One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of Ϸ6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.One-third of the world's population is infected with Mycobacterium tuberculosis, and 3 million human deaths annually are attributed to the organism (1, 2). Although there is a very large global pool of infected individuals and considerable chromosomal heterogeneity based on restriction fragment length polymorphism (RFLP) patterns generated by probing with mobile insertion elements (3, 4), studies of drug resistance and pathogenesis have raised the possibility that synonymous (silent) nucleotide substitutions in structural genes may be limited (5). To investigate this apparent discrepancy from the perspective of molecular population genetics, we sequenced two megabases in 26 structural genes or loci in strains of M. tuberculosis and the three closely related members of the M. tuberculosis complex (M. africanum, M. bovis, and M. microti) collected worldwide. MATERIALS AND METHODSBacterial Isolates. The study is based on a sample of 842 M. tuberculosis complex isolates recovered from diverse geographic localities. The organisms include M. tuberculosis (n ϭ 715), M. bovis (n ϭ 109), and M. africanum and M. microti (n ϭ 9 each). M. tuberculosis isolates were recovered from diseased patients in the United States (five states), Latin America (Mexico, Honduras, Ecuador, Peru, Venezuela, Brazil, and Chile), Europe (Portugal, Spain, The Netherlands, Belgium, Germany, Switzerland, Italy, former Yugoslavia, and Romania), Africa (Kenya, Rwanda, Guinea, Algeria, Som...

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Escherichia coli O157:H7, a toxin-producing food and waterborne bacterial pathogen, has been linked to large outbreaks of gastrointestinal illness for more than two decades. E. coli O157 causes a wide range of clinical illness that varies by outbreak, although factors that contribute to variation in disease severity are poorly understood. Several recent outbreaks involving O157 contamination of fresh produce (e.g., spinach) were associated with more severe disease, as defined by higher hemolytic uremic syndrome and hospitalization frequencies, suggesting that increased virulence has evolved. To test this hypothesis, we developed a system that detects SNPs in 96 loci and applied it to >500 E. coli O157 clinical strains. Phylogenetic analyses identified 39 SNP genotypes that differ at 20% of SNP loci and are separated into nine distinct clades. Differences were observed between clades in the frequency and distribution of Shiga toxin genes and in the type of clinical disease reported. Patients with hemolytic uremic syndrome were significantly more likely to be infected with clade 8 strains, which have increased in frequency over the past 5 years. Genome sequencing of a spinach outbreak strain, a member of clade 8, also revealed substantial genomic differences. These findings suggest that an emergent subpopulation of the clade 8 lineage has acquired critical factors that contribute to more severe disease. The ability to detect and rapidly genotype O157 strains belonging to such lineages is important and will have a significant impact on both disease diagnosis and treatment guidelines.pathogens ͉ polymorphisms ͉ population genetics E nterohemorrhagic Escherichia coli (EHEC) includes a diverse population of Shiga toxin-producing E. coli that causes outbreaks of food and waterborne disease (1-3). EHEC often resides in bovine reservoirs and is transmitted via many food vehicles including cooked meat, such as hamburger (4) and salami (5), and raw vegetables, such as lettuce (6, 7) and spinach (8). In North America, E. coli O157:H7 is the most common EHEC serotype contributing to Ͼ75,000 human infections (9) and 17 outbreaks (3) per year.It is not clear why outbreaks of EHEC O157 vary dramatically in the severity of illness and the frequency of the most serious complication, hemolytic uremic syndrome (HUS) (10-12). The 1993 outbreak in western North America (4) and the large 1996 outbreak in Japan (13) had low rates of hospitalization and HUS (14, 15), whereas the 2006 North American spinach outbreak (8) had high rates of both hospitalization (Ͼ50%) and HUS (Ͼ10%). One hypothesis is that outbreak strains differ in virulence as a result of variation in the presence and expression of different Shiga toxin (Stx) gene combinations (16)(17)(18)(19).To assess the genetic diversity and variability in virulence among E. coli O157 strains, we developed a real-time PCR system for identifying synonymous and nonsynonymous mutations as SNPs (20-23). Although molecular subtyping methods, such as pulsedfield gel electrophoresis (PFGE), ...

Multicopy single-stranded DNA (msDNA), a branched DNA-RNA molecule, has been shown in Escherichia coli B and clinical strain Cl-1 to be synthesized by reverse transcriptase. We report that 13% of the strains of the ECOR collection, a sample of 72 E. coli isolates representing the breadth of genetic variation of the species, produce msDNA. Three of the four major subspecific groups include msDNA-producing strains. Screening of 25 isolates that are genetically related to msDNA-producing clinical strains uncovered 22 additional msDNA-producing strains. A phylogenetic tree based on allelic variation detected electrophoretically at 20 enzyme-encoding loci revealed two major clusters and several deep branches composed of strains that synthesize msDNA. Although E. coli K-12 does not harbor msDNA, other closely related strains of the K-12 family do. The results support the hypothesis that msDNA-synthesizing systems, including reverse transcriptase genes, were acquired recently and independently in different lineages of E. coli.The recent discovery of bacterial reverse transcriptases (RTs) was engendered by the observation of a peculiar satellite DNA, now called multicopy single-stranded DNA (msDNA), in the soil bacterium Myxococcus xanthus (26). The satellite (msDNA-Mxl62) is unusual because it consists of a 162-base single DNA strand linked to a 77-base RNA (msdRNA) sequence by a 2',5'-phosphodiester linkage and occurs in hundreds of copies per cell (4). An msDNA of a similar structure (msDNA-Sa163) has been found in the closely related myxobacterium Stigmatella aurantiaca (5, 6). Recently it has been reported that M. xanthus contains another species of msDNA (msDNA-Mx65) in addition to msDNA-Mxl62 (3). Most recently, msDNA molecules with structural features similar to those of myxobacterial msDNAs have been found in clinical isolate Cl-1 (9), strain B (10), and several other clinical isolates (21) of Escherichia coli.In M. xanthus (8), E. coli clinical isolate Cl-1 (9), and E. coli B (10), the synthesis of msDNA depends on RT encoded by a sequence adjacent to the chromosomal region specifying msdRNA. Although the three bacterial RTs are substantially divergent in size and amino acid sequence, as inferred from the nucleotide sequences, they exhibit the common amino acid motifs of retroviral reverse transcriptases (22).The objective of the study reported here was to determine the frequency of occurrence and genetic relationships among strains that harbor msDNA synthesized by RT in natural populations of E. coli. To accomplish this, we examined the 72 strains of the ECOR collection (13), a sample of natural isolates chosen to represent the major subspecific groups of the E. coli species as a whole. These groups have been identified on the basis of allelic variation at enzyme-encoding genes detected by multilocus enzyme electrophoresis (14, 23) and include isolates recovered from a variety of human and animal hosts in diverse geographic areas (13). In addition, we characterized the multilocus genotypes of previously repor...

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