Manuel Simões

Antibacterial Activity and Mode of Action of Ferulic and Gallic Acids Against Pathogenic Bacteria

The increased resistance of pathogenic microorganisms is frequently attributed to the extreme and inadequate use of antibiotics and transmission of resistance within and between individuals. To counter the emergence of resistant microorganisms, considerable resources have been invested in the search for new antimicrobials. Plants synthesize a diverse array of secondary metabolites (phytochemicals) known to be involved in defense mechanisms, and in the last few years it is recognized that some of these molecules have health beneficial effects, including antimicrobial properties. In this study, the mechanism of action of gallic (GA) and ferulic (FA) acids, a hydroxybenzoic acid and a hydroxycinnamic acid, was assessed on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Listeria monocytogenes. The targets of antimicrobial action were studied using different bacterial physiological indices: minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), membrane permeabilization, intracellular potassium release, physicochemical surface properties, and surface charge. It was found that FA and GA had antimicrobial activity against the bacteria tested with MIC of 500 μg/mL for P. aeruginosa, 1500 μg/mL for E. coli, 1750 μg/mL for S. aureus, and 2000 μg/mL for L. monocytogenes with GA; 100 μg/mL for E. coli and P. aeruginosa, 1100 μg/mL and 1250 μg/mL for S. aureus and L. monocytogenes, respectively, with FA. The MBC for E. coli was 2500 μg/mL (FA) and 5000 (GA), for S. aureus was 5000 μg/mL (FA) and 5250 μg/mL (GA), for L. monocytogenes was 5300 μg/mL (FA) and 5500 μg/mL (GA), and 500 μg/mL for P. aeruginosa, with both phytochemicals. GA and FA led to irreversible changes in membrane properties (charge, intra and extracellular permeability, and physicochemical properties) through hydrophobicity changes, decrease of negative surface charge, and occurrence of local rupture or pore formation in the cell membranes with consequent leakage of essential intracellular constituents. The overall study emphasizes the potential of plant-derived molecules as a green and sustainable source of new broad spectrum antimicrobial products.

Plants as sources of new antimicrobials and resistance-modifying agents

Infections caused by multidrug-resistant bacteria are an increasing problem due to the emergence and propagation of microbial drug resistance and the lack of development of new antimicrobials. Traditional methods of antibiotic discovery have failed to keep pace with the evolution of resistance. Therefore, new strategies to control bacterial infections are highly desirable. Plant secondary metabolites (phytochemicals) have already demonstrated their potential as antibacterials when used alone and as synergists or potentiators of other antibacterial agents. The use of phytochemical products and plant extracts as resistance-modifying agents (RMAs) represents an increasingly active research topic. Phytochemicals frequently act through different mechanisms than conventional antibiotics and could, therefore be of use in the treatment of resistant bacteria. The therapeutic utility of these products, however, remains to be clinically proven. The aim of this article is to review the advances in in vitro and in vivo studies on the potential chemotherapeutic value of phytochemical products and plant extracts as RMAs to restore the efficacy of antibiotics against resistant pathogenic bacteria. The mode of action of RMAs on the potentiation of antibiotics is also described.

Species association increases biofilm resistance to chemical and mechanical treatments.

The study of biofilm ecology and interactions might help to improve our understanding of their resistance mechanisms to control strategies. Concerns that the diversity of the biofilm communities can affect disinfection efficacy have led us to examine the effect of two antimicrobial agents on two important spoilage bacteria. Studies were conducted on single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens. Biofilms were formed on a stainless steel rotating device, in a bioreactor, at a constant Reynolds number of agitation (Re(A)). Biofilm phenotypic characterization showed significant differences, mainly in the metabolic activity and both extracellular proteins and polysaccharides content. Cetyl trimethyl ammonium bromide (CTAB) and glutaraldehyde (GLUT) solutions in conjunction with increasing Re(A) were used to treat biofilms in order to assess their ability to kill and remove biofilms. B. cereus and P. fluorescens biofilms were stratified in a layered structure with each layer having differential tolerance to chemical and mechanical stresses. Dual species biofilms and P. fluorescens single biofilms had both the highest resistance to removal when pre-treated with CTAB and GLUT, respectively. B. cereus biofilms were the most affected by hydrodynamic disturbance and the most susceptible to antimicrobials. Dual biofilms were more resistant to antimicrobials than each single species biofilm, with a significant proportion of the population remaining in a viable state after exposure to CTAB or GLUT. Moreover, the species association increased the proportion of viable cells of both bacteria, comparatively to the single species scenarios, enhancing each others survival to antimicrobials and the biofilm shear stress stability.

Biofilm Interactions between Distinct Bacterial Genera Isolated from Drinking Water

ABSTRACT In the environment, multiple microorganisms coexist as communities, competing for resources and often associated as biofilms. In this study, single- and dual-species biofilm formation by, and specific activities of, six heterotrophic intergeneric bacteria were determined using 96-well polystyrene plates over a 72-h period. These bacteria were isolated from drinking water and identified by partial 16S rRNA gene sequencing. A series of planktonic studies was also performed, assessing the bacterial growth rate, motility, and production of quorum-sensing inhibitors (QSI). This constituted an attempt to identify key attributes allowing bacteria to effectively interact and coexist in a drinking-water environment. We observed that in both pure and dual cultures, all of the isolates formed stable biofilms within 72 h, with specific metabolic activity decreasing, in most cases, with an increase in biofilm mass. The largest single- and dual-biofilm amounts were found for Methylobacterium sp. and the combination of Methylobacterium sp. and Mycobacterium mucogenicum, respectively. Evidences of microbial interactions in dual-biofilm formation, associated with appreciable biomass variation in comparison with single biofilms, were found for the following cases: synergy/cooperation between Sphingomonas capsulata and Burkholderia cepacia, S. capsulata and Staphylococcus sp., and B. cepacia and Acinetobacter calcoaceticus and antagonism between S. capsulata and M. mucogenicum, S. capsulata and A. calcoaceticus, and M. mucogenicum and Staphylococcus sp. A neutral interaction was found for Methylobacterium sp.-M. mucogenicum, S. capsulata-Staphylococcus sp., M. mucogenicum-A. calcoaceticus, and Methylobacterium sp.-A. calcoaceticus biofilms, since the resultant dual biofilms had a mass and specific metabolic activity similar to the average for each single biofilm. B. cepacia had the highest growth rate and motility and produced QSI. Other bacteria producing QSI were Methylobacterium sp., S. capsulata, and Staphylococcus sp. However, only for S. capsulata-M. mucogenicum, S. capsulata-A. calcoaceticus, and M. mucogenicum-Staphylococcus sp., dual-biofilm formation seems to be regulated by the QSI produced by S. capsulata and Staphylococcus sp. and by the increased growth rate of S. capsulata. The parameters assessed by planktonic studies did not allow prediction and generalization of the exact mechanism regulating dual-species biofilm formation between the drinking-water bacteria.

Influence of the Diversity of Bacterial Isolates from Drinking Water on Resistance of Biofilms to Disinfection

ABSTRACT Single- and multispecies biofilms formed by six drinking water-isolated bacterial species were used to assess their susceptibilities to sodium hypochlorite (SHC). In general, multispecies biofilms were more resistant to inactivation and removal than single biofilms. Total biofilm inactivation was achieved only for Acinetobacter calcoaceticus single-species biofilms and for those multispecies biofilms without A. calcoaceticus. Biofilms with all bacteria had the highest resistance to SHC, while those without A. calcoaceticus were the most susceptible. A. calcoaceticus formed single biofilms susceptible to SHC; however, its presence in multispecies biofilms increased their resistance to disinfection.

The activity of ferulic and gallic acids in biofilm prevention and control of pathogenic bacteria

The activity of two phenolic acids, gallic acid (GA) and ferulic acid (FA) at 1000 μg ml−1, was evaluated on the prevention and control of biofilms formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. In addition, the effect of the two phenolic acids was tested on planktonic cell susceptibility, bacterial motility and adhesion. Biofilm prevention and control were tested using a microtiter plate assay and the effect of the phenolic acids was assessed on biofilm mass (crystal violet staining) and on the quantification of metabolic activity (alamar blue assay). The minimum bactericidal concentration for P. aeruginosa was 500 μg ml−1 (for both phenolic acids), whilst for E. coli it was 2500 μg ml−1 (FA) and 5000 μg ml−1 (GA), for L. monocytogenes it was >5000 μg ml−1 (for both phenolic acids), and for S. aureus it was 5000 μg ml−1 (FA) and >5000 μg ml−1 (GA). GA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. FA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. Colony spreading of S. aureus was completely inhibited by FA. The interference of GA and FA with bacterial adhesion was evaluated by the determination of the free energy of adhesion. Adhesion was less favorable when the bacteria were exposed to GA (P. aeruginosa, S. aureus and L. monocytogenes) and FA (P. aeruginosa and S. aureus). Both phenolics had preventive action on biofilm formation and showed a higher potential to reduce the mass of biofilms formed by the Gram-negative bacteria. GA and FA promoted reductions in biofilm activity >70% for all the biofilms tested. The two phenolic acids demonstrated the potential to inhibit bacterial motility and to prevent and control biofilms of four important human pathogenic bacteria. This study also emphasizes the potential of phytochemicals as an emergent source of biofilm control products.

Parametric study of a brewery effluent treatment by microalgae Scenedesmus obliquus

This work analyses the potential of using microalgae Scenedesmus obliquus (So) for a brewery wastewater treatment and biomass production. The chemical oxygen demand (COD), total nitrogen (TN) and total carbon (TC) was followed in time, and the influence of light exposure, light intensity and culture aeration was studied. Results show that the most adequate conditions for cultivating So in this effluent are the aerated cultures, exposed to a 12h period of daily light, at 12000 Lux intensity. At these conditions it is obtained a maximum of 0.9 g of dry biomass per liter of culture, after 9 days, for a maximum reduction of 57.5% and 20.8% of COD and TN, respectively, after 14 days, and 56.9% of TC, after 13 days, corresponding to the final values of 1692 mg O(2)/L COD, 47 mg N/L TN, and 1mg C/L TC.

Potential of the adhesion of bacteria isolated from drinking water to materials

Heterotrophic bacteria (11 genera, 14 species, 25 putative strains) were isolated from drinking water, identified either biochemically or by partial 16s rDNA gene sequencing and their adherence characteristics were determined by two methods: i. thermodynamic prediction of adhesion potential by measuring hydrophobicity (contact angle measurements) and ii. by measuring adherence to eight different substrata (ASI 304 and 316 stainless steel, copper, polyvinyl chloride, polypropylene, polyethylene, silicone and glass). All the test organisms were hydrophilic and inter‐species variation in hydrophobicity occurred only for Comamonas acidovorans. Stainless steel 304 (SS 304), copper, polypropylene (PP), polyethylene (PE) and silicone thermodynamically favoured adhesion for the majority of test strains (>18/25), whilst adhesion was generally less thermodynamically favorable for stainless steel 316 (SS 316), polyvinyl chloride (PVC) and glass. The predictability of thermodynamic adhesion test methods was validated by comparison with 24‐well microtiter plate assays using nine reference strains and three adhesion surfaces (SS 316, PVC and PE). Results for Acinetobacter calcoaceticus, Burkolderia cepacia and Stenotrophomonas maltophilia sp. 2 were congruent between both methods whilst they differed for the other bacteria to at least one material. Only A. calcoaceticus had strongly adherent properties to the three tested surfaces. Strain variation in adhesion ability was detected only for Sphingomonas capsulata. Analysis of adhesion demonstrated that in addition to physicochemical surface properties of bacterium and substratum, biological factors are involved in early adhesion processes, suggesting that reliance on thermodynamic approaches alone may not accurately predict adhesion capacity. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Antimicrobial activity of phenolics and glucosinolate hydrolysis products and their synergy with streptomycin against pathogenic bacteria.

The purpose of the present study was to evaluate the in vitro antibacterial effects of different classes of important and common dietary phytochemicals (5 simple phenolics - tyrosol, gallic acid, caffeic acid, ferulic acid, and chlorogenic acid; chalcone - phloridzin; flavan-3-ol - (-) epicatechin; seco-iridoid - oleuropein glucoside; 3 glucosinolate hydrolysis products - allylisothiocyanate, benzylisothiocyanate and 2-phenylethylisothiocyanate) against Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes and Staphylococcus aureus. Another objective of this study was to evaluate the effects of dual combinations of streptomycin with the different phytochemicals on antibacterial activity. A disc diffusion assay was used to evaluate the antibacterial activity of the phytochemicals and 3 standard antibiotics (ciprofloxacin, gentamicin and streptomycin) against the four bacteria. The antimicrobial activity of single compounds and dual combinations (streptomycin-phytochemicals) were quantitatively assessed by measuring the inhibitory halos. The results showed that all of the isothiocyanates had significant antimicrobial activities, while the phenolics were much less efficient. No antimicrobial activity was observed with phloridzin. In general P. aeruginosa was the most sensitive microorganism and L. monocytogenes the most resistant. The application of dual combinations demonstrated synergy between streptomycin and gallic acid, ferulic acid, chlorogenic acid, allylisothiocyanate and 2-phenylethylisothiocyanate against the Gram-negative bacteria. In conclusion, phytochemical products and more specifically the isothiocyanates were effective inhibitors of the in vitro growth of the Gram-negative and Gram-positive pathogenic bacteria. Moreover, they can act synergistically with less efficient antibiotics to control bacterial growth.

Antimicrobial Strategies Effective Against Infectious Bacterial Biofilms

Bacteria are able to adapt to undesirable changes in nutrient availability, environmental conditions and presence of antimicrobial products, as well as to immunological defenses. One particularly important example of bacterial adaptation is the ability to grow as part of a sessile community, commonly referred to as biofilm. It is a natural tendency of microorganisms to attach to biotic or abiotic surfaces, to multiply and to embed themselves in a slimy matrix, resulting in biofilms. Biofilms are the leading example of physiological adaptation and are one of the most important sources of bacterial resistance to antimicrobials. It is now recognized that most bacterial-associated infections, including endocarditis, dental caries, middle ear infections, osteomyelitis, medical device-related infections and chronic lung infections in cystic fibrosis patients are problematic because of biofilms. Bacteria in biofilms demonstrate intrinsic resistance to antimicrobial stress more effectively than the planktonic counterparts. Antimicrobial concentrations necessary to inhibit bacterial biofilms can be up to 10-1000 times higher than those needed to inhibit the same bacteria grown planktonically. Thus, in the presence of therapeutically available antibiotic concentrations biofilms remain viable after treatment. Therefore, the identification of new antimicrobials that inhibit or destroy biofilms is needed. The aim of this review is to cover the recent advances on the studies of antimicrobial strategies effective against infectious bacterial biofilms, including the current developments in the structure-activity relationship of those effective antimicrobials.