Date Published: September 19, 2017
Publisher: Springer Berlin Heidelberg
Author(s): Ayomi Dilhari, Asanga Sampath, Chinthika Gunasekara, Neluka Fernando, Deepaka Weerasekara, Chris Sissons, Andrew McBain, Manjula Weerasekera.
Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profiling of complex ecosystems including infected chronic wounds. Six different DNA extraction methods were compared for profiling of the microbial community associated with chronic wound infections using PCR-DGGE. Tissue debris obtained from chronic wound ulcers of ten patients were used for DNA extraction. Total nucleic acid was extracted from each specimen using six DNA extraction methods. The yield, purity and quality of DNA was measured and used for PCR amplification targeting V2–V3 region of eubacterial 16S rRNA gene. QIAGEN DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA compared to the other five DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the five conventional methods, bead beater/phenol–chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with a high purity and resulted in a higher DGGE band diversity. Although DNA extraction using heat and NaOH had the lowest purity, DGGE revealed a higher bacterial diversity. The findings suggest that the quality and the yield of genomic DNA are influenced by the DNA extraction protocol, thus a method should be carefully selected in profiling a complex microbial community.
Microorganisms associated with polymicrobial infections such as chronic wound infections are diverse and often unculturable (Han et al. 2011; Oates et al. 2012; Wadinamby 2013). Molecular biological methods based on 16S rDNA and other genetic markers have been used effectively to analyze diversity of microbial populations in environmental samples (Nakatsu 2007; Øvreås et al. 1997) as well as clinical specimens (Ariefdjohan et al. 2010; Oates et al. 2012; Walter et al. 2001). Molecular methods can bypass the culture isolation and can generate a comprehensive and precise set of data which is important to understand the role of these microorganisms in polymicrobial infections (Oates et al. 2012; Tannock 2002). As a molecular fingerprinting technique, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) has been successfully applied to profile diverse microbial communities of various clinical specimens (Ariefdjohan et al. 2010; Oates et al. 2012; Walter et al. 2001; Weerasekera et al. 2017, 2013).
All ten patients included in the study had chronic foot wound infections. The mean age of this group of patients were 64.4 years and the range was between 54 and 80 years. Male:female ratio was 1:1.
Among the five tested conventional methods, bead beater/phenol–chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with high purity. Further it resulted in broad diversity of bacterial communities in chronic wounds using PCR-DGGE. This method is a less time consuming process and less expensive, therefore it can be easily applied for the settings where expediency and cost effectiveness is essential. This reinforces the BP1 method as an appropriate, conventional DNA extraction method to study microbial communities in human chronic wounds. The DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA with appreciably greater yield compared to the quality and yield of the other five conventional DNA extraction methods. Further K method gave a broad diversity of bacterial communities in chronic wounds using PCR-DGGE.