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