Publications

Abstract Multidrug-resistant bacteria in biofilms are a growing public health threat, due to their resistance to conventional antibiotics. Phytochemicals are attractive candidates because of their structural diversity and ability to potentiate antimicrobial activity. This study investigated the antibiofilm and resistance-modifying effects of two monoterpenes, menthol and linalool, alone and in combination with ten antibiotics, against Escherichia coli and Staphylococcus epidermidis. Menthol exhibited MIC and MBC of 800 mu g/mL against E. coli and the same MIC against S. epidermidis, while linalool showed MICs of 800 mu g/mL and 400 mu g/mL, respectively. Combination assays revealed enhanced activity of erythromycin with both monoterpenes against E. coli and of amoxicillin with menthol against S. epidermidis, although sessile cells were largely unaffected. When applied individually, both monoterpenes caused a 3-log reduction in culturable E. coli biofilm cells. The overall findings highlight the antibiofilm activity of linalool and, particularly, menthol, supporting their role as antibiotic adjuvants against biofilm-associated infections.

Abstract Quorum sensing (QS) is a bacterial cell-to-cell communication induced at high cell density, commonly involved in regulating gene expression of spoilage and pathogenic virulence factors. In dairy products, Pseudomonas species grow under cold conditions and produce thermostable proteases. The QS pathway in Pseudomonas represents a potential target to limit protease synthesis during raw milk storage prior to heat processing. This study aims to characterize AHL-mediated QS system in highly proteolytic Pseudomonas isolated from raw milk and to explore the use of polyphenols as a strategy to control proteolytic activity. Six isolates with high proteolytic activity were identified as Pseudomonas gessardii by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and 16S rRNA sequencing. Biosensor strains, thin layer chromatographyoverlay assay, and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis were used to explore QS, revealing C4-HSL as the main type of AHL produced by P. gessardii. Salicylic acid (SA), cinnamaldehyde (CIN), and tannic acid (TA) were tested as QS inhibitors (QSIs) and the mechanism verified by in silico analysis. Various degrees of proteolytic activity inhibition were observed at 4 degrees C and 25 degrees C by using QSIs (15-58 % by SA, 10-60 % by CIN), with no antibacterial effect. The mechanism behind that is the competition with the C4-HSL to bind with the receptor protein (LuxR) as corroborated by the in silico analysis. The results highlight the potential to employ polyphenols to restrict proteolytic activity by psychrotrophic Pseudomonas in dairy products.

Abstract Pseudomonas aeruginosa biofilm-associated infections present higher recalcitrance to antimicrobial treatments, contributing to persistent and difficult-to-treat infections. Quorum sensing (QS) regulates various cellular processes that are important for the establishment and survival of microbial communities on the host. However, QS inhibitors for the treatment of P. aeruginosa biofilms remain under-researched, partly due to the complexity of QS signalling pathways and the challenge of developing non-toxic inhibitors. Herein, the bioactivity of 2-{(2E)-2-[1-(pyridin-2-yl)ethylidene]hydrazinyl}-1,3-thiazole-4-carboxylic acid (TTNF37), a novel pyridine-based thiazolyl-hydrazone (PTH), was investigated. The compound antimicrobial activity was evaluated against a broad spectrum of microorganisms, its antioxidant potential was assessed using different assays, and its QS-inhibitory effect on P. aeruginosa was studied using bioreporter strains. The effect on P. aeruginosa biofilm formation was analysed in terms of biomass, culturability, and metabolic activity, structure, and cell membrane integrity, while virulence factors were evaluated through absorbance measurements. In addition, molecular docking studies were performed to predict the drug's interactions with essential QS proteins and biological targets. TTNF37 exhibited potent antimicrobial activity with low to moderate minimum inhibitory concentrations against clinically relevant Gram-negative and Gram-positive bacteria, as well as fungi and yeasts. It also showed antioxidant activity, with variable effectiveness across different radicals and systems. TTNF37 inhibited the 3-oxo-C12-HSL-dependent QS system of P. aeruginosa in a dose-dependent manner, with reductions ranging from 26% to 98%. It also impaired the production and detection of 3-oxo-C12-HSL, resulting in a 56% and 65% decrease in bioluminescence, respectively. Molecular docking studies revealed strong binding interactions with LasI and LasR proteins, with affinity values exceeding those of furvina, a known potent QS inhibitor. Molecular dynamics simulations validated stable TTNF37 binding to LasR and LasI. Both experimental and docking data indicate a significant interaction with human serum albumin (HSA). TTNF37 also significantly reduced pyocyanin production and prevented biofilm set-up with a reduction of 50% in biomass with pronounced alterations in biofilm structure. These results indicate the potential of TTNF37 and related PTHs for treating biofilm-associated infections.

Abstract Diabetic foot ulcers (DFUs) represent a significant global burden, associated with high morbidity and increased mortality. More than half of DFUs become infected by polymicrobial communities, in which Pseudomonas aeruginosa and Staphylococcus aureus form resilient biofilms. Antimicrobial photodynamic inactivation (aPDI) using blue light is promising, its efficacy against polymicrobial biofilms remains suboptimal in infected DFUs. This study evaluated, for the first time, the activity of a berberine-gentamicin (Ber-Gen) combination under blue light photoactivation against dual-species P. aeruginosa MJMC568-A and S. aureus MJMC568-B biofilms, both isolated from a DFU patient. First, the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) for each agent against pre-formed dual-species biofilms were determined. Ber and Gen alone did not reach MBIC or MBEC at concentrations <2000 & micro;g mL(-1), but in combination, MBIC values decreased two-fold to 1000 & micro;g mL(-1) for Ber and 1024 & micro;g mL(-1) for Gen. The combinatorial effect was assessed by checkerboard (CKB), with Ber-Gen resulting in a synergistic effect for MBIC values. The optimised concentrations from CKB were tested under one, two, and three irradiation cycles (with a 24 h interval between irradiation cycles) of blue light at 30 mW cm(-2) for 10 min per cycle (18 J cm(-2)). Antibiofilm activity was quantitatively assessed by biomass (crystal violet), metabolic activity (alamar blue), and culturability (colony-forming unit (CFU cm(-2)) counts). Photoactivated Ber-Gen produced strong reductions in biomass, metabolic activity, and culturability after one cycle (approximate to 50%, approximate to 70%, and approximate to 5 log CFU cm(-2), respectively), near-complete eradication after two cycles (approximate to 60%, approximate to 80%, and approximate to 6 log CFU cm(-2), respectively), and a further effect after three cycles (approximate to 90%, approximate to 95%, and approximate to 10 log CFU cm(-2), respectively). Regrowth assays showed full recovery after one cycle, about half recovery after two, and less than 10% recovery after three cycles. Mechanistic assays on the antibiofilm effect included measurement of reactive oxygen species (ROS) by fluorometry, membrane integrity by flow cytometry and confocal microscopy, matrix components by confocal microscopy, spectrophotometric and fluorometric assays, and architecture by optical coherence tomography. Biofilm structure was markedly disrupted, with strong reductions in thickness, extracellular matrix components such as proteins, polysaccharides, and eDNA. These structural changes coincided with a decrease in biofilm cells' membrane integrity and increased ROS production. Overall, Ber-Gen-mediated blue light aPDI exhibits strong activity against dual-species biofilms of P. aeruginosa and S. aureus.

Abstract Aims Antimicrobial resistance, particularly in healthcare-associated infections (HAIs), highlights the need for more effective and sustainable disinfection strategies. This study aimed to evaluated the bactericidal effects of two conventional biocides-benzalkonium chloride (BAC) and peracetic acid (PAA)-and two phytochemicals-salicylic acid (SAL) and eugenol (EUG)-against Escherichia coli and Staphylococcus aureus, focusing on potential synergistic interactions.Methods and results Antimicrobial efficacy was assessed through standardized dose- and time-response assays (EN 1276) and kinetic modeling using the Chick-Watson and Weibull equations. BAC and PAA achieved complete bacterial inactivation at 3 mg/L and 1 mg/L, respectively, within 5-15 min, confirming rapid and potent activity. Conversely, SAL and EUG required substantially higher concentrations (500-1700 mg/L) to achieve total loss of culturability. Chick-Watson modeling demonstrated high disinfection rate coefficients for BAC and PAA, while SAL and EUG exhibited markedly lower values. Synergy testing revealed a strong interaction between BAC and EUG, with fractional bactericidal concentration indices of 0.350 for E. coli and 0.309 for S. aureus, whereas other combinations were additive. Weibull modeling further indicated that bacterial tolerance was dependent on compound type and concentration, with S. aureus generally more susceptible than E. coli.Conclusions These findings collectively confirm the enhanced efficacy of selected biocide/phytochemical combinations, allowing lower concentrations and promoting more sustainable antimicrobial practices.

Abstract Introduction Staphylococcus aureus is an important opportunistic pathogen whose ability to form biofilms and acquire multidrug resistance limits the effectiveness of conventional antibiotics. Although some antibiotic combinations have shown promise, their clinical use is limited by pharmacokinetic and pharmacodynamic issues, chemical instability and formulation challenges. Objectives This study aimed to develop novel hybrid molecules combining ciprofloxacin with plant-derived monoterpenoids to improve antibacterial and antibiofilm efficacy against S. aureus strains. Methods Based on preliminary antibiotic-terpenoids combination studies in S. aureus CECT 976, citronellol and geraniol were selected for hybrid synthesis. The minimum inhibitory concentration (MIC) of the hybrids was evaluated in ciprofloxacin-susceptible ( S. aureus CECT 976) and resistant strains ( S. aureus 8093). Furthermore, the antibiofilm efficacy was analysed in the prevention and eradication of pre-established biofilms by quantifying biomass, metabolic activity and culturability. Moreover, their impact on biofilm structure and membrane integrity was investigated using optical coherence tomography, flow cytometry and epifluorescence microscopy, respectively. Furthermore, matrix components were analysed by spectrophotometry and confocal laser scanning microscopy (CLSM). The cytotoxic and haemolytic profile of the hybrids were evaluated using mammalian cells and red blood cells, respectively. Results For the resistant strain (ciprofloxacin MIC > 1024 µg mL−1), the hybrids showed MICs of 16 µg mL−1, indicating a limited shift in activity compared to the susceptible strain (MIC = 8 µg mL−1). For pre-established biofilms of the S. aureus resistant strain, where ciprofloxacin was ineffective, the hybrids reduced up to 40 % of biomass, inhibited 90 % of metabolic activity and reduced culturability by ∼ 2.3 log(CFU cm−2). They also disrupted the biofilm structure, with an average thickness of less than 10 µm in both strains tested. Furthermore, the hybrids caused significant damage to the cell membrane (90 % in the susceptible strain and about 35 % in the resistant strain) and exhibited low toxicity in HepG2 cells. Consistent with confocal observations, alterations in matrix composition were also observed in the presence of the hybrid compounds. Conclusion This study highlights the potential of monoterpenoid-ciprofloxacin hybrids that combine the antibacterial effects of ciprofloxacin with the bioactive properties of monoterpenes, potentially affecting S. aureus biofilms through multiple mechanisms. Copyright © 2026. Published by Elsevier B.V.

Abstract Antimicrobial photodynamic inactivation (aPDI) is a promising strategy for combating chronic wound infections, particularly those involving biofilms. However, its efficacy against Pseudomonas aeruginosa biofilms is limited by poor light penetration and photosensitisers through the biofilm matrix. This study explores, for the first time, the combination of subinhibitory colistin (Col) concentrations with berberine/curcumin (Ber/Cur) and gentamicin (Gen) to enhance aPDI activity against P. aeruginosa biofilms, including a clinical strain from a diabetic foot ulcer. Membrane integrity changes induced by Col were assessed by flow cytometry with propidium iodide. Synergistic concentrations of Ber/Cur-Gen-Col were determined via checkerboard assay. The effects of blue light (420 nm, 30 mW/cm(2), 10 min) on these combinations were evaluated for both biofilm prevention and eradication assays. For established biofilms, one or three irradiation cycles were applied at 24 h intervals, followed by an assessment of regrowth after 48 and 72 h. Biomass, metabolic activity, and culturability were quantified using crystal violet, resazurin, and colony-forming units (CFU). The mode of action was investigated via ROS production, membrane disruption, and biofilm structural changes using fluorometry, microscopy, and optical coherence tomography. Col at subinhibitory concentrations reduced membrane integrity (approximate to 30 %), enhancing Ber/Cur-Gen efficacy. Photoactivated Ber/Cur-Gen-Col inhibited biofilm formation (>90% reduction in biomass and metabolic activity and 6-log reduction in CFU). Against pre-formed biofilms, this combination achieved a 7-log reduction in CFU and a 90% reduction in biomass and metabolic activity. Three irradiation cycles sustained inhibition for up to 72 h. Ber-Gen-Col showed superior performance, highlighting the synergistic potential of subinhibitory Col in enhancing aPDI as a promising strategy for managing persistent wound infections.

Abstract Aim This study investigates the mechanisms of action of a promising series of previously synthesized quaternary ammonium (QASs) and phosphonium (QPSs) salts, which have shown potent activity against Staphylococcus aureus, including methicillin-resistant strains (MRSA).Methods and results The effects of QASs and QPSs on S. aureus surface charge, total surface hydrophobicity, intracellular potassium release, membrane integrity, and ultrastructure were examined. QASs and QPSs significantly altered bacterial surface properties by reducing negative surface charge, disrupting membrane integrity, and inducing potassium leakage and propidium iodide uptake. Furthermore, S. aureus became less hydrophilic due to changes in surface hydrophobicity. Transmission electron microscopy revealed cytoplasmic leakage and the presence of electron-dense extracellular material around damaged bacterial cells upon exposure to high concentrations of these salts.Conclusions The antimicrobial activity of QASs and QPSs is driven by their ability to alter bacterial surface properties, destabilizing and disrupting membranes.

Abstract Iron is essential for the formation, maturation and dispersal of bacterial biofilms, playing a crucial role in the physiological and metabolic functions of bacteria as well as in the regulation of virulence. Limited availability of iron can impair the formation of robust biofilms by altering cellular motility, hydrophobicity and protein composition of the bacterial surface. In this study, the antibiofilm activity of two natural iron chelating agents, kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) and maltol (3-hydroxy-2-methyl-4-pyrone), were investigated against Staphylococcus aureus and Pseudomonas aeruginosa. In addition, the ability of these 2-hydroxy-4-pyrone derivatives in preventing and eradicating S. aureus and P. aeruginosa biofilms through the enhancement of the efficacy of two antibiotics (tobramycin and ciprofloxacin) was explored. The iron binding capacity of the kojic acid and maltol was confirmed by their affinity for iron (III) which was found to be about 90 %, comparable to the regular chelating agent ethylenediaminetetraacetic acid (EDTA, 89 %). The antibiofilm efficacy of 2-hydroxy-4-pyrone derivatives, alone and in combination with antibiotics, was evaluated by measuring the total biomass, metabolic activity, and culturability of biofilm cells. Furthermore, their impact on the membrane integrity of S. aureus biofilm cells was investigated using flow cytometry and epifluorescence microscopy with propidium iodide staining. It was also examined the ability of 2-hydroxy-4-pyrone derivatives and 2-hydroxy-4-pyrone derivate-antibiotic dual-combinations in inhibiting the production of virulence factors (total proteases, lipases, gelatinases and siderophores) by S. aureus. Regarding biofilm formation, the results showed that 2-hydroxy-4-pyrone derivatives alone reduced the metabolic activity of S. aureus biofilm cells by over 40 %. When combined with tobramycin, a 2-log (CFU cm-2) reduction in S. aureus biofilm cells was observed. Moreover, the combination of maltol and kojic acid with ciprofloxacin prevented P. aeruginosa biomass production by 60 %, compared to 36 % with ciprofloxacin alone. In pre-established S. aureus and P. aeruginosa biofilms, selected compounds reduced the metabolic activity by over 75 %, and a 3-log (CFU cm-2) reduction in the culturability of biofilm cells was noted when kojic acid and maltol were combined with antibiotics. Moreover, 2-hydroxy-4-pyrone derivatives alone and in combination with tobramycin, damaged the cell membranes of pre-established biofilms and completely inhibited total proteases production. Despite the increasing of reactive oxygen species production caused by the cellular treatment of maltol, both 2-hydroxy-4-pyrone derivatives showed good safe profile when tested in human hepatocarcinoma (HepG2) cells. The pre-treatment of HepG2 cells with both compounds was crucial to prevent the cellular damage caused by iron (III). This study demonstrates for the first time that the selected 2-hydroxy-4-pyrone derivatives significantly enhance the antibiofilm activity of tested antibiotics against S. aureus and P. aeruginosa, highlighting their potential as antibiotic adjuvants in preventing and eradicating biofilm-related infections.

Abstract Polypharmacological approaches have significant potential for the treatment of various complex diseases, including infectious bacteria-related diseases. Actually, multitargeting agents can achieve better therapeutic effects and overcome the drawbacks of monotherapy. Although multidrug multitarget strategies have demonstrated the ability to inactivate infectious bacteria, several challenges have been pointed out. In this way, multitarget direct ligands approaches appear to be a rational and sustainable strategy to combat antibiotic resistance. By combining different pharmacophores, antibiotic hybrids stand out as a promising application in the field of bacterial infections. These new chemical entities can achieve synergistic interactions that allow to extend the spectrum of action and target multiple pathways. In addition, antibiotic hybrids can reduce the likelihood of resistance development and provide improved chemical stability. It is worth highlighting that despite the efforts of the scientific community to discover new solutions for the most complex diseases, there is a significant lack of studies on biofilm-associated infections. This review describes the different polypharmacological approaches that can be used to treat bacterial infections with a particular focus, whenever possible, on those promoted by biofilms. By exploring these innovative approaches, we aim to inspire further research and progress in the search for effective treatments for infectious bacteria-related diseases, including biofilm-related ones. Significance Statement: The importance of the proposed topic lies in the escalating challenge of antibiotic resistance, particularly in the context of infectious bacteria-related infections. Polypharmacological approaches, such as antibiotic hybrids, represent innovative strategies to combat bacterial infections. By targeting multiple signaling pathways, these approaches not only enhance therapeutic effect but also reduce the development of resistance while improving the drug's chemical stability. Despite the urgent need to combat bacterial infectious diseases, there is a notable research gap, in particular in biofilmrelated ones. This review highlights the critical importance of exploring polypharmacological approaches with the aim of motivating further research and advances in effective treatments for infectious bacteria, including biofilm related infections.