Diletta Mazzantini

Auranofin (AF), an FDA-approved drug for rheumatoid arthritis, exhibits strong antibacterial activity against Gram-positive bacteria, while Gram-negative species remain largely tolerant. This study assessed the antimicrobial activity of AF and three analogues against clinically relevant Gram-negative pathogens and explored tolerance mechanisms in Pseudomonas aeruginosa. Broth microdilution assays were performed on reference strains and clinical isolates of Escherichia coli, Klebsiella pneumoniae, and P. aeruginosa. Synergy studies with the most active analogue, PEt₃AuCl (AF-Cl), were conducted against P. aeruginosa using polymyxin B (PMB), two efflux-pump inhibitors, and two glutathione (GSH) depletors. Gold compounds showed MICs between 4 and >64 µg/mL, with AF-Cl displaying the highest activity. AF-Cl activity was markedly enhanced by PMB and efflux-pump inhibitors, indicating that outer membrane permeability and efflux contribute to tolerance. Additionally, GSH depletion significantly potentiated AF-Cl, implicating redox homeostasis in resistance. Overall, AF-Cl shows potential against Gram-negative bacteria when combined with agents targeting membrane integrity, efflux systems, or redox balance, supporting combinatorial strategies to overcome resistance in P. aeruginosa and related pathogens.

Gut microbiota play crucial roles in host health, including immune regulation, metabolism, and nutrient absorption. Its dysregulation has been linked to various diseases. When administered in sufficient amounts, probiotics can contribute to restoring the gut microbial balance and maintain homeostasis. Species of the genus Bacillus and related genera (Alkalihalobacillus and Heyndrickxia) have been extensively studied and commercialized as probiotics due to their ability to form endospores, the dormant cell forms that provide remarkable resistance to adverse conditions. Understanding the mechanisms of the action of spore-forming probiotics is essential for harnessing their therapeutic potential. This review explores the mechanisms underlying the action of spore-forming probiotics, with a special focus on Alkalihalobacillus clausii. Many beneficial properties such as resilience in extreme conditions, multiplication in the gut, gut adhesion, immunomodulatory effects, the production of bioactive and antimicrobial compounds, as well as efficacy in human health and diseases are extensively dissected. In parallel, this review underscores the limitations of spore-forming probiotics, focusing on safety concerns, issues related to dose standardization and quality control, as well as potential off-target effects and risks in immunocompromised individuals.

Objectives: Gold(I) complexes, such as the drug Auranofin (AF) which is approved for treating rheumatoid arthritis, have attracted significant interest as a potential treatment for bacterial infections due to their promising, broad-spectrum antimicrobial activity. In this study, we investigated the anti-staphylococcal activity of three AF analogues [PEt ₃ AuCl (AF-Cl), PEt ₃ AuI (AF-I) and PPh ₃ AuCl (TPP-AuCl)] with the aim of discovering new weapons in the fight against antibiotic resistance. Methods: The antimicrobial activity and cytotoxicity of the gold compounds were evaluated by broth microdilution and the WST-1 assay, respectively. Time-kill assays were used to investigate killing kinetics, and the crystal violet (CV) assay was used to evaluate biofilm formation. Eradication of mature biofilms was assessed using the crystal violet assay, a plate count of biofilm-associated cells and scanning electron microscopy. The anti-virulence effect was tested by the hemolysis and agar diffusion assays. Results: All of the AF analogues were active against staphylococci, including antibiotic-resistant strains, with minimum inhibitory concentrations (MICs) ranging from 0.063 to 4 µg/mL. Additionally, they exhibited lower toxicity towards the A549 lung cell line and the spontaneously immortalized human keratinocyte line HaCaT than AF. AF-Cl was identified as the most promising compound and was selected for further biological investigations. Time-kill experiments revealed that AF-Cl was rapidly bactericidal against clinical staphylococci, causing at least a 3-log reduction in the number of viable cells within six hours. At sub-inhibitory concentrations, the compound inhibited biofilm formation and reduced the secretion of hemolysins and phospholipases, representing key virulence factors in S. aureus infections. Furthermore, AF-Cl was able to eradicate mature S. aureus biofilms at non-cytotoxic concentrations. Conclusion: Overall, our findings highlight the potential of AF-Cl as a promising candidate for treating staphylococcal infections, including those caused by antibiotic-resistant strains. In addition, the compound exhibited anti-biofilm and anti-virulence properties, which could be advantageous in treating toxin-mediated and biofilm-associated staphylococcal diseases.

Probiotics are microorganisms with recognized beneficial properties that are used to improve host health. In particular, probiotics administered as spores, such as those belonging to the genera Bacillus and Alkalihalobacillus, are attracting great interest due to their high tolerance to gastrointestinal conditions. This in vitro study aimed to assess the probiotic attributes potentially contributing to the in vivo beneficial effects of a commercial spore-based probiotic formulation composed of four Alkalihalobacillus clausii strains. The tolerance and survival of the spores from the formulation in simulated gastrointestinal fluids, as well as their germination rate and adhesion to mucins, were analyzed. Furthermore, metabolic properties of spore-derived vegetative cells were assessed, including lactose degradation and biosynthesis of antioxidant enzymes (catalase and superoxide dismutase), group B vitamins (B₂, B₈, B₉, and B₁₂), short-chain fatty acids (acetate, propionate, and butyrate), and D-lactate. A. clausii spores were shown to survive in artificial gastric juice, adhere to mucins and germinate in vitro, and replicate in simulated intestinal fluid, suggesting their potential resilience in the gastrointestinal tract, where they can exert beneficial effects after germination. A. clausii was also able to produce beneficial enzymes and metabolites, including β-galactosidase, catalase, superoxide dismutase, group B vitamins, and short-chain fatty acids, but it was unable to produce D-lactic acid. Our findings highlight the probiotic properties and potential of such A. clausii strains in both their spore and vegetative forms, reinforcing the clinical relevance of this multi-strain spore-based formulation for enhancing intestinal health.

The flagellar MS‐ring, uniquely constituted by FliF, is essential for flagellar biogenesis and functionality in several bacteria. The aim of this study was to dissect the role of FliF in the Gram‐positive and peritrichously flagellated Bacillus cereus. We demonstrate that fliF forms an operon with the upstream gene fliE. In silico analysis of B. cereus ATCC 14579 FliF identifies functional domains and amino acid residues that are essential for protein functioning. The analysis of a ΔfliF mutant of B. cereus, constructed in this study using an in frame markerless gene replacement method, reveals that the mutant is unexpectedly able to assemble flagella, although in reduced amounts compared to the parental strain. Nevertheless, motility is completely abolished by fliF deletion. FliF deprivation causes the production of submerged biofilms and affects the ability of B. cereus to adhere to gastrointestinal mucins. We additionally show that the fliF deletion does not compromise the secretion of the three components of hemolysin BL, a toxin secreted through the flagellar type III secretion system. Overall, our findings highlight the important role of B. cereus FliF in flagella‐related functions, being the protein required for complete flagellation, motility, mucin adhesion, and pellicle biofilms.

Biofilms are surface-associated microbial communities embedded in a matrix that is almost impenetrable to antibiotics, thus constituting a critical health threat. Biofilm formation on the cornea or ocular devices can lead to serious and difficult-to-treat infections. Nowadays, natural molecules with antimicrobial activity and liposome-based delivery systems are proposed as anti-biofilm candidates. In this study, the anti-biofilm activity of a formulation containing citrus polyphenols encapsulated in liposomes was evaluated against Staphylococcus aureus and Staphylococcus epidermidis, the most common agents in ocular infections. The formulation activity against planktonic staphylococci was tested by broth microdilution and sub-inhibitory concentrations were used to evaluate the effect on biofilm formation using the crystal violet (CV) assay. The eradicating effect of the preparation on mature biofilms was investigated by the CV assay, plate count, and confocal laser scanning microscopy. The product was bactericidal against staphylococci at a dilution of 1:2 or 1:4 and able to reduce biofilm formation even if diluted at 1:64. The formulation also had the ability to reduce the biomass of mature biofilms without affecting the number of cells, suggesting activity on the extracellular matrix. Overall, our results support the application of the used liposome-encapsulated polyphenols as an anti-biofilm strategy to counter biofilm-associated ocular infections.

Examining the interplay between intestinal pathogens and the gut microbiota is crucial to fully comprehend the pathogenic role of enteropathogens and their broader impact on human health. Valid alternatives to human studies have been introduced in laboratory practice to evaluate the effects of infectious agents on the gut microbiota, thereby exploring their translational implications in intestinal functionality and overall health. Different animal species are currently used as valuable models for intestinal infections. In addition, considering the recent advances in bioengineering, futuristic in vitro models resembling the intestinal environment are also available for this purpose. In this review, the impact of the main human enteropathogens (i.e., Clostridioides difficile, Campylobacter jejuni, diarrheagenic Escherichia coli, non-typhoidal Salmonella enterica, Shigella flexneri and Shigella sonnei, Vibrio cholerae, and Bacillus cereus) on intestinal microbial communities is summarized, with specific emphasis on results derived from investigations employing animal and in vitro models.

In vitro models for culturing complex microbial communities are progressively being used to study the effects of different factors on the modeling of in vitro-cultured microorganisms. In previous work, we validated a 3D in vitro model of the human gut microbiota based on electrospun gelatin scaffolds covered with mucins. The aim of this study was to evaluate the effect of Bacillus cereus, a pathogen responsible for food poisoning diseases in humans, on the gut microbiota grown in the model. Real-time quantitative PCR and 16S ribosomal RNA-gene sequencing were performed to obtain information on microbiota composition after introducing B. cereus ATCC 14579 vegetative cells or culture supernatants. The adhesion of B. cereus to intestinal mucins was also tested. The presence of B. cereus induced important modifications in the intestinal communities. Notably, levels of Proteobacteria (particularly Escherichia coli), Lactobacillus, and Akkermansia were reduced, while abundances of Bifidobacterium and Mitsuokella increased. In addition, B. cereus was able to adhere to mucins. The results obtained from our in vitro model stress the hypothesis that B. cereus is able to colonize the intestinal mucosa by stably adhering to mucins and impacting intestinal microbial communities as an additional pathogenetic mechanism during gastrointestinal infection.

This study investigated B. cereus presence in 122 samples belonging to 34 typologies of fresh or short-ripened cheeses made from cow, sheep, goat, or buffalo pasteurized milk, and sold on the Italian market. B. cereus was isolated at a prevalence of 9.8%, with a marked variability among cheese categories, and at low counts (always below 2.26 Log CFU/g). Twelve isolates were identified by MALDI-TOF analysis and typified by RAPD PCR as belonging to different B. cereus strains. All the strains were tested for the production of hemolysin BL, phosphatidylcholine-specific phospholipase C, proteases, and biofilm formation, and for the presence of chromosomal toxin-encoding genes (sph, plcA, cytK, entFM, bcet, nheA, nheB, nheC). Overall, 92% of strains harbored bcet, 75% the three genes nheA, nheB, and nheC, as well as plcA and sph, 67% entFM, and 33% cytK. All strains showed biofilm-forming ability. A chemical-physical characterization of the cheeses was also performed to show their suitability as substrates for B. cereus growth, showing high heterogeneity in terms of pH, aw, salt content, and concentration of organic acids. Finally, the ability to support spore germination and vegetative cell growth of a selected cheese was investigated in spores-inoculated samples maintained at 10 °C and 15 °C, showing the inhibitory effect of low storage temperatures.