Frontiers in Cellular and Infection Microbiology

ObjectiveRecurrent urinary tract infections (rUTIs) significantly decrease quality of life and antibiotics are becoming increasingly less effective due to antimicrobial resistance. Alternative effective treatment strategies are urgently needed for rUTIs. Prior studies have indicated that women can experience resolved or improved rUTI following electrofulguration (EF). To further investigate these findings, we report on the design and methodology behind a randomized trial examining two treatment arms: standard prolonged antibiotic treatment with nitrofurantoin (NF) alone or in combination with EF. Patients and MethodsThe aim of this randomized trial is to determine, at two institutions, the efficacy of two interventions for rUTI associated with early stages of chronic cystitis (stages 1 and 2): conventional 6 months low-dose (100mg) NF daily antibiotic suppression alone (NF) or conventional NF with EF (EF + NF). The study is also designed to analyze changes in the urinary microbiomes in the two different treatment arms and to determine the durability of clinical outcomes in both treatment arms at 2 years after the end of each intervention. The primary outcomes will be obtained from 6 to 18 months, as well as 18 - 30 months following completion of the original 6-month intervention. Failure is defined based on UTI symptoms documented by a validated questionnaire with a documented urine culture confirming a bacterial strain at each UTI episode following the end of the 6-month intervention. ConclusionsThis randomized trial is designed to examine the efficacy and durability of treating women with rUTIs using the standard of care of NF alone, or an EF procedure with NF.

Hyalomma lusitanicum is a characteristic tick species of the western Mediterranean region, with a well-established distribution across the Iberian Peninsula. It is strongly associated with wild ungulates, particularly red deer, as well as livestock, to which it can transmit a wide range of pathogens, including viruses, bacteria, and protozoa. Here, we present three genomic resources for H. lusitanicum: a scaffold-scale nuclear genome, the complete mitochondrial genome, and the complete genome of its associated Francisella bacterial endosymbiont. The nuclear genome assembly spans 1.81 Gb and comprises 59 scaffolds, with a scaffold N50 of 153.6 Mb (L50 = 5) and no gaps, indicating high contiguity and completeness with a gene annotation completeness BUSCO score of 97.1 %. Genome annotation of the nuclear assembly identified 20,638 protein-coding genes, 1,422 non-coding genes, and 5,775 pseudogenes. A total of 18 scaffolds were assembled as putative chromosomes, exceeding the 11 chromosomes inferred as ancestral; however, synteny analyses suggest that several scaffolds likely represent fragmented portions of the same chromosome, probably due to incomplete Hi-C scaffolding. Despite this, the assembly represents one of the most complete tick nuclear genomes generated to date. In addition, we report the complete genome of a Francisella endosymbiont (1.51 Mb, 1,679 genes), characterized by a high proportion of pseudogenes and reduced genome size, consistent with patterns of genome reduction associated with obligate symbiosis. Together, these genomic resources provide a framework to investigate local adaptation and host-symbiont evolution, and to support improved surveillance, control, and management strategies for species of public health relevance.

Background: Recurrent urinary tract infections(rUTI) represent a major clinical challenge due to persistent clinical symptoms, repeated antibiotic exposure, and increased risk of multidrug resistance. Further clinical management of rUTI remains challenging, as existing diagnostic and treatment guidelines are largely designed for uncomplicated, acute infections. Though uropathogenic Escherichia coli (UPEC) is the predominant cause of community-acquired UTIs, pathogen-derived genomic features that may predispose certain E. coli strains to repeatedly establish infection are not fully understood. Methods: To comprehensively dissect distinct genetic signals across genomic compartments that distinguish rUTI-associated isolates from those causing sporadic infection, the pan-genome analysis in three different frameworks (i) Combined genomes (chromosome + plasmid), (ii) bacterial chromosomes only and (iii) plasmid-only was conducted. A comprehensive evaluation of population structure was performed using Gubbins, recombination-aware phylogeny IQTree, phylogroup distribution, pan-genome openness using Heaps law, and plasmidome architecture using MOBSUITE. Findings: Supervised machine learning models showed that the highest discriminatory performance was achieved using the combined genomic dataset (accuracy ~0.98), and integration of feature-selected genes with PanGWAS (Pyseer and Scoary) identified a robust set of recurrence-associated genes, namely cbtA, cbeA, and ldrD, which were consistently detected across machine learning and association frameworks. Subsequent association rule mining further revealed cooperative gene networks enriched in rUTI isolates, particularly involving toxin-antitoxin modules and metabolic regulators. Interpretation: Overall, this integrated ML-PanGWAS approach demonstrates that rUTI is a lineage-independent, polygenic phenotype encoded within a combined chromosomal-plasmid genomic context, providing new insights into the bacterial genomic architecture underlying recurrent disease and offering candidate biomarkers for future diagnostic and therapeutic development.

PurposeBacterial vaginosis (BV) is associated with disruption of the vaginal microbiome and extracellular matrix (ECM) remodeling, yet the contribution of host proteases to this process remains unclear. This study investigated whether expression and activity of cathepsins K, L, S, and V differ by BV diagnosis and community state type (CST). We hypothesized that BV and BV associated CSTs would exhibit increased expression and activity of collagen and elastin-degrading cathepsins. MethodsVaginal fluid samples were collected and classified by BV diagnosis and CST. Cathepsin expression was evaluated by Western blotting to distinguish inactive and active enzyme forms. Proteolytic activity was assessed using multiplex cathepsin zymography. Statistical analyses compared cathepsin expression and activity across diagnoses and CSTs. Principal component analysis and linear regression were performed to assess associations between cathepsin activity, microbial diversity, and CST. ResultsProcathepsin K expression was significantly increased in BV-positive and CST IV samples, while total cathepsin L expression was significantly elevated in samples with Nugent-intermediate scores. Cathepsins S and V showed variation in inactive and active forms in Nugent-intermediate and CST III samples. In contrast, total cathepsin activity, including cathepsins K and V, did not significantly differ across BV diagnoses or CSTs. Overall, cathepsin activity varied between individuals rather than by clinical classification. ConclusionsCathepsin expression and maturation state differ by microbiome composition, suggesting that the vaginal microbiome may regulate post-translational processing of cathepsins. As a result, cathepsin activity appears to be regulated at the individual level rather than strictly by BV diagnosis or CST. These findings link vaginal microbiome composition to ECM remodeling and potential adverse reproductive outcomes.

Urinary tract infections (UTIs) are the most prevalent bacterial infections globally, and their management increasingly challenged by antimicrobial resistance (AMR). Probiotics offer a promising approach to mitigate AMR by competitively excluding uropathogens and enhancing host immunity by producing immune modulators. Despite being potential, key gaps persist between the discovery of uroprotective probiotic strains and optimization of formulations for urinary tract delivery. Here, we analyzed the urinary microbiome of UTI patients and healthy individuals to identify potential probiotic candidates for the prevention and management of UTIs. Publicly available 16S rRNA amplicon sequencing data of the urinary tract were processed using a standardized pipeline for sequence quality assessment, taxonomic assignment, and microbial function prediction. Comparative analysis showed a significant shift in microbial composition between UTI patients and healthy controls. The dominated phyla identified included Acidobacteriota, Actinobacteriota, Bacteroidota, Campylobacterota, Cyanobacteria, Firmicutes, Fusobacteriota, Patescibacteria, Proteobacteria, and Synergistota. Overall differential abundance analysis revealed Escherichia coli as the predominant UTI-associated species, while Lactobacillus crispatus was enriched in healthy samples. Additionally, predictive functional analysis indicated that metabolic pathways associated with beneficial microbes were enriched in the healthy group. Overall, the study highlights the association of distinct urinary microbiome signatures with infection status, which supports L. crispatus as the most promising probiotic for UTI prevention and control.

Nocardiosis is a human infectious disease caused by several species of Nocardia and primarily affecting the skin, lungs and central nervous system. The first line treatment is based on cotrimoxazole, combining trimethoprim and sulfamethoxazole. These two drugs target respectively the dihydrofolate synthase (DHFR) and the dihydropteroate synthase (DHPS) involved in the essential folate synthesis pathway. The occurrence of drug resistance to these two drugs is however frequent. While the molecular mechanisms of trimethoprim resistance are well documented in other bacteria, they remain poorly explored and documented in Nocardia. This is partly because few biochemical structural or genetic studies have been conducted on DHFR from this genus. In this study, we report the biochemical and structural characterization of DHFR from Nocardia asteroides (DHFRNad). We show that overexpression of DHFRNad in N. asteroides confers strong resistance to trimethoprim. We recombinantly expressed and purified active DHFRNad and determined its inhibition constant for trimethoprim. We solved the crystal structure of DHFRNad bound to trimethoprim at high resolution. Further, biochemical studies of mutant DHFR variants pinpointed the role of important residues for trimethoprim binding and drug-resistance. HighlightsFirst biochemical and structural characterization of Nocardia asteroides DHFR. Overexpression of DHFRNad induces high-level trimethoprim resistance in N. asteroides. Crystal structure of DHFRNad reveals key residues for trimethoprim binding. Mutagenesis confirms residues critical for trimethoprim susceptibility. IC50 data confirm strong DHFRNad inhibition by trimethoprim and methotrexate

Entamoeba histolytica is a protozoan parasite that can cause severe liver disease known as amoebic liver abscess. However, only a subset of infected individuals develops invasive disease, indicating that host-parasite interactions are critical determinants of disease outcome. In this study, we investigated the clone-specific modulation of hepatic immune responses using non-pathogenic A1np and pathogenic B2p E. histolytica clones. Time-resolved transcriptome analyses (6, 12, 24 hours post-infection) in a murine model revealed distinct immune trajectories. Both clones activated innate immune pathways early after infection, but their responses differed markedly in magnitude and composition. A1np infection induced a rapid and controlled inflammatory response associated with antimicrobial activity and resolution-promoting signalling. In contrast, B2p infection triggered a stronger and more complex immune response characterised by pronounced cytokine and chemokine expression, activation of stress and redox pathways, and tissue remodelling processes. The B2p induced response exhibited features of excessive immune activation, accompanied by the upregulation of counter-regulation genes such as Ackr2. These findings indicate that liver pathology is not solely determined by parasite presence, but rather may also be influenced by the nature and regulation of the host immune response. Overall, the observed differences between A1np and B2p infections suggest that parasite-specific properties shape hepatic immune activation and may influence disease progression. Author summaryAlthough infection with the parasite Entamoeba histolytica can lead to severe liver disease, most infected individuals remain asymptomatic. This suggests that the outcome of the disease is not determined solely by the parasite, but also by how the host responds to the infection. In this study, we used a mouse model to compare how the liver reacts to infection with two E. histolytica clones that differ in their ability to cause amoebic liver abscesses. Using this model and time-resolved transcriptome analysis, we found that both clones trigger an early immune response; however, the nature of this response differs markedly. The non-pathogenic clone induced a rapid and controlled reaction associated with antimicrobial defence and tissue protection. In contrast, the pathogenic clone provoked a stronger and more prolonged inflammatory response accompanied by cellular stress and tissue remodelling processes. Notably, this heightened response also activated regulatory mechanisms that attempted to limit excessive inflammation. Our findings demonstrate that differences in disease severity are linked to the activation and regulation of the host immune system, rather than simply to the presence of the parasite.

Soil-transmitted helminths (STH) are a plausible but under-characterized comorbidity in tuberculosis. In this prospective South Indian cohort, multiplex stool PCR detected STH in 43% of 137 adults with pulmonary tuberculosis and 34% of 230 household contacts. Food insecurity independently predicted co-infection. Current adult deworming gaps warrant evaluation.

Burkholderia cepacia (B. cepacia) is an opportunistic pathogen with versatile virulence mechanisms. The pathogenesis of B.cepacia in the immunocompetent host following intranasal exposure largely remains ambiguous. Male BALB/c mice were intranasally inoculated with B. cepacia strain 20209 (1x10{square} CFU) and evaluated on days 3, 7, 14, and 21 post-infection. Histopathology of lung, liver, spleen, and kidney tissues were performed using H&E and PAS staining. Plasma cytokines were quantified using commercial multiplex assays and ELISA. Matrix metalloproteinase-2 (MMP-2) activity was assessed via gelatin zymography and metabolomic profiling by high-resolution mass spectrometry (HRMS). Histopathological analysis revealed organ-specific pathological indices such as interstitial pneumonitis, bronchitis, leukocyte infiltration, hepatic inflammation, as well as splenic hyperplasia. Similarly, MMP-2 activity revealed time-dependent modulation, reflecting dynamic proteolytic responses. Plasma and tissue IL-18 and IL-1{beta} levels demonstrated a temporal regulation, with IL-18 peaking on day 7 post-infection, while IL-1{beta} showed a biphasic expression peaking on day 3 and 14. Untargeted metabolomics revealed differential expression of lipid metabolism, and energy pathways, with higher expression of phospholipids and sphingolipids. Together, our study portrayed a physiologically relevant intranasal BALB/c model that captures both localized and systemic inflammatory responses to B. cepacia. Our findings highlight organ-specific pathologic progression and sustained inflammation providing key insights into host-pathogen interactions.

Objective: To characterize pregnancy outcomes and menstrual irregularities in Mexican women with systemic lupus erythematosus (SLE) and identify clinical factors associated with adverse pregnancy outcomes and early-onset menopause. Methods: We conducted a cross-sectional study of women with SLE enrolled in the Mexican Lupus Registry (LupusRGMX) between May 2021 and September 2024. Clinical and reproductive data were collected using standardized questionnaires. Menopause was defined as the absence of menstruation for [≥]12 consecutive months, and early menopause as onset before age 40. Univariable and multivariable logistic regression analyses were used to identify factors associated with pregnancy complications and early menopause. Results: A total of 210 women were included. Median age was 38 years (IQR 29-46) and median disease duration was 4 years (IQR 1-10). Among women with a history of pregnancy (47%), full-term delivery predominated (61%), while pregnancy loss occurred in 26% and preterm delivery in 13%. Pregnancy complications were reported in 9.6%, most commonly preeclampsia (6.7%). Younger maternal age was independently associated with pregnancy complications (OR 0.89, 95% CI 0.83-0.95) and adverse outcomes (OR 0.95, 95% CI 0.92-0.98). Higher disease activity was associated with complications in univariable analysis. Most pregnancies (68.3%) occurred before diagnosis. Early menopause was observed in 6.2% and independently associated with longer disease duration and older age. Conclusion: Younger maternal age was independently associated with adverse pregnancy outcomes, whereas disease activity showed an association in univariable analysis. Most pregnancies occurred prior to SLE diagnosis. Early menopause was associated with longer disease duration, suggesting impact of cumulative disease burden on ovarian function.

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI

Actin superfamily members are critical for the biology of eukaryotes and archaea. Actin-related proteins (Arps) are a subgroup within the actin superfamily and play essential roles in trafficking, replication and motility. The genome of the malaria parasite Plasmodium contains a set of Arps unique to apicomplexans, termed actin-like proteins (Alps). However, the importance and specific roles of many of these Alps in Plasmodium progression are not yet understood. Here, we determined the functional contribution of Plasmodium berghei Alp3 and Alp5a (recently relabelled as Arp3) by generation of knock-out (KO) lines and their subsequent characterisation across different life cycle stages. Deletion of either Alp did not affect blood stage growth, gametogenesis and ookinete gliding motility. However, deletion of Alp5a lead to smaller and fewer oocysts as well as severely impaired sporozoite formation. The Alp3KO line had highly reduced oocyst loads compared to wild-type parasites. This striking decrease was due to impaired ookinete penetration of the mosquito midgut epithelium. Our study shows that both Alp3 and Alp5a are indispensable for Plasmodium transmission at different steps of initial mosquito infection, provides insights into the role of specific unique members of the actin superfamily during parasite progression and the requirements for efficient midgut penetration.

Background: Tuberculosis (TB) remains the leading infectious cause of death worldwide, with India accounting for nearly one-fourth of global TB cases. Ni-kshay, the countrys digital case-based TB notification platform is rich in data pertaining to the continuum of care of TB patients. This study aims to develop a standardized analytical approach to programmatic data to identify predictors of unfavourable treatment outcomes and mortality among adult drug-sensitive TB patients at the state level for Maharashtra during 2021 and 2022. Methods: Two separate analyses were undertaken comparing treatment success with: (1) unfavourable outcomes (death, treatment failure, loss to follow-up, regimen change, or not evaluated); and (2) mortality. Multivariate logistic regression was used to compute adjusted odds ratios (aOR) for key risk factors, adjusting for age, gender, and weight. Results: The final cohort included 323,124 cases for unfavourable outcome analysis and 315,579 cases for mortality analysis. Increasing age, male gender, lower body weight, known HIV and diabetes comorbidities, tobacco and alcohol consumption, and "unknown" status for behavioural risks and comorbidity status were significantly associated with increased odds of both unfavourable outcomes and mortality. Conclusions: This study highlights the utility of programmatic data in identifying high-risk TB patients and offers a reproducible analytic framework.

Tick-borne pathogens can reshape vector microbiomes in ways that influence pathogen colonisation and transmission, yet the interplay between Ehrlichia ruminantium and the microbiota of its tick vectors remains uncharacterised. We profiled bacterial communities in haemolymph, midgut, and salivary glands of infected (n = 11) and uninfected (n = 12) Am. gemma ticks, a vector of E. ruminantium in East Africa, collected from cattle in Kajiado County, Kenya, using near-full-length 16S rRNA gene amplicon sequencing on the Oxford Nanopore platform. Community composition, alpha and beta diversity, co-occurrence networks, keystone taxa, and PICRUSt2-inferred functional profiles were compared across tissue-infection status groups. We identified 226 bacterial genera dominated by Coxiella, Pseudomonas, Acinetobacter, Proteus, and Rickettsia. Infection was associated with tissue-specific shifts in community composition (PERMANOVA R{superscript 2} = 0.14, p < 0.001) and co-occurrence network structure, with midgut networks showing complete hub taxon turnover (Jaccard = 0.000, p = 0.043). Haemolymph communities converged around Luteimonas as a keystone taxon, while opportunistic Proteobacteria, including Acinetobacter and Serratia, emerged as keystones in infected midgut. Endosymbiotic Rickettsia was near-absent in infected tissues (0.3% vs 9.3% mean relative abundance in midgut), consistent with competitive exclusion. Functional inference identified FDR-significant enrichment of predicted aerobactin siderophore biosynthesis, antimicrobial efflux, and oxidative stress response gene families in infected microbiota. These findings show tissue-specific restructuring of the Am. gemma microbiome associated with E. ruminantium infection and point to candidate targets for microbiome-based interventions against heartwater. ImportanceHeartwater, caused by the bacterium Ehrlichia ruminantium and transmitted by Amblyomma ticks, kills up to 90% of susceptible ruminants and is one of the most devastating tick-borne diseases in sub-Saharan Africa. Controlling heartwater requires understanding how the pathogen interacts with the microbial communities living inside its tick vector. In this exploratory study, we show that E. ruminantium infection is associated with tissue-specific shifts in the Amblyomma tick microbiome, including reduced abundance of beneficial symbionts, elevated representation of opportunistic bacteria among community hubs, and enrichment of iron acquisition and antimicrobial resistance functions. The midgut, the first tissue colonised during infection, showed the most marked structural reorganisation. These tissue-resolved microbiome signatures point to potential targets for novel control strategies, such as anti-microbiota vaccines or approaches that reinforce natural colonisation resistance, offering new strategies to reduce heartwater transmission and protect livestock livelihoods across Africa.

Non-typeable Haemophilus influenzae (NTHi) is a prominent opportunistic pathogen relevant to chronic respiratory diseases. NTHis metabolic diversity enables its survival in a wide range of environmental conditions within the host. As such, deeper research into the metabolic pathways of NTHi may open an avenue for novel therapies aimed at combating NTHi-associated respiratory diseases. Draft genome sequences from nine NTHi clinical strains from three isolation sites - ear (ear sample, ES), pharynx (pharynx sample, PS), and lower respiratory tract (Lungs) - were analyzed and annotated using RAST, PROKKA, KEGG KAAS, and antiSMASH. Pathway module coverage per-strain was computed and summarized by per-group for significant annotated metabolites. Metabolites were analyzed by LC/HRMS, identified by Metaboscape, and statistically compared using MetaboAnalyst and R software. Gene content across the tested NTHi strains was largely conserved, with limited core-genome SNP variation. Gene annotation for metabolite-related pathways revealed that all nine strains possessed largely similar sets of metabolic pathway genes, despite minor nucleotide-level differences, indicating broadly comparable metabolic capacities. In contrast, metabolomics data revealed differential metabolic profiles among the body-site groups. In a principal component analysis (PCA), the ES group was significantly separated from both the PS and Lung groups, which overlapped considerably. Detailed metabolite analyses showed that inosine, hypoxanthine, and uracil were highly significant in the ES group compared to the PS and Lung groups. For the first time, our study sheds light on the extent of metabolic differences associated with NTHi inhabiting diverse host niches. The observed metabolic differences suggest that NTHi may modulate its metabolism in a site-specific manner that is affected by environmental factors. These findings add to our understanding of how NTHi metabolism contributes to site-specific colonization. Author summaryHaemophilus influenzae is widely recognized as a causative agent of meningitis and pneumonia. In particular, H. influenzae strains with a polysaccharide capsule--known as H. influenzae type b (Hib)--were historically a major cause of invasive disease. However, Hib has been largely eradicated following implementation of the Hib vaccine. Nonetheless, there are H. influenzae strains that lack this capsule and are therefore not targeted by the vaccine. These are known as non-typeable H. influenzae (NTHi). Following the decline of Hib, NTHi has rapidly occupied the ecological niche in the lower respiratory tract, becoming the most prominent pathogen in patients with chronic respiratory infections--particularly in those with chronic obstructive pulmonary disease (COPD), where it frequently triggers exacerbations. Importantly, NTHi is also a common component of the normal microbiome in healthy individuals, typically residing in the upper respiratory tract without causing disease. In our study, we investigated the metabolic characteristics of NTHi isolates obtained from different body sites in patients to better understand what distinguishes strains capable of colonizing specific anatomical niches. We successfully identified several distinct metabolic features associated with NTHi strains from the ear, pharynx, and lung. These findings may serve as a foundation for future research into patient-tailored biomarkers and targeted therapies, ultimately aiming to eradicate NTHi in chronic lung infections.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating immune responses and have emerged as potential biomarkers and therapeutic targets in complex diseases. Leishmaniasis is a neglected disease that compromises host immunity and is associated with challenging treatments regimens. Leishmania amazonensis (L. amazonensis), an intracellular protozoan parasite, causes cutaneous leishmaniasis by replicating inside mammalian macrophages to establish infection. In this context, miRNAs have emerged as vital post-transcriptional factors that regulate the inflammatory landscape during infection. In this study, we aimed to analyze the function of miR-721 in macrophages during L. amazonensis infection by integrating in silico miR-721 target prediction with RNAseq data from macrophages of two distinct mouse genotypes, resistant C57BL/6 and susceptible BALB/c. We found that miR-721 is induced in macrophages infected with L. amazonensis, but is not in LPS-stimulated macrophages, suggesting a TLR4-independent activation. Integrating miR-721 target prediction with comparative transcriptomic analyses in resistant C57BL/6 and susceptible BALB/c models revealed the TNF-IRF1 axis as a primary miR-721-associated regulatory network. Specifically, miR-721 is predicted to target the 3UTRs of Tnf and Irf1 to suppress the inflammatory response. Functional inhibition of miR-721 successfully restored Tnf and Irf1 expression and reduced the amastigote burden over 24 hours. Furthermore, we showed that the miR-721/TNF-IRF1 axis regulates downstream genes associated with macrophage response, such as Serpine1, Csf1, Cd69 and Maf. Our work demonstrated that Leishmania induces miR-721, which negatively modulates the TNF-IRF1 axis, thereby suppressing the immune response and favoring parasite persistence. While C57BL/6 macrophages exhibit a robust activation of the TNF-IRF1 network, promoting inflammatory response, BALB/c macrophage showed a breakdown of this network. This was associated with post-transcriptional suppression of inflammatory responses, thereby favoring parasite persistence. These findings link miR-721 to the establishment of macrophage polarization, providing relevant insights into the mechanisms of parasite subversion of the host immune response.

Trichophyton mentagrophytes genotype VII (TmVII) is an emerging sexually transmitted dermatophyte that causes skin infections characterized by inflammatory, erythematous-squamous, painful, and persistent lesions. This genotype is part of the T. interdigitale/T. mentagrophytes Species Complex (TiTmSC), which comprises 28 genotypes. To enable rapid and specific differentiation of TmVII from other genotypes, a real-time polymerase chain reaction (rt-PCR) assay was developed targeting three unique single-nucleotide polymorphisms in the ITS1 region of TmVII. Assay specificity was further improved by introducing an additional mismatch at the 3 ends of both forward and reverse primers. The rt-PCR assay demonstrated high sensitivity, with a detection limit of 0.0002 ng of TmVII genomic DNA. The assay was highly specific, with no cross-reactivity observed with either closely or distantly related fungal pathogens when a cycle threshold (Ct) cutoff of 37 was applied. Among 497 mold isolates tested, 47 were confirmed as TmVII by rt-PCR, and the results were fully concordant with conventional ITS-PCR/Sanger sequencing. The rt-PCR assay demonstrated high sensitivity, specificity, reproducibility, and speed, with a turnaround time of one day after DNA extraction, compared with seven to ten days for Sanger sequencing. The first rapid molecular assay developed using TaqMan chemistry for TmVII identification is expected to enhance patient care and support infection control measures.

The human small intestine (SI) plays a central role in nutrient processing, host-microbe interactions, and immune regulation, yet remains poorly characterized due to the lack of minimally disruptive sampling methods. Here, we present a protocol for deploying, recovering, and analyzing samples collected using an ingestible device that enables multi-region, lumen-targeted SI sampling during normal digestion. The device incorporates a ~30-cm collapsible tube wound into pH- or time-responsive layers that sequentially unfurl in situ, typically capturing three spatially ordered samples with high yield and reliable retrieval. This protocol outlines study design, participant handling, device recovery, contamination control, and standardized workflows for analyses, including cell quantification, culturomics, sequencing, and metabolomics. We further describe benchmarking approaches for evaluating spatial resolution and strategies for assay prioritization when sample volume is limiting. By reducing participant burden and facilitating integration with stool, saliva, and clinical metadata, this approach enables longitudinal and large-cohort studies linking SI microbial ecology and host physiology to human health.

Tuberculosis (TB) continues to pose a significant global public health challenge with substantial patient morbidity and mortality. Current TB patient biomarkers lack sufficient resolution to inform treatment response and patient stratification. This necessitates the development of sensitive and reliable host biomarkers. We previously demonstrated the efficacy of TruCulture whole blood stimulation for differentiating asymptomatic TB from active pulmonary TB disease patients in endemic regions. Our systems immunology study expands upon this previous work by evaluating the potential of TruCulture to monitor longitudinal responses to TB treatment in patients from the Predict-TB trial before, during, and after 6 months of antibiotic therapy. We stimulated whole blood from TB patients (n=40) using TruCulture under four conditions (Null, Mycobacterium tuberculosis-antigen, LPS, and IL-1{beta}) at baseline (week 0), during treatment (weeks 16 and 24), and one-year follow-up post- treatment (week 72). 20/25 measured cytokines exhibited significant changes throughout treatment, with several continuing to evolve during post-therapy follow-up. Machine learning based analysis identified Mtb-Ag-induced IL-1RA (AUC = 0.90, 0.92, 0.95 at weeks 16, 24, 72) and LPS-induced NLRP3 (AUC = 0.94 at week 16) as the best protein and transcriptional biomarkers for distinguishing treated from untreated patients, strongly implicating the inflammasome response. Combining these results with the extent of lung disease assessed by FDG PET/CT scans, we showed direct disease relevance for these blood-based biomarkers. The identified biomarker profiles hold promise for improving TB patient care through early prediction of treatment responses, real-time therapy monitoring, and informed development of host-directed therapeutic strategies for clinical decision-making. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/723467v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@14a32eforg.highwire.dtl.DTLVardef@55f3d4org.highwire.dtl.DTLVardef@fb0137org.highwire.dtl.DTLVardef@10cf39e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Predict-TB clinical study overview and summary of TB-specific biomarkers identified from TruCulture whole blood stimulation system. C_FIG

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium that causes scrub typhus, a potentially life-threatening disease. To systematically identify host factors regulating early stages of infection, we performed a microscopy-based genome-wide siRNA screen in HeLa cells. This approach identified 2,989 genes grouped into 55 functional networks that modulate bacterial entry and intracellular translocation. In addition to confirming previously described pathways, including endocytosis and microtubule-dependent trafficking, the screen revealed an association between Ot infection and host cell cycle regulation. We found that Ot preferentially infects and/or replicates in host cells in the S and G2 phases, where intracellular bacterial accumulation is increased relative to G1. Early infection was associated with a shift in host cell cycle distribution, consistent with a delay in progression through S and G2 phases. Longitudinal analysis further showed that these cell cycle states support enhanced bacterial expansion. In parallel, infected cells exhibited reduced proliferation compared to uninfected cells, suggesting that Ot infection alters host cell division dynamics. Together, these findings support a model in which host cell cycle state influences susceptibility to Ot infection and intracellular growth. This work provides a systems-level map of host pathways involved in early infection and identifies cell cycle regulation as an important component of host-pathogen interactions in scrub typhus. Author SummaryScrub typhus is a potentially life-threatening disease caused by the bacterium Orientia tsutsugamushi, which can only survive and replicate inside human cells. Although some host factors involved in infection have been identified, many remain unknown. In this study, we used a large-scale screening approach to systematically identify human genes that influence the bacteriums ability to enter and move within host cells. Our analysis uncovered multiple pathways required for infection, including a role for the host cell cycle. We found that O. tsutsugamushi preferentially accumulates in cells during specific stages of the cell cycle, particularly when cells are preparing to divide. At the same time, infection slows host cell division, suggesting that the bacterium alters the cellular environment to support its own growth. These findings provide new insight into how O. tsutsugamushi interacts with human cells and identify potential host processes that could be targeted to limit infection.