Anna Sajduda

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

BackgroundThe 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.ResultsThe 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.ConclusionOur 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.

DNA fingerprinting as an indicator of active transmission of multidrug-resistant tuberculosis in Poland☆

OBJECTIVES To use genetic fingerprinting to investigate the epidemiology of tuberculosis (TB) caused by Mycobacterium tuberculosis in Poland, a country with a relatively high incidence of tuberculosis, to improve TB control. DESIGN One hundred M. tuberculosis isolates from 98 patients in the Institute of Tuberculosis and Lung Diseases in Warsaw from 1993 to 1995 and 85 isolates obtained from 50 patients in the Hospital of Lung Diseases in Lodz in 1996 were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the insertion sequence IS6110 as a probe. RESULTS IS6110-associated banding patterns of the M. tuberculosis isolates originating from different localities varied considerably, but isolates from Lodz had a higher degree of similarity. Strains with identical RFLP types were identified in patients of the same family or patients living in the same area, indicating active transmission. Of strains isolated in Warsaw, 45% were resistant to at least one drug, and 35% were resistant to two or more drugs and were classified as multidrug-resistant (MDR). Some drug-resistant isolates were found to have identical banding patterns and originated from epidemiologically linked cases. CONCLUSIONS Active transmission of TB, including MDR TB, is occurring in Poland. Active measures must be taken to prevent the spread of drug-resistant TB in Poland and potentially, the rest of Europe.

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

OBJECTIVES The 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. METHODS PCR-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. RESULTS PRA performed very well in comparison with the two other methods (96.4% concordance). Among 27 M. kansasii isolates, this method detected five genetic types, of which type 1 represented the most common clinical isolate, as it is worldwide. PRA differentiated 29 M. 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. CONCLUSIONS PRA 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.

Profiling Mycobacterium xenopi with restriction fragment length polymorphism of insertion element IS1395

OBJECTIVES The aim of the present study was to assess the usefulness of insertion element IS1395 for differentiation of Mycobacterium xenopi, an increasingly common opportunistic human pathogen. METHODS Fifty-two isolates obtained from 51 patients in Poland in 1996, 1997, and 1999, were analyzed by IS1395 restriction fragment length polymorphism (RFLP), and their susceptibilities to 11 anti-tuberculosis drugs were also determined. RESULTS IS1395-associated banding patterns of the isolates were not highly polymorphic; the RFLP patterns displayed several bands in common. Nevertheless, 44 of the 52 isolates were clearly distinguishable from each other. Only eight strains (15.4%) occurred in four clusters of two identical clones, one of which comprised two isolates obtained from one patient with a 12-month interval. The remaining six patients with clustered strains showed no apparent epidemiologic links with the other patients from the same cluster, and they were most likely infected by the same environmental source. No noticeable difference in RFLP pattern or IS1395 copy number between drug-sensitive and drug-resistant strains was shown. A high proportion (84.6%) of strains resistant to at least one drug was found, and 7.7% were resistant to more than three drugs. CONCLUSIONS The stability and utility of IS1395 for further detailed epidemiological investigations of M. xenopi was confirmed and extended.

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.