Use of whole-genome sequencing to distinguish relapse from reinfection in a completed tuberculosis clinical trial.

BACKGROUND: RIFAQUIN was a tuberculosis chemotherapy trial in southern Africa including regimens with high-dose rifapentine with moxifloxacin. Here, the application of whole-genome sequencing (WGS) is evaluated within RIFAQUIN for identifying new infections in treated patients as either relapses or reinfections. WGS is further compared with mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) typing. This is the first report of WGS being used to evaluate new infections in a completed clinical trial for which all treatment and epidemiological data are available for analysis. METHODS: DNA from 36 paired samples of Mycobacterium tuberculosis cultured from patients before and after treatment was typed using 24-loci MIRU-VNTR, in silico spoligotyping and WGS. Following WGS, the sequences were mapped against the reference strain H37Rv, the single-nucleotide polymorphism (SNP) differences between pairs were identified, and a phylogenetic reconstruction was performed. RESULTS: WGS indicated that 32 of the paired samples had a very low number of SNP differences (0-5; likely relapses). One pair had an intermediate number of SNP differences, and was likely the result of a mixed infection with a pre-treatment minor genotype that was highly related to the post-treatment genotype; this was reclassified as a relapse, in contrast to the MIRU-VNTR result. The remaining three pairs had very high SNP differences (>750; likely reinfections). CONCLUSIONS: WGS and MIRU-VNTR both similarly differentiated relapses and reinfections, but WGS provided significant extra information. The low proportion of reinfections seen suggests that in standard chemotherapy trials with up to 24 months of follow-up, typing the strains brings little benefit to an analysis of the trial outcome in terms of differentiating relapse and reinfection. However, there is a benefit to using WGS as compared to MIRU-VNTR in terms of the additional genotype information obtained, in particular for defining the presence of mixed infections and the potential to identify known and novel drug-resistance markers.

Clinical use of whole genome sequencing for Mycobacterium tuberculosis.

Drug-resistant tuberculosis (TB) remains a major challenge to global health and to healthcare in the UK. In 2014, a total of 6,520 cases of TB were recorded in England, of which 1.4 % were multidrug-resistant TB (MDR-TB). Extensively drug-resistant TB (XDR-TB) occurs at a much lower rate, but the impact on the patient and hospital is severe. Current diagnostic methods such as drug susceptibility testing and targeted molecular tests are slow to return or examine only a limited number of target regions, respectively. Faster, more comprehensive diagnostics will enable earlier use of the most appropriate drug regimen, thus improving patient outcomes and reducing overall healthcare costs. Whole genome sequencing (WGS) has been shown to provide a rapid and comprehensive view of the genotype of the organism, and thus enable reliable prediction of the drug susceptibility phenotype within a clinically relevant timeframe. In addition, it provides the highest resolution when investigating transmission events in possible outbreak scenarios. However, robust software and database tools need to be developed for the full potential to be realized in this specialized area of medicine.

Clinical application of whole-genome sequencing to inform treatment for multidrug-resistant tuberculosis cases.

The treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time frame, with one case sequenced retrospectively. WGS identified mutations in the M. tuberculosis genes associated with antibiotic resistance that are likely to be responsible for the phenotypic resistance. Thus, an evidence base was developed to inform the clinical decisions made around antibiotic treatment over prolonged periods. All strains in this study belonged to the East Asian (Beijing) lineage, and the strain relatedness was consistent with the expectations from the case histories, confirming one contact transmission event. We demonstrate that WGS data can be produced in a clinically relevant time scale some weeks before drug sensitivity testing (DST) data are available, and they actively help clinical decision-making through the assessment of whether an isolate (i) has a particular resistance mutation where there are absent or contradictory DST results, (ii) has no further resistance markers and therefore is unlikely to be XDR, or (iii) is identical to an isolate of known resistance (i.e., a likely transmission event). A small number of discrepancies between the genotypic predictions and phenotypic DST results are discussed in the wider context of the interpretation and reporting of WGS results.

BµG@Sbase--a microbial gene expression and comparative genomic database.

The reducing cost of high-throughput functional genomic technologies is creating a deluge of high volume, complex data, placing the burden on bioinformatics resources and tool development. The Bacterial Microarray Group at St George's (BµG@S) has been at the forefront of bacterial microarray design and analysis for over a decade and while serving as a hub of a global network of microbial research groups has developed BµG@Sbase, a microbial gene expression and comparative genomic database. BµG@Sbase (http://bugs.sgul.ac.uk/bugsbase/) is a web-browsable, expertly curated, MIAME-compliant database that stores comprehensive experimental annotation and multiple raw and analysed data formats. Consistent annotation is enabled through a structured set of web forms, which guide the user through the process following a set of best practices and controlled vocabulary. The database currently contains 86 expertly curated publicly available data sets (with a further 124 not yet published) and full annotation information for 59 bacterial microarray designs. The data can be browsed and queried using an explorer-like interface; integrating intuitive tree diagrams to present complex experimental details clearly and concisely. Furthermore the modular design of the database will provide a robust platform for integrating other data types beyond microarrays into a more Systems analysis based future.

A Novel Microfluidic Dielectrophoresis Technology to Enable Rapid Diagnosis of Mycobacteria tuberculosis in Clinical Samples.

To achieve the global efforts to end tuberculosis, affordable diagnostics suitable for true point-of-care implementation are required to reach the missing millions. In addition, diagnostics with increased sensitivity and expanded drug susceptibility testing are needed to address drug resistance and to diagnose low-bacterial burden cases. The laboratory-on-a-chip technology described herein used dielectrophoresis to selectively isolate Mycobacterium tuberculosis from sputum samples, purifying the bacterial population ahead of molecular confirmation by multiplex real-time quantitative PCR. After optimization using a panel of 50 characterized sputum samples, the performance of the prototype was assessed against the current gold standards, screening 100 blinded sputum samples using characterized and biobanked sputum provided by Foundation for Innovative New Diagnostics. Concordance with culture diagnosis was 100% for smear-negative samples and 87% for smear-positive samples. Of the smear-positive samples, the high burden sample concordance was 100%. Samples were diagnosed on the basis of visual assessment of the dielectrophoresis array and by multiplex real-time quantitative PCR assay. The results described herein demonstrate the potential of the CAPTURE-XT technology to provide a powerful sample preparation tool that could function as a front-end platform for molecular detection. This versatile tool could equally be applied as a visual detection diagnostic, potentially associated with bacterial identification for low-cost screening or coupled with an expanded PCR assay for genotypic drug susceptibility testing.

Role of the DinB homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis.

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the beta-clamp, consistent with its canonical C-terminal beta-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N(2)-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

Cholesterol utilization in mycobacteria is controlled by two TetR-type transcriptional regulators: kstR and kstR2.

Mycobacterium tuberculosis is able to use a variety of carbon sources in vivo and current knowledge suggests that cholesterol is used as a carbon source during infection. The catabolized cholesterol is used both as an energy source (ATP generation) and as a source of precursor molecules for the synthesis of complex methyl-branched fatty acids. In previous studies, we described a TetR-type transcriptional repressor, kstR, that controls the expression of a number of genes involved in cholesterol catabolism. In this study, we describe a second TetR-type repressor, which we call kstR2. We knocked this gene out in Mycobacterium smegmatis and used microarrays and quantitative RT-PCR to examine the effects on gene expression. We identified a palindromic regulatory motif for KstR2, showed that this motif is present in three promoter regions in mycobacteria and rhodococcus, and demonstrated binding of purified KstR2 to the motif. Using a combination of motif location analysis, gene expression analysis and the examination of gene conservation, we suggest that kstR2 controls the expression of a 15 gene regulon. Like kstR, kstR2 and the kstR2 regulon are highly conserved among the actinomycetes and studies in rhodococcus suggest a role for these genes in cholesterol catabolism. The functional significance of the regulon and implications for the control of cholesterol utilization are discussed.

Inositol monophosphate phosphatase genes of Mycobacterium tuberculosis.

BACKGROUND: Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome. RESULTS: Mutants lacking either impA (Rv1604) or suhB (Rv2701c) were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c) was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137) when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained. CONCLUSIONS: We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.

The case for hypervirulence through gene deletion in Mycobacterium tuberculosis.

Deletion of genes in a pathogen is commonly associated with a reduction in its ability to cause disease. However, some rare cases have been described in the literature whereby deletion of a gene results in an increase in virulence. Recently, there have been several reports of hypervirulence resulting from gene deletion in Mycobacterium tuberculosis. Here, we explore this phenomenon in the context of the interaction between the pathogen and the host response.

RNA profiling in host-pathogen interactions.

The development of novel anti-bacterial treatment strategies will be aided by an increased understanding of the interactions that take place between bacteria and host cells during infection. Global expression profiling using microarray technologies can help to describe and define the mechanisms required by bacterial pathogens to cause disease and the host responses required to defeat bacterial infection.

Screening of highly expressed mycobacterial genes identifies Rv3615c as a useful differential diagnostic antigen for the Mycobacterium tuberculosis complex.

Tuberculous infections caused by mycobacteria, especially tuberculosis of humans and cattle, are important both clinically and economically. Human populations can be vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), and control measures for cattle involving vaccination are now being actively considered. However, diagnostic tests based on tuberculin cannot distinguish between genuine infection and vaccination with BCG. Therefore, identification of differential diagnostic antigens capable of making this distinction is required, and until now sequence-based approaches have been predominant. Here we explored the link between antigenicity and mRNA expression level, as well as the possibility that we may be able to detect differential antigens by analyzing quantified global transcriptional profiles. We generated a list of 14 candidate antigens that are highly expressed in Mycobacterium tuberculosis and M. bovis under a variety of growth conditions. These candidates were screened in M. bovis-infected and naïve cattle for the ability to stimulate a gamma interferon (IFN-gamma) response. We identified one antigen, Rv3615c, which stimulated IFN-gamma responses in a significant proportion of M. bovis-infected cattle (11 of 30 cattle [37%] [P < 0.01]) but not in naïve or BCG-vaccinated animals. Importantly, the same antigen stimulated IFN-gamma responses in a significant proportion of infected cattle that did not respond to the well-characterized mycobacterial antigens ESAT-6 and CFP-10. Therefore, use of the Rv3615c epitope in combination with previously described differential tests based on ESAT-6 and CFP-10 has the potential to significantly increase diagnostic sensitivity without reducing specificity in BCG-vaccinated populations.

A universally applicable method of operon map prediction on minimally annotated genomes using conserved genomic context.

An important step in understanding the regulation of a prokaryotic genome is the generation of its transcription unit map. The current strongest operon predictor depends on the distributions of intergenic distances (IGD) separating adjacent genes within and between operons. Unfortunately, experimental data on these distance distributions are limited to Escherichia coli and Bacillus subtilis. We suggest a new graph algorithmic approach based on comparative genomics to identify clusters of conserved genes independent of IGD and conservation of gene order. As a consequence, distance distributions of operon pairs for any arbitrary prokaryotic genome can be inferred. For E.coli, the algorithm predicts 854 conserved adjacent pairs with a precision of 85%. The IGD distribution for these pairs is virtually identical to the E.coli operon pair distribution. Statistical analysis of the predicted pair IGD distribution allows estimation of a genome-specific operon IGD cut-off, obviating the requirement for a training set in IGD-based operon prediction. We apply the method to a representative set of eight genomes, and show that these genome-specific IGD distributions differ considerably from each other and from the distribution in E.coli.

A GAF domain in the hypoxia/NO-inducible Mycobacterium tuberculosis DosS protein binds haem.

The majority of the Mycobacterium tuberculosis response to hypoxia and nitric oxide is through the DosRS (DevRS) two-component regulatory system. The N-terminal input domain of the DosS sensor contains two GAF domains. We demonstrate here that the proximal GAF domain binds haem, and identified histidine 149 of DosS as critical to haem-binding; the location of this histidine residue is similar to the cGMP-binding site in a crystal structure of cyclic nucleotide phosphodiesterase 2A. GAF domains are frequently involved in binding cyclic nucleotides, but this is the first GAF domain to be identified that binds haem. In contrast, PAS domains (similar to GAF domains in structure but not primary sequence) frequently use haem cofactors, and these findings further illustrate how the functions of these domains overlap. We propose that the activation of the DosS sensor is controlled through the haem binding of molecular oxygen or nitric oxide.

A derivative of Mycobacterium smegmatis mc(2)155 that lacks the duplicated chromosomal region.

The genome of Mycobacterium smegmatis mc(2)155 contains a 56kb duplicated region. We isolated a mutant of mc(2)155 lacking this duplication (DeltaDRKIN). This mutation did not affect the growth rate, surface properties or transformation efficiency of the organism, confirming the potential utility of DeltaDRKIN for the study of genes contained within the duplicated region.

Crystal structure of inositol 1-phosphate synthase from Mycobacterium tuberculosis, a key enzyme in phosphatidylinositol synthesis.

Phosphatidylinositol (PI) is essential for Mycobacterium tuberculosis viability and the enzymes involved in the PI biosynthetic pathway are potential antimycobacterial agents for which little structural information is available. The rate-limiting step in the pathway is the production of (L)-myo-inositol 1-phosphate from (D)-glucose 6-phosphate, a complex reaction catalyzed by the enzyme inositol 1-phosphate synthase. We have determined the crystal structure of this enzyme from Mycobacterium tuberculosis (tbINO) at 1.95 A resolution, bound to the cofactor NAD+. The active site is located within a deep cleft at the junction between two domains. The unexpected presence of a zinc ion here suggests a mechanistic difference from the eukaryotic inositol synthases, which are stimulated by monovalent cations, that may be exploitable in developing selective inhibitors of tbINO.

What do microarrays really tell us about M. tuberculosis?

Bacterial pathogens adapt to their host environments to a large extent through switching on complex transcriptional programmes, and whole-genome microarray experiments promise to reveal this complexity. There has been a recent burst of articles reporting transcriptome analyses of Mycobacterium tuberculosis, including for the first time studies in macrophages and mice. We review gene expression reports, and compare them with each other and with microarray-based gene essentiality studies, revealing at times a startling lack of correlation. Additionally, we suggest a standardization format for the submission of processed data for publication, to facilitate cross-experiment analyses.

Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis.

The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.

The influence of reduced oxygen availability on pathogenicity and gene expression in Mycobacterium tuberculosis.

We investigated how Mycobacterium tuberculosis responded to a reduced oxygen tension in terms of its pathogenicity and gene expression by growing cells under either aerobic or low-oxygen conditions in chemostat culture. The chemostat enabled us to control and vary the oxygen tension independently of other environmental parameters, so that true cause-and-effect relationships of reduced oxygen availability could be established. Cells grown under low oxygen were more pathogenic for guinea pigs than those grown aerobically. The effect of reduced oxygen on global gene expression was determined using DNA microarray. Spearman rank correlation confirmed that microarray expression profiles were highly reproducible between repeat cultures. Using microarray analysis we have identified genes that respond to a low-oxygen environment without the influence of other parameters such as nutrient depletion. Some of these genes appear to be involved in the biosynthesis of cell wall precursors and their induction may have contributed to increased infectivity in the guinea pig. This study has shown that a combination of chemostat culture and microarray presents a biologically robust and statistically reliable experimental approach for studying the effect of relevant and specific environmental stimuli on mycobacterial virulence and gene expression.

The Mycobacterium tuberculosis sigJ gene controls sensitivity of the bacterium to hydrogen peroxide.

Sigma factors are important global regulators which control bacterial gene expression during growth and in response to stress. Previous work showed that mRNA of the sigJ gene was up-regulated in late stationary-phase and after rifampicin treatment. In order to verify the function of SigJ, we constructed a Mycobacterium tuberculosis mutant lacking the sigJ gene. In a microaerophilic stationary-phase model, the sigJ mutant showed the same growth pattern as the wild-type strain. In an immune stasis murine model in which the bacterial number plateaued between the second and the 15th week, the mutant showed a similar growth curve to the wild-type strain. However, the sigJ mutant was more susceptible to killing by H2O2 than its parental strain. The parental level of sensitivity to H2O2 was recovered in the sigJ complemented strain. These data suggest that the SigJ protein is not essential for survival in long-term stationary phase or in bacterial stasis in mice. However, the sigJ gene may control an alternative H2O2 resistance pathway.

bkaR is a TetR-type repressor that controls an operon associated with branched-chain keto-acid metabolism in Mycobacteria.

This study describes how bkaR, a highly conserved mycobacterial TetR-like transcriptional repressor, regulates a number of nearby genes that have associations with branched-chain keto-acid metabolism. bkaR (MSMEG_4718) was deleted from the nonpathogenic species Mycobacterium smegmatis, and changes in global gene expression were assessed using microarray analysis and reporter gene studies. bkaR was found to directly control the expression of 10 genes in M. smegmatis, and its ortholog in Mycobacterium tuberculosis (Rv2506) is predicted to control at least 12 genes. A conserved operator motif was identified, and binding of purified recombinant M. tuberculosis BkaR to the motif was demonstrated. Analysis of the stoichiometry of binding showed that BkaR binds to the motif as a dimer.