Anabela Portela Borges

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 <jats:title>Abstract</jats:title> <jats:sec> <jats:title>Aims</jats:title> <jats:p>Antimicrobial resistance, particularly in healthcare-associated infections (HAIs), highlights the need for more effective and sustainable disinfection strategies. This study 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.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods and Results</jats:title> <jats:p>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 minutes, 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 modelling 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 modelling further indicated that bacterial tolerance was dependent on compound type and concentration, with S. aureus generally more susceptible than E. coli.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>These findings collectively confirm the enhanced efficacy of selected biocide/phytochemical combinations, allowing lower concentrations and promoting more sustainable antimicrobial practices.</jats:p> </jats:sec>

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.