Microorganisms

BACKGROUNDEndophytic fungi are naturally inhabiting plant organs without causing disease symptoms. They can also contribute to their hosts pest and disease resistance by displaying entomopathogenic and/or antifungal traits. In this study, we evaluated the ability of 11 strains of avocado fungal endophytes to antagonize three important avocado plant pathogens: Colletotrichum gloeosporioides, Fusarium solani, and Phytophthora cinnamomi, and two insect pests: Sitophilus zeamais and Xyleborus bispinatus. RESULTSThe results show that Trichoderma spp. strains were the most effective against the evaluated plant pathogens in terms of growth inhibition, in direct contact assays or through metabolite production. Other fungi, such as Purpureocillium sp. and Pochonia sp., only exhibited pathogen inhibition through diffusible metabolites but displayed strong insecticidal capacity against the evaluated pests, hence being identified as promising multi-target biocontrol agents in the integrative analysis. CONCLUSIONOur findings evidence the potential of avocado fungal endophytes and their metabolites as multi-target biocontrol agents of crop pests and pathogens.

Toxoplasma gondii is a widespread human pathogen that has multiple, clinically relevant stages in its complex life cycle, including fast-replicating tachyzoites and latent bradyzoites. Bradyzoite differentiation is triggered by stress responses that lead to changes in transcription, translation, and metabolism. Two aspects of this process are addressed in this report: first, whether proteins that play roles in bradyzoite differentiation are specific to T. gondii and other bradyzoite-forming parasites of the Sarcocystidae family, and second, whether new bradyzoite differentiation proteins can be identified in T. gondii. To answer these questions, a phylogenetic approach was used, comparing proteomes of select members of the Sarcocystidae family that form morphologically different bradyzoite cysts and members of the Eimeriidae family that do not form cysts. This approach resulted in 8 distinct clusters of T. gondii proteins that reflected different conservation patterns; for example, one cluster showed conservation among all organisms, while another showed conservation in bradyzoite cyst-forming organisms. Known T. gondii proteins involved in bradyzoite differentiation were found in all clusters, indicating that this process uses both highly conserved pathways as well as bradyzoite-specific pathways. Importantly, the cluster containing proteins that are conserved in bradyzoite-forming organisms contained several known regulators of bradyzoites, and will be a source for identifying novel T. gondii proteins that are involved in bradyzoite differentiation.

Monascus spp. are economically important filamentous fungi that have been utilized in the production of beneficial metabolites such as Monascus pigments and monacolin K, as well as in the brewing of some Asian fermented foods. The delimitation of Monascus species has traditionally relied on phenotypic traits; however, this morphological classification approach is susceptible to subjective judgments and variations in cultural conditions and also may not necessarily be related to the actual genetic relationship. Consequently, synonymy and misidentification frequently occur in Monascus taxonomy, highlighting the urgent need for a convenient and reliable classification system for this genus. In this study, a phylogenetic analysis of 82 representative Monascus strains, encompassing all previously recognized species of the genus, was conducted based on the concordance of five gene genealogies (BenA, CaM, ITS, LSU, and RPB2) to clarify species delimitation and resolve phylogenetic relationships within Monascus. The results revealed that the genus Monascus is resolved into 11 species, which are clustered into two sections: Floridani (including M. argentinensis, M. flavipigmentosus, M. floridanus, M. lunisporas, M. mellicola, M. pallens, and M. recifensis) and Rubri (including M. pilosus, M. purpureus, M. ruber, and M. sanguineus). M. pilosus and M. sanguineus were reaffirmed as distinct species due to their well-supported and divergent phylogenetic lineages. Additionally, M. albidulus, M. anka, M. barkeri, and M. fumeus are synonymized with M. pilosus, while M. aurantiacus and M. rutilus are synonyms of M. purpureus. Finally, a comprehensive list of accepted Monascus species along with their corresponding barcode sequence data is provided.

This study investigates the seasonal dynamics of the microbiome within the marine sponge Halichondria panicea from Baltic coastal waters, focusing on its symbiotic relationship with Candidatus Halichondribacter symbioticus. Over 16 months, we observed distinct summer and winter microbial communities, transitioning rapidly between these states during spring and fall. Marine sponges host complex microbiomes composed of diverse microbial taxa that play critical roles in host metabolism and nutrient cycling within marine ecosystems. While our understanding of sponge microbiomes has traditionally been based on static characterizations, the temporal dynamics of these associations across seasonal cycles remain poorly understood. In this study, we investigated temporal variation in bacterial symbionts of Halichondria panicea over 16 months in Baltic coastal waters using high-throughput amplicon sequencing of bacterial 16S rRNA gene sequences. The microbiota of H. panicea exhibited host-specific structure and a high degree of stability across seasons, despite fluctuations in environmental factors such as temperature, salinity, photoperiod intensity, and inorganic nutrient availability. In contrast, bacterial communities in surrounding seawater displayed large seasonal shifts which potentially mix with the sponge bacterial community, suggesting that different degrees of ecological pressures act on free-living and symbiotic marine bacteria. These findings establish an empirical baseline for identifying abnormal shifts in symbiont communities, which could be indicative of environmental stress or biological disturbance events.

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.

A multiplex diagnostic test is evaluated for self-reported long COVID associated persistent symptoms and a poor recovery from a SARS-CoV-2 infection. A mass-standardised concentration of total antibodies (AC), high-quality (HQ) antibodies and percentage of HQ antibodies (HQ%) is assessed against a spectrum of spike proteins to the SARS-CoV-2 variants: Wuhan, , {delta}, and the Omicron variants BA.1, BA.2, BA.2.12.1, BA.2.75, BA.5, CH.1.1, BQ.1.1 and XBB.1.5 in three cohorts. A cohort of control patients (n = 46) recovered (CC) and a cohort of self-declared long COVID patients (n = 113) (LCC). A nested Receiver Operating Characteristic (ROC) analysis, performed for the variant with lowest HQ concentration in the spectrum, produced an area under the curve and AUC = 0.61 (0.53-0.70) for the CC vs LCC cohorts. For the LCC cohort, the cut-off thresholds for AC = 0.8 mg/L, HQ = 1.5 mg/L and HQ% of 34% were determined, leading to a 71% sensitivity and 66% specificity derived by the Youden metric. The cohorts may be fully classified based on ROC and outlier analysis to give an incidence of persistent virus 62% (95% CI 52% - 71%), hyperimmune 12% (95% CI 7% - 20%) and unclassified, 26% (95% CI 18% - 35%). The overall diagnostic accuracy for both the hyper and hypo immune is 69%. All clinical interventions can now be tailored for the heterogenous long COVID patient cohort.

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.

Urinary tract infections (UTIs) are among the most common infectious diseases worldwide, with Escherichia coli being the predominant uropathogen. The increasing prevalence of extended-spectrum beta-lactamase (ESBL)-producing strains and their association with fluoroquinolone resistance pose a significant challenge to empirical therapy, particularly in community settings. The aim of this study was to determine the epidemiology and predictive factors associated with ESBL-producing E. coli and its concomitant fluoroquinolone resistance in community-acquired clinical isolates. A retrospective cross-sectional study was conducted analyzing 244 clinical E. coli isolates. Demographic and microbiological data were collected, including age, sex, sample type, and antibiotic susceptibility. Associations between variables and ESBL production were assessed using Pearsons chi-squared test, and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Of the isolates, 165 (68%) were ESBL-producing. A significant association was observed between age group and ESBL production (p < 0.001), with the highest frequency in the 20-39 age group. Most ESBL-positive isolates were obtained from women (73%), although odds ratio (OR) analysis suggested a non-significant trend toward a higher probability in men (OR = 1.29; 95% CI: 0.72-2.31). High rates of fluoroquinolone resistance were identified among the ESBL-producing isolates, with 30% resistance to levofloxacin and 35% to ciprofloxacin (p < 0.001). Urine samples showed the highest concentration of ESBL-positive isolates, with a significant association between sample type and resistance (p < 0.001). The high prevalence of ESBL-producing E. coli and its concomitant resistance to fluoroquinolones highlight a critical challenge for the empirical treatment of urinary tract infections in Mexico, underscoring the need to strengthen antimicrobial use management and local surveillance strategies.

Objectives: To identifiy risk factors for antimicrobial resistance (AMR) in seven pathogen-antimicrobial combinations in patients with cancer and cancer survivors. Methods: Using data from patients with recent or past cancer diagnostic codes in Oxfordshire, UK, we examined associations between 22 potential risk-factors and AMR in blood culture isolates, collected between 1-April-2015 and 31-March-2025. Results: Among 5,975 bacteraemias in 4,365 adults, we analysed 3,141 (52.6%) due to Enterobacterales and 620 (10.4%) due to Enterococcus faecalis/faecium in 2,752 patients. Fourteen risk-factors for antimicrobial-resistant bacteraemia were identified, varying across pathogen-antimicrobial combinations. Compared with no previous antimicrobial susceptibility test result, prior resistance to the same antibiotic in any culture in the last year was strongly associated with AMR across all pathogen-antimicrobial combinations (all p<=0.001). Prior antibiotic exposure and younger age were also positively associated with AMR in four and five combinations, respectively. Cancer type showed modest effects; lymphoid/haematopoietic malignancies were associated with higher odds (vs colorectal cancer) of trimethoprim-sulfamethoxazole-resistant Enterobacterales (aOR=2.07 95%CI 1.40-3.06) and vancomycin-resistant Enterococcus bacteraemia (aOR=6.68, 1.21-36.91). Conclusions: Previous resistance was the greatest risk factor for bacteraemia with AMR in cancer patients and survivors, with prior antibiotic exposure and age also contributing. Lymphoid/haematopoietic malignancies increased risk of resistance to specific antimicrobials. Keywords: antimicrobial resistance, bacteraemia, cancer, risk factors

Russian wheat aphid (Diuraphis noxia Homoptera; Aphididae) is a major pest that significantly reduces chlorophyll content and photosynthetic capacity in wheat (Triticum aestivum L.), leading to substantial crop yield losses. Jasmonic acid (JA) is a plant signaling molecule known to activate defense mechanisms against herbivorous insects. This study examined the effectiveness of exogenous jasmonic acid application in maintaining chlorophyll content during Russian wheat aphid infestation. A pot experiment was conducted with four treatments: control (no treatment), aphid infestation only, jasmonic acid application only, and jasmonic acid with aphid infestation. Results demonstrated that aphid infestation significantly reduced chlorophyll a (F = 42.565, P = 0.0001), chlorophyll b (F = 52.565, P = 0.0001), and total chlorophyll (F = 32.565, P = 0.0002) contents compared to healthy plants. Jasmonic acid treatment at 2 mM concentration effectively preserved all forms of chlorophyll, significantly counteracting aphid-induced chlorophyll depletion (P < 0.01). The protective effect of jasmonic acid was evident through the statistically significant interaction between aphid stress and JA application for all chlorophyll parameters. These findings suggest that foliar application of jasmonic acid can serve as an effective strategy to maintain photosynthetic capacity and plant vigor under Russian wheat aphid attack, thereby contributing to sustainable crop management and improved wheat production.

Grapevine (Vitis vinifera L.) is highly prone to viral infections that pose a significant threat to global viticulture sustainability. Traditional detection methods, such as PCR and ELISA, are limited to well-known pathogens, highlighting the need for more comprehensive and unbiased approaches. Here, we present the development of a cost-effective viral enrichment system adapted to next-generation sequencing (NGS) for the detection and characterization of grapevine viruses. Our strategy leverages hybridization-based capture using biotin-labeled cDNA probes hereafter named "Chloro-Zero") designed to selectively deplete highly abundant host transcripts particularly plastid and ribosomal RNAs while preserving viral RNA. Probe design was informed by transcriptomic analysis of V. vinifera. We evaluated different subtractor-to-target RNA ratios, observing a consistent reduction of host RNA and a moderate enrichment of viral sequences. NGS analysis revealed improved recovery of low-abundance viral transcripts, with coverage levels comparable, to a certain extent, to those obtained using previously available commercial kits, but at a significantly lower cost. Although variability in depletion efficiency was observed, the results demonstrate the potential of this scalable and locally adaptable protocol for virome profiling in grapevines. By addressing key limitations of current depletion methods, our approach facilitates the detection of emerging viral threats and supports the development of more effective certification programs and sustainable management practices. Ongoing improvements in probe design and bioinformatic workflows are expected to enhance performance, providing a robust platform for broader applications in plant virology.

Phage therapeutics are re-emerging as adjuncts or alternatives to antibiotics and their clinical translation will be enhanced with production methods that minimise downstream processing. We evaluated whether an endotoxin-reduced E. coli strain developed for production of recombinant proteins, ClearColi(R), can serve as a useful, safe phage production host without compromising yield and whether targeted receptor complementation can expand its utility. The parent strain BL21(DE3), and its lipid A modified derivative, ClearColi(R), were compared with respect to infection and generation of phage. Across a panel of 31 phage, a similar host range was observed between BL21(DE3) and ClearColi(R). To expand host range ompC was genetically engineered into the chromosome of ClearColi(R), thereby adding OmpC-dependent phage to its production capacity. Production metrics were broadly comparable between the hosts; efficiency of plating and final titres for representative phage were not significantly different; burst size varied by phage but without consistent host bias. Endotoxin activity in ClearColi(R)-propagated lysates was reduced by over 1000-fold relative to BL21(DE3), reaching the low hundreds of endotoxin units (EU) versus hundreds of thousands for BL21(DE3). Intravesical administration of ClearColi(R)-derived phage (LUC4) into pigs elicited no clinical abnormalities and no significant increases in circulating cytokines up to 48 hours after administration. ClearColi(R) allows efficient production of diverse phage with low endotoxin, reducing the requirement for downstream processing. Although its minimal LPS reduces its capacity for producing some LPS-dependent phage and its growth is slower than BL21(DE3), requiring optimisation for maximal phage titre, the safety and simplified manufacturing process support further development of endotoxin modified strains for phage production. Impact statementAntibiotic resistance is a current global problem and treatments based on phage and phage products already have a proven track record with particular bacterial infections, especially in the urinary tract. While progress is being made on in vitro phage synthesis, large scale bacteriophage preparations require a bacterial host for production, consequently toxic components in the initial lysate need to be removed or significantly diluted for safe clinical use. This is a study of the potential to utilise an endotoxin-reduced E. coli strain, ClearColi(R), to produce safer phage therapeutics. Such endotoxin modified strains should minimise the processing steps required and reduce overall production costs of a phage preparation. The research demonstrates that the endotoxin-reduced strain was able to produce a wide range of phage and for studied examples at phage titres equivalent to the more toxic parent strain. We also show that the strain can be modified to increase its host range and confirm the very low endotoxicity of basic phage lysates produced by the strain. Replicating this process to engineer additional low-toxicity bacterial production strains will accelerate the development of safer, more cost-effective phage therapeutics.

O_LIWD40 is a highly conserved protein domain in eukaryotes, playing a critical role in various cellular process. C_LIO_LIWe conducted genome-wide functional analysis of WD40 genes in Fusarium graminearum--a phytopathogenic fungus that causes severe yield loss and mycotoxin contamination in major cereal crops. C_LIO_LIComprehensive phenome analysis of 119 WD40 gene deletion mutants across 22 distinct phenotypic traits revealed phenotypic divergence within the phenome, establishing a strong correlation between virulence and sexual reproduction. Notably, 21 "core WD40 genes" were identified, offering valuable insights into divergent biological processes. C_LIO_LIPilot interactome studies of Fgwd101 and Fgwd133 provided further insights into their potential pathobiological functions. Our investigation contributes to broadening our knowledge of the biological mechanisms underlying fungal pathogenesis and may assist in the identification of targets for antifungal agents. C_LI

Emerging infectious diseases and antimicrobial resistance (AMR) have surfaced as two major public health threats over the past two decades. Consequently, integrative surveillance systems capable of detecting both emerging pathogens and resistance-carrying bacteria are crucial. With advances in next-generation sequencing, simultaneous detection of pathogens and AMR is increasingly feasible. In this study, we used short-read metatranscriptomics complemented by total 16S rRNA metagenomic long-read sequencing to analyze paired oral and plasma samples from a cohort of febrile individuals at two locations in Senegal. Oral microbiomes differed in community composition between locations, and reduced diversity and richness were significantly associated with high fever. We identified at least one known pathogen in 15.33 % (23/150) of samples, with Borrelia crocidurae as the most frequently detected pathogen. We detected both pathogenic and non-pathogenic viruses in oral (10/72) and plasma (09/78) samples. Finally, we observed a high frequency of genes associated with resistance and virulence: 10% of samples expressed at least one AMR gene (ARG), and 24% expressed virulence factor genes. Resistance to widely used beta-lactam antibiotics was the most prevalent. Our findings provide critical data on oral and plasma microbiomes in the context of acute febrile illness in Senegal while expanding understanding of circulating ARGs.

Background: Since September 2024, France has implemented a national reform allowing prescription-free access (PFA) to sexually transmitted infection (STI) screening in medical biological laboratories (MBLs). This study aims to characterize the populations undergoing STI testing according to their access modality and evaluate the probability of test positivity in relation to testing pathway, sex, and age groups. Methods: We conducted a cross-sectional analysis of all individuals screened for Chlamydia trachomatis, Gonorrhoea, human immunodeficiency virus (HIV), hepatitis B virus (HBV), and syphilis by treponemal-specific immunoassay (TSI) in Cerballiance MBLs between Mars 2025 and February 2026. Multivariable logistic regression models stratified by sex and adjusted for age and region assessed associations between screening modality and STI positivity. Results: Among 1,008,737 individuals included, 27.8% were under PFA and 72.2 under prescription-based access (PBA). PFA users were more frequently male (47.4% vs. 36.3%, p<0.001) and aged 20-39 years (34.0%, p<0.001). Overall positivity rates differed by modality: PFA was associated with higher detection of Chlamydia (4.6% vs. 3.6%). PBA group showed more positive cases of syphilis (3.4% vs. 1.2%), HBV (1.3% vs. 0.4%), and HIV infections (0.3% vs. 0.2%, all p<0.001). Co-infection and gonorrhoea proportions did not significantly differ between modalities. Conclusions: PFA substantially increased STI screening uptake, particularly among young adults and men, and enhanced detection of bacterial STIs. PBA remains essential for diagnosing viral and chronic infections. These findings highlight the complementary roles of both access strategies and support PFA screening as an effective public health intervention to broaden STI detection and reduce transmission.

Endophytic fungi residing within medicinal plants are emerging as prolific sources of structurally diverse bioactive secondary metabolites with applications in drug discovery. Azadirachta indica (Neem) and Melia azedarach (Melia), members of the Meliaceae family, are renowned for their rich phytochemical composition; however, the contribution of their endophytic fungi communities to this chemical diversity remains largely unexplored. Herein, endophytic fungi were isolated from leaves and bark of Neem and Melia collected in Kenya and cultured under distinct physical conditions, solid (plates) and liquid (broth) media to assess how culture environment influences compound production. Compounds were extracted and analyzed using gas chromatography-mass spectrometry (GCMS) to profile the chemical diversity associated with each endophytic fungi, physical culturing state and host plant. GCMS analysis revealed that while the host plant identity influences the presence of specific compounds, the dominant determinant of chemical diversity was intrinsic biosynthetic capacity of the endophytic fungi themselves. Several compounds were unique to endophytic fungi cultures, highlighting their role as independent sources of bioactive compounds. Culture conditions moderately influence metabolite profiles, demonstrating the importance of optimizing growth environments in experimental design and natural product bioprospecting. From the Neem samples, we found 53 compounds uniquely present in the broth samples (consisting of Neem powder and endophytic fungi), 22 found exclusively with the endophytic fungi from the Neem, and 31 compounds shared between the broth and the endophytic fungi samples. In Melia samples, 109 compounds were uniquely present in broth samples from Melia plant (consisting of Melia powder and endophytic fungi), 22 compounds were found exclusively with the endophytic fungi from the Melia, and 55 were shared between the broth and the endophytic fungi samples. Our comparative analysis assessed the Neem and Melia endophytic fungi exclusive samples and reported 12 shared compounds. 10 compounds were unique to Neem and 10 unique to Melia; however, their identities varied between the two categories. While GCMS enabled the identification of volatile and semi-volatile metabolites, future studies employing complementary metabolomic approaches, such as liquid chromatography-mass spectrometry (LCMS), ultra-high-performance liquid chromatography MS/MS (UHPLC MS/MS), or nuclear magnetic resonance (NMR) spectroscopy, would expand coverage to non-volatile, polar, and high molecular weight compounds, providing a more comprehensive understanding of endophyte-derived chemical diversity. These findings provide insights into the interplay between medicinal plants and their endophytes and establish a foundation for leveraging endophytic fungi from Neem and Melia as scalable sources of structurally complex natural products for pharmaceutical and biotechnological applications while minimizing ecological impact.

Recent advances in high-throughput sequencing and novel culture techniques have revolutionized our understanding of the human microbiota. However, most studies primarily focused on bacterial communities, often overlooking the fungal component. Building upon our previous metagenomic analysis of the Inuit oropharyngeal microbiome 1, this study used culturomics to provide a more comprehensive view of both bacterial and fungal communities. We analyzed oropharyngeal swabs from the Qanuilirpitaa? 2017 Inuit Health Survey 2, demonstrating the complementarity of metagenomic and culturomic approaches. Our findings highlight the importance of culturomics in revealing low-abundance microorganisms, particularly fungi, which are often underrepresented in metagenomics data. Moreover, we designed an approach to isolate previously uncultivated species. We described two Pauljensenia sp., and provided insights into the phylogenetic relationship between Schaalia and Pauljensenia genera. This study underscores the necessity of a holistic approach to microbiome research, combining multiple techniques to fully elucidate microbial diversity in unique populations like the Inuit.

Background and AimsThe rising incidence of Crohns disease (CD) in Westernized countries has been linked to changes in diet and increased consumption of food additives, yet the mechanisms by which these factors fuel intestinal inflammation remain unclear. Adherent-invasive Escherichia coli (AIEC), a pathobiont involved in CD pathogenesis, lacks a clear genetic hallmark but exhibits intestinal colonization and virulence traits, raising questions about the evolutionary forces promoting its emergence among select individuals. Here, we investigated how chronic exposure to two common dietary emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (P80), along with host inflammation, drives AIEC genomic evolution and pathogenic potential. MethodsWild-type and Il10-deficient mice were monocolonized with AIEC and chronically exposed to CMC, P80, or water. Bacterial isolates were collected and analyzed for genomic diversification, mutations, and phenotype both in vitro and in vivo. ResultsEmulsifiers accelerated AIEC genomic diversification and selected for mutations linked to increased motility, invasion, and pro-inflammatory activity. Moreover, dietary emulsifier-evolved strains displayed a marked fitness advantage in vivo, outcompeting their counterparts in murine hosts, with the greatest advantage observed when evolution occurred under inflammatory conditions. Notably, evolutionary pathways and phenotypic outcomes were shaped by both emulsifier and the hosts inflammatory status, highlighting synergy between diet and host genetics in fostering pro-inflammatory pathobionts. ConclusionThese findings provide an evolutionary framework connecting modern dietary habits to the emergence of pathogenic AIEC strains, and underscore the importance of dietary interventions in individuals at risk for inflammatory bowel disease.

Sequencing the respiratory tract transcriptome has the potential to provide insights into infectious pathogens and the hosts immune response. While DNA-based sequencing is more standard in clinical laboratories due to its stability, RNA assays offer unique advantages. RNA reflects dynamic physiological changes, and for RNA viruses, viral RNA particles directly represent copies of the viral genome, enabling greater diagnostic sensitivity. However, RNAs susceptibility to degradation remains a significant challenge, particularly in RNase-rich specimens like saliva. To address this, we conducted a systematic, combinatorial evaluation of 24 distinct mNGS workflows, crossing eight nucleic acid extraction methods with three RNA-Seq library preparation protocols. Remnant saliva samples (n = 6) were pooled and spiked with MS2 phage as a control. The SARS-CoV-2 virus was spiked into half of the samples, which were extracted using the eight different extraction methods (n = 3) and compared using RNA Integrity Number equivalent (RINe) scores and RNA concentration. The extracted RNA was then processed across the three library construction methods and subjected to short-read sequencing to assess all 24 combinations head-to-head. We compared methods based on viral read recovery and found that RINe and concentration did not correlate with viral detection. The Zymo Quick-RNA Magbead kit and the Tecan Revelo RNA-Seq High-Sensitivity RNA library kit were the extraction and library-preparation kits that yielded the most SARS-CoV-2 reads, respectively. Importantly, our combinatorial analysis revealed that any small variability attributable to different nucleic acid extraction methods was heavily overshadowed by differences in quality attributable to the RNA-Seq library preparation methods. These findings challenge the reliance on conventional RNA quality metrics for clinical metagenomics and underscore the need to redefine extraction quality standards for mNGS applications. IMPORTANCEmNGS is a powerful and unbiased approach towards pathogen detection that has mostly been applied to blood and cerebrospinal fluid samples. However mNGS has recently been applied to more areas including the respiratory pathogen detection space, with potential applications in both in-patient diagnostics and public health surveillance. Saliva samples are an ideal sample type for these use cases since they can be collected non-invasively. However, saliva is also a challenging sample type due to its high RNase activity and often yields low-quality nucleic acid. This study explores the feasibility of using saliva specimens in mNGS with contrived SARS-CoV-2 samples to optimize the combination of two factors: nucleic acid extraction and RNA-seq library preparation. Exploration in this area could enhance the sensitivity of saliva-based mNGS assays, with the goal of future expansion of this specimen type in clinical diagnostics and public health surveillance. Key PointsO_LIThe choice of RNA-Seq library preparation kit has a greater impact on pathogen detection than the nucleic acid extraction method. C_LIO_LIThe combination of Zymo Quick-RNA Magbead extraction kit and TECAN Revelo RNA-Seq High Sensitivity RNA library kit recovered the highest percentage of total SARS-CoV-2 reads. C_LIO_LIRNA quantity and RINe score do not correlate with viral read capture, indicating a need for an alternative metric to assess RNA quality for downstream mNGS clinical diagnostics. C_LI

Despite the evolution of epidemiological analysis and modeling tools, difficulties still remain, especially in developing countries, regarding the availability and use of these tools. Often expensive, requiring high technical expertise, demanding constant connectivity of several or sometimes even significant resources, these tools, although efficient, present a major gap with the operational realities of health districts. It is in this context that we introduce Episia, an open-source Python library designed and conceived to provide a framework to facilitate epidemiological analysis and modeling. It integrates a suite of compartmental epidemic models (SIR, SEIR, SEIRD) with a sensitivity analysis using the Monte Carlo method, a complete biostatistics suite validated against the OpenEpi reference standard, as well as a native DHIS2 client for automated data ingestion. Developed in Burkina Faso, it is optimized and aims not only to address these health challenges encountered in Africa but also remains a versatile tool for global health informatics.