OBJECTIVES: To characterize by restriction fragment length polymorphism (RFLP) patterns, the distribution of different Mycobacterium tuberculosis strains isolated consecutively from 75 tuberculosis patients who resided in Prague and had culture-confirmed cases during a 4-month period in 1995. METHODS: The insertion sequence IS6110-based RFLP analysis of M. tuberculosis isolates was carried out. RESULTS: There were a total of 75 patients with various forms of tuberculosis (54 males; 21 females). The sources of M. tuberculosis isolates were sputum (n = 64), pleura or lymph node drainage (n = 8), and urine (n = 3). Fifty-three of the patients (70.7%) had isolates with unique RFLP patterns, while 22 (29.3%) had isolates that belonged to seven clusters of related RFLP patterns. The seven clusters consisted of four groups of two patients, two groups of four patients, and one group of six patients. Most of the patients whose isolates fell within a clustered RFLP pattern lived in different quarters of the city and had no identifiable contacts with other patients whose isolates had the same pattern. CONCLUSIONS: The finding that isolates from most patients (70.7%) had unique rather than clustered RFLP patterns suggests that endogenous reactivation rather than exogenous transmission is the major determinant of most of the tuberculosis cases in Prague. The occurrence of seven distinct clusters comprising 29.3% of the isolates suggests that approximately one third of cases developed active tuberculosis from recent exogenous transmission.
- MeSH
- DNA, Bacterial * analysis MeSH
- DNA Fingerprinting MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Lymph Nodes microbiology MeSH
- Urine microbiology MeSH
- Molecular Epidemiology MeSH
- Mycobacterium tuberculosis * genetics isolation & purification MeSH
- Polymorphism, Restriction Fragment Length * MeSH
- Aged MeSH
- Sputum microbiology MeSH
- Tuberculosis * epidemiology microbiology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.
- MeSH
- Amplified Fragment Length Polymorphism Analysis methods standards MeSH
- DNA, Fungal chemistry genetics MeSH
- Fungi chemistry genetics isolation & purification MeSH
- Nucleic Acid Conformation MeSH
- Phalaris microbiology MeSH
- Polymorphism, Restriction Fragment Length MeSH
- Transition Temperature MeSH
- Base Composition MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
URA5-RFLP is one of the most widely used genotyping methods relating to Cryptococcus neoformans and C. gattii consensus genotype nomenclature. In order to identify a molecular type, this method uses a visual comparison of digested PCR products of tested and reference strains, therefore any anomaly in RFLP patterns of studied isolates makes recognition difficult or impossible. This report describes a strain of VNIV type showing an atypical URA5-RFLP pattern as well as a group of AD hybrids displaying the same anomaly. The atypical RFLP pattern is the result of a point mutation and emergence of a new restriction site. Emergence of the allele presenting a new banding pattern may lead to misidentification using the URA5-RFLP technique; the results of this study as well as the literature data may suggest the spread of the allele in the environment.
- MeSH
- Cryptococcus neoformans classification genetics MeSH
- Genotype MeSH
- Genes, Fungal genetics MeSH
- Environmental Microbiology MeSH
- Mutation MeSH
- Mycological Typing Techniques MeSH
- Orotate Phosphoribosyltransferase genetics MeSH
- Polymorphism, Restriction Fragment Length MeSH
- Base Sequence MeSH
- Publication type
- Journal Article MeSH
- MeSH
- Archaea genetics metabolism MeSH
- Bacteria genetics metabolism MeSH
- Chlorine metabolism MeSH
- Fungi genetics metabolism MeSH
- Microbial Consortia physiology MeSH
- Polymorphism, Restriction Fragment Length MeSH
- Soil analysis MeSH
- Soil Microbiology MeSH
- RNA, Ribosomal, 16S analysis MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Drug Resistance, Bacterial physiology MeSH
- Adult MeSH
- Phylogeny MeSH
- Humans MeSH
- Mycobacterium avium Complex isolation & purification drug effects MeSH
- Lung Diseases microbiology MeSH
- Polymerase Chain Reaction MeSH
- Polymorphism, Restriction Fragment Length MeSH
- Sputum microbiology MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
Streptococcus suis is an important pathogen of pigs but is also transmissible to humans, with potentially fatal consequences. Among 29 serotypes currently recognized, some are clinically and epidemiologically more important than others. This is particularly true for serotypes 2 and 14, which have a large impact on pig production and also on human health. Conventional PCR-based serotyping cannot distinguish between serotype 1/2 and serotype 2 or between serotype 1 and serotype 14. Although serotype 1/2 and serotype 2 have a very similar cps locus, they differ in a single-nucleotide substitution at nucleotide position 483 of the cpsK gene. Similarly, serotypes 1 and 14 have a very similar cps locus but also differ in the same nucleotide substitution of the cpsK gene. Fortunately, this cpsK 483G→C/T substitution can be detected by BstNI restriction endonuclease. A PCR-restriction fragment length polymorphism (RFLP) detection method amplifying a fragment of the cpsK gene digested by BstNI restriction endonuclease was developed and tested in reference strains of these serotypes and also in field isolates.
- MeSH
- Polymerase Chain Reaction MeSH
- Polymorphism, Restriction Fragment Length MeSH
- Swine MeSH
- Serogroup MeSH
- Serotyping MeSH
- Streptococcus suis * genetics MeSH
- Streptococcal Infections * diagnosis veterinary MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
