Rapid Identification of Key Pathogens in Wound Infection by Molecular Means

Background: Wound infections are typically mixed, with both anaerobic and non-anaerobic bacteria present and there are usually more anaerobes than aerobes (for example, in perforated or gangrenous appendicitis, we have found an average of 9 anaerobes and 3 aerobes). Many clinical laboratories do limited anaerobic bacteriology, using commercial kits based on inadequate phenotypic characteristics and inaccurate taxonomy. As a result, physicians and surgeons must treat wound infections empirically and tend to use drugs active against the most resistant anaerobes which leads to increased resistance to antimicrobials. Molecular techniques now available permit accurate and rapid characterization of microorganisms, both aerobic and anaerobic.

Objective/Hypothesis: The use of molecular biology techniques will provide identification of the microorganisms responsible for wound infection both more rapidly and more accurately. The current conventional method of culture and identification by phenotypic testing is slow and inaccurate.

Specific Aims: 1) Develop a rapid identification method, to include quantitation, for important wound pathogens, 2) Analyze bacterial flora of wound infection by molecular means and by conventional culturing; both tissue biopsies and pus will be studied by both methodologies, 3) Determine the incidence and significance of the so-called "uncultivable" flora.

Study Design: We will study 50 postoperative and traumatic wound infections and 50 closed soft tissue abscesses in intravenous drug abusers annually, from a general surgical service at Olive View County Hospital, affiliated with UCLA and the VA Medical Center West Los Angeles, in collaboration with Dr. Robert Bennion. Whenever possible, we will obtain tissue biopsies from the active edge of the wound for processing, along with pus from the wound.

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.

The two molecular approaches will be compared with the conventional cultural approach in terms of organisms detected; each specimen will be studied by all techniques, for comparative purposes.

Relevance: The speed of the various identification schemes indicated above shows clearly that we would be able to provide accurate, quantitative identification (more accurate than by the conventional cultural approach) a great deal sooner than has been possible in the past. This should lead to much earlier application of the most appropriate therapy and minimize empiric use of our most powerful agents. This is likely to result in improved clinical outcome, less chance for antimicrobial resistance, and cost savings as well. It will provide us with a much more accurate picture of the infecting flora of various surgical wounds and the antimicrobial susceptibility pattern of these organisms. We will learn whether, as in burn wound infections, quantitative study of tissue biopsies will provide a better picture of the true infecting flora than conventional wound cultures. We will learn whether "uncultivable" bacteria (which actually make up the greatest percentage of the indigenous flora of the bowel and the upper respiratory tract) are important clinically or not. Finally, we will develop a more complete set of primers/probes to improve the molecular approach.