Anabela Portela Borges

Abstract The increase of bacterial resistance to antibiotics is a growing concern worldwide and the search for new therapies could cost billions of dollars and countless lives. Inert surfaces are major sources of contamination due to easier adhesion and formation of bacterial biofilms, hindering the disinfection process. Therefore, the objective of this study was to develop a photoactivatable and anti-adhesive kappa-carrageenan coating using proanthocyanidin as a photosensitizer. The complete reduction (>5-log(10) CFU/cm(3)) of culturable cells of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa pathogens was achieved after 30 min of exposure to visible light (420 nm; 30 mW/cm(2)) with 5 % (w/v) of the photosensitizer. Cell membrane damage was confirmed by measuring potassium leakage, epifluorescence microscopy and bacterial motility analysis. Overall, visible light irradiation on coated solid surfaces mediated by proanthocyanidin showed no cytotoxicity and inactivated clinically important pathogens through the generation of reactive oxygen species, inhibiting bacterial initial adhesion. The developed coating is a promising alternative for a wide range of applications related to surface disinfection and food biopreservation.

Abstract Quorum sensing (QS) has a central role in biofilm lifestyle and antimicrobial resistance, and disrupting these signaling pathways is a promising strategy to control bacterial pathogenicity and virulence. In this study, the efficacy of three structurally related benzaldehydes (4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde)) in disrupting the las and pqs systems of Pseudomonas aeruginosa was investigated using bioreporter strains and computational simulations. Additionally, these benzaldehydes were combined with tobramycin and ciprofloxacin antibiotics to evaluate their ability to increase antibiotic efficacy in preventing and eradicating P. aeruginosa biofilms. To this end, the total biomass, metabolic activity and culturability of the biofilm cells were determined. In vitro assays results indicated that the aromatic aldehydes have potential to inhibit the las and pqs systems by > 80 %. Molecular docking studies supported these findings, revealing the aldehydes binding in the same pocket as the natural ligands or receptor proteins (LasR, PQSA, PQSE, PQSR). Benzaldehydes were shown to act as virulence factor attenuators, with vanillin achieving a 48 % reduction in pyocyanin production. The benzaldehyde-tobramycin combination led not only to a 60 % reduction in biomass production but also to a 90 % reduction in the metabolic activity of established biofilms. A similar result was observed when benzaldehydes were combined with ciprofloxacin. 4-Hydroxybenzaldehyde demonstrated relevant action in increasing biofilm susceptibility to ciprofloxacin, resulting in a 65 % reduction in biomass. This study discloses, for the first time, that the benzaldehydes studied are potent QS inhibitors and also enhancers of antibiotics antibiofilm activity against P. aeruginosa.

Abstract Staphylococcus aureus is characterized by its high resistance to conventional antibiotics, particularly methicillin-resistant (MRSA) strains, making it a predominant pathogen in acute and chronic wound infections. The persistence of acute S. aureus wound infections poses a threat by increasing the incidence of their chronicity. This study investigated the potential of photodynamic activation using phytochemical-antibiotic combinations to eliminate S. aureus under conditions representative of acute wound infections, aiming to mitigate the risk of chronicity. The strategy applied takes advantage of the promising antibacterial and photosensitising properties of phytochemicals, and their ability to act as antibiotic adjuvants. The antibacterial activity of selected phytochemicals (berberine, curcumin, farnesol, gallic acid, and quercetin; 6.25–1000 μg/mL) and antibiotics (ciprofloxacin, tetracycline, fusidic acid, oxacillin, gentamicin, mupirocin, methicillin, and tobramycin; 0.0625–1024 μg/mL) was screened individually and in combination against two S. aureus clinical strains (methicillin-resistant and -susceptible–MRSA and MSSA). The photodynamic activity of the phytochemicals was assessed using a light-emitting diode (LED) system with blue (420 nm) or UV-A (365 nm) variants, at 30 mW/cm2 (light doses of 9, 18, 27 J/cm2) and 5.5 mW/cm2 (light doses of 1.5, 3.3 and 5.0 J/cm2), respectively. Notably, all phytochemicals restored antibiotic activity, with 9 and 13 combinations exhibiting potentiating effects on MSSA and MRSA, respectively. Photodynamic activation with blue light (420 nm) resulted in an 8- to 80-fold reduction in the bactericidal concentration of berberine against MSSA and MRSA, while curcumin caused 80-fold reduction for both strains at the light dose of 18 J/cm2. Berberine and curcumin-antibiotic combinations when subjected to photodynamic activation (420 nm light, 10 min, 18 J/cm2) reduced S. aureus culturability by ≈9 log CFU/mL. These combinations lowered the bactericidal concentration of antibiotics, achieving a 2048-fold reduction for gentamicin and 512-fold reduction for tobramycin. Overall, the dual approach involving antimicrobial photodynamic inactivation and selected phytochemical-antibiotic combinations demonstrated a synergistic effect, drastically reducing the culturability of S. aureus and restoring the activity of gentamicin and tobramycin. © 2023

Abstract Shake flask cultivation is a routine technique in microbiology and biotechnology laboratories where cell growth can be affected by the hydrodynamic conditions, which depend on the agitation velocity, shaking diameter, and shake flask size. Liquid agitation is implemented inherently to increase aeration, substrate transfer to the cells, and prevent sedimentation, disregarding the role of hydrodynamics in microbial growth and metabolism. Here, we present a simple approach to help standardize the hydrodynamic forces in orbital shakers to increase the experimental accuracy and reproducibility and give students a better knowledge of the significance of the agitation process in microbial growth.

Abstract The treatment of bacterial infections has been troubled by the increased resistance to antibiotics, instigating the search for new antimicrobial therapies. Phytochemicals have demonstrated broad-spectrum and effective antibacterial effects as well as antibiotic resistance-modifying activity. In this study, perillyl alcohol and hydrocinnamic acid were characterized for their antimicrobial action against Escherichia coli. Furthermore, dual and triple combinations of these molecules with the antibiotics chloramphenicol and amoxicillin were investigated for the first time. Perillyl alcohol had a minimum inhibitory concentration (MIC) of 256 mu g/mL and a minimum bactericidal concentration (MBC) of 512 mu g/mL. Hydrocinnamic acid had a MIC of 2048 mu g/mL and an MBC > 2048 mu g/mL. Checkerboard and time-kill assays demonstrated synergism or additive effects for the dual combinations chloramphenicol/perillyl alcohol, chloramphenicol/hydrocinnamic acid, and amoxicillin/hydrocinnamic acid at low concentrations of both molecules. Combenefit analysis showed synergism for various concentrations of amoxicillin with each phytochemical. Combinations of chloramphenicol with perillyl alcohol and hydrocinnamic acid revealed synergism mainly at low concentrations of antibiotics (up to 2 mu g/mL of chloramphenicol with perillyl alcohol; 0.5 mu g/mL of chloramphenicol with hydrocinnamic acid). The results highlight the potential of combinatorial therapies for microbial growth control, where phytochemicals can play an important role as potentiators or resistance-modifying agents.