Jacques Bou Khalil

Introduction: Clostridioides difficile infection (CDI) remains one of the leading healthcare-associated infections. CDI management is increasingly complicated due to rising antimicrobial resistance, variability in treatment response, and the lack of standardized antimicrobial susceptibility testing (AST) in routine practice. The current reference method, agar dilution, is laborious and time-consuming, leaving therapy largely empirical. Methods: We present the development and proof-of-concept for a novel accelerated AST assay for C. difficile using tabletop scanning electron microscopy (SEM). Following confirmation of C. difficile growth in liquid medium, a broth microdilution-based AST protocol was established and evaluated using SEM imaging at early time points. Susceptibility to fidaxomicin and vancomycin was assessed for eight isolates with different toxigenic profiles. Results: Analysis of SEM images enabled the detection and quantification of antibiotic effects, including bacterial growth inhibition, reduced bacterial viability, and pronounced morphological alteration as early as three hours following exposure. This facilitated the determination of SEM-based minimum inhibitory concentration (SEM-MIC) ranges, which concorded with MICs determined using standard agar dilution. Conclusion: The SEM-AST assay achieved a substantial reduction in turnaround time from 48 hours to approximately 7 hours, without compromising accuracy. These results demonstrate that combining optimized culture conditions with SEM imaging at early time points allows for rapid and reliable MIC estimation, establishing the first SEM-based AST approach for strict anaerobes and providing promising potential for future CDI diagnostics.

La co-culture d’amibes est utilisée afin d’isoler des microorganismes. Ainsi le premier virus géant,fut découvert. Cependant, les méthodes de culture sur protozoaires sont délicates et fastidieuses. De ce fait, le développement de ces cultures représente une difficulté pour les microbiologistes, limitant ainsi l’analyse d’un nombre important d’échantillons et la caractérisation de nouveaux virus. De nouvelles stratégies et des améliorations des modèles actuels sont donc nécessaires. Notre travail a été de développer de nouvelles stratégies permettant l’isolement de microorganismes associés aux amibes. Dans la 1ere partie nos travaux ont permis une amélioration des cultures avec le développement de nouveaux milieux de culture et l’utilisation ciblée d’antimicrobiens.La clé de ces stratégies est l’association des techniques rapides aux étapes améliorées de culture et leur application à un large panel de protozoaires pouvant abriter des microorganismes. Les résultats ont permis le développement d’un système d’isolement à haut débit très efficace. Nous avons notamment mis au point des techniques de tri de virus géants par cytométrie.Dans la seconde partie, nos travaux ont porté sur la description et la caractérisation des nouveaux isolats.Les résultats obtenus démontrent l’importance de poursuivre l’isolement et la caractérisation de ces microorganismes afin de mieux appréhender l’évolution de ces microorganismes, leur biotope et leur pathogénicité.De nouveaux outils sont nécessaires,notre manque d’imagination et l’absence de systèmes automatisés seront les limites aux nouvelles stratégies dans le monde de la microbiologie.

Coxiella burnetii , the causative agent of Q fever, is a highly infectious obligate intracellular bacterium. In consequence, cultivating this pathogen was fastidious and resource-consuming, requiring 3 weeks for isolation in cell culture. In recent years, axenic media allowing C. burnetii growth without eukaryotic cells were developed. These media reduced the complexity of its manipulation. However, despite this advancement, traditional growth monitoring methods such as cell culture, colony counts, and immunofluorescence remain time-consuming, requiring an average of 10–14 days to detect growth. However, this is still considered too long in the context of research, epidemiologic surveillance, and treatment failure. Here, we present a novel method that coupled axenic growth to early detection using the TM4000Plus II-Tabletop scanning electron microscopy (SEM) (Hitachi, Japan). For this, we used eight strains of C. burnetii that were cultured in axenic medium and then prepared for SEM observation. Growth was assessed by the calculation of the percentage area coverage. We were able to detect growth within only 3 days with a percentage area coverage superior to 60%. This result was reproducible among the eight strains. Finally, the use of SEM for early growth detection of C. burnetii in axenic medium is simple and feasible and may reduce detection time by 70%. This promising approach may also provide deeper insights into the morphological details, metabolic, and growth kinetics of this intracellular bacterium. IMPORTANCE: Coxiella burnetii is the causative agent of Q fever, a serious disease affecting humans and other mammals. Q fever outbreaks are reported worldwide, posing significant public health and economic concerns. While cultivating this pathogen is not typically required for diagnosis, it remains valuable in cases involving new clinical presentation, treatment failure, or atypical epidemiological situations. This highlights the need for novel techniques that are both simple and capable of enabling early growth detection. Here, we present an innovative and simple approach allowing early growth detection of C. burnetii in axenic media using scanning electron microscopy.

Scanning electron microscopy (SEM) is re-emerging as an accessible method in bacteriology, driven by technological advances that produced the powerful and compact tabletop SEM. This review highlights recent advances (2015–2025) demonstrating how novel tabletop SEM delivers rapid, high-resolution, and accurate results that can transform both fundamental and clinical bacteriology. Several studies consistently demonstrate the utility of tabletop SEM in basic research, such as studying biofilms, building antibacterial coated material, and describing bacteria-environment interactions. In clinical bacteriology, diverse applications have emerged over the past few years placing the tabletop SEM at the forefront of bacterial visualization from clinical samples, reaching accurate descriptions of bacteria-antibiotic interactions and the accurate detection of bacterial morphologic changes following exposure to antimicrobial agents with dramatically reduced turnaround times. When combined with energy-dispersive X-ray spectroscopy (EDX), tabletop SEM offers insights into bacterial metabolic states and chemical composition under stress or antimicrobial treatment. In this new era of bacteriology, tabletop SEM truly marks the return of a forgotten ally, empowering the investigative arsenal with speed, robustness, and accuracy in both research and clinical practice.

The Gut Microbiota (GM) remains a complex microbial ecosystem with many unknown facets despite significant technologic advancement. This study introduces a novel rapid technique using tabletop scanning electron microscopy (SEM) for investigating GM composition, focusing on Clostridioides difficile infection (CDI) as a representative model for dysbiosis-related diseases. Six stool sample preparation protocols were tested on 40 stool samples to develop an optimized SEM protocol. Protocol stability was evaluated after four-month storage. The optimized protocol produced high-resolution micrographs while maintaining sample integrity over time. SEM investigation of GM was done by analyzing ten stool samples (5-control and 5-C. difficile groups), imaged at low and high magnifications. Object detection analysis generated a SEM-based GM components database helping describe and compare microbial diversity variation between the groups. CDI group revealed a reduction in microbial diversity, compared to the controls. Epithelial and red blood cells were more prevalent in CDI group. Statistical analyses of objects proved clear clustering of samples into CDI and control groups. This study pioneers the proof-of-concept for using tabletop SEM to investigate GM components in a dysbiosis-related disease model. This concept emerges as a complementary technique capable of providing deeper insight to describe GM components previously elusive with other methods.

Introduction: Our work aims at establishing a proof-of-concept for a method that allows the early prediction of the bactericidal and bacteriostatic effects of antibiotics on bacteria using scanning electron microscopy (SEM) as compared to traditional culture-based methods.Methods: We tested these effects using Imipenem (bactericidal) and Doxycycline (bacteriostatic) with several strains of sensitive and resistant Escherichia coli. We developed a SEM-based predictive score based on three main criteria: Bacterial Density, Morphology/Ultrastructure, and Viability. We determined the results for each of these criteria using SEM micrographs taken with the TM4000Plus II-Tabletop-SEM (Hitachi, Japan) following an optimized, rapid, and automated acquisition and analysis protocol. We compared our method with the traditional culture colony counting gold standard method and classic definitions of the two effects. Results: Our method revealed total agreement with the CFU method and classic definition by visualizing the effect of the antibiotic at 60 minutes and 120 minutes using SEM. Discussion: This early prediction allows a rapid and early identification of the bactericidal and bacteriostatic effects as compared to culture that would take a minimum of 18 hours. This has several future applications in the development of SEM-automated assays coupled to machine learning models that identify the antibiotic effect and facilitate determination of bacterial susceptibility.

Background. Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram‐negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM‐AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Methods. Forty‐four Gram‐negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼10⁷ CFU/mL were exposed to Colistin at EUCAST‐ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII‐Tabletop‐SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA‐staining and morphologic changes. Results. We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard‐based broth microdilution method. Conclusions. We provide an extended application of the proof of concept for the utilization of the SEM‐AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale.Significance:In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli–specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies.This article is highlighted in the In This Issue feature, p. 2221

Supplementary Figure from Escherichia coli–Specific CXCL13-Producing TFH Are Associated with Clinical Efficacy of Neoadjuvant PD-1 Blockade against Muscle-Invasive Bladder Cancer

Supplementary Figure from Escherichia coli–Specific CXCL13-Producing TFH Are Associated with Clinical Efficacy of Neoadjuvant PD-1 Blockade against Muscle-Invasive Bladder Cancer