Vibeke Børsholt Rudkjøbing

The bacteriology of chronic venous leg ulcer examined by culture-independent molecular methods.

The bacterial microbiota plays an important role in the prolonged healing of chronic venous leg ulcers. The present study compared the bacterial diversity within ulcer material from 14 skin graft operations of chronic venous leg ulcers using culture‐based methods and molecular biological methods, such as 16S rRNA gene sequencing, fingerprinting, quantitative polymerase chain reaction, and fluorescence in situ hybridization. Each wound contained an average of 5.4 species but the actual species varied between wounds. The diversity determined by culture‐based methods and the molecular biological methods was different. All the wounds contained Staphylococcus aureus, whereas Pseudomonas aeruginosa was in six out of 14 wounds. Molecular methods detected anaerobic pathogens in four ulcers that were not detected with anaerobic culture methods. Quantitative polymerase chain reaction was used to compare the abundance of S. aureus and P. aeruginosa at different locations in the ulcers and their numbers varied greatly between samples taken at different locations in the same ulcer. This should be considered when ulcers are investigated in routine clinical care. The differences between the results obtained with culture‐based and molecular‐based approaches demonstrate that the use of one approach alone is not able to identify all of the bacteria present in the wounds.

True Microbiota Involved in Chronic Lung Infection of Cystic Fibrosis Patients Found by Culturing and 16S rRNA Gene Analysis

ABSTRACT Patients suffering from cystic fibrosis (CF) develop chronic lung infection. In this study, we investigated the microorganisms present in transplanted CF lungs (n = 5) by standard culturing and 16S rRNA gene analysis. A correspondence between culturing and the molecular methods was observed. In conclusion, standard culturing seems reliable for the identification of the dominating pathogens.

An exploratory study of microbial diversity in sinus infections of cystic fibrosis patients by molecular methods

BACKGROUND For the first time microorganisms in CF sinuses are investigated by molecular methods in response to an absence of anaerobes in CF sinus samples during a two-year period at the Copenhagen CF center. METHODS Endoscopic sinus surgery was performed in 19 CF patients. DNA from intact bacterial cells was investigated by 16S rRNA gene analysis and quantitative PCR. Results were compared to culture-dependent routine diagnosis. RESULTS Molecular methods showed a large microbial diversity, which included undetected anaerobes that may play a pathogenic role. Importantly, the culture methods did not always detect known CF pathogens. Quantitative PCR showed generally a higher abundance of classic CF pathogens e.g. Pseudomonas aeruginosa and Staphylococcus aureus compared with the anaerobe Propionibacterium acnes. CONCLUSIONS The results indicate that the culture methods in some cases may not be suitable as stand-alone method for this patient group, as diversity may be underestimated and important species undetected.