Conventional and molecular approaches for bacterial identification and quantification in chronic wounds

Wounds present an ideal environment for the growth of bacteria as they are usually moist and warm. The impact of bacteria on wound healing and developing infection is debatable and only partially understood. Some clinicians believe that the number of bacteria is a crucial factor in determining whether the wound is likely to heal. Others argue that the presence of specific pathogens and their interactions are the main cause of non-healing wounds. Also, the methodology of wound culture has been prone to controversy for many years. Most diagnostic laboratories use conventional microbiological techniques to indicate if there are pathogens in a wound. Some specialists still argue that greater than 105 organisms per gram of tissue is diagnostic for infection. Introduction of new molecular techniques have shown that only a small percentage of bacteria are identified and they grown in biofilms, which makes sampling difficult. In this project, the aspect of sample collection and transport was investigated as well as the conventional and molecular approaches for bacteria identification and quantification. Four different swab transport systems were tested for their ability to maintain viability of the most common wound bacteria during transport and their performance with molecular methods in order to establish the best swab transport devices for further testing on patients. The most satisfactory results were achieved with Sigma dry swabs and this swab remained the best choice for further in vivo studies involving both conventional and molecular techniques of bacteria identification and quantification. The semi-quantitative swab and biopsy culture was compared with quantitative culture to establish the best method for bacteria culture. Our findings demonstrated a statistically strong significant correlation between semi-quantitative and quantitative swab and biopsy methods and use of semi-quantitative count as a cost effective method compared to quantitative serial dilutions. However, when time is important rapid methods should be employed thus Real-Time PCR (RT-PCR) assay was developed for the direct and rapid detection of MRSA and compared with conventional methods. The diagnostic values of the RT-PCR assay for the detection of mecA and femB genes were as follows: sensitivity 83.3%, specificity 88.5%, PPV 62.5% and NPV 95.8%. Quantitative analysis revealed that the average difference between the MRSA counts obtained using the RT-PCR and conventional culture results was 0.61 log. These findings show the potential of the RT-PCR assay in rapid detection and quantification of MRSA. Development of a RT-PCR assay for MRSA detection was the first step in developing a multiplex RT-PCR assay for chronic wound samples. In further studies, a DGGE-sequencing method was developed for the analysis of the diversity of microflora in chronic wounds and healthy feet and compared with conventional methods. DGGE-sequencing allowed identification of a number of strains not detected by culture techniques with 43% of the DGGE fragments representing organisms not cultured from the wound from which they had been amplified. This highlights the fact that a significant proportion of the resident microflora was not able to be analysed by culture. Development of PCR-DGGE sequencing and investigation of the diversity of microflora in chronic wounds allowed us to select the panel of microorganisms for the further development of multiplex RT-PCR assay for the rapid detection of bacteria in chronic wounds.