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BackgroundFoot infections are a major cause of morbidity in people with diabetes and the most common cause of diabetes-related hospitalization and lower extremity amputation. Staphylococcus aureus is by far the most frequent species isolated from these infections. In particular, methicillin-resistant S. aureus (MRSA) has emerged as a major clinical and epidemiological problem in hospitals. MRSA strains have the ability to be resistant to most β-lactam antibiotics, but also to a wide range of other antimicrobials, making infections difficult to manage and very costly to treat. To date, there are two fifth-generation cephalosporins generally efficacious against MRSA, ceftaroline and ceftobripole, sharing a similar spectrum.Biofilm formation is one of the most important virulence traits of S. aureus. Biofilm growth plays an important role during infection by providing defence against several antagonistic mechanisms. In this study, we analysed the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains isolated from diabetic foot infections. The antibiotic minimum inhibitory concentration (MIC) was determined for ten antimicrobial compounds, along with the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC), followed by PCR identification of genetic determinants of biofilm production and antimicrobial resistance.ResultsResults demonstrate that very high concentrations of the most used antibiotics in treating diabetic foot infections (DFI) are required to inhibit S. aureus biofilms in vitro, which may explain why monotherapy with these agents frequently fails to eradicate biofilm infections. In fact, biofilms were resistant to antibiotics at concentrations 10–1000 times greater than the ones required to kill free-living or planktonic cells. The only antibiotics able to inhibit biofilm eradication on 50 % of isolates were ceftaroline and gentamicin.ConclusionsThe results suggest that the antibiotic susceptibility patterns cannot be applied to biofilm established infections. Selection of antimicrobial therapy is a critical step in DFI and should aim at overcoming biofilm disease in order to optimize the outcomes of this complex pathology.

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.

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BackgroundPeriodontal disease (PD) is caused by the development of a microbial biofilm (dental plaque) in the periodontium, affecting approximately 80% of dogs. Several bacterial species present in the canine oral cavity can be implicated in the development of this disease, including Enterococcus spp. To decrease antibiotic administration, a possible control strategy for dog’s enterococcal PD may involve the use of the antimicrobial peptide (AMP) nisin.Nisin’s inhibitory activity was evaluated against a collection of previously characterized enterococci obtained from the oral cavity of dogs with PD (n = 20), as well as the potential of a guar-gum gel and a veterinary toothpaste as topical delivery systems for this AMP. The Minimum Inhibitory (MIC) and Bactericidal Concentrations (MBC) and the Minimum Biofilm Eradication (MBEC) and Inhibitory Concentrations (MBIC) were determined for nisin and for the supplemented guar-gum gel. For the supplemented veterinary toothpaste an agar-well diffusion assay was used to evaluate its inhibitory potential.ResultsNisin was effective against all isolates. Independently of being or not incorporated in the guar-gum gel, its inhibitory activity on biofilms was higher, with MBIC (12.46 ± 5.16 and 13.60 ± 4.31 μg/mL, respectively) and MBEC values (21.87 ± 11.33 and 42.34 ± 16.61 μg/mL) being lower than MIC (24.61 ± 4.64 and 14.90 ± 4.10 μg/mL) and MBC (63.09 ± 13.22 and 66.63 ± 19.55 μg/mL) values. The supplemented toothpaste was also effective, showing inhibitory activity against 95% of the isolates.ConclusionsThe inhibitory ability of nisin when incorporated in the two delivery systems was maintained or increased, demonstrating the potential of these supplemented vehicles to be applied to PD control in dogs.

BackgroundDiabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes are foot infections that may be colonized by pathogenic and antimicrobial resistant bacteria, harboring several virulence factors, that could impair its successful treatment. Staphylococcus aureus is one of the most prevalent isolate in diabetic foot infections, together with aerobes and anaerobes.MethodsIn this study, conducted in the Lisbon area, staphylococci isolated (n = 53) from diabetic foot ulcers were identified, genotyped and screened for virulence and antimicrobial resistance traits. Genetic relationship amongst isolates was evaluated by pulsed-field-gel-electrophoresis with further multilocus sequence typing of the identified pulsotypes. PCR was applied for detection of 12 virulence genes and e-test technique was performed to determine minimal inhibitory concentration of ten antibiotics.ResultsAmong the 53 isolates included in this study, 41 Staphylococcus aureus were identified. Staphylococcal isolates were positive for intercellular adhesins icaA and icaD, negative for biofilm associated protein bap and pantone-valentine leucocidin pvl. S. aureus quorum sensing genes agrI and agrII were identified and only one isolate was positive for toxic shock syndrome toxin tst.36 % of staphylococci tested were multiresistant and higher rates of resistance were obtained for ciprofloxacin and erythromycin. Clonality analysis revealed high genomic diversity and numerous S. aureus sequence types, both community- and hospital-acquired, belonging mostly to clonal complexes CC5 and C22, widely diffused in Portugal nowadays.ConclusionsThis study shows that diabetic foot ulcer staphylococci are genomically diverse, present resistance to medically important antibiotics and harbour virulence determinants. These properties suggest staphylococci can contribute to persistence and severity of these infections, leading to treatment failure and to the possibility of transmitting these features to other microorganisms sharing the same niche. In this context, diabetic patients may become a transmission vehicle for microorganisms’ clones between community and clinical environments.

Biofilm formation is considered a selective advantage for staphylococci mastitis isolates, facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers and enclosing in a polymeric matrix, being regulated by several loci. As biofilm formation can proceed through different pathways and time ranges, its detection may differ according to the time of observation. This study aimed at evaluating the time course evolution of biofilm production in Staphylococcus aureus (n = 26) and Staphylococcus epidermidis (n = 29) mastitis isolates by Fluorescent In Situ Hybridisation. Biofilm-forming ability increased with incubation time for both species: for S. aureus, 34.6%, 69.2% and 80.8% of the isolates were able to produce biofilm at 24, 48 and 72 h, respectively. For S. epidermidis, 44.8%, 62.1% and 75.9% of the isolates were biofilm-positive at 24, 48 and 72 h, respectively. No significant difference was found between species at each time point (Friedman's test, p > 0.05). For S. aureus, although a significant difference was found between 24 and 48 h (Wilcoxon matched paired test, p < 0.05), no significant difference was found between 24 and 48 h (p > 0.05). For S. epidermidis, significant differences were found between each time point (p < 0.05). Bacterial biofilms may impair eradication of chronic mastitis, rendering antibiotherapy less effective. Detection of biofilm-forming ability in mastitis isolates may provide useful information for the establishment of a more adequate therapeutic regimen, in view of the antimicrobial concentrations required for bacterial control. However, it is essential that biofilm formation time course is taken into consideration.

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.