| 2302 Detection of Sulfate-Reducing Bacteria (SRB) using RT-PCR and MALDI-TOF MS | ||
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W.-D. GRIMM1, F. SHIHABI1, G. GASSMANN1, and V. HOMANN2, 1University of Witten/Herdecke, Germany, 2Max Planck Institute of Molecular Physiology, Dortmund, Germany Objectives: The term sulfate-reducing bacteria (SRB) describes strictly anaerobic microbes that accomplish the dissimilatory reduction of sulfate to hydrogen sulfide. In our pilot studies the frequency of SRB presence was significantly higher in periodontal diseases in comparison with healthy oral sites. Thus 90% of the isolated periodontal SRB were identified as members of a new species Desulfomicrobium orale. Material and methods: The bacterial strains were characterized by DNA extraction (High pure PCR template preparation kit) and DNA amplification with 16S rDNA sequencing. Nearly full length (350 bp) 16 rRNA gene sequences were amplified using general bacterial primers (Paster et al. 2001). RT-PCR was performed using Gene Amp 5700 Sequence Detection System, and Quantitect PCR Probe KIT. Additionally, Matrix-Assisted-Laser Desorption /Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was used for comparative characterization. Bacteria were cultured in Balmelli's broth for 24 h at 35° C, harvested and rinsed in water. 107 bacterial cells were co-crystallized with -cyano-4-hydroxy-cinnamic acid on a stainless steel target. MALDI-TOF MS was carried out with a BrukerBiflex III operated in the linear mode. Spectra were recorded 5-fold. Results: Analysis of 16S rDNA sequences of all strains showed a high similarity among these oral strains, forming a distinct lineage within the genus Desulfomicrobium. Characteristic and reproducible spectra were found for the two SRB strains displaying characteristic peaks in MALDI-TOF MS. Conclusions: Our clinical pilot studies indicate the SRB as “late colonizers” in the subgingival biofilm formation. Therefore, the presence of SRB may indicate pockets in a state of risk for progressive destructive periodontal disease. RT-PCR and MALDI-TOF MS proved to be well suited for the differentiation of SRB strains. The two SRB strains (Langendijk, 2001) have been submitted to the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Germany, and are referred to as DSM 12803 and DSM 12838T, respectively. | ||
| Seq #249 - Microbes, Models, and Responses 2:00 PM-4:00 PM, Friday, 11 March 2005 Baltimore Convention Center Exhibit Hall E-F | ||
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