Pathogens最新文献

Antimicrobial drug resistance is an escalating global health burden, often driven by multiple genetic changes within key resistance-associated genes. Achieving multiplex capability of mutation detection while maintaining simplicity and affordability is critical, particularly in point-of-care and resource-limited settings. Here, we introduce a strategy for multi-site mutation detection using single isothermal amplification of a nucleic acid fragment spanning multiple mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene, encompassing codons 516 and 526 in Mycobacterium tuberculosis. This unified design eliminates competition among targets amplified by multiple primer sets. Site-specific hybridization probes enable accurate discrimination between wild-type and mutant sequences, while an integrated self-calibration probe provides normalization to mitigate variability from sample concentration and sample matrix interference. To validate this approach, we applied it to detect rifampicin (RIF) resistance mutations at codons 516 and 526 of the rpoB gene in Mycobacterium tuberculosis, which are two key targets for molecular diagnostics and surveillance. Using 42 artificial DNA fragments, which included both wild-types and mutants with single- or two-site mutations, the assay achieved 100% accuracy in discriminating between wild-type and mutant sequences for codon 516. On the other hand, sequences harboring mutations at codon 526 were identified with 100% accuracy, compared to 94% accuracy for wild-type sequences. Overall, the system achieved a 100% positive percent agreement (PPA) for drug-resistance sequences and 97% negative percent agreement (NPA) for drug-sensitive sequences based on these 42 samples. These findings suggest that this method has the potential to provide a reliable framework for multi-site mutation detection.

Bacterial biofilms play a major role in delayed wound-healing and in the development of chronic, non-healing wounds. Natural, plant-based agents, which can eradicate bacterial biofilms, are alternatives to antibiotics and antiseptics in the treatment of bacterially contaminated wounds. Bacterial wound biofilms are three-dimensional and complex microbial communities. Therefore, in this study, we used a three-dimensional fibrin-gel wound biofilm (FGWB) model to compare a commonly used natural agent in wound care, hypochlorous acid (HOCl), to a combination of two natural plant-based agents, polygalacturonic acid (PG) and caprylic acid (CAP) (PG + CAP), for their abilities to eradicate resistant bacterial biofilms of common wound pathogens methicillin resistant Staphylococcus aureus (MRSA), multi-drug resistant (MDR) Pseudomonas aeruginosa, metallo β-Lactamase Escherichia coli, and Streptococcus pyogenes. PG + CAP produced a significantly greater reduction in viable organisms when compared to HOCL (p ≤ 0.05) against all tested bacterial isolates. PG + CAP was highly effective against biofilms of all resistant bacterial isolates and is a promising option that merits further study for treating chronic wounds contaminated with bacterial biofilms.

Multidrug-resistant Pseudomonas aeruginosa is a major cause of healthcare-associated infections, particularly in high-burden clinical settings where rapid tools to capture clinically relevant resistance and virulence phenotypes are needed. In this study, we applied an integrated whole-genome sequencing (WGS) and Fourier-transform infrared (FTIR) spectroscopy approach to evaluate genotype-phenotype relationships in multidrug-resistant P. aeruginosa isolates collected during the COVID-19 pandemic. High-quality WGS data were used to characterize antimicrobial resistance determinants, mobile genetic elements, and virulence gene repertoires, while FTIR spectroscopy provided culture-based phenotypic fingerprints reflecting cell envelope composition. Genomic analyses revealed a conserved efflux-centered intrinsic resistance backbone, variably supplemented by acquired β-lactamases and aminoglycoside-modifying enzymes, alongside a largely conserved core virulome with heterogeneity driven primarily by type III secretion system effector profiles. Comparison of FTIR- and WGS-derived distance matrices revealed a weak but statistically significant global association, indicating a non-linear relationship between genomic relatedness and phenotypic similarity. Cluster-level concordance was strongly scale-dependent, with high agreement emerging only at finer clustering resolutions, consistent with FTIR capturing phenotypic variation linked to regulatory, metabolic, and cell envelope adaptations rather than deep phylogenetic structure. Together, these findings show that multidrug resistance and virulence in P. aeruginosa are shaped by a modular genomic architecture that manifests as distinct, measurable phenotypic states. The observed scale-dependent concordance supports FTIR spectroscopy as a rapid, cost-effective phenotypic screening tool for outbreak-oriented surveillance, complementing WGS in integrated antimicrobial resistance monitoring workflows.

Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) and is linked to cases of cutaneous leishmaniasis in dogs. Dogs often develop severe systemic disease and serve as the primary reservoir of L. infantum. Although several vaccine candidates are under development, no vaccine for visceral leishmaniasis has been approved for human use to date. Chemotherapeutic treatment is hampered by toxicity, cost, and the emergence of parasite-resistant strains. Immunotherapy, combining chemotherapy with modulation of Th1 responses, is a promising therapeutic approach. Helicobacter pylori neutrophil-activating protein (HP-NAP), an immunomodulatory protein from Helicobacter pylori, is known to promote Th1 immune responses. A Th1 response activates macrophage promoting parasite killing, while a Th2 response favors disease progression. Macrophages are central for infection, either eliminating parasites (Th1 response) or supporting their persistence (Th2 response). IL-12 is a crucial cytokine in driving Th1 immunity and counteracting Th2 responses. We therefore investigated the role of HP-NAP in an in vitro model of L. infantum macrophage infection. Canine monocyte-derived macrophages from seven dogs were incubated with L. infantum promastigotes. More than 85% of macrophages from all donors were infected, with approximately seven amastigotes per cell. HP-NAP treatment significantly reduced all infection parameters and induced IL-12 production. Collectively, these findings suggest that HP-NAP may represent a promising candidate for adjuvant immunotherapies and vaccine development against L. infantum.

Herpes simplex keratitis (HSK) is classically described as an immunopathological disease driven by recurrent herpes simplex virus type 1 (HSV-1) infection and chronic inflammation. So far, immune-mediated tissue damage has not fully explained the molecular mechanisms governing disease progression toward corneal neovascularization (CNV), a major cause of corneal blindness and vision loss worldwide. Increasing evidence indicates that CNV results from complex interactions that extend beyond leukocyte-driven inflammation, as the host cell machinery, including key pathways and molecular markers, is hijacked by the invading virus to establish and perpetuate replication and lifelong latency. These host-cell interactions regulate angiogenic imbalance, vascular privilege, and tissue remodeling, which collectively promote pathological vascular invasion. This review re-examines HSK by focusing on molecular mechanistic pathways and drivers that regulate disease progression towards CNV, upstream of immune response drivers. Specifically, we discuss the roles of endothelial growth factors, matrix metalloproteinases, Heparanase, and Syndecan-1 signaling, as well as microRNA-mediated regulation, and key signaling axes, including JAK2/STAT3, PI3K/AKT/mTOR, and hypoxia signaling. By integrating these pathways and molecular markers, we propose an updated mechanistic framework, including a conceptual model for the underexplored role of heparanase, and identify pathway-level targets with potential therapeutic relevance for HSK-associated CNV.

HTD1265 is a newly identified antifungal compound that displays potent activity against Candida krusei, a clinically challenging non-albicans species. To elucidate its mechanism of action, we applied an integrative phenotypic approach combining high-resolution morphological profiling, pathway inference, and genetic validation in Saccharomyces cerevisiae. Morphological signature extraction revealed a characteristic defect in nuclear positioning upon HTD1265 treatment. Integration of nuclear positioning traits with global morphological similarity highlighted 36 genes enriched for the Gene Ontology term "tubulin complex assembly." Consistent with this prediction, HTD1265 impaired mitotic spindle elongation without directly inhibiting tubulin polymerization. HTD1265 further induced hallmarks of GimC (prefoldin) deficiency, including aberrant chitin accumulation, actin disorganization, and nuclear mispositioning, and caused hypersensitivity in GimC subunit mutants. These converging observations suggest that HTD1265 exerts antifungal activity by disrupting GimC-dependent cellular processes rather than by directly targeting tubulin. Our findings highlight GimC-dependent cytoskeletal and cell wall regulatory processes as a critical vulnerability for fungal growth and position HTD1265 as a functional tool for dissecting this pathway.

Recent advances in next-generation sequencing have revealed that the virome-the set of viruses residing in the gastrointestinal tract-is a fundamental yet still underexplored component of the human intestinal ecosystem. Despite the prevalence of research focused on bacterial alterations, recent findings suggest a significant role for viral elements within the intestinal microbiota, namely latent viruses, bacteriophages and eukaryotic viruses, in influencing brain health. Alterations in the gut virome may, in particular, contribute to the processes underlying brain aging, cognitive decline, and neurodegenerative diseases such as Alzheimer's, Parkinson's, and multiple sclerosis (MS). This review highlights the potential of intestinal viruses to modulate gut barrier integrity, systemic immune response and neuroimmune inflammation. Such interactions could promote conditions of chronic neuroinflammation, alterations in synaptic plasticity, and neuronal vulnerability. A more comprehensive understanding of the role of the gut virome could potentially result in novel approaches to the early detection and treatment of neurocognitive disorders in adults and older individuals.

Malaria remains a global health threat, with Plasmodium falciparum causing most deaths, especially in sub-Saharan Africa. Although artemisinin-based therapies reduce the burden, drug-resistant parasites threaten control efforts. Mapping the distribution and evolution of molecular resistance markers is vital for evidence-based strategies. This systematic review mapped the global distribution, pooled prevalence, and temporal trends of key P. falciparum antimalarial resistance markers. Following the PRISMA methodology (PROSPERO: CRD4202511098991), databases (PubMed, Web of Science, Scopus, and Google Scholar) and gray sources were searched (July 2005-July 2025). Data were extracted in Rayyan, assessed via the JBI prevalence tool, and analyzed using Python v3.13 for WHO regional distribution, temporal trends, and treatment outcome trends. Of the 1972 records, 261 studies from 64 countries qualified for inclusion in this review. The pooled prevalence was highest for pfdhfr (85.7%), followed by pfcrt (78.0%), pfdhps (73.7%), pfmdr1 (60.5%), and pfk13 (45.0%). High heterogeneity (I² > 95%) and rising pfk13 since 2012 highlight emerging artemisinin resistance, while persistent pfdhfr/pfdhps mutations show that ongoing sulfadoxine-pyrimethamine (SP) pressure on P. falciparum drug resistance, decreased parasite clearance, and treatment failure remain widespread and evolving in Africa. Integrating molecular surveillance into national malaria programs is essential to guide treatment modalities and support progress toward malaria elimination.

Latent tuberculosis infection (LTBI) represents a major global health concern as it constitutes the principal reservoir for future tuberculosis (TB) disease. Its identification is particularly important in Bacille Calmette-Guérin (BCG)-vaccinated populations, where cross-reactivity of purified protein derivative limits the specificity of the tuberculin skin test and hampers targeted preventive therapy. Early Mycobacterium tuberculosis antigens encoded within the RD1 region, especially ESAT-6, CFP-10 and TB7.7, have enabled the development of antigen-specific interferon-gamma release assays (IGRAs) and recombinant skin tests with improved BCG-independent specificity. This narrative review integrates and critically appraises current evidence on the immunobiological properties of early and latency-associated antigens, the cellular mechanisms underlying T-cell-dependent immune reactivity, and the diagnostic performance of IGRAs and ESAT-6/CFP-10-based skin tests, rather than merely summarizing individual studies. Although these platforms rely on different assay principles (in vitro cytokine release versus in vivo delayed-type hypersensitivity), both measure antigen-specific T-cell memory and do not define the biological stage of infection or reliably distinguish latent from incipient or active TB. Across most adult populations, IGRAs demonstrate high specificity and acceptable sensitivity, whereas reduced sensitivity and higher rates of indeterminate results are observed in young children and immunocompromised individuals. ESAT-6/CFP-10-based skin tests show diagnostic accuracy comparable to IGRAs and may offer operational advantages in resource-limited settings. Latency-associated antigens and host biomarkers such as IP-10, together with multi-analyte immune signatures, represent promising avenues for improving diagnostic sensitivity and prognostic stratification but currently lack sufficient validation for routine clinical use. Overall, RD1-encoded antigens remain central to LTBI immunodiagnosis, while future research should focus on developing stage-resolving and prognostic biomarkers, optimized antigen panels, and standardized interpretive frameworks.

Respiratory syncytial virus (RSV) resurged in many regions after the relaxation of stringent non-pharmaceutical interventions (NPIs) implemented during the COVID-19 pandemic. Here, we characterized the epidemiological patterns and molecular evolution of RSV among pediatric inpatients with acute respiratory tract infections (ARTIs) on tropical Hainan Island, China. We retrospectively analyzed 32,329 children (≤18 years) hospitalized at Hainan Women and Children's Medical Center from January 2021 to December 2024. RSV positivity was determined using targeted next-generation sequencing. In total, 4483/32,329 (13.86%) patients were RSV-positive, with a high positivity in 2021 (20.27%, 957/4721), marked suppression in 2022 (2.03%, 106/5227) during intensive NPIs, and a rebound in 2023-2024 (15.31%, 1490/9732; 15.26%, 1930/12,649). RSV positivity was higher in boys than girls (14.42% vs. 13.00%). Seasonality shifted from a summer-autumn peak in 2021 to a spring-summer predominance in 2023-2024. Among 56 sequenced RSV-positive specimens (29 RSV-A; 27 RSV-B), all RSV-A strains belonged to genotype ON1 (lineages A.D.3 and A.D.5.2), and all RSV-B strains belonged to genotype BA9 (lineages B.D.4.1.1, B.D.E.1, and B.D.E.2). Subtype dominance transitioned from RSV-A (2021-2023; mainly A.D.3) to RSV-B in 2024 (all B.D.E.1). Lineage-specific amino-acid and predicted N-glycosylation changes were observed, including loss of the N179 site in A.D.5.2 and acquisition of N258 in B.D.E.1. These findings indicate that RSV circulation on tropical Hainan was strongly suppressed during intensive NPIs and re-established after policy relaxation, accompanied by earlier seasonal activity and clear lineage replacement, underscoring the need for sustained genomic surveillance to inform locally tailored clinical preparedness and immunization strategies.