Frontiers in Cellular and Infection Microbiology

Urinary tract infection (UTI) remains the most common infectious complication among individuals with neurogenic lower urinary tract dysfunction (NLUTD) due to spinal cord injury or disease (SCI/D). Despite widespread reliance on microbiological and symptom-based criteria for UTI diagnosis, significant ambiguity persists--especially in distinguishing clinically meaningful change from normal variability in urinary analysis results. This uncertainty contributes to overdiagnosis, inappropriate antibiotic use, and antimicrobial resistance. The present study seeks to operationalize "normal variability" of the urinary microbiome (urobiome) among adults with SCI/D. Using repeated samples collected from asymptomatic individuals over time, we analyzed inter- and intra-individual microbial composition to determine stability and fluctuation under baseline conditions. We observed wide intra- and inter-individual variability, substantial overlap between asymptomatic and pre-symptomatic states, and a consistent predominance of genera conventionally labeled as "uropathogens" even in the absence of symptoms. These findings suggest that assumptions drawn from cross-sectional studies--linking particular taxa or diversity values to health or disease--are not supported within individuals over time, at least in people with NLUTD. This study provides a foundation for distinguishing expected variation from those potentially related to infection, supporting development of precision-based diagnostic thresholds. Results offer critical insight into the ecological dynamics of the urobiome among people with NLUTD who are asymptomatic, establishes a methodological precedent for urobiome-informed clinical decision-making in SCI/D populations, and provides a foundation for distinguishing expected variation from those potentially related to infection, supporting development of precision-based diagnostic thresholds. By identifying personalized baselines and patterns of change, we aim to support research designed to obtain actionable information from the urobiome to enhance the accuracy and stewardship of UTI diagnosis and treatment in this high-risk population.

The genus Gardnerella comprises a group of fastidious bacteria associated with the female urogenital tract and has undergone extensive taxonomic revision in recent years. In this study, a bacterial strain, designated CCPDSM, was isolated from the female urinary microbiome and subjected to a comprehensive polyphasic taxonomic characterization. The 16S rRNA gene sequence confirmed that this strain is a member of the genus Gardnerella, and phylogenetic analyses based on cpn60 sequences, together with phylogenomic reconstruction placed strain CCPDSM within the genus Gardnerella as a distinct and well-supported lineage. Genome-based relatedness indices (ANIb, ANIm, TETRA and dDDH), demonstrated clear separation of CCPDSM from all validly published Gardnerella species. In contrast, comparisons with two publicly available closely related genomes yielded values above accepted species delineation thresholds, supporting their assignment to the same taxon. Phenotypic characterization, together with genome-based functional predictions, revealed a fastidious, fermentative metabolic profile that further differentiated CCPDSM from its closest relatives, while remaining consistent with traits characteristic of the genus. On the basis of combined phylogenetic, genomic and phenotypic evidence, strain CCPDSM is proposed as representing a novel species within the genus Gardnerella, for which the name Gardnerella fastidiominuta sp. nov. is proposed, with strain CCPDSM (=CECT 31324=CCP 588) designated as the type strain. This study expands the recognized diversity of Gardnerella and highlights the female urinary tract as a reservoir of previously uncharacterized species within this genus.

Helicobacter pylori is a prevalent bacterial pathogen that chronically colonizes the human gastric epithelium, but the bacteriums physiological mechanisms that promote this are understudied. Dormancy and low growth are known to facilitate other microbial chronic infections. A critical feature of low growth states is the down regulation of ribosome translational activity via regulation factors. The H. pylori genome is predicted to encode only one ribosome regulation factor, called RsfS (Ribosomal Silencing Factor S). In other bacterial species, RsfS prevents ribosome assembly by binding to a protein called L14 on the 50S large ribosomal subunit. Although H. pylori RsfS has not been experimentally investigated prior to this work, it conserves key residues, suggesting it is a bona fide RsfS homolog. To investigate phenotypes associated with rsfS, the gene was deleted and mutant phenotypes characterized. H. pylori rsfS null mutants had no defects during exponential phase but had viability defects in stationary phase and low growth factor conditions. Additionally, rsfS null mutants could not form biofilms, and instead were only able to form monolayers of multicellular aggregates. These defects were corrected by the re-introduction of rsfS in a second site on the chromosome. To explore whether rsfS is required in vivo, a mouse model was employed. rsfS mutants initially colonized in low numbers in both the glands and total stomach but were unable to develop robust long-term colonization. This work supports that H. pylori requires RsfS for survival in low growth states and to maintain chronic infections in the host. ImportanceH. pylori chronic infections are difficult to cure in part because H. pylori is proposed to adopt low-growth states known to render bacteria tolerant to antibiotics. One key signature of a low growth state includes low translation via ribosome regulation factors. Unlike other bacterial species, H. pylori contain only one known ribosome regulation factor called Ribosomal Silencing Factor S (RsfS). This gene was previously found to be transcriptionally upregulated in at least one low growth state, biofilms. In this work, we found that H. pylori rsfS is required for this microbe to thrive in low growth states and during infection. This study is one of only two studies that investigates the phenotypes of rsfS knockout mutants in any bacterial species and the first to address knowledge gaps in ribosomal regulation by H. pylori in vivo.

BackgroundComprehensive data on the prevalence of Helicobacter pylori infection and its associated risk factors among patients with gastrointestinal symptoms remain limited. Generating this evidence would help inform clinical management and improve antibiotic stewardship. H. pylori infection affects a substantial proportion of the global population, with prevalence varying widely across regions. In Uganda, previous studies have documented the presence of H. pylori infection. However, data specific to symptomatic patients are scarce. This study therefore aimed to determine the prevalence of H. pylori infection and associated factors among patients with gastrointestinal symptoms attending Mulago National Referral Hospital in Kampala, Uganda. MethodsA cross-sectional study was conducted among 353 patients with gastrointestinal symptoms attending Mulago Hospital. Data on socio-demographic characteristics, lifestyle and dietary habits, and medical history were collected using a semi-structured questionnaire. H. pylori infection status was determined using stool antigen tests. Proportions were used to determine the prevalence of H. pylori, and associated factors analyzed using STATA version 14 software by performing bivariate and multivariate analyses. ResultsAmong the 353 participants, majority were between 16 and 25 years old (69%), female (58%), and residing in peri-urban areas (74%). The prevalence of H. pylori infection in this population was 308 (87.3%). Multivariate analysis showed that H. pylori infection was significantly associated with having more than five income dependents (aPRR = 1.104, 95% CI: 1.025-1.189, p = 0.008), a history of previous H. pylori treatment (aPRR = 3.459, 95% CI: 2.138-5.595, p < 0.001), and a family history of H. pylori infection or gastrointestinal ulcers (aPRR = 1.135, 95% CI: 1.055-1.221, p = 0.001). ConclusionThis study demonstrated a high prevalence of Helicobacter pylori infection among patients presenting with gastrointestinal symptoms, with nearly nine out of ten individuals testing positive. The high burden observed suggests that routine screening for H. pylori, or carefully guided empirical treatment, may be clinically justified in symptomatic patients. These findings underscore the need for integrated clinical and public health strategies to improve diagnosis, treatment, and prevention of H. pylori infection in this setting.

BackgroundAcinetobacter baumannii is a common bacterial pathogen in nosocomial infections. It has become one of the greatest threats to human health for its growing resistance to last resort antibiotics, which has led to a revival of phage therapy as a potential treatment. However, conventional methods for isolating A. baumannii-infecting phages are labor-intensive and often unsuccessful. MethodsOur approach involves a computational pipeline to identify temperate phages (prophages) integrated into A. baumannii genomes, followed by mitomycin C (MMC) induction of those strains to screen for active prophages. ResultsHere we show a prophage analysis for nearly 900 A. baumannii genomes. We observed MMC-triggered excision of nine prophages from eight A. baumannii strains by PCR and sequencing. Further we show four prophage form virions detectable by transmission electron microscopy, and two which can plaque on other A. baumannii isolates. ConclusionThis work demonstrates the utility and diversity of prophages for further development as therapeutics for antibiotic resistant A. baumannii.

This pilot study assessed the composition and diversity of the urinary microbiome from clinically confirmed UTI samples using 16S rRNA sequencing, whilst also exploring inter-individual variability of microbial community structure. We examined ten urine samples from patients with culture-positive UTIs. Demographic and clinical metadata, including age, sex, body mass index (BMI), diabetes status and recent antibiotic exposure was recorded per sample. Metagenomic DNA was extracted from microbial samples and sequenced to generate genus-level taxonomic profiling through 16S rRNA gene sequencing. Relative abundance tables were generated for each of the samples to identify dominant bacterial genera within each sample and summarize cohort level microbial patterns. To evaluate within-sample richness and evenness, alpha diversity indices (Shannon, Simpson, observed features and Chao1) were computed; beta diversity was measured using Bray-Curtis dissimilarity with principal coordinates analysis (PCoA) for graphical representation. The studys findings revealed the sex and moderate clinical diversity of the study sample; all samples were confirmed as having been taken from a UTI patient and exhibited a wide level of heterogeneity regarding the microbial composition of each urine sample. Overall, Pseudomonas was the dominant genus present, however, specific samples had approximately 50% of their microbiomes composed of Klebsiella, Proteus, and Escherichia species as well as approximately 25% of their total microbes were made up of Burkholderia spp., which are closely related to the genus of interest used during the course of this study. The observed alpha diversity of each sample displayed considerable variation for the included samples with a continuum of samples ranging from a single dominant microbe to a highly diverse mixed population producing a highly diverse polymicrobial population/bacterial composition, with some ratios of individual taxa to collective taxa of many samples repeatedly illustrating the exact nature of the specimen. Furthermore, a significant degree of Beta diversity was found between the patients, providing compelling evidence of identifiable differences among urinary microbiomes between patients with UTI. This pilot project provides a clear indication of the diversity and overall heterogeneity of urinary microbiota found in the UTI patients studied. In addition, the results of this study support the notion that the ecological complexities present within a urinary microbiome cannot necessarily be established through conventional culture methods, and that combined with molecular techniques such as 16S rRNA sequencing of bacterial DNA could be used to quantify and characterize the ecologic condition of urinary microbiota separate from the traditional high prevalence of identifiable uropathogens.

Microbial keratitis is a sight-threatening corneal infection with varying etiological agents, primarily bacteria and fungi. Assessing and contrasting the virulence factors of microorganisms isolated from a non-contact lens-associated keratitis (NCLAK) and contact lens-associated keratitis (CLAK) is the goal of the current investigation. Samples were collected from over 60 patients and analysed using standard microbiological techniques, including culture, Gram staining, KOH mount, biochemical tests, antimicrobial susceptibility testing, and biofilm assays. The results demonstrated that CLAK isolates were predominantly bacterial, especially Pseudomonas aeruginosa, known for strong biofilm production and high multidrug resistance. In contrast, NCLAK showed a higher incidence of fungal infections, particularly Candida albicans. The results highlight the significance of early diagnosis, tailored and improved awareness regarding contact lens hygiene to prevent complications associated with keratitis.

Latent tuberculosis infection (LTBI) remains a significant barrier to global TB control and elimination efforts. The QuantiFERON-TB Gold (QFT) assay is commonly used for the diagnosis of LTBI. However, blood collected in QFT tubes is seldom utilized for molecular and genetic analysis due to the presence of heparin and a dense gel barrier that hinders efficient DNA extraction. To address this limitation, we aimed to develop a method for directly isolating high-quality DNA from blood in QFT tubes, eliminating the need for additional blood sampling and enabling their use in both diagnostic and molecular workflows. In this study, DNA was extracted from blood in EDTA and QFT tubes using a hybrid approach that combined manual lysis with three commercial kits: Thermo Scientific GeneJET, QIAamp DNA Blood Kit, and FavorPrep Blood Genomic DNA Extraction Kit. DNA concentration and purity were measured with a Multiskan SkyHigh Microplate Spectrophotometer, while integrity was assessed through agarose gel electrophoresis. Two nucleic acid amplification techniques (NAATs), ARMS-PCR and whole exome sequencing (WES) were performed to validate applicability of extracted DNA for molecular biology applications. We did not find any differences in the quantity, quality, or application of PCR or sequencing for DNA extracted from EDTA or QFT tubes. The extracted DNA from both EDTA and QFT tubes exhibited A260/280 ratios of 1.7-1.9 and concentrations ranging from 4.9 to 118.5 {micro}g/mL, indicating an adequate yield and purity. Intact genomic DNA and PCR product bands on agarose gel indicated suitability for downstream applications. Additionally, WES produced 6.47-8.71 GB of data per sample, with 42.8-57.7 M reads and GC content between 49.29% and 52.54%. Sequencing metrics were consistently strong, with Q20 values exceeding 98.6% and Q30 values above 95%. Our study presents an optimized and reproducible protocol for extracting high-quality DNA from QFT tubes, producing DNA suitable for both PCR and sequencing technologies. This protocol provides a cost-effective and practical strategy to integrate LTBI diagnosis with genomic research, particularly beneficial in resource-limited settings. This study introduces a novel analytical workflow applicable to diagnostic laboratory settings, enabling the integration of routine LTBI immunodiagnostic testing with downstream genomic analysis. The approach supports improved utilization of clinical specimens in laboratory medicine and may facilitate future biomarker and precision diagnostics research.

IntroductionAcinetobacter is a highly diverse genus which includes a range of common pathogenic species such as A. baumannii, A. lwoffii etc. Acinetobacter species causes bacteremia, pneumonia, wound infections, Urinary tract infections in community as well as hospital settings. A. baumannii is one of the ESKAPE pathogen which makes it even more lethal as antibiotics cannot action on this. AimTo isolate Acinetobacter species from various clinical samples and to check their antimicrobial susceptibility pattern by VITEK {square} Compact in SGT Hospital, Gururam, Haryana. ResultsOut of total 6673 samples 595 were the positive isolates from which 35 were Acinetobacter isolates which were received from various wards of the hospital. Occurrence of Acinetobacter was seen more in males(57.14%) as compare to females (46.8%). A total of 31 strains were A. baumannii, 3 were A. lwoffi and 1 strain was of A. haemolyticus. Prominent presence of Acinetobacter was seen in Blood (48.57%) specimen along with pus(22.85%), endotracheal (22.85%), tracheal (2.85%) and eye swabs (2.85%). All the isolates were resistant to piperacillin/tazobactam (100%), ceftriazone (100%), amikacin (100%), gentamicin (100%) ciprofloxacin (91.42%), ceftazidime (91.42%), cefepime (88.57%), levofloxacin (88.57%) and trimethoprim/sulfamethoxazole (80%). Colistin susceptibility was observed in 88.57% of the isolates. ConclusionAcinetobacter is a common pathogen in hospital acquired as well as in community acquired infections as it is a opportunistic pathogen hence to identify the Acinetobacter species and to understand their antimicrobial resistance pattern this study was conducted.

Abstarct Chlamydia trachomatis (CT) is a sexually transmitted infection (STI). In 2020, an estimated 128.5 million new CT infections were reported among adults worldwide. The global prevalence was 4.0% in women and 2.5% in men. Young adults are the most frequently infected. This bacterial STI is often asymptomatic but can lead to serious complications, particularly in pregnant women. The objective of this study was to determine the prevalence and sociodemographic profile of CT infection in pregnant women living in resource-limited settings. Methods We conducted a cross-sectional study between June 2023 and december 2023 in a maternity ward located west of Kinshasa in an impoverished area with a low-income population. During the study period, we collected 239 cervical swab samples from pregnant women. CT DNA was extracted using the QIAamp DNA Mini Kit. Molecular diagnosis was performed by amplification of a 201 bp fragment of bacterial 16S rDNA from the cryptic plasmid. Results The age group most affected by CT in our cohort was 25 to 35 years (65.83%); married women were more represented than single women (83.22% versus 16.77%). The incidence of CT infection was 18%. The most common vaginal symptoms associated with the infection were vaginal itching and abnormal vaginal discharge, while the most common hypogastric symptom was chronic pelvic pain. Prematurity and spontaneous abortions were the most frequently observed pregnancy complications. Conclusion Chlamydia trachomatis (CT) infection is common among pregnant women living in poverty in Kinshasa. The infection particularly affects the most sexually active age group. It is associated with vaginal and hypogastric symptoms, as well as complications related to the progression of the pregnancy.

Cryptosporidium spp. are protozoan parasites responsible for diarrheal diseases. In humans, cryptosporidiosis is predominantly caused by the human-specific Cryptosporidium hominis and by Cryptosporidium parvum. This second species has been classically reported as zoonotic, with a host preference for ruminants. However, the recently described subspecies C. parvum anthroponosum has been found to be restricted to humans. Here, we generated novel whole genome sequences from West African samples of C. p. anthroponosum, and analyzed them together with all those already available, originating from East Africa, Europe, North America and Asia. Phylogenomics showed that all C. p. anthroponosum isolates are strongly clustered together, forming the sister clade of the zoonotic C. parvum representatives. The phylogenetic variations within C. p. anthroponosum did not present a clear geographic structure, consistent with C. hominis, primarily transmitted in humans. To elucidate the evolution of host species adaptation in C. p. anthroponosum, we then investigated genetic exchanges with C. hominis, detecting an ancestral introgression present in all C. p. anthroponosum isolates. This introgression involved a single gene, encoding for an extracellular galectin-like protein, which we predicted with high confidence to form a protein complex with the human insulin-degrading enzyme, a key metabolic regulator. Considering the role of host insulin metabolism in the proliferation of parasites as well as its known intrinsic differences between humans and ruminants, this molecular interaction could represent a plausible mechanism for an important role of the galectin-like protein in host-parasite interactions and in the host specificity of C. p. anthroponosum.

Treponema pallidum subsp. pallidum, the causative agent of syphilis, remains difficult to study owing to long-standing limitations in in vitro cultivation. Although a rabbit epithelial cell co-culture system (Sf1Ep) enabled major advances in recent years, the lack of human cell-based models restricts clinical relevance and mechanistic insight into host-pathogen interactions. Here, we sought to establish a co-culture system that uses human epithelial cell lines capable of supporting T. pallidum growth in vitro. Six human epithelial or epithelial-like cell lines from diverse tissue origins were evaluated under microaerophilic conditions using the standard T. pallidum cultivation medium. Among these, CAL-39 (vulva) and HepG2 (liver) supported T. pallidum survival, replication, characteristic growth behaviours, and long-term passage at levels comparable to Sf1Ep cells. Growth kinetics, attachment dynamics, and motility of T. pallidum were quantified over extended culture periods. Using live-cell imaging in this co-culture system, for the first time we were able to define two distinct T. pallidum host-cell interaction behaviours; surface-associated crawling and stable single-polar attachment. Both behaviours were observed across all tested host cell lines and persisted over time only in cell lines permissive for sustained growth. Together, our findings establish clinically-relevant human epithelial co-culture models for T. pallidum, provide new insights into host-cell-dependent growth and motility, and create a platform for future mechanistic studies of syphilis pathogenesis and vaccine target discovery.

Achromobacter xylosoxidans (Ax) is an emerging pathogen with a strong capacity to adapt to different niches, but its pathogenesis is poorly understood. To investigate the virulence of this versatile bacterium, alternative infection models are valuable. Galleria mellonella wax moth presents cost and ethical advantages as an in vivo infection model. Here, we investigate the utility of Galleria as a model of Ax infection and demonstrate that mortality following Ax infection in Galleria recapitulates survival outcomes observed in infected mice. We further show that the Galleria infection model can be used to examine antimicrobial activity against Ax. Visualization of hemocytes suggested that Ax was internalized into immune cells, similar to what is observed in vertebrate models. Overall, our work establishes Galleria mellonella as a model of Ax infection that mirrors disease severity and innate immune cell interactions in murine models.

Toxoplasma gondii is an obligate intracellular parasite that infects virtually all warm-blooded animals, progressing through acute and chronic stages. The Akt/mTOR signaling axis plays critical roles in cell survival, proliferation, and metabolism, making it a key target for intracellular pathogens. This study investigated how T. gondii infection modulates this pathway during both infections. Outbred CD-1 mice were infected intraperitoneally with the virulent GT1 strain of T. gondii. Mice for acute studies were sacrificed five days post-infection, while those for chronic studies were treated with sulfadiazine and sacrificed five months post-infection. Phosphoprotein expression of eight Akt/mTOR pathway components was measured in liver tissues using a multiplexed bead-based immunoassay. Acute T. gondii infection caused broad suppression of Akt/mTOR signaling, with 6 of 8 markers significantly downregulated, including pS6RPSer235/236, pAKTS473, pBADSer136, pIRS1S636/639, pPTENSer380, and pGSK-3/{beta}Ser21/9. In contrast, chronic infection selectively activates specific nodes of the pathway in a cyst burden-dependent manner, including pBADSer136, pmTORSer2448, and pGSK-3/{beta}Ser21/9. There are strong correlations in signaling changes between inter-components, which reflect coherent and coordinated pathway-level reprogramming rather than random perturbation. These findings show that acute and chronic T. gondii infections have opposing effects on host Akt/mTOR signaling for their own benefit, which may present new therapeutic targets. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/716682v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@8c5021org.highwire.dtl.DTLVardef@1e0cdcaorg.highwire.dtl.DTLVardef@1e690eaorg.highwire.dtl.DTLVardef@342c0b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAcute T. gondii infection broadly suppresses hepatic Akt/mTOR signaling C_LIO_LIChronic infection exerts cyst burden-dependent activation of specific Akt/mTOR nodes C_LIO_LIT. gondii has distinct strategies to manipulate host survival based on its life stages. C_LIO_LIThe Akt/mTOR pathway may serve as a therapeutic target for the treatment of T. gondii. C_LI

BackgroundPeriodontitis (Perio) is a progressive oral disease characterized by inflammation and degradation of the periodontal apparatus and is associated with local and systemic morbidity including loss of teeth, cardiovascular disease, and diabetes mellitus, among others. Perio is highly prevalent in domestic canines and exhibits certain parallels in pathogenesis and pathophysiology to Perio in humans, although standard treatments are less effective. In both species, a complex interplay between oral microbiota and host immune response is implicated in the etiology of Perio but is not fully understood. ResultsUsing shotgun metagenomics and RNA-seq on oral samples from companion dogs, we identify features of the oral microbiome and host transcriptional profile that are associated with Perio and its progression. We observe differences in microbiota composition between Perio and non-Perio animals that are largely consistent with what has been described in humans but also identify several species that are distinctly associated with canine Perio. We observe an abrupt shift in host gene expression related to immune response and tissue structure that is associated with disease severity, specifically the progression from mild periodontal disease (PD) to more severe Perio and the initiation of clinical attachment loss. The gingival plaque microbiota exhibits a parallel dynamic, with distinct compositional profiles in mild, moderate, and severe PD. We then examine several of the known mechanistic components of the keystone pathogen hypothesis of PD, identifying specific commonalities between canine and human pathologies, including the involvement of Porphyromonas species and related virulence factors. Additionally, we show infiltration of gingival tissue by Porphyromonas and Tannerella spp. via fluorescence microscopy. Finally, we assess correlations between host gene expression and microbial metabolic pathways which suggest additional potential virulence factors. ConclusionsThis work elucidates the metagenomic and transcriptomic signatures of Perio in companion dogs with the goals of informing veterinary medicine, evaluating the potential of canines as a model organism for the study of Perio, and clarifying the relationship between Perio development and progression, the oral microbiota, and the localized host response. Our findings provide insight into the etiopathogenesis of canine Perio and its relationship to human Perio and suggest novel targets of potential translational interest.

AO_SCPLOWBSTRACTC_SCPLOWOrnithodoros phacochoerus are nidicolous soft ticks of the Ornithodoros moubata complex of species known to be vectors of the African swine fever (ASF) virus. These Ornithodoros ticks depend on endosymbionts to produce essential nutrients necessary for their development. However, endosymbionts are only a part of the complex microbiota hosted by the tick. This microbiota often includes primary or secondary endosymbionts, commensal species from the environment, and, most of the time, some pathogens. The present study was performed to understand the organization and spatial distribution of the microbiota of O. phacochoerus. One of the objectives was to investigate if the pathogen of interest (ASF virus) is involved in the organization of the microbiota through pathogen-induced dysbiosis or other interactions. For this purpose, 704 O. phacochoerus ticks were collected from two conservation areas in Mozambique. Sequencing was performed targeting the V3-V4 region of the 16S rRNA gene, and the resulting dataset was processed using FROGS to characterize the bacterial microbiota hosted by the ticks. The results indicate that the microbiota of Ornithodoros phacochoerus contains very low bacterial diversity, with one primary endosymbiont (Francisella-like endosymbiont), one potential secondary endosymbiont (Rickettsiella), and very few environmental or pathogenic bacterial species. We found a clear spatial structure of the microbiota, with ticks from the same sampling site showing similar patterns. On the contrary, no association with the infectious status for African swine fever virus was detected, suggesting that this pathogen does not shape Ornithodoros microbial communities. Our results on tick - microbiota - pathogen - environment interactions in nidicolous soft ticks, showed patterns that differ from most hard tick studies.

Bilophila wadsworthia is a gut pathobiont implicated in dysbiosis-driven inflammation, yet its pathogenic mechanisms remain poorly investigated. Here, we evaluated the suitability of Galleria mellonella larvae as an in vivo model to study B. wadsworthia infection. Two infection routes were compared: oral inoculation to mimic gastrointestinal colonization and hemolymph injection to model systemic infection. Oral challenge had minimal impact on larval health, whereas hemolymph injection caused marked morbidity, including reduced mobility, impaired cocoon formation, and progressive melanization, indicating that access to the circulatory system is required for overt disease. Infection required live bacteria, with B. wadsworthia capable of intracellular replication within hemocytes, leading to transient depletion of circulating immune cells followed by compensatory hemocyte proliferation. These findings reveal tight coupling between bacterial proliferation and host immune dynamics. Comparison with other sulfidogenic bacteria suggests that Bilophila pathogenicity is likely to involve host-specific interactions. Overall, our results establish G. mellonella as a practical and ethically favorable model to investigate B. wadsworthia virulence, host-pathogen interactions, and mechanisms relevant to gut-associated infection.

Whipworms (Trichuris spp.) are intracellular intestinal parasites that develop within the host caecal epithelium, yet the host signals that regulate their growth and developmental progression remain poorly understood. Progress in studying these processes has been limited by the lack of physiologically relevant in vitro systems capable of supporting sustained whipworm development. Here, we established an in vitro infection system using caecal organoids (caecaloids) and evaluated their capacity to support sustained growth and morphological development of Trichuris muris larvae. To rigorously validate this system, we generated a comprehensive and up-to-date anatomical and biometrical reference dataset describing the whole-body growth and tissue-level morphogenesis of T. muris throughout its life cycle in vivo. Quantitative analysis across larval and adult stages confirmed that the trajectory of parasite growth is largely conserved across host mouse strains and provided a detailed contextualised description of the development of key anatomical structures of T. muris. Using this reference framework, we evaluated parasite growth and development in long-term T. muris-caecaloid co-cultures. Larvae invading the caecaloid epithelium remained intracellular within syncytial tunnels and exhibited sustained growth over extended culture periods. in vitro parasites developed increasing anatomical complexity, including formation of the bacillary band, stichosome, intestine, and rectum. Importantly, quantitative comparisons revealed that larvae developing within caecaloids follow growth trajectories and morphological developmental patterns closely resembling those observed in vivo. This study therefore presents the first detailed anatomical and morphometric framework for validating whipworm development in an organoid system and provides concrete evidence that the caecaloid epithelium is sufficient to trigger and sustain whipworm growth and morphogenesis, establishing caecaloids as a powerful experimental platform for investigating Trichuris infection and development.

BackgroundUntil recently, the urine and urinary tract were considered sterile. Over the past decade, the discovery of a urinary microbiome has prompted growing interest in its potential role in urinary symptoms and disease. However, the composition and diversity of the urinary microbiome in the population remain poorly defined and prior studies have been limited by small sample sizes, selected patient populations, and low-resolution sequencing. Here, we characterize urine samples and their microbial content in a large population-based Swedish cohort using shotgun metagenomics. MethodsWe performed shotgun metagenomic sequencing of non-invasively collected (voided) urine samples from 2,062 participants of the population-based Swedish CArdioPulmonary bioImage Study (SCAPIS; 55% women, age range 50-65 years). These samples are considered genitourinary as they can contain microbiota of the urinary tract but also from the surrounding skin and genital tract. ResultsOf the 3,668 identified species, 90% were assigned at the species level taxonomy. Only 26 species were present in more than half of samples. Males had higher Shannon and inverse Simpson indices although no significant differences in species richness were observed by sex. Age showed only modest associations with alpha diversity. Beta diversity was significantly associated with sex, with females exhibiting greater within-group dispersion than males, and age had only a minimal effect in microbial composition. Microbial communities differed substantially between sexes, with several lactobacilli and bifidobacteria enriched in females, while Cutibacterium acnes, Enterococcus and Propionimicrobium lymphophilum were in males. Well-known urinary tract pathogens as Enterococcus faecalis or Escherichia coli were common even though all participants were asymptomatic and reported no urinary tract symptoms at the time of sampling. ConclusionsIn this large population-based study, we provide the most detailed characterization of voided urine profiles and their genitourinary microbiota to date, revealing substantial sex-specific differences and frequent occurrence of recognized uropathogens. These findings broaden the concept of a normal genitourinary microbiome and highlight the need to account for sex when studying the microbial content of urine samples. This sex-specificity may be key to define the functional role of the urinary or genitourinary microbiomes in health and disease.

Background: Mucormycosis is a rapidly progressive and often fatal invasive fungal infection caused by moulds in the order, Mucorales. Early diagnosis is essential for effective clinical management; however, conventional diagnostic approaches such as culture and histopathology are slow, insensitive, and require specialist mycological expertise. Although molecular methods are available for disease detection, they are not widely accessible. At present, no enzyme immunoassay (EIA) exists for the detection of mucormycosis. Methods: A murine IgG1 monoclonal antibody (mAb), FH12, was generated against extracellular polysaccharides (EPSs) produced by Mucorales pathogens during active growth. The antibody was characterised for specificity, epitope stability, and antigen localisation using ELISA, immunoblotting, and immunofluorescence techniques. The mAb was incorporated into a Sandwich-ELISA and evaluated using culture filtrates, purified EPSs spiked into human serum, and tissue homogenates from a patient with cutaneous mucormycosis caused by Lichtheimia ramosa. Results: mAb FH12 demonstrated pan-Mucorales specificity and no cross-reactivity with other clinically relevant yeasts and moulds. The epitope recognised by FH12 is periodate-insensitive and moderately heat-stable. The Sandwich-ELISA detected EPS antigens in human serum with limits of detection ranging from pg/mL to low ng/mL levels, and successfully identified the EPS biomarker in patient tissue homogenates. Conclusion: The FH12-based Sandwich-ELISA shows high sensitivity and specificity, and has the potential to be used as a laboratory-based adjunct diagnostic test for the detection of mucormycosis in humans.