Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.
A commercially available DNA strip assay (Genotype MTBDR; Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect mutations conferring resistance to rifampin (RMP) and isoniazid (INH) in clinical Mycobacterium tuberculosis complex isolates. A total of 103 multidrug-resistant (MDR; i.e., at least resistant to RMP and INH) and 40 fully susceptible strains isolated in Germany in 2001 in which resistance mutations have been previously defined by DNA sequencing and real-time PCR analysis were investigated. The Genotype MTBDR assay identified 102 of the 103 MDR strains with mutations in the rpoB gene (99%) and 91 strains (88.4%) with mutations in codon 315 of katG. All 40 susceptible strains showed a wild-type MTBDR hybridization pattern. The concordance between the MTBDR assay and the DNA sequencing results was 100%. Compared to conventional drug susceptibility testing, the sensitivity and specificity were 99 and 100% for RMP resistance and 88.4 and 100% for INH resistance, respectively. In conclusion, the MTBDR assay is a rapid and easy-to-perform test for the detection of the most common mutations found in MDR M. tuberculosis strains that can readily be included in a routine laboratory work flow.The rapid determination of drug resistance in clinical isolates of Mycobacterium tuberculosis is the prerequisite for the initiation of effective chemotherapy ensuring successful treatment of the patient and preventing further spread of drugresistant isolates (17). The development of drug resistance in M. tuberculosis isolates is the result of random genetic mutations in particular genes conferring resistance (30). Based on this knowledge, molecular assays have been established that allow the prediction of drug resistance in clinical M. tuberculosis isolates within one working day and potentially are the most rapid methods for the detection of drug resistance (16). The utility of such assays depends on different aspects, such as the technical requirements of each method and the ability of different tests to detect the most common drug resistance mutations in a given area.The mutations that predominate in rifampin (RMP)-resistant M. tuberculosis isolates are located in an 81-bp "core region" of the rpoB gene (ca. 95% of all RMP-resistant strains (1, 27). In contrast, the mutations causing isoniazid (INH) resistance are located in several genes and regions (24). Approximately 50 to 95% of INH-resistant strains have been found to contain mutations in codon 315 of the katG gene (14,15,26), 20 to 35% contain mutations in the inhA regulatory region (15,18,26), and an additional 10 to 15% have mutations in the ahpC-oxyR intergenic region (8,18,26), often in conjunction with katG mutations outside of codon 315 (25).In order to allow the detection of the majority of mutations causing RMP resistance and also a high proportion of mutations causing INH resistance in M. tuberculosis isolates, a DNA strip assay was developed (Genotype MTBDR; Hain Lifescience, Nehren, Germany). It is based on a multiplex PCR in combinat...
Background: The reasons for recurrent adenotonsillitis are poorly understood. Methods: The in situ composition of microbiota of nasal (5 children, 25 adults) and of hypertrophied adenoid and tonsillar tissue (50 children, 20 adults) was investigated using a broad range of fluorescent oligonucleotide probes targeted to bacterial rRNA. None of the patients had clinical signs of infection at the time of surgery. Results: Multiple foci of ongoing purulent infections were found within hypertrophied adenoid and tonsillar tissue in 83% of patients, including islands and lawns of bacteria adherent to the epithelium, with concomitant marked inflammatory response, fissures filled with bacteria and pus, and diffuse infiltration of the tonsils by bacteria, microabscesses, and macrophages containing phagocytosed microorganisms. Haemophilus influenzae mainly diffusely infiltrated the tissue, Streptococcus and Bacteroides were typically found in fissures, and Fusobacteria, Pseudomonas and Burkholderia were exclusively located within adherent bacterial layers and infiltrates. The microbiota were always polymicrobial. Conclusions: Purulent processes persist during asymptomatic periods of adenotonsillitis. Most bacteria involved in this process are covered by a thick inflammatory infiltrate, are deeply invading, or are located within macrophages. The distribution of the bacteria within tonsils may be responsible for the failure of antibiotic treatment.
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