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 Diabetic foot ulcers (DFUs) remain a major therapeutic challenge due to persistent biofilms, with Staphylococcus aureus as the predominant pathogen. Antimicrobial photodynamic inactivation (aPDI) offers an alternative to antibiotics but shows limited efficacy against biofilms. This study introduces a novel combinative strategy in which berberine (Ber) acts simultaneously as a photosensitiser and an adjuvant to restore gentamicin (Gen) activity, enabling enhanced aPDI efficacy against S. aureus biofilms. Two clinical strains, a methicillin-resistant (MRSA, MJMC568-B) and a methicillin-susceptible (MSSA, CECT 976), were used. The Ber-Gen interaction was first evaluated by checkerboard assay, followed by blue light activation (420 nm, 30 mW/cm2, 10 min, 18 J/cm2) to test biofilm prevention and eradication. In pre-formed biofilms, single or triple irradiation cycles (every 24 h) were applied, and regrowth was monitored for up to 72 h, allowing assessment of post-treatment biofilm recovery dynamics. Quantitative analyses included biomass (crystal violet), metabolic activity (resazurin), and culturability (CFU/cm2). Mechanistic insights were obtained through reactive oxygen species (ROS) quantification, flow cytometry, confocal microscopy, and optical coherence tomography. Ber restored Gen susceptibility, showing additive effects in MRSA and synergism in MSSA. Photoactivated Ber-Gen prevented biofilm formation with over 90% reduction in biomass, metabolic activity, and culturability (6-log CFU/cm2). In mature biofilms, reductions reached 7-log CFU/cm2 with over 90% reduction in biomass and metabolic activity. While single irradiation allowed partial regrowth, triple cycles prevented recovery for 72 h. Mechanistic analyses confirmed enhanced ROS generation, membrane disruption, and biofilm matrix disruption. These findings demonstrate that Ber-Gen-mediated aPDI suggests a potential light-activated therapeutic strategy, supporting a dual-functionally integrated approach that combines antibiotic potentiation with photodynamic activity to overcome biofilm tolerance and effectively suppress post-treatment regrowth in chronic DFUs.

Abstract Chronic wound infections are a major healthcare challenge, especially with the continued rise of antibiotic resistance. Antibacterial photodynamic inactivation (aPDI) is a localized, non-antibiotic approach in which light-activated photosensitizers (PSs) generate reactive oxygen species (ROS) that damage essential bacterial structures. Natural PSs, including harmine (HA), riboflavin 5 '-phosphate (RB), palmatine (PM), and ferulic acid (FA), have gained attention for their bioactivity and photoreactivity. This study investigated for the first time the potential of combining these natural PSs with the membrane-active antiseptics, octenidine dihydrochloride (OCT), polyhexamethylene biguanide (PHMB), and hydrogen peroxide (H2O2), to improve aPDI efficacy against a clinical Staphylococcus aureus strain (MJMC568-B) isolated from a diabetic foot ulcer. Antibacterial activity of PSs and antiseptics, alone and in combination, was assessed by broth microdilution (minimum inhibitory and bactericidal concentration (MIC/MBC) determination), checkerboard, and disk diffusion assays. All PSs, except RB, demonstrated intrinsic antibacterial activity, and most combinations showed indifference under non-irradiated conditions. Photodynamic activity was evaluated using LED irradiation at 420 nm (30 mW cm-2; 9.23-27.68 J cm-2) for RB and PM, and 365 nm (5.5 mW cm-2; 4.95-19.90 J cm-2) for HA and FA. After photoactivation, specific PS-antiseptic combinations achieved complete bacterial inactivation (approximate to 8 log (CFU mL-1) reduction in culturability) at concentrations below individual MBCs: HA (100 & micro;g mL-1)-OCT (0.489-0.978 & micro;g mL-1) and HA (25 & micro;g mL-1)-PHMB (7.81 & micro;g mL-1) at 18.45 J cm-2, and PM (200 & micro;g mL-1)-OCT (1.955 & micro;g mL-1) at 19.90 J cm-2. FA-based combinations did not achieve bactericidal effects when irradiated. ROS generation capacity was evaluated using 2 ' 7 '-dichlorodihydrofluorescein diacetate (DCFH-DA) and singlet oxygen production was evaluated using 9,10-anthracenediyl-bis (methylene) dimalonic acid (ADMA), which indicated an increase in oxidative stress when bacterial suspensions were irradiated as well as the capacity to produce singlet oxygen. Overall, HA and PM substantially enhanced the antibacterial performance of OCT and PHMB under photodynamic activation, enabling complete S. aureus inactivation. These findings highlight PS-antiseptic combinations as a promising non-antibiotic strategy for chronic wound management while reducing the required PS and antiseptic concentrations.

Abstract Developing innovative and efficient approaches to enhance the antimicrobial activity of conventional biocides is a promising strategy for improving microbial control for healthcare disinfection purposes. In this context, we describe a detailed protocol for evaluating the potentiating effects of plant-derived phytochemicals on synthetic biocides. These plant-based products are attractive candidates as biocide adjuvants due to their structural diversity, bioactivity, and often lower toxicity. The workflow here described allows for the determination of minimum bactericidal concentrations (MBCs) and the systematic generation of both dose-response and time-response curves for each product tested individually (biocides and phytochemicals) and for their dual or triple combinations. The resulting data allow for advanced mathematical modelling, including determining the fractional bactericidal concentration index (FBCI) and assessing the interactions between the tested products regarding synergistic, additive, or antagonistic effects using the software Combenefit. In that sense, it is possible to identify promising natural products as biocide enhancers, optimize antimicrobial efficacy, and design more sustainable and safer disinfection strategies that may reduce the concentrations required of synthetic biocides. This improves microbial control and minimizes environmental and public health risks associated with excessive biocide use.