| Variable-number tandem repeats (VNTRs) are being increasingly used to genotype bacterial pathogens. Due to the low genetic diversity of many recently emerged pathogens, VNTR markers are often the only means of detecting sufficient sequence variation to discriminate among isolates. This is true for both Bacillus anthracis and Yersinia pestis, etiologic agents of anthrax and plague, respectively. Although VNTR markers have been useful in B. anthracis, more information is needed concerning mutation rates and the factors influencing mutation rates for these markers to be fully utilized. Despite its potential, VNTR analysis has not been applied as widely in Y. pestis, and more information concerning the usefulness of these markers on different geographical and/or temporal scales is needed. This dissertation expands the usefulness of a set of VNTRs for B. anthracis by estimating the mutation rate for each one. In addition, the usefulness of VNTR markers for examining the molecular evolution and diversity of Y. pestis is shown on two scales: one scale is the country of Madagascar, and the other is a single, recently infected city within Madagascar.; Spontaneous mutation rates ranging from 5 × 10−5 to 5.1 × 10−4 mutations/generation were determined for a set of highly variable VNTRs in B. anthracis (known as single nucleotide repeats [SNRs]) using a parallel, serial, passage experiment. Spontaneous point mutation rates for nfampin and ciprofloxacin resistance ranging from 3.4 × 10−11 to 7.3 × 10−9 also were determined in B. anthracis using Luria-Delbrück fluctuation tests. These point mutation rates suggest that sequence variation in DNA coding regions of B. anthracis occurs at a rate similar to other bacteria and that the low genetic diversity of B. anthracis is not due to a slower mutation rate. The SNR mutation rates were 104 to 107 faster than point mutation rates in rpoB, gyrA, gyrB, or parC and were used to generate a probability model to estimate the number of generations separating isolates exhibiting SNR differences. These SNR mutation rates combined with the probability model could be very useful for epidemiological tracking of anthrax outbreaks.; A multi-locus VNTR analysis (MLVA) system employing 43 VNTRs was used to genotype 203 Y. pestis isolates from Madagascar. This analysis revealed 168 unique genotypes that segregated into 11 principal phylogenetic clusters when analyzed using a weighted neighbor-joining algorithm. These clusters exhibited only low geographic grouping, possibly due to cryptic niches or to rapid transmission from unknown geographically separated foci. (Abstract shortened by UMI.)... |
