Microbiology Spectrum

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder characterized by recurrent wounds and frequent bacterial colonization. Clinically applicable methods that provide information on bacterial composition, viable burden, and spatial distribution are needed to support wound assessment and therapeutic evaluation. In this exploratory four-week longitudinal study, standard-of-care-treated wounds from five patients with RDEB were examined using autofluorescence imaging, quantitative and chromogenic culture, MALDI-TOF MS, 16S rRNA and staphylococcal tuf2 amplicon sequencing, and spatial Bactogram analysis. Sequencing showed that staphylococci dominated 22/23 communities, with Staphylococcus aureus being the most abundant staphylococcal species (21/23). Culture likewise identified S. aureus as the most frequent species in all 14 swab samples. Aerobic bacterial burden showed no association with open wound area and remained high in several contracting wounds. The Bactogram provided a spatial representation of viable bacterial growth and, when combined with chromogenic agar, reflected the predominance of S. aureus identified by sequencing, MALDI-TOF MS, and quantitative culture. Together, these complementary methods captured the dynamic nature of RDEB wounds through heterogeneous healing trajectories and longitudinal variation in bacterial burden, while consistently identifying a low-diversity, S. aureus-dominated microbiota. Bactogram spatial imprinting and autofluorescence imaging warrant further evaluation as minimally invasive tools for longitudinal EB assessment.

Background: Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridioides difficile infections, yet the diversity of FMT formulations and delivery routes hampers comparisons across studies. Oral frozen capsules are widely used and current guidelines recommend storage at -80C for up to two years. Despite extensive use of FMT, data on the long-term persistence and maintenance of their microbial composition remains limited. Method: In this prospective study, we assessed the temporal stability of bacterial profiles in frozen FMT capsules derived from 48 donations of 10 healthy donors. Using metabarcoding, we longitudinally profiled one capsule per donation thawed within a month of production and after 3, 6, 12, and 24 months of storage. We used linear mixed effect models to evaluate changes in alpha diversity and community composition over time. Results: Species richness remained stable across all timepoints, whilst species evenness decreased slightly. Although changes in community composition were detectable, they were small and mostly affected low-abundance genera. Clinical efficacy, assessed in a subset of recipients, was not associated with storage duration. Conclusion: Our findings demonstrate that frozen FMT capsules preserve their bacterial community structure for at least two years of storage at -80C, supporting their suitability for long-term biobanking and standardised clinical or research use.

The recent invasion of the two-spot cotton leaf hopper (TSCL) (Amrasca biguttula) into the U.S. now threatens row and vegetable crops as well as ornamentals. TSCL ecology and biology, including microbial diversity, are understudied. Insect-specific microbes could influence host biology and ecology including insect-plant interactions. Here, microbiota (bacteria and viruses) within TSCL along with its feeding behavior were characterized using high throughput sequencing, microscopy, and Electrical penetration graph (EPG). Results revealed low diversity of bacteria dominated by Wolbachia and confirm the absence of obligate (primary) symbionts. Two distinct and differentially abundant Wolbachia strains (B-supergroup) were found co-infecting midgut tissues of 60% of TSCL tested, and the remaining 40% were free of symbionts. Virome analysis uncovered a diverse complex of at least eight dsDNA genomes (12-17 kb) of Adintoviruses (Eupolintoviridae), but no RNA viruses commonly found in other leafhoppers were identified in TSCL. The mesophyll feeding nature of TSCL was identified by observing chlorophyll derived autofluorescence in midguts and higher abundance of chloroplast transcripts (photosystem I, II, Rubisco, cytochrome b6 and ATP synthase complex) in TSCL (whole insects) compared with thrips and aphids. EPG recordings show a cell-rupture style of feeding with differences between adults and nymphs. Lastly, consistent with its cell-rupturing feeding style, no evidence for the presence of plant-pathogenic bacteria or viruses that could induce hopperburn was found. These results overall provide insights into the microbial diversity and feeding pattern of TSCL and lay a foundation for future research. Key messageThis work characterizes the TSCL microbiome and its mesophyll feeding pattern. Symbionts within insects often complement the nutritional deficiencies of its host and can aid insect spread by boosting immunity. The findings of this study lay the foundation for future research on the biology, ecology, and management of this invasive pest.

Necrotizing soft tissue infections (NSTIs) carry 10 to 30% mortality. Current empirical antimicrobial guidance derives almost entirely from Western cohorts dominated by Streptococcus pyogenes and aerobic-anaerobic consortia, yet whether this microbial paradigm applies to tropical, high-antimicrobial-pressure settings has not been tested with culture-independent methods. We did a prospective cohort study of 169 patients with intraoperatively confirmed NSTI at a North Indian tertiary center (2021 to 2024). Wound tissue underwent aerobic and anaerobic culture, QIIME2-based 16S rRNA gene amplicon sequencing (V3-V4), and targeted SYBR Green quantitative PCR (qPCR) for Acinetobacter baumannii and S. pyogenes. The wound microbiota was overwhelmingly Gram-negative and polymicrobial, anchored by A. baumannii (culture, 33.7%; metagenomics, 49.1%; qPCR, 37.9%), Escherichia coli (32.0%), and Klebsiella pneumoniae (20.7%); S. pyogenes contributed only 4.7% of culture-positive cases. Polymicrobial wounds had higher Shannon diversity (2.59 versus 2.33; P = 0.048) and discrete community composition (PERMANOVA R2 = 0.511; P = 0.010). Culture-metagenomics agreement ranged from almost perfect for Escherichia ({kappa} = 0.849) to slight for Streptococcus ({kappa} = 0.131). North Indian NSTIs present a microbial picture distinct from the Western paradigm, with implications for empirical therapy.

Identification of acyclovir-resistant herpes simplex virus (HSV) infections is critical for directing appropriate antiviral therapy, particularly for immunocompromised patients where resistance rates can reach 30%. In 2020, the University of Washington Clinical Virology Laboratory launched the first clinical genotypic HSV drug resistance test in the United States. While genotypic testing offers significantly faster turnaround times than traditional phenotypic assays, interpretation depends on established mutational databases and remains challenging when novel variants are identified. Here, we retrospectively reviewed all HSV acyclovir resistance Sanger sequencing tests performed from January 2020 to November 2025 at this primary national reference laboratory. Mutations identified via clinical sequencing were compared against published databases of HSV UL23 mutations to determine their phenotypic effects. Over the nearly six-year study period, 136 samples were sequenced with a median turnaround time of 10.6 days. Among these, 65 samples (47.8%) harbored acyclovir resistance mutations, including 45 frameshift mutations. Notably, across the 100 samples (73.5%) displaying mutations not known to cause acyclovir resistance at the time of clinical testing, we identified 56 distinct mutations, including 23 without prior characterization. Our national experience demonstrates that genotypic testing accelerates actionable results in clinical practice and confirms that frameshift mutations remain a primary driver of acyclovir resistance. Furthermore, by uncovering these 23 novel variants, this work provides critical targets for future biochemical and phenotypic characterization of HSV UL23 mutations.

Pseudomonas aeruginosa is a major opportunistic pathogen, responsible for healthcare-associated urinary tract infections. Its metabolic flexibility and genomic plasticity promote its survival and adaptation in complex environments. Here, we conducted an in-depth analysis of three pairs of sequential P. aeruginosa urinary isolates, named "early" and "late", from three patients to investigate metabolic and phenotypic changes during urinary tract adaptation. An integrated multi-omics approach combining RNA sequencing and metabolomics was performed on isolates grown in human urine (HU) and trypticase soy (TS) medium, and compared with previously published proteomics data. Late isolates showed down-regulation of genes encoding type VI secretion system in both media, while oxidative phosphorylation associated-genes were up-regulated in HU. These late isolates also displayed significant down-regulation of amino-acid metabolism suggesting an increased use of carbon sources available in urine. As previously observed in proteomics, siderophore- and iron-related genes were significantly down-regulated for all late isolates in HU but not in TS, supporting convergent adaptation to the low-iron urinary environment. Metabolomic profiles of HU supernatants from late isolates clustered together, showing common metabolite production in HU. The differential metabolic profile between early and late isolates included phosphatidylcholines, acylcarnitines, biogenic amines and amino-acids, highlighting their importance in urinary adaptation. While long-term survival in HU and TS was similar between isolates, biofilm formation was reduced or lost in late isolates in line with the down-regulation of biofilm-related genes. These findings highlight the transcriptomic and metabolic reprogramming as well as the phenotypic changes occurring during P. aeruginosa adaptation to the urinary tract. ImportancePseudomonas aeruginosa is a major opportunistic bacterial pathogen responsible for healthcare difficult-to-treat urinary tract infections. Understanding how this bacterium adapts and persists in the human urinary tract is essential for improving the management of persistent infections. Through an integrated analysis of six sequential clinical isolates from three patients, this study shows that P. aeruginosa undergoes coordinated metabolic and phenotypic changes that promote survival in the urinary environment. During adaptation, the bacterium reduces traits involved in bacterial competition and biofilm formation while reprogramming its metabolism to the nutrients available in urine. These changes emerged independently in different patients, revealing convergent adaptation to this challenging environment. By identifying biological processes consistently associated with urinary tract persistence, this work provides new insight into the mechanisms that support long-term colonization and highlights adaptive metabolic pathways that may represent future targets for therapeutic intervention.

The respiratory microbiome plays important roles in poultry health, immune regulation, and pathogen resistance, yet its development throughout the broiler production cycle remains poorly understood. This study investigated temporal changes in the tracheal microbiome of broiler chickens across production phases. Tracheal samples were collected during the starter (day 12), grower (day 21), early finisher (day 26), and late finisher (day 35) phases and analyzed using 16S ribosomal RNA gene sequencing. Tracheal microbial richness, diversity, community structure, and taxonomic composition changed significantly across broiler production stages, including starter, grower, early finisher, and late finisher feeding phases. Alpha diversity increased progressively throughout production, with significant increases in richness, diversity, and phylogenetic diversity during later stages. Beta diversity analysis revealed distinct microbial communities associated with each production phase, with starter-phase samples clearly separated from later phases. Taxonomic profiling showed dominance of Proteobacteria during the starter and grower phases, with enrichment of Methylobacterium-Methylorubrum and Pseudomonas during the starter phase and of Escherichia-Shigella during the grower phase. In contrast, the finisher phases exhibited reduced Proteobacteria abundance and increased Firmicutes and Actinobacteriota, including Lactobacillus, Ligilactobacillus, Faecalibacterium, Streptococcus, Staphylococcus, Romboutsia, and Corynebacterium. Overall, the tracheal microbiome underwent progressive maturation, shifting from a Proteobacteria-dominated community to a more diverse, complex, Firmicutes-rich ecosystem. These findings provide new insights into the development of the respiratory microbiome in broiler chickens and may support strategies to improve poultry respiratory health. Because dietary transitions occurred concurrently with age progression, the observed microbiome shifts should be interpreted as production-stage-associated changes rather than diet-specific effects.

The innate immune system controls bacterial growth and modulates inflammation during wound healing. TCP-25 is a synthetic thrombin-derived host-defense peptide that combines direct antibacterial activity with neutralization of microbial products and modulation of CD14-dependent inflammatory signaling. We investigated whether this dual mechanism translates to human wounds using longitudinal samples from 24 healthy volunteers enrolled in a randomized, double-blind, within-participant, placebo-controlled phase I dose-escalation study of topical TCP-25 gel in matched epidermal suction blister wounds. We assessed inflammatory cytokines, neutrophil-derived proteins, wound exudation, cultivable bacterial burden, spatial bacterial distribution, and microbiome composition. TCP-25 reduced multiple cytokines, myeloperoxidase, and heparin-binding protein, with the strongest effects observed during the peak inflammatory phase. These changes were accompanied by reduced wound exudation and significant reductions in cultivable bacterial burden. Despite this antibacterial effect, microbiome composition and diversity remained largely unchanged, and participant-specific microbial profiles were preserved. TCP-25 therefore coordinated bacterial control, modulation of the physiological inflammatory response, and reduced wound leakage without major disruption of the resident microbiota composition. These findings provide clinical support for translating nature's endogenous host-defense principles into new therapies for complex wounds.

L-Serine is an important metabolic and immunomodulatory biomolecule with promising role in managing infections, and autoimmune diseases. L-Serine provides the energy requirements and triggers the toll-like receptor signalling collaterally. However, the role of L-Serine in host antimicrobial response against Mycobacterium tuberculosis (Mtb) remains unexplored. In this study, we investigated whether this metabolite could modulate the antibiotics efficacy against Mtb. Although L-Serine exhibits limited intrinsic anti-mycobacterial activity, but L-Serine demonstrates a synergistic effect when combined with rifampicin and moxifloxacin against both drug-sensitive and multidrug-resistant Mtb. Moreover, L-Serine particularly in combination with palmitic acid showed the enhanced intracellular bacterial clearance in a dose- and time-dependent manner in murine and human macrophages. This synergistic effect was accompanied by increased nitric oxide production and modulation of the host immune response. We identified elevated levels of pro-inflammatory cytokines and reduced IL-10 expression. Furthermore, the metabolic supplementation demonstrated enhanced antimicrobial activity in isolated primary CD14+ monocytes from TB patients. Similarly, the metabolic supplementation of L-Serine in combination with isoniazid and rifampicin significantly reduced bacterial burdens in the lungs and spleen, while improving tissue architecture in murine infection model. Our observations suggest that L-Serine contributes to the observed therapeutic effects. Collectively, this study concludes that L-Serine acts as a promising host-directed therapeutic adjunct, which enhances antimicrobial immunity and potentiating antibiotic efficacy, providing a potential strategy for improving tuberculosis treatment outcomes.

BackgroundMetagenomic next-generation sequencing (mNGS) has the potential to transform clinical diagnostics for bloodstream infections (BSIs). However, its clinical utility is currently limited by several challenges, including the extraction of DNA from blood cultures. Our aim was to develop, evaluate, and optimize an in-house method for host depletion and bacterial DNA extraction from positive blood cultures to enable rapid mNGS-based pathogen detection and antimicrobial resistance profiling, informing clinical management of BSIs. Methods151 clinical blood cultures (115 positive and 36 negative) were processed for DNA extraction using an in-house-developed SEPSINN method for host depletion and bacterial DNA extraction. mNGS on the MinION was performed, and the results for pathogen identification and antimicrobial susceptibility predictions were compared with the routine clinical workflow. SEPSINN was also evaluated against a commercial DNA extraction method to assess its effectiveness in depleting host DNA and recovering bacterial DNA. ResultsThe SEPSINN method achieved up to 1000-fold depletion of host DNA and outperformed the commercial DNA extraction method. At the sample level, mNGS achieved 100% accuracy, specificity, and sensitivity, identifying at least one pathogen in all 115 positive blood cultures. At the pathogen level, mNGS showed 98% accuracy (120/123), specificity, and sensitivity. For antimicrobial susceptibility predictions, mNGS achieved an accuracy of 95% (1382/1451), a sensitivity of 88% (203/230), and a specificity of 97% (1179/1221). Moreover, the method also identified fungi, indicating a wider taxonomic range. mNGS resulted in an approximately 4-hour turnaround time for pathogen identification and resistance profiling. ConclusionsThe method can provide information on BSI clinical management within approximately 24 hours of receiving the sample, including the time required for culture positivity. This represents an important advancement in the clinical management of BSIs, with the potential to save lives and promote antibiotic stewardship.

Alongshan virus (ALSV) is an emerging tick-borne segmented RNA virus belonging to the Jingmenvirus group and has been reported in humans, ticks, and vertebrates across Asia and Europe. Despite its potential public health relevance, its distribution and genetic diversity remain poorly characterized in several European regions where tick-borne pathogens are endemic. In this study, we developed a specific TaqMan-based real-time RT-PCR assay targeting a conserved region of ALSV segment 2 and used it to investigate the presence of ALSV RNA in Ixodes ricinus ticks collected in northeastern Italy. The assay showed high linearity over a broad dynamic range and no cross-reactivity with related flaviviruses. A total of 212 archival tick samples collected between March 2021 and November 2022 were screened, and 28 samples (13.2%) tested positive for ALSV RNA. Positive ticks were detected in the provinces of Belluno and Vicenza and included individual adult males and nymph pools. A subset of positive samples was further characterized by nested PCR and Sanger sequencing of all four genomic segments. Phylogenetic analyses showed that Italian ALSV sequences clustered within the broader European ALSV diversity and were closely related to strains from Central and Northern Europe, without forming a distinct country-specific lineage. Sequence comparisons suggested purifying selection and revealed differences in predicted structural proteins between European and Chinese strains. These findings provide the first molecular evidence of ALSV circulation in Italy and support further studies to clarify its epidemiology, host range, genetic diversity, and potential clinical relevance. IMPORTANCEAlongshan virus (ALSV) is an emerging tick-borne virus identified in febrile patients in China and subsequently detected in ticks in Russian Federation and several European countries. Although severe disease has not yet been reported in humans, surveillance and elucidation of the virus distribution are essential to assess its pathogenicity and potential public health impact. We developed a specific real-time RT-PCR protocol and detected ALSV in Ixodes ricinus ticks collected in northeastern Italy. Sequence analyses suggested multiple introductions and revealed differences in structural proteins between European and Chinese strains, suggesting potential adaptation and differences in pathogenicity. Since the clinical signs of ALSV infection in humans may overlap with those of tick-borne encephalitis (TBE), differential diagnostic procedures should be developed to improve patient management, particularly in TBE-endemic regions such as northeastern of Italy.

Background Sequencing of monkeypox virus (MPXV) from antigen rapid diagnostic tests (Ag-RDTs) could expand genomic surveillance during outbreaks in decentralized settings where sequencing equipment and cold chain transportation are unavailable. We aimed to evaluate the efficacy of MPXV sequencing from MPXV antigen Ag-RDTs. Methods We tested MPXV Ag-RDTs from three different brands using serial dilutions of cultured MPXV subclade Ib. Positive Ag-RDTs with different intensities of the test band were stored for 19 days, either at room temperature or at +4 degree C, after which viral DNA was extracted from the pads of the test cassettes. Metagenomic and tiled amplicon-based Oxford Nanopore technology sequencing methods were then performed. Results Viral DNA extraction from MPXV Ag-RDTs showed a consistent decrease in viral load of 3 logs compared to the initial viral load of the applied viral dilution. Both sequencing methods were able to reach high coverage but the tiled amplicon-based demonstrated more consistent results with a coverage always above 85%. Conclusion This proof-of-concept supports the development of this approach in the field, with the aim of combining genomic surveillance with decentralized testing, including in remote areas.

Background: Latent tuberculosis - infection (LTBI) reactivation accounts for most tuberculosis (TB) cases in the United States (U.S.). Expanding primary care LTBI services can improve detection and treatment, but real-world implementation is not well described. Objective: Describe how LTBI screening and treatment workflows are operationalized into practice in diverse U.S. primary care health systems. Design: Descriptive, multi-site study. Setting: Four U.S. primary care health systems participating in the U.S. Centers for Disease Control and Prevention (CDC) Tuberculosis Epidemiologic Studies Consortium III (TBESC-III, 2020-2022). Patients: Patients visiting any study site (N = 3,522,077). Measurements: Electronic medical record (EMR) data on patient demographics, TB test type, and results. Using narrative reporting and study documentation, LTBI care workflows were summarized by site into six domains: patient registration, TB testing evaluation, TB testing, TB disease assessment, LTBI diagnosis, and LTBI treatment. Results: Sites implemented recommended practices for testing and treatment: interferon-gamma release assays were the predominant test, and short-course rifamycin-based regimens were the primary treatment. Follow-up care practices and documentation of treatment outcomes varied. Country of birth was not consistently recorded at all sites, limiting TB screening capability based on nativity. Physician training, EMR decision-support tools, and patient education resources differed in scope and availability. Limitations: Descriptive design using self-reported workflows; may not generalize to all primary care settings or reflect physician-level variations. Conclusion: We provide a descriptive snapshot showing how LTBI care workflows varied in approach and consistency at four primary care systems with integrated LTBI services. These real-world examples may serve as templates for U.S. primary care systems seeking to expand TB testing and treatment. Primary Funding Source: U.S. Centers for Disease Control and Prevention (TBESC-III).

Trachoma programs are increasingly using serological tools to complement clinical indicators and to measure current and historical transmission of Chlamydia trachomatis (Ct). In January 2024, the Amhara Trachoma Program in Ethiopia implemented the Pgp3 lateral flow assay (LFA) for trachoma serosurveillance through the regional public health laboratory system. Three Amhara Public Health Institute Trachoma Molecular Laboratory scientists were trained and passed competency testing on the assay. The scientists then conducted LFA testing on dried blood spot (DBS) samples collected during a December 2023 trachoma impact survey in Tach Gaynt, Amhara. During testing, 2,542 DBS samples were processed over 12 days. Age-specific seroprevalence and seroconversion rates (SCRs) were estimated. Among children ages 1-5 years, seroprevalence was 20.8% (95% confidence interval [CI]): 17.4-24.7%), and the SCR was 7.9 per 100 child-years (95% CI: 4.6-13.5). Among children ages 1-9 years, seroprevalence was 26.0% (95% CI: 22.9-29.3%), and the SCR was 7.0 per 100 child-years (95% CI: 4.6-10.6). Seroprevalence among individuals ages 15 years and older was 85.8% (95% CI: 83.9-87.4%). Trachomatous inflammation-follicular prevalence among children ages 1-9 years (TF1-9) was 28.6% (95% CI: 22.1-35.4%), and Ct infection among children ages 1-5 years was 5.7%, suggesting ongoing transmission. The concordance between LFA results, TF1-9 prevalence, and Ct infection prevalence supports the utility of the LFA for trachoma surveillance in Ethiopia, with Pgp3 LFA testing performed in a regional public health laboratory. Integrating LFA-based serology into the Ethiopia national trachoma surveillance systems offers a scalable approach to guide programmatic decisions and support trachoma elimination as a public health problem.

The gut microbiota is crucial for immune development and overall health in chickens. In commercial production, birds routinely receive multiple vaccines during early life. While individual vaccines are known to affect microbial composition, the impact of complex, multi-vaccine programs, as used in the poultry industry, is not well understood. This longitudinal study examined the impact of multiple live and inactivated vaccines, given at commercially relevant times from an early age, on gut microbial diversity and composition in layer chickens. We characterised microbiota profiles using 16S rRNA gene sequencing at pre- and post-vaccination timepoints across different vaccine groups. Overall, microbial diversity remained stable across most vaccines, indicating strong resilience of the gut microbiota to repeated immunological interventions. Differential abundance analyses identified changes in selected bacterial taxa following vaccination, with responses varying among vaccine groups. Notably, these changes were not sustained, as the gut microbial community returned to a stable state after the vaccination schedule. These findings underscore the robustness of the chicken gut ecosystem and lay a foundation for future research into microbiome-vaccine interactions and their implications for poultry health, immunity, and production efficiency.

Trachoma, a chronic ocular disease caused by Chlamydia trachomatis (Ct), is the leading infectious cause of blindness worldwide. Despite WHOs SAFE (Surgery, Antibiotics, Facial cleanliness, Environmental improvement) strategy, [~]100M are at risk of blindness. Using metagenomic shotgun sequencing, we characterized the ocular microbiome of 680 villagers in Amhara Ethiopia, identifying 10 Community State Types (CSTs) associated with different population characteristics. Children with the highest prevalence of inflammatory trachoma and Ct were in CST10, dominated by Haemophilus influenzae and four other Haemophilus spp. Adults with the highest prevalence of scarring trachoma were in CST3 and CST6, dominated by Corynebacterium macginleyi. CST5, dominated by Mesomycoplasma hyorhinis and Staphylococcus aureus, had the lowest prevalence of Ct and trachoma, and was the only CST without zoonotic Chlamydia spp. Both M. hyorhinis, a zoonotic porcine bacterium, and S. aureus are capable of forming biofilms, which may competitively prevent/down-regulate chlamydial infections. Other CSTs were dominated by environmental species like Vibrio. This is the first microbiome study to develop CSTs for trachoma. Pathogenic and potentially protective microbes showed distinct associations with demographic, clinical, and chlamydial characteristics, which will guide the design of microbial therapeutics as alternatives to antibiotics and strategies for WHOs global elimination of blinding trachoma.

The global prevalence of Extended-spectrum {beta}-lactamase-producing Enterobacterales (ESBL-E) colonization is increasing. However, it is unclear whether ESBL-E persist and if that is associated with an altered gut microbial ecology especially in early life where the developing microbiome may not provide the same colonization resistance as in adults. In this study, we collected longitudinal infant gut microbiome samples at delivery and in the nonclinical home setting in Chicago, Illinois, U.S.A, aiming to disentangle how genetic factors pertaining to the ESBL-E, as well as the surrounding gut ecology, influences persistence in the infant gut microbiome. We observed not only a higher-than-expected prevalence of ESBL-E in healthy infant gut microbiomes, but also a trend of ESBL-E persistence once colonized. Microbial communities showed higher dissimilarity between ESBL-E positive and negative infant gut microbiome at earlier time points. Although dissimilarity decreased over time, we present evidence that ESBL-E persist even when traditional detection methods are negative.

The rhizosphere microbiome, one of the most diverse and metabolically active microbial ecosystems known, plays fundamental roles in plant health and productivity. However, the ecological dynamics occurring during the transition between germination and the establishment of the first true leaves, a developmental window associated with the onset of active photosynthesis and rapid root expansion, remain poorly understood. Here, we investigated rhizosphere microbiome assembly during the first four weeks of tomato development by sampling communities arising from seven distinct natural soil inocula twice weekly to obtain fine-scale temporal resolution. Bacterial load, richness, evenness and phylogenetic diversity all increased significantly during plant development, indicating progressive increases in rhizosphere ecosystem complexity. In addition, diverse initial microbial communities differentially influenced both host plant development and the bacterial carrying capacity of the resulting rhizosphere ecosystem. Although temporal effects on rhizosphere microbiome composition were significant, assembly trajectories remained strongly constrained by the initial inoculum. Temporal analysis nevertheless revealed significant taxonomic turnover despite limited global compositional restructuring. In particular, Proteobacteria and Pseudomonadaceae decreased over time, whereas Actinobacteria, Acidobacteria and Streptomycetaceae increased. However, communities did not become progressively more similar or divergent over time. Altogether, our results indicate that early rhizosphere microbiome assembly involves rapid ecological succession within inoculum-constrained compositional trajectories, with early copiotrophic Proteobacteria progressively giving rise to more diverse and phylogenetically structured communities. These findings suggest that the first weeks of plant development may represent a critical ecological window for microbiome-based manipulation strategies in agriculture.

Clostridioides difficile is the leading cause of healthcare acquired infections (HAIs). Healthcare systems lack scalable strategies to identify patients colonized with toxigenic strains to inform prevention programs. We developed a scalable program using rectal swabs, collected for vancomycin resistance Enterococci surveillance, with ex vivo amplification of C. difficile in Cdiff Banana Broth (BB). Confirmatory testing of BB-positive tubes used the rapid C. difficile QuickCheck assay with Cepheid's Xpert(R) C. difficile/Epi (Xpert) to confirm toxin gene carriage for QuickCheck results that were C. difficile glutamate dehydrogenase-positive (GDH) and antigenic toxin-negative. BB with confirmation demonstrated 92% specificity and 100% sensitivity, to a limit of <5 colony forming units (CFU)/swab, versus published values of 100/swab by chromID(R) C. difficile agar and 460/swab by direct Xpert swab testing. The two-step confirmation detected toxigenic C. difficile in cases missed by these other methods. Prospective testing of 797 VRE swabs from 538 ICU and inpatient oncology patients seen at academic and community hospitals demonstrated 11% positivity for toxigenic and 4% for non-toxigenic C. difficile. Among swabs, 32% of toxigenic C. difficile were resulted at 24 hours and 68% at 48 hours. Addition of 17% glycerol to BB-positive aliquots, for -80C storage, supported 100% retrieval of C. difficile >6 months later. BB with confirmation provides a more sensitive method to detect patient colonization with toxigenic C. difficile and, by using VRE surveillance swabs, does not require additional patient samples. The simplified approach can be deployed across healthcare settings to support C. difficile-prevention programs.

Pseudomonas aeruginosa remains one of the most important clinical pathogens for which new drugs are needed, due to its resistance machinery. Consequently, there is an increasing effort to develop new and effective treatments against this pathogen. We recently showed that tetrasodium ethylenediaminetetraacetic acid (tEDTA) exhibits promising antibacterial and antibiofilm activity against P. aeruginosa in advanced biofilm models. tEDTA is known to chelate divalent cations, with predicted effects on the outer membrane; however, a full understanding of how this kills P. aeruginosa is lacking. Also, it is currently not clear how slowly or rapidly P. aeruginosa will evolve resistance to this treatment. Using membrane disruption assays and RNA-seq, we showed that tEDTA disrupts bacterial membrane potential and permeabilises P. aeruginosa membranes. RNA-seq revealed the significant upregulation of genes involved in the transport of iron, phosphate, potassium, and magnesium ion. The arnABCD operon which is involved in lipid A biosynthesis was also upregulated. Using a 7-day evolutionary ramp approach, we showed that P. aeruginosa could not evolve resistance to tEDTA under strong selection. Lastly, we carried out a cytotoxicity assay with Human Epithelial type 2 (HEp-2) cells and showed that there was reduced cytotoxicity of tEDTA compared to meropenem. This study provides good insight into the mechanism of action of tEDTA and further evidence of its potential as an alternative to antibiotics for P. aeruginosa infections.