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- Resveratrol against Arcobacter butzleri and Arcobacter cryaerophilus: Activity and effect on cellular functionsPublication . Ferreira, Susana; Silva, Filomena; Queiroz, João; Oleastro, Mónica; Domingues, F.C.The frequent isolation of Arcobacter butzleri and Arcobacter cryaerophilus from food samples makes it imperative to search for potential compounds able to inhibit the development of these bacteria. Taking this into consideration, this study focuses on the antimicrobial activity of resveratrol and its mechanism of action against A. butzleri and A. cryaerophilus. The activity of resveratrol was assessed by a microdilution method and time-kill curves. Resveratrol effect on cellular functions was assessed by flow cytometry evaluating intracellular DNA content and metabolic activity. Ethidium bromide (EtBr) accumulation in the presence of resveratrol was also evaluated, as well as the susceptibility to resveratrol in the presence of phenylalanine-arginine β-naphthylamide (PAβN). Scanning electron microscopy (SEM) was used to further evaluate cell damage caused by resveratrol. Resveratrol presented MIC values of 100 and 50μg/mL to A. butzleri and A. cryaerophilus, respectively. Based on the time-kill curves, resveratrol exhibited bactericidal activity, leading to a ≥3log10CFU/mL reduction of initial inoculums, for A. butzleri exponential phase cells incubated for 6h with 1× MIC or with 2× MIC after 24h for stationary phase cells. For A. cryaerophilus cells in exponential growth phase, 99.9% killing was achieved after 24h incubation with 2× MIC, whereas, for stationary phase cells, bactericidal activity was only detected after incubation with 4× MIC. Incubation with resveratrol led to a decrease in both intracellular DNA content and metabolic activity. An increase in the accumulation of EtBr was observed in the presence of resveratrol, and the efflux pump inhibitor PAβN reduced the MIC of resveratrol. SEM analysis revealed disintegration of A. butzleri cells treated with resveratrol, whereas no morphological alteration was observed for A. cryaerophilus cells. Resveratrol has a good anti-Arcobacter activity, and the results obtained suggest that this compound could act through several different mechanisms in the inhibition of this microorganism. The results encourage the use of this compound for the development of potential strategies to control Arcobacter in food products.
- The use of DRAQ5 to monitor intracellular DNA in Escherichia coli by flow cytometryPublication . Silva, Filomena; Lourenço, Olga; Vaz, Cidália Pina; Rodrigues, Acácio Gonçalves; Queiroz, João; Domingues, F.C.Flow cytometry provides a rapid and high-content multiparameter analysis of individual microorganisms within a population. In the past years, several fluorescent stains were developed in order to monitor DNA content distribution and cell-cycle phases, mainly in eukaryotic cells. Recently, due to its low detection limits, several of these fluorescent stains were also applied to prokaryotic cells. In this study, the ability of a novel far-red fluorescent stain DRAQ5 in assessing intracellular DNA content distribution in Escherichia coli DH5alpha was evaluated. The results showed that a DRAQ5-labelled live E. coli suspension can be obtained by incubation of 1 x 10(6) cells/mL with 5 microM DRAQ5 in PBS buffer supplemented with EDTA (pH = 7.4) during 30 min at 37 degrees C. Flow cytometric analysis of fixed E. coli cells revealed that ethanol should be used in detriment of glutaraldehyde for DRAQ5 labelling. After the analysis of RNase and DNase digested samples, DRAQ5 was proven to be a specific DNA labelling stain. The present study demonstrates that the use of DRAQ5 as a DNA-labelling stain provides an easy assessment of intracellular DNA content and cell-cycle phases in gram-negative bacteria such as E. coli.
- Resveratrol inclusion complexes: Antibacterial and anti-biofilm activity against Campylobacter spp. and Arcobacter butzleriPublication . Duarte, Andreia; Alves, Ana C.; Ferreira, Susana; Silva, Filomena; Domingues, F.C.Worldwide, the consumption or handling of contaminated food has been described as one of the major causes of foodborne illness with campylobacteriosis being the most commonly reported zoonosis. Campylobacter jejuni and Campylobacter coli are considered the major cause of bacterial gastroenteritis, while Arcobacter spp. are also known to be human and animal pathogens. Furthermore, these bacteria are able to form biofilms which have become a relevant issue in a wide range of food industries since they are more resistant to disinfectants and so, more difficult to eliminate. This question gives rise to the research on the use of alternative substances that can effectively prevent biofilm formation or eradicate the biofilm already formed. Given this, the aim of this study was to evaluate the antimicrobial and anti-biofilm activity of resveratrol-hydroxypropyl-γ-cyclodextrin inclusion complexes (IC) against C. jejuni, C. coli and A. butzleri as well as their quorum sensing (QS) inhibition activity. Besides improving resveratrol solubility, the ICs showed anti-Campylobacter and anti-Arcobacter activity, inhibited biofilm formation and promoted the biofilm dispersion even at sub-MIC concentrations for both genera. It was also demonstrated the anti-QS activity of the ICs through the inhibition of violacein production by Chromobacterium violaceum. In conclusion, since the use of natural compounds can improve the safety and security of foods, our results showed that this IC could be developed as a new anti-biofilm agent and QS inhibitor to enhance the shelf-life and safety of foods.
- Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dyePublication . Silva, Filomena; Lourenço, Olga; Queiroz, João; Domingues, F.C.As common microbiological methods for the assessment of bacteriostatic or bactericidal activities are very time-consuming, in this work we describe that the use of a novel far-red fluorescent stain, Vybrant DyeCycle Ruby (DCR) for the flow cytometric analysis of fluoroquinolone (ciprofloxacin) bacteriostatic and bactericidal activities in Escherichia coli proved to be specific for bacterial DNA and, after ciprofloxacin exposure, DNA distribution analysis was achieved using a 7.5 μM DCR concentration to stain 5 × 10⁵ ethanol-fixed bacterial cells. The analysis of the bacterial DNA histograms obtained from the ciprofloxacin concentrations tested, enabled the distinction between ciprofloxacin bacteriostatic and bactericidal activities.
- Resveratrol production in bioreactor: Assessment of cell physiological states and plasmid segregational stabilityPublication . Afonso, Margarida S.; Ferreira, Susana; Domingues, F.C.; Silva, FilomenaResveratrol is a plant secondary metabolite commonly found in peanuts and grapevines with significant health benefits. Recombinant organisms can produce large amounts of resveratrol and, in this work, Escherichia coli BW27784 was used to produce resveratrol in bioreactors while monitoring cell physiology and plasmid stability through flow cytometry and real-time qPCR, respectively. Initially, the influence of culture conditions and precursor addition was evaluated in screening assays and the data gathered was used to perform the bioreactor assays, allowing the production of 160 μg/mL of resveratrol. Cellular physiology and plasmid instability affected the final resveratrol production, with lower viability and plasmid copy numbers associated with lower yields. In sum, this study describes new tools to monitor the bioprocess, evaluating the effect of culture conditions, and its correlation with cell physiology and plasmid segregational stability, in order to define a viable and scalable bioprocess to fulfill the need for larger quantities of resveratrol.
- Biofunctionalization of cellulosic fibres with l-cysteine: Assessment of antibacterial properties and mechanism of action against Staphylococcus aureus and Klebsiella pneumoniaePublication . Caldeira, Estela; Piskin, Erhan; Granadeiro, Luiza Breitenfeld ; Silva, Filomena; Gouveia, Isabel C.The main purpose of this work is to obtain a cotton-based textile material functionalized with l-cysteine (l-cys) to achieve an antimicrobial effect with potential application in biomedical, geriatric or pediatric textiles. The binding capacity of l-cys to cotton fibres was assessed through different functionalization strategies—surface activation and exhaustion processes. A subsequent analysis of the possible antibacterial action against Staphylococcus aureus and Klebsiella pneumoniae was performed according with the Japanese International standard (JISL, 2008). To determine the mechanism of action of l-cys on the selected strains, flow cytometry was used. The results revealed that the exhaustion process was performed with success to confer bioactivity to the treated fabric, as assessed by an effective antibacterial effect against both Gram-positive and Gram-negative bacteria, and successfully linkage of l-cys was observed via FTIR with a durable effect demonstrated after the washing tests (fastness to washing). It was also observed that l-cys exerts a bacteriostatic effect against both bacterial strains, since there were alterations in the metabolic activity ofthe microorganisms after the application of the bioactive textile which was shown by the CTC (cyanoditolyl tetrazolium chloride) staining used in flow cytometry. This study shows a new and successful biotechnological process to develop antibacterial textiles through the functionalization of cotton fibres with l-cys which presents a broad range of applications in healthcare, since l-cys is a natural antibacterial compound, non-toxic and affects pathogenic bacteria related to hospital infections.