Constipation is linked to gut microbiota dysbiosis, but causal microbial contributors and tractable bacterial interventions remain incompletely defined. This study first performed two-sample Mendelian randomization using Dutch Microbiome Project gut-microbiome Genome-Wide Association Study (GWAS) data ( N = 7,738) and a European constipation GWAS ( N = 411,623) to identify gut taxa linked to constipation. Then, in vitro metabolomics and a loperamide-induced constipation mouse model were used in male C57BL/6 mice to exploratorily test whether the commercial Rothia mucilaginosa strain BNCC363031 prevents loperamide-induced constipation. In an exploratory two-sample MR screen across 207 gut taxa, genetically predicted Rothia was nominally associated with lower constipation risk [odds ratio ( OR ) = 0.916, p = 0.0041], and the species-level Rothia mucilaginosa showed a concordant nominal protective association ( OR = 0.921, p = 0.0232). In mice ( n = 8 per group), preventive gavage partially restored loperamide-suppressed defecation: the cross-day mean fecal pellet count in the last 3 h per day was higher in the Rothia mucilaginosa group (RG) than in model controls (MCs) (7.94 vs. 4.41; p = 0.0306) but lower than in normal controls (NCs) (13.16; p = 0.00162), whereas body weight; small-intestinal transit; and immunohistochemical zonula occludens-1 (ZO-1), Claudin-1, and mucin 2 (MUC2) H-scores were unchanged. A fecal 16S V4 signal corresponding to R. mucilaginosa was detected at a very low abundance in only 2/8 RG mice; in vitro metabolomics identified candidates of secreted metabolites, including acetic acid. This study provides a reference for research on Rothia mucilaginosa in the context of constipation.
Long Chen, Sixiang Xu, Xiaoyue Li, Pei Li, Mengmeng Su, Peifan Liu, Yilin Fang, Wei Zhang
Bacteriophage therapy is a promising strategy against multidrug-resistant (MDR) extraintestinal pathogenic Escherichia coli (ExPEC), yet bacterial resistance evolution remains a major clinical challenge. In this study, we isolated and comprehensively characterized a novel lytic phage, vB_EcoP_P61425, belonging to the genus Suseptimavirus . The phage has a latent period of 15 min and a burst size of approximately 68 PFU/cell. Phage vB_EcoP_P61425 exhibits stability across temperatures of 4 °C to 50 °C and a pH range of 3 to 10, displaying potent lytic activity against common ExPEC serotypes including O25 and O75. Genomic analysis revealed a 77933 bp double-stranded DNA genome with a GC content of 42.60% and 118 predicted coding sequences (CDSs), devoid of known lysogeny-associated, toxin, or antimicrobial resistance genes. In vitro assays confirmed its strong bactericidal efficacy, and it significantly reduced bacterial colonization in a medical catheter model. To elucidate the genetic mechanisms underlying bacterial resistance, spontaneous phage-resistant mutants were isolated and analyzed using whole-genome resequencing, targeted gene deletion, and genetic complementation assays. We demonstrated that mutational inactivation or directed deletion of the lipopolysaccharide (LPS) biosynthesis genes wbbL and waaG significantly impaired phage adsorption, thereby conferring resistance to vB_EcoP_P61425 in EC1649. Crucially, these resistant mutants exhibited phenotypic and virulence-associated trade-offs, manifested by altered cell morphology, markedly impaired adherence to host epithelial cells, and significantly attenuated virulence in a Galleria mellonella larval infection model. Collectively, this study demonstrates that mutations in the LPS biosynthesis genes wbbL and waaG in E. coli EC1649 confer phage resistance while imposing an evolutionary trade-off that attenuates bacterial pathogenicity, providing new insights for optimizing phage therapy regimens against ExPEC.
Yixin Zhang, Nanyan Bian, Zhuojun Dong, Biao Ren, Juan Li
In the oral microecosystem, cross-kingdom interactions between bacteria and fungi are pivotal ecological events that determine the equilibrium between oral health and disease. Historically, technical limitations have confined research to describing correlations in microbial community composition, hindering the elucidation of underlying pathogenic mechanisms. This review aims to systematically outline the evolution of detection technologies for oral bacterial-fungal interactions and critically discuss the paradigm shift from identifying “associative correlations” to deciphering “causal mechanisms.” The literature search encompassed studies ranging from traditional culture and molecular identification to high-throughput sequencing for ecological network construction, as well as cutting-edge multi-omics integration and high-resolution imaging technologies. Beyond cataloguing technical advances, we critically evaluate the level of causal inference supported by each methodological class, with particular attention to temporality, confounding, ecological simplification, and the need for perturbational validation in disease-relevant models. Representative studies are used to illustrate how evidence can progress from co-occurrence and spatial proximity to functional dependency and mechanism.
Carbapenemase-producing Enterobacterales (CPE) pose a major therapeutic challenge because antimicrobial susceptibility measured at standard inoculum may not reflect activity at higher bacterial burdens. This Mini Review examines the biological basis, carbapenemase dependence, and agent-specific expression of inoculum effects among contemporary β-lactams. Increasing bacterial density can enhance β-lactamase burden and extracellular carbapenemase activity, alter the effective drug-to-enzyme relationship, and interact with permeability defects, bacterial physiology, and antimicrobial exposure. Available evidence shows substantial heterogeneity across carbapenemases and agents. Ceftazidime-avibactam appears comparatively stable in several CPE studies, whereas cefiderocol, aztreonam-avibactam, and other β-lactam/β-lactamase inhibitor combinations can show marked density-dependent reductions in activity under specific conditions. However, in vitro MIC increases do not consistently translate into reduced in vivo efficacy. Standardized testing approaches, mechanism-informed susceptibility assessment, and clinically relevant pharmacokinetic and pharmacodynamic studies are needed to clarify the therapeutic significance of inoculum-dependent β-lactam activity.
Manaf AlMatar, Raja Lakhal, Kamaleldin H. Kamaleldin, Arig Ibrahim-Hashim
Antimicrobial resistance (AMR) is increasing rapidly, necessitating the development of non-traditional therapeutic approaches. Overexpression of bacterial efflux pumps mediates active extrusion of drugs and is a major mechanism underlying multidrug resistance (MDR) in both Gram-negative and Gram-positive pathogens. Inhibition of efflux pumps (EPIs) can restore the activity of conventional antibiotics that have lost efficacy due to resistance. Here, we examine two classes of non-antibiotic adjuvants: halogenated small molecules and metal-based coordination complexes or nanoparticles. Halogen bonding and related chemical modifications can modulate membrane permeability and influence binding kinetics within bacterial transporters. Metal-based antimicrobial compounds utilize specific three-dimensional geometries and iron-mimicry to disrupt microbial transport systems. Notably, although microbial efflux pumps and mammalian multidrug transporters are structurally and mechanistically distinct, they share a functional role in reducing intracellular drug accumulation. This functional convergence provides a rationale for investigating whether selected EPI scaffolds developed in antimicrobial systems may also modulate resistance-associated transport processes in cancer. However, evidence supporting such cross-domain activity remains predominantly preclinical, and functional activity in one biological system should not be interpreted as evidence of therapeutic equivalence or clinical transferability to the other. By integrating structure–activity relationships, transporter mechanisms, microenvironmental pH effects, and translational limitations, this review highlights the potential of halogenated and metal-based scaffolds as experimental EPI platforms while emphasizing the substantial pharmacological, safety, and validation challenges that remain before clinical application.
