Clostridioides difficile is a nosocomial pathogen that is associated with the use of antibiotics. One of the most worrying aspects of C. difficile infection is its ability to resist antimicrobial therapies due to spore formation. In several bacterial pathogens, proteases of the Clp family participate in phenotypes associated with persistence and virulence. This suggests that these proteins could be involved in virulence-related traits. In this study, we analyzed the role of ClpC chaperone-protease of C. difficile in virulence-related traits by comparing the phenotypes of a wild-type and a mutant strain lacking the clpC gene (ΔclpC). We performed biofilm, motility, spore formation, and cytotoxicity assays. Our results show significant differences between the wild-type and ΔclpC strains in all analyzed parameters. Based on these findings, we conclude that clpC plays a role in the virulence properties of C. difficile.

A new Schiff base, (E)-2-(((1,10-phenanthrolin-5-yl)imino)methyl)-4,6-di-tert-butylphenol (Fen-IHB), was designed to incorporate an intramolecular hydrogen bond (IHB) between the phenolic OH and the azomethine nitrogen with the goal of modulating its physicochemical and biological properties. Fen-IHB was synthesized by condensation of 5-amino-1,10-phenanthroline with 3,5-di-tert-butyl-2-hydroxybenzaldehyde and exhaustively characterized by HR-ESI-MS, FTIR, 1D/2D NMR (¹H, ¹³C, DEPT-45, HH-COSY, CH-COSY, D₂O exchange), and UV–Vis spectroscopy. Cyclic voltammetry in anhydrous CH₃CN revealed a single irreversible cathodic peak at −1.43 V (vs. Ag/Ag⁺), which is consistent with the intramolecular reductive coupling of the azomethine moiety. Density functional theory (DFT) calculations, including MEP mapping, Fukui functions, dual descriptor analysis, and Fukui potentials with dual descriptor potential, identified the exocyclic azomethine carbon as the principal nucleophilic site and the phenolic ring (hydroxyl oxygen and adjacent carbons) as the main electrophilic region. Noncovalent interaction (NCI) analysis further confirmed the strength and geometry of the intramolecular hydrogen bond (IHB). In vitro antimicrobial assays indicated that Fen-IHB was inactive against Gram-negative facultative anaerobes (Salmonella enterica serovar Typhimurium and Typhi, Escherichia coli) and strictly anaerobic Gram-positive species (Clostridioides difficile, Roseburia inulinivorans, Blautia coccoides), as any growth inhibition was indistinguishable from the DMSO control. Conversely, Fen-IHB displayed measurable activity against Gram-positive aerobes and aerotolerant anaerobes, including Bacillus subtilis, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus haemolyticus. Overall, these comprehensive characterization results confirm the distinctive chemical and electronic properties of Fen-IHB, underlining the crucial role of the intramolecular hydrogen bond and electronic descriptors in defining its reactivity profile and selective biological activity.

Clostridioides difficile is a Gram-positive pathogen known for its toxin production and spore formation. It is primarily responsible for most cases of antibiotic-associated diarrhea. Bacterial persisters are a small subset of the population that exhibits transient tolerance to bactericidal substances, and they are of significant medical concern due to their association with the emergence of antibiotic resistance and difficult-to-treat chronic or recurrent infections. Vancomycin, the predominant antibiotic utilized in the management of C. difficile infection, is extensively applied in the realm of clinical practice. Previous studies have demonstrated a persister-like phenotype with treatments involving this antibiotic. However, the mechanism in C. difficile remains largely unknown, primarily due to the challenge of isolating this small population at any given time. To better characterize C. difficile persister cells, we present a study that enables the enrichment and characterization of persister cells from bacterial cultures in both the exponential and stationary phases. Moreover, we could differentiate between triggered (induced using antibiotics such as vancomycin) and spontaneous (stochastic) persister cells. Additionally, we observed the involvement of toxin-antitoxin systems and Clp proteases in persister cell formation.

Clostridioides difficile is a Gram-positive bacterium recognized for its ability to produce toxins and form spores. It is mainly accountable for the majority of instances of antibiotic-related diarrhea. Background. Bacterial persister represent a minor fraction of the population that shows temporary tolerance to bactericidal agents, and they pose considerable medical issues because of their link to the rise of antibiotic resistance and challenging chronic or recurrent infections. Our previous research has shown a persister-like phenotype associated with treatments that include pefloxacin. Nonetheless, the mechanism is still mostly unclear, mainly because of the difficulty in isolating this small group of cells. Objectives. To enhance the understanding of C. difficile persister cells, we made an enrichment and characterization of these cells from bacterial cultures during the exponential phase under pefloxacin treatment and lysis treatment. Results. We demonstrate the appearance of cells with lower metabolism and DNA damage. Furthermore, we noted the participation of toxin–antitoxin systems and Clp proteases in the generation of persister cells. Conclusions. This work demonstrates the formation of C. difficile persister cells triggered by a lethal concentration of pefloxacin.

Clostridioides difficile is a Gram-positive pathogen known for its toxin production and spore formation. It is primarily responsible for most cases of antibiotic-associated diarrhea. Bacterial persisters are a small subset of the population that exhibits transient tolerance to bactericidal substances, and they are of significant medical concern due to their association with the emergence of antibiotic resistance and difficult-to-treat chronic or recurrent infections. Vancomycin, the predominant antibiotic utilized in the management of C. difficile infection, is extensively applied in the realm of clinical practice. Previous studies have demonstrated a persister-like phenotype with treatments involving this antibiotic. However, the mechanism in C. difficile remains largely unknown, primarily due to the challenge of isolating this small population at any given time. To better characterize C. difficile persister cells, we present a study that enables the enrichment and characterization of persister cells from bacterial cultures in both exponential and stationary phases. Moreover, we could differentiate between triggered (induced by antibiotics, such as vancomycin) and spontaneous (stochastic) persister cells. Additionally, we observed the involvement of toxin-antitoxin systems and Clp proteases in persister cell formation.

Metal-free aminobenzoic acid-derived Schiff bases are attractive antimicrobial leads because their azomethine (–C=N–) functionality enables tunable electronic properties and target engagement. We investigated whether halogenation on the phenolic ring would modulate the redox behavior and enhance antibacterial potency, and hypothesized that heavier halogens would favorably tune physicochemical and electronic descriptors. We synthesized three derivatives (SB-3/Cl, SB-4/Br, and SB-5/I) and confirmed their structures using FTIR, ¹H- and ¹³C-NMR, UV-Vis, and HRMS. For SB-5, single-crystal X-ray diffraction and Hirshfeld analysis verified the intramolecular O–H⋯N hydrogen bond and key packing contacts. Cyclic voltammetry revealed an irreversible oxidation (aminobenzoic ring) and, for the halogenated series, a reversible reduction associated with the imine; peak positions and reversibility trends are consistent with halogen electronic effects and DFT-based MEP/LHS descriptors. Antimicrobial testing showed that SB-5 was selectively potent against Gram-positive aerobes, with low-to-mid micromolar MICs across the panel. Among anaerobes, activity was more substantial: Clostridioides difficile was inhibited at 0.1 µM, and SB-3/SB-5 reduced its sporulation at sub-MICs, while Blautia coccoides was highly susceptible (MIC 0.01 µM). No activity was detected against Gram-negative bacteria at the tested concentrations. In the fungal assay, Botrytis cinerea displayed only a transient fungistatic response without complete growth inhibition. In mammalian cells (HeLa), the compounds displayed clear concentration-dependent behavior. Overall, halogenation, particularly iodination, emerges as a powerful tool to couple redox tuning with selective Gram-positive activity and a favorable cellular tolerance window, nominating SB-5 as a promising scaffold for further antimicrobial optimization.