Open Access Presidential Scholars Thesis
Tuberculosis; Drug resistance in microorganisms;
In a recent study of tuberculosis cases in Texas it was found that while the incidence of tuberculosis is fairly low (compared to developing countries), of the cases reported, almost 10% were resistant to at least one of the four first line drugs (Taylor, 1999). When tuberculosis is diagnosed in a patient, especially one who may have contracted an antibiotic resistant strain, discovery of the type and extent of the resistance is imperative to a successful treatment regimen. For years, BACTEC (a broth dilution method, where a microbe is incubated in serial dilutions of an antibiotic to see what is the minimum concentration that prohibits growth-- called a Minimum Inhibitory Concentration, or MIC) has been used, along with similar agar dilution methods for determining the resistance or susceptibility of strains for certain drugs. However, most of these methods are fairly costly, requiring specific machines to read concentrations of bacteria in broth; complicated, requiring multiple plates and dilutions to be kept track of; inaccurate, giving only a general ballpark MIC number; and simply cumbersome, requiring a great deal of lab equipment and glassware. With M. tuberculosis, there is the added complication of the 2-3 week time window required for full growth. A few alternatives for MIC determination have appeared recently including Etest (AB Biodisk, Sweden), a method that the lab I did research in this summer has helped to test and standardize. Etest simply is a strip of paper with a gradient of antibiotic dry-adhered to it that is placed on a plated lawn of bacteria. The antibiotic diffuses into the agar, creating an ellipse of inhibited bacterial growth. (See figures 5-6). The MIC can be read where the ellipse crosses the strip. Etest appears to be a simple alternative to many of the more unwieldy methods, taking less time (especially with M. Tuberculosis, some plates can be read in five days rather then three weeks) and in the case of ethambutol, MIC determination may actually be a more accurate method. Ethambutol is a bacteriostatic antibiotic (see figure 4) and has been found to be unstable in solution at common incubation temperatures and thus, may have shown a higher MIC (more resistant) for a strain then may have actually been true (e.g. since the antibiotic was actually already broken down, it didn't effect the bacteria which then appeared to have resistance to it). However, this had never been quantified, thus, the first research project that I selected was to use the Etest method on strains collected from local hospitals between 1991-96 (Mycobacterium stay alive preserved in glycerol and frozen indefinitely) that had been labeled as Ethambutol resistant to test for MIC in a new way. This experiment would hopefully establish which method was the most accurate in determining the actual resistance or susceptibility of a strain.
Date of Award
Department of Biology
Presidential Scholar Designation
A paper submitted in partial fulfillment of the requirements for the designation Presidential Scholar
1 PDF file (50 pages)
©1999 - Marla DeWitt
DeWitt, Marla, "Tuberculosis and the genetic basis of antibiotic resistance in mycobacterium" (1999). Presidential Scholars Theses (1990 – 2006). 61.