Molecular and Culture-Based Assessment of the Microbial Diversity of Diabetic Chronic Foot Wounds and Contralateral Skin Sites

Abstract

Wound debridement samples and contralateral (healthy) skin swabs acquired from 26 patients attending a specialist foot clinic were analyzed by differential isolation and eubacterium-specific PCR-denaturing gradient gel electrophoresis (DGGE) in conjunction with DNA sequencing. Thirteen of 26 wounds harbored pathogens according to culture analyses, with Staphylococcus aureus being the most common (13/13). Candida (1/13), pseudomonas (1/13), and streptococcus (7/13) were less prevalent. Contralateral skin was associated with comparatively low densities of bacteria, and overt pathogens were not detected. According to DGGE analyses, all wounds contained significantly greater eubacterial diversity than contralateral skin (P < 0.05), although no significant difference in total eubacterial diversity was detected between wounds from which known pathogens had been isolated and those that were putatively uninfected. DGGE amplicons with homology to Staphylococcus sp. (8/13) and S. aureus (2/13) were detected in putatively infected wound samples, while Staphylococcus sp. amplicons were detected in 11/13 noninfected wounds; S. aureus was not detected in these samples. While a majority of skin-derived DGGE consortial fingerprints could be differentiated from wound profiles through principal component analysis (PCA), a large minority could not. Furthermore, wounds from which pathogens had been isolated could not be distinguished from putatively uninfected wounds on this basis. In conclusion, while chronic wounds generally harbored greater eubacterial diversity than healthy skin, the isolation of known pathogens was not associated with qualitatively distinct consortial profiles or otherwise altered diversity. The data generated support the utility of both culture and DGGE for the microbial characterization of chronic wounds.

A clinico-microbiological study of diabetic foot ulcer patients to identify risk factors and their correlation with prognosis in tertiary care hospital in India

“…Wound infection is commonly defined according to the presence of pathogens and colonization densities exceeding ≥106 organisms per gram of tissue and the development of significant tissue damage and clinical signs of infection. 12,13 In the present study, 50 patients were studied for over a period of 18 months. The demographics and patient characteristics have been described in ( Table 2).…”

New Molecular Techniques to Study the Skin Microbiota of Diabetic Foot Ulcers

“…In the past few years, however, molecular microbiology techniques have demonstrated the presence of greater numbers and varieties of species in various types of wounds than had previously been recognized. 24,28,29,[37][38][39] The goal now is to better understand the role of the many organisms we now know that comprise the cutaneous microbiota in DFU.…”

“…28 Defining the full microbiota of both intact and wounded skin should improve our understanding of the microbial nature of DFU. 29 …”

The bacteria profiles of wounds in diabetic patients hospitalized in northern Kwazulu-Natal, South Africa

“…Routine analysis of wound specimen normally involves the use of traditional culture methods such as selective and differential agar media to culture the anaerobic and aerobic bacteria [4]. The organisms are classified by means of similarities and differences based on their phenotypic characteristics such as cell appearance, cell shape, size, pigmentation [11][12][13]. Gram staining, biochemical tests (catalase and oxidase) and controlled growth conditions are required for definitive grouping of bacteria [12].…”

“…However, with the aid of molecular diagnostic techniques, identification has shown that most chronic wounds are polymicrobial [11]. Culture-based techniques alone often fail to identify fastidious bacteria that are important in diagnosis and they may underestimate microbial diversity [11] while culture-independent methods are able to detect bacterial species that were omitted by culture-based techniques [16].…”