Mycobacterium tuberculosis - the 10 years of epidemiological and diagnostics studies

Tuberculosis (ТВ) is the main bacterial pathogen that causes more deaths than AIDS, malaria and all infectious diseases. The unusual long doubling time (about 24h), highly hydrophobic cell envelope resistant to chemical lysis was the reason to delay the molecular study of this bacteria. Fifteen years ago, we did not have any molecular tools and methods for genetic manipulation or isolation and analysis of intracellular protein and nucleic acids. Today we have many useful shuttle or integration vectors for basic study of mycobacteria. The full sequence of M. tuberculosis genome is already known. At the present time the diagnosis of tuberculosis is supported with fast-culture system BACTEC and molecular techniques based on PCR and DNA hybridization. The mechanisms of resistance to antituberculosis drugs were described, and first identification of resistance profile is available by using PCR and sequencing or real time PCR methods. In our group in the Center for Microbiology and Virology Polish Academy of Sciences and in the Dept, of Genetics of Microorganisms, University Łódź we have characterized new insertion sequences from M. tuberculosis complex- 18990 and IS1607. In diagnostic studies we have proposed the DIG-PCR ELISA assay as a reliable, specific and sensitive test to identify M. tuberculosis directly in clinical samples. We have performed wide epidemiological studies of M. tuberculosis strains isolated from Polish ТВ patients including drug - and multidrug - resistant strains. Finally we identified the most frequently present mutations responsible for drug resistance of polish clinical isolates of M. tuberculosis.Zadanie pt. „Digitalizacja i udostępnienie w Cyfrowym Repozytorium Uniwersytetu Łódzkiego kolekcji czasopism naukowych wydawanych przez Uniwersytet Łódzki” nr 885/P-DUN/2014 dofinansowane zostało ze środków MNiSW w ramach działalności upowszechniającej naukę

Resistant mutants of Mycobacterium tuberculosis selected in vitro do not reflect the in vivo mechanism of isoniazid resistance

The high prevalence of isoniazid-resistant Mycobacterium tuberculosis is often explained by a high mutation rate for this trait, although detailed information to support this theory is absent. We studied the development of isoniazid resistance in vitro, making use of a laboratory strain of M. tuberculosis. Spontaneous isoniazid-resistant mutants were characterized by molecular methods allowing identification of the most commonly encountered resistance-conferring mutations. Additionally, we determined the in vitro mutation rates for isoniazid and rifampicin resistance, and characterized the genome of a triple-resistant strain. Results confirm that the in vitro mutation rate for isoniazid resistance (3.2 x 10(-7) mutations/cell division) is much higher than the rate for rifampicin resistance (9.8 x 10(-9) mutations/cell division). However, in the majority of the in vitro mutants katG was partially or completely deleted and neither of the two most common in vivo mutations, katG-S315T or inhA-C(-)15T, were found in 120 isogenic mutants. This implies that clinically prevalent resistance mutations were present in <0.8% of isoniazid-resistant strains selected in vitro (95% CI 0%-2.5%). The triple-resistant strain had acquired isoniazid resistance via a 49 kbp deletion, which included katG. Apart from previously identified resistance-conferring mutations, three additional point mutations were acquired during sequential selection steps. These outcomes demonstrate that the in vivo mechanism of isoniazid resistance is not reflected by in vitro experiments. We therefore conclude that the high in vitro mutation rate for isoniazid resistance is not a satisfactory explanation for the fact that isoniazid monoresistance is significantly more widespread than monoresistance to rifampici

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology

BACKGROUND: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database. RESULTS: The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network. CONCLUSION: Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress

hsp65 PCR-restriction analysis (PRA) with capillary electrophoresis in comparison to three other methods for identification of Mycobacterium species

We developed a scheme for rapid identification of Mycobacterium species using an automated fluorescence capillary electrophoresis instrument. A 441-bp region of the hsp65 gene was examined using PCR-restriction analysis (PRA). The assay was initially evaluated on 38 reference strains. The observed sizes of restriction fragments were consistently smaller than the real sizes for each of the species as deduced from the sequence analysis (mean variance=7bp). Nevertheless, the obtained PRA patterns were highly reproducible and resulted in correct species identifications. A blind test was then successfully performed on 64 test isolates previously characterized by conventional biochemical methods, a commercial INNO-LiPA Mycobacteria assay and/or sequence determination of the 5' end of 16S rRNA gene. A total of 14 of 64 isolates were erroneously identified by conventional methods (78% accuracy). In contrast, PRA performed very well in comparison with the LiPA (89% concordance) and especially with DNA sequencing (93.3% of concordant results). Also, PRA identified seven isolates representing five previously unreported hsp65 alleles. We conclude that hsp65 PRA based on automated capillary electrophoresis is a rapid, simple and reliable method for identification of mycobacteria. Copyright 2010 Elsevier B.V. All rights reserved

Evaluation of multiple genetic markers for typing drug-resistant Mycobacterium tuberculosis strains from Poland

In the present study, 77 drug-resistant Mycobacterium tuberculosis strains isolated in Poland in 2000 were characterized by the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and our novel method based on PCR amplification of DNA regions between IS6110 and 16-bp GC-rich frequent repeats (designated IS6110-Mtb1/Mtb2 PCR). The results were compared with previous data of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The discriminatory power of IS6110-Mtb1/Mtb2 method was only slightly lower than that of IS6110 RFLP, whereas MIRU-VNTR typing was the least discriminative among the 4 methods used. Clustering of strains by using results of IS6110-Mtb1/Mtb2 PCR correlated well with RFLP-defined clusters, further confirming epidemiologic relationships among patients. These results indicate that the novel genotyping method could be an attractive alternative for other PCR-based typing procedures, such as spoligotyping and MIRU-VNTR typing. Also, it seems to be a valuable adjunct to the reference IS6110 RFLP method for studying the genetic diversity of drug-resistant M. tuberculosis strains in Poland

hsp65 PCR-restriction analysis (PRA) with capillary electrophoresis for species identification and differentiation of Mycobacterium kansasii and Mycobacterium chelonae-Mycobacterium abscessus group

SummaryObjectivesThe aim of the present study was to identify and differentiate Mycobacterium kansasii and Mycobacterium chelonae–Mycobacterium abscessus group strains isolated from clinical and environmental sources in different countries.MethodsPCR-restriction analysis of the hsp65 gene (PRA) with automated capillary electrophoresis was applied to the isolates previously identified by conventional biochemical testing and the molecular INNO-LiPA MYCOBACTERIA assay.ResultsPRA performed very well in comparison with the two other methods (96.4% concordance). Among 27M. kansasii isolates, this method detected five genetic types, of which type 1 represented the most common clinical isolate, as it is worldwide. PRA differentiated 29M. chelonae–M. abscessus group isolates into Mycobacterium immunogenum type 2 (n=13), M. chelonae (n=12), and M. abscessus types 1 (n=1) and 2 (n=1). M. immunogenum was the most frequent (69%) isolate from humans, but only one of 11 cases was clinically significant. M. chelonae was the most commonly (83%) recovered from water. PRA also identified two isolates with hsp65 alleles representing previously unreported patterns.ConclusionsPRA based on automated capillary electrophoresis is a rapid, simple, and reliable method for the identification and differentiation of both clinically relevant and environmental isolates of M. kansasii and M. chelonae–M. abscessus group

Genotyping of Clinical Mycobacterium tuberculosis Isolates Based on IS6110 and MIRU-VNTR Polymorphisms

In this study, 155 clinical Mycobacterium tuberculosis isolates were subject to genotyping with fast ligation-mediated PCR (FLiP). This typing method is a modified mixed-linker PCR, a rapid approach based on the PCR amplification of HhaI restriction fragments of genomic DNA containing the 3′ end of IS6110 and resolving the amplicons by polyacrylamide gel electrophoresis. The results were compared with previous data of the more commonly used methods, 15-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and, to verify combined FLiP/MIRU-VNTR clusters, the reference IS6110 restriction fragment length polymorphism (RFLP). FLiP banding patterns were highly reproducible and polymorphic. This method differentiated 119 types among the study set compared to 108 distinct MIRU-VNTR profiles. The discriminatory power of FLiP was slightly higher than that of MIRU-VNTR analysis (Hunter-Gaston Discriminatory Index = 0.991 and 0.990, resp.). Detailed comparison of the clusters defined by each of the methods revealed, however, a more apparent difference in the discriminatory abilities that favored FLiP. Clustering of strains by using combined results of these two PCR-based methods correlated well with IS6110 RFLP-defined clusters, further confirming high discriminatory potential of FLiP typing. These results indicate that FLiP could be an attractive and valuable secondary typing technique for verification of MIRU-VNTR clusters of M. tuberculosis strains

Comparison of Ligation-Mediated PCR Methods in Differentiation of Mycobacterium tuberculosis Strains

Fast and inexpensive identification of epidemiological links between limited number of Mycobacterium tuberculosis strains is required to initially evaluate hospital outbreaks, laboratory crosscontaminations, and family or small community transmissions. The ligation-mediated PCR methods (LM-PCR) appear sufficiently discriminative and reproducible to be considered as a good candidate for such initial, epidemiological analysis. Here, we compared the discriminative power of the recently developed in our laboratory fast ligation amplification polymorphism (FLAP) method with fast ligation-mediated PCR (FLiP). Verification of the results was based on analyzing a set of reference strains and RFLP-IS6110 typing. The HGDI value was very similar for both LM-PCR methods and RFLP-IS6110 typing. However, only 52% of strains were correspondingly grouped by both FLiP and FLAP methods. Differentiation by FLAP method demonstrated a limited similarity to IS6110-RFLP (37,7%). As much as 78,7% of strains were grouped identically when differentiated by FLiP and IS6110-RFLP methods. The analysis differentiated 31, 35, and 36 groups when using FLAP, FLiP, and RFLP-IS6110 methods, respectively