Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.

PLASMA MEMBRANE GLYCOPROTEINS OF RAT HEPATOCELLULAR CARCINOMA CELLS

Previous investigations have demonstrated qualitative differences in the plasma membrane glycoproteins of normal and malignant rat liver cells. The present investigations were designed to identify and characterize the spectrum of glycoproteins present on the surface of Novikoff and AS-30D hepatocellular carcinoma cells. Three cell-surface radiolabeling techniques were employed to tag specifically the plasma membrane glycoproteins: lactoperoxidase catalyzed iodination, specific for tyrosine residues; galactose oxidase/NaB{('3)H}(,4), specific for galactosyl residues; and NaIO(,4)/NaB{('3)H}(,4), specific for sialic acids. The glycoproteins were resolved by one- and two-dimensional gel electrophoresis and visualized by fluorography or autoradiography. It was found that these glycoproteins are a complex population of molecules. The complexity of this system is reflected not only in the number of individual components that can be detected (> 25), but in the charge heterogeneity of individual glycoproteins due to variable sialic acid content. Certain glycoproteins behaved anamolously on SDS-polyacrylamide gel electrophoresis; the apparent molecular weight decreasing with increasing acrylamide concentrations suggesting a high % carbohydrate. Cell-surface radiolabeling techniques were employed in combination with lectin affinity chromatography, using lectins of different saccharide specificity, to analyze the saccharide determinants present on the spectrum of cell-surface molecules. It was also found that particular glycoproteins differed in their lability to protease or neuraminidase digestion and in their extractability by non-ionic detergents. From these studies, detailed models of the plasma membrane of Novikoff and AS-30D cells were constructed which incorporates information concerning the structure and accessibility of heterosaccharide and peptide moieties, the relationship of the glycolipids, and the interaction of particular glycoproteins with the lipid bilayer. These investigations provide basic information concerning the molecular composition and properties of the plasma membrane of glycoproteins of malignant rat liver cells and lay the groundwork for future comparison to normal hepatocytes. ^