Journal of Microbiological Methods

Antibiotic resistance is an increasingly serious problem worldwide. Identifying the bacterial species can help guide antibiotic selection and improve patient outcomes. In sepsis, where every minute counts, rapid identification is particularly important, and new approaches are needed. In this study, deep learning models were trained to identify bacterial species from phase-contrast time-lapse images of cells growing in microfluidic traps, without fluorescent labels. We demonstrated this approach in two previous studies using laboratory strains. Here, the previous work is extended to clinical bacterial isolates, evaluating performance on isolates not seen during training. Furthermore, it was tested whether autofluorescence could aid species identification. Two validation sets were acquired in separate experiments from the training data, the first containing clinical-isolate traps with five species (Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus aureus and Enterobacter cloacae), imaged every two minutes, and the second containing clinical isolates of two species (E. coli and K. pneumoniae) recovered from spiked donor blood, imaged every minute. Training with the best previous CNN method achieved unsatisfactory results. Therefore, vision transformers were explored, and a self-supervised adaptation procedure was developed for pretrained DINOv3 using a frozen teacher, followed by supervised classification using time-lapse montages. Mean macro-F1 was 93.6 {+/-} 1.3% for isolates precultured in Mueller-Hinton medium using 60 frames (118 minutes) and 87.2 {+/-} 2.7% for isolates precultured in blood using 55 frames (54 minutes) (mean {+/-} sample standard deviation across training seeds). Performance was evaluated over time by increasing the number of frames available to the model during testing, and Fourier-domain filtering approximated reduced numerical aperture. The results are promising and suggest that the aperture can be decreased while retaining good results. However, further evaluation on independent isolates, experiments and patient samples is needed to establish generalization. Together with previous findings, these results bring us closer to rapid image-based diagnostics that could transform the initial treatment of acute bacterial infections by enabling better-informed antibiotic selection.

Primer design is a fundamental component of molecular diagnostics in both quantitative polymerase chain reaction qPCR and loop-mediated isothermal amplification LAMP assays. However, assay design is often performed manually as nucleotide databases, sequence alignment tools and resources are found at different places on the Internet. In this study, an AI-orchestrated bioinformatics workflow was developed to automate the end-to-end qPCR and LAMP primers and probes. The workflow was implemented using LangGraph, LangChain and Biopython, where a series of specialized agents were coordinated to execute sequential bioinformatics tasks with minimal human intervention. Target sequences were then retrieved based on the user request from the National Center for Biotechnology Information nucleotide database and the requested sequence records were then subjected to multiple sequence alignment for the identification of conserved genomic regions. The multi-agentic primer design system can be used for assay development for applications in infectious disease diagnostics, outbreak surveillance and environmental monitoring. This study also demonstrates how multi-agentic systems can be combined with established bioinformatics methods to automate qPCR and LAMP assay design.

Balamuthia mandrillaris is a free-living amoeba that can cause cutaneous skin lesions, systemic disease, and a severe life-threatening CNS infection, Balamuthia amoebic encephalitis (BAE), which has a high mortality rate (~92%). The current diagnostic methods for B. mandrillaris have many limitations including the long detection time, low sensitivity, and specific diagnostic tests only available in highly specialized centers. The early stage of diagnosis is important for the treatment because of how quickly BAE infection then develops. Therefore, we developed, optimized, and evaluated a novel loop-mediated isothermal amplification (LAMP) assay for the 18S rRNA gene of B. mandrillaris, which can be observed by naked eyes with phenol red. This novel diagnostic assay can detect ten different B. mandrillaris isolates with no cross-reactivity with the DNA of other free-living amoeba, protozoa, fungi, or bacteria tested. Compared with conventional PCR, our LAMP assay displayed ~10-100-fold higher sensitivity from extracted DNA, heat-treated samples, and non-treated samples, with the limit of detection (LOD) of 10 fg/L DNA, 10 trophozoite/100 L for non-treated cells, and 1 trophozoite/100 L for heat-treated cells, respectively. This method was further validated using two newly developed qPCR and dPCR methods to determine the LOD of approximately 1 copy/L. In addition, there is no tolerance for complicated matrices, that LAMP maintained efficacy in cerebrospinal fluid (CSF), urine, and blood-spiked samples. Due to the simplicity, robustness, and high sensitivity of the developed LAMP method, it will be useful for quick clinical detection and diagnosis of B. mandrillaris, particularly in non-specialized centers and resource-limited areas. Here we report the first LAMP diagnostic method for B. mandrillaris; publicly disclosed on Tuesday, November 4th, 2025 at the 20th (XX) International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM 2025) at Puerto Morelos, Mexico.

The coprophilous agaricomycete Coprinopsis cinerea is a model organism for antagonistic fungal-bacterial interactions. Previous studies showed that C. cinerea responds to antagonistic bacteria with strong induction of a set of genes encoding secreted antibacterial molecules. However, little is known about the elicitors of this response. Key open questions in this respect include whether individual antibacterial defence genes are induced by different bacteria and/or by specific bacterial soluble molecules. Here, we present a new C. cinerea reporter system to monitor antibacterial defence induction and address related outstanding issues with minimal hands-on time. In this system, the promoter of the endogenous bacterial-induced gene cclys1 drives the expression of cnluc, which encodes a secreted variant of the deep-sea shrimp luciferase Nluc. We show that cNluc allows to detect and quantify cclys1 induction by measuring luminescence directly in the culture medium of reporter strain colonies. Building on these features, we successfully leveraged the inducible cNluc reporter strain for the development of a novel 96-well plate assay that allows the high-throughput screening of antibacterial defence elicitors. As cNluc can be subject to degradation by secreted proteases of fungal or bacterial origin in the culture medium, we coupled this assay to confirmatory qRT-PCR. Testing this set-up by confronting the reporter strain with several different bacteria revealed that cclys1 induction occurs independently of the bacterial ecological niche. Based on these results, we also recommend qRT-PCR exclusively for validation of negative results. We conclude that cNluc offers significant advantages over cytoplasmic reporter proteins, especially for preliminary rapid screening of multiple conditions.

Beech leaf disease (BLD), caused by the migratory plant pathogenic nematode, Litylenchus crenatae (LC, Anguinidae) is presently one of the most concerning forest diseases in the United States. BLD has spread rapidly across the landscape and is currently found in 15 U.S. states and Ontario, Canadian, making monitoring a top priority. Here, we report on the development and testing of a probe-based qPCR detection assay that integrates previously reported LC-specific primers with a newly designed probe, enabling sensitive and reproducible quantification of nematodes in plant and non-plant matrices. This assay exhibited strong in silico and in planta specificity and high quantitative performance (R2 = 0.988-0.996), with amplification efficiencies of 90.0-96.8%. The assay also performed consistently on LC-spiked spore trap membranes used to monitor the fungal aerobiome, despite matrix-associated shifts in amplification efficiency. These results support the utility of the assay for monitoring and surveillance. The reliability was further evaluated by two independent diagnostic laboratories, demonstrating high inter-laboratory concordance in detection and strong agreement in relative quantification. The analytical sensitivity and reproducible performance of the assay align with key validation principles outlined by the APS Diagnostic Assay Validation Network (DAVN) and support its application as a molecular tool in experimental, diagnostic, and surveillance contexts, with important implications for national and international LC monitoring and disease management.

Antimicrobial resistance (AMR) represents a major global health threat, with plasmid-borne mcr genes driving colistin resistance and exposing critical gaps in One-Health surveillance across human, animal, and environmental reservoirs. The most prevalent variant, mcr-1, remains difficult to monitor in resource-limited settings due to the lack of rapid, affordable, and field-deployable molecular tools. Here, we developed C12amcr, an integrated molecular toolbox that combines pre-amplification PCR with a fluorescent CRISPR-Cas12a assay targeting a conserved region of mcr-1 and a custom low-cost, hand-held 3D-printed portable fluorometer. Under optimized conditions, the assay achieved a limit of detection of 630 cells/mL. In poultry feces spiked with mcr-1-positive E. coli, C12amcr detected as few as 1,800 cells/mL. When tested on 22 community-derived E. coli isolates, the assay showed 100% concordance with both next-generation sequencing for mcr-1 detection and phenotypic colistin susceptibility testing by broth microdilution. The accompanying portable fluorometer performed equivalently to a laboratory microplate reader while enabling fully decentralized workflows compatible with portable PCR platforms. By integrating locally produced molecular reagents, straightforward protocols, and an accessible field-ready fluorescence reader, C12amcr overcomes key barriers to decentralized AMR surveillance and provides a practical, scalable solution for One-Health monitoring in resource-limited settings.

Invasive candidiasis has become more common in recent decades, particularly within ICUs. Identifying specific Candida spp., and testing their sensitivity to antifungal drugs is crucial for effective treatment that helps healthcare providers to detect potential treatment challenges early. This study aimed to identify Candida spp. at their spp. level present in blood, assess their susceptibility to antifungal agents, and apply a random forest classifier to predict the best antifungal drug for a given species. The isolates were classified at the species complex level through MALDI-TOF MS. The strains' susceptibility to antifungal agents such as AMB, FLZ, VOZ, ITZ, POZ, CAS, MFN, and ANF was assessed using CLSI guidelines. Out of the 354 Candida spp. isolated from blood, C. utilis was found to be the predominant species, followed by C. tropicalis, and C. albicans. This study demonstrated that MFN and ANF exhibited high potency, with an impressive MIC90 of 0.12 {micro}g/mL against all isolates, surpassing CAS, which had a slightly higher overall MIC90 of 0.25 {micro}g/mL. FLZ exhibited the highest MIC90 value among the azoles at 16 {micro}g/mL, while VOZ, ITZ, and POZ all had MIC90 values of 0.5 {micro}g/mL. AMB MIC90 values were 2 {micro}g/mL against all isolated Candida species. The Random Forest AI/ML model showed perfect overall prediction, with the actual and predicted best antifungal drugs matching for all fungal species. This study is particularly essential because rare Candida spp. becoming more common and exhibiting higher MICs to AMB and FLZ and required suitable remedies for controlling the infection.

Sporulation is an integral process in the lifecycle of many fungal pathogens, including Histoplasma, a primary human pathogen that causes respiratory infections. Histoplasma conidia, or asexual spores, are the primary infectious particle but very little is known about them, in part due to the need for Biosafety Level 3 containment and inconsistency in generating viable conidia under laboratory conditions. Here, we identify media that consistently promote Histoplasma conidiation, yielding both micro- and macroconidia, and conditions that promote high levels of germination. We show that conidiation media and duration affect the proportion of macroconidia produced, and we demonstrate that Histoplasma strains vary in their response to these conidiation parameters. Finally, imaging studies of chitin, exposed chitin, and cell wall mannoproteins show that while micro- and macroconidia have similar cell wall compositions, strain type and conidiation media variation result in qualitative differences in staining. These optimized methods for Histoplasma conidial preparations will enable more detailed investigations into this understudied aspect of the biology of an important human fungal pathogen.

To explore adapting qPCR systems for end-point nucleic acid quantification using dyes such as SYTO-9, we quantified serial dilutions of DNA and RNA standards in the range of 0.75 - 200 ng/{micro}l on 384-well qPCR devices. SYTO-9 fluorescence was successfully measured using standard SYBR Green settings. Blank-subtracted relative SYTO-9 signal showed a logarithmic dependence on DNA/RNA concentration (R2 > 0.95). Measurements were highly stable with different incubation times, temperatures of up to 95{degrees}C, and photobleaching. The described approach is a valuable QC option for high-throughput DNA/RNA isolations and could be adapted to additional fluorometric assays beyond nucleic acids.

The Enterobacteriaceae pathogens like E. coli, Shigella, Klebsiella and Salmonella cause a wide range of gastrointestinal and other mucosal infections when the contaminated food and water are consumed. Conventional microbiological methods are time-consuming and laborious process to detect these pathogens. Molecular assays are fast and sensitive to detect the pathogens. But a potential target for the detection is very essential to develop a molecular assay for the rapid detection. In this study, we explored the cpn60 UT region as a potential target for the detection of significant Enterobacteriaceae members like E. coli, Shigella sp, Salmonella, Klebsiella etc. We did the phylogenetic analysis of cpn60 UT region among the Enterobacteriaceae pathogenic groups. This analysis has revealed the presence of 13 clusters and two oligonucleotide regions with highest nucleotide conservation. Targeting these regions we have designed a set of oligonucleotide primer pairs and developed a SYBR green real time PCR assay to detect the Enterobacteriaceae pathogenic members. The degenerate oligonucleotide primers designed amplified a 132 bp UT region of cpn60 gene present in Enterobacteriaceae pathogens. The sensitivity of the assay was initially investigated by 10-fold dilution series of overnight broth culture of E. coli ATCC 10536 and had a detection limit of 101 CFU/mL. The specificity was assessed by comparing the amplification plot of a set of Enterobacteriaceae pathogens and non-Enterobacteriaceae members like Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes) and found to detect all the Enterobacteriaceae cultures included in the study whereas amplification was not seen in S. aureus and L. monocytogenes.

The crystal violet (CV) microtiter plate assay is widely used to compare microbial biofilm formation because it is inexpensive, simple, and readily adapted to 96-well formats. Its high-throughput design enables multiple strains, growth conditions, and treatments to be tested in parallel, but increasing experimental complexity can make downstream data handling difficult. Plate layouts, absorbance measurements, blank correction, replicate analysis, and visualization are often managed using separate spreadsheets or software, requiring repeated transfer and organization of experimental information. This can compromise consistency in linking measurements with their corresponding conditions and controls. We developed BiofilmQ-HT, a standalone, browser-based workflow that integrates plate annotation with direct import of plate-reader data, condition-specific blank correction, replicate summarization, growth-normalized specific biofilm formation (SBF) analysis, percentage-inhibition analysis, visualization, and data export. The workflow was applied to three representative datasets: a comparison of biofilm formation in 25% and 100% media, an analysis of bacteriophage- and antibiotic-associated antibiofilm activity, and a screening of biofilm formation among natural microbial isolates. These applications demonstrate a common analytical framework for comparative biofilm analysis, antibiofilm screening, and high-throughput isolate screening while retaining experimental conditions and controls. By linking plate organization to quantitative analysis and reporting, BiofilmQ-HT provides a reproducible and traceable approach for processing condition-rich CV assay datasets and facilitates consistent comparisons across experimental groups. The utility therefore improves consistency in data handling without altering the underlying experimental assay or requiring changes to the established assay.

The effect of microcurrents on facultative microbial pathogens remains controversial. Solid cultures in Sabouraud Glucose Agar of the ubiquitous mold Aspergillus fumigatus were repeatedly treated with a commercially available device performing wireless conductive microelectrostimulation by 3.5 A microcurrent routed by spraying negatively charged air particles onto solid cultures in modified petri dishes. The treated cultures displayed increased growth compared to standard ones, but only as a function of mycelial density and total surface; the radial growth rate of the mycelium remained unaltered. The increased growth was positively related to the duration of the treatment. At the same time, secondary development (new mycelial loci within the dish) was greatly upheld due to treatment, as the spraying created microairstreams dislocating the fungal spores. These results imply perplexed kinetics of mycelial growth both with and without treatment, since the folding of the mycelial mat is observed regularly. Both the fungus response to the ES and the possible revision of growth kinetics create prospects for biotechnological and bioremediation applications but also imply biomedical considerations, regarding infection dynamics of mycelial fungi and their in situ resistance to immune responses and treatment.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease gene (Cas), originating from the bacteria acquired immune system, have revolutionized gene editing technology. In this regard, type II (Cas9) been extensively studied and widely applied CRISPR system so far. The mechanism for precise manipulation of genomic sequences is guided by small RNA called CRISPR RNA (crRNA). In this study we devised and optimized CRISPR-Cas9 screening system based on Cas9 gene detection, targeting a conserved part of recognition domain (REC) consisting of arginine rich bridge helix (BH). We used hemi-nested PCR approach for screening sensitivity and reproducibility. The recombinant E. coli DH5 alpha containing the pRGEB32 vector (DH5 alpha/pRGEB32) with the Cas9 gene was used for system optimization. Subsequently, the screening system was applied and validated on different environmental bacterial strains including Alcaligenes faecalis and Pseudomonas stutzeri, isolated from sewerage samples. The optimized hemi-nested PCR resulted in amplification of targeted region in environmental bacterial strains and results were reproduced successfully. Furthermore, nucleotides and amino acid sequence, motif and domain analysis of PCR products, confirmed the targeted Cas9 REC-BH domain. Presently, no rapid and cost effective CRISPR-Cas screening system is available except expensive whole genome sequencing approach. Our investigation aimed to device rapid and cost effective screening system for identification of new variants of Cas9 proteins in environmental bacterial species. In this context, the developed Cas9 gene-based CRISPR-Cas screening system (C9CSS) may be a potential rapid screening tool to identify new Cas9 orthologs in different bacterial genomes with improved functions.

Bacterial cellulose (BC) is a biopolymer that comes from natural sources. It has high purity, crystallinity, mechanical strength, and biocompatibility, which makes it suitable for various biomedical and industrial uses. Unlike plant cellulose, BC does not contain lignin or hemicellulose. This results in better material quality and simpler processing. However, producing BC on a large scale is limited by high production costs, expensive culture media, and dependence on a few bacterial strains. It is essential to find cost-effective production methods and alternative microbial sources to expand its commercial use. This study looked at the cellulose-producing ability of Acetobacter diazotrophicus, a safe and relatively unexplored bacterium, under different growth conditions. We compared bacterial growth and cellulose production using Hestrin-Schramm (HS) medium, the standard for BC production, and LB supplemented with glucose (LB+Glucose), which we explored as a more affordable option. We analyzed growth rates, inoculum age, and pH levels to find the best conditions for cellulose production. We observed faster bacterial growth in HS medium, with a doubling time of 3.906 hours, compared to 6.241 hours in LB+Glucose medium. Cellulose production was greatly affected by inoculum age, with successful synthesis from cultures that were agitated for 36 to 40 hours. The highest cellulose yield was at pH 6.0 in HS medium (4.6 mg/mL) and at pH 5.5 in LB+Glucose medium (3.6 mg/mL). FTIR analysis confirmed the presence of characteristic functional groups of bacterial cellulose. These results suggest that Acetobacter diazotrophicus is a promising and cost-effective option for producing bacterial cellulose and highlight the importance of medium composition, inoculum age, and pH for optimizing production.

Health monitoring is essential to ensure laboratory animal welfare and the reliability of experimental data in zebrafish research facilities. Conventional surveillance strategies based on resident or sentinel fish have limitations in terms of sensitivity and animal use, highlighting the need for alternative approaches consistent with the 3Rs principles. In this perspective, the present study evaluated the use of sludge collected from recirculating aquaculture systems as an environmental matrix for molecular health monitoring. Because sludge accumulates microorganisms and organic material from the entire system, it represents a promising sample for pathogen detection. Following an initial environmental surveillance phase to detect pathogens present in the system, the study aimed to optimise a molecular protocol for sludge analysis. To this end, four commercial DNA extraction kits were evaluated to assess their effectiveness in recovering bacterial DNA from sludge. Their performance was analysed by quantitative PCR in terms of extraction efficiency, repeatability, and limit of detection. The results highlighted the strengths and limitations of each DNA extraction protocol and confirmed the suitability of sludge as a non-lethal matrix for pathogen detection. These findings support the implementation of environmental monitoring as a practical and cost-effective alternative to sentinel-based surveillance, improving pathogen detection while reducing animal use in accordance with the 3Rs principles and Directive 2010/63/EU.

GeneCapture is developing a novel, point-of-care, rapid AST technique that, when fully automated, can accomplish AST within 2 hours directly from a raw sample, representing a significant advancement in diagnostic capabilities. For this study, a panel of uropathogen isolates was collected from clinical specimens positive for urinary tract infection from various laboratories in the southern US. A selection of these isolates were then spiked into fresh clean-catch urine samples to produce mock samples covering a variety of antibiotic responses. Testing was performed on 20 strains of Escherichia coli, the cause of over 80% of UTIs, and 6 strains each of Klebsiella pneumoniae and Proteus mirabilis, the next most common causes. Each strain was tested against the susceptible, intermediate and resistant (S-I-R) breakpoints of up to 5 appropriate antibiotics including Amoxicillin-Clavulanate, Cefpodoxime, Ciprofloxacin, Nitrofurantoin and Trimethoprim-Sulfamethoxazole. The new method uses a phenotypic assessment of bacterial growth, comparing responses from bacteria treated with antibiotics at S-I-R breakpoint concentrations to untreated controls as the metric for determining susceptibility. Exposure to the drugs for only 90 minutes gave a Categorical Agreement of 98% to gold standard culture results across the resulting 247 bacteria/antibiotic pairs. Testing on separate days of nine of these strains in triplicate gave a reproducibility of 97%. The sequential steps of this novel AST - sample processing, growth, labeling, and counting - can readily be performed on an automated cartridge that is currently under development. The presented work, which took place in a laboratory setting, demonstrates the efficacy of the assay.

BackgroundInnovative vector control strategies are urgently needed to combat malaria transmission by Anopheles gambiae, given the limitations of current insecticides--namely resistance, human toxicity, environmental damage, and high costs. This study evaluated the larvicidal efficacy of endophytic bacteria isolated from Cola nitida against third-instar larvae of Anopheles gambiae. MethodsEndophytic bacteria were isolated from nine Cola nitida plant parts (leaves, roots, stems, fruits, flowers, stem bark, branches, root bark, whole plant extracts) using standard surface sterilization and culturing protocols. Thirty-two morphologically distinct strains were purified and screened for larvicidal activity against third-instar An. gambiae larvae using the WHO-recommended turbidity method at McFarland 4 standard. Six active isolates underwent dose-response testing (McFarland 0.5-4) with mortality recorded at 24 and 48 hours. Environmental safety was assessed via Lemna minor growth inhibition, and molecular identification employed the API 20E biochemical gallery. ResultsScreening identified six strains with significant larvicidal activity (LC < McFarland 2). At McFarland 2 concentration, all six strains achieved >50% mortality within 24-48 hours, with Lemna minor EC >100% indicating minimal phytotoxicity. API 20E identification (96-99.99% similarity) revealed three strains (6164, 6211, 6501) matching Photobacterium damselae, two (6186-1, 6512-1) matching Salmonella enterica subsp. arizonae, and one (6600) matching Pseudomonas sp., confirmed by distinctive biochemical profiles. ConclusionThis study provides the first evidence of potent, environmentally safe larvicidal activity from Cola nitida endophytic bacteria against Anopheles gambiae. These strains represent promising biocontrol candidates to address insecticide resistance, offering a sustainable alternative for malaria vector management in endemic regions.

Cronobacter sakazakii is an opportunistic foodborne pathogen that affects neonates, and has ability to produce dry surface biofilm (DSB). However, little is known about its resistance to dry sanitizers. This study aimed to evaluate the efficiency of dry sanitizers compared with sodium hypochlorite (SH) on DSB of C. sakazakii. DSB were formed on stainless steel or polypropylene coupons (10 cm{superscript 2}) using two cycles of hydrated/dry phases at 25 {degrees}C: T1 (48/48 h) or T2 (24/120 h). At DSB endpoint, the coupons were sanitized with 70% ethanol, a commercial product based on isopropyl alcohol (25%) and quaternary ammonium (0.015%), hot air (90{+/-}2 {degrees}C), UV-C light (254 nm), gaseous ozone (45{+/-}2 mg/L), or SH (200 mg/L, pH 6.5). All dry sanitizers showed limited antimicrobial activity (p > 0.05), with reductions [&le;] 0.8 log CFU/cm{superscript 2} over 30 min exposure. In contrast, SH was effective against DSB regardless of the DSB formation protocol, with reductions [&ge;]2 and >5 log CFU/cm{superscript 2} after 10 and 30 min, respectively. Interestingly, DSB formed with shorter hydrated phase and longer dry phase (T2) had greater sensitivity to SH, but it was not noted to dry sanitizers. CLSM images suggested the presence of VBNC cells, particularly after SH treatment. In conclusion, our results emphasize the importance of implementing stringent hygiene measures to control C. sakazakii DSB in the low moisture food industry, and indicate that SH is an effective sanitation strategy when the drying line is promptly addressed.

Pore forming toxins (PFTs) are cytotoxins secreted in water-soluble form by pathogenic bacteria. They have the ability to form pores in the membrane of host cells, ultimately leading to cell death by lytic activity. Staphylococcus aureus produces a variety of bi-component PFTs, the leukocidins, which target and lyse particular leukocytes, erythrocytes and endothelial cells through specific interactions with membrane receptors. Most of these receptors belong to the family of complement or chemokine receptors that are G protein-coupled receptors (GPCRs). Gamma-hemolysins (Hlgs) are the major leukocidins secreted by S. aureus, and form receptor-dependent hetero-octameric pores through mechanisms that are not fully elucidated. Studying these molecular mechanisms is technically challenging due to the requirement of specific receptors in a lipid bilayer environment. In the present article, we developed a simple and highly sensitive method allowing cell surface expression of a large diversity of target receptors and recording in real-time, currents generated by neo-formed pores. This method is based on the heterologous expression of receptors in Xenopus oocytes and on the two-electrode voltage-clamp technique with electrophysiological robots. Using this approach, we characterized the concentration dependent-kinetics of pore formation, determined the receptor density as a limiting factor, showed specific response to non-cognate pairing of PFTs, observed cell surface binding of F subunits preceding pore formation and propose a hybrid model of subunit oligomerization. This method could be easily implemented for the in vitro characterization of various PFTs on a wide diversity of membrane receptors, to decipher early mechanisms of pore formation or to screen therapeutic agents blocking the cytotoxicity of receptor-dependent PFTs. Author SummaryStaphylococcus aureus is a bacterial species naturally present in our external flora and environment, but it is also one of the main pathogens responsible for nosocomial infections in hospital, with strains having highly problematic multi-resistance to antibiotics. S. aureus is able to secrete various virulence factors, some of which can specifically target and lyse our immune cells, making us more vulnerable to this pathogen. Thus, leukotoxins bind to receptors on the cell surface, drastically change their conformation and form cytotoxic pores in the membrane. Studying the molecular mechanisms underlying the formation of these pores is technically challenging due to their requirement for specific receptors. Here, we tested a simple electrophysiological method enabling the real-time measurement of pore formation on model cells (Xenopus oocytes), which express the receptors of interest. We were thus able to elucidate the kinetics of pore formation, the limiting role of receptors in this process, and propose a complementary model to the standard model. We also demonstrated the ability of this method to detect pore formation of non-cognate pairs of subunits and suggest further applications to characterize pore-forming properties of other toxins, to identify new target receptors, or to screen therapeutic agents inhibiting the formation of pores.

Measurement uncertainty can affect the classifications of individual as positive or negative, and thus, the diagnostic performances of a test. Existing methods to assess measurement uncertainty and its impact on diagnostic performance are difficult to apply in the absence of a gold standard. We proposed a method applicable to any quantitative diagnostic test and in the absence of a gold standard, and applied it to ELISA tests for Q fever serology in ruminants. We assessed measurement uncertainty using a mixed-effects model on data from an inter-laboratory proficiency testing. Then, we estimated the sensitivities and specificities accounting for measurement uncertainty. To do so, we combined, for each individual of a sample representative of the population, the probability of being truly seropositive and the probability of being positive when retested in another laboratory. We also estimated the sensitivity and specificity of each laboratory and of each batch, taking into account their bias. While the sensitivities and specificities of the ELISA tests were slightly affected by the measurement uncertainty, they varied between laboratories and between batches. This highlights the importance of harmonising analytical practices across laboratories and calibrating batches. The extent to which measurement uncertainty impacts diagnostic performance depends not only on the values of the inter- and intra-laboratory standard deviations, but also on the position of the cut-off in the population's measurand distribution. Therefore, in addition to the analytical performance of a test, the position of the cut-off relative to the distribution of test value is an essential parameter to consider.