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.

Emergence of phenotypic and genotypic antimicrobial resistance in Mycobacterium tuberculosis.

Concentration dependency of phenotypic and genotypic isoniazid-rifampicin resistance emergence was investigated to obtain a mechanistic understanding on how anti-mycobacterial drugs facilitate the emergence of bacterial populations that survive throughout treatment. Using static kill curve experiments, observing two evolution cycles, it was demonstrated that rifampicin resistance was the result of non-specific mechanisms and not associated with accumulation of drug resistance encoding SNPs. Whereas, part of isoniazid resistance could be accounted for by accumulation of specific SNPs, which was concentration dependent. Using a Hollow Fibre Infection Model it was demonstrated that emergence of resistance did not occur at concentration-time profiles mimicking the granuloma. This study showed that disentangling and quantifying concentration dependent emergence of resistance provides an improved rational for drug and dose selection although further work to understand the underlying mechanisms is needed to improve the drug development pipeline.

Anticipating the impact of the COVID-19 pandemic on TB patients and TB control programmes.

The COVID-19 pandemic has currently overtaken every other health issue throughout the world. There are numerous ways in which this will impact existing public health issues. Here we reflect on the interactions between COVID-19 and tuberculosis (TB), which still ranks as the leading cause of death from a single infectious disease globally. There may be grave consequences for existing and undiagnosed TB patients globally, particularly in low and middle income countries (LMICs) where TB is endemic and health services poorly equipped. TB control programmes will be strained due to diversion of resources, and an inevitable loss of health system focus, such that some activities cannot or will not be prioritised. This is likely to lead to a reduction in quality of TB care and worse outcomes. Further, TB patients often have underlying co-morbidities and lung damage that may make them prone to more severe COVID-19. The symptoms of TB and COVID-19 can be similar, with for example cough and fever. Not only can this create diagnostic confusion, but it could worsen the stigmatization of TB patients especially in LMICs, given the fear of COVID-19. Children with TB are a vulnerable group especially likely to suffer as part of the "collateral damage". There will be a confounding of symptoms and epidemiological data through co-infection, as happens already with TB-HIV, and this will require unpicking. Lessons for COVID-19 could be learned from the vast experience of running global TB control programmes, while the astonishingly rapid and relatively well co-ordinated response to COVID-19 demonstrates how existing programmes could be significantly improved.

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.

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.

Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses.

Mannose-capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAM observed in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine-induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co-stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL-10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low-dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.

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.

Cholesterol metabolism in Mycobacterium smegmatis.

The metabolism of cholesterol in Mycobacterium smegmatis mc(2) 155 has been investigated by using a microarray approach. The transcriptome of M. smegmatis growing in cholesterol was compared with that of cells growing in glycerol as the sole carbon and energy sources during the middle exponential phase. Microarray analyses revealed that only 89 genes were upregulated at least threefold during growth on cholesterol compared with growth on glycerol. The upregulated genes are scattered throughout the 7 Mb M. smegmatis genome and likely reflect a general physiological adaptation of the bacterium to grow on this highly hydrophobic polycyclic compound. Nevertheless, 39 of the catabolic genes are organized in three specific clusters. These results not only supported the role of KstR and KstR2 as auto-regulated repressors of cholesterol catabolism, and revealed some metabolic similarities and differences on actinobacteria, but more important, they have facilitated the identification of new catabolic genes, opening a research scenario that might provide important clues on the role of cholesterol in tuberculosis infection.

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning.

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis.

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.

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.

Construction of a severely attenuated mutant of Mycobacterium tuberculosis for reducing risk to laboratory workers.

The ability to construct defined deletions of Mycobacterium tuberculosis has allowed many genes involved in virulence to be identified. Deletion of nutritional genes leads to varying levels of attenuation, presumably reflecting the need for a particular molecule, and the availability (or lack) of that molecule in vivo. We have previously shown that M. tuberculosis mutants lacking either the trpD or ino1 gene are highly attenuated in mouse models of infection, but can grow when supplemented with tryptophan or inositol, respectively. In this paper we have constructed a double Delta trpDDelta ino1 mutant, and show that this is severely attenuated in SCID mouse and guinea pig models. As the strain will grow in the presence of supplements, we propose that this strain could be used for research and antigen preparative purposes, with reduced risks to laboratory workers.

Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages.

BACKGROUND: Transcriptional profiling using microarrays provides a unique opportunity to decipher host pathogen cross-talk on the global level. Here, for the first time, we have been able to investigate gene expression changes in both Mycobacterium tuberculosis, a major human pathogen, and its human host cells, macrophages and dendritic cells. METHODOLOGY/PRINCIPAL FINDINGS: In addition to common responses, we could identify eukaryotic and microbial transcriptional signatures that are specific to the cell type involved in the infection process. In particular M. tuberculosis shows a marked stress response when inside dendritic cells, which is in accordance with the low permissivity of these specialized phagocytes to the tubercle bacillus and to other pathogens. In contrast, the mycobacterial transcriptome inside macrophages reflects that of replicating bacteria. On the host cell side, differential responses to infection in macrophages and dendritic cells were identified in genes involved in oxidative stress, intracellular vesicle trafficking and phagosome acidification. CONCLUSIONS/SIGNIFICANCE: This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments.

Investigating the function of the putative mycolic acid methyltransferase UmaA: divergence between the Mycobacterium smegmatis and Mycobacterium tuberculosis proteins.

Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.