The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the aetiology of these diseases. Common wisdom is that lactobacilli dominate the normal vaginal microflora of post-pubertal women. However, this conclusion is based on methods that require cultivation of microbial populations; an approach that is known to yield a biased and incomplete assessment of microbial community structure. In this study cultivation-independent methods were used to analyse samples collected from the mid-vagina of five normal healthy Caucasian women between the ages of 28 and 44. Total microbial community DNA was isolated following resuspension of microbial cells from vaginal swabs. To identify the constituent numerically dominant populations in each community 16S rRNA gene libraries were prepared following PCR amplification using the 8f and 926r primers. From each library, the DNA sequences of approximately 200 16S rRNA clones were determined and subjected to phylogenetic analyses. The diversity and kinds of organisms that comprise the vaginal microbial community varied among women. Species of Lactobacillus appeared to dominate the communities in four of the five women. However, the community of one woman was dominated by Atopobium sp., whereas a second woman had appreciable numbers of Megasphaera sp., Atopobium sp. and Leptotrichia sp., none of which have previously been shown to be common members of the vaginal ecosystem. Of the women whose communities were dominated by lactobacilli, there were two distinct clusters, each of which consisted of a single species. One class consisted of two women with genetically divergent clones that were related to Lactobacillus crispatus, whereas the second group of two women had clones of Lactobacillus iners that were highly related to a single phylotype. These surprising results suggest that culture-independent methods can provide new insights into the diversity of bacterial species found in the human vagina, and this information could prove to be pivotal in understanding risk factors for various infectious diseases. INTRODUCTIONPrevious studies on the microbial flora of the human vagina indicate that micro-organisms normally present in the human vagina play a key role in preventing successful colonization by 'undesirable' organisms, including those responsible for bacterial vaginosis, yeast infections, sexually transmitted diseases and urinary tract infections (Donders et al., 2000;Gupta et al., 1998;Sobel, 1999;van De Wijgert et al., 2000). Moreover, epidemiologic studies have clearly established that abnormal vaginal microbial communities and lower genital tract infections are significantly associated with an increased risk of HIV infection (Cohen et al., 1995;Martin et al., 1999;Sewankambo et al., 1997). Based on this, researchers have speculated that the normal vaginal microbial ecosystem may play a pivotal role in lo...
The analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes has proven to be a facile means to compare microbial communities and presumptively identify abundant members. The method provides data that can be used to compare different communities based on similarity or distance measures. Once communities have been clustered into groups, clone libraries can be prepared from sample(s) that are representative of each group in order to determine the phylogeny of the numerically abundant populations in a community. In this paper methods are introduced for the statistical analysis of T-RFLP data that include objective methods for (i) determining a baseline so that 'true' peaks in electropherograms can be identified; (ii) a means to compare electropherograms and bin fragments of similar size; (iii) clustering algorithms that can be used to identify communities that are similar to one another; and (iv) a means to select samples that are representative of a cluster that can be used to construct 16S rRNA gene clone libraries. The methods for data analysis were tested using simulated data with assumptions and parameters that corresponded to actual data. The simulation results demonstrated the usefulness of these methods in their ability to recover the true microbial community structure generated under the assumptions made. Software for implementing these methods is available at http://www.ibest.uidaho.edu/tools/trflp_stats/index.php.
Molecular microbial community analysis methods have revolutionized our understanding of the diversity and distribution of bacteria, archaea and microbial eukaryotes. The information obtained has adequately demonstrated that the analysis of microbial model systems can provide important insights into ecosystem function and stability. However, the terminology and metrics used in macroecology must be applied cautiously because the methods available to characterize microbial diversity are inherently limited in their ability to detect the many numerically minor constituents of microbial communities. In this review, we focus on the use of indices to quantify the diversity found in microbial communities, and on the methods used to generate the data from which those indices are calculated. Useful conclusions regarding diversity can only be deduced if the properties of the various methods used are well understood. The commonly used diversity metrics differ in the weight they give to organisms that differ in abundance, so understanding the properties of these metrics is essential. In this review, we illustrate important methodological and metric-dependent differences using simulated communities. We conclude that the assessment of richness in complex communities is futile without extensive sampling, and that some diversity indices can be estimated with reasonable accuracy through the analysis of clone libraries, but not from community fingerprint data.
Since its first description in coastal Connecticut in 1976, both the incidence of Lyme disease and the geographic extent of endemic areas in the US have increased dramatically. The rapid expansion of Lyme disease into its current distribution in the eastern half of the US has been due to the range expansion of the tick vector, Ixodes scapularis, upon which the causative agent, Borrelia burgdorferi is dependent for transmission to humans. In this study, we examined the phylogeographic population structure of B. burgdorferi throughout the range of I. scapularis-borne Lyme disease using multilocus sequence typing based on bacterial housekeeping genes. We show that B. burgdorferi populations from the Northeast and Midwest are genetically distinct, but phylogenetically related. Our findings provide strong evidence of prehistoric population size expansion and east-to-west radiation of descendent clones from founding sequence types in the Northeast. Estimates of the time scale of divergence of northeastern and midwestern populations suggest that B. burgdorferi was present in these regions of North America many thousands of years before European settlements. We conclude that B. burgdorferi populations have recently reemerged independently out of separate relict foci, where they have persisted since precolonial times.geography ͉ phylogeny ͉ ticks ͉ multilocus sequence typing
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