Precise differentiation between closely related species is crucial for effective clinical diagnosis and treatment. Haemophilus influenzae and Haemophilus aegyptius are phenotypically similar but differ in pathogenic potential, with H. influenzae involved in respiratory and invasive infections, and H. aegyptius mainly associated with ocular disease and, rarely, severe systemic outcomes. Traditional phenotypic methods and gene-based analyses often struggle to reliably distinguish them due to high sequence similarity and shifting taxonomic boundaries. In this study, we present a high-resolution proteotyping method that combines whole-cell MALDI-TOF MS, nano-LC-MS/MS, and comparative genomics to identify and verify species-specific protein biomarkers across a broad mass range (m/z 3,000-40,000). Using sequenced reference strains and extensive in silico analyses, we found 31 unique protein biomarkers that allow reliable differentiation between H. influenzae and H. aegyptius. These markers were confirmed through LC-MS/MS peptide mapping and comparative genomics, showing consistent sequence differences and phylogenetic clustering. Our results demonstrate that combining advanced proteomic profiling with genome-based validation effectively addresses the limitations of conventional identification methods. This approach enhances diagnostic accuracy for closely related pathogens and facilitates the ongoing refinement of spectral and genomic reference databases, which are essential for precise microbial identification in clinical microbiology.IMPORTANCESpecies-level resolution of clinically relevant pathogens remains a challenge in clinical diagnostics. Haemophilus influenzae and Haemophilus aegyptius are particularly problematic because of their high genetic similarity, overlapping phenotypes, and distinct clinical implications. Conventional techniques often fail to reliably distinguish them, leading to frequent misclassification. Although diagnostic issues within the H. influenzae complex are well recognized, differentiation between H. influenzae and H. aegyptius remains underexplored. Accurate and rapid identification is essential, as these opportunistic pathogens differ in disease manifestations and epidemiology, and misidentification can result in suboptimal treatment. Our study presents a robust high-resolution nano-LC-MS/MS proteotyping approach, combined with whole-cell MALDI-TOF MS and comparative genomics, to confidently classify H. influenzae and H. aegyptius at species and strain levels. By identifying species-unique peptide biomarkers, this method fills a diagnostic gap and improves species-level microbial diagnostics, understanding of pathogenicity, and epidemiological surveillance.

The study highlighted the biosynthetic potential of Streptomyces chrestomyceticus B5, which is a promising strain for producing various bioactive metabolites and is rich in biosynthetic gene clusters responsible for encoding multiple metabolites and producing an antifungal compound, a streptimidone derivative or isomer, active against drug-resistant Candida auris.

This study analyzes the demographic-dependent variability of the intratumor mycobiome, providing a novel understanding of fungal abundance across different cancer types and patient demographics. By analyzing over 5,000 tumor samples from The Cancer Genome Atlas, the research identified 24 fungal species with significant abundance variations linked to demographic factors such as race, age, sex, and body mass index. These findings underscore the complexity of the tumor microenvironment and the importance of accounting for demographic diversity in cancer research. The study emphasizes the necessity of using robust data normalization and batch correction techniques to avoid spurious associations in order to ensure the reliability of mycobiome analysis. This work highlights the mycobiome as a new frontier in precision oncology and paves the way for future personalized cancer diagnostics and treatments that account for the influence of demographic factors on tumor biology.

Bovine respiratory disease (BRD) remains a significant economic challenge in dairy cattle despite extensive vaccination programs that have been developed and implemented during the last few decades. This study investigated the nasopharyngeal microbiome and resistome of dairy cattle across various life stages to understand the roles of microbial communities associated with BRD. A case-control study was conducted on three commercial dairy farms in Northern California, collecting nasopharyngeal swabs from 69 animals, including preweaned calves, weaned heifers, and lactating cows with and without BRD. Shotgun metagenomic sequencing was used to characterize both microbiome and resistome profiles observed at the time of BRD diagnosis. Results revealed that BRD is associated with distinct microbial community patterns, rather than the increased abundance of a specific pathogen. Age was a critical factor influencing microbial diversity, with adult cows showing the highest diversity and weaned heifers with BRD showing the lowest. A total of 1,164 bacterial species were identified, with BRD cases harboring 14 unique species compared to control animals. BRD cases were characterized by the co-occurrence of multiple respiratory pathogens, including Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mesomycoplasma species, which showed positive correlation with BRD cases but negative correlations in BRD controls, while BRD control animals showed significantly higher abundance of commensal Staphylococcus species. Resistome analysis identified 65 antimicrobial resistance genes, with BRD cases harboring more unique resistance genes than BRD controls. These findings challenge traditional single-pathogen models and demonstrate that BRD is likely the result of complex microbial community interactions and changes in community abundance, providing new potential targets to explore when considering prevention strategies toward promoting microbial communities that prevent or reduce the risk of BRD.IMPORTANCEBovine respiratory disease (BRD) represents one of the most economically challenging conditions in cattle production, with an estimated direct cost that exceeds $165 million annually in the United States alone. Despite decades of vaccination efforts targeting known pathogens, BRD prevalence remains unchanged, indicating an incomplete understanding of disease pathogenesis. This study provides critical insights by shifting focus from individual pathogens to entire microbial communities, revealing that BRD involves complex bacterial interactions, as well as the role of the understudied nasal commensal microbiome in healthy animals. The identification of distinct "pathobiomes" associated with disease and protective commensal communities in healthy animals fundamentally changes approaches to BRD prevention and treatment. The discovery that age significantly influences microbiome stability highlights critical intervention periods. Furthermore, the association between BRD and increased antimicrobial resistance genes raises concerns about current treatment and overall management practices, selecting for drug-resistant communities. This research provides a foundation for developing microbiome-based diagnostic tools and interventions supporting healthy microbial ecosystem development.

Analyzing gene expression data from three separate clinical trials, we find that the antiretroviral drug tenofovir, which belongs to the class of nucleotide analogue reverse transcriptase inhibitors, induces the type I/III interferon system of innate immunity in the mucosa. This effect occurs in the absence of HIV infection and manifests itself over various treatment durations and after both oral and topical drug delivery. Tenofovir and other related medications are important components of long-term antiretroviral treatment taken by people living with HIV. Therefore, this unexpected immunological effect might need to be considered as a potential contributor to comorbidities in people living with HIV, as well as an immunopharmacological co-factor when testing novel HIV cure interventions. This study is registered with ClinicalTrials.gov as NCT01768962, NCT01687218, and NCT01232803.

Ventilator-associated pneumonia (VAP) remains a leading complication in mechanically ventilated patients, yet the contribution of the respiratory microbiota remains poorly understood. The PULMIVAP study is a multicenter, longitudinal cohort investigation of respiratory microbiota composition and host immune responses in critically ill adults intubated for non-pulmonary conditions. A total of 146 intubated adult patients were enrolled across eight Italian ICUs, forming matched groups of 73 with VAP and 73 without. Oropharyngeal swabs and endotracheal aspirates were collected at intubation and either at VAP diagnosis or at a matched point in controls for a total of 584 biological samples. Metataxonomic analyses revealed substantial temporal shifts in microbial communities across both upper and lower respiratory compartments, with a trend toward reduced microbial richness in patients who developed VAP. Several genera, such as Corynebacterium, were more abundant in no-VAP patients, whereas Escherichia-Shigella and Peptoniphilus were enriched in VAP samples. Cytokine-microbiota correlation analysis suggested a pro-inflammatory signature in VAP patients, with Citrobacter positively associated with IFN-γ and TNF-α, while several commensal genera were inversely correlated with inflammatory mediators. Additionally, taxa associated with VAP correlated with lower PaO2/FiO2 ratios, implicating them in disease severity. Consistently, several bacteria, such as Corynebacterium, appeared to be linked to better respiratory outcomes, suggesting protective or risk-associated microbial profiles. Overall, these findings highlight the complex interplay between microbial communities and mucosal immunity in the pathogenesis of VAP. The identification of condition-associated microbial and immunological signatures may inform future strategies for risk stratification and targeted prevention.IMPORTANCEVentilator-associated pneumonia (VAP) remains a major complication of mechanical ventilation, yet most microbiome studies have focused on late-stage infection or single airway compartments, limiting insight into early microbial dynamics associated with VAP risk. By longitudinally characterizing upper and lower airway microbiota before and during VAP development, this study provides new insights into microbial and immune patterns associated with susceptibility and disease severity in humans. These findings contribute to the current understanding of VAP pathogenesis by suggesting a role for early airway dysbiosis and local immune responses alongside clinical factors. Remarkably, the identification of taxa associated with risk or protection supports the potential for microbiota-informed monitoring and future risk stratification strategies during mechanical ventilation.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT04849039.

Mycobacterium avium-intracellulare complex (MAC) infections are the most common nontuberculous mycobacterial infections, and ethambutol (EMB) is one of the main therapeutic agents used to treat MAC infections. However, the EMB resistance profile and its resistance mechanisms in M. avium remain poorly understood. This study determined the minimum inhibitory concentration (MIC) of 40 M. avium clinical strains, revealing that 97.5% (39/40) of the strains were intermediate (MIC = 4 µg/mL) or resistant (MIC ≥ 8 µg/mL) to EMB. One susceptible clinical isolate strain 245 (MIC = 2 µg/mL) was picked for EMB-resistant mutant isolation, and a total of 121 resistant mutants were isolated and subjected to whole-genome sequencing or Sanger sequencing. Integrated analysis revealed that 94.21% (114/121) of the mutants carried mutations in the ubiA gene, which encodes decaprenylphosphoryl-β-D-5-phosphoribose (DPPR) synthase-an enzyme involved in cell wall biosynthesis that has been associated with high-level EMB resistance in Mycobacterium tuberculosis. Complementation with the wild-type ubiA gene restored EMB susceptibility in EMB-resistant mutants and clinical strain 322 (EMB MIC = 64 µg/mL) with ubiA mutation, reducing the MICs from 32 to 4 μg/mL and from 64 to 8 μg/mL, respectively. This study indicates that the ubiA mutation is the major mechanism of EMB resistance in M. avium, which should facilitate the development of molecular tests for rapid detection of resistance in this organism.IMPORTANCEThis study identified ubiA as a key gene associated with ethambutol (EMB) resistance in Mycobacterium avium, a finding which has not previously been reported. Furthermore, although ubiA has been linked to EMB resistance in Mycobacterium tuberculosis (MTB), mutations in this gene account for only a small proportion of EMB-resistant MTB isolates. In contrast, our study showed that ubiA played a major role in EMB resistance in M. avium. Our findings contribute to the development of molecular assays for rapid detection of EMB resistance in M. avium and highlight the distinct main resistance mechanisms across different bacterial species to the same antibiotic.

Volume 14, no. 1, e01690-25, 2026, https://doi.org/10.1128/spectrum.01690-25. Figure 3 caption: “…panel A showing growth for 24 h to highlight growth in glucose M9 media and panel B showing the full 96 h to highlight the growth in xanthan M9 media” should read “…panel A showing the full 96 h of growth and panel B showing the first 24 h of growth to highlight the faster growth in glucose M9 media.”

Evidence suggests the persistence of non-spore-forming Acinetobacter johnsonii in high-stakes controlled and nutrient-limited environments. Here, we investigated the mechanisms underlying this adaptability through a comprehensive genomic analysis of 22 isolates of A. johnsonii from NASA's Payload Hazardous Servicing Facility (PHSF) and one carbapenem-resistant strain (E154408A) from patient colonization in Ireland. Core-genome phylogeny revealed clustering of PHSF-originating isolates in a monophyletic clade divergent from the main species lineage. Species-wide virulence-associated genes and metabolic reconstruction indicated the exclusive presence in PHSF-originating isolates of two complete efflux pumps and a conserved allantoin racemase, suggesting adaptability for multiple environmental stresses. The ubiquity of blaOXA in genomes analyzed (n = 112) and the phenotypically validated multidrug-resistant profile of the E154408A strain highlight A. johnsonii's potential as an antimicrobial resistance (AMR) reservoir. Plasmidome analysis suggested gain/loss events across the monophyletic population and potential AMR acquisition pathways. Genome-to-metagenome mapping identified genomic signatures of A. johnsonii in PHSF >10 years post-initial isolation.IMPORTANCEAcinetobacter johnsonii is increasingly recognized as an emerging human pathogen, with growing evidence of its ability to persist in controlled, high-stakes environments, posing risks as both a persistent environmental contaminant and an antimicrobial resistance (AMR) reservoir. Yet, gaps remain in our understanding of its AMR profile and the mechanisms that enable its enhanced environmental adaptability. This knowledge is necessary in contexts where biological cleanliness is a priority, such as clinical settings and spacecraft assembly facilities' cleanrooms, where contamination of hardware with terrestrial microorganisms is concerning. In this study, we aim to address some of the key knowledge gaps by providing genomic insights into a rare multidrug-resistant clinical isolate and 22 NASA cleanroom isolates that persisted for over a decade in extremely clean conditions. Our findings will help assess the contamination risk of A. johnsonii in high-stakes environments and ultimately strengthen our ability to manage this microbial contaminant across terrestrial and extraterrestrial settings.Cleanroom-derived A. johnsonii genomes show traits consistent with increased adaptability.Genomic signatures of A. johnsonii persisted in the cleanrooms for over 10 years.blaOXA is ubiquitously found in all 112 A. johnsonii genomes analyzed.Isolate E154408A is the first reported patient colonization case by carbapenem-resistant A. johnsonii in Europe.