Biohydrogen is a leading candidate in the global shift toward renewable energy, offering a sustainable solution to reduce reliance on fossil fuels. This review underscores the use of microbial electrolysis cells (MECs) as a viable strategy to address increasing energy demands. In MECs, extracellular electron transfer (EET) is a vital mechanism that enables microorganisms to channel electrons (e⁻) from organic donors to external acceptors. Exo-electrogenic bacteria, viz., Geobacter, Shewanella, Pseudomonas, Nitrospira, and Desulfovibrio, are frequently reported for their ability to utilize Iron: Fe (III) and Manganese: Mn (III/IV) as electron acceptors, which can be replaced with electrodes to drive proton reduction and hydrogen (H₂) generation. The use of renewable substrates in fermentation processes further strengthens the commercial feasibility of biohydrogen production, while the molecular traits of microbes provide insights into optimizing H₂ production. Despite encouraging laboratory-scale demonstrations, challenges persist due to variability in recovery methods and the limited microbial diversity explored. Expanding the repertoire of exo-electrogens and refining MEC configurations are critical to achieving higher yields. Overall, biohydrogen stands out as a clean, eco-friendly energy resource, with microbial electrolysis offering a sustainable energy technology.

Lactate, particularly the D-isomer, which is produced exclusively by microbial fermentation, is usually low or undetectable in the healthy colon but can accumulate when there is a microbial imbalance, contributing to low pH and inflammation. Intestinal lactate-utilizing bacteria, such as Anaerobutyricum soehngenii, Anaerostipesspecies, and Coprococcus catus, convert lactate to short- chain fatty acids, including butyrate, potentially important for health. Here, we mined genomes of lactate utilizers for genes involved in propionate formation and conducted metabolic network modeling to investigate the cross-feeding mechanism. Genomes were also mined for antibiotic resistance genes, important when considering strains for biotherapeutics. Metabolic models were computed to elucidate the potential cross-feeding mechanisms between lactate utilizers in the human gut. Bioinformatics analysis revealed that the butyryl CoA:acetate CoA-transferase route was the dominant pathway for butyrate formation. The propanediol utilization compartment was annotated in A. halli, A. soehngenii, Eubacterium limosum, and C. catus. Of the genomes of key lactate utilizers assessed for antibiotic resistance genes, several possessed genes for multidrug resistance; however, other strains of Anaerostipes, C. catus, and Megasphaera elsdenii exhibited no antibiotic resistance genes. Metabolic modeling revealed that lactate-utilizing strains, including Anaerotignum, Eubacterium, and Anaerostipes, are key symbionts in maximizing the metabolic capabilities of the gut microbial community. Clinical interest in lactate-utilizers as next-generation probiotics is developing, and these data underscore the exciting therapeutic potential of targeting lactate metabolism to restore microbial and metabolic balance, in addition to potentially mitigating inflammatory diseases.

Neutrophil extracellular traps (NETs) contribute to innate immune defense by trapping and exposing microorganisms to DNA-associated antimicrobial proteins. However, some pathogens can tolerate NET-associated antimicrobial activity and persist within the host. Staphylococcus haemolyticus is an emerging multidrug-resistant coagulase-negative staphylococcal pathogen associated with healthcare-related infections, but its interaction with neutrophil-mediated defenses remains poorly understood. This study investigated whether a multidrug-resistant clinical S. haemolyticus isolate modulates neutrophil antimicrobial activity and NET-associated responses, using a clinical Staphylococcus aureus isolate as a comparator. Extracellular DNA release and NET-associated structures were assessed by fluorescence microscopy, scanning electron microscopy, fluorometric DNA quantification, microscopic enumeration, and MPO-DNA complex ELISA. The tested S. haemolyticus isolate induced extracellular DNA release and MPO-DNA complex formation; however, these responses were lower than those induced by PMA and were attenuated compared with the S. aureus comparator. S. haemolyticus also showed reduced susceptibility to neutrophil-mediated killing and greater resistance to NET-mediated killing than S. aureus. NE and MPO mRNA expression was not significantly altered following bacterial exposure. Collectively, these findings provide initial evidence that a clinical S. haemolyticus isolate can induce attenuated NET-associated responses while resisting NET-mediated antimicrobial activity, highlighting a potential immune-evasion mechanism in this emerging opportunistic pathogen.

Wolffia, a rootless duckweed in the family Lemnaceae, is increasingly recognized as a sustainable bioresource for food, animal feed, bioenergy, and biomaterials. Its productivity depends strongly on the associated microbiome, yet the functional roles of individual duckweed-associated bacteria (DAB) remain poorly understood. In this study, we evaluated the effects of DAB isolated from Wolffia globosa microbiomes on host plant growth and characterized their physiological traits. A total of 98 DAB strains were isolated from seven W. globosa microbiomes and individually co-cultivated with W. globosa, exhibiting diverse effects on host growth ranging from promotion to inhibition, with −14.6 to +12.9% of final effects on plant growth. Ten strains consistently promoted host growth and nine strains inhibited growth, which were identified as plant growth-promoting and growth-inhibiting bacteria, respectively. Of these, seven and four strains were identified within the core taxa of W. globosa microbiomes. At the community level, growth-promoting strains tended to be enriched in the growth-promoting microbiomes, whereas neutral or negative strains appeared to be enriched in the growth-inhibiting microbiomes. Taxonomic evaluation demonstrated that prominent promotive and inhibitory phenotypes occurred across six and five genera, respectively, with strain-level characteristics likely determining plant growth effects. Indole-related compound production by DAB emerged as a key physiological determinant associated with Wolffia growth. These findings provide a traceable strain set to advance our understanding of Wolffia–microbiome interactions and offer practical clues on microbiome engineering strategies to enhance Wolffia biomass productivity.

The growing need for alternatives to conventional antibiotics has intensified interest in essential oils as multitarget antimicrobial agents. This study investigated the global transcriptional response of Bacillus subtilis to a novel essential oil blend (EOB1) composed of garlic, lemongrass, cumin, lavender, and nutmeg. The minimum inhibitory concentration (MIC) of EOB1 against B. subtilis was determined to be 25% (v/v), and a subinhibitory concentration of 12.5% (v/v) was used for transcriptomic analysis. Exposure to EOB1 caused widespread transcriptional changes, with 2178 genes identified as differentially expressed, including 1044 upregulated and 1134 downregulated transcripts. After filtering at a false discovery rate (FDR) of <0.05 and an absolute log₂ fold change of >1, 1682 differentially expressed genes remained, comprising 981 upregulated and 701 downregulated genes. This corresponds to approximately 41% of the 4100 annotated protein-coding genes of the B. subtilis 168 genome. Principal component analysis showed clear separation between treated and control samples, with the first principal component explaining 97% of the total variance, indicating a strong global transcriptional effect of EOB1 on bacterial gene expression; because library preparation and sequencing were performed in a single batch and replicates within each condition clustered tightly, this separation is attributable to the treatment rather than to technical or batch-related variability. Among the most significantly induced genes were arsB, copA, cadA, pchE, htrB, cypX, arsC, and nfrAB, suggesting the activation of metal detoxification pathways, oxidative and envelope stress responses, secretion stress pathways, and secondary metabolite-associated responses. In contrast, genes involved in cell wall remodeling and nutrient uptake, including cwlO and gltT, were strongly downregulated. Gene Ontology enrichment analysis further supported these patterns, with significant overrepresentation of fatty acid β-oxidation, oxidoreductase activity acting on nicotinamide adenine dinucleotide phosphate (NAD(P)H), stress response pathways, amino acid biosynthetic processes, and cytoplasmic components. Collectively, these findings indicate that EOB1 disrupts multiple aspects of the physiology of B. subtilis and triggers coordinated metabolic, redox, and envelope-associated stress responses. This study provides mechanistic insight into the antimicrobial activity of EOB1 and supports the potential of essential oil blends as alternative antimicrobial agents.

Background: Autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD) are among the most prevalent neurodevelopmental conditions worldwide, yet the biological mechanisms driving their onset and phenotypic variability remain incompletely understood. The gut–brain axis (GBA), a bidirectional network linking intestinal microbial communities to central nervous system function through endocrine, immune, neural, and metabolic pathways, has emerged as a candidate mechanism through which environmental factors interact with genetic susceptibility to shape neurodevelopmental outcomes. Objective: To systematically map the peer-reviewed evidence on gut microbiome-related mechanistic pathways in ASD and ADHD, we delineated features common to both disorders and identified areas of divergence and knowledge gaps requiring further investigation. Methods: A scoping review was performed following the PRISMA Extension for the Scoping Reviews (PRISMA-ScR) framework. PubMed, Scopus, and Google Scholar were searched for peer-reviewed studies published January 2018 through March 2025. Studies addressing gut microbiome composition, GBA signaling, or microbiome-directed interventions in ASD or ADHD were eligible for inclusion. Extracted data were synthesized using inductive thematic analysis via Covidence software. Results: Forty-five studies satisfied the eligibility criteria. Inductive analysis produced five mechanistic themes: Endocrine dysregulation and microbial metabolite signaling; neuronal signaling and neurotransmitter modulation; immune activation and neuroinflammation; gene–microbiota–environment interactions; and microbiota-targeted interventions. Studies on both disorders demonstrated modifications involving taxa with short-chain-fatty-acid-producing capacity; however, findings were varied and did not prove a common reduction in butyrate production. ASD was marked by pronounced dysregulation of the serotonergic system and tryptophan–kynurenine pathway and increased hypothalamic-pituitary-adrenal (HPA) axis responsiveness. On the other hand, the results in ADHD were majorly cross-sectional or preliminary, and the widely-m recognized dopaminergic dysfunction in ADHD has not been associated to specific microbiota alterations to date. Conclusions: Evidence demonstrates the GBA as a useful research conceptual for investigating ASD and ADHD, although the strength of mechanistic evidence differs substantially between the two disorders. Translating these insights into therapeutic strategies will require longitudinal, multi-omic research designs capable of establishing causality and enabling patient stratification for precision microbiome-based interventions.

Bacillus species, which are among the most extensively studied and widely used rhizobacteria, have demonstrated potent antagonistic activity against a broad spectrum of phytopathogenic fungi, making them ideal microorganisms for biological control. Phytopathogenic fungi cause severe preharvest and postharvest losses in global crop production, significantly reducing yield and quality. The prolonged and heavy reliance on traditional chemical fungicides has resulted in environmental pollution and poses risks to human health. In this context, Bacillus-based biological control has emerged as an environmentally friendly, cost-effective, and sustainable alternative strategy. In this review, we provide a comprehensive overview of the screening of Bacillus strains for the biocontrol of phytopathogenic fungi. The possible mechanisms, including the production of a wide array of antifungal metabolites, the induction of systemic resistance in host plants, and indirect suppression through competition for nutrients and space, were also evaluated. Furthermore, the effects of multistrain synergistic consortia and the development of multifunctional Bacillus strains on biocontrol efficacy are discussed, and research advances in the application of Bacillus spp. in the control of soilborne diseases and phytopathogenic fungi are summarized. Finally, this review provides suggestions for the development of efficient Bacillus-based biocontrol agents, thereby promoting sustainable agricultural development.

Urolithiasis is a prevalent global health burden characterized by high recurrence rates and marked sexual dimorphism. While the gut microbiome’s role in oxalate degradation is established, the functional capacity of the local urinary microbiome remains largely unexplored. This study investigated the composition and function of the urinary microbiome in 40 healthy volunteers and 61 urolithiasis patients using 16S rRNA amplicon sequencing, alongside a retrospective analysis of stone composition in 2027 patients. We identified a diverse, ecologically structured microbiome in healthy urine dominated by genera such as Lactobacillus, Streptococcus, and Gardnerella. Significant sex-dependent differences were observed: While males exhibited higher microbial α-diversity, females demonstrated a distinct compositional structure with superior predicted functional capacity for stone degradation. Specifically, the female microbiome was enriched with enzymes including oxalyl-CoA decarboxylase, formyl-CoA transferase, urate oxidase, and purine nucleosidase. Correlation analysis identified Lactobacillus, Enterococcus, and Gardnerella as key contributors to this litholytic potential. Conversely, urolithiasis patients displayed significant dysbiosis, characterized by disrupted microbial networks and a marked reduction in these stone-degrading enzymes. Clinical data supported these findings, revealing a significantly higher prevalence of uric acid stones in males, consistent with their lower microbial degradation capacity. These findings suggest that the healthy urinary microbiome functions as a metabolically active defense system against stone formation. The observed sex-specific variations in microbial composition and enzymatic function provide a novel mechanistic explanation for the epidemiological sex disparities in urolithiasis, highlighting the urinary–kidney axis as a potential therapeutic target.

The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae undermines treatment efficacy, while limited genomic epidemiological data constrain understanding of local transmission dynamics. We therefore aimed to investigate the genomic epidemiology and the antimicrobial resistance landscape of K. pneumoniae in tertiary-care settings in Peshawar, Pakistan. A total of 497 clinical samples (urine, blood, sputum, pus, wound swabs, sterile body fluids) were collected from 01 August 2024 to 31 July 2025. K. pneumoniae was identified using the API 20E kit, followed by antimicrobial susceptibility testing (AST), with colistin and tigecycline susceptibility determined by broth microdilution. Whole-genome sequencing (WGS) was carried out on 43 MDR and XDR isolates to resolve clonal diversity, resistome composition, virulence-associated loci, and phylogenetic relationships. Among 497 samples, 152 K. pneumoniae isolates were identified, exhibiting widespread resistance to β-lactams, cephalosporins, fluoroquinolones, aminoglycosides, and carbapenems, while colistin and tigecycline retained substantial in vitro activity. Overall, 37% and 24% of isolates were classified as MDR and XDR, respectively. Forty-three MDR/XDR isolates were subjected to WGS, of which 34 high-quality K. pneumoniae genomes passed quality control and species verification for downstream analysis. Genomic analysis identified 18 sequence types, with ST147 (7/34, 20.6%), ST37 (4/34, 11.8%), ST2629 (3/34, 8.8%), ST39 (3/34, 8.8%), and ST870 (2/34, 5.9%) representing the predominant lineages. The dominant high-risk clones, particularly ST147, ST11, ST231, and ST42-3LV, harbored multiple antimicrobial resistance determinants, including blaCTX-M-15 (20/34) or blaCTX-M-14 (2/34), blaKPC-2 (2/34), blaOXA-48 (1/34), blaOXA-181 (1/34), blaNDM-5 (1/34), aminoglycoside-modifying enzymes, fluoroquinolone resistance determinants, porin disruptions (ompK35/ompK36; 9/34), and colistin resistance–associated mutations in pmrB in 4 isolates. ST147 was the predominant lineage and was consistently associated with the KL64 capsule type and O1/O2-associated O-loci. Virulence profiling demonstrated a predominantly low-virulence population, with 19/34 (55.9%) isolates carrying yersiniabactin as the only detected virulence determinant, whereas aerobactin, colibactin, and rmpA/rmpA2 were absent from all genomes. Collectively, these findings demonstrate that MDR K. pneumoniae circulating in Peshawar are dominated by internationally disseminated high-risk clones characterized by extensive antimicrobial resistance but limited virulence potential, underscoring the importance of genomic surveillance to strengthen infection prevention and antimicrobial stewardship.

Gut microbiota dysbiosis driven by pathogenic and opportunistic bacteria, including Clostridioides difficile, adherent-invasive Escherichia coli (AIEC), Salmonella enterica serovar Typhimurium, Fusobacterium nucleatum, Klebsiella pneumoniae, Enterococcus faecalis, and Campylobacter spp., disrupts colonization resistance, epithelial barrier integrity, and immune homeostasis, thereby contributing to the development of inflammatory and metabolic diseases. In this review, we summarize the mechanisms through which these microorganisms promote gut dysbiosis and critically evaluate current microbiome-targeted therapeutic strategies, including fecal microbiota transplantation (FMT), live biotherapeutic products (LBPs), probiotics, and bacteriophage (phage) therapy. We review the preclinical and emerging clinical evidence supporting phage-based targeting of major dysbiosis-associated pathogens and examine the principal challenges limiting clinical translation, including phage resistance, immune-mediated clearance, safety considerations related to temperate phages, manufacturing and delivery constraints, and regulatory barriers. Although evidence remains predominantly preclinical, advances in phage engineering, artificial intelligence-guided phage selection, and systems-level microbiome modeling are expanding the potential of personalized phage therapeutics. Collectively, these developments position bacteriophage therapy as a promising addition to future microbiome-directed strategies for restoring gut microbial homeostasis and managing dysbiosis-associated disease.

Microbiological N fertilizers containing native highly effective and competitive rhizobial bacteria placed in a suitable carrier are the best choice for sustainable plant production. This study evaluated the ability of various peat-based formulations with different zeolite/CaCO₃ amendments to support the survival and preservation of symbiotic efficiency of alfalfa rhizobia. 15 different inoculants were tested, including five nitrogen-fixing efficient Ensifer meliloti strains (218, 225, 252, 4148ss and 4193cs) in three different carriers made of peat and perlite and supplemented with micro-zeolite (B1), nanozeolite (B2), and micro-zeolite + CaCO₃ (B3). The number of rhizobia after 90 days of storage at room temperature was above 10¹⁰ CFU/g of inoculant in most of the combinations, after 180 days of storage was 10⁹ CFU/g in all combinations, and in 330^th day stayed above 10⁸ CFU/g of inoculant for strains 218, 4148ss, and 252. Considering the whole period of storage, the most performant carriers were B3, with the addition of micro-zeolite + CaCO₃and B2 with nanozeolite amendment. The efficiency of all inoculants, measured by the nodulation ability and shoot dry weight (SDW), was confirmed in inoculated alfalfa plants after six and eleven months of storage. The SDW of inoculated alfalfa plants increased from 2.5 up to 5 times compared to non-inoculated plants, while the nitrogen content (mg/plant) was also significantly increased. The SDW was influenced by strain, while the type of carrier and storage of inoculant did not have an effect. The inoculant formulations with zeolite and calcium carbonate showed good potential for rhizobia survival and effectiveness preservation during long-term storage, which should be further tested under field conditions.

Viral proteins are dynamic molecular machines that couple molecular-scale events to microbiological phenotypes, including host-cell tropism, entry efficiency, environmental persistence, strain-specific immune escape, and antiviral resistance. In this review, we critically evaluated how experimental, computational, and biochemical approaches define these links and support clinical translation. We organized the evidence around four key observables: Structure, dynamics, mechanics, and thermodynamics. We then assessed which conclusions remained robust across methods and viral families. Nuclear magnetic resonance spectroscopy, cryogenic electron microscopy, atomic force microscopy, calorimetry, all-atom molecular dynamics (MD), coarse-grained MD, structural bioinformatics, artificial intelligence, and biochemical reconstitution were compared in terms of resolution, sampling, assumptions, and validation requirements. Human immunodeficiency virus type 1 and severe acute respiratory syndrome coronavirus 2 were used as data-rich exemplars, but the resulting conclusions were tested against influenza virus, hepatitis B virus (HBV), human papillomavirus (HPV), Ebola virus, adenovirus, bacteriophages, and other systems. Particular attention was given to force-field selection, enhanced sampling, free-energy calculations, convergence, experimentally restrained modeling, and the multiscale relationship between all-atom and coarse- grained simulations. We also examined integrative workflows, the licensed HBV and HPV virus-like particle (VLP) vaccines, emerging dengue and coronavirus VLP platforms, and clinically validated structure-guided antivirals and prefusion-stabilized antigens. The analysis identified where evidence converges, where method-specific biases or limited taxonomic sampling constrain generalization, and which measurements are required to connect a molecular mechanism to infection, transmission- relevant traits, surveillance, or therapeutic performance. This critical, experimentally anchored framework defines the distinctive contribution of the review.

Pseudomonas aeruginosa is a prominent biofilm-forming pathogen associated with chronic infections that are difficult to eradicate due to the protective biofilm matrix. Inducing biofilm dispersion has emerged as a potential therapeutic strategy. Although researchers have compared selected biofilm dispersion mechanisms, the characteristics of cells generated by mechanistically distinct release strategies, particularly enzymatic matrix degradation, remain uncharacterized. In this study, we compared P. aeruginosa cells released from biofilms using four distinct dispersion mechanisms: An external signaling molecule ( cis-2-decenoic acid; cis-DA), a nutrient cue (glutamate), an environmental toxin (sodium nitroprusside; SNP), and a matrix-degrading enzyme (amylase). Released cells exhibited distinct antibiotic susceptibility, attachment, and gene expression depending on the release mechanism. Glutamate-released cells exhibited greater tobramycin tolerance and attachment, whereas glutamate and amylase-released populations exhibited differences in the expression of selected genes. Additionally, the release mechanism influenced subsequent biofilm recovery and reformation, and cis-DA significantly enhanced tobramycin-mediated killing of mature biofilms. Collectively, these findings demonstrate that the mechanism used to induce release may influence subsequent bacterial behavior and antibiotic susceptibility. These findings highlight the importance of considering release mechanism when developing dispersion-based strategies for treating biofilm-associated infections.

Lignocellulosic resources are naturally renewable, low-priced, and abundant. They comprise three kinds of polymers, cellulose, hemicelluloses, and lignin, bonded to each other with the help of covalent cross-linking and non-covalent forces. Cellulose is the world’s most abundant organic material and the chief constituent of biomass energy. Moreover, production of calluses from Bacillus valezensis strain 2H was statistically optimized by substrate carrot in submerged fermentation using Response Surface Methodology (RSM) and Artificial Neural Network (ANN). The RSM and ANN were used to successfully optimize Carboxymethyl cellulase (CMCase) and Filter paper (FPase). Moreover, RSM was utilized for optimization of the target enzyme using a newly identified Bacillus velezensis strain. Plackett-Burman design (PBD) was adopted for screening of the most significant factors affecting cellulase production. Central composite design (CCD) was utilized to optimize enzyme production. Biochemical characterization revealed that both enzymes were optimally active under acidic conditions (pH 6.0), with the optimum temperature observed to be 60 ℃, and stability was maintained up to 60 ℃. The tested metal ions affected CMCase and FPase activities to varying degrees but did not enhance enzyme activity compared with the control. Similarly, the tested organic solvents generally decreased enzyme activities, although the extent of inhibition varied depending on the enzyme and solvent. R² values for the CMCase were 95.43% and for FPase 99.58%, calculated by RSM analysis. R² values for CMCase were 91.44% (Overall R = 0.95622), and for FPase were 53.70% (Overall R = 0.7328), calculated by ANN analysis. Our results showed that the RSM and ANN models provided different predictive performances for CMCase and FPase. This study elucidates the potential of Bacillus velezensis for efficient cellulase production under optimized submerged fermentation conditions.