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.

Characterization of multidrug-resistant diabetic foot ulcer enterococci / Caracterización de enterococos multirresistentes aislados de infecciones del pie diabético

INTRODUCTION: Diabetes mellitus is a highly prevalent chronic progressive disease with complications that include diabetic-foot ulcers. METHODS: Enterococci isolated from diabetic-foot infections were identified, evaluated by macro-restriction analysis, and screened for virulence traits and antimicrobial resistance. RESULTS: All isolates were considered multidrug-resistant, cytolysin and gelatinase producers, and the majority also demonstrated the ability to produce biofilms. CONCLUSIONS: These results indicate the importance of enterococci in diabetic-foot infection development and persistence, especially regarding their biofilm-forming ability and resistance to clinically relevant antibiotics

INTRODUCCIÓN: La diabetes mellitus es una enfermedad crónica progresiva de alta prevalencia cuyas complicaciones incluyen úlceras del pie. Procedimiento: Se han identificado enterococos aislados de infecciones del pie diabético, evaluados mediante análisis de macrorrestricción y búsqueda de marcadores de virulencia y de resistencia antimicrobiana. RESULTADOS: Todos los aislados analizados fueron considerados multirresistentes, productores de citolisina y gelatinasa, y la mayoría fueron capaces de formar biofilms. CONCLUSIONES: Estos resultados permiten conjeturar sobre la importancia de los enterococos en el desarrollo y la persistencia de la infección del pie diabético, fundamentalmente debido a la capacidad de formación de biofilm y de resistencia a antibióticos de relevancia clínica

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.

Characterization of multidrug-resistant diabetic foot ulcer enterococci.

INTRODUCTION: Diabetes mellitus is a highly prevalent chronic progressive disease with complications that include diabetic-foot ulcers. METHODS: Enterococci isolated from diabetic-foot infections were identified, evaluated by macro-restriction analysis, and screened for virulence traits and antimicrobial resistance. RESULTS: All isolates were considered multidrug-resistant, cytolysin and gelatinase producers, and the majority also demonstrated the ability to produce biofilms. CONCLUSIONS: These results indicate the importance of enterococci in diabetic-foot infection development and persistence, especially regarding their biofilm-forming ability and resistance to clinically relevant antibiotics.

First report of Corynebacterium pseudotuberculosis from caseous lymphadenitis lesions in Black Alentejano pig (Sus scrofa domesticus).

BACKGROUND: Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a common disease in small ruminant populations throughout the world and responsible for a significant economic impact for producers. CASE PRESENTATION: To our knowledge, this is the first characterization of C. pseudotuberculosis from caseous lymphadenitis lesions in Black Alentejano pig (Sus scrofa domesticus). In this study, phenotypic and genotypic identification methods allocated the swine isolates in C. pseudotuberculosis biovar ovis. The vast majority of the isolates were able to produce phospholipase D and were susceptible to most of the antimicrobial compounds tested. Macrorestriction patterns obtained by Pulsed Field Gel Electrophoresis (PFGE) grouped the C. pseudotuberculosis in two clusters with a high similarity index, which reveals their clonal relatedness. Furthermore, swine isolates were compared with C. pseudotuberculosis from caprines and PFGE patterns also showed high similarity, suggesting the prevalence of dominant clones and a potential cross-dissemination between these two animal hosts. CONCLUSIONS: This work represents the first report of Corynebacterium pseudotuberculosis from caseous lymphadenitis lesions in Black Alentejano pig and alerts for the importance of the establishment of suitable control and sanitary management practices to control the infection and avoid further dissemination of this important pathogen to other animal hosts.

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.

In vitro antiviral activity of fluoroquinolones against African swine fever virus.

African swine fever (ASF) is a viral swine disease against which neither an effective vaccine nor a treatment is available. The antiviral effect of thirty fluoroquinolones on the infectivity of African swine fever virus (ASFV) was screened in vitro. There was a severe reduction of the cytopathic effect in ASFV-infected Vero cells when exposed to six independent fluoroquinolones, or to some of their combinations, from an early phase of infection. Moreover, after 7-day treatments, ASFV genome could not be detected by PCR, and the culture supernatants were unable to infect new cell cultures. Pulsed field gel electrophoresis (PFGE) analysis revealed a diminished viral DNA replication without identifiable genome fragmentation in cells exposed to fluoroquinolones. In parallel, altered patterns of viral protein synthesis were observed from early infection. The overall results indicate that bacterial topoisomerase inhibitors interfere with the ASFV replication cycle probably by targeting a putative ASFV-topoisomerase II, opening a new window for antiviral treatments.