Biofilm formation is considered one of the primary virulence mechanisms in Gram-positive and Gram-negative pathogenic species, particularly those responsible for chronic infections and promoting bacterial survival within the host. In recent years, there has been a growing interest in discovering new compounds capable of inhibiting biofilm formation. This is considered a promising antivirulence strategy that could potentially overcome antibiotic resistance issues. Effective antibiofilm agents should possess distinctive properties. They should be structurally unique, enable easy entry into cells, influence quorum sensing signaling, and synergize with other antibacterial agents. Many of these properties are found in both natural systems that are isolated from plants and in synthetic systems like nanoparticles and nanocomposites. In this review, we discuss the clinical nature of biofilm-associated infections and some of the mechanisms associated with their antibiotic tolerance. We focus on the advantages and efficacy of various natural and synthetic compounds as a new therapeutic approach to control bacterial biofilms and address multidrug resistance in bacteria.

Cystic fibrosis (CF) is a life-limiting autosomal recessive disorder affecting a large number of individuals in Europe. The disease arises from mutations in the CFTR gene encoding the cystic fibrosis transmembrane conductance regulator, a chloride ion channel crucial for maintaining epithelial ion and fluid homeostasis. Dysfunctional CFTR disrupts mucociliary clearance, particularly in the respiratory tract, resulting in persistent bacterial colonization, chronic inflammation, and progressive pulmonary damage—ultimately leading to respiratory failure, the principal cause of mortality in CF patients. Early diagnosis and advances in therapy have substantially improved both survival and quality of life. A hallmark of CF pathology is the establishment of polymicrobial infections within the thickened airway mucus. Pseudomonas aeruginosa is the dominant pathogen in chronic CF lung infections and demonstrates a remarkable capacity for adaptation via biofilm formation, metabolic reprogramming, and immune evasion. Biofilms confer increased tolerance to antimicrobial agents and facilitate long-term persistence in hypoxic, nutrient-limited microenvironments. P. aeruginosa exhibits a wide range of virulence factors, including exotoxins (e.g., ExoU, ExoS), pigments (pyoverdine, pyochelin), and motility structures (flagella and pili), which contribute to tissue invasion, immune modulation, and host damage. During chronic colonization, P. aeruginosa undergoes significant genotypic and phenotypic changes, such as mucoid conversion, downregulation of acute virulence pathways, and emergence of hypermutator phenotypes that facilitate rapid adaptation. Persistent cells, a specialized subpopulation characterized by metabolic dormancy and antibiotic tolerance, further complicate eradication efforts. The dynamic interplay between host environment and microbial evolution underlies the heterogeneity of CF lung infections and presents significant challenges for treatment. Elucidating the molecular mechanisms driving persistence, hypermutability, and biofilm resilience is critical for the development of effective therapeutic strategies targeting chronic P. aeruginosa infections in CF.

Traditional milk products, widely consumed in many countries for centuries, have been drawing renewed attention in recent years as sources of bacteria with possible bioprotective properties. One such product for which only limited information exists is the traditional Bulgarian “katak”. This fermented yogurt-like product, renowned for its taste and long-lasting properties, possesses specific sensory characteristics. In this study, 18 lactic acid bacteria (LABs) were isolated from artisanal samples made in the Northwest part of Bulgaria. A polyphasic taxonomic approach combining classical phenotypic and molecular taxonomic methods, such as multiplex PCR, 16S rDNA sequencing, and MALDI-TOF MS, was applied, leading to the identification of 13 strains. The dominance of Lactiplantibacillus plantarum was confirmed. In vitro tests with the identified strains in model systems showed a promising broad strain-specific spectrum of activity against food-borne and human pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli). Non-purified Lactobacillus postbiotics, produced during fermentation in skimmed and soya milks and in MRS broth, were estimated as limiting agents of virulence factors. The LAB’s production of lactate, acetate, and butyrate is a promising probiotic feature. A further characterization of the active strains and analysis of the purified post-metabolites are needed and are still in progress.

Biofilms are a well-known multifactorial virulence factor with a pivotal role in chronic bacterial infections. Their pathogenicity is determined by the combination of strain-specific mechanisms of virulence and the biofilm extracellular matrix (ECM) protecting the bacteria from the host immune defense and the action of antibacterials. The successful antibiofilm agents should combine antibacterial activity and good biocompatibility with the capacity to penetrate through the ECM. The objective of the study is the elaboration of biofilm-ECM-destructive drug delivery systems: mixed polymeric micelles (MPMs) based on a cationic poly(2-(dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA₃₅-b-PCL₇₀-b-PDMAEMA₃₅) and a non-ionic poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO₁₀₀-b-PPO₆₅-b-PEO₁₀₀) triblock copolymers, loaded with ciprofloxacin or azithromycin. The MPMs were applied on 24 h pre-formed biofilms of Escherichia coli and Pseudomonas aeruginosa (laboratory strains and clinical isolates). The results showed that the MPMs were able to destruct the biofilms, and the viability experiments supported drug delivery. The biofilm response to the MPMs loaded with the two antibiotics revealed two distinct patterns of action. These were registered on the level of both bacterial cell-structural alterations (demonstrated by scanning electron microscopy) and the interaction with host tissues (ex vivo biofilm infection model on skin samples with tests on nitric oxide and interleukin (IL)-17A production).

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium and also an opportunistic pathogen for both humans and animals, causing acute or chronic infections. It has been frequently detected in healthy and diseased reptiles, more commonly in captive ones. Since most studies are primarily on clinical isolates, the pathogenic potential of strains originating from wild animals is poorly explored. We isolated the strain P. aeruginosa PM1012 from the cloacal microbiota of a common wall lizard (Podarcis muralis Laurenti, 1768) from a free-living population. The effect of temperature, pH and salinity on its growth was evaluated. Antibiotic resistance, the expression of several virulence factors as some extracellular enzymes, pyocyanin production and biofilm formation were also assessed. Apart from intrinsic resistance, the newly isolated strain P. aeruginosa PM1012 presented an antibiotic susceptibility profile with a low resistance rate limited to meropenem and intermediate to ceftazidime and aztreonam. Protease, lipase and gelatinase secretion was detected. Strong pyocyanin production was observed in the optimal range of growth conditions. An excellent biofilm-forming capacity was manifested.

This study examines the impact of inhaled tobramycin therapy on the within-host changes in P. aeruginosa strains isolated from Bulgarian patients with CF prior to and post treatment. Genotypic comparison by RAPD-PCR indicated that most of the pre-treatment isolates had a high similarity and were genetically comparatively close to strains from other countries with known increased morbidity or treatment requirements. Most of the post-treatment isolates were, however, genetically distant from their pre-treatment counterparts, showing genotypic diversification after the treatment. Phenotypic comparisons showed a lower ODmax reached during groswth and an increased lag-time in the post-treatment isolates. All strains were capable of invasion and intracellular reproduction within A549 cultured cells. The addition of sub-inhibitory amounts (1/4 or 1/2 MIC) of tobramycin during growth showed the higher relative fitness (as a percentage of the untreated control) of the post-treatment strains. The effects of sub-MICs on biofilm growth did not show such a pronounced trend. However, when a resazurin-based viability test was applied, the advantage of the post-treatment strains was confirmed for both broth and biofilm cultures. In spite of that, according to the determined MIC values, all isolates were tobramycin-sensitive, and the data from this study imply the development of tolerance to the antibiotic in the strains that survived the treatment.