Development and validation of a reference marker for identification of aerobic and anaerobic bacteria associated with diabetes chronic wound ulcers using PCR denaturing gradient gel electrophoresis

Abstract

Introduction: Diabetes chronic wounds consist with a diverse microbial community and unculturablespecies may be highly prevalent.Objectives: This study aimed to establish a bacterial reference marker consisting of a group ofchronic wound related bacteria, using polymerase chain reaction-denaturing gradient gelelectrophoresis (PCR-DGGE) for profiling of bacteria in diabetes chronic wound infections.Methods: DNA was extracted from the known wound bacterial strains. PCR–DGGE was performedusing eubacterial specific primers targeting V2-V3 region of 16S rDNA. DGGE was performed usinga 30-55% denaturing gradient. Migration position of each organism was detected on DGGE gel andimportant organisms were selected. Equal volume from PCR products of each selected organism wasmixed, diluted with gel loading dye in 1:1.5 ratio and used for all DGGE gels. The ladder was thensubjected to species identification of fifteen tissue debridement specimens obtained from diabeteschronic wound ulcers. The identification efficacy was tested by sequencing.Results: DNA of bacterial pathogens which showed different migration distances on the gel werecombined and used as a reference panel. This bacterial ladder consisted of eleven different bacterialspecies including Bacteroides sp., S. aureus, Acineto bacter sp., P. aeruginosa, Streptococcus Group Aand Group B sp., E. faecalis, Providencia sp., Veillonella sp., E .coli and Enterobacter sp. Accordingto the reference panel, Pseudomonas species were abundant. Further the results were confirmed bysequencing.Conclusion: Reference marker allows comparative analysis of DGGE patterns and can be used as atool for presumptive identification of polymicrobial microbiota in chronic wound infections