Identity and quantity of microorganisms in necrotising fasciitis determined by culture and molecular methods

Necrotising fasciitis (NF), commonly known as flesh eating disease is a fast progressing, potentially lethal infection of the subcutaneous tissue/fascia. Treatment includes high doses of intravenous antibiotics and aggressive surgical debridement. Accurate identification of the microbial community involved in the disease may add to the knowledge of NF pathogenesis and influence the administration of antibiotics, thereby potentially improving the outcome for the patients. In this study the aim was to investigate the applicability of different molecular methods as compared to standard culture-based methods.
We investigated the microbial communities in 21 samples obtained during debridement of NF patients (n=8). Samples were examined by standard bacteriological examination (culture and microscopy) at Rigshospitalet (Copenhagen, Denmark) and a range of molecular methods. The best DNA extraction protocol was chosen based on tests of 5 protocols performed on 5 NF samples. Molecular methods included full-length 16S rRNA gene analysis, real-time PCR, the Ibis T5000 Biosensor System, and 454 pyrosequencing of partial 16S rRNA genes.
In 9 samples (43%) no growth of microorganisms was observed whereas molecular methods identified microorganisms in all samples. Culture identified Streptococcus sp. in most cases (48%). By molecular methods the incidence was increased (90%) and identification to species level was possible, with S.
pyogenes being dominant (85% of samples with Streptococcus). Real-time PCR showed dominance of Streptococcus sp. in the samples, however some additional bacteria were found. The remaining samples were found to contain yeast by culture and a mixture of Mycoplasma sp., Fusobacterium necrophorum and Candida albicans by molecular methods. Overall findings of polymicrobial communities were more common by molecular methods than culture.
Correspondence between findings by culture and molecular methods indicates that the latter may be an appropriate method. The advantages of using molecular methods are: 1) identification of the pathogen(s) even when antibiotics have been administered, and 2) less time-consumption by some of the molecular
methods than conventional culture. The challenge is the interpretation of the significance of the findings by molecular methods. The use of the molecular methods will potentially enable a more rapid adjustment from empiric antibiotic treatment to defined treatment directed at identified microorganisms.