Large Sequence Polymorphisms Research Articles

Mycobacterium ulcerans and Other Mycolactone-Producing Mycobacteria Should Be Considered a Single Species

Mycobacterium ulcerans and Other Mycolactone-Producing Mycobacteria Should Be Considered a Single Species

Genomic Signatures of the Haarlem Lineage of Mycobacterium tuberculosis : Implications of Strain Genetic Variation in Drug and Vaccine Development

Tuberculosis is the world's leading cause of death due to a single infectious agent, and efforts aimed at its control require a better understanding of host, environmental, and bacterial factors that govern disease outcome. Growing evidence indicates that certain Mycobacterium tuberculosis strains of distinct phylogeographic lineages elicit unique immunopathological events. However, identifying the genetic basis of these phenotypic peculiarities has proven difficult. Here we report the presence of six large sequence polymorphisms which, together with two single-nucleotide changes previously described by our group, consistently differentiate Haarlem strains from the remaining M. tuberculosis lineages. The six newly found Haarlem-specific genetic events are four deletions, which altogether involve more than 13 kb, and two intragenic insertions of the element IS6110. The absence of the genes involved in these polymorphisms could have an important physiological impact on Haarlem strains, i.e., by affecting key genes, such as Rv1354c and cyp121, which have been recently proposed as plausible drug targets. These lineage-specific polymorphisms can serve as genetic markers for the rapid PCR identification of Haarlem strains, providing a useful tool for strain surveillance and molecular epidemiology studies. Strain variability such as that described here underscores the need for the definition of a core set of essential genes in M. tuberculosis that are ubiquitously present in all circulating lineages, as a requirement in the development of effective antituberculosis drugs and vaccines.

Concordance of variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses of Mycobacterium tuberculosis strains

Variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses were compared to determine whether VNTR analysis was effective for population genetic analysis of Mycobacterium tuberculosis strains. A total of 682 strains, 510 Beijing genotype and 172 non-Beijing genotype strains, were studied. The number of repeats was investigated for 24 VNTR loci: the 15 loci of “optimized miru”, the 8 loci of “Beijing option”, and 1 locus for “JATA12”. Six loci (miru31, Mtub4, QUB4156c, QUB3232, VNTR3820, and VNTR4120) showed significantly different median numbers of repeats in strains belonging to different lineages defined by LSP ( P < 0.01, Mann–Whitney U test). When a minimum-spanning tree (MST) was reconstructed using these 6 loci, most strains clustered in the expected branches in the MST branches. However, topology of the MST was not congruent with the evolutional hypothesis of M. tuberculosis, indicating that MST analysis using VNTR data should not use for phylogeny of the organism. When the standardized index of association (s I A) was calculated using data for the 6 VNTR loci, the value of s I A was significantly different from zero (Monte Carlo simulation with 10,000 resamplings) in every lineage, indicating the linkage disequilibrium in different lineage strains of M. tuberculosis. These results were consistent with the hypothesis that clonal evolution of lineages of the organism has occurred. Therefore, the 6 loci identified in this study would be effective for M. tuberculosis population genetic analysis due to their significantly different median numbers of repeat and linkage disequilibrium though VNTR data was not effective for phylogeny of the organism.

Influence of M. tuberculosis Lineage Variability within a Clinical Trial for Pulmonary Tuberculosis

Recent studies suggest that M. tuberculosis lineage and host genetics interact to impact how active tuberculosis presents clinically. We determined the phylogenetic lineages of M. tuberculosis isolates from participants enrolled in the Tuberculosis Trials Consortium Study 28, conducted in Brazil, Canada, South Africa, Spain, Uganda and the United States, and secondarily explored the relationship between lineage, clinical presentation and response to treatment. Large sequence polymorphisms and single nucleotide polymorphisms were analyzed to determine lineage and sublineage of isolates. Of 306 isolates genotyped, 246 (80.4%) belonged to the Euro-American lineage, with sublineage 724 predominating at African sites (99/192, 51.5%), and the Euro-American strains other than 724 predominating at non-African sites (89/114, 78.1%). Uneven distribution of lineages across regions limited our ability to discern significant associations, nonetheless, in univariate analyses, Euro-American sublineage 724 was associated with more severe disease at baseline, and along with the East Asian lineage was associated with lower bacteriologic conversion after 8 weeks of treatment. Disease presentation and response to drug treatment varied by lineage, but these associations were no longer statistically significant after adjustment for other variables associated with week-8 culture status.

MIRU-VNTRplus: a web tool for polyphasic genotyping of Mycobacterium tuberculosis complex bacteria

Harmonized typing of bacteria and easy identification of locally or internationally circulating clones are essential for epidemiological surveillance and disease control. For Mycobacterium tuberculosis complex (MTBC) species, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible and discriminatory typing method. However, no specialized bioinformatics web tools are available for analysing MLVA data in combination with other, complementary typing data. Therefore, we have developed the web application MIRU-VNTRplus (http://www.miru-vntrplus.org). This freely accessible service allows users to analyse genotyping data of their strains alone or in comparison with a reference database of strains representing the major MTBC lineages. Analysis and comparisons of genotypes can be based on MLVA-, spoligotype-, large sequence polymorphism and single nucleotide polymorphism data, or on a weighted combination of these markers. Tools for data exploration include search for similar strains, creation of phylogenetic and minimum spanning trees and mapping of geographic information. To facilitate scientific communication, an expanding genotype nomenclature (MLVA MtbC15-9 type) that can be queried via a web- or a SOAP-interface has been implemented. An extensive documentation guides users through all application functions.

Associations betweenMycobacterium tuberculosisStrains and Phenotypes

To inform development of tuberculosis (TB) control strategies, we characterized a total of 2,261 Mycobacterium tuberculosis complex isolates by using multiple phenotypic and molecular markers, including polymorphisms in repetitive sequences (spoligotyping and variable-number tandem repeats [VNTRs]) and large sequence and single-nucleotide polymorphisms. The Beijing family was strongly associated with multidrug resistance (p = 0.0001), and VNTR allelic variants showed strong associations with spoligotyping families: >or=5 copies at exact tandem repeat (ETR) A, >or=2 at mycobacterial interspersed repetitive unit 24, and >or=3 at ETR-B associated with the East African-Indian and M. bovis strains. All M. tuberculosis isolates were differentiated into 4 major lineages, and a maximum parsimony tree was constructed suggesting a more complex phylogeny for M. africanum. These findings can be used as a model of pathogen global diversity.

Genotyping of Genetically Monomorphic Bacteria: DNA Sequencing in Mycobacterium tuberculosis Highlights the Limitations of Current Methodologies

Because genetically monomorphic bacterial pathogens harbour little DNA sequence diversity, most current genotyping techniques used to study the epidemiology of these organisms are based on mobile or repetitive genetic elements. Molecular markers commonly used in these bacteria include Clustered Regulatory Short Palindromic Repeats (CRISPR) and Variable Number Tandem Repeats (VNTR). These methods are also increasingly being applied to phylogenetic and population genetic studies. Using the Mycobacterium tuberculosis complex (MTBC) as a model, we evaluated the phylogenetic accuracy of CRISPR- and VNTR-based genotyping, which in MTBC are known as spoligotyping and Mycobacterial Interspersed Repetitive Units (MIRU)-VNTR-typing, respectively. We used as a gold standard the complete DNA sequences of 89 coding genes from a global strain collection. Our results showed that phylogenetic trees derived from these multilocus sequence data were highly congruent and statistically robust, irrespective of the phylogenetic methods used. By contrast, corresponding phylogenies inferred from spoligotyping or 15-loci-MIRU-VNTR were incongruent with respect to the sequence-based trees. Although 24-loci-MIRU-VNTR performed better, it was still unable to detect all strain lineages. The DNA sequence data showed virtually no homoplasy, but the opposite was true for spoligotyping and MIRU-VNTR, which was consistent with high rates of convergent evolution and the low statistical support obtained for phylogenetic groupings defined by these markers. Our results also revealed that the discriminatory power of the standard 24 MIRU-VNTR loci varied by strain lineage. Taken together, our findings suggest strain lineages in MTBC should be defined based on phylogenetically robust markers such as single nucleotide polymorphisms or large sequence polymorphisms, and that for epidemiological purposes, MIRU-VNTR loci should be used in a lineage-dependent manner. Our findings have implications for strain typing in other genetically monomorphic bacteria.

Single Nucleotide Polymorphisms on the Road to Strain Differentiation in Mycobacterium ulcerans

The genomic fine-typing of strains of Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer, is difficult due to the clonal population structure of geographical lineages. Although large sequence polymorphisms (LSPs) resulted in the clustering of patient isolates originating from across the globe, differentiation of strains within continents using conventional typing methods is very limited. In this study, we analyzed M. ulcerans LSP haplotype-specific insertion sequence elements among 83 M. ulcerans strains and identified single nucleotide polymorphisms (SNPs) that differentiate between regional strains. This is the first genetic discrimination based on SNPs of M. ulcerans strains from African countries where Buruli ulcer is endemic, resulting in the highest geographic resolution of genotyping so far. The findings support the concept of genome-wide SNP analyses as tools to study the epidemiology and evolution of M. ulcerans at a local level.

Lack of Insertional-Deletional Polymorphism in a Collection of Mycobacterium ulcerans Isolates from Ghanaian Buruli Ulcer Patients

Mycobacterium ulcerans causes the devastating infectious skin disease Buruli ulcer and has a monomorphic population structure. The resolution of conventional genetic fingerprinting methods is therefore not sufficient for microepidemiological studies aiming to characterize transmission pathways. In a previous comparative genomic hybridization analysis with a microarray covering part of the M. ulcerans genome, we have found extensive insertional-deletional sequence polymorphisms among M. ulcerans isolates of diverse geographic origins that allowed us to distinguish between strains coming from different continents. Since large numbers of insertion sequences are spread over the genome of African M. ulcerans strains, we reasoned that these may drive large sequence polymorphisms in otherwise clonal local mycobacterial populations. In this study, we used a printed DNA microarray covering the whole genome of the Ghanaian M. ulcerans reference strain Agy99 for comparative genomic hybridization. The assay identified multiple regions of difference when DNA of a Japanese M. ulcerans strain was analyzed. In contrast, not a single insertional-deletional genomic variation was found within a panel of disease isolates coming from an area of Ghana where Buruli ulcer is endemic. These results indicate that, despite the expectations deduced from other mycobacterial pathogens, only analyses of single nucleotide polymorphisms will have the potential to differentiate local populations of M. ulcerans.

Beijing genotype of Mycobacterium tuberculosis is significantly associated with high-level fluoroquinolone resistance in Vietnam.

Consecutive fluoroquinolone (FQ)-resistant isolates (n = 109) identified at the Pham Ngoc Thach Hospital for Tuberculosis, Ho Chi Minh City, Vietnam, were sequenced in the quinolone resistance-determining regions of the gyrA and gyrB genes and typed by large sequence polymorphism typing and spoligotyping to identify the Beijing genotype of Mycobacterium tuberculosis. Beijing genotype prevalence was compared with 109 consecutive isolates from newly presenting patients with pulmonary tuberculosis from the hospital outpatient department. Overall, 82.6% (n = 90/109) of isolates had mutations in gyrAB. Nine novel mutations were identified in gyrB (S486F, N538T, T539P, D500A, D500H, D500N, G509A, E540V, and E540D). The influence of these novel gyrB mutations on FQ resistance is not proven. The Beijing genotype was significantly associated with FQ resistance (odds ratio [OR], 2.39 [95% confidence interval {CI}, 1.34 to 4.25]; P = 0.003). Furthermore, Beijing genotype FQ-resistant isolates were significantly more likely than FQ-resistant isolates of other genotypes to have gyrA mutations (OR, 7.75 [95% CI, 2.84 to 21.15]; P = 0.0001) and high-level (>8 microg/ml) FQ resistance (OR, 11.0 [95% CI, 2.6 to 47.0]; P = 0.001). The underlying mechanism of the association of the Beijing genotype with high-level FQ resistance in this setting remains to be determined. The association of the Beijing genotype with relatively high-level FQ resistance conferred by specific gyrA mutations reported here is of grave concern given the epidemic spread of the Beijing genotype and the current hopes for shorter first-line treatment regimens based on FQs.

Large Sequence Polymorphisms Unveil the Phylogenetic Relationship of Environmental and Pathogenic Mycobacteria Related to Mycobacterium ulcerans

Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum, indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans, we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.

Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III

Mycobacterium avium subsp. paratuberculosis (MAP) strains with two new IS900 restriction fragment length polymorphism (RFLP) BstEII types intermediate suspected to belong to the MAP Type III group were isolated from migrating sheep in Germany. Such strains have only been sporadically identified in a few studies. For a better understanding of the genomic diversity of MAP with regard to specific host associations, geographic origin, and the discussed classification into Type I, Type II and Type III, these isolates were further characterized.Using IS900-RFLP, the isolates showed unique fingerprint patterns after BstEII-, PstI-, PvuII- and BamHI-digestion which had not been published before. Additionally, using gyrB-PCR-restriction endonuclease analysis (PCR/REA) and mycobacterial interspersed repetitive unit (MIRU)-PCR, the two strains showed differences to known patterns of the Type I as well as the Type II group. Unique genotypes were also obtained with multilocus short sequence repeat (MLSSR) sequencing and MIRU-variable-number tandem-repeat (VNTR) typing.As expected, genomic profiles identical to the Type I and different from the Type II group were detected by IS1311-PCR/REA, IS1311 sequencing as well as by Large Sequence Polymorphism analysis (LSPA 8, 17, 20, 4-II, and 18).In addition to distinct growth characteristics, the unique genotypes of the studied sheep strains support their affiliation to the assumed third group within the MAP subspecies and suggest the existence of different genotypes within this Type III group. The results could serve as further evidence that Type I and Type III groups are more closely related to each other than to the bovine Type II group.

Major Mycobacterium tuberculosis Lineages Associate with Patient Country of Origin

Over recent years, there has been an increasing acknowledgment of the diversity that exists among Mycobacterium tuberculosis clinical isolates. To facilitate comparative studies aimed at deciphering the relevance of this diversity to human disease, an unambiguous and easily interpretable method of strain classification is required. Presently, the most effective means of assigning isolates into a series of unambiguous lineages is the method of Gagneux et al. (S. Gagneux et al., Proc. Natl. Acad. Sci. USA 103:2869-2873, 2006) that involves the PCR-based detection of large sequence polymorphisms (LSPs). In this manner, isolates are classified into six major lineages, the majority of which display a high degree of geographic restriction. Here we describe an independent replicate of the Gagneux study carried out on 798 isolates collected over a 6-year period from mostly foreign-born patients resident on the island of Montreal, Canada. The original trends in terms of bacterial genotype and patient ethnicity are remarkably conserved within this Montreal cohort, even though the patient distributions between the two populations are quite distinct. In parallel with the LSP analysis, we also demonstrate that "clustered" tuberculosis (TB) cases defined through restriction fragment length polymorphism (RFLP) analysis (for isolates with >or=6 IS6110 copies) or RFLP in combination with spoligotyping (for isolates with <6 IS6110 copies) do not stray across the LSP-defined lineage boundaries. However, our data also demonstrate the poor discriminatory power of either RFLP or spoligotyping alone for these low-IS6110-copy-number isolates. We believe that this independent validation of the LSP method should encourage researchers to adopt this system in investigations aimed at elucidating the role of strain variation in TB.

Use of Spoligotyping and Large Sequence Polymorphisms To Study the Population Structure of the Mycobacterium tuberculosis Complex in a Cohort Study of Consecutive Smear-Positive Tuberculosis Cases in The Gambia

Mycobacterium africanum, first described in Senegal in 1968, causes up to half of the smear-positive pulmonary tuberculosis cases in West Africa, but it has not been found in other geographical areas except among recent West African migrants. The reasons for the geographic restriction of M. africanum are unknown. We used molecular tools to determine the population structure of the Mycobacterium tuberculosis complex in a cohort study of consecutive smear-positive tuberculosis cases in The Gambia. We collected and genotyped 386 clinical isolates using spoligotype analysis and PCRs for large sequence polymorphisms (LSPs) and compared the genotype patterns to the patterns in an international database. The results of spoligotyping and LSP analysis for the study population were also compared to determine the correlation between them. The main lineages within the Mycobacterium tuberculosis complex identified in The Gambia included M. africanum type I (38.4%), characterized by an LSP in region of difference 702 (RD702; West African type 2). Among the M. tuberculosis sensu stricto isolates, lineages characterized by RD182 and by RD174 were the most common. We also detected a gradient in the prevalence of M. africanum that extended from neighboring Guinea-Bissau. The genotypic diversity of the spoligotype patterns was greater among the isolates of M. africanum than among the isolates of M. tuberculosis. We postulate that M. africanum became endemic in West Africa first, before the introduction of different lineages within M. tuberculosis sensu stricto.

MyBASE: a database for genome polymorphism and gene function studies of Mycobacterium

BackgroundMycobacterial pathogens are a major threat to humans. With the increasing availability of functional genomic data, research on mycobacterial pathogenesis and subsequent control strategies will be greatly accelerated. It has been suggested that genome polymorphisms, namely large sequence polymorphisms, can influence the pathogenicity of different mycobacterial strains. However, there is currently no database dedicated to mycobacterial genome polymorphisms with functional interpretations.DescriptionWe have developed a mycobacterial database (MyBASE) housing genome polymorphism data and gene functions to provide the mycobacterial research community with a useful information resource and analysis platform. Whole genome comparison data produced by our lab and the novel genome polymorphisms identified were deposited into MyBASE. Extensive literature review of genome polymorphism data, mainly large sequence polymorphisms (LSPs), operon predictions and curated annotations of virulence and essentiality of mycobacterial genes are unique features of MyBASE. Large-scale genomic data integration from public resources makes MyBASE a comprehensive data warehouse useful for current research. All data is cross-linked and can be graphically viewed via a toolbox in MyBASE.ConclusionAs an integrated platform focused on the collection of experimental data from our own lab and published literature, MyBASE will facilitate analysis of genome structure and polymorphisms, which will provide insight into genome evolution. Importantly, the database will also facilitate the comparison of virulence factors among various mycobacterial strains. MyBASE is freely accessible via http://mybase.psych.ac.cn.

Differential Gene Repertoire in Mycobacterium ulcerans Identifies Candidate Genes for Patho-Adaptation

BackgroundBased on large genomic sequence polymorphisms, several haplotypes belonging to two major lineages of the human pathogen Mycobacterium ulcerans could be distinguished among patient isolates from various geographic origins. However, the biological relevance of insertional/deletional diversity is not understood.MethodologyUsing comparative genomics, we have investigated the genes located in regions of difference recently identified by DNA microarray based hybridisation analysis. The analysed regions of difference comprise ∼7% of the entire M. ulcerans genome.Principal FindingsSeveral different mechanisms leading to loss of functional genes were identified, ranging from pseudogenization, caused by frame shift mutations or mobile genetic element interspersing, to large sequence polymorphisms. Four hot spot regions for genetic instability were unveiled. Altogether, 229 coding sequences were found to be differentially inactivated, constituting a repertoire of coding sequence variation in the rather monomorphic M. ulcerans.Conclusions/SignificanceThe differential gene inactivation patterns associated with the M. ulcerans haplotypes identified candidate genes that may confer enhanced adaptation upon ablation of expression. A number of gene conversions confined to the classical lineage may contribute to particular virulence of this group comprising isolates from Africa and Australia. Identification of this spectrum of anti-virulence gene candidates expands our understanding of the pathogenicity and ecology of the emerging infectious disease Buruli ulcer.

Discovery of Stable and Variable Differences in the Mycobacterium avium subsp. paratuberculosis Type I, II, and III Genomes by Pan-Genome Microarray Analysis

Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.

Does the lipR gene of tubercle bacilli have a role in tuberculosis transmission and pathogenesis?

Mycobacterium tuberculosis lipases, a diverse class of enzymes involved in lipid metabolism, may have an important role in tuberculosis (TB) pathogenesis. We explored the association of large sequence polymorphism (LSP) in one of the M. tuberculosis lipase-encoding genes, lipR (Rv3084), with patient characteristics using a population-based sample of clinical isolates to elucidate the potential role of lipR in TB pathogenesis. LSP in lipR was found in 104 (15.6%) of 665 isolates, of which 96% belonged to principal genetic group 3. When linkage by molecular type and epidemiologic evidence were compared, molecularly clustered cases infected with a lipR LSP isolate were more often epidemiologically linked than clustered cases infected with a lipR wild-type isolate. Further epidemiologic and functional studies are necessary to determine if the association between this lipR LSP and recent transmission we identified in this population reflects a functional role of lipR in TB transmission and pathogenesis or other unidentified mechanisms.

Insertion and Deletion Events That Define the Pathogen Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium comprises genetically related yet phenotypically distinct subspecies. Consistent with their common origin, whole-genome sequence comparisons have revealed extensive synteny among M. avium organisms. However, the sequenced strains also display numerous regions of heterogeneity that likely contribute to the diversity of the individual subspecies. Starting from a phylogenetic framework derived by multilocus sequence analysis, we examined the distribution of 25 large sequence polymorphisms across a panel of genetically defined M. avium strains. This distribution was most variable among M. avium subsp. hominissuis isolates. In contrast, M. avium subsp. paratuberculosis strains exhibited a characteristic profile, with all isolates containing a set of genomic insertions absent from other M. avium strains. The emergence of the pathogen from its putative M. avium subsp. hominissuis ancestor entailed the acquisition of approximately 125 kb of novel genetic material, followed by a second phase, characterized by reductive genomics. One genomic deletion is common to all isolates while additional deletions distinguish two major lineages of M. avium subsp. paratuberculosis. For the average strain, these losses total at least 38 kb (sheep lineage) to 90 kb (cattle lineage). This biphasic pattern of evolution, characterized by chromosomal gene acquisition with subsequent gene loss, describes the emergence of M. avium subsp. paratuberculosis and may serve as a general model for the origin of pathogenic mycobacteria.

Novel genome polymorphisms in BCG vaccine strains and impact on efficacy

Bacille Calmette-Guérin (BCG) is an attenuated strain of Mycobacterium bovis currently used as a vaccine against tuberculosis. Global distribution and propagation of BCG has contributed to the in vitro evolution of the vaccine strain and is thought to partially account for the different outcomes of BCG vaccine trials. Previous efforts by several molecular techniques effectively identified large sequence polymorphisms among BCG daughter strains, but lacked the resolution to identify smaller changes. In this study, we have used a NimbleGen tiling array for whole genome comparison of 13 BCG strains. Using this approach, in tandem with DNA resequencing, we have identified six novel large sequence polymorphisms including four deletions and two duplications in specific BCG strains. Moreover, we have uncovered various polymorphisms in the phoP-phoR locus. Importantly, these polymorphisms affect genes encoding established virulence factors including cell wall complex lipids, ESX secretion systems, and the PhoP-PhoR two-component system. Our study demonstrates that major virulence factors are different among BCG strains, which provide molecular mechanisms for important vaccine phenotypes including adverse effect profile, tuberculin reactivity and protective efficacy. These findings have important implications for the development of a new generation of vaccines.