Malassezia pachydermatis is a yeast pathogen associated with recurrent skin and ear infections in dogs, often complicated by biofilm formation and reduced antifungal susceptibility. We aimed to evaluate the in vitro antifungal activity of essential oils and nanoemulsions of Zingiber cassumunar and Cymbopogon citratus compared with conventional antifungal agents against planktonic and biofilm forms of M. pachydermatis. Preliminary screening of six plant extracts was performed using 12 clinical isolates identified Z. cassumunar and C. citratus for nanoemulsion formulation. Antifungal susceptibility testing of conventional antifungal agents and nanoemulsions was subsequently conducted using 31 clinical isolates, and nanoemulsions were prepared by high-pressure homogenization. Both essential oils exhibited antifungal activity, and nanoemulsion formulations showed enhanced inhibitory effects compared with the crude oils. Biofilm-associated cells demonstrated reduced susceptibility, particularly to conventional antifungal agents. Terbinafine was the most potent agent against planktonic cells but showed reduced efficacy in biofilms. Nanoemulsions of Z. cassumunar and C. citratus exhibited improved activity against both forms. These findings suggest that nanoemulsification may enhance the in vitro antifungal performance of essential oils against M. pachydermatis biofilms. However, further studies, including mechanistic investigations and in vivo evaluations, are required to confirm their therapeutic potential and safety.

Comparative fermentation performance of biofilm and planktonic Saccharomyces cerevisiae under standard and high-carbon media.

Effect of surfactants on the antimicrobial activity of hydrogen peroxide and chlorine dioxide foams against Listeria monocytogenes on stainless steel.

Cleaning and disinfection of food production environments (FPE) are fundamental components of food safety programmes designed to control microbial pathogens and prevent food contamination. Yet, FPE can still harbour foodborne pathogens, including Listeria monocytogenes, a significant concern to food manufacturers and health authorities due to the high mortality rate associated with invasive listeriosis. Mechanisms contributing to L. monocytogenes persistence in FPE include biofilm formation and reduced susceptibility to biocides, such as benzalkonium chloride (BC), for which several mechanisms are known. We hypothesized that prolonged exposure to disinfectants and other FPE-associated stressors would drive L. monocytogenes adaptation, resulting in the accumulation of genetic mutations linked to biofilm formation and reduced biocide susceptibility. To test this, we developed a biofilm persistence model, which studied 30 consecutive passages of biofilm-associated cells grown on stainless steel under sub-inhibitory BC concentrations. Whole-genome sequencing of evolved populations identified mutations that were associated with biofilm lineages and/or BC exposure. Non-synonymous mutations were identified in genes and pathways involved in metal homeostasis, stress response and pyrimidine biosynthesis. In addition, reduced susceptibility to BC arose through multiple independent mutations within the fepRA operon, encoding FepR transcriptional repressor and FepA MATE efflux pump. These mutations were observed across both planktonic and biofilm lifestyles, resulting in a comparable level of reduced susceptibility to BC in both states. Several loci with fixed mutations associated with biofilm lineages were identified, including the ykoK riboswitch leader, the pyrimidine synthesis operon and the stress response-related gene rsbU. Collectively, these findings provide new insights into the genetic mechanisms underlying L. monocytogenes biofilm persistence and reduced biocide susceptibility in the context of FPE and reveal novel targets potentially exploited by L. monocytogenes to establish and maintain niches in unfavourable environments.

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 [PEt3AuCl (AF-Cl), PEt3AuI (AF-I) and PPh3AuCl (TPP-AuCl)] with the aim of discovering new weapons in the fight against antibiotic resistance. 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. 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. 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.

Vibrio cholerae, the causative agent of cholera, poses a continuous threat to global public health, especially in regions with poor sanitation. Its ability to form biofilms and rapidly acquire antimicrobial resistance (AMR) complicates therapeutic interventions. In this study, the quorum-sensing (QS) response regulator LuxO was targeted for anti-virulence therapy using a synthesized pyridine-based compound, QSIpy. Pyridine derivatives are explored as inhibitors that can target the LuxO protein, a key regulator in the QS pathway of V. cholerae. By inhibiting LuxO, these compounds reduced the virulence factor expression and biofilm formation, offering a novel antivirulence strategy without promoting resistance. Molecular docking showed that QSIpy exhibited a strong binding affinity, with a glide score of −5.046 at the ATP-binding domain of LuxO. In vitro evaluation revealed that QSIpy had no inhibitory effect on planktonic bacterial growth, indicating a non-bactericidal mechanism. However, it showed significant inhibition of biofilm formation, as confirmed by crystal violet assay and quantified through MBIC determination. Pellicle CFU enumeration demonstrated a reduction in viable biofilm-associated cells, particularly at the lowest concentrations of 15.6 µg mL−1 and 31.2 µg mL−1, while fluorescence microscopy validated the loss of pellicle integrity. Additionally, checkerboard synergy testing with azithromycin (AZM) revealed a high synergy score based on Bliss independence modelling, whereas no synergy was observed with ciprofloxacin or doxycycline. These results suggest that QSIpy potentiates macrolide antibiotic activity by interfering with quorum-sensing-regulated biofilm and virulence expression.

Bacterial biofilms are resilient multicellular communities that underlie persistent infections and environmental survival. Dispersal from biofilms is a pivotal event for transmission and pathogenesis, yet the host signals and bacterial mechanisms orchestrating this transition remain poorly understood. Here, we show that nitric oxide (NO), a ubiquitous host-derived signaling molecule, acts as a rapid trigger for biofilm dispersal in Vibrio cholerae, a highly motile gram-negative bacterium and the etiologic agent of cholera, by promoting the development of motility. NO exposure induces broad upregulation of flagellar biosynthesis genes, increases flagellin production, and reduces intracellular cyclic-di-GMP levels, thereby priming aflagellated biofilm-associated cells for active swimming and dispersion. Using single-cell imaging in custom microfluidic devices, we directly visualize NO-stimulated biofilm detachment and development of robust swimming motility within minutes. In vivo, biofilm-derived V. cholerae colonize more efficiently in NO-rich environments, and NO produced by epithelial cells enhances bacterial detachment from epithelial surfaces. Our findings reveal a host-pathogen interface in which NO serves as a morphogenetic cue, orchestrating the rapid transition from sessility to motility.

A rapid and efficient method for visualisation of microbial biofilms on natural, and industrially- and medically-relevant surfaces, using field emission- scanning electron microscopy.

Transcriptomic analysis of mature biofilm and planktonic cells of Listeria monocytogenes under nutritional stress.

Candida auris promotes Pseudomonas aeruginosa tolerance to meropenem in a mature dual-species biofilm.

A fundamental technical challenge in detecting pathogenic bacteria in aquatic reservoirs is the inability to accurately estimate biofilm-associated cells in water. Considering the role of biofilms in environmental persistence and waterborne transmission of bacterial pathogens, there is an increasing interest in substances that can effectively degrade bacterial biofilms. The biofilm-dispersing Vibrio cholerae phage JSF7 was analyzed by whole genome sequencing and found to carry a gene predicted to encode an enzyme for degrading complex polysaccharides. The gene was cloned in Escherichia coli DH5α, and crude extract from the recombinant E. coli enhanced the dispersion of diverse bacterial biofilms, including those of E. coli, Shigella dysenteriae, Pseudomonas aeruginosa, and V. cholerae. The crude extract was fully active at a temperature of 37°C and pH of 7.0 but was inactivated by proteinase-K treatment. Analysis of environmental water samples for the presence of V. cholerae O1 by enrichment culture detected significantly more V. cholerae O1-positive samples when the enrichment medium was supplemented with the extract, as compared with typical enrichment without the extract. These results suggest that a crude preparation of the phage-encoded biofilm-degrading factor expressed in E. coli has potential application in degrading bacterial biofilms and enhancing bacteriological analysis of water.IMPORTANCEIn their aquatic reservoirs, bacteria often exist as biofilms and are difficult to accurately detect by culturing water samples. Such biofilms have been implicated in waterborne transmission of pathogenic bacteria. We identified a bacteriophage that can disintegrate biofilms and disperse biofilm-associated bacteria. The putative phage gene responsible for this activity was cloned in an E. coli strain, and the crude cellular extract of the recombinant E. coli was found to promote dispersion of a variety of bacterial biofilms. Supplementation of bacterial growth medium with the crude extract also enhanced detection of V. cholerae O1, the causative agent of cholera in environmental water samples. The ability of a phage-derived biofilm-degrading factor to disperse diverse bacterial biofilms provides a novel approach for enhancing detection of waterborne bacterial pathogens in water beyond traditional enrichment methods.

Legionella affects biofilm structural response to detachment upon shear stress increase

Dynamics of mono and dual-species biofilms of Escherichia coli and Salmonella Typhimurium: Interspatial interactions and novel inhibition strategies.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant threat in oncology settings due to its multidrug resistance and ability to form biofilms on indwelling medical devices. This study investigated the in vitro and in vivo activity of meropenem/vaborbactam (MEV) against two CRKP isolates recovered from catheter-related bloodstream infections in patients undergoing orthopedic oncologic surgery. Whole-genome sequencing identified the isolates as ST101 and ST307, harboring resistance determinants including blaKPC-3 and blaOXA-1 , distributed across IncFII and IncFIB plasmid replicons. Both isolates exhibited extensive resistance to β-lactams, aminoglycosides, and fluoroquinolones but remained susceptible to MEV. Phenotypic assays revealed enhanced biofilm formation and metabolic activity compared to the reference strain Kp ATCC 13883 in the absence of hypervirulence-associated genes. MEV demonstrated bactericidal activity against both planktonic and biofilm-associated cells, with minimum bactericidal concentration (MBC90) and minimum biofilm eradication concentration (MBEC90) values of 0.5/8 μg/ml for CRKP ST101, 0.12/8 μg/ml for CRKP ST307, and 0.25/8 μg/ml for the Kp ATCC 13883 strain. In the Galleria mellonella infection model, MEV significantly improved larval survival following the CRKP challenge. These findings demonstrate that MEV exhibits activity against planktonic and biofilm-associated CRKP cells and highlight the need for further investigation in managing catheter-related bloodstream infections caused by multidrug-resistant K. pneumoniae.

Conventional antibiotic drug discovery selects leads based on bacterial growth inhibition. This approach is ineffective against growth-arrested persister cells. When the treatment stops, persister cells revert to normal cells, causing the infection to relapse. To address the challenge of persistent infections, a paradigm shift in antibiotic development is needed to identify new leads that can eradicate dormant cells. Based on our foundational study, we recently proposed a set of principles for developing new persister killing agents. Here, we report the discovery of new leads that are effective against persister cells using a tailored chemoinformatic clustering algorithm based on these principles. We focused on persister penetration using a small compound library that has known antimicrobial activities against normal cells. Experimental testing of eleven compounds identified from clustering led to the discovery of five new compounds that can effectively penetrate and kill persister cells of Escherichia coli HM22. The top leads were further tested and also found active against persister cells of Pseudomonas aeruginosa and uropathogenic E. coli (UPEC), as well as UPEC biofilms and biofilm-associated persister cells. This rather high yield demonstrates the potential of this new rational approach in identifying effective agents against dormant cells, a root cause of persistent infections that is largely missed in conventional screening.

Clostridioides difficile is an anaerobic, spore-forming pathogen responsible for illnesses ranging from mild diarrhea to life-threatening colitis. Current treatments rely on antibiotics such as vancomycin and metronidazole (MTZ), but high doses can disrupt gut microbiota, contributing to recurrent infections. Mitomycin C (MMC), a Food and Drug Administration-approved anticancer agent, is known to induce prophage activation in lysogenic bacteria. Given that over 70% of C. difficile strains harbor prophages, we evaluated MMC's potential to enhance antibiotic efficacy against C. difficile infection (CDI). In vitro, MMC synergized with MTZ to inhibit strain R20291 and clinical isolates of RT027 and RT078 while reducing the minimum bactericidal concentration of MTZ against biofilm-associated cells. Ex vivo assays using mouse fecal suspensions confirmed the enhanced killing effect of the combination. In a murine recurrence model, low-dose MTZ + MMC treatment significantly improved survival and reduced fecal spore counts compared to monotherapies or vancomycin. Importantly, the combination did not cause greater liver or kidney toxicity than other antibiotics and resulted in less colonic epithelial damage. Microbiota profiling revealed that MTZ + MMC better preserved gut microbial composition than standard treatments. These findings suggest that low-dose MTZ combined with MMC enhances antimicrobial efficacy while reducing toxicity and microbiota disruption, offering a promising strategy for CDI management.

The biofilm-associated infections pose a great threat to human health. The available drugs are not effective due to the formation of biofilm and limited access to underlying pathogens. The initiation of biofilm formation occurs through adhesion, facilitated by the adhesin protein MrkD1P in the fimbriae tip. This study targeted the MrkD1P protein and employed plant phenols to inhibit biofilm formation in Escherichia coli, Salmonella typhi, and Klebsiella pneumoniae, as major Enterobacteriaceae species. A homology model was constructed for the MrkD1P protein, and 44 phenolic derivatives were assessed for their interaction with this protein. Caffeic acid and 3-hydroxybenzoic acid exhibited the best binding-free energies of 29.61kcal/mol and 24.24kcal/mol, respectively. Using a microtiter plates-based minimum biofilm inhibitory concentration assay, it was found that doses of these compounds ranging from 2 to 256mg/mL effectively reduced biofilm formation. The biofilm inhibition assay demonstrated over 80% reduction of biofilms in all tested species at inhibitory doses. Further analysis through field emission gun scanning electron micrographs revealed that the compounds disintegrated fimbriae on cell surfaces. Additionally, the re-formation assay demonstrated the inability of biofilm-associated cells to re-form the biofilm on fresh surfaces due to fimbriae inhibition. This study highlights the antibiofilm capabilities of caffeic acid and 3-hydroxybenzoic acid, indicating their potential as effective treatments for illnesses caused by Enterobacteriaceae biofilms.

Biofilms are structured microbial communities embedded in a self-produced extracellular matrix. This lifestyle provides significant protection against environmental stressors such as desiccation, chemical treatments and even ionizing radiation. Radiation, while a well-established antibacterial strategy, can be less effective in biofilms. Biofilm superior resilience is due to several advantages such as the shielding provided by the matrix, the metabolic heterogeneity and adaptive stress responses of biofilm-associated cells. To address this challenge, researchers are increasingly employing combination strategies in antibiofilm treatment. Radiosensitizers, compounds originally developed to enhance the efficacy of radiation therapy in cancer treatment, have also garnered attention for their potential in antimicrobial applications. These compounds act by amplifying the effects of radiation, often through mechanisms such as increased oxidative stress or inhibition of DNA repair pathways. However, research on radiosensitizers in bacterial systems has focused on planktonic cultures, with limited studies exploring their effects on biofilms. Given the complexity and unique characteristics of biofilms, their response to radiosensitization remains poorly understood and requires further investigation. The use of radiosensitizers in conjunction with radiation presents a promising approach to overcome the inherent resilience of biofilms. By enhancing the susceptibility of biofilm-associated bacteria to radiation and simultaneously disrupting their protective structures, such approaches could lead to more effective and comprehensive solutions. Understanding the nuanced responses of biofilms to these combined treatments is essential for advancing both medical and environmental applications and addressing the challenge of biofilm persistence.

Chlorhexidine, an antimicrobial with a broad inhibitory spectrum, is commonly used to treat oral infections as an active ingredient in mouthwash. While typically used at high concentrations (1–2 mg/ml), oral bacteria can be exposed to sublethal concentrations due to the bioavailability and protective barrier of biofilms (dental plaques). Sublethal concentrations can cause transcriptional remodelling of bacteria such as Streptococcus mutans, a key player in dental caries. Using an RNA-seq approach, this report provides a compendium on the effect of sublethal concentrations of chlorhexidine on the transcriptome of S. mutans as planktonic cells and in biofilm states. Streptococcus mutans showed major transcriptional remodelling between planktonic and biofilm states. The transcriptional response towards chlorhexidine was more pronounced in planktonic cells compared to sessile cells. However, the response observed for biofilm-associated cells was not specific to chlorhexidine, as the transcriptional response in biofilms exposed to the β-lactam amoxicillin was similar to those observed for chlorhexidine. Furthermore, we found that S. mutans modulates the transcription of a multitude of ABC transporters in both planktonic and biofilm-associated cells upon exposure to these antimicrobials.

Characterisation of four novel bacteriophages targeting carbapenem-resistant Klebsiella pneumoniae and their lytic activity alone and in combination