Frontiers in Cellular and Infection Microbiology

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.

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.

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.

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.

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.

Background: Female genital schistosomiasis (FGS) is a neglected gynaecological manifestation of Schistosoma haematobium (S. haematobium) infection, resulting from the deposition of parasite eggs in the female genital tract. Although urogenital schistosomiasis is highly prevalent in parts of coastal Kenya, including Kilifi County, the burden of FGS among women of reproductive age remains poorly characterised. Routine diagnosis of S. haematobium infection relies largely on urine microscopy, which may underestimate genital involvement. This study aimed to assess the prevalence, diagnostic concordance, and risk factors for FGS among women of reproductive age in Kilifi County, Kenya. Methodology: In this cross-sectional study, 320 randomly selected women aged 15-50 years were recruited from rural Kilifi County; 261 provided complete data for analysis. A structured questionnaire was administered to collect sociodemographic and behavioural information. Urinary schistosomiasis was assessed using triplicate urine microscopy over three consecutive days, and FGS was evaluated using real-time polymerase chain reaction (PCR) targeting the S. haematobium Dra1 gene sequence on self-collected high vaginal swabs. Results: Overall, the prevalence of PCR-confirmed FGS was 36.0% (94/261), while urinary egg excretion was detected in 13.0% (34/261) of participants. Concordance between urine microscopy and genital PCR was 70.9%. Notably, 72% of women with PCR-confirmed FGS had no detectable parasite eggs in their urine. In bivariate analyses, factors such as urinary infection severity, water contact behaviours, haematuria, dysuria, age group, place of residence, and prior history of schistosomiasis were found to be associated with female genital schistosomiasis (FGS). However, in the multivariable logistic regression, only sub-location and urinary infection severity remained independently associated with the infection. Additionally, PCR cycle threshold (Ct) values showed a non-linear relationship with mean urinary egg counts, indicating that the detection of genital parasite DNA does not directly correspond to the urinary egg burden. Conclusion: FGS prevalence among women in Kilifi County was substantially higher than indicated by urine microscopy alone. The majority of women with genital schistosomiasis did not exhibit detectable urinary egg excretion, highlighting the limitations of routine parasitological screening for identifying genital disease. These findings underscore the need to incorporate genital sampling and molecular diagnostics into schistosomiasis control strategies targeting women of reproductive age in endemic settings.

Despite multiple treatment strategies and extensive research on resistance mechanisms, tuberculosis (TB) remains a major global health threat, largely because of the rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. Among various mechanisms complicating the situation, active antibiotic export via efflux pumps is particularly significant, yet largely unexplored. Mycobacterium sp. encodes numerous transporters, many of which are overexpressed in clinical isolates or under drug stress. Here, we examined the possible role of Rv0783c, a putative transporter that is reportedly overexpressed in drug-stressed conditions. Rv0783c conferred resistance to multiple structurally diverse antibiotics, fluoroquinolones and anti-TB drugs in the heterologous hosts, namely, Escherichia coli and Mycobacterium smegmatis. Reduced drug accumulation and active efflux of ethidium bromide (EtBr) confirmed its transport activity, which in turn gets nullified upon using the proton-motive force blocker, CCCP. On the other hand, its expression enhanced biofilm formation, linking antibiotic resistance to persistence-associated phenotype. Furthermore, site-directed mutagenesis confirmed the presence of crucial interacting residues with antibiotics that were identified by in silico analysis. Overall, we demonstrate the role of Rv0783c in the extrusion of first and second-line anti-TB drugs and enhancing biofilm formation.

1Canine atopic dermatitis (CAD) is a chronic inflammatory skin condition sometimes associated with microbial dysbiosis, including alterations in colonization by the lipophilic yeast Malassezia pachydermatis. This study investigated the population diversity of M. pachydermatis in the ear canals of healthy and CAD-affected dogs using Fourier-transform infrared (FTIR) spectroscopy and whole genome sequencing (WGS). Among 60 dogs, M. pachydermatis prevalence was significantly higher in CAD cases than in healthy controls. FTIR spectroscopy revealed greater strain heterogeneity in CAD-affected dogs, often with distinct genotypes in each ear, while healthy dogs exhibited more homogeneous populations. Using a previously developed FTIR-based artificial neural network classifier, we assigned strains to three phylogroups. Strains from phylogroups I and III were significantly enriched in CAD-affected dogs, while phylogroup II was most prevalent overall and the dominant phylogroup in healthy controls. This suggests that CAD-associated inflammation may favor specific M. pachydermatis phylogroups and sub-clusters within phylogroups, shaping colonization dynamics. FTIR-based typing showed full concordance with WGS across 35 sequenced isolates, recapitulating relationships among phylogenetically related isolates and their similar phenotypic profiles. Overall, our findings reveal strain-level shifts in M. pachydermatis populations associated with CAD and establish FTIR spectroscopy as a rapid, cost-effective tool for large-scale epidemiological studies.

Background: Reusable menstrual products provide sustainable and cost effective alternatives to disposable sanitary products; however, their adoption remains limited, even among healthcare professionals. Objectives: To assess awareness, knowledge, perceptions, and utilisation of reusable menstrual products among female medical students and healthcare professionals, and to identify predictors of willingness and use. Design: Cross sectional analytical study. Setting: An online survey was conducted among female medical students and healthcare professionals in Nigeria. Participants: A total of 203 female respondents aged 15 to 55 years. Intervention: Not applicable. Primary Outcome Measures: Utilisation of reusable menstrual products and willingness to adopt their use. Secondary Outcome Measures: Awareness, knowledge, perceptions, and barriers. Methods: Data were collected using a structured questionnaire and analysed using descriptive statistics, chi square tests, and logistic regression. Results: Awareness was high (96.06%), but utilisation was low, with 5.42% ever using and 4.43% currently using reusable products. About 31.53% were willing to use them. Respondent type was not associated with willingness (p = 0.735), although healthcare professionals had higher knowledge (p = 0.024). Positive perception predicted willingness (AOR = 7.58, 95% CI: 3.18 to 18.03, p < 0.001). Good knowledge (AOR = 14.96, p = 0.014) and increasing age (AOR = 1.28, p = 0.004) predicted utilisation. Conclusion: Despite high awareness, utilisation remains low. Perception influences willingness, while knowledge drives use. Targeted behavioural and educational interventions are needed. Keywords: Menstrual hygiene, reusable menstrual products, menstrual cup, sustainability, healthcare professionals

Introduction : The World Health Organization (WHO) recommends testing for sexually transmitted infections (STIs), but laboratory-based and rapid or point-of-care (POC) testing are often unavailable and unaffordable, especially in low-resource settings, leading to empiric (usually antibiotic) treatment. Community pharmacies (CPs) are often the first point of contact for persons with symptoms of STIs, where pharmacists dispense treatment without diagnostic testing or prescriptions. This study evaluated clients, providers, and policymakers perspectives on POC testing for STIs in CPs for targeted treatment. Methods : We nested a qualitative study into a study of participants seeking both STI and non-STI treatments in CPs. They were tested for HIV, syphilis, trichomonas, chlamydia, and gonorrhea using both rapid POC tests and a central reference lab. A purposive sample of 50 participants from September 2020 to June 2022 consented to participate in in-depth and key informant interviews. Data were analyzed thematically using an inductive approach. Results: Clients (n=35), health care providers (n=9), and policy makers (n=6) highlighted the benefits of POC tests for HIV and STIs at CPs, including affordability, accessibility, and ensuring convenience. The impact of POC testing for STI diagnosis and treatment was promoting behavioral change, rapid results turnaround time, leading to faster treatment access compared to conventional laboratory methods, and supporting sustainable antimicrobial resistance (AMR) control. Barriers to POC testing included a lack of awareness among clients and health workers, inadequate privacy and space, long wait times, unclear self-sample collection instructions, stigma around HIV testing, and reluctance to test for STIs beyond HIV. To address these, participant recommendations included raising STI awareness, providing more explanation of test results, increasing test access, addressing stigma, provider training, and ensuring a sustainable supply chain for testing kits. Conclusions : POC testing for STIs and HIV in CP settings was found to be highly acceptable to both pharmacy clients and providers. Integrating POC testing in CPs could be beneficial for national STI management programs. If the existing barriers are addressed, POC tests could improve accessibility to STI diagnostics and facilitate better linkage to care.

HIV remains among the worlds most serious healthcare challenges, with adolescent girls and young women in sub-Saharan Africa at particularly high risk of infection. Bacterial vaginosis (BV) is a key risk factor for HIV acquisition, however current treatment strategies are limited. Optimal vaginal Lactobacillus spp. protect against BV and HIV, largely through immunoregulatory and antimicrobial activities mediated in part by lactic acid. Towards the development of a Lactobacillus-containing live biotherapeutic for African women, we sampled 181 vaginal Lactobacillus isolates from 25 BV-negative South African women. Fifty isolates were selected for evaluation of inflammatory responses using vaginal epithelial cells, D- and L-lactate and lactic acid production and culture acidification. Aside from a single Lactobacillus salivarius strain, L. crispatus isolates acidified the culture media the most and produced the most D- and L-lactic acid. Inflammatory cytokine responses to Lactobacillus strains were variable, with L. crispatus eliciting the lowest levels of cytokine production. When all properties were evaluated collectively, L. crispatus strains exhibited the most desirable biotherapeutic characteristics. Whole genome sequence analysis of ten L. crispatus isolates showed that the majority were more closely related to one another than to isolates from other geographical regions. This supports the need for live biotherapeutics to be tailored for the population of intended use. No antimicrobial resistance elements were detected, while putative bacteriocins and intact prophage sequences were identified in all isolates. L. crispatus isolates displayed characteristics essential for optimal live biotherapeutic performance, however additional analysis is required to determine the functionality of identified putative prophages. ImportanceHIV is a leading cause of morbidity and mortality in sub-Saharan Africa, where adolescent girls and young women are three times more likely to acquire HIV than their male counterparts. A key risk factor for HIV is bacterial vaginosis (BV), a condition characterised by the loss of beneficial Lactobacillus species and increased abundance of non-optimal, inflammatory bacteria. Although BV affects approximately 25% of women in sub-Saharan Africa, effective therapeutics are lacking. Live biotherapeutics containing optimal Lactobacillus spp. represent a promising strategy to improve BV treatment outcomes and reduce HIV infection risk. We isolated 181 vaginal Lactobacillus spp. from 25 BV-negative South African women and characterized 50 selected isolates. This led to the identification of live biotherapeutic candidates for African women with distinct genomes compared to isolates from other geographical regions. This study contributes to current knowledge of the characteristics that should be considered when screening novel isolates for this purpose.

Background: The enterotype concept proposed that gut microbiomes cluster into discrete types, but subsequent critiques demonstrated that such clustering depends on methodological choices, that the number of clusters is not fixed, and that faecal samples cannot capture spatial heterogeneity along the gastrointestinal tract. The stomach remains particularly understudied, and no systematic classification exists for gastric microbial community types. Methods: We assembled a multi-cohort dataset of 566 gastric mucosal samples spanning healthy controls to gastric cancer, with both Helicobacter pylori (HP)-negative and HP-positive individuals. Critically, we applied the key methodological lessons of the enterotype debate: we used a variational autoencoder (VAE) for dimensionality reduction to learn a continuous latent representation without forcing discrete structure, determined the optimal number of clusters using the Silhouette index (an absolute validation measure) across K=2 to K=10 rather than arbitrarily selecting a cluster number, and performed transparent evaluation of multiple clustering solutions. This VAE-plus-silhouette workflow directly addresses the critiques leveled against the original enterotype analysis. Results: Four gastotypes were identified, with K=4 achieving the highest mean silhouette score, indicating good cluster cohesion and separation. Two gastotypes (Variovorax-type and Trabulsiella-type) were significantly enriched in HP-positive samples, while two gastotypes (Bacteroides-type and Streptococcus-type) were significantly enriched in HP-negative samples. Random Forest and Gradient Boosting achieved excellent baseline performance for predicting HP infection (AUC = 0.990 and 0.993). Conclusions: The VAE-plus-silhouette workflow provides a robust, data-driven approach for identifying gastotypes without forcing discrete structure or arbitrarily fixing cluster numbers. Using this framework, we identified four gastotypes with significantly different HP infection rates. Variovorax-type and Trabulsiella-type showed strong HP-positive enrichment, while Bacteroides-type and Streptococcus-type showed strong HP-negative enrichment. These findings demonstrate that methodological advances from the enterotype controversy can be successfully transferred to the stomach, offering a reproducible taxonomy for stratifying HP infection status with potential clinical utility.

Helminthiases remain a major global health burden, and limitations of current anthelmintic therapies highlight the need for new pharmacological targets. In this study, we examined the effects of ion-channel and cytoskeletal modulators on bovine lung protoscoleces (PSCs) of Echinococcus granulosus sensu lato. Compounds acting on ion channels (praziquantel, amiloride, and amlodipine) and cytoskeletal components (albendazole and cytochalasin D) were evaluated using a semi-automated motility assay, methylene blue exclusion to assess viability, and scanning electron microscopy (SEM) to characterize structural damage. All compounds produced concentration-dependent reductions in PSCs motility. Amlodipine was the most potent inhibitor of motility, whereas praziquantel and cytochalasin D produced pronounced tegumental alterations and strong correlations between motility impairment and parasite death. In contrast, amiloride markedly reduced motility with comparatively minor effects on viability, indicating a primarily paralytic effect. Cytoskeletal disruption induced severe structural damage and parallel declines in motility and viability. SEM analysis revealed extensive tegumental collapse, loss of glycocalyx, and microtrichial damage in PSCs exposed to cytoskeletal and calcium-modulating agents. These findings highlight cytoskeletal organization and calcium-dependent ion fluxes as key physiological vulnerabilities in E. granulosus. Comparative analysis of these pharmacological targets provides mechanistic insight into how disruptions in cytoskeletal dynamics and cation homeostasis compromise parasite motility and survival.

Leishmaniases remain a significant global public health threat, with Leishmania amazonensis and Leishmania infantum representing the etiological agents of the cutaneous and visceral forms in the Americas, respectively. Building on our previous identification of Phospholipase A1 (PLA1) in Leishmania braziliensis, this study provides a comprehensive molecular, immunological, and biochemical characterization of PLA1 in L. amazonensis and L. infantum promastigotes. We analyzed PLA1 activity and expression, purified the recombinant enzyme from L. amazonensis, and validated protein expression using a specific anti-PLA1 serum. The major contribution of this research is the first description of the subcellular localization of a PLA1 within the Leishmania genus. Moreover, our results reveal an unprecedented association between PLA1 and lipid droplets within the parasites. This discovery is of particular interest as it provides the first evidence linking this enzyme to lipid storage organelles in Leishmania. Given that PLA1 is an established virulence factor in other trypanosomatids, these findings suggest a specialized role for the enzyme in parasite lipid metabolism and potentially in its pathogenic mechanisms, opening new perspectives for understanding Leishmania biology.

The lack of a reliable chronic murine model limits drugs evaluation against Mycobacterium abscessus. Models show discrepancies, especially regarding host factors (mouse strain, sex and age). Using beads-model, we compared BALB/cJRJ and C57BL/6NCrl across sexes and ages. BALB/cJRJ showed more sustained infection and lower variability, with no significant sex- or age-related differences. Considering these results and the higher prevalence of NTM pulmonary infections in female patients, 5-6 weeks-old female BALB/cJRJ are appropriate for M. abscessus beads-model.

Mycetoma is a neglected tropical disease caused by various bacterial and fungal pathogens that has a significant health impact across a broad geographically defined "mycetoma belt" spanning South America, Africa and Asia. Histologically, mycetoma is characterised by invasive and destructive granuloma development in the skin, deep tissues and bone, leading to tissue destruction, deformities and high morbidity. The presence of macroscopic, highly compacted pathogen microcolonies, or "grains," is a key diagnostic feature, and the formation of grains supports pathogen persistence and disease chronicity. However, there is a paucity of information on immune responses in mycetoma patients and on the relative importance of phylogeny and/or grains in establishing the local immune landscape. Here, we used spatial proteomics to examine the distribution of 43 immune-related proteins in surgical biopsies from 11 patients with mycetoma of bacterial (Actinomycetoma; Actinomadura pelletierii and Streptomyces somaliensis; n=6) and fungal (Eumycetoma; Madurella mycetomatis; n=5) origin. Using mixed-effects modelling, an exploratory analysis across species and pathogen classes revealed few significant differences in immune marker expression. In contrast, and independently of pathogen class, the cellular infiltrate closest to grain boundaries had higher per-cell expression of CD66b+, ARG1, and VISTA. The preferential accumulation of CD66b+ARG1+VISTA+ cells at grain boundaries was confirmed by quantitative immunofluorescence analysis. Hence, the local tissue microenvironment surrounding the mycetoma grain represents a specialised immunosuppressive niche, with parallels to the tumour microenvironment.

Red Mark Syndrome (RMS) is a widespread skin disease affecting rainbow trout (Onchorhynchus mykiss). It provokes substantial economic losses in aquaculture, and is putatively caused by a Rickettsiales bacterium named Midichloria-like organism (RMS-MLO), which is strongly associated with RMS lesions. However, RMS-MLO ecology and epidemiology in aquaculture systems remain poorly understood. In this study, we analysed environmental DNA to monitor the presence of RMS-MLO and its putative vector Ichthyophthirius multifiliis in a trout farm in Northern Italy over one year. Water and sediment samples were monthly collected from multiple water tanks. RMS-MLO was consistently detected by PCR throughout the study in all trout-containing tanks, both in water and sediment samples, but never in the trout-free inflow tank. We did not observe an increase in RMS-MLO abundance during the single RMS outbreak recorded nor in relation with the co-occurrence of I. multifiliis. Our findings indicate a long-term persistence of RMS-MLO in the aquaculture, possibly as a consequence of infections with low prevalence or abundance, rather than its entry from the external environment at the time of RMS outbreaks. Additionally, hints were recorded for a potential role of free-living aquatic microeukaryotes as additional occasional reservoirs. In contrast, I. multifiliis was negatively related with RMS-MLO, while it significantly increased in abundance during the RMS outbreak, particularly in the inflow tank. This supports that, rather than a stable reservoir, I. multifiliis may act as a facilitator of RMS outbreaks, which might indeed be triggered by the entry of this parasite in trout farms.

Gastrointestinal disorder caused by the ingestion of (oo)cysts of Cryptosporidium and Giardia is one of the major health problems in developing countries. Fruits and vegetables that are usually consumed unpeeled, poorly washed and or cooked and are the major modes of transmission. Frequent large-scale screening of the food samples is necessary to prevent outbreaks but screening of vegetables for such microbes is limited in Nepal. In this study, we used a smartphone microscopy system to study prevalence and quantification of (oo)cysts of Cryptosporidium and Giardia in 651 vegetable samples collected from nine major vegetable collection sites across Nepal. The overall prevalence rate of vegetable samples was 37.5% with at least with one of the parasites. We found that 23.2% samples were contaminated with Giardia and 33.3% samples were contaminated with Cryptosporidium. Among eight vegetable types, the prevalence rate was lowest in carrot (20%) and highest in spinach (48%). The prevalence rate of vegetable samples at different sites ranged from 13% in Dhading to 61% in Dhangadi. The contamination rate was 28% for winter, 43% for summer and 33% for monsoon seasons in samples collected from Kathmandu. These vegetables should be considered as a potential source of parasitic contamination in people. These vegetables can cause infection if consumed poorly washed and or cooked, posing a potential source of parasitic contamination in people.