion process using a substrate mixture developed based on the actual feedstock composition of a full-scale agricultural biogas plant. The daily feed mixture consisted of maize silage (8.2%), sugar beet pulp (4.9%), cellulose pulp (6.6%), distillery stillage (38.6%), corn syrup (6.4%), cattle slurry (22.5%) and sterilised animal by-products (12.8%). Digestate was recirculated separately as part of the reactor operation. Laboratory-scale experiments were conducted in a two-stage anaerobic digestion system operated at 42 °C and 50 °C. The physicochemical properties of the substrates and digestate were determined, biogas quantity and composition were monitored, and the microbial community structure was assessed using 16S rRNA gene amplicon sequencing. The process remained stable throughout the experimental period, with pH values ranging from 8.23 to 8.53, alkalinity between 2600 and 2940 mg CaCO₃/dm³, and a VFAs/alkalinity ratio of 0.17–0.93. Despite ammonium nitrogen concentrations reaching 4346 mg N-NH₄⁺/L, no clear concurrent reduction in gas or methane production was observed. Methane accounted for approximately 70–80% of the biogas produced. The overall specific methane yield reached 346.4 NL CH₄ kg⁻¹ VS added. 16S rRNA gene amplicon sequencing revealed a diverse microbial community containing taxa previously associated with hydrolysis, fermentation and syntrophic interactions, including Proteiniphilum and Syntrophaceticus. The results demonstrate stable process performance and methane production in this site-specific laboratory-scale case study based on the feedstock composition and process configuration of a full-scale agricultural biogas plant. Full article

Non-IgE-mediated food allergy is characterized by delayed gastrointestinal manifestations and the absence of reliable non-invasive biomarkers. Increasing evidence suggests that gut microbiota may contribute to this disease pathogenesis. The aim of this study was to characterize the gut microbiome composition in thirty pediatric patients diagnosed with non-IgE-mediated food allergy, in comparison to fifteen healthy controls children, and to investigate its association with fecal calprotectin, eosinophil-derived neurotoxin (EDN) and IgA. Gut microbiota profiling was performed by 16S rRNA gene sequencing targeting the V3–V4 region. Compared with healthy controls, higher mean relative abundances of Bacteroides, Faecalibacterium, Alistipes, Parabacteroides, and Sutterella were observed in patients. Conversely, healthy children showed higher mean relative abundances of Pseudobutyrivibrio, Roseburia, Bifidobacterium, Collinsella, Clostridium, Eubacterium, Streptococcus, and Barnesiella. Several genera (Escherichia–Shigella, Agathobacter, and Enterococcus/Streptococcus) were detected only in the allergy cohort. Shannon diversity was higher in patients compared to controls and in the subgroups of patients with elevated fecal calprotectin (p = 0.028), previous antibiotic exposure (p = 0.015), and atopic dermatitis (p = 0.021). Stratification according to inflammatory biomarkers identified a distinct inflammatory microbiome endotype characterized by increased fecal calprotectin and EDN together with enrichment of Veillonellaceae and depletion of Bifidobacteriaceae and Lachnospiraceae. Correlation analyses further revealed positive associations between Veillonella abundance and both fecal calprotectin and EDN. These findings suggest that pediatric non-IgE-mediated food allergy is characterized by distinct microbiome–inflammation relationships rather than a single dysbiotic signature. Full article

The population genetic diversity and demographic history of Hypomeces pulviger in Thailand were examined based on mitochondrial cytochrome c oxidase subunit 1 (CO1) and 16S ribosomal DNA (16S rDNA) sequence data. A total of 171 and 104 individuals from multiple populations were analyzed using CO1 and 16S rDNA sequences, respectively. The CO1 sequence dataset revealed high haplotype diversity (Hd = 0.999) and moderate nucleotide diversity (Nd = 0.0323), whereas the 16S rDNA showed lower diversity (Hd = 0.750, Nd = 0.0026). Population structure analyses showed low to moderate differentiation in CO1 sequences with a significant isolation-by-distance pattern, suggesting distance-limited gene flow, while 16S rDNA sequences showed weaker structure. Neutrality tests and mismatch distribution analyses supported a recent population expansion, as indicated by significantly negative Fu’s Fs and a unimodal distribution. Haplotype network and phylogenetic analyses further demonstrated greater resolution in the CO1 gene compared to the 16S rRNA gene. Collectively, H. pulviger populations in Thailand are genetically diverse, connected, and expanding, likely facilitated by both natural dispersal and agricultural activities. These findings provide important insights for understanding pest dynamics and developing effective management strategies. Full article

Spontaneously fermented camel milk from Xinjiang is valued for its nutrition but faces quality inconsistency due to complex microbial ecosystems. This study investigated how regional microbial communities correlate with volatile flavor profiles. Nine samples from three distinct areas—Dabancheng, Hami, and Keping—were analyzed using bacterial 16S rRNA gene and fungal ITS region amplicon sequencing combined with comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC-TOF MS). Bacterial communities were dominated by Firmicutes and Proteobacteria, while Ascomycota and Basidiomycota prevailed among fungi. Dominant genera varied by region: Lactobacillus and Dekkera in Dabancheng; Lactobacillus and Dipodascus in Hami; Lactococcus and Cutaneotrichosporon in Keping. Flavor profiles also diverged, with Dabancheng being rich in esters, alcohols, and ketones; Hami featuring hydrocarbons and alcohols; and Keping containing elevated hydrocarbons, alcohols, and esters. Key statistical associations were identified: Lactobacillus, Rahnella, and Acetobacter were positively associated with propene and 3-decyn-2-ol (VIP > 1.0, |r| > 0.8), while Pseudomonas, Lactococcus, and Klebsiella showed negative correlations with vinyl n-caproate. These findings reveal statistical associations between geographic origin, specific microbial taxa, and flavor attributes, providing a scientific foundation that may inform future efforts toward developing targeted starter cultures pending microbial isolation and functional validation. Full article

Blastocystis is a common intestinal protist whose clinical and ecological significance remains uncertain. This secondary exploratory analysis examined Blastocystis PCR detection in paired baseline and Week 12 stool samples collected during a randomized pilot intervention comparing daily goat-milk and cow-milk consumption. Nineteen healthy Greek Cypriot adults provided 38 stool samples. Stool DNA was screened by PCR targeting a partial 18S rRNA gene fragment, and positive amplicons were Sanger sequenced for subtype assignment. Blastocystis was detected at both time points in 3 of 19 participants (15.8%); ST2 was identified in two participants and ST4 in one. No Cryptosporidium spp. or Giardia duodenalis PCR-positive samples were identified. In exploratory 16S rRNA gene analyses, median Shannon diversity index was 6.10 versus 4.85 at baseline (p = 0.171) and 5.67 versus 4.17 at Week 12 (p = 0.085) in repeat-detection and no-detection groups, respectively. Bray–Curtis PERMANOVA was non-significant at baseline (R² = 0.0946, p = 0.0815) and Week 12 (R² = 0.0597, p = 0.8576); Aitchison-distance sensitivity analyses were likewise non-significant (p = 0.0547 and p = 0.3232, respectively). No selected genus-level comparison met the false-discovery-rate-adjusted threshold. The three-participant repeat-detection subgroup precluded reliable analysis of dairy-arm effects, subtype-specific differences, cardiometabolic outcomes, or bacterial-community associations. In this small pilot cohort, ST2 and ST4 were identified in paired baseline and Week 12 stool samples from three healthy adults, providing molecular evidence of repeat detection over the sampled interval. Larger studies with more frequent sampling, quantitative assays, and adequately powered, prespecified analyses are needed to investigate the temporal dynamics and host-associated correlates of Blastocystis detection. Full article

Minicitrus (Fortunella hindsii) is a wild kumquat with potential nutritional and medicinal value, but its tissue-specific bioactive metabolites, antioxidant activity, and endophytes remain poorly characterized. Here, we integrated LC-MS profiling, HPLC quantification, chemical and cellular antioxidant assays, 16S rRNA gene sequencing, bacterial isolation, and treatment experiments across five tissues. Among all the metabolites determined by HPLC, 13 were quantified using authentic standards and 24 were semi-quantified as linarin equivalents. Leaves, stems, and fruits accumulated abundant flavone glycosides, with phloretin-3′,5′-di-C-glucoside reaching 1823.19 ± 278.71 μg/g FW in leaves and 847.11 ± 27.28 μg/g FW in fruits, while roots and seeds showed distinct coumarin- and furanocoumarin-rich profiles, respectively. Leaf extracts showed the strongest chemical and cellular antioxidant activities, followed by fruit extracts. Isoorientin 2″-O-rhamnoside and diosmin exhibited strong chemical antioxidant capacity, and several flavone glycosides showed protective effects in cellular assays. Endophytic bacterial communities differed markedly among tissues, with fruit harboring a distinct community dominated by Weissella and Pantoea, which accounted for 65.64% and 19.13% of the relative abundance, respectively. Among two culturable fruit-associated isolates, Weissella sp. SJG-1 treatment was associated with 32.92–104.45% increases in six HPLC-quantified metabolites and higher antioxidant activity of fruit extracts. These findings highlight minicitrus as a source of bioactive metabolites and suggest a potential association between fruit-associated bacteria and fruit functional properties. Full article

In 2024, an accident involving two tankers in the Kerch Strait resulted in the release of approximately 2400 tons of fuel oil into the Black Sea, causing significant contamination of seawater and the coastal zone. This study presents the first microbiological and molecular–ecological assessment of prokaryotic community composition and hydrocarbon-oxidizing bacteria (HOB) in coastal seawater and sand near Anapa (Russian Federation) following the spill. The taxonomic composition of nine samples was analyzed using high-throughput sequencing of 16S rRNA genes (V3–V4 regions), identifying Bacteria as the dominant domain (85.3–99.8%). In seawater samples, bacteria of the phyla Pseudomonadota, Cyanobacteriota, and Bacteroidota and archaea of the phyla Thermoplasmatota and Crenarchaeota predominated. Eighteen aerobic bacterial strains, including members of the genera Shewanella, Pseudoalteromonas, Halopseudomonas, Marinomonas, Pseudomonas, Vibrio, Alcanivorax, Ectopseudomonas, Nitratireductor, and Echinicola, were isolated from the zone of fuel oil spill. Several isolates demonstrated heavy oil degradation and biosurfactant production. Screening of collection strains isolated from other habitats revealed that Rhodococcus erythropolis TG65 and Marinobacter lutaoensis Pd1 and Pd2 degraded 92–94% of fuel oil n-alkanes. Elevated dissolved iron concentrations in the seawater indicate the possibility of a metabolic coupling between hydrocarbon oxidation and microbial iron reduction, mediated by indigenous Shewanella and Pseudomonas species. These findings indicate that indigenous HOB may contribute to the natural attenuation of aliphatic hydrocarbons in fuel oil. Full article