Predicting tuberculosis drug resistance with machine learning-assisted Raman spectroscopy

Tuberculosis (TB) is the world's deadliest infectious disease, with 1.5 million annual deaths and half a million annual infections. Rapid TB diagnosis and antibiotic susceptibility testing (AST) are critical to improve patient treatment and to reduce the rise of new drug resistance. Here, we develop a rapid, label-free approach to identify Mycobacterium tuberculosis (Mtb) strains and antibiotic-resistant mutants. We collect over 20,000 single-cell Raman spectra from isogenic mycobacterial strains each resistant to one of the four mainstay anti-TB drugs (isoniazid, rifampicin, moxifloxacin and amikacin) and train a machine-learning model on these spectra. On dried TB samples, we achieve > 98% classification accuracy of the antibiotic resistance profile, without the need for antibiotic co-incubation; in dried patient sputum, we achieve average classification accuracies of ~ 79%. We also develop a low-cost, portable Raman microscope suitable for field-deployment of this method in TB-endemic regions

RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities

With rising rates of drug-resistant infections, there is a need for diagnostic methods that rapidly can detect the presence of pathogens and reveal their susceptibility to antibiotics. Here we propose an approach to diagnosing the presence and drug-susceptibility of infectious diseases based on direct detection of RNA from clinical samples. We demonstrate that species-specific RNA signatures can be used to identify a broad spectrum of infectious agents, including bacteria, viruses, yeast, and parasites. Moreover, we show that the behavior of a small set of bacterial transcripts after a brief antibiotic pulse can rapidly differentiate drug-susceptible and -resistant organisms and that these measurements can be made directly from clinical materials. Thus, transcriptional signatures could form the basis of a uniform diagnostic platform applicable across a broad range of infectious agents

Regulation of Tumor Necrosis Factor Alpha Gene Expression by Mycobacteria Involves the Assembly of a Unique Enhanceosome Dependent on the Coactivator Proteins CBP/p300

Tumor necrosis factor alpha (TNF-α) plays an important role in host containment of infection by Mycobacterium tuberculosis, one of the leading causes of death by an infectious agent globally. Using the pathogenic M. tuberculosis strain H37Rv, we present evidence that upon stimulation of monocytic cells by M. tuberculosis a unique TNF-α enhanceosome is formed, and it is distinct from the TNF-α enhanceosome that forms in T cells stimulated by antigen engagement or virus infection. A distinct set of activators including ATF-2, c-jun, Ets, Sp1, Egr-1 and the coactivator proteins CBP/p300 are recruited to the TNF-α promoter after stimulation with M. tuberculosis. Furthermore, the formation of this enhanceosome is dependent on inducer-specific helical phasing relationships between transcription factor binding sites. We also show that the transcriptional activity of CBP/p300 is potentiated by mycobacterial stimulation of monocytes. The identification of TNF-α regulatory elements and coactivators involved in M. tuberculosis-stimulated gene expression thus provides potential selective molecular targets in the modulation of TNF-α gene expression in the setting of mycobacterial infection

Benzoic Acid-Inducible Gene Expression in Mycobacteria

<div><p>Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the <i>Pseudomonas putida</i>-derived positively regulated XylS/<i>Pm</i> expression system to control inducible gene expression in <i>Mycobacterium smegmatis</i> and <i>Mycobacterium tuberculosis</i>, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/<i>Pm-</i>regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer <i>m</i>-toluate, we achieve a robust, time- and dose-dependent reversible induction of <i>Pm</i>-mediated expression in mycobacteria, with low background expression levels. XylS/<i>Pm</i> is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.</p></div

<i>Pm</i>–mediated basal expression is low and compares favorably to <i>Ptet-</i>mediated basal expression in <i>Mtb</i>.

<p>(A-B) <i>Mtb</i> transformed with pMDX-luc or pUV15tetORm::luciferase was diluted to OD₆₀₀ 0.005 in the presence or absence of m-toluate (1.5 mM) or atc (200 ng/ml), respectively. Samples were grown in triplicates and monitored for 7 days registering OD₆₀₀ at 2, 4 and 7 days. Basal expression from <i>Pm</i> and <i>Ptet</i> is presented by level of luciferase produced by <i>Mtb</i> pMDX-luc and <i>Mtb</i> pUV15tetORm::luciferase. (A) shows the luciferase expression over time in induced sample and (B) shows the basal expression from uninduced samples over time. The results represent two independent experiments.</p

<i>Pm</i>–mediated basal expression is low and compares favorably to <i>Ptet</i>-mediated basal expression in <i>Msmeg</i>.

<p>(A-B) <i>Msmeg</i> transformed with pMDX-zeo or pTET-zeo was diluted to OD₆₀₀ 0.005 in the presence or absence of m-toluate (1.5 mM) or atc (200 ng/ml), respectively, and increasing amounts of zeocin (0, 0.5, 2.5, 5, 7.5, 10, 15, 25, 50, 100, 150, 200, 250 or 500 μg/ml). Samples were grown in triplicates and monitored for 120 hours by a Bioscreen, registering OD₆₀₀ every other hour. Basal expression from <i>Pm</i> and <i>Ptet</i> is presented by growth of <i>Msmeg</i> pMDX-zeo and <i>Msmeg</i> pTET-zeo in increasing concentrations of zeocin, when the respective sample grown in the <i>absence</i> of zeocin reached mid log phase (A) or stationary phase (B). (C) Induced and uninduced samples of pMDX-zeo strain in late log phase. (D) Induced and uninduced samples of pTET-zeo strain in mid log phase. The results represent two independent experiments.</p

Benzoic acid-inducible expression system, XylS/<i>Pm</i>, for regulation of genes in mycobacteria.

<p>The inducible <i>Pm</i> promoter and its activator XylS regulate the expression of “your favorite gene” (YFG). XylS is constitutively expressed under control of <i>Ptet</i> in the absence of anhydro-tetracycline (atc) and binds the <i>Pm</i> promoter in the presence of the inducer <i>m</i>-toluate. This facilitates expression of YFG and leaves the expression system ON (upper panel). In the absence of <i>m</i>-toluate, XylS is not activated, leaving the expression system OFF, as expression from <i>Pm</i> is not induced (middle panel). Reverse TetR is constitutively expressed by <i>Psmyc</i>, and binds the operator in <i>Ptet</i> in the presence of atc blocking transcription of <i>xylS</i>. Addition of atc leaves the system in a more fully OFF mode as potential basal <i>Pm</i>-mediated transcription caused by excessive levels of XylS is abolished (lower panel).</p

Regulation of the zeocin resistance gene in the presence of zeocin.

<p>(A) <i>Msmeg</i> transformed with pMDX-zeo was grown to OD₆₀₀ 0.05–0.1 before addition of 1.5 mM <i>m</i>-toluate (induced) or ethanol carrier (uninduced) was added. Cells were then incubated for 5 hours at 30°C. The cells were normalized by OD₆₀₀, serially diluted, and spotted on plates containing increasing amounts of zeocin and 1.5 mM <i>m</i>-toluate (induced) or ethanol (uninduced) and incubated at 30°C for 2 days. (B) <i>Msmeg</i> transformed with pMDX-zeo was pre-induced for 5 hours as described above, then diluted to OD₆₀₀ 0.005 and grown in triplicates in micro-plate wells in the presence of increasing concentrations of zeocin (0, 0.5, 2.5, 5, 7.5, 10, 15, 25, 50, 100, 150, 200 and 250 μg/ml) and 1.5 mM <i>m</i>-toluate (induced) or ethanol carrier (uninduced) shaking at 37°C. Growth was monitored by Bioscreen, registering OD₆₀₀ every other hour. The samples are presented by the OD₆₀₀ of uninduced or induced <i>Msmeg</i> pMDX-zeo in increasing concentrations of zeocin, when the respective sample grown in the <i>absence</i> of zeocin reached mid log phase. Error bars represent standard deviations and the results represent three independent experiments.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p