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      Research Roadmap for Tuberculosis Transmission Science: Where Do We Go From Here and How Will We Know When We’re There?

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          Abstract

          High rates of tuberculosis transmission are driving the ongoing global tuberculosis epidemic, and there is a pressing need for research focused on understanding and, ultimately, halting transmission. The ongoing tuberculosis–human immunodeficiency virus (HIV) coepidemic and rising rates of drug-resistant tuberculosis in parts of the world add further urgency to this work. Success in this research will require a concerted, multidisciplinary effort on the part of tuberculosis scientists, clinicians, programs, and funders and must span the research spectrum from biomedical sciences to the social sciences, public health, epidemiology, cost-effectiveness analyses, and operations research. Heterogeneity of tuberculosis disease, both among individual patients and among communities, poses a substantial challenge to efforts to interrupt transmission. As such, it is likely that effective interventions to stop transmission will require a combination of approaches that will vary across different epidemiologic settings. This research roadmap summarizes key gaps in our current understanding of transmission, as laid out in the preceding articles in this series. We also hope that it will be a call to action for the global tuberculosis community to make a sustained commitment to tuberculosis transmission science. Halting transmission today is an essential step on the path to end tuberculosis tomorrow.

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          Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis.

          Although progress has been made to reduce global incidence of drug-susceptible tuberculosis, the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis during the past decade threatens to undermine these advances. However, countries are responding far too slowly. Of the estimated 440,000 cases of MDR tuberculosis that occurred in 2008, only 7% were identified and reported to WHO. Of these cases, only a fifth were treated according to WHO standards. Although treatment of MDR and XDR tuberculosis is possible with currently available diagnostic techniques and drugs, the treatment course is substantially more costly and laborious than for drug-susceptible tuberculosis, with higher rates of treatment failure and mortality. Nonetheless, a few countries provide examples of how existing technologies can be used to reverse the epidemic of MDR tuberculosis within a decade. Major improvements in laboratory capacity, infection control, performance of tuberculosis control programmes, and treatment regimens for both drug-susceptible and drug-resistant disease will be needed, together with a massive scale-up in diagnosis and treatment of MDR and XDR tuberculosis to prevent drug-resistant strains from becoming the dominant form of tuberculosis. New diagnostic tests and drugs are likely to become available during the next few years and should accelerate control of MDR and XDR tuberculosis. Equally important, especially in the highest-burden countries of India, China, and Russia, will be a commitment to tuberculosis control including improvements in national policies and health systems that remove financial barriers to treatment, encourage rational drug use, and create the infrastructure necessary to manage MDR tuberculosis on a national scale. Copyright 2010 Elsevier Ltd. All rights reserved.
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            Epidemiology of extrapulmonary tuberculosis in the United States, 1993-2006.

            Almost one-fifth of United States tuberculosis cases are extrapulmonary; unexplained slower annual case count decreases have occurred in extrapulmonary tuberculosis (EPTB), compared with annual case count decreases in pulmonary tuberculosis (PTB) cases. We describe the epidemiology of EPTB by means of US national tuberculosis surveillance data. US tuberculosis cases reported from 1993 to 2006 were classified as either EPTB or PTB. EPTB encompassed lymphatic, pleural, bone and/or joint, genitourinary, meningeal, peritoneal, and unclassified EPTB cases. We excluded cases with concurrent extrapulmonary-pulmonary tuberculosis and cases of disseminated (miliary) tuberculosis. Demographic characteristics, drug susceptibility test results, and risk factors, including human immunodeficiency virus (HIV) status, were compared for EPTB and PTB cases. Among 253,299 cases, 73.6% were PTB and 18.7% were EPTB, including lymphatic (40.4%), pleural (19.8%), bone and/or joint (11.3%), genitourinary (6.5%), meningeal (5.4%), peritoneal (4.9%), and unclassified EPTB (11.8%) cases. Compared with PTB, EPTB was associated with female sex (odds ratio [OR], 1.7; 95% confidence interval [CI], 1.7-1.8) and foreign birth (OR, 1.5; CI, 1.5-1.6), almost equally associated with HIV status (OR, 1.1; CI, 1.1-1.1), and negatively associated with multidrug resistance (OR, 0.6; CI, 0.5-0.6) and several tuberculosis risk factors, especially homelessness (OR, 0.3; CI, 0.3-0.3) and excess alcohol use (OR, 0.3; CI, 0.3-0.3). Slower annual decreases in EPTB case counts, compared with annual decreases in PTB case counts, from 1993 through 2006 have caused EPTB to increase from 15.7% of tuberculosis cases in 1993 to 21.0% in 2006. EPTB epidemiology and risk factors differ from those of PTB, and the proportion of EPTB has increased from 1993 through 2006. Further study is needed to identify causes of the proportional increase in EPTB.
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              Cytological and Transcript Analyses Reveal Fat and Lazy Persister-Like Bacilli in Tuberculous Sputum

              Introduction Mycobacterium tuberculosis infects one in three worldwide and kills more people each year than any other bacterial pathogen. Routine treatment of tuberculosis requires combination antibiotic therapy for a minimum of six months, and places a substantial burden on health care systems, particularly in resource-poor countries. Over eight million new cases every year testify to this obligate pathogen's ongoing success in transmission [1], yet we know little about what the organism needs to achieve this essential step. Expectorated tubercle bacilli have been thought to originate from rapid and extensive bacterial growth at the margins of liquefied lesions in the lung [2,3]. Sputum provides a tractable sample of the bacterial population that must be targeted by antibiotic therapy and a snapshot of the organism on its way to a new host. It follows that the bacilli in microscopy smear-positive tuberculosis sputum express properties required for transmission—properties that might explain the existence of drug-tolerant persister subpopulations and account for the prolonged antibiotic therapy necessary for relapse-free treatment [4]. Since transmission is required for evolutionary survival, we may assume that M. tuberculosis experiences powerful selection pressures to maintain and express these as-yet unidentified properties. Thus, any bacillary phenotype recognised preferentially in sputum could provide clues to these properties. We have previously shown that nonpathogenic mycobacteria readily accumulate intracellular triacylglycerol lipid bodies in vitro [5]; these bodies could not be demonstrated under similar conditions with M. tuberculosis, yet anecdotally have been seen in acid-fast bacilli (AFB) in tuberculous sputum [5]. The recent discovery of a novel class of diacylglycerol acyl transferase enzymes in Acinetobacter [6] and the subsequent characterisation of 15 members of this class as triacylglycerol synthase-encoding genes (tgs1–tgs15) in M. tuberculosis [7] provide a biochemical basis for the presence of lipid bodies in this organism. Intriguingly, Tgs1, the most active of these enzymes, is a member of the DosR regulon [8], a set of genes responsive to hypoxia and linked to long-term survival of M. tuberculosis in animal hosts [9–13]. It has recently been shown that triacylglycerol is accumulated by M. tuberculosis following hypoxic and other stresses [7,14] and may contribute to long-term mycobacterial survival. These observations raise the possibility that lipid body–positive cells in sputum may be in a nonreplicating persistent (NRP) state, which, given that NRP bacilli display antibiotic tolerance [11,13,15], would have implications for chemotherapy. Defining the phenotypes of bacterial pathogens in their natural environments remains a key challenge. Accurate knowledge of the properties expressed at different stages of infection enables precise targeting of therapeutic and preventive measures. While much has been learnt about bacterial pathogens from in vitro and in vivo (animal model) transcriptome studies [16,17] as well as from human lung tissue [18], there have, to the best of our knowledge, been no published studies of transcript profiles in sputum samples—a clinically tractable sample. Such methods as rapidly stabilised RNA, differential cell lysis, and RNA amplification have enabled us to report here the transcriptome of M. tuberculosis in the sputum of patients prior to treatment. Methods Patients Patients attending the public clinic at the MRC Laboratories, Fajara, The Gambia and identified as sputum smear–positive by routine microscopy were invited to provide early-morning samples for transcriptome analysis. Patients who agreed to participate gave informed oral consent (study nos. L2002.52 and L2006.60, ethical committee, MRC Laboratories, Fajara, The Gambia). Sputum from nine patients yielded sufficient mycobacterial RNA for analysis by microarray or PCR; these were designated sputum samples 1–9. Mycobacterial Strains and Growth Conditions M. tuberculosis complex for direct microarray transcriptome analysis was isolated from an aliquot of sputum 1 using standard methods [19]. M. tuberculosis complex was grown on 7H10 agar with oleic acid-albumin-dextrose-catalase [20] supplement or in 7H9 broth with albumin-dextrose-catalase supplement [20], 0.2% glycerol and 0.05% Tween-80. For hypoxic (nonreplicating persistence) cultures M. tuberculosis strains H37Rv and CH [21] were grown in Dubos Tween-albumin broth. Routine Culturing of Smear-Positive Sputum Samples Diagnostic sputum specimens were stained with auramine-phenol [19], and positive smears confirmed and scored by Ziehl-Neelsen staining after initial examination by fluorescence microscopy. Smears were scored as either 1+ (1–10 AFB in 100 fields of view), 2+ (1–10 AFB in ten fields of view), or 3+ (1–10 AFB in one field of view). Decontamination of specimens was performed by the NaOH-NALC method [19]. Each decontaminated specimen was inoculated into one vial of BACTEC 9000 MB medium for isolation of M. tuberculosis. The time to positivity of the BACTEC culture was recorded in days. All mycobacterial cultures were identified and confirmed as M. tuberculosis complex using standard procedures. Auramine-Nile Red Labelling of Sputum Samples Whole sputum (∼1–4 ml) was digested for 15 min with an equal volume of 0.5% w/v N-acetyl L-cysteine in 50 mM sodium citrate [19]. Phosphate buffer (67 mM [pH 6.8]) was added to a final volume of 20 ml, and bacteria were concentrated (1,398g, 20 min). The pellet was resuspended in 0.5 ml of phosphate-buffered saline and a smear prepared with ∼10 μl of the suspension. Heat-fixed smears were labelled with auramine-Nile red as previously described [5]. Preparations were observed by epifluorescence microscopy using a Nikon Diphot 300 inverted microscope with a 100 W mercury light source. Images were recorded using a 12/10bit, high speed Peltier-cooled CCD camera (FDI, Photonic Science) using Image-Pro Plus (Media Cybernetics) software. The 11001V2 Blue (excitation 470 ± 40 nm; emission > 515 nm; Chroma Technology) and the G-2A (excitation 510–560 nm; emission: 590 ± 10 nm, Nikon) filter sets were used for epifluorescence microscopy. Nile Red Labelling of Nonreplicating Persistence M. tuberculosis Cultures M. tuberculosis H37Rv and strain CH [21] were grown as agitated, aerated cultures (370 rpm) to mid-log phase in Dubos liquid medium, supplemented with Dubos medium albumin. M. tuberculosis NRP1/2 (nonreplicating persistence stage 1 and 2) cultures were incubated with continuous stirring at 37 °C for 168, 288, and 504 h, respectively, according to Wayne and Hayes [15]. At these time points one tube of each culture was destructively sampled for microscopic analysis. 10 μl of each sample was spread on a slide, heat fixed, and labelled with Nile red as previously described [5]. RNA Extraction from Tuberculous Sputa With the exception of sputum sample 1, which was frozen in liquid nitrogen within 10 min of expectoration, approximately four volumes of GTC solution (5 M guanidinium thiocyanate, 0.5% w/v sodium N-lauryl sarcosine, 25 mM trisodium citrate, 0.1 M 2-mercaptoethanol, 0.5% w/v Tween 80 [pH 7.0]) [22] were added to sputum within 5 min of collection. Mycobacteria were harvested by centrifugation (1398g, 30 min), resuspended in 400 μl of sterile deionized water, and added to 1 ml of Trizol LS (Invitrogen). RNA was extracted using a method modified from that of DesJardin et al. [23] with chloroform replacing chloroform:isoamyl alcohol washes and the Cleanascite step omitted. The mixture was transferred to a glass matrix tube for cell lysis (Lysing matrix B; Q-Biogene) and processed in a spin/rotation instrument for cell lysis (Ribolyser; Hybaid), with a speed setting of 6.5 and a time setting of 45 s. After processing, 200 μl of chloroform was added to the mixture and it was vortex-mixed for 2 min. The aqueous and organic layers were separated by microcentrifugation for 15 min at room temperature at 16,000g. The aqueous phase containing the RNA was washed once with an equal volume of chloroform. The aqueous phase was removed to a fresh tube and 1 μl of glycoblue (Ambion), 0.1 volume of 5 M ammonium acetate, and an equal volume of isopropanol were added. The RNA was precipitated overnight at −20 °C. The resulting RNA pellet was washed once with 70% v/v and once with 95% v/v ethanol, dried and resuspended in 100 μl of RNase-free H2O (Sigma). The cognate M. tuberculosis complex isolate from sputum sample 1 was cultured for 6 d in 100ml 7H9 broth at 37 °C, 200 rpm at which time the absorbance was 0.22 at 580 nm. Mycobacterial RNA was stabilised with GTC solution and extracted as previously described. Total RNA from sputum samples (5, 7, 8, and 9) for amplification was quantified using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies) and Agilent 2100 Bioanalyser (Agilent Technologies). Microarray Analysis of RNA from Sputum 1 RNA from sputum 1 and the in vitro-grown cognate isolate was cleaned using the RNeasy kit (Qiagen). A M. tuberculosis whole genome microarray, generated by the Bacterial Microarray Group at St. George's (University of London) and consisting of 3,924 gene-specific PCR products (designed with minimal cross-homology) to the M. tuberculosis H37Rv [24], was utilised (ArrayExpress accession number A-BUGS-1; http://bugs.sgul.ac.uk/A-BUGS-1). Hybridisations were conducted as previously described [25] with 15 μg of Cy5-labelled cDNA derived from M. tuberculosis RNA against 1 μg Cy3-labelled M. tuberculosis H37Rv genomic DNA. The hybridised slides were scanned sequentially at 532 nm and 635 nm corresponding to Cy3 and Cy5 excitation maxima using the Affymetrix 428 Array Scanner (MWG). Comparative spot intensities from the images were calculated using Imagene 5.5 (BioDiscovery), and imported into GeneSpring GX 7.2 (Agilent Technologies) for further analysis. After local background subtraction the measured intensity in the cDNA channel for each gene was divided by its intensity in the genomic DNA control channel. The array data were normalised to the 50th percentile of all genes detected to be present on the array and filtered to remove unreliable low intensity data (below a value of 500 in either channel).Genes were identified as differentially expressed in sputum with a cut-off of >3-fold relative to in vitro growth. Growth Conditions and RNA Extraction for Microarray Analysis M. tuberculosis H37Rv was grown as agitated, aerated cultures (370 rpm) to mid-log phase at 37 °C in Dubos liquid medium, supplemented with Dubos medium albumin. M. tuberculosis NRP1/2 cultures were set up and cultured in a stirred model for 72 h and 240 h, respectively, according to Wayne and Hayes [15]. Mycobacterial RNA was extracted from in vitro models (collected straight into GTC solution) using the GTC/Trizol method as developed by Mangan et al. [26]; RNA was DNase-treated and purified using RNeasy columns (Qiagen). Total RNA was quantified using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies) and Agilent 2100 Bioanalyser (Agilent Technologies). RNA Amplification An aliquot of 5 ng of total M. tuberculosis RNA was amplified using an Eberwine T7-oligo-dT based system after an initial polyadenylation step (MessageAmp II Bacteria, Ambion). Using this method, bacterial RNA was polyadenylated before priming the first-strand cDNA synthesis reaction with T7-linked oligo-dT. Amplified RNA was generated after second-strand cDNA synthesis and cDNA purification by in vitro run-off transcription (IVT) using T7 polymerase. Single rounds of amplification were performed, with an in vitro transcription reaction of 16 h at 37 °C. This amplification method has been previously demonstrated to be reproducible and capable of identifying representative changes in gene expression [27,28]. The yield and size distribution of amplified products was assessed spectrophotometrically at OD260 and using the Agilent 2100 Bioanalyser (Agilent Technologies). Microarray Analyses of Samples 5, 7, 8, and 9 An M. tuberculosis whole-genome microarray, generated by the Bacterial Microarray Group at St. George's (ArrayExpress accession number A-BUGS-23; http://bugs.sgul.ac.uk/A-BUGS-23), and consisting of 4,410 gene-specific PCR products (designed with minimal cross-homology) to the M. tuberculosis H37Rv [24], CDC1551 [29], and M. bovis AF2122/97 [30] genomes was utilised. Hybridisations were conducted as previously described [25] except for the use of M. tuberculosis genomic DNA as a common reference [31]. Using genomic DNA reduced technical variation between replicate hybridisations and allowed RNA profiles to be used in multiple comparisons. 5 μg of Cy5-labelled cDNA derived from amplified M. tuberculosis RNA was hybridised with 2 μg of Cy3-labelled M. tuberculosis H37Rv genomic DNA. A lower ratio of test cDNA to comparator gDNA was used than with sample 1, as we were able to confirm the purity of our preparations with the Bioanalyser at the same time and perform technical replicates. The M. tuberculosis H37Rv reference DNA was kindly provided by Colorado State University (http://www.cvmbs.colostate.edu/microbiology/tb/top.htm). Two biological replicates of each in vitro growth condition and four sputum samples (5, 7, 8, and 9) were hybridised in triplicate. The microarrays were scanned and spot intensities calculated as described above. The array data were normalised to the 50th percentile of all genes detected to be present on the array. The dataset was filtered to include only cDNA elements flagged to be present on 80% of the arrays. Significantly differentially expressed genes were identified using ANOVA (p 2.5. The significantly differentially expressed genes were hierarchically clustered using Cluster and the results displayed using Treeview software [32]. The hypergeometric distribution was used to determine if functional categories of genes were significantly enriched in the sputum profile [33]. Fully annotated microarray data are deposited in BμG@Sbase and ArrayExpress. Quantitative Real-Time RT-PCR Mycobacterial RNA (0.5 μg) from sputum sample 1 and its cognate isolate and eight further sputum samples (samples 2–9) were reverse transcribed in a total volume of 30 μl using random primers and Superscript II (Invitrogen Technologies) according to manufacturer's instructions. To estimate DNA contamination of samples, all were subjected to a no reverse transcriptase control, which was then subtracted from the RNA result. A no-reverse transcriptase threshold of 10% of the test value was taken, with the exception of four reactions in which values which were 100 assessable bacilli, the frequency of lipid body–positive cells varied from 3% to 86% (mean 45%, standard deviation 20%), and these contained between two and eight lipid bodies per cell (Figure 1). Thus, lipid body–positive tubercle bacilli are readily demonstrable in smear-positive samples from tuberculosis patients in two well-separated geographic locations and are present in a subpopulation of mycobacterial cells. Figure 1 Lipid Bodies in Tuberculous Sputum Samples Auramine/Nile red-fixed sputum smears [5] and aerobic M. tuberculosis growth. Variation in lipid bodies per cell: (A) none, (B) three, (C) five, and (D) eight. Samples are shown with (E) low and (F) high proportions of lipid body–positive cells. (G) Aerobically grown mid-log M. tuberculosis H37Rv contained negligible lipid bodies. Scale bar 2μm. Lipid Bodies Are Readily Observed in M. tuberculosis Cells in Nonreplicating Persistence The discovery and characterisation of tgs1 [6,7] raised the possibility that lipid bodies might be formed in response to the hypoxic growth shift-down conditions that have been described by Wayne and Hayes [15], conditions known to cause up-regulation of the DosR regulon [9,10]. When M. tuberculosis H37Rv, a laboratory strain, and CH, a recent clinical isolate responsible for a large outbreak [21], were exposed in vitro to these conditions, abundant Nile red-staining lipid bodies were observed in both strains; respectively, 29% and 42% in NRP1 (168 h), 50% and 65% in NRP2 (288 h), and 41% and 56% in late NRP2 (504 h). An average of two lipid bodies per cell (range one to five) was observed in all samples except for the H37Rv NRP1 sample in which only one (range one to three) was seen in positive cells. Thus, NRP M. tuberculosis cultures contain lipid bodies at levels comparable to those seen in sputum. Expression Profiling of M. tuberculosis Recovered from Sputum If lipid bodies are a biomarker for cells in an NRP state, then the M. tuberculosis transcripts present in sputum should be compatible with those observed in NRP in vitro studies [9,10]. We therefore compared the transcriptome of M. tuberculosis recovered from human sputum to that obtained from in vitro aerobic cultures and NRP-inducing conditions [15]. Twenty sputa were collected from known microscopy-positive Gambian patients before they started antibiotic treatment, and the samples were rapidly stabilized against RNA degradation. Five samples (designated 1, 5, 7, 8, and 9) were analysed by microarray hybridisation, four with and one without prior polyadenylation/oligo-dT based amplification. Although the results from sputum 1, the single direct (nonamplified) array, are not discussed further, they confirm the essential details of the amplified analyses (Tables S1 and S2). This high-volume sample (∼30 ml) had an exceptionally high bacterial load, and we estimate that >1010 bacilli were present. The data from the four amplified samples were analysed with array hybridisations of amplified RNA extracted from M. tuberculosis H37Rv under different conditions: log-phase aerobic growth, the two stages of NRP (NRP1 t = 72 h; NRP2 t = 240 h) [15], and a mixed preparation containing RNA from aerobic and NRP2 cells mixed in the proportion 70:30 (w/w total RNA). This latter preparation was included because this mixture was representative of the lipid body–positive population in sputum. This preparation therefore enabled us to test the hypothesis that sputum comprises a mixture of the rapidly and aerobically growing bacilli expected at the margins of liquefying caseous lesions [2] with the NRP-like cells indicated by our lipid body studies. Microarray data analysis revealed that, after filtering to remove genes with low signals in either channel, 182 genes were significantly induced in sputum compared to aerobic growth, and 334 genes were significantly repressed (Tables S3 and S4). Figure 2 displays the results of gene cluster analysis of array data from the biological and technical replicates for these genes across the sputa, NRP and mixed aerobic:NRP2 sample sets. Boxes 1 and 2 highlight gene clusters similarly regulated in NRP2 and sputum relative to aerobic growth. We note the large cluster of strongly down-regulated signals in sputum, a feature lost in box 1 in the 70:30 mix, presumably due to the aerobic signals obscuring the NRP2 signals. A similar pattern of differential expression is apparent for the amplified RNA from the four sputum samples, even though they came from separate, untreated patients. Figure 2 Display of Genes Differentially Regulated in Sputum and NRP versus Aerobic Culture Clustering of 648 genes significantly differentially expressed in either sputum, NRP1, NRP2, or a 70:30 mix of aerobic:NRP2 compared to aerobic growth. Biological and technical replicates of conditions are displayed as columns, genes as rows. Red represents the induction of gene expression relative to aerobic growth, green repression. Asterisked columns mark the conditions in which genes were identified as significantly differentially expressed compared to aerobic growth. Boxes 1 and 2 highlight clusters of genes similarly regulated in NRP2 and sputum. The data show that none of our comparator conditions, including the 70:30 aerobically replicating:NRP2 mixture, closely parallel the sputum transcriptome. While significant overlaps between the genes differentially expressed in sputum were revealed by hypergeometric probability values (Tables S5 and S6), no single or obvious combination of defined conditions herein, nor previously reported in vitro or in vivo, correspond to the signature we have obtained from sputum. Amongst the different functional categories of genes, relative to aerobic growth there were significant decreases in expression of genes required for aerobic respiration and ribosomal function and an increase in transcripts associated with cholesterol utilisation (Figure 3) [38]. We note also that genes previously observed to be repressed during bacillary stasis in a chronic murine infection model [37], nuoB, ctaD, qcrC, atpA, and atpD, followed this pattern in our data while narK2 was up-regulated, as was the case in the murine studies. DosR was the most prominently activated regulon in sputum (box 2 in Figure 2; Tables S2 and S5), although the level of activation was lower than in the comparator conditions. Figure 3 Genes Required for Aerobic Respiration and Ribosomal Function Show Decreased Expression in Sputum Compared with Aerobic Growth while Genes Involved in Lipid Metabolism Were Induced Box and whisker plots showing the distribution of expression ratios (log2 scale) of (A) 21 aerobic respiration genes and (B) 45 ribosomal genes in NRP2 and sputum relative to aerobic growth using functional classifications defined by Cole et al., 1998 [24]; also (C) 64 genes that may be involved in cholesterol catabolism [38] and (D) 45 genes in the fadB, echA, fadE, and fadA families, which may be involved in the β-oxidation of fatty acids. *Significant difference (p 100 bacilli were analysable. Our further in vitro studies revealed nonreplicating persistence, as defined by Wayne and colleagues [46], to be a condition in which M. tuberculosis cells are induced to form lipid bodies at frequencies comparable to those observed among tubercle bacilli in sputum. Consistent with this finding, transcriptome analysis of M. tuberculosis in sputum revealed signals compatible with slow or non-growth and absence of aerobic respiration. Moreover, the time to positivity in diagnostic liquid culture was shown to be directly related to sputum lipid body content, adding further weight to the view that lipid body–positive cells are not replicating. While other explanations remain possible, we conclude that the lipid body–positive cells in sputum have a persister-like phenotype, with important implications for the treatment and transmission of tuberculosis. Further studies should elucidate the impact of chemotherapy on the frequency of lipid body–positive populations of M. tuberculosis in patient sputum, and the relationship between this candidate biomarker and both infectivity and the clinical response to treatment. The analysis of tuberculous sputum has played a central role in the diagnosis and management of tuberculosis. While the presence of acid-fast bacilli in sputum is the feature most prominently linked to the potential of a patient to disseminate infection, there are other influential factors. Setting aside those associated with human behaviour and the immediate atmospheric conditions, a transmitted tubercle bacillus must survive transit and master new environmental pressures if it is to establish infection in a new individual. From what we know about bacterial adaptation, it is highly probable that specific traits are expressed to achieve this ability. Furthermore, it is recognised that in the treatment of tuberculosis and other bacterial infections, bacterial burden correlates not only with increased potential for onward transmission, but also with the duration of chemotherapy required for a cure [47]. Bacterial populations often show heterogeneous properties. The presence of a slow or nongrowing subpopulation of bacteria phenotypically resistant to antibiotics has been proposed to account for the extended time required for treatment of tuberculosis [4,48]. Although never directly identified, the presence of such a population is inferred from the biphasic reduction of viable bacterial counts recovered from serial sputum samples collected during therapy [4]. Such antibiotic-tolerant “persister” populations have been recognised in many bacterial infections [49]; a greater bacterial burden being associated with a higher frequency of phenotypic resistance. The results we present here are a first step towards defining the transmission phenotype of M. tuberculosis and also reveal directly, to our knowledge for the first time, a substantial population of persister-like bacilli in sputum prior to commencement of therapy. Lipid bodies must now be recognised as a universal feature of smear-positive tuberculosis, and the significance of this finding and of the variation in the proportion of positive cells between samples must be established. While lipid bodies are a well-established feature of eukaryotic cell biology [50], their recognition in prokaryotes is relatively recent [51]. The link between tgs1, the DosR regulon, the hypoxia-induced NRP state, and lipid bodies that is strengthened and made clinically relevant by our findings, relates these structures to a coherent set of laboratory studies. Lipid body–positive cells must now be factored into the debate about mycobacterial dormancy and persistence. Fourteen functional Tgs enzymes that are not DosR regulated have been identified [7]. However, none of the mRNAs encoding these enzymes was found to be up-regulated in our transcriptional studies, while Tgs1, the most active enzyme, and the DosR regulon itself were. While the microarray results can be analysed in several different contexts, we focus here on the data that have a bearing on the growth state and lipid body content of our samples. The transcriptome clearly shows that the sputum bacillary population is dominated by slowly or nonreplicating bacilli, a contention further supported by two lines of comparative evidence. Firstly, two in vitro transcriptome datasets can be robustly argued to represent nonreplicating cell populations: the nutrient deprivation studies of Betts et al. [52] and our NRP2 results. Of the repressed 33 genes common to both of these datasets, 20 were found to be down-regulated in our sputum samples (hypergeometric p-value 2.56 × 10−11), a feature that is further supported by strong correlations with the recently published reduced growth rate dataset (Figure 5) [53]. Secondly, Shi and colleagues studied specific gene expression in chronic mouse infections [37] under conditions in which there is clear evidence for lack of replication [54]. In common with this study, our results show repression of nuoB, ctaD, qcrC, atpA, and atpD and up-regulation of narK2. This supports the view that our sputum samples contained many nonreplicating bacilli in respiratory state III defined by these authors [37], that is, a shift from oxygen electron transfer to anaerobic electron transfer. If the frequency of lipid body–positive mycobacteria in sputum provides an estimate of the NRP cells present, then other NRP-related features should be correlated. Remarkably, we found this to be the case with time to positivity in routine diagnostic cultures performed on samples that we had analysed for their lipid body content (Figure 6). These results provide direct evidence that the frequency of lipid body–positive cells provide an estimate of the nonreplicating mycobacterial population in sputum in these samples. Drawing all these results together, we now reject the commonly held belief that smear-positive sputum is dominated by aerobically replicating Mycobacterium tuberculosis. The transcriptome data in particular show that such cells could only be a minor component in the samples analysed in this way. In contrast, we conclude that our samples contained nonreplicating mycobacteria at levels proportional to the lipid body-positive cells therein. While the significance of this finding to clinical tuberculosis will only be established by long-term studies, several important implications can be recognised at this stage. First, it is clear that the large numbers of tubercle bacilli observed in sputum are not a direct sample from extensive and rapid aerobic growth at the margins of open cavities. Rather, we propose that, as with all growth in restricted environments, this aerobic growth results in the buildup of larger and larger numbers of stationary phase nonreplicating bacilli and that this accords with the mature “colony-like” growth of tubercle bacilli reported in caseous lesions by Canetti [2]. Second, the cidal action of many antibiotics is proportional to the growth rate of bacteria, with those growing slowly or in a nonreplicating state showing phenotypic tolerance [49,55,56]. In particular, Wayne type M. tuberculosis NRP cultures are tolerant to isoniazid and rifampin [15]. Interestingly, while such cultures provide the closest available transcriptome match to the signals we have obtained from sputum, our own studies on phenotypic resistance have so far consistently shown that the presence of M. tuberculosis lipid bodies accumulated following growth-arresting stimuli, is correlated with tolerance to the cidal action of these antibiotics (see Figure S2). Such phenotypic resistance is widely believed to underpin the persister phenomenon in tuberculosis, in which a residual and antibiotic-recalcitrant population requires extended chemotherapy for its elimination [4]. We emphasise that hypoxia is not the only stress capable of inducing lipid body formation. This is exemplified by our preliminary nitric oxide data (Figure S2). This latter effect is probably mediated via DosR [57]. The relationship between DosR induction and growth rate is clearly multifactorial, with the up-regulation of DosR perhaps a general indicator of mycobacterial stress, for example the DosR regulon is induced during the exponential phase of growth in mice [31]. It is the slow/nongrowth transcriptional profile and our time-to-positivity results that indicate the presence of a nonreplicating population in sputum rather than dosR expression, which is found in both growing and nongrowing populations. However, it should be noted that bacterial populations are evidently nonuniform. Thus lipid body–positive cells may also represent a slower or nonreplicating population within a growing culture. We cannot say whether the expectorated persister-like population we report here reflects the persister population revealed during chemotherapy. Initial establishment of persisters in growing populations is probably random and at a low level; however, during infection these populations will be influenced by specific conditions, including the development of colonial/biofilm-like growth [2] and inflammatory responses that may increase the numbers of persister-like cells observed [49,57]. We note that all the sputum samples we examined were collected prior to the commencement of chemotherapy; the status of bacilli within patients treated with antibiotics is not clear. Nonetheless, this question is amenable to further study through the analysis of the responses of patients to therapy and serial analyses of the lipid body content of their sputum samples. Finally, returning to the proposal that the bacilli in sputum display traits that underpin the transmission of tuberculosis, the relative resistance of nonreplicating bacteria, including M. tuberculosis, to a variety of stresses is well established [59–61]. Global stress resistance will promote survival that is essential for transmission. More specifically, we note that formation of lipid bodies in Rhodococcus, another actinomoycete, has been linked to improved survival during desiccation [62]. Even more intriguing is the observation that hypoxically grown M. tuberculosis cultures, in which we have demonstrated ∼34% lipid body–positive cells (unpublished data), are 10-fold more infectious for guinea pigs by the aerosol route than their aerobically grown counterparts [63]. Moreover, recent investigation of Beijing strains of M. tuberculosis revealed that they accumulate more triacylglycerol and express tgs1 at levels 10-fold higher than laboratory strains, during aerobic log-phase growth [64]. The enhanced transmissibility, evidenced by the rapid global spread of these strains, may reflect a greater propensity for lipid body formation in vivo. We propose that lipid body positive (fat) acid fast bacilli are a biomarker for nonreplicating (lazy) M. tuberculosis cells in sputum; their further study offers exciting and tractable avenues for research into the treatment and prevention of tuberculosis. Supporting Information Figure S1 Overexpression of tgs1 in M. smegmatis Leads to Enhanced Accumulation of Triacylglycerol and Lipid Bodies We cloned tgs1 under the control of the acetamide-inducible promoter pSD26 [65] and expressed it in Mycobacterium smegmatis, which readily forms tiacylglycerol (TAG) lipid bodies in vitro. Test and control cells were exposed to radiolabelled oleic acid for 10 min and incorporation into TAG determined [66]. (A) Triacylglycerol synthase (TGS) activity of induced MspSD26-tgs1 and MspSD26 vector control, p 3-Fold Up-regulated in Sputum 1 Compared to In Vitro Cognate Isolate Growth by Direct Microarray (85 KB DOC) Click here for additional data file. Table S3 Genes Determined to be Greater than 2.5-Fold Up-regulated in Sputum Compared with In Vitro Growth by Amplified Microarray (403 KB DOC) Click here for additional data file. Table S4 Genes Determined to be Greater than 2.5-fold Down-regulated in Sputum Compared with In Vitro Growth by Amplified Microarray (664 KB DOC) Click here for additional data file. Table S5 Hypergeometric Probability Analysis of Genes Determined to be >2.5-Fold Up-regulated in Sputum Compared to In Vitro H37Rv Growth by Amplified Microarray (104 KB DOC) Click here for additional data file. Table S6 Hypergeometric Probability Analysis of Genes Determined to be >2.5-Fold Down-regulated in Sputum Compared to In Vitro H37Rv Growth by Amplified Microarray (86 KB DOC) Click here for additional data file. Accession Numbers The fully annotated microarray data from this study are deposited in BμG@Sbase (accession number: E-BUGS-52; http://bugs.sgul.ac.uk/E-BUGS-52) and ArrayExpress (accession number: E-BUGS-52; http://www.ebi.ac.uk/arrayexpress/experiments/E-BUGS-52).
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                Author and article information

                Journal
                0413675
                4830
                J Infect Dis
                J. Infect. Dis.
                The Journal of infectious diseases
                0022-1899
                1537-6613
                26 January 2018
                03 November 2017
                03 November 2018
                : 216
                : Suppl 6
                : S662-S668
                Affiliations
                [1 ]School of Medicine and Rollins School of Public Health, Emory University, Atlanta, Georgia
                [2 ]Division of Global HIV and Tuberculosis, Centers for Disease Control and Prevention, Atlanta, Georgia
                [3 ]Bill and Melinda Gates Foundation, Seattle, Washington
                [4 ]Bloomberg School of Public Health, Johns Hopkins University, Baltimore
                [5 ]Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland
                [6 ]Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, New York
                [7 ]Aurum Institute and the School of Public Health, University of Witwatersrand, Johannesburg, South Africa
                [8 ]Advancing Care for tuberculosis and HIV, South African Medical Research Council, Johannesburg, South Africa
                Author notes
                Correspondence: S. C. Auld, MD, MSc, Rollins School of Public Health, Emory University, 1518 Clifton Rd NE, Claudia Nance Rollins Bldg, Rm 3002, Atlanta, GA 30322 ( sauld@ 123456emory.edu )
                Article
                HHSPA937271
                10.1093/infdis/jix353
                5793854
                29112744
                d58c5d18-07f8-49bc-a73f-c1d4b3677184

                This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 IGO (CC BY 3.0 IGO) License ( https://creativecommons.org/licenses/by/3.0/igo/) which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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                Infectious disease & Microbiology
                tuberculosis,transmission,public health
                Infectious disease & Microbiology
                tuberculosis, transmission, public health

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