Staphylococcus aureus Toxins and Diabetic Foot Ulcers: Role in Pathogenesis and Interest in Diagnosis.
ABSTRACT: Infection of foot ulcers is a common, often severe and costly complication in diabetes. Diabetic foot infections (DFI) are mainly polymicrobial, and Staphylococcus aureus is the most frequent pathogen isolated. The numerous virulence factors and toxins produced by S. aureus during an infection are well characterized. However, some particular features could be observed in DFI. The aim of this review is to describe the role of S. aureus in DFI and the implication of its toxins in the establishment of the infection. Studies on this issue have helped to distinguish two S. aureus populations in DFI: toxinogenic S. aureus strains (harboring exfoliatin-, EDIN-, PVL- or TSST-encoding genes) and non-toxinogenic strains. Toxinogenic strains are often present in infections with a more severe grade and systemic impact, whereas non-toxinogenic strains seem to remain localized in deep structures and bone involving diabetic foot osteomyelitis. Testing the virulence profile of bacteria seems to be a promising way to predict the behavior of S. aureus in the chronic wounds.
Project description:Background:The polymicrobial nature of diabetic foot infection (DFI) and the emergence of antimicrobial resistance have complicated DFI treatment. Current treatment guidelines for deep DFI recommend coverage of methicillin-resistant Staphylococcus aureus (MRSA) and susceptible Enterobacteriaceae. This study aimed to describe the epidemiology of DFI and to identify predictors for DFI associated with multidrug-resistant organisms (MDROs) and pathogens resistant to recommended treatment (PRRT). Methods:Adult patients admitted to Detroit Medical Center from January 2012 to December 2015 with DFI and positive cultures were included. Demographics, comorbidities, microbiological history, sepsis severity, and antimicrobial use within 3 months before DFI were obtained retrospectively. DFI-PRRT was defined as a DFI associated with a pathogen resistant to both vancomycin and ceftriaxone. DFI-MDRO pathogens included MRSA in addition to PRRT. Results:Six-hundred forty-eight unique patients were included, with a mean age of 58.4 ± 13.7 years. DFI-MDRO accounted for 364 (56%) of the cohort, and 194 (30%) patients had DFI-PRRT. Independent predictors for DFI-PRRT included history of PRRT in a diabetic foot ulcer, antimicrobial exposure in the prior 90 days, peripheral vascular disease, and chronic kidney disease. Long-term care facility residence was independently associated with DFI due to ceftriaxone-resistant Enterobacteriaceae, and recent hospitalization was an independent predictor of DFI due to vancomycin-resistant Enterococcus. Conclusions:An unexpectedly high prevalence of DFI-PRRT pathogens was identified. History of the same pathogen in a prior diabetic foot ulcer and recent antimicrobial exposure were independent predictors of DFI-PRRT and should be considered when selecting empiric DFI therapy.
Project description:Bacterial biofilms do serious harm to the diabetic foot ulcer (DFU) because they play a crucial role in infection invasion and spread. Staphylococcus aureus, the predominant Gram-positive bacteria in diabetic foot infection (DFI), is often associated with colonization and biofilm formation. Through biofilm formation tests in vitro, we observed that S. aureus bacteria isolated from DFU wounds were more prone to form biofilms than those from non-diabetic patients, while there was no difference in blood sugar between the biofilm (+) diabetics (DB+) and biofilm (-) diabetics (DB-). Furthermore, we found that advanced glycation end products (AGEs) promoted the biofilm formation of S. aureus in clinical isolates and laboratory strains in vitro, including a methicillin-resistant strain. Analysis of biofilm components demonstrated that the biofilms formed mainly by increasing extracellular DNA (eDNA) release; remarkably, the S. aureus global regulator sigB was upregulated, and its downstream factor lrgA was downregulated after AGE treatments. Mechanism studies using a sigB-deleted mutant (Newman-?sigB) confirmed that AGEs decreased expression of lrgA via induction of sigB, which is responsible for eDNA release and is a required component for S. aureus biofilm development. In conclusion, the present study suggests that AGEs promote S. aureus biofilm formation via an eDNA-dependent pathway by regulating sigB. The data generated by this study will provide experimental proof and theoretical support to improve DFU infection healing.
Project description:Management of foot salvage therapy (FST) for diabetic foot infections (DFI) is challenging due to the absence of reliable diagnostics to identify the etiologic agent and prognostics to justify aggressive treatments. As Staphylococcus aureus is the most common pathogen associated with DFI, we aimed to develop a multiplex immunoassay of IgG in serum and medium enriched for newly synthesized anti-S. aureus antibodies (MENSA) generated from cultured peripheral blood mononuclear cells of DFI patients undergoing FST. Wound samples were collected from 26 DFI patients to identify the infecting bacterial species via 16S rRNA sequencing. Blood was obtained over 12 weeks of FST to assess anti-S. aureus IgG levels in sera and MENSA. The results showed that 17 out of 26 infections were polymicrobial and 12 were positive for S. aureus While antibody titers in serum and MENSA displayed similar diagnostic potentials to detect S. aureus infection, MENSA showed a 2-fold-greater signal-to-background ratio. Multivariate analyses revealed increases in predictive power of diagnosing S. aureus infections (area under the receiver operating characteristic curve [AUC] > 0.85) only when combining titers against different classes of antigens, suggesting cross-functional antigenic diversity. Anti-S. aureus IgG levels in MENSA decreased with successful FST and rose with reinfection. In contrast, IgG levels in serum remained unchanged throughout the 12-week FST. Collectively, these results demonstrate the applicability of serum and MENSA for diagnosis of S. aureus DFI with increased power by combining functionally distinct titers. We also found that tracking MENSA has prognostic potential to guide clinical decisions during FST.
Project description:Diabetic foot infection (DFI) is a major complication of diabetic foot that lead to nontraumatic lower-extremity amputation (LEA). Such distal infection of the body having systemic inflammatory response syndrome (SIRS) is rarely reported. Consecutive patients treated for limb-threatening DFI in a major diabetic foot center in Taiwan were analyzed between the years 2014 to 2017. Clinical factors, laboratory data, perfusion, extent, depth, infection and sensation (PEDIS) wound score in 519 subjects with grade 3 DFI and 203 presenting SIRS (28.1%) were compared. Major LEA and in-hospital mortality were defined as poor prognosis. Patients presenting SIRS had poor prognosis compared with those with grade 3 DFI (14.3% versus 6.6% for major LEA and 6.4% versus 3.5% for in-hospital mortality). Age, wound size, and HbA1c were independent risk factors favoring SIRS presentation. Perfusion grade 3 (odds ratio 3.37, p = 0.044) and history of major adverse cardiac events (OR 2.41, p = 0.036) were the independent factors for poor prognosis in treating patients with DFI presenting SIRS. SIRS when presented in patients with DFI is not only limb- but life-threatening as well. Clinicians should be aware of the clinical factors that are prone to develop and those affecting the prognosis in treating patients with limb-threatening foot infections.
Project description:INTRODUCTION:Diabetic foot infections (DFIs) are the leading cause of non-traumatic lower extremity amputations in the United States. Antimicrobials active against methicillin-resistant Staphylococcus aureus (MRSA) are recommended in patients with associated risk factors; however, limited data exist to support these recommendations. Due to the changing epidemiology of MRSA, and the consequences of unnecessary antibiotic therapy, guidance regarding the necessity of empirical MRSA coverage in DFIs is needed. We sought to 1) describe the prevalence of MRSA DFIs at our institution and compare to the proportion of patients who receive MRSA antibiotic coverage and 2) identify risk factors for MRSA DFI. METHODS:This was a retrospective cohort study of all adult, culture-positive DFI patients managed at University Hospital, San Antonio, TX between January 1, 2010 and September 1, 2014. Patient eligibility included a principal ICD-9-CM discharge diagnosis code for foot infection and a secondary diagnosis of diabetes. The primary outcome was MRSA identified in the wound culture. Independent variables assessed included patient demographics, comorbidities, prior hospitalization, DFI therapies, prior antibiotics, prior MRSA infection, and laboratory values. Multivariable logistic regression was used to identify risk factors for MRSA DFI. RESULTS:Overall, 318 patients met inclusion criteria. Patients were predominantly Hispanic (79%) and male (69%). Common comorbidities included hypertension (76%), dyslipidemia (52%), and obesity (49%). S. aureus was present in 46% of culture-positive DFIs (MRSA, 15%). A total of 273 patients (86%) received MRSA antibiotic coverage, resulting in 71% unnecessary use. Male gender (OR 3.09, 95% CI 1.37-7.99) and bone involvement (OR 1.93, 1.00-3.78) were found to be independent risk factors for MRSA DFI. CONCLUSIONS:Although MRSA was the causative pathogen in a small number of DFI, antibiotic coverage targeted against MRSA was unnecessarily high.
Project description:Pexiganan, a 22-amino-acid synthetic cationic peptide, is currently in phase 3 clinical trials as a topical antimicrobial agent for the treatment of mild infections associated with diabetic foot ulcers. Bacterial isolates from the 2013 SENTRY Antimicrobial Surveillance Program designated as pathogens from diabetic foot infections (DFI) and Gram-negative and -positive pathogens from various infection types that harbored selected resistance mechanisms/phenotypes were tested against pexiganan in reference cation-adjusted Mueller-Hinton broth. The MIC50 and MIC90 against all organisms tested from DFI were 16 and 32 μg/ml, respectively. Escherichia coli, Klebsiella pneumoniae, Citrobacter koseri, Enterobacter cloacae, Acinetobacter species, and Pseudomonas aeruginosa MIC values ranged from 8 to 16 μg/ml. Pexiganan MIC values among Staphylococcus aureus (methicillin-resistant S. aureus [MRSA] and methicillin-susceptible S. aureus [MSSA]), beta-hemolytic streptococci, and Enterococcus faecium ranged from 8 to 32 μg/ml. Pexiganan activity was not adversely affected for members of the family Enterobacteriaceae or P. aeruginosa that produced β-lactamases or resistance mechanisms to other commonly used antimicrobial agents. Decreased susceptibility to vancomycin did not affect pexiganan activity against S. aureus or E. faecium. Enterococcus faecalis appears to be intrinsically less susceptible to pexiganan (MIC, 32 to 256 μg/ml). The "all organism" MIC90 of 32 μg/ml for pexiganan in this study was >250-fold below the pexiganan concentration in the cream/delivery vehicle being developed for topical use.
Project description:BACKGROUND:Diabetic foot infections (DFIs) are a frequent complication of Diabetes mellitus and a major cause of nontraumatic limb amputations. The Gram-positive bacterium Staphylococcus aureus, known for its resilient biofilms and antibiotic resistant profile, is the most frequent DFI pathogen. It is urgent to develop innovative treatments for these infections, being the antimicrobial peptide (AMP) nisin a potential candidate. We have previously proposed the use of a guar gum biogel as a delivery system for nisin. Here, we evaluated the potential of the nisin-biogel to enhance the efficacy of conventional antibiotics and antiseptics against DFIs S. aureus clinical isolates. METHODS:A collection of 23 S. aureus strains isolated from DFI patients, including multidrug- and methicillin-resistant strains, was used. The antimicrobial activity of the nisin-biogel was tested alone and in different combinations with the antiseptic chlorhexidine and the antibiotics clindamycin, gentamicin and vancomycin. Isolates' in vitro susceptibility to the different protocols was assessed using broth microdilution methods in order to determine their ability to inhibit and/or eradicate established S. aureus biofilms. Antimicrobials were added to the 96-well plates every 8 h to simulate a typical DFI treatment protocol. Statistical analysis was conducted using RCBD ANOVA in SPSS. RESULTS:The nisin-biogel showed a high antibacterial activity against biofilms formed by DFI S. aureus. The combined protocol using nisin-biogel and chlorhexidine presented the highest efficacy in biofilm formation inhibition, significantly higher (p<0.05) than the ones presented by the antibiotics-based protocols tested. Regarding biofilm eradication, there were no significant differences (p>0.05) between the activity of the combination nisin-biogel plus chlorhexidine and the conventional antibiotic-based protocols. CONCLUSIONS:Results provide a valuable contribution for the development of complementary strategies to conventional antibiotics protocols. A combined protocol including chlorhexidine and nisin-biogel could be potentially applied in medical centres, contributing for the reduction of antibiotic administration, selection pressure on DFI pathogens and resistance strains dissemination.
Project description:Background:Multidrug-resistant organisms (MDROs) are important diabetic foot infection (DFI) pathogens. This study evaluated the impact of DFIs associated with MDRO pathogens (DFI-MDRO) on clinical outcomes. Methods:Adults admitted to Detroit Medical Center from January 2012 to December 2015 with culture-positive DFI were included. Associations between outcomes and DFI-MDRO (evaluated as a single group that included methicillin-resistant Staphylococcus aureus [MRSA], vancomycin-resistant enterococci, Enterobacteriaceae resistant to third-generation cephalosporin [3GCR-EC], Acinetobacter baumannii, and Pseudomonas aeruginosa) were analyzed. Outcomes included above- and below-knee lower extremity amputation (LEA), readmissions, and mortality within a year after DFI. A propensity score predicting the likelihood of having DFI-MDRO was computed by comparing patients with DFI-MDRO with patients with DFI with non-MDRO pathogens (DFI-non-MDRO). Using conditional logistic regression, DFI-MDRO was analyzed as an independent variable after patients in the MDRO and non-MDRO groups were matched by propensity score. Results:Six hundred forty-eight patients were included, with a mean age ± SD of 58.4 ± 13.7. Most patients in the cohort presented with chronic infection (75%). DFI-MDRO occurred in greater than one-half of the cohort (n = 364, 56%), and MRSA was the most common MDRO (n = 224, 62% of the DFI-MDRO group). In propensity-matched analyses, DFI-MDRO was not associated with 1-year LEA or readmissions, but was associated with recurrent DFI episodes (odds ratio, 2.1; 95% confidence interval, 1.38-3.21). Conclusions:DFI-MDRO was associated with a 2-fold increased risk of recurrent DFI compared with patients with DFI-non-MDRO.
Project description:This study assessed the clonal diversity, the resistance profile and the virulence potential of <i>Escherichia coli</i> strains isolated from diabetic foot infection (DFI) and diabetic foot osteomyelitis (DFOM). A retrospective single-centre study was conducted on patients diagnosed with <i>E. coli</i> isolated from deep DFI and DFOM at Clinique du Pied Diabétique Gard-Occitanie (France) over a two-year period. Phylogenetic backgrounds, virulence factors (VFs) and antibiotic resistance profiles were determined. Whole-genome analysis of <i>E. coli</i> strains isolated from same patients at different periods were performed. From the two-years study period, 35 <i>E. coli</i> strains isolated from 33 patients were analysed; 73% were isolated from DFOM. The majority of the strains belonged to the virulent B2 and D phylogenetic groups (82%). These isolates exhibited a significant higher average of VFs number than strains belonging to other groups (<i>p</i> < 0.001). <i>papG2</i> gene was significantly more detected in strains belonging to B2 phylogroup isolated from DFI compared to DFOM (<i>p</i> = 0.003). The most prevalent antibiotic resistance pattern was observed for ampicillin (82%), cotrimoxazole (45%), and ciprofloxacin (33%). The genome analysis of strains isolated at two periods in DFOM showed a decrease of the genome size, and this decrease was more important for the strain isolated at nine months (vs. four months). A shared mutation on the putative acyl-CoA dehydrogenase-encoding gene <i>aidB</i> was observed on both strains. <i>E. coli</i> isolates from DFOM were highly genetically diverse with different pathogenicity traits. Their adaptation in the bone structure could require genome reduction and some important modifications in the balance virulence/resistance of the bacteria.
Project description:OBJECTIVE:The purpose of this study was to assess the virulence potential of Staphylococcus aureus strains isolated from diabetic foot ulcers and to discriminate noninfected from infected ulcers. RESEARCH DESIGN AND METHODS:Diabetic patients hospitalized in a diabetic foot department with a foot ulcer were prospectively enrolled if they had been free of antibiotic treatment over the previous 6 months. At admission, ulcers were classified as infected or noninfected on the basis of clinical examination, according to the International Working Group on the Diabetic Foot system. Only patients carrying S. aureus as the sole pathogen were included. In individuals with a grade 1 ulcer, a second bacterial specimen was obtained 1 month later. Using virulence genotyping markers, clonality tools, and an in vivo Caenorhabditis elegans model, we correlated the virulence of 132 S. aureus strains with grade, time of collection, and ulcer outcome. RESULTS:Among virulence genes, the most relevant combination derived from the logistic regression was the association of cap8, sea, sei, lukE, and hlgv (area under the curve 0.958). These markers were useful to distinguish noninfected (grade 1) from infected (grades 2-4) ulcers and to predict wound status at the follow-up. With use of the nematode model, S. aureus strains isolated from grade 1 ulcers were found to be significantly less virulent than strains from ulcers at or above grade 2 (P < 0.001). CONCLUSIONS:This study highlights the coexistence of two S. aureus populations on diabetic foot ulcers. A combination of five genes that may help distinguish colonized grade 1 from infected grade >or=2 wounds, predict ulcer outcome, and contribute to more appropriate use of antibiotics was discovered.