Search Results (263)

The increasing detection of Riemerella anatipestifer in chickens poses severe challenges to the poultry industry. However, the genomic differences among R. anatipestifer isolates from different poultry hosts remain poorly understood. Here, we investigated host-associated genomic differences among 158 R. anatipestifer strains isolated from different hosts in China, using pan-genome, phylogenetic, virulence factor, and antimicrobial resistance analyses. The core-genome phylogeny showed no apparent separation according to host, sampling period, or geographic origin. Conserved genes including groEL, tufA, and katB were detected in all strains, whereas tviB and ureG displayed host-associated distribution patterns. More than 40 putative antimicrobial resistance (AMR) genes were detected across 158 R. anatipestifer strains, indicating the presence of multiple putative AMR determinants. Among these, bla_RAD-1, bla_RASA-1, and dfrA36 exhibited host-associated distribution patterns. Pan-genome analysis further identified host-associated genomic differences in R. anatipestifer. These included genes contributing to cell-surface modification pathways mediated by pglH, and genes involved in toxin–antitoxin system-dependent stress regulation via parD1/parE1 and yefM-yoeB. Overall, our comparative genomic characterization of R. anatipestifer characterizes host-associated genomic differences and provides a basis for further investigation of their biological significance. Full article

Staphylococcus aureus frequently colonizes the skin and upper airways and produces a broad repertoire of immunomodulatory molecules. In asthma, the most consistent evidence concerns staphylococcal enterotoxins (SEs), which can act both as superantigens and as allergens, and IgE sensitization to SEs (SE-sIgE). SE-sIgE is associated with severe asthma, type 2 inflammation, chronic rhinosinusitis with nasal polyps (CRSwNP), exacerbations, and, in some longitudinal studies, persistent airflow obstruction. However, this relationship is not necessarily causal: SE-sIgE may reflect exposure, an immune response, or a biologically active endotype, whereas colonization, local toxin production, and systemic sensitization are not equivalent. SEs simultaneously bind class II MHC molecules and Vbeta regions of the T-cell receptor, activating large fractions of T lymphocytes; they also promote IL-4, IL-5, and IL-13 production, polyclonal B-cell activation, local IgE synthesis, mast-cell degranulation, eosinophilia, and IL-8/neutrophil circuits. Alpha-toxin (Hla) and SEB can damage the epithelial barrier, facilitating allergen penetration and alarmin signalling. These observations support an interaction model in which dysbiosis, barrier dysfunction, and type 2 immunity mutually reinforce one another along the nasobronchial axis. Corticosteroids and antibiotics may modify selected nodes in this circuit, but current evidence is insufficient to recommend decolonization or antitoxin therapy in stable asthma. Biologics interrupt downstream pathways potentially fuelled by toxins: omalizumab neutralizes free IgE; mepolizumab and benralizumab reduce the eosinophilic axis; dupilumab blocks IL-4/IL-13 signalling; and tezepelumab acts upstream on TSLP. Nevertheless, randomized trials stratified by SE-sIgE are lacking, and no evidence demonstrates that these treatments eliminate colonization or toxin production. SE-sIgE therefore appears to be a promising biomarker, particularly in severe asthma with CRSwNP, but it is not yet an autonomous criterion for biologic selection. A broader barrier-organ analysis also identifies nasal, cutaneous, and intestinal colonization as distinct ecological states; atopic dermatitis as a complementary model of toxin-amplified type 2 inflammation; and biofilms and extracellular vesicles as candidate mechanisms of persistent toxin delivery. These data increase biological plausibility but remain indirect for asthma. Full article

Mechanisms for the maintenance of chronic viruses are poorly understood, particularly for archaeal viruses. Here, we identify the product of Sulfolobus spindle-shaped virus 1 (SSV1) ORF a291 as a putative virally encoded toxin required for growth inhibition but dispensable for viral replication and virion production. Viruses lacking ORF a291 replicated their genomes and formed morphologically normal spindle-shaped particles, yet failed to inhibit the growth of uninfected Saccharolobus solfataricus. Substitution of residues at a predicted N-terminal signal peptide cleavage site abolished growth suppression without affecting replication, suggesting that secretion is essential for toxin function. Despite primary sequence divergence among fusellovirus toxin candidates, analysis of protein structure predictions revealed a conserved hydrolase-like fold across SSV1, SSV9 and SSV10 toxins. These findings demonstrate functional separation of viral replication and host growth suppression and support a model in which chronic archaeal viruses modulate host competition through antagonistic factors. This work expands the known diversity of putative viral toxins and suggests that fuselloviruses employ conserved strategies to promote persistence in extreme environments. Full article

Background/Objectives: Cholera remains an important global health problem. Inactivated oral cholera vaccines (OCVs) are essential in the WHO/GTFCC (World Health Organization/Global Task Force on Cholera Control) strategy to end cholera by 2030; however, global supply is insufficient, they require partial cold-chain storage, and their formulation and antigen contents leave room for improvement. We describe here the development and preclinical evaluation of DuoChol OCV, a next-generation thermostable oral vaccine designed to address these gaps. Methods: DuoChol is a lyophilized dry-powder formulation in enteric capsules containing formalin-inactivated Vibrio cholerae O1 El Tor Ogawa and Inaba isogenic bacteria, recombinant cholera toxin B subunit (rCTB), and sucrose as stabilizer. Methods describe the construction of the novel vaccine strains, processes for the preparation and characterization of vaccine components, and the final dry formulation in enteric capsules, and in vitro and in vivo vaccine stability analyses. Results: The newly engineered vaccine strains, together with a high-yield mixed-mode chromatography process for rCTB purification, enabled efficient and cost-effective vaccine production. Stability studies demonstrated complete preservation of O1 LPS and rCTB antigens for at least 21 months across temperatures of 4–40 °C. Moreover, regardless of storage duration or temperature, oral immunization of mice with DuoChol elicited strong serum and mucosal antibacterial and antitoxin responses that were similar to those induced by the licensed Dukoral^® OCV. Conclusions: Its heat stability, practical enteric capsule formulation, and potential for improved efficacy compared to inactivated whole-cell only OCVs support positioning DuoChol as a promising next-generation OCV, suitable for national cholera control programs and particularly advantageous for outbreak response, where rapid deployment and early, robust protection are essential. Full article

We previously demonstrated that botulinum neurotoxin A (BoNT-A) exerts bilateral antinociceptive effects, involving trans-synaptic transport at the level of the lumbar spinal cord. However, the potential distribution of the toxin to supraspinal sites has not yet been investigated. In the present study, we examined the distribution of cleaved SNAP-25 (cl-SNAP-25), a marker of BoNT-A activity, in the rat brain following peripheral unilateral BoNT-A administration. Brain tissues from rats treated with BoNT-A (7 U/kg, into the hind paw) were analyzed using immunofluorescent tyramide signal amplification to detect cl-SNAP-25. To assess the contribution of trans-synaptic transport, a BoNT-A-neutralizing antitoxin (2 IU) was administered intrathecally 24 h after BoNT-A injection. Signal intensity was evaluated using a semi-quantitative immunohistochemical scoring method based on cl-SNAP-25-positive nerve fibers. Bilateral cl-SNAP-25 immunoreactivity was observed in multiple supraspinal regions, most prominently within the trigeminal complex and the facial and gracile nuclei. Signal intensity was significantly reduced by intrathecal antitoxin, indicating that trans-synaptic transport contributes to central BoNT-A distribution. Peripherally administered BoNT-A reaches distant supraspinal regions, possibly via neuronal retrograde and trans-synaptic transport. Further studies are warranted to clarify exact pathways and alternative distribution routes, determine the functional relevance of central BoNT-A presence, and assess its clinical implications. Full article

Hemolytic Uremic Syndrome (HUS) is a severe clinical manifestation primarily triggered by Shiga toxin-producing Escherichia coli (STEC). While Shiga toxins (Stx) are central to the development of systemic endothelial damage, current recombinant antibody developments have overwhelmingly focused on neutralizing the Stx2 subtype. However, numerous STEC isolates produce Stx1 either independently or alongside Stx2, revealing a critical need to diversify the antibody repertoire for comprehensive antitoxin therapies. To address this, we characterized two novel, fully human recombinant Fabs targeting Stx1 (FabB6:Stx1 and FabC8:Stx1) selected from a synthetic library via phage display. We evaluated their binding specificity and neutralizing activity in Vero and human proximal tubular epithelial (HK-2) cells, as well as in primary human glomerular endothelial cells (HGEC exposed to HUS-derived STEC supernatants. Both Fabs exhibited high specificity and nanomolar affinity for Stx1. Notably, they displayed cell-type-dependent neutralization profiles, with FabC8:Stx1 demonstrating superior and more consistent neutralization in HK-2 cells. Crucially, when evaluated alongside previously characterized anti-Stx2 antibodies (FabC11:Stx1/Stx2 and FabF8:Stx2), the Stx1-specific Fabs conferred complementary protection against clinical STEC isolates. These findings support the inclusion of Stx1-targeting recombinant antibodies into broader multi-toxin neutralization strategies, thereby expanding the therapeutic potential against STEC-associated diseases. Full article

Antibiotic resistance is a growing global health threat. However, its evolution cannot be fully understood without considering antibiotic tolerance and persistence. Persister cells are phenotypic variants that survive lethal antibiotic exposure without heritable resistance, primarily through growth arrest, metabolic slowdown, and stress-adaptive states. Although persistence has been viewed as a transient survival phenomenon, increasing evidence suggests that it may also have a genetic basis by preserving populations during antibiotic-induced bottlenecks and enabling regrowth, mutation, and selection under certain conditions. This review examines the molecular mechanisms underlying persister formation, including toxin–antitoxin systems, stringent-response signaling, ATP depletion, translational arrest, and stress-response networks. We discuss how persistence contributes to antibiotic tolerance in biofilms, host environments, and recurrent infections, and how repeated antibiotic exposure may promote stepwise evolution from phenotypic survival to stable resistance in specific contexts. Evidence from experimental evolution, clinical observations, and system-level analyses supports a potential but context-dependent link between persistence and resistance. We also highlight therapeutic strategies targeting persister cells, including antipersister compounds, metabolic activation, combination therapies, bacteriophages, and alternative approaches. Finally, we outline future research directions, emphasizing single-cell technologies, systems biology, longitudinal clinical studies, and evolution-informed treatment design. A comprehensive understanding of persistence and its evolutionary implications is essential for improving treatment efficacy and limiting the emergence of long-term antibiotic resistance. Full article

Salmonella persistence contributes to infection relapse, chronic carriage, and reduced antibiotic efficacy. Traditionally viewed as dormant subpopulations that passively survive antibiotic exposure, persister cells are now increasingly recognized as dynamic, heterogeneous, and context-dependent physiological states shaped by bacterial regulatory programs and host microenvironmental pressures. This review examines Salmonella persistence from a multiscale perspective. We first clarify key antibiotic survival phenotypes, including resistance, heteroresistance, tolerance, persistence, and viable but non-culturable states. We then discuss how host-derived stressors, such as phagosomal acidification, nutritional restriction, metal perturbation, and reactive oxygen and nitrogen species, promote growth-restricted, persistence-associated bacterial states. At the bacterial level, we summarize stress-response networks involving the stringent response, SOS response, toxin–antitoxin systems, and auxiliary regulators that coordinate metabolic remodeling, growth restriction, and antibiotic survival. At the host level, we highlight how organ reservoirs, immune cell subsets, metabolic cues, and Salmonella-mediated immune niche remodeling shape persistence-associated phenotypes in vivo. Finally, we discuss clinical and translational implications, including endogenous relapse, resistance evolution, and emerging anti-persistence strategies. Together, this review provides a framework for understanding Salmonella persistence as a multiscale, niche-dependent process relevant to recurrent and chronic infection. Full article

Type II toxin–antitoxin (TA) systems are significantly expanded in the Mycobacterium tuberculosis complex; however, the functional role of the VapBC40 system in Mycobacterium bovis (M. bovis) pathogenesis remains poorly characterized. This study aimed to investigate the role of VapBC40 in mycobacterial virulence and evaluate its potential as a target for rational vaccine attenuation. We performed evolutionary analysis and yeast two-hybrid assays to characterize VapBC40 system specificity, conducted in vitro macrophage infection models and in vivo murine studies to assess virulence contribution, and evaluated the immunoprotective efficacy of a VapC40 knockout strain. Evolutionary analysis revealed progressive sequence conservation and stringent homologous pairing specificity within the VapBC40 system. The VapC40 toxin correlates with enhanced intracellular bacterial survival, increased host cell death, and more severe pulmonary pathology with systemic dissemination. Based on these findings, we evaluated the vaccine potential of a vapC40 knockout strain. Immunization with this attenuated strain elicited a Th1 cellular immune response, characterized by enhanced IFN-γ production and increased frequency of CD4⁺IFN-γ⁺ T cells. Upon challenge with virulent M. bovis, the knockout strain conferred superior protection compared to the conventional BCG vaccine, significantly reducing lung pathology and restricting extrapulmonary bacterial dissemination. Although the molecular mechanisms underlying VapC40-mediated effects remain to be fully elucidated, our findings suggest an important role of the VapBC40 system in mycobacterial-host interactions and support its potential as a target for next-generation tuberculosis vaccine development. Full article

A highly potent antitoxin for botulinum neurotoxin (BoNT) serotypes A and B has been developed that comprises three monoclonal antibodies (mAbs) targeting BoNT/A and three targeting BoNT/B. These oligoclonal antibody combinations neutralize toxin by simultaneously binding non-overlapping epitopes, thereby promoting rapid toxin clearance. All six mAbs use the same human Fc and framework and have been individually manufactured using the same expression platform and purification process. To minimize the time and labor required to produce the divalent antitoxin, we tested a co-expression and co-purification strategy for the three mAbs per serotype. The mAbs were expressed in CHO-K1 cells, and the media were optimized for co-expression in 10 L bioreactors. Chromatographic co-purification consisted of Protein A capture, followed by strong anion exchange chromatography in flow-through mode and cation-exchange chromatography in bind-elute mode. Co-expression experiments demonstrated that expression of the three anti-BoNT/A antibodies remained within approximately ±30% of the optimal equimolar ratio, whereas the anti-BoNT/B antibodies showed greater variability. Downstream purification steps achieved recoveries greater than 95% per chromatographic step, resulting in overall process yields of approximately 63–75%. This strategy provided sufficient purity of all six mAbs while largely preserving their relative ratios. These results demonstrate the feasibility of producing oligoclonal antitoxin antibodies using co-expression and shared purification strategies. Such approaches may simplify the manufacturing of antibody cocktails while maintaining product quality and biological activity. Full article

Bacterial persistence has been extensively studied as a possible explanation for strain survival under stress; however, in Enterococcus spp., this ability is still an understudied phenomenon. In this study, 40 Enterococcus spp. isolates of human clinical (n = 10), veterinary commensal (n = 10), veterinary clinical (n = 10) and veterinary environmental (hospital surfaces) (n = 10) origins, were exposed to a high concentration of ciprofloxacin. Time–kill curves were established, after which antimicrobial susceptibility profiles were reassessed. Subsequently, the only presumptive persister was selected for Whole-Genome Sequencing, together with one isolate showing no evidence of persister formation. Comparative genomic analyses were conducted to identify genetic variations between exposed and non-exposed isolates and to explore potential genetic determinants associated with persistence. Observed genetic features present in the persister isolate included toxin–antitoxin systems, a cold-shock protein and the tyrosine-type recombinase/integrase XerC, which may represent putative candidates for further investigation. Interestingly, the majority of toxin–antitoxin system-associated genes were found in plasmids. This study represents an important step towards a better understanding of persistence development in Enterococcus spp.; however, validation using other methodologies such as RNA-sequencing is an important next step. Full article

A recent publication proposed that the main biological function of chromosomal toxin–antitoxin systems (TASs) in free-living bacteria is to optimize fitness by mediating K Sensing and Control via a Nutrient-Responsive Cybernetic System. Viable cell density data were consistent with analog (continuous) regulation of population dynamics and cellular physiology throughout the life cycle; however, exactly how bacteria utilize TASs to regulate this was not explained in that publication. Two different concepts of injury have been proposed in the field of microbiology: (1) injury due to external physical and chemical stresses, which lead to sublethal (reversible) or lethal (irreversible) injury depending on the degree of injury, and (2) injury due to internal, self-inflicted stresses mediated by TA toxins. While self-inflicted injury due to TA toxins has been recognized as playing a role in growth arrest and dormancy, which can be reversed by repair, there is little support for TA toxins causing irreversible programmed cell death under normal physiological conditions. The purpose of the present paper was to explain how merging the above two concepts of injury might reveal how TASs optimize the fitness of free-living bacteria under normal physiological conditions by continuously regulating the ratio of injury: repair throughout the life cycle. Full article

Background: Persistence represents a critical evolutionary reservoir for the development of antimicrobial resistance in Acinetobacter baumannii (Ab). Understanding the basal mechanisms that enable this survival strategy is crucial for elucidating how high-risk clones evolve resistance during therapy. Methods: High-dose colistin time-kill assays were performed in ten ST2 clinical colistin-susceptible (COL-S) Carbapenem-Resistant Ab (CRAB) developing in vivo stable and full-colistin resistance to detect persisters. Genomics and basal transcriptomics of chromosomal/plasmid toxin–antitoxin systems (T/As) were performed, as duplicates for each sample, in two ST2 COL-S CRAB to investigate the genomics and basal T/A transcriptomic backgrounds. Results: Phenotypically, all strains showed a persistent subpopulation (~1% survival at 8 h) under 5× COL MIC exposure. Genomics identified 10 type-II and one type-IV T/A systems. Basal transcriptomics revealed active expression patterns mainly of GNAT superfamily T/A systems, with consistently low toxin mRNA levels associated with toxin- or antitoxin-directed asRNAs in chromosomal modules. This architecture defined new dual-combined regulatory models in which asRNAs acted as primary T/A mRNA balance modulators, putatively impacting on the T/A mRNA ratio. Conversely, the plasmid-encoded BrnT/A module showed a highly balanced expression. Conclusions: Our findings revealed, for the first time, the role of the type-II GNAT T/A superfamily as putative molecular switchers via a fine-tuning transcript balance regulation, impacting the transition from a metabolically active cell state to a dormant one in developing colistin persistence and in vivo resistance CRAB. Full article

Brucella anthropi is an emerging opportunistic pathogen characterized by intrinsic resistance to most $\beta$-lactams. However, the acquisition of carbapenem resistance in this species has rarely been documented in environmental, animal, or clinical settings. In this study, a multidrug-resistant strain, SBA01, was isolated from wastewater-irrigated soil. SBA01 exhibited phenotypic resistance to carbapenems and colistin, the latter being independent of mcr genes. Genomic analysis localized bla_IMP-1 on a stable 21 kb plasmid maintained by a Type II toxin–antitoxin system. While non-self-transmissible, this plasmid was mobilized to Escherichia coli and Klebsiella pneumoniae via an unclassified 50 kb helper plasmid. Additionally, a 217 kb prophage-bearing megaplasmid was identified, enhancing genomic plasticity. Genomic screening identified 32 putative virulence determinants, including markers associated with host interaction. Risk profiling indicated an elevated hazard index for SBA01, driven by the convergence of multidrug resistance, cryptic mobilization capacity, and opportunistic survival traits. These findings position B. anthropi as a resilient environmental reservoir for clinically relevant carbapenemases. Expanding surveillance frameworks to include such adaptive hosts is necessary to better evaluate potential occupational exposures at the wastewater–soil interface. Full article

Tetanus is a zoonotic disease posing significant threats to both humans and animals, particularly horses, sheep, and ruminants. Current antitoxin therapies rely on animal-derived immunoglobulins, presenting challenges including animal welfare concerns, pathogen contamination risks, and manufacturing complexity. Alpaca-derived nanobodies (VHH) are promising alternatives owing to their high antigen-binding affinity, thermostability, and potential for microbial production. We developed highly active trivalent VHH antibodies (tVHH) that target multiple epitopes of tetanus neurotoxin (TeNT). Following alpaca immunization with tetanus toxoid, 41 VHH clones were isolated using phage display. Six VHH clones were selected through in vivo neutralization assays, from which three clones of VHH (8, 11, 36) were selected to construct tVHH-8/11/36 and tVHH-8/36/11. Using an improved 21-day mouse neutralization assay, tVHH-8/11/36 demonstrated exceptional neutralizing activity of approximately 1580 IU/mg against 4000 LD₅₀ of toxin, substantially exceeding current human and veterinary anti-tetanus immunoglobulin preparations. Surface plasmon resonance and ELISA confirmed that each VHH recognizes different TeNT domains, producing synergistic neutralizing effects through multimerization. Since antitoxin therapy challenges are common to both animals and humans, this tVHH technology supports One Health by providing a unified therapeutic platform applicable across species through sustainable microbial production. Full article