Polymicrobial interactions influence the agr copy number in Staphylococcus aureus isolates from diabetic foot ulcers.

Diabetic foot ulcers are a major complication of diabetes and are often colonised by complex bacterial communities, where Staphylococcus aureus is frequently co-present with Pseudomonas aeruginosa. These bacteria interact through quorum sensing, encoded in S. aureus by the accessory gene regulator (agr). Typing and copy number of S. aureus agr were assessed here to give insights on strain variability and possible interspecies influence. As agr is classified in four genetic groups, agr-I, agr-II, agr-III and agr-IV, the agr type of 23 S. aureus diabetic foot ulcers isolates was evaluated by PCR and gene copy number determined by qPCR, including in S. aureus present in polymicrobial infections. agr-I and agr-II were found to be present in 52 and 39% of the isolates, respectively. In two isolates, no agr type was identified, and types III and IV were not detected. Interestingly, agr-II copy number was higher in dual suspensions than in S. aureus single suspension. We conclude that agr type I was the most frequent in clinical centers in Lisbon, and variations in agr-I and agr-II copy numbers were strain specific. Variations in agr copy number in dual suspensions suggests that P. aeruginosa may influence S. aureus agr-II gene regulation, confirming an interaction between these two bacteria. This is a first approach to characterise agr variation in S. aureus from diabetic foot ulcers in vitro.

Susceptibility patterns of Staphylococcus aureus biofilms in diabetic foot infections.

BACKGROUND: Foot 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 ß-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. RESULTS: Results 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. CONCLUSIONS: The 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.

Molecular typing, virulence traits and antimicrobial resistance of diabetic foot staphylococci.

BACKGROUND: Diabetes 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. METHODS: In 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. RESULTS: Among 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. CONCLUSIONS: This 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.

Polymicrobial biofilms by diabetic foot clinical isolates.

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.

In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections.

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.

Wound healing potential of topical bacteriophage therapy on diabetic cutaneous wounds.

Chronic wounds that fail to heal are a common complication of diabetes mellitus and the most common precipitating reason for nontraumatic lower limb amputation. Unfortunately, the bacterial species that cause these infections are becoming more resistant to antibiotics, making them increasingly difficult to treat. We assessed the feasibility of combating chronic bacterial infections with a topically delivered bacteriophage cocktail in two animal models of diabetes mellitus. Microbiological, planimetric, and histological parameters were compared in debrided infected wounds with or without topical bacteriophage treatment. We determined that bacteriophage treatment effectively decreased bacterial colony counts and improved wound healing, as indicated by smaller epithelial and dermal gaps, in Staphylococcus aureus and Pseudomonas aeruginosa infections but was not as effective against Acinetobacter baumannii. Although the improvements were more significant in the rodent model than in the porcine model, our results suggest that topically administered bacteriophage treatment may be effective in resolving chronic infections, especially when applied in conjunction with wound debridement. These findings have important implications for the feasibility of using topical antimicrobial therapies to safely treat chronic infections in diabetes mellitus patients.

A rat model of diabetic wound infection for the evaluation of topical antimicrobial therapies.

Diabetes mellitus is an epidemic multisystemic chronic disease that frequently is complicated by complex wound infections. Innovative topical antimicrobial therapy agents are potentially useful for multimodal treatment of these infections. However, an appropriately standardized in vivo model is currently not available to facilitate the screening of these emerging products and their effect on wound healing. To develop such a model, we analyzed, tested, and modified published models of wound healing. We optimized various aspects of the model, including animal species, diabetes induction method, hair removal technique, splint and dressing methods, the control of unintentional bacterial infection, sampling methods for the evaluation of bacterial burden, and aspects of the microscopic and macroscopic assessment of wound healing, all while taking into consideration animal welfare and the '3Rs' principle. We thus developed a new wound infection model in rats that is optimized for testing topical antimicrobial therapy agents. This model accurately reproduces the pathophysiology of infected diabetic wound healing and includes the current standard treatment (that is, debridement). The numerous benefits of this model include the ready availability of necessary materials, simple techniques, high reproducibility, and practicality for experiments with large sample sizes. Furthermore, given its similarities to infected-wound healing and treatment in humans, our new model can serve as a valid alternative for applied research.