Comparing culture and molecular methods for the identification of microorganisms involved in necrotizing soft tissue infections.

BACKGROUND: Necrotizing soft tissue infections (NSTIs) are a group of infections affecting all soft tissues. NSTI involves necrosis of the afflicted tissue and is potentially life threatening due to major and rapid destruction of tissue, which often leads to septic shock and organ failure. The gold standard for identification of pathogens is culture; however molecular methods for identification of microorganisms may provide a more rapid result and may be able to identify additional microorganisms that are not detected by culture. METHODS: In this study, tissue samples (n = 20) obtained after debridement of 10 patients with NSTI were analyzed by standard culture, fluorescence in situ hybridization (FISH) and multiple molecular methods. The molecular methods included analysis of microbial diversity by 1) direct 16S and D2LSU rRNA gene Microseq 2) construction of near full-length 16S rRNA gene clone libraries with subsequent Sanger sequencing for most samples, 3) the Ibis T5000 biosensor and 4) 454-based pyrosequencing. Furthermore, quantitative PCR (qPCR) was used to verify and determine the relative abundance of Streptococcus pyogenes in samples. RESULTS: For 70 % of the surgical samples it was possible to identify microorganisms by culture. Some samples did not result in growth (presumably due to administration of antimicrobial therapy prior to sampling). The molecular methods identified microorganisms in 90 % of the samples, and frequently detected additional microorganisms when compared to culture. Although the molecular methods generally gave concordant results, our results indicate that Microseq may misidentify or overlook microorganisms that can be detected by other molecular methods. Half of the patients were found to be infected with S. pyogenes, but several atypical findings were also made including infection by a) Acinetobacter baumannii, b) Streptococcus pneumoniae, and c) fungi, mycoplasma and Fusobacterium necrophorum. CONCLUSION: The study emphasizes that many pathogens can be involved in NSTIs, and that no specific "NSTI causing" combination of species exists. This means that clinicians should be prepared to diagnose and treat any combination of microbial pathogens. Some of the tested molecular methods offer a faster turnaround time combined with a high specificity, which makes supplemental use of such methods attractive for identification of microorganisms, especially for fulminant life-threatening infections such as NSTI.

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.

Bacterial diversity in suspected prosthetic joint infections: an exploratory study using 16S rRNA gene analysis.

Formation of biofilm is a prominent feature of prosthetic joint infections (PJIs) and constitutes a challenge to current sampling procedures and culture practices. Molecular techniques have a potential for improving diagnosis of biofilm-adapted, slow-growing and non-culturable bacteria. In this exploratory study we investigated the bacterial diversity in specimens from 22 patients clinically suspected of having PJIs. Bacteriological cultures were performed according to standard practice. A total of 55 specimens from 25 procedures ('specimen sets') were submitted to broad range 16S rRNA gene PCR, cloning, sequencing and phylogenetic analysis. More than 40 bacterial taxa within six phyla were identified in 14 specimen sets originating from 11 patients. Direct observation of biofilm was made in selected specimens by fluorescence in situ hydridization. 16S rRNA gene analysis and bacteriological cultures were concordant for 15/25 specimen sets (60%; five positive, 10 negative); additional taxa were detected in four sets by gene analysis, and discrepant results were obtained for six sets, five of which were negative on culture. Polymicrobial communities were revealed in 9/14 sets by gene analysis and 1/10 sets by culture (P < 0.05). Although our study was not conclusive, these findings are consistent with a primary role of biofilm formation in PJIs.