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Virulence characterization and antimicrobial resistance of major bacterial genera from diabetic foot infections

Publication . Mottola, Carla; Oliveira, Maria Manuela Castilho Monteiro de; Montez, Patrícia Maria Cavaco Silva de Sá

Diabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes is the development of foot ulcers, colonized by pathogenic and antimicrobial resistant bacteria, which may be responsible for impairing its successful treatment. Diabetic foot ulcer (DFU) bacterial communities can be organized in polymicrobial biofilms, which may be responsible for its chronicity. The ability of these communities to produce biofilm was evaluated and was higher when compared to biofilm formation by individual species. Staphylococcus aureus is one of the most prevalent species in diabetic foot infections (DFI). Staphylococci isolated from DFU in patients from the Lisbon area were identified, genotyped and screened for virulence and antimicrobial resistance traits. The isolates showed high genomic diversity, were resistant to important clinically antibiotics and expressed relevant virulence determinants. As biofilm formation is one of the most important virulence traits of S. aureus, the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains were also analysed. The minimum biofilm inhibitory and eradication concentrations were determined for ten antimicrobial compounds. Staphylococci biofilms were resistant to antibiotic concentrations ten to thousand times higher than those effective for planktonic cells. Furthermore, the enterococci frequently isolated from DFI, were also identified and characterized, showing high antimicrobial resistance and important virulence traits. Since DFI are often caused by resistant bacteria, it is necessary to find alternatives to antibiotic therapy, such as phage therapy. The inhibitory potential of five bacteriophages, previously characterized, was evaluated against established biofilms formed by S. aureus, P. aeruginosa and A. baumannii. A significant cell reduction after phage exposure was observed, mainly after multiple treatments. DFI are very complex and studies on this topic are scarce. It is necessary to intensify research in order to develop more adequate therapeutic protocols for this type of infection.

2017-07-21Doctoral thesis

Open access

Polymicrobial biofilms by diabetic foot clinical isolates

Publication . Mottola, Carla; Mendes, João J.; Cristino, José Melo; Cavaco Silva, Patrícia; Tavares, Luis; Oliveira, Manuela

Diabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes is foot ulceration that may be colonized by pathogenic and antimicrobial resistant bacteria, which may express several virulence factors that could impair treatment success. These bacterial communities can be organized in polymicrobial biofilms, which may be responsible for diabetic foot ulcer (DFU) chronicity. We evaluated the influence of polymicrobial communities in the ability of DFU isolates to produce biofilm, using a microtiter plate assay and a multiplex fluorescent in situ hybridization, at three time points (24, 48, 72 h), after evaluating biofilm formation by 95 DFU isolates belonging to several bacterial genera (Staphylococcus, Corynebacterium, Enterococcus, Pseudomonas and Acinetobacter). All isolates were biofilm-positive at 24 h, and the amount of biofilm produced increased with incubation time. Pseudomonas presented the higher biofilm production, followed by Corynebacterium, Acinetobacter, Staphylococcus and Enterococcus. Significant differences were found in biofilm formation between the three time points. Polymicrobial communities produced higher biofilm values than individual species. Pseudomonas + Enterococcus, Acinetobacter + Staphylococcus and Corynebacterium + Staphylococcus produced higher biofilm than the ones formed by E. faecalis + Staphylococcus and E. faecalis + Corynebacterium. Synergy between bacteria present in dual or multispecies biofilms has been described, and this work represents the first report on time course of biofilm formation by polymicrobial communities from DFUs including several species. The biological behavior of different bacterial species in polymicrobial biofilms has important clinical implications for the successful treatment of these infections.

2015Journal article

Restricted access

Guar gum as a new antimicrobial peptide delivery system against diabetic foot ulcers Staphylococcus aureus isolates

Publication . Santos, Raquel; Gomes, Diana; Macedo, Hermes; Barros, Diogo; Tibério, Catarina; Veiga, Ana Salomé; Tavares, Luís; Castanho, Miguel A. R. B.; Oliveira, Manuela

Diabetic patients frequently develop diabetic foot ulcers (DFUs), particularly those patients vulnerable to Staphylococcus aureus opportunistic infections. It is urgent to find new treatments for bacterial infections. The antimicrobial peptide (AMP) nisin is a potential candidate, mainly due to its broad spectrum of action against pathogens. Considering that AMP can be degraded or inactivated before reaching its target at therapeutic concentrations, it is mandatory to establish effective AMP delivery systems, with the natural polysaccharide guar gum being one of the most promising. We analysed the antimicrobial potential of nisin against 23 S. aureus DFU biofilm-producing isolates. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were determined for nisin diluted in HCl and incorporated in guar gum gel. Statistical analysis was performed using the Wilcoxon matched-pair test. Nisin was effective against all isolates, including some multidrug-resistant clinical isolates, independent of whether it is incorporated in guar gum. While differences among MIC, MBC and MBIC values were observed for HCl- and guar gum- nisin, no significant differences were found between MBEC values. Inhibitory activity of both systems seems to differ only twofold, which does not compromise guar gum gel efficiency as a delivery system. Our results highlight the potential of nisin as a substitute for or complementary therapy to current antibiotics used for treating DFU infections, which is extremely relevant considering the increase in multidrug-resistant bacteria dissemination. The guar gum gel represents an alternative, practical and safe delivery system for AMPs, allowing the development of novel topical therapies as treatments for bacterial skin infections.

2016Journal article

Open access