We will develop species-specific primers/probes for organisms that are encountered in different types of wound infections based on information about the flora of wound infection obtained from previous studies by our group and others and supplemented with primers/probes made from cultural isolates not previously encountered. The primers and probes will be used in a high-throughput procedure (real-time PCR) that permits rapid identification and quantitation of organisms. The entire procedure, including both DNA extraction and real-time PCR, can be completed within 5 hours for one sample. Multiple samples can be done concurrently. While it may be necessary to start therapy empirically, it will be possible to change to more appropriate antimicrobial agents when the bacteriologic data becomes available in 5 hours. Concurrently, we will utilize another molecular approach, 16S rRNA gene cloning, to analyze the total flora of the sample. We will first amplify 16S rRNA genes using universal (all bacteria) primers by PCR of DNA extracted from the sample. Then, a clone library will be constructed in E. coli and all clones will be studied by 16S rDNA sequencing of ~1500 base pairs. This permits detection of organisms called "uncultivable", as well as organisms that can be cultured successfully. Any "uncultivable" organisms that are encountered frequently may be important; therefore, we will develop primers and probes for their rapid identification and we will use a variety of techniques to attempt to cultivate these (we have done this successfully in a few cases already). At the same time as the molecular approaches are being done, we will use conventional cultural processing. The cultural approach will provide organisms for additional study (especially for antimicrobial susceptibility testing) and will serve as the current standard for comparison with the newer molecular approaches. Culture will be done semiquantitatively and identification will be done by 16S rDNA sequencing supplemented by phenotypic testing as indicated. The cultural approach typically takes several days to isolate the organisms in pure culture.