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 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.

Abstract Antimicrobial photodynamic inactivation (aPDI), using photosensitisers in combination with antibiotics, is a promising multi-target strategy to address antibiotic resistance, particularly in wound infections. This study aimed to elucidate the antibacterial mode of action of combinations of berberine (Ber) or curcumin (Cur) with selected antibiotics (Ber-Ab or Cur-Ab) under blue light irradiation (420 nm) against Staphylococcus aureus, including methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains. Multiple physiological parameters were assessed using complementary assays (fluorometry, epifluorescence microscopy, flame emission and atomic absorption spectroscopy, zeta potential, flow cytometry, and the plate agar method) to examine the effect on ROS production, membrane integrity, DNA damage, motility and virulence factors of S. aureus. Results indicated that blue light photoactivated Ber-Ab and Cur-Ab combinations led to substantial ROS generation, even at low concentrations, causing oxidative stress that severely impacted bacterial membrane integrity (approximately 90 % in MRSA and 40 % in MSSA). Membrane destabilization was further confirmed by elevated intercellular potassium release (approximate to 2.00 and 2.40 mu g/mL in MRSA and MSSA, respectively). Furthermore, significant DNA damage was observed in both strains (approximate to 50 %). aPDI treatment with blue light also reduced S. aureus pathogenicity by impairing motility and inhibiting key virulence factors such as proteases, lipases, and gelatinases, all of which play key roles in the infectious process. Overall, Ber-Ab combinations demonstrated the highest efficacy across all parameters tested, highlighting for the first time the multi-target therapeutic potential of this phytochemical-based aPDI strategy to combat antibiotic-resistant S. aureus infections and improve wound infection treatment outcomes.

Abstract The ability of Salmonella enterica subsp. enterica to persist and form biofilms on different surfaces can constitute a source of food contamination, being an issue of global concern. The objective of this study was to understand the biofilm formation profile of 14 S. enterica strains among different serovars and sources and to evaluate the ability of essential oil (EO) components (carveol, citronellol, and citronellal) to disinfect the biofilms formed on stainless steel and polypropylene surfaces. All the strains were able to form biofilms with counts between 5.34 to 6.78 log CFU cm(-2). Then, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EO components were evaluated on two selected strains. All compounds inhibited the growth of Salmonella Typhimurium (strain 1; MIC = 800-1000 mu g ml(-1)) and Salmonella Enteritidis (strain 5; MIC = 400-1000 mu g ml(-1)) and only carveol showed bactericidal activity against strains 1 and 5 (MBC = 1200 mu g ml(-1)). Biofilms were exposed to the EO components at 10 x MIC for 30 min and polypropylene surfaces were more difficult to disinfect showing reductions between 0.9 and <1.2 log CFU cm(-2). In general, the S. enterica biofilms demonstrated a significant tolerance to disinfection, demonstrating their high degree of recalcitrance on food processing surfaces.

Abstract Introduction: Quorum sensing (QS) is a bacterial communication mechanism that regulates gene expression, playing a crucial role in various physiological processes. Interfering with this signalling pathway is a promising strategy to control bacterial pathogenicity and virulence. Objectives: This study evaluated the potential of two cinnamic acid derivatives, ferulic and sinapic acids, to inhibit the las and pqs systems in Pseudomonas aeruginosa. Their effects on biofilm architecture, virulence factor production and bacterial motility were also investigated. Methods: Bioreporter strains and bioluminescence-based assays were used to evaluate the modulation of QS-activity by cinnamic acid-type phenolic acids. In addition, in silico docking analysis was performed to validate the binding interactions of the cinnamic acid derivatives with QS-receptors. The biofilm architecture was analysed by optical coherence tomography, and virulence factors production (pyoverdine, pyocyanin, total proteases, lipases, gelatinases and siderophores) and motility were measured by absorbance measurement and plate agar method. Results: Ferulic and sinapic acids at 1000 µg mL−1 inhibited the las and pqs systems by 90 % and 80 %, respectively. The N-3-oxododecanoyl-homoserine lactone production was reduced by 70 % (6.25 µg mL-¹). In silico analysis demonstrated that cinnamic acid derivatives exhibited comparable interactions and higher docking scores than reference ligands and inhibitors. Biofilm thickness decreased from 96 µm to 11 µm, and virulence factors and swarming motility were significantly impaired. The comparable anti-QS activity of cinnamic acid derivatives suggests that the additional methoxy group in sinapic acid does not directly contribute to its anti-QS effect. Conclusion: Ferulic and sinapic acids compromised the biofilm architecture and virulence of P. aeruginosa through QS inhibition. © 2025

Abstract Manuel Simões was included as a corresponding author in the original publication [1] by mistake. The corrected corresponding author should be Lúcia C. Simões, and the email is updated accordingly. The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated. © 2024 by the authors.

Abstract Antibacterial resistance poses a critical public health threat, challenging the prevention and treatment of bacterial infections. The search for innovative antibacterial agents has spurred significant interest in quaternary heteronium salts (QHSs), such as quaternary ammonium and phosphonium compounds as potential candidates. In this study, a library of 49 structurally related QHSs was synthesized, varying the cation type and alkyl chain length. Their antibacterial activities against Staphylococcus aureus, including antibiotic-resistant strains, were evaluated by determining minimum inhibitory/bactericidal concentrations (MIC/MBC) <= 64 mu g/mL. Structure-activity relationship analyses highlighted alkyl-triphenylphosphonium and alkyl-methylimidazolium salts as the most effective against S. aureus CECT 976. The length of the alkyl side chain significantly influenced the antibacterial activity, with optimal chain lengths observed between C-10 and C-14. Dose-response relationships were assessed for selected QHSs, showing dose-dependent antibacterial activity following a non-linear pattern. Survival curves indicated effective eradication of S. aureus CECT 976 by QHSs at low concentrations, particularly compounds 1e, 3e, and 5e. Moreover, in vitro human cellular data indicated that compounds 2e, 4e, and 5e showed favourable safety profiles at concentrations <= 2 mu g/mL. These findings highlight the potential of these QHSs as effective agents against susceptible and resistant bacterial strains, providing valuable insights for the rational design of bioactive QHSs.