Clearly, the military is subject to traumatic wounds of various types and severity, both in formal military combat and in guerilla-type warfare as well as in civil uprisings and ordinary accidents. Such wounds are predisposed to infection because they (1) tend to be extensive and deep; (2) may affect areas of normal carriage of high counts of potentially pathogenic bacteria in the gastrointestinal tract, oral cavity and upper respiratory tract, and the female genital tract; (3) typically produce a great deal of tissue damage, thus interfering with the local defenses against infection; (4) may introduce foreign bodies; (5) may interfere with local blood supply; (6) tend to produce ischemia, edema, and hemorrhage; (7) may be complicated by fractures or burns; and (8) may lead to shock and overwhelming of the body's systemic defenses. It will not always be possible in the military setting to cleanse and debride the wound promptly and effectively or to promptly provide major surgery in the event of damage to viscera or other vital structures. The setting for initial management of the wound may be such as to increase the possibility of introduction of microorganisms from the environment or from the hands of people reacting promptly to life-threatening injuries. In the active military setting, then, the probability of wound infection following trauma is relatively high.

In addition to problems that may exist to interfere with prompt and effective surgical management of wounds, normally the most important first step in prevention or treatment of wound infection, there are problems relating to use of the optimum antimicrobial prophylaxis or management. These relate to incomplete knowledge of the indigenous flora at various sites in the body to some extent, but primarily to our inability to rapidly and accurately define the infecting flora in an established wound infection. Aerobic bacteria are typically easy to grow, grow rapidly, and can be identified accurately in relatively short order. More important in this regard are the anaerobic bacteria, which often outnumber the aerobic wound flora, grow more slowly, need to be isolated in pure culture from the often complex mixtures in which they are found, and for which the usual phenotypic tests used for identification are not only slow but also often inaccurate. Clinicians usually do not get information on the anaerobic flora in less than two days, and it may take several more days to even weeks for full identification of complex wound floras. Clearly, this is of no value to clinicians who must act quickly to provide appropriate antimicrobial therapy. Good microbiologists can provide some clues that may be important, but many microbiologists do not function that way, especially in laboratories that are overworked and underfunded, a not uncommon situation. In any case, definitive identification of anaerobes can be provided very quickly and that, along with information on usual antimicrobial susceptibility patterns in various settings, can be life-saving or can shorten the course of the infection considerably. In the absence of rapid identification of infecting flora and provision of information on antimicrobial susceptibility, clinicians must resort to empiric therapy rather than the tailored therapy that should be possible. This is not only justified but totally appropriate, pending information from the laboratory, in patients who are seriously ill but there is a tendency for clinicians to use one of the top available agents that would likely be active against the vast majority of anaerobes for all patients. This leads to further increases in antimicrobial resistance, an important problem already.

The most promising technique, that we wish to evaluate under this proposal, is real-time PCR (polymerase chain reaction). This procedure can be applied directly to appropriate material from the wound without any need for first growing the organisms. It can be used to define the total flora of the wound within 5 hours. It is first necessary to develop sets of primers/probes that will detect each of the various some of these required primers/probes and would develop the others that may be needed bacteria anticipated in a given wound in a certain location. We will develop these primers and probes early in the course of our studies under this proposal so that we can start evaluating real-time PCR for very rapid and accurate identification of the total wound flora as early in the course of our grant as possible. The information obtained with real-time PCR can be quantitated so that one may judge by this (along with known virulence of various organisms) the relative importance of different isolates. Analysis of the bacterial flora by molecular means will provide further information about the flora of various wounds (including the so-called "uncultivable" flora) and use of conventional cultures will provide bacterial cultures for antimicrobial susceptibility testing as well as provide comparisons with the molecular approaches in terms of accuracy, time involved for obtaining answers, and relative cost.

In terms of usefulness to the Armed Forces, there should be value even as early as the end of the first year of the study and much more by the end of the second year. Clearly, there will be long-term usefulness, particularly as more and more medical facilities in the various branches of the service become equipped and trained in the new approaches. Modifications of the procedures we expect to develop would certainly come, particularly in the area of automation.