Virulence potential of Staphylococcus aureus strains isolated from diabetic foot ulcers: a new paradigm

Abstract

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.

Figure 1

Flow of patients with diabetic foot ulcers (DFU) through the study and the genotyping determination obtained during grade 1 inclusion and follow-up. In grade 1 ulcers, two genotyping profiles were obtained: profile 1, without the four infection markers and with the noninfected ulcer marker; and profile 2, with presence of infection markers. During the follow-up, if the wound healed, the genotyping profiles (profile 1) are the same as the inclusion profiles. Also if a wound presented a slowly worsening evolution, the genotyping profiles (profile 4) are different from the inclusion profile (profile 1), with the four infection markers in all of the follow-up cases. On the other hand, if the wound presented a rapidly worsening evolution, the genotyping profiles (profile 2) are exactly the same in inclusion and follow-up with the four infection markers. *Grades according to the IWGDF classification system (3,4). Number of monomicrobial cultures for S. aureus (132 = 118 + 12 + 2) is in bold type.

Figure 2

C. elegans killing assay. A: Comparison of LT50 according to IWGDF grade and susceptibility of S. aureus to methicillin. B: Comparison of LT50 of strains isolated from grade 1 ulcers at inclusion and on the same wound after follow-up: ulcer 1, healing ulcer (grade 1 followed by grade 1); ulcer 2, rapid worsening ulcer (grade 1 followed by grade 2); and ulcer 3, slow worsening ulcer (grade 1 followed by grade 3). Virulent bacteria (isolated from ulcers grades 2–4) kill worms more quickly than avirulent strains (isolated from grade 1 ulcers). No statistically significant differences in LT50 were noted between MSSA and MRSA isolated from ulcers of the same grade. LT50 corresponds to the time for half of the worms to die. A: , MRSA; ▪, MSSA. 1, NSA6759 (n = 6 virulence genes); 2, NSA43233 (n = 3); 3, NSA56348 (n = 2); 4, NSA443564 (n = 15); 5, NSA16210 (n = 15); 6, NSA454193 (n = 13); 7, NSA369602 (n = 14); 8, NSA26758 (n = 15); 9, NSA19308 (n = 14); 10, NSA4281 (n = 18); 11, NSA29197 (n = 15); and 12, NSA41007 (n = 10). B: □, at admission; ▪, after follow-up. 1, NSA11260 (n = 4 virulence genes); 2, NSA739 (n = 6); 3, NSA22465 (n = 9); 4, NSA27333 (n = 9); 5, NSA18026 (n = 5); and 6, NSA388104 (n = 12). The results are representative of at least five independent trials for each group of strains. I, strains isolated at inclusion; F, strains isolated at follow-up.