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The genus Acinetobacter is vast and diverse regarding its hosts. However, it is best known as an opportunistic pathogen that causes hard-to-treat nosocomial infections. Yet, some species of the genus can be beneficial for some hosts. Such is the case of Acinetobacter calcoaceticus, which can have a significant impact on tomato plants, as was recently shown in a paper by Robertson et al. (S. Robertson, A. Mosca, S. Ashraf, A. Corral, et al., mSphere 11:e00842-25, 2026, https://doi.org/10.1128/msphere.00842-25). Importantly, that study also exemplifies how metagenomics in general, but metagenome-assembled genomes in particular, can be employed to understand the functional specialization and identity of the bacterial species dwelling in particular environments.

Viruses are obligate parasites that rely extensively on host cellular machinery to complete their life cycles. Therefore, examining the fundamental nature behind how viruses interact with their hosts can teach us more about the pathogenesis of these microbes while also contributing to our overall understanding of essential cell biological processes. In a 2021 mSphere of Influence article, the use of live-cell imaging to uncover a unique mechanism of viral assembly was discussed. In this Full Circle review, we highlight the high-resolution imaging techniques currently revolutionizing virology research and discuss how they can be utilized to advance our ability to identify and interrogate novel virus-host interactions.

Candidozyma auris is a high-priority, emerging fungal pathogen frequently isolated from urine in healthcare settings. These isolates are often associated with indwelling urinary catheters, a primary risk factor for catheter-associated urinary tract infections (CAUTIs). Despite its clinical prevalence, the mechanisms of C. auris colonization and pathogenesis within the bladder remain poorly understood. In this study, we screened C. auris isolates from diverse clades using an in vitro biofilm model and in vivo murine models of uncomplicated UTI and CAUTI. While in vitro biofilm formation varied among isolates, the presence of a catheter in vivo significantly enhanced fungal burden in the bladder. Notably, one strain (B11103) caused rapid systemic dissemination and mortality. To address this, we evaluated a liquid-infused silicone (LIS) catheter coating, which has previously shown efficacy against other uropathogens. The LIS coating significantly reduced C. auris attachment in vitro and, crucially, mitigated fungal burden on both the catheter and bladder tissue in vivo across all tested strains. For the hypervirulent B11103 strain, LIS catheters also significantly reduced dissemination to the kidneys and bloodstream. Furthermore, cytokine analysis revealed that C. auris CAUTI upregulates IL-6, CSF3, and CXCL1; importantly, this damaging inflammatory response was also dampened by the LIS catheter. These findings demonstrate that catheterization potentiates C. auris pathogenicity and identify LIS catheters as a promising, antimicrobial-sparing strategy to prevent colonization, systemic spread, and inflammation during C. auris CAUTI.IMPORTANCEThis research addresses the critical public health challenge posed by the emergence of Candidozyma auris, elucidating its pathogenesis in the urinary tract, the second-most common yet understudied reservoir. Here, we find that C. auris exhibits plasticity in its ability to form biofilms in urine and cause uncomplicated urinary tract infections (UTIs) and catheter-associated UTIs (CAUTIs). Importantly, we show that our liquid-infused silicone (LIS) catheters effectively disrupt this cycle by reducing fungal burden, preventing systemic spread, and dampening the damaging host inflammatory response. This work establishes the urinary tract as a critical niche for systemic entry and provides a validated strategy for infection prevention. Urinary catheters make C. auris dangerous, but this liquid-infused silicone coating is fighting back.

Patients with chronic kidney disease (CKD) frequently experience constipation, often linked to gut microbiota disruptions. We hypothesized that constipation is not merely a comorbidity but a driver of adverse outcomes through a specific host-microbial metabolic axis. Employing a multi-cohort design for evidence triangulation, we first employed a large U.S. database and mortality data up to 31 December 2019 to investigate the association between constipation and overall mortality in patients with CKD using weighted Cox proportional hazards models. To explore potential microbial and metabolic mechanisms that could underlie such clinical observations, we then used genetic data from the independent FINRISK cohort to investigate causal relationships between specific gut microbiota, their metabolites, and CKD. In the observational cohort, we identified functional constipation as a potent, independent predictor of both all-cause (HR: 1.33; 95% CI: 1.11-1.58) and cardiovascular mortality. This association remained robust across sensitivity analyses and showed a distinct U-shaped dose-response relationship with stool frequency. In the causal inference phase, while the direct causal effect of constipation on CKD was not significant, MR identified a significant causal depletion of the genus Herbidospora driven by constipation. Further analyses revealed that Herbidospora exerts a protective effect against CKD. Crucially, mediation analysis demonstrated that phosphatidylcholine (14:0_18:2) [PC 14:0_18:2] metabolites mediate 12.5% of the protective pathway from Herbidospora to CKD. Constipation is a key and independent predictor of mortality in patients with CKD. We found that constipation leads to a reduction in the number of Herbidospora, thereby disrupting the protective function of the kidney. This is because PC (14:0_18:2) is lacking. This study emphasizes the importance of restoring this microbial metabolic axis as a novel therapeutic strategy for CKD.IMPORTANCEFor millions living with chronic kidney disease (CKD), a common issue such as constipation can be a hidden danger, increasing their mortality risk by over one-third. Our research uncovers why: an unhealthy gut, often indicated by constipation, lacks specific "good"" bacteria essential for producing a protective fat molecule. This natural molecule acts as a key, activating the kidney's own defense and repair systems. This discovery of a "gut-lipid-kidney" connection offers a groundbreaking new strategy: therapies aimed at restoring gut health and supplementing this key protective fat could provide a powerful new way to slow disease progression and improve survival in CKD patients.

The yeast pathogen Candidozyma (Candida) auris can form biofilms, which contribute to its virulence and nosocomial transmission. In this study, we identified the transcription factor Wor2 as a negative regulator of biofilm formation in C. auris. Wor2 hyperactivation in a strain of clade IV via the use of a protein tagging strategy resulted in downregulation of two important adhesins, SCF1 and ALS4112, and decreased biofilm-forming capacity. We showed that the impact on biofilm was predominantly mediated via decreased SCF1 expression in this strain. However, results of adhesion assays on inert surfaces and human keratinocytes found relatively modest roles of Wor2 and Scf1 in this process, suggesting that their effect on biofilm formation is complex and not limited to the adhesion step. Finally, analyses of other strains from different clades identified three distinct WOR2 genotypes, with variable WOR2 expression levels and distinct impacts of WOR2 deletion on biofilm formation. Notably, Wor2 negatively regulated biofilm in strains of clades I, III, and IV with distinct profiles of SCF1/ALS4112 expression, while it had no impact on biofilm in a clade II strain. Taken together, this study showed that Wor2 exhibited some distinct genotypic evolution in C. auris resulting in clade- or strain-specific regulatory roles and pathways in biofilm formation.IMPORTANCECandidozyma (Candida) auris is a pathogenic yeast exhibiting a particular capacity for interhuman transmission via medical instruments, which was the cause of nosocomial outbreaks of candidemia. Adhesion to inert surfaces and subsequent biofilm formation is therefore important for C. auris propagation. This work highlights the role of the transcription factor Wor2 as a negative regulator of biofilm formation in C. auris. In a strain of clade IV, Wor2 was shown to downregulate two important adhesins (SCF1 and ALS4112). Interestingly, Wor2 exhibited different genotypes across C. auris clades and strains, which were associated with distinct differential expression of WOR2, ALS4112, and SCF1, and possibly distinct roles in biofilm formation.

To investigate the shared molecular mechanisms between Parkinson's disease (PD) and COVID-19 through integrated bioinformatics analysis and single-cell RNA sequencing (scRNA-seq). We conducted a comprehensive analysis of bulk RNA-seq data from publicly available databases, along with scRNA-seq data from brain tissues of COVID-19 patients. Differential expression analysis identified 725 differentially expressed genes (DEGs) in COVID-19 and 633 in PD samples. A total of 77 overlapping DEGs were identified, highlighting common pathways associated with neuroinflammation and dopaminergic neuron dysfunction. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed significant enrichment in inflammation-related pathways. The protein-protein interaction network analysis identified CHI3L1 as a key gene linking PD and COVID-19. ScRNA-seq analysis revealed a significant increase in CHI3L1-expressing astrocytes in COVID-19 samples, indicating a potential mechanism by which COVID-19 may exacerbate PD symptoms. Furthermore, cell-cell communication analysis revealed enhanced interactions between astrocytes and microglia, excitatory neurons, or oligodendrocytes through signaling molecules such as phosphoprotein 1, CADM1, NCAM1, NRG, and NRXN1, suggesting that astrocytes play a central role in regulating neuronal excitability, synaptic plasticity, and immune responses in the context of COVID-19. These findings suggest a complex interplay between COVID-19 and PD, emphasizing the need for further investigation into the shared pathogenic mechanisms and potential therapeutic targets.IMPORTANCEThis study demonstrates the critical role of neuroinflammation and dopaminergic neuron damage in the shared pathogenesis of COVID-19 and Parkinson's disease. CHI3L1 emerges as a key target, highlighting its potential involvement in modulating neuroinflammatory pathways and synaptic plasticity. The functional significance of CHI3L1, along with its pathological relevance, warrants further investigation through larger studies. Additionally, the active intercellular communication among astrocytes, microglia, and excitatory neurons underscores the profound impact of COVID-19 on neural circuitry. Collectively, these results provide important insights into the mechanisms driving the neurodegenerative consequences of COVID-19, emphasizing the need for continued exploration of therapeutic interventions and the long-term neurological effects of viral infection.

Aspergillus fumigatus is a globally distributed fungal pathogen that can infect humans and is commonly found in the environment. Azole antifungals are the primary treatment for A. fumigatus infections; however, the emergence of azole resistance has become an important public health issue. This resistance is often linked to the use of agricultural fungicides and is primarily caused by tandem repeats (TRs) in the cyp51A promoter, along with mutations in the coding region, leading to pan-azole resistance. Azole-resistant A. fumigatus has been identified in patients across the United States and has been found in environmental soil samples from 38 states. A previous study conducted in the southeastern United States detected azole-resistant A. fumigatus in the environment. To further investigate the persistence of these resistant strains, soil samples were collected over three time periods. Whole genome sequencing was performed on isolates from each sampling date, and results were compared to publicly available isolates using phylogenetic, principal component, and ADMIXTURE analyses. These findings demonstrated that the same strains of TR-based azole-resistant A. fumigatus persisted in the same environmental hotspot over time. Additionally, we observed high levels of recombination between different clades, which may contribute to the ongoing presence of A. fumigatus in the United States. These findings highlight the environmental persistence of azole-resistant strains and underscore the need for monitoring antifungal resistance in environmental settings to better understand its potential clinical implications.IMPORTANCEAspergillus fumigatus is a fungal pathogen that poses a substantial threat to humans, particularly in high-risk populations, with mortality rates reaching 90%. It is also a saprophyte that is commonly found in agricultural settings. Azoles are the primary antifungal treatment for A. fumigatus infections; however, azole-resistant strains have emerged, particularly in Europe and Asia, over the past two decades. Recently, such strains have also been isolated from clinical cases in the United States and identified in environmental soil samples from 38 states. In this study, azole-resistant A. fumigatus was isolated with three different variants of tandem repeat mutations in the cyp51A promoter region from southeastern U.S. soil samples collected over three time periods. These findings demonstrate that resistant strains can persist in the environment for at least 12 months, suggesting that established environmental hotspots can serve as ongoing reservoirs for resistant strains.

Entamoeba histolytica, a protozoan parasite, causes amebiasis, which is a global public health problem. Clinical manifestation and pathogenesis of amebiasis are closely associated with the proliferation and tissue invasion abilities of E. histolytica trophozoites. At the same time, E. histolytica trophozoites are studied in a broad range of biology research fields. As such, the development of in vitro culture of E. histolytica trophozoites greatly facilitated amebic research. Now, a standard method for cryopreservation of E. histolytica trophozoites is required because available methods either do not give a high enough revival rate, which can significantly delay projects, or they are not widely adopted. Here, we attempted to optimize the conditions for E. histolytica trophozoite cryopreservation, including cell density, cooling rate, cell freezing reagent, and freezing profile. We found an optimized condition that reproducibly yielded >30% revival, regardless of storage period in liquid nitrogen (up to 365 days). This optimized condition is that E. histolytica trophozoites are suspended in 0.5 mL CELLBANKER 2 in a 1 mL cryotube at 2 × 106 cells/mL and frozen from 4 to -40°C at a rate of -0.2°C/min using VIA Freeze Uno. This cryopreservation method can minimize the risk of losing E. histolytica trophozoite lines required to continue projects, facilitating amebic research. Amebiasis, which is caused by Entamoeba histolytica infection, is the third deadliest parasitic disease globally. Proliferation of E. histolytica trophozoites and their invasion into the host tissues cause amebiasis symptoms and pathogenesis. E. histolytica trophozoites are also important in multiple biology research topics. Therefore, E. histolytica trophozoites are a common subject in academic as well as clinical fields. A standard method for in vitro culture of E. histolytica trophozoites is well established. By contrast, a widely adopted practical method for cryopreservation of E. histolytica trophozoites is not yet available. This hampers the advancement of amebic research, as the required E. histolytica trophozoite lines sometimes cannot be revived from cryopreservation. In this study, we varied parameters critical to the revival rate, namely, cell density, cooling rate, freezing reagent, and freezing profile, and present an optimized cryopreservation method for E. histolytica trophozoites, which gives reproducibly high revival rates.

Pneumococcal vaccination is essential to prevent invasive Streptococcus pneumoniae infections in immunocompromised individuals, including kidney transplant recipients. Current recommendations favor single-dose immunization with higher-valent conjugate vaccines, including PCV20 or PCV21. Five years ago, sequential administration of the 13-valent conjugate vaccine (PCV13) followed by the 23-valent polysaccharide vaccine (PPSV23) represented standard of care. Long-term data on antibody persistence after this regimen remain limited. In this prospective 5-year follow-up study, 46 kidney transplant recipients previously vaccinated sequentially with PCV13 and PPSV23 were re-evaluated. Nine patients were lost to follow-up, and 11 had died during the observation period. The remaining 26 participants were re-enrolled and completed the 5-year assessment. Global and serotype-specific IgG anti-pneumococcal antibody concentrations were quantified and compared with baseline and 12-month post-vaccination levels. Clinical outcomes, including pneumococcal infections and allograft status, were recorded. Five years after vaccination, antibody concentrations remained above baseline levels in most participants. Mean IgG levels were still approximately threefold higher than pre-vaccination values. Even for serotype-specific responses, mean antibody levels showed minimal changes compared with those measured 12 months after the first vaccination, although absolute titers remained considerably lower than those observed in healthy individuals. No cases of pneumococcal pneumonia or vaccine-associated allograft rejection occurred during follow-up. Sequential vaccination elicits durable immune responses in kidney transplant recipients, persisting up to 5 years post-immunization. With the availability of new vaccines covering additional serotypes, and given the generally lower antibody responses in this high-risk population, a booster with PCV20 or PCV21 appears advisable to enhance and broaden protection.IMPORTANCEKidney transplant recipients are at high risk for invasive pneumococcal disease, yet long-term vaccine-induced immunity in this population remains poorly defined. This study provides one of the longest longitudinal assessments of humoral responses following sequential PCV13 and PPSV23 vaccination, extending to 5 years post-immunization. We demonstrate sustained but heterogeneous antibody persistence and serotype-dependent responses to PCV20 booster vaccination. These results are directly relevant to transplant clinicians, vaccinologists, and public health policy, offering critical insight into long-term pneumococcal immunity in immunocompromised hosts and guiding future vaccine scheduling in solid organ transplantation.

Mass drug administration (MDA) of azithromycin (AZ) and case finding and treatment of children with cutaneous ulcers (CUs) have been trialed as strategies to eliminate yaws. Metagenomic sequencing of CU swabs obtained from children 3 to 4 years after the initiation of a yaws elimination campaign on Lihir Island showed enrichment for Treponema pallidum subsp. pertenue (TPE), Haemophilus ducreyi (HD), Streptococcus pyogenes (SP), and several anaerobic bacteria. Whether these results were influenced by AZ pressure or are generalizable to other populations is unknown. Here, we performed quantitative PCR (qPCR) for TPE, HD, and SP DNAs and metagenomic sequencing on 260 CU specimens collected from children on New Britain Island, whose inhabitants had not received MDA of AZ. Based on qPCR results, specimens were classified as containing no pathogens, a single pathogen, or multiple pathogens. 31.9% of the specimens contained SP, 28.1% HD, and 25.4% TPE DNAs; mixed infections with two or three pathogen DNAs occurred in 16.5% of cases. No pathogen DNA was detected in 31.5% of the specimens, which were defined as idiopathic ulcers (IUs). In most categories, the same pathogen(s) used to classify the ulcers by qPCR were the most abundant taxa present in the specimens. In IU, the most abundant taxon was Arcanobacterium haemolyticum; however, its relative abundance was similar across all groups, suggesting that this organism may adapt to the CU environment rather than represent a cause of IU. These data indicate that TPE, HD, and SP are the primary causes of CU in this untreated population.IMPORTANCECutaneous ulcers (CUs) affect ~100,000 children annually in tropical regions. After mass drug administration (MDA) of azithromycin (AZ) failed to eradicate yaws, the World Health Organization proposed an integrated disease management strategy to control CU, which emphasizes identifying the causative pathogens in each population. This is critical because organisms associated with CU vary geographically, with Treponema pallidum subsp. pertenue (TPE), Haemophilus ducreyi (HD), Streptococcus pyogenes (SP), and Leishmania spp. predominating in different countries. We previously found that TPE, HD, and SP DNAs were enriched in CU specimens from children on Lihir Island in Papua New Guinea (PNG), a population heavily exposed to AZ. Here, we show that these three organisms were also the major pathogens in CU specimens from children on New Britain Island in PNG, whose population had not received MDA of AZ, suggesting that our previous findings are generalizable within PNG and not a consequence of AZ exposure.

Utilizing treatment strategies based on collateral sensitivity (CS) represents a promising approach to suppressing antibiotic resistance. Although the mechanism of CS between numerous drugs has been researched, the mechanism of CS between tigecycline and colistin remains unclear. Therefore, the purpose of our research is to investigate the possible mechanism by which tigecycline affects colistin CS in the Enterobacter cloacae complex. Tigecycline induction significantly reduced the minimum inhibitory concentration of carbapenem-resistant Enterobacter cloacae complex (CRECC) to colistin, and sequencing revealed a single-base deletion at the RamR binding site. Complementation experiments demonstrated that deletion of the RamR binding site increased the resistance of CRECC417 to colistin and tigecycline by 2-fold and 4-fold, respectively. Transcriptomic comparison analysis of strains before and after CRECC417 induction revealed a total of 1,977 genes with significant differences in expression. Genes associated with carbohydrate, amino acid, and inorganic ion metabolism were the most highly enriched. Furthermore, the observed increase in colistin susceptibility in CRECC417R can be attributed to the inhibition of quorum sensing and biofilm formation pathways, as well as increased expression levels of genes associated with lipopolysaccharide biosynthesis and modification.IMPORTANCEDue to the overuse of antibiotics, antimicrobial resistance (AMR) has become a serious threat to global public health. Dosage regimens based on bacterial CS can reduce antibiotic use without reducing efficacy, thereby reducing antibiotic-related toxicity risks, expanding the scope of antibiotic application, and limiting the development of antibiotic resistance. In this study, we analyzed the drug resistance mutations and global transcriptional changes in CRECC after tigecycline induction through genomics and transcriptomics. Our study showed that tigecycline exposure significantly inhibited quorum sensing pathways and biofilm formation. There were significant changes in the transcriptional levels of genes related to cell membrane lipopolysaccharide synthesis and modification, but no mutations were found in genes related to colistin resistance. These findings provide valuable insights for further investigation into the CS between tigecycline and colistin.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections pose a major challenge to effective treatment. Understanding genomic adaptations during antimicrobial therapy in patients infected with this pathogen is crucial for preventing therapeutic failure. Here, we investigated the population diversity and evolution of P. aeruginosa collected longitudinally from six patients who evolved multidrug-resistant infections. Serial clinical P. aeruginosa single-colony isolates (n = 63) and culture-enriched metagenomic population samples (n = 39) were collected and subjected to whole-genome sequencing. The resulting data were used to characterize and compare the species composition, multi-locus sequence types (STs), and resistance-associated mutations present within each sample type. Single-colony isolate sequencing showed that each patient was infected with a single P. aeruginosa strain that accumulated mutations and became increasingly more antibiotic-resistant over time. Mutations in genes associated with β-lactam resistance, including ampC, ftsI, and mexR, arose over time and corresponded with changes in antimicrobial susceptibility in single-colony isolates. Species profiling of culture-enriched metagenomic populations revealed that all samples contained P. aeruginosa, but also additional gram-negative pathogens. Metagenomic analysis of culture-enriched populations identified resistance-associated mutations at low frequency, many of which were not identified in single-colony isolates from the same sample. In some cases, resistance-associated mutations initially detected at low frequency rose to fixation after antimicrobial treatment. Overall, this study shows that population-based metagenomic sequencing effectively captures the within-patient genomic diversity of P. aeruginosa during antimicrobial therapy and could aid the detection and interpretation of resistance-associated mutations in this pathogen. Pseudomonas aeruginosa infections are notoriously difficult to treat and are associated with high rates of morbidity and mortality. While the genetic basis of resistance in P. aeruginosa is well documented in vitro, less is known about how resistance evolves within patients during antibiotic therapy. Standard approaches based on analysis of clonal isolates may miss within-patient diversity, potentially overlooking low-frequency mutations that contribute to treatment failure. In this study, we compared single-colony isolate whole-genome sequencing with culture-enriched metagenomic sequencing to monitor the evolution of P. aeruginosa populations in patients receiving antibiotic therapy. The culture-enriched metagenomic approach enabled the detection of emerging resistance mutations, such as low-frequency variants in ampC and ftsI, before these variants rose to fixation. It also revealed genetically resistant subpopulations missed by isolate sequencing alone. Overall, our findings highlight the value of population-based metagenomic sequencing in capturing bacterial adaptation during infection and underscore its potential to improve resistance surveillance and guide personalized antimicrobial therapy.

Invasive infections caused by Streptococcus pyogenes (iGAS) have increased in Europe over the past decade, with a marked upsurge after the COVID-19 pandemic. Here, we examined whether the increase in iGAS infections in Norway was associated with the spread of variants that had acquired new virulence factors. A collection of 1,163 iGAS isolates submitted to the National Reference Laboratory between January 2017 and April 2023, representing 87% of all cases recorded by the Norwegian Surveillance System for Infectious Diseases, was analyzed by whole genome sequencing. Resistance to one or more antibiotics was found in 15.6% of the isolates: 14.1% were resistant to tetracycline, 6.4% to erythromycin, and 4.0% to clindamycin. Resistance to other antibiotics was < 1%. The dominating emm types were emm1 (30.9%), emm12 (13.8%), emm89 (9.3%), emm28 (8.3%), emm4 (6.0%), and emm87 (4.5%), with the remaining isolates belonging to 55 other emm types; 62.3% of emm1 belonged to the hypervirulent lineage M1UK. Genetic characterization of the virulence factors of the dominant six emm types demonstrated extensive competition between related phages, leading to phage switching and interplay between integration and excision of temperate phages carrying virulence factors. Bacteriophages carrying virulence factors speC and spd1 displayed a particularly high turnover rate, with several pairs of otherwise genomically identical isolates exhibiting different phage-carrying status. We identified and characterized four new speC and spd1-carrying phages. The rapid turnover pattern of these, as well as other phages carrying superantigens and DNAses suggests an important role in pathogenesis.IMPORTANCEThis analysis of 1,163 iGAS isolates collected between January 2017 and April 2023 aimed to map virulence factor content to understand the observed increased incidence of iGAS in Norway. Our findings indicate that 15.6% of the isolates were resistant to at least one antibiotic, with tetracycline resistance being the most common. The dominant emm types were emm1, emm4, emm12, emm28, emm87, and emm89, which together accounted for 72.7% of the isolates. The study highlights the dynamic nature of virulence factor-carrying temperate phages, particularly the ones carrying speC and spd1, which frequently integrate and excise within emm types. Four previously unseen phages carrying speC and spd1 were identified and characterized. This research underscores the complexity of iGAS epidemiology and the need for continuous surveillance to understand the evolving landscape of bacterial virulence factors, antibiotic resistance, and circulating emm types.

To evaluate the association between respiratory tract microorganisms at birth and the subsequent development and severity of bronchopulmonary dysplasia (BPD) in preterm infants. This prospective cohort study enrolled 98 preterm infants (gestational age < 32 weeks, birth weight < 2,000 g). Tracheal aspirate samples were collected through endotracheal intubation within 2 h after birth. Using 16S rRNA sequencing, we characterized the airway microbiome and performed co-occurrence network analysis with compositionally robust methods. Among 98 preterm infants analyzed, the incidence of BPD was 68.4%, comprising 31 grade I, 20 grade II, and 16 grade III cases. Airway microbiota in infants with BPD exhibited distinct severity-stage patterns: Escherichia-Shigella and Streptococcus were significantly enriched in grade I, while Chryseobacterium increased markedly in grade III, accompanied by a significant reduction in Streptococcus. Microbial co-occurrence network analysis yielded three key insights. (i) Network complexity declined sharply with BPD severity, being sparsest in grade III. (ii) Distinct keystone taxa were identified across different groups: Acinetobacter and Fusobacterium in the non-BPD group; Brevundimonas and Fusobacterium in grade I; Fusobacterium and Acinetobacter at grade II and grade III. (iii) In a multivariable model adjusted for key clinical confounders, a higher microbial network density at birth was independently associated with a substantially reduced risk (OR = 0.12, P < 0.05). The ecological architecture of the neonatal airway microbiome at birth, defined by network complexity and keystone taxa, is associated with BPD severity. This highlights microbial network stability as a novel factor and ecological interactions as a target for future research. Bronchopulmonary dysplasia (BPD) remains the most common chronic lung disease in preterm infants. While its pathogenesis is incompletely understood, the role of the early respiratory microbe is increasingly recognized. Previous studies have largely focused on individual pathogenic taxa, overlooking the complex ecological interactions within microbial communities. Our analysis reveals that the architecture of microbial co-occurrence networks in the neonatal airway varies significantly with BPD severity. Notably, network complexity decreased markedly as BPD severity increased. We identified specific keystone taxa uniquely associated with disease outcomes, suggesting that microbial ecosystem stability rather than individual species may be a critical factor in BPD pathogenesis. These findings shift the focus from single microbes to the stability of the microbial ecosystem as a novel risk factor for severe BPD, offering new avenues for risk stratification and early intervention.

New effectors of spore properties have been identified in two Bacillus subtilis strains-bacillithiol, the major low-molecular-weight thiol in B. subtilis, and sporulene, a multi-ring compound synthesized from curcumene in spores' outer layers. The absence of either bacillithiol or sporulene caused faster germination of spores of one wild-type B. subtilis strain, PS832, with two germinant receptors (GR)-dependent germinants, L-valine for the GerA GR, and the AGFK mixture for the cooperative action of the GerB and GerK GRs, as well as the GR-independent germinant dodecylamine. However, these effects on PS832 spore germination were due to the presence of the antibiotic marker used to replace the genes for bacillithiol or sporulene synthesis. The absence of bacillithiol also caused reduced spore resistance to wet heat but not to hydrogen peroxide or UV radiation, while sporulenes' absence had no effect on spore wet heat resistance, but reduced spore resistance to hydrogen peroxide as found previously (T. Bosak, R. M. Losick, and A. Pearson, Proc Natl Acad Sci USA 105:6725-6729, 2008, https://doi.org/10.1073/pnas.0800199105) and to UV radiation, but the presence of the antibiotic markers in these mutant strains was not responsible for these effects. Notably, spores lacking sporulene were much whiter than wild-type (wt) spores, suggesting that sporulene and its precursor curcumene contribute to spore pigmentation that can absorb UV, thus reducing spores' UV resistance. Analyses of reasons for the effects of bacillithiol's or sporulene's absence on spore resistance, specifically core water content and/or altered spore inner membrane (IM) permeability, found no major differences from values in wt spores. However, other analyses showed that sporulene's absence led to slight changes in spore IM fluidity. The work in this paper has identified two new factors, bacillithiol and sporulene, as modulators of the resistance and germination of spores of two Bacillus subtilis strains, and by extension, probably spores of other Bacillota. Since spores of some species can give rise to cells that can cause food spoilage and/or disease, new knowledge about spore resistance and germination could have applied utility.

Trypanosoma brucei is a model unicellular parasite for cellular and molecular genetic studies, but tools for more multiplexed experiments are limited. Tetracycline (Tet)-inducible gene regulation has been a long-standing and effective approach for overexpression, RNAi, and genome-wide screens and has been foundational for studying essential genes that are required for biological processes and identifying potential drug targets. To achieve greater flexibility in experimental design, we capitalized upon previously described dual inducer systems that combined vanillic acid (Van) or cumate (Cym) with Tet as inducers. Here, we report the development of a triple inducible system combining Cym, Van, and Tet repressors to selectively regulate the expression of three genes within a single cell line called PHITER. To demonstrate independent control, we adapted the previously characterized Van/Tet dual inducible RNAi complementation cell line, IBCompVaT, for additional Cym inducible expression of an eGFP reporter. We provide evidence that each inducer operates independently with no evidence of leaky expression. In this system, Cym induction is specific and tunable. As proof of principle, we used triple induction for POLIB RNAi, complementation with a recoded ectopic POLIB-PTP variant, and overexpression of the maxicircle helicase. We confirmed that Cym inducible PIF2 overexpression resulted in massive overreplication of maxicircles that is suppressed when POLIB is silenced, thereby demonstrating a requirement for POLIB during replication stress. The triple inducible system enables the study of complex and pleiotropic phenotypes through a more sophisticated experimental design that could not previously be achieved and can be applied for the development of multiplexed genomic screens.IMPORTANCETrypanosoma brucei is a protist parasite that causes significant health and economic burden for sub-Saharan Africa and serves as a key model organism for a group of eukaryotes called the kinetoplastids. T. brucei and related parasites contain an interlocked mtDNA network called kinetoplast DNA (kDNA), composed of maxicircles and minicircles. Although T. brucei is highly genetically tractable, limitations exist for more complex experimental designs and multiplexed forward genetic screens. Here, we describe the first triple inducible system in T. brucei or any eukaryote that combines three independent repressors (cumate [Cym], tetracycline [Tet], and vanillic acid [Van]) to selectively regulate three genes within a single cell line. We demonstrate the utility of the system using an eGFP reporter and robust RNAi complementation of kDNA polymerase POLIB. Furthermore, we investigate the role of POLIB in maxicircle replication by overexpressing the maxicircle helicase during POLIB RNAi, revealing an early requirement for POLIB during replication stress.