Congran Li

Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous microorganism and common gram-negative bacterium [1]. P. aeruginosa induces various severe infections in hospitals, especially in immunocompro...

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with limited therapeutic options. To investigate the roles of the pulmonary microbiota and metabolism in fibrosis, we established a bleomycin (BLM)-induced mouse model at 14- and 28-day timepoints and performed integrated 16S rRNA gene amplicon sequencing and untargeted metabolomic analyses. Histological and Western blot analyses confirmed significant fibrotic changes and the upregulation of fibrotic markers. Microbiome profiling revealed marked dysbiosis after BLM exposure, characterized by reduced microbial diversity and enrichment of Klebsiella. LC-MS–based metabolomic analysis identified substantial perturbations in the lung tissue metabolome, particularly in lipid metabolism, amino acid metabolism, and energy pathways. Correlation analysis indicated a strong positive association between the abundance of Klebsiella and the levels of specific dipeptides, including Ala-Hyp-Gly, Asp-His, and Asp-Asn. The accumulation of these dipeptides may reflect increased collagen degradation and turnover in fibrotic lungs. Collectively, our findings demonstrate that BLM-induced pulmonary fibrosis is accompanied by coordinated alterations in the lung microbiome and metabolome. Notably, microbial dysbiosis, particularly the expansion of Klebsiella, may be associated with alterations in amino acid metabolism and fibrotic progression.

Background: Staphylococcus epidermidis , typically regarded as a harmless commensal, has become one of the major causes of nosocomial infections, including ocular, skin, medical device-associated and bloodstream infections. Therefore, we analyze its population structure through genomic analysis integrated with metadata. Results: We performed whole-genome sequencing-based population genomic analyses by integrating 1742 publicly available S. epidermidis genomes (accessed by August 2025) with 94 newly sequenced isolates. Our analyses revealed that S. epidermidis represents a species complex composed of four phylogenetic lineages (phylogroups 1-4) with diverse clonal backgrounds and a broad global distribution. The species harbors an open pan-genome and demonstrates a strong capacity to acquire novel genetic traits through mobile genetic elements. Extensive antimicrobial resistance and substantial virulence potential were observed across lineages. Notably, phylogroup 1, dominated by ST 2, exhibited a 97.8% detection rate of the methicillin resistance gene mecA , likely driven by clonal expansion and horizontal gene transfer, identifying it as a high-risk lineage. The analysis of enriched genes in blood-derived strains showed that the adaptability of S. epidermidis in bloodstream-associated environments is controlled by multiple genes, involving antimicrobial resistance, cell wall remodeling, environmental adaptation, and core metabolism. Conclusions: This study provides a comprehensive population genomic framework for S. epidermidis , elucidating its population structure, genomic diversity, antimicrobial resistance, and virulence-associated genetic features. These findings offer valuable insights into the evolutionary dynamics and pathogenic potential of S. epidermidis and provide an important genomic resource to inform infection control strategies and clinical management.

Staphylococcus aureus remains a leading cause of morbidity and mortality worldwide, with persistent and relapsing infections posing a major global health threat. Here, we report that baloxavir, an FDA-approved influenza antiviral, exhibits antibacterial activity against S. aureus. Baloxavir demonstrated potent activity against both MSSA and MRSA clinical isolates with MICs of 2–4 μg/mL and exhibited concentration-dependent antibacterial activity in time-kill assays. Notably, baloxavir effectively eliminated intracellular S. aureus in both A549 alveolar epithelial cells and RAW264.7 macrophages at 10 μg/mL and achieved complete eradication in A549 cells at 50 μg/mL. In vivo, baloxavir (20–40 mg/kg) significantly improved survival in MRSA-infected mice from 12.5% to 75–87.5%. Transcriptomic analysis revealed significant downregulation of purine de novo biosynthesis genes, including purF and purK, which was validated by RT-qPCR (r = 0.862, p = 0.027). This study demonstrates for the first time that baloxavir possesses significant antibacterial activity against S. aureus including MRSA, positioning it as a promising repurposed candidate for treating persistent intracellular infections and post-viral superinfections.

The genetic connection between virulence and antibiotic resistance remains poorly understood. Our previous RNA-Seq analysis of a polymyxin-resistant Klebsiella pneumoniae ATCC BAA2146 mutant identified a highly expressed VirK/YbjX family gene (kpn2146_RS17285), encoding a conserved membrane protein, designated virK (virulence required for Klebsiella pneumoniae). While PhoP-dependent antibiotic resistance is mediated through established pathways such as arn/pmr, we identify VirK as a PhoP-regulated factor specifically contributing to virulence. VirK localizes to the outer membrane and, although not involved in lipopolysaccharide modification, its deletion modestly reduced bacterial virulence in a mouse systemic infection model. Transcriptional and electrophoretic mobility shift assays demonstrated that virK is directly activated by the PhoP protein. A strong positive correlation between virK and phoP expression (r = 0.98) was also observed in multidrug-resistant clinical isolates. Since the PhoP/PhoQ two-component system mediates polymyxin resistance, its direct regulation of VirK uncovers an adaptive mechanism coupling enhanced virulence with antibiotic resistance. These findings reveal a previously unrecognized PhoP/VirK regulatory pathway that contributes to pathogenicity in polymyxin-resistant Klebsiella pneumoniae, offering new insights into bacterial evolution and suggesting that targeting PhoP/PhoQ could provide an effective strategy to combat multidrug-resistant K. pneumoniae infections.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a serious clinical threat, and D-Serine (D-Ser) showed significant sensitization effects on β-lactams against MRSA in our previous study. Quantitative PCR analysis found the elevated expression of the dlt operon with D-Ser combination, which is responsible for wall teichoic acid (WTA) modification involving D-Alanine (D-Ala). This study aims to verify the effect of D-Ser on WTA modification through the dlt pathway and explore the related effects on bacteria. The DltA and DltC were recombined, and enzyme kinetic evaluations with different D-amino acids were then conducted; it was found that D-Ser is the second-best substrate for DltA (just after D-Ala), no matter whether DltC is present or not. D-Ser treatment also lowered WTA generation as demonstrated by WTA phosphate quantification and native-PAGE electrophoresis, increased the susceptibility of S. aureus to polymyxins, and elevated the mouse survival rate in the MRSA intraperitoneal infection model without affecting the bacterial loads in the main organs, indicating possible effects of D-Ser on MRSA virulence through WTA modification. In conclusion, the current study provided evidence for D-Ser modification of WTA via the dlt pathway, and its possible involvement in D-Ser sensitization deserves further investigation.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the mechanism of non-carbapenemase carbapenem resistance is still unclear. In the current study, we investigated the contributions of point mutations in mexR , oprD , and ftsI to carbapenem resistance in P. aeruginosa during in vivo evolution studies with consecutive clinical isolates. Real-time qPCR and Electrophoretic Mobility Shift Assay demonstrated that MexR (Gln55Pro) mutation increased MexAB efflux pump genes expression by altering MexR’s binding capacity, leading to a four- to eight-fold increase in meropenem MIC in the Pae d1 Green ∆ mexR and PAO1∆ mexR mutants. The OprD (Trp415*) truncation affected porin structure, and the constructed mutant Pae d1 Green oprD Trp415* increased meropenem MIC by 16-fold (from 0.25 to 4 µg/mL). The contribution of ftsI mutation to meropenem resistance was confirmed by clinical linkage analysis and was estimated to cause a two-fold increase in meropenem MIC by comparing the resistant clinical isolate with the Pae d1 Green oprD Trp415*∆ mexR double mutant. The study found that the oprD Trp415* allele alone accounts for the imipenem MIC in clinical isolates, while the ∆ mexR and ftsI Arg504Cys alleles do not contribute to imipenem resistance. In conclusion, we identified and explored the contributions of mexR , oprD, and ftsI mutations to high level non-carbapenemase carbapenem resistance in P. aeruginosa . These findings highlight the interplay of different mutations in causing non-carbapenemase carbapenem-resistance in P. aeruginosa . IMPORTANCE: The emergence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant global health threat, complicating treatment options for infections caused by this pathogen. Understanding the mechanisms behind non-carbapenemase carbapenem resistance is critical for developing effective therapeutic strategies. This study provides crucial insights into how specific point mutations in key genes- mexR , oprD , and ftsI -contribute to carbapenem resistance, particularly the MexR (Gln55Pro) mutation’s effect on efflux pump expression and the OprD (Trp415*) truncation’s impact on porin structure. The findings elucidate the complex interplay of these mutations, highlighting their roles in conferring high-level resistance, and underscore the imperative for continued research to inform therapeutic strategies against CRPA infections.