High-Resolution Typing by Integration of Genome Sequencing Data in a Large Tuberculosis Cluster

Abstract

To investigate whether genome sequencing yields more useful markers than those currently used to study the epidemiology of tuberculosis, it was applied to three Mycobacterium tuberculosis isolates of the Harlingen outbreak. Our findings suggest that single nucleotide polymorphisms can be used to identify transmission chains in restriction fragment length polymorphism clusters.Molecular typing contributes significantly to our understanding of the epidemiology of tuberculosis. A variety of genetic markers, such as IS6110 restriction fragment length polymorphism (RFLP) and variable-number tandem repeat (VNTR) typing, are currently used for DNA fingerprinting of Mycobacterium tuberculosis isolates (2,7,(12)(13)(14)23). Unfortunately, these markers do not distinguish primary and subsequent sources of infection in long-term DNA fingerprinting surveillance, as the turnover of these markers is not in range with the pace of transmission (4-6). Therefore, molecular typing is inaccurate when applied for extended time periods in a given area.In the Netherlands, IS6110 RFLP typing has been routinely used for molecular epidemiology since the early 1990s. A remarkably large outbreak began in the city of Harlingen in 1992, and this cluster grew to over 100 cases in 2008 and is still expanding (10, 11). Although a small subset of isolates of this cluster exhibited a single transposition or deletion of IS6110, it soon became impossible to distinguish sources of infection and secondary and subsequent cases in the cluster. Some contact chains in the Harlingen cluster were suggested by contact tracing, performed according to the stone-in-the-pond principle (15, 25), but the exact transmission chains could not be validated by fingerprinting of the M. tuberculosis isolates, as most of the isolates revealed the same DNA fingerprints.For this study, three isolates from two chains of transmission in the Harlingen cluster that could be accurately determined by contact tracing were selected for genome sequencing (Fig. 1). The bacterial isolates exhibited no change in antituberculosis drug resistance or any other observable change in phenotype. Sequencing and analysis of strains SH1 and SH5, as well as the tempo and mode of evolutionary changes between these two isolates, were described in one of our earlier studies (19). The DNA of strain SH9, purified according to the method of van Soolingen et al. (24), was de novo sequenced on a GS FLX Titanium system, and assembly of raw sequencing reads with an average read length of 400 bases was performed by using the Genome Sequencer software, version 2.0.0.22. Sequence reads, contigs, and quality scores were provided by Microsynth AG, Switzerland. The SH9 sequence consisted of 214,283,462 high-quality bases assembled in 401 contigs with 4,207,440 bases (50.9-fold coverage). In total, 95.4% of the theoretical genome size of 4.41 Mb was available for analysis. From the in silico comparison of the three genomes, eight polymorphic single nucleotide polymorphisms (SNPs) were verified by subsequent rese...

Combination of Single Nucleotide Polymorphism and Variable-Number Tandem Repeats for Genotyping a Homogenous Population of Mycobacterium tuberculosis Beijing Strains in China

“…One way to address this challenge is whole-genome sequencing (WGS), which provides the highest discrimination power and where homoplasy is not an issue (7). In recent years, with the development of high-throughput next-generation sequencing technology, several studies have applied WGS to reconstruct transmission chains between VNTR-defined cluster cases (15,22,40), and some clustered strains defined by traditional molecular genotyping methods were identified as distantly related isolates (37). However, because of the relatively high cost and the complexity of data analysis, the application of this technology to study the molecular epidemiology of M.tuberculosis will be limited for some time, especially in developing countries.…”

Clustering of Tuberculosis Cases Based on Variable-Number Tandem-Repeat Typing in Relation to the Population Structure of Mycobacterium tuberculosis in the Netherlands

“…Various studies have shown that not all cases in DNA fingerprint clusters have epidemiological links with other cases in the cluster (4,5). Moreover, epidemiological links have been found between cases caused by bacteria with slightly different DNA fingerprints (6). Clustering results among cases in the immigrant population especially should be interpreted with caution (7,8), as isolates from these patients often belong to genetically compact strain lineages predominating in the countries of origin (9,10,11,12).…”

Preferential Deletion Events in the Direct Repeat Locus of Mycobacterium tuberculosis

“…IS6110 RFLP typing identified the so-called Harlingen cluster in The Netherlands, which is, with over 100 tuberculosis patients identified since 1993, one of the largest tuberculosis clusters found in the country (16,17,25,26). The initial outbreak took place in the harbor town of Harlingen, but the strain soon spread through The Netherlands.…”

“…This locus is a member of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR), which provides acquired immunity against viruses and plasmids (13). The DR locus is a hot spot region for integration of IS6110 insertion sequences (11,20,34), and several preferential insertion sites in this genomic region have been identified (22,34).IS6110 RFLP typing identified the so-called Harlingen cluster in The Netherlands, which is, with over 100 tuberculosis patients identified since 1993, one of the largest tuberculosis clusters found in the country (16,17,25,26). The initial outbreak took place in the harbor town of Harlingen, but the strain soon spread through The Netherlands.…”

“…Therefore, we subjected the DR loci of these four isolates to DNA sequencing and compared the obtained sequences to the sequence of the DR locus of the precursor strains. These data were combined with contacttracing information, IS6110 RFLP, and single-nucleotide polymorphism (SNP) typing (25), providing evidence for three independent convergent deletions in the DR locus.…”