D'Anjou pears are routinely stored for up to nine months under controlled-atmosphere (CA) conditions to meet market demands. While this practice maintains fruit quality, limited information exists on pears' natural microbiota throughout storage. The objective of this study was to describe fungal and bacterial composition on marketable and unmarketable conventional, whole, intact pears under two storage practices (bulk vs wrapped) at 3, 6, and 9 months in long-term CA cold storage. Storage practices had a significant effect on the composition and succession of both fungal and bacterial communities. No significant differences in Chao1 index were found between the bacterial and fungal communities on marketable or unmarketable pears. Trends in Chao1 indices of fungal and bacterial communities peaked at mid-storage and declined by 9 months, with wrapped pears showing parallel trends, and bulk pears exhibiting a sharper late-stage reduction. No distinct clusters could be found for 3- and 6-month fungal communities, irrespective of marketability, or whether bulk or wrapped. The principal coordinate analysis of the bacterial communities showed tight clustering by time point for the individually wrapped pears, irrespective of their marketability. Bacterial communities included genera common in food-processing and plant environments, such as Pseudomonas (19.2% relative abundance [RA]) and Acinetobacter (3.31% RA). Fungal communities shifted over time, with spoilage-associated genera like Aureobasidium (23.3% RA), Penicillium (9.28% RA), Botrytis (0.33% RA), and Mucor (0.14% RA) present at different storage stages.IMPORTANCEThis study highlights the influence of storage duration and packaging on microbial succession, establishing initial benchmarks of pear surface microbiomes. The observed lack of significant differences in microbial diversity between marketable and unmarketable pears suggests that these baseline community profiles can serve as critical reference points for identifying other influential factors. Variables such as handling practices may exert a more direct effect on microbial dynamics and, consequently, product quality. Establishing these baselines is essential because they provide a foundation for detecting deviations linked to spoilage or safety risks. Moreover, understanding these patterns can guide the development of targeted microbial control strategies in postharvest systems, enabling interventions that maintain fruit quality, reduce losses, and possibly improve food safety throughout the supply chain.

Enterococcus faecalis (E. faecalis) is an opportunistic pathogen capable of causing various life-threatening infections, including urinary tract infections, bloodstream infections, infective endocarditis, and meningitis. As a major etiological agent of healthcare-associated infections (HAIs), its global prevalence continues to rise, a trend closely linked to the increasing problem of multidrug resistance driven by overuse of antibiotics. Therefore, rapid and accurate detection is essential for timely treatment and improved prognosis. In this study, the pheS gene of E. faecalis was rapidly amplified using recombinase polymerase amplification (RPA), and detection was achieved via a CRISPR/Cas12a system. The Cas12a-crRNA complex specifically recognized the amplification product and triggered nonspecific cleavage of a single-stranded DNA (ssDNA) reporter, generating a fluorescent signal that could be quantified in a real-time PCR system or visualized directly under ultraviolet (UV) light. After optimization of key parameters-including RPA primers, reaction conditions, crRNA sequence, and the crRNA/Cas12a combination-the assay achieved a limit of detection (LOD) of 10-2 ng/μL within a short turnaround time, and showed no cross-reactivity with other common pathogen detection results from clinical isolates and spiked samples were fully consistent with those obtained through PCR/qPCR, confirming high reliability. In summary, the RPA-CRISPR/Cas12a detection method established in this study is sensitive, specific, and reliable. Its simplicity, minimal equipment requirements, and cost-effectiveness make it a promising tool for rapid clinical detection of E. faecalis.IMPORTANCEEnterococcus faecalis is a major opportunistic pathogen responsible for severe healthcare-associated infections, with rising prevalence linked to antibiotic resistance. Rapid and accurate detection is critical for timely treatment and infection control. Conventional methods are often time-consuming or require complex laboratory infrastructure, limiting their use at the point of care. This study developed a rapid detection assay by integrating recombinase polymerase amplification with the CRISPR/Cas12a system, targeting the pheS gene of E. faecalis. The method is sensitive and specific, providing visual results under UV light within a short turnaround time. It offers a simple, cost-effective, and requires minimal equipment, suitable for clinical and resource-limited settings, potentially improving diagnostic efficiency and supporting antimicrobial stewardship.

Clostridioides difficile infection (CDI) is a serious and recurrent intestinal disease for which current treatment options remain limited and often disrupt the protective gut microbiota, increasing the risk of relapse. In this study, we show that the antifungal drugs miconazole, econazole, and tioconazole exhibit rapid and sustained activity against C. difficile under conditions that mimic the gastrointestinal environment. Unlike existing therapies, these agents display minimal activity against commensal gut bacteria and provide prolonged post-antibiotic effects. We also demonstrate that cholesterol, a common host-derived molecule, modulates bacterial susceptibility to these drugs, revealing a previously underappreciated link between host factors and therapeutic efficacy. Finally, these compounds confer significant protection in an in vivo Caenorhabditis elegans model of CDI, highlighting their potential as candidates for repurposing and further development.

Ocular infections caused by Pseudomonas aeruginosa often lead to severe vision loss due to antibiotic-resistant bacterial biofilms, which shield bacteria from conventional treatments. This study introduces (LLKK)3C, that uniquely attacks biofilms through a dual-action mechanism: physically disrupting bacterial membranes and binding to bacterial DNA. Unlike traditional antibiotics like amikacin, (LLKK)3C maintains robust antibacterial activity even after prolonged exposure, reducing the risk of resistance development. In a rabbit endophthalmitis model, (LLKK)3C significantly reduced intraocular bacterial loads, demonstrating its potential as a targeted therapy for sight-threatening infections. By addressing the dual challenges of biofilm resistance and drug stability, (LLKK)3C offers a promising strategy to improve clinical outcomes for patients with difficult-to-treat eye infections.

This study compared the analytical performance of three commercial real-time PCR assays for the detection of monkeypox virus (MPV) DNA, on their own platforms and/or on open platforms, were evaluated at the virology laboratory of Grenoble-Alpes University Hospital (CHUGA): the VIASURE monkeypox virus real-time PCR Reagents on the BD MAX System; the MPXV AMP Kit on the Alinity m system; and the Altona RealStar Zoonotic Orthopoxvirus PCR Kit 1.0 on the Argene System (EMAG, ESTREAM [Biomérieux], and LC480 [Roche]). Results obtained on these platforms were compared to those from the French National Reference Center (NRC) for orthopoxviruses. A total of 52 samples were selected by the NRC and the CHUGA: 8 dilutions of MPV culture supernatant, 2 supernatants from cowpox and vaccinia virus cultures, 32 clinical samples (20 positive/12 negative), and 10 external quality control samples from the "Quality Control for Molecular Diagnostics" (QCMD) program. The sensitivity and specificity of the three assays, compared to the NRC assay, were 100% on the samples tested. All positive samples were correctly identified as positive with all three assays, and all negative samples were negative for MPV detection. Overall, the cycle threshold (Ct) values obtained with the VIASURE, Abbott, and Altona assays were lower than those obtained with the NRC PCR in 84.38%, 81.25%, and 75.86% of positive samples, respectively. In conclusion, all three assays demonstrated good analytical performance and were suitable for MPV detection.IMPORTANCEMpox is an emerging zoonotic disease caused by the monkeypox virus, mostly reported in Africa. In 2022, an outbreak characterized mainly by anogenital rash in MSM (men having sex with men) occurred in Europe. In 2024, many cases of infection were reported, presenting with generalized skin rashes. Detection of monkeypox virus DNA in biological samples is crucial for diagnosis, interruption of transmission chains, treatment initiation, and vaccination. During the European outbreak in 2022, numerous companies rapidly developed qPCR kits. In France, the performance of these kits has been evaluated by the French National Reference Center (NRC) for orthopoxviruses. The Grenoble-Alpes University Hospital collaborated with the NRC to evaluate three available assays on its own platforms and/or on open platforms available in its virology laboratory. All three assays demonstrated good analytical performance and can be used with confidence.

The degradation of organic matter (OM) by microorganisms in thawing permafrost produces greenhouse gases. Terrestrial OM is transported into fjords through hydrological runoff, but it is unclear whether the microbial mechanisms of OM degradation on land persist after soils enter marine environments, which differ greatly in conditions and microbial communities. This question is particularly relevant for low-OM soils, which dominate Arctic landscapes and are more exposed to oxidants. Here, we compared OM-degrading capacity in permafrost-affected active layer soils and adjacent fjord sediments from Kongsfjorden, Svalbard, focusing on carbohydrate-active enzymes (CAZymes), which target some of the most abundant types of organic matter in soils. Using multi-omics approaches-metagenomics, metagenome-assembled genomes (MAGs), metabolomics, metatranscriptomics, and metaproteomics-we examined CAZyme presence, distribution, and activity. Despite environmental differences, both soils and sediments harbored diverse glycoside hydrolases and polysaccharide lyases, most of which showed evidence of activity. Verrucomicrobia expressed the highest number of CAZyme transcripts, indicating that they dominated active carbohydrate degradation in fjord sediments, while Acidobacteria and Actinobacteria were more active in soils. Notably, CAZymes in fjord sediments targeted primarily soil-derived OM, and the proportions of enzymes degrading terrestrial OM, marine OM, and microbial necromass-remnants of dead microbial cells were similar across both environments. These results suggest that microbial communities in both soils and fjord sediments are equipped to degrade carbohydrates, and that burial of terrestrial-derived OM in fjord sediments may not protect it from microbial breakdown under Arctic warming.IMPORTANCEPermafrost thaw may be a critical climate feedback because microbial degradation of organic matter (OM) can release greenhouse gases. While fjords serve as major carbon burial sites, our results show that burial of terrestrial-derived OM in these sediments does not ensure protection from microbial degradation. Microbial communities in both active layer soils and fjord sediments harbor a broad arsenal of carbohydrate-active enzymes, with evidence of activity across diverse taxa. This functional continuity indicates that once terrestrial material is washed into fjords, it remains vulnerable to microbial breakdown despite different environmental conditions. Understanding these cross-system continuities in microbial function is essential for predicting the fate of OM in a rapidly warming Arctic and highlights the importance of including fjord sediments in global carbon cycle models.

Functional constipation (FC) is a common gastrointestinal disorder that can seriously affect patients' quality of life. Probiotics have emerged as a promising strategy for preventing and managing gastrointestinal diseases. Previously, we isolated Lactobacillus acidophilus C4 from the feces of a healthy child, and here, we investigated its probiotic efficacy against FC in a mouse model. Prophylactic treatment with L. acidophilus C4 prior to FC onset significantly reduced impairment of defecation function and increased intestinal transit rate. L. acidophilus C4 also increased the expression of tight junction proteins ZO-1 and occludin, suggesting a protective effect on the intestinal barrier. In addition, L. acidophilus C4 was able to modulate the inflammatory response by decreasing the pro-inflammatory factor IL-1β while increasing the anti-inflammatory cytokine IL-10. These results reveal that L. acidophilus C4 can improve constipation through anti-inflammation and restoration of intestinal barrier function, which can be a candidate strain for the development of novel interventions for constipation.IMPORTANCEFunctional constipation (FC) requires novel therapeutic approaches. Probiotics have attracted attention in the prevention and management of gastrointestinal diseases; however, their efficacy and mechanisms of action against FC need to be verified. This study provides experimental evidence for this purpose, supporting its potential as a non-invasive and well-tolerated intervention. The core of this study is Lactobacillus acidophilus C4, which exhibits high compatibility with the human intestinal microenvironment and lays a foundation for clinical research. L. acidophilus C4 can enhance intestinal peristalsis, protect the intestinal barrier (by upregulating tight junction proteins ZO-1 and occludin), and regulate inflammation (by balancing the pro-inflammatory cytokine IL-1β and anti-inflammatory cytokine IL-10). This strain provides a new direction for research on probiotic-based treatment of FC.

During the 2023 Australian winter respiratory virus season, 2,000 clinical samples were prospectively tested over 47 days (July-August 2023) using the BioFire Respiratory Panel 2.1 plus (RP2.1+) assay, which detects 19 viral and 4 bacterial targets. These samples were retrospectively tested with the RespiV PlexPlus (RespiV) assay, which detects 14 viral targets. RespiV demonstrated high overall percentage agreement with RP2.1+ (99.2%), with a negative percent agreement exceeding 99% for the majority of shared targets. Positive percent agreement was more variable across targets, with more positive discordant results for high-prevalence pathogens. Positive discordant results had higher quantification cycle values (median 37.2) than concordant positives (median 27.8), consistent with near-limit-of-detection inter-assay discordance. Discordant results for influenza A virus, influenza B virus, respiratory syncytial virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were retested with Xpert Xpress Flu/RSV and Xpress SARS-CoV-2 to generate a consensus result. RP2.1+ and RespiV showed 99.6% overall agreement with the consensus result for these targets. RespiV detected 14 additional influenza A-positive samples, all confirmed by amplicon sequencing. RespiV also identified 19 enterovirus-positive samples, 17 of which were RP2.1+ rhinovirus/enterovirus positive. Conversely, RP2.1+ detected pathogens not included in the RespiV panel, such as 79 non-SARS-CoV-2 coronaviruses and 1 Chlamydophila pneumoniae, with 62 samples negative across all RespiV targets. In summary, both assays show a high degree of concordance for all shared targets. RP2.1+ offers rapid test turnaround and broader pathogen coverage, making it valuable for urgent diagnostics, while RespiV provides higher throughput and is well-suited for centralized, high-volume testing environments.IMPORTANCERapid detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other treatable respiratory pathogens are critical for guiding therapy and infection control. Sample-to-answer multiplex panels, such as the BioFire Respiratory Panel 2.1 plus, allow simultaneous detection of multiple pathogens. These assays are fast, user-friendly, and support random-access testing for small batches, but they are costly and limited in throughput by instrument availability. Conversely, conventional multiplex assays like RespiV PlexPlus are slower and more labor-intensive for small batches but optimized for large batch processing on standard molecular diagnostic platforms. They offer higher throughput with lower consumable costs, making them suitable for centralized laboratories. Recent innovations in both sample-to-answer and conventional multiplexing technologies have expanded detectable targets, with comparable technical performance between formats. Ultimately, the optimal choice depends on healthcare system resources and testing demands.

Reverse genetic approaches in molds are complicated by their recalcitrance to transformation, multicellular growth during much of the vegetative life cycle, and frequent off-target integration of deletion constructs. Thus, protocols for gene deletion in filamentous fungi are predominantly confined to established model organisms. Adapting an existing protocol developed for gene deletion in the related fungus Aspergillus fumigatus, we employed an expression-free CRISPR/Cas9 directed mutagenesis strategy to the non-model environmental mold Aspergillus calidoustus. As a test case, we have deleted A. calidoustus pyrG, encoding orotidine-5'-phosphate decarboxylase, using short regions of homology to guide on-target integration of a nourseothricin resistance cassette (NatR) to CRISPR/Cas9-induced double strand breaks. We genotypically and phenotypically validated two A. calidoustus ΔpyrG deletion strains generated using this methodology, with whole-genome sequencing revealing one ΔpyrG strain to have integrated the resistance cassette by homologous recombination, and another strain by non-homologous end joining. Thus, distinct modes of double-strand break repair were responsible for on-target integration of the homology-bearing NatR cassette at the pyrG locus in A. calidoustus.IMPORTANCEIn the environment, filamentous fungi play essential roles in soil health and agriculture, decomposition, and nutrient cycling. The study of these organisms is often limited by an inability to dissect the functions of their genes and the roles that they play by modifying the genomes of these organisms. Here, we adapt and validate a tool for genome modification to Aspergillus calidoustus, a soil fungus that forms a partnership with a bacterium (Paraburkholderia edwinii) to resist natural toxins found in the soil.

In this study, through the establishment of a complete research chain of "virus titer-enhanced cell model-drug effect evaluation-signaling pathway analysis", we confirmed that ursodeoxycholic acid (UDCA) can inhibit feline infectious peritonitis virus (FIPV) infection and revealed that UDCA can activate the JAK-STAT signaling pathway by promoting the production of large amounts of interferon-β in host cells to protect against FIPV infection. It not only provides a new experimental tool (CRFK-PHBLV-N cell line) and drug candidate (UDCA) for FIP prevention and control, but also lays a theoretical foundation for the development of novel therapeutic strategies targeting the host antiviral pathway through the elucidation of the JAK-STAT pathway mechanism.