Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia.

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as Stenotrophomonas maltophilia. Using a Tn7-based genomic integration system, we report the construction of improved mini-Tn7 delivery plasmids for labeling of S. maltophilia with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different S. maltophilia wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in Galleria mellonella. It is also shown that the mini-Tn7 elements remained stably integrated in the genome of S. maltophilia over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn7 delivery plasmids are valuable tools for generating fluorescently labeled S. maltophilia strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium S. maltophilia is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of S. maltophilia infections worldwide. One important virulence-related trait of S. maltophilia is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn7-based labeling system for S. maltophilia to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions.

Deficiency of the Intramembrane Protease SPPL2a Alters Antimycobacterial Cytokine Responses of Dendritic Cells.

Signal peptide peptidase-like 2a (SPPL2a) is an aspartyl intramembrane protease essential for degradation of the invariant chain CD74. In humans, absence of SPPL2a leads to Mendelian susceptibility to mycobacterial disease, which is attributed to a loss of the dendritic cell (DC) subset conventional DC2. In this study, we confirm depletion of conventional DC2 in lymphatic tissues of SPPL2a-/- mice and demonstrate dependence on CD74 using SPPL2a-/- CD74-/- mice. Upon contact with mycobacteria, SPPL2a-/- bone marrow-derived DCs show enhanced secretion of IL-1ß, whereas production of IL-10 and IFN-ß is reduced. These effects correlated with modulated responses upon selective stimulation of the pattern recognition receptors TLR4 and Dectin-1. In SPPL2a-/- bone marrow-derived DCs, Dectin-1 is redistributed to endosomal compartments. Thus, SPPL2a deficiency alters pattern recognition receptor pathways in a CD74-dependent way, shifting the balance from anti- to proinflammatory cytokines in antimycobacterial responses. We propose that in addition to the DC reduction, this altered DC functionality contributes to Mendelian susceptibility to mycobacterial disease upon SPPL2a deficiency.

Evaluation of Myeloperoxidase as Target for Host-Directed Therapy in Tuberculosis In Vivo.

Due to the rise of tuberculosis cases infected with multi and extensively drug-resistant Mycobacterium tuberculosis strains and the emergence of isolates resistant to antibiotics newly in clinical use, host-directed therapies targeting pathogenesis-associated immune pathways adjunct to antibiotics may ameliorate disease and bacterial clearance. Active tuberculosis is characterized by neutrophil-mediated lung pathology and tissue destruction. Previously, we showed that preventing M. tuberculosis induced necrosis in human neutrophils by inhibition of myeloperoxidase (MPO) promoted default apoptosis and subsequent control of mycobacteria by macrophages taking up the mycobacteria-infected neutrophils. To translate our findings in an in vivo model, we tested the MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH) in C3HeB/FeJ mice, which are highly susceptible to M. tuberculosis infection manifesting in neutrophil-associated necrotic granulomas. MPO inhibition alone or as co-treatment with isoniazid, a first-line antibiotic in tuberculosis treatment, did not result in reduced bacterial burden, improved pathology, or altered infiltrating immune cell compositions. MPO inhibition failed to prevent M. tuberculosis induced neutrophil necrosis in C3Heb/FeJ mice in vivo as well as in murine neutrophils in vitro. In contrast to human neutrophils, murine neutrophils do not respond to M. tuberculosis infection in an MPO-dependent manner. Thus, the murine C3HeB/FeJ model does not fully resemble the pathomechanisms in active human tuberculosis. Consequently, murine infection models of tuberculosis are not necessarily adequate to evaluate host-directed therapies targeting neutrophils in vivo.

Mycobacterium tuberculosis Acetyltransferase Suppresses Oxidative Stress by Inducing Peroxisome Formation in Macrophages.

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal ß-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11ß, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy, Synergy, Resistance and Mechanistic Studies.

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.

Mycobacterium tuberculosis Complex Lineage 3 as Causative Agent of Pulmonary Tuberculosis, Eastern Sudan1.

Pathogen-based factors associated with tuberculosis (TB) in eastern Sudan are not well defined. We investigated genetic diversity, drug resistance, and possible transmission clusters of Mycobacterium tuberculosis complex (MTBC) strains by using a genomic epidemiology approach. We collected 383 sputum specimens at 3 hospitals in 2014 and 2016 from patients with symptoms suggestive of TB; of these, 171 grew MTBC strains. Whole-genome sequencing could be performed on 166 MTBC strains; phylogenetic classification revealed that most (73.4%; n = 122) belonged to lineage 3 (L3). Genome-based cluster analysis showed that 76 strains (45.9%) were grouped into 29 molecular clusters, comprising 2-8 strains/patients. Of the strains investigated, 9.0% (15/166) were multidrug resistant (MDR); 10 MDR MTBC strains were linked to 1 large MDR transmission network. Our findings indicate that L3 strains are the main causative agent of TB in eastern Sudan; MDR TB is caused mainly by transmission of MDR L3 strains.

Phenotypic and Transcriptomic Analyses of Seven Clinical Stenotrophomonas maltophilia Isolates Identify a Small Set of Shared and Commonly Regulated Genes Involved in the Biofilm Lifestyle.

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces S. maltophilia lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings.IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by S. maltophilia, particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of S. maltophilia infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms.

TB sequel: incidence, pathogenesis and risk factors of long-term medical and social sequelae of pulmonary TB - a study protocol.

BACKGROUND: Up to fifty percent of microbiologically cured tuberculosis (TB) patients may be left with permanent, moderate or severe pulmonary function impairment. Very few studies have systematically examined pulmonary outcomes in patients to understand the pathophysiologic basis and long-term socio-economic consequences of this injury. The planned multi-country, multi-centre observational TB cohort study, aims to advance the understanding of the clinical, microbiological, immunological and socio-economic risk factors affecting long-term outcome of pulmonary TB. It will also determine the occurrence of reversible and irreversible socio-economic consequences to patients, their households and the health sector related to pulmonary TB disease and its treatment. METHODS: We will enrol up to 1.600 patients with drug sensitive and multidrug-resistant pulmonary TB who are treated according to the local standard of care by the respective National TB Program. Recruitment is taking place at the time of TB diagnosis at four African study clinics located in The Gambia, Mozambique, South Africa and Tanzania. The primary outcome is the proportion of TB patients with severe lung impairment measured by spirometry at 24 months after TB treatment initiation. Biological samples, including sputum, urine and blood, for studying host- and pathogenic risk factors will be collected longitudinally and examined in a nested case-control fashion. A standardized quality of life questionnaire will be used together with a novel version of WHO's generic patient cost instrument which has been adapted for the longitudinal study design. DISCUSSION: This study is an integral part of an overall strategy to fill a knowledge gap needed to improve TB treatment outcomes globally. The main scientific goal is to identify the major pathogenic mechanisms associated with poor TB treatment outcomes, so that such pathways can be interrupted to avert long term TB sequelae. National as well as supra-national stakeholders and decision makers have been integrated early in the study planning process to inform future treatment guidelines and national health policies. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03251196 , August 16, 2017.

Macrophage defense mechanisms against intracellular bacteria.

Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics.

Targeting neutrophils for host-directed therapy to treat tuberculosis.

M. tuberculosis is one of the prime killers from infectious diseases worldwide. Infections with multidrug-resistant variants counting for almost half a million new cases per year are steadily on the rise. Tuberculosis caused by extensively drug-resistant variants that are even resistant against newly developed or last resort antibiotics have to be considered untreaTable Susceptible tuberculosis already requires a six-months combinational therapy which requires further prolongation to treat drug-resistant infections. Such long treatment schedules are often accompanied by serious adverse effects causing patients to stop therapy. To tackle the global tuberculosis emergency, novel approaches for treatment need to be urgently explored. Host-directed therapies that target components of the defense system represent such a novel approach. In this review, we put a spotlight on neutrophils and neutrophil-associated effectors as promising targets for adjunct host-directed therapies to improve antibiotic efficacy and reduce both, treatment time and long-term pathological sequelae.

Analysis of Staphylococcus aureus proteins secreted inside infected human epithelial cells.

Staphylococcus aureus, an opportunistic pathogen is able to invade into and persist inside non-professional phagocytic cells. To do so, this bacterium possesses a wide range of secreted virulence factors which enable attachment to the host as well as intracellular survival. Hence, a monitoring of virulence factors specifically produced upon internalization might reveal targets for prevention or therapy of S. aureus infections. However, previous proteome approaches enriching S. aureus from lysed host cells after infection did not cover secreted virulence factors. Therefore, we used density gradient centrifugation and mass spectrometry to identify S. aureus HG001 proteins which were secreted into compartments of infected human bronchial epithelial S9 cells. Because shotgun mass spectrometry revealed only few bacterial proteins amongst 1905 host proteins, we used highly sensitive and selective single reaction monitoring mass spectrometry as an alternative approach and quantified 37 bacterial proteins within the S. aureus containing host cell compartment 2.5 h and 6.5 h post infection. Among them were secreted bacterial virulence factors like lipases, pore forming toxins, and secreted adhesins which are usually hard to detect from infected sample material by proteomics approaches due to their low abundance. S. aureus adapted its proteome to improve its response to oxidative and cell wall stress occurring inside the host, but also, increased the amounts of some adhesins and pore-forming toxins, required for attachment and host cell lysis.

Lysosomal phospholipase A2: a novel player in host immunity to Mycobacterium tuberculosis.

Phospholipases catalyze the cleavage of membrane phospholipids into smaller bioactive molecules. The lysosomal phospholipase A2 (LPLA2 ) is specifically expressed in macrophages. LPLA2 gene deletion in mice causes lysosomal phospholipid accumulation in tissue macrophages leading to phospholipidosis. This phenotype becomes most prominent in alveolar macrophages where LPLA2 contributes to surfactant phospholipid degradation. High expression of LPLA2 in alveolar macrophages prompted us to investigate its role in host immunity against the respiratory pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis. Here we report that adaptive immune responses to M. tuberculosis were impaired in LPLA2 deficient mice. Upon aerosol infection with M. tuberculosis, LPLA2 deficient mice showed enhanced mycobacterial counts but less lung immunopathology and pulmonary inflammatory responses. Compromised T-cell priming in the lymph nodes was associated with impaired pulmonary T-cell recruitment and activation. Together with reduced Th1 type cytokine production, these results indicate that LPLA2 is indispensable for the induction of adaptive T-cell immunity to M. tuberculosis. Taken together, we identified an unexpected and novel function of a lysosomal phospholipid-degrading enzyme.

A novel method for non-transferrin-bound iron quantification by chelatable fluorescent beads based on flow cytometry.

The reliable measurement of non-transferrin-bound iron (NTBI) in serum has proved to be difficult and generally time consuming. We have sought a simple and fast method for such a determination. We adopted a fluorescence assay and designed a fluorescent dye with a chelating agent attached to sense iron. To avoid autofluorescence from serum samples, the iron probes were linked to beads and the autofluorescence could be separated and excluded from the measurement by flow cytometry due to the size difference between beads and serum proteins. Fluorescent beads containing both fluorescent and chelating moieties have been synthesized. The nature of the chelating function has been systematically investigated using four different chelators: bidentate hydroxypyranone, bidentate hydroxypyridinone, hexadentate hydroxypyranone and hexadentate hydroxypyridinone, each with different iron affinity constants. Competition studies demonstrate that the hexadentate hydroxypyridinone-based beads are capable of scavenging most of low molecular mass and albumin-bound iron but negligible amounts of iron from transferrin and ferritin. Serum samples from 30 patients with different types of disease and normal volunteers were measured. The concentrations of NTBI fall in the range -0.41 to +6.5 µM. The data have been compared with those obtained from the traditional 'NTA' method.

Isoniazid@Fe2 O3 Nanocontainers and Their Antibacterial Effect on Tuberculosis Mycobacteria.

Isoniazid-filled Fe2 O3 hollow nanospheres (INH@Fe2 O3 , diameter <30 nm, 48 wt % INH-load) are prepared for the first time and suggested for tuberculosis therapy. After dextran-functionalization, the INH@Fe2 O3 @DEX nanocontainers show strong activity against Mycobacterium tuberculosis (M.tb.) and M.tb.-infected macrophages. The nanocontainers can be considered as "Trojan horses" and show efficient, active uptake into both M.tb.-infected macrophages and even into mycobacterial cells.

Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for ß-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.

L-Thyroxine and L-thyroxine-based antimicrobials against Streptococcus pneumoniae and other Gram-positive bacteria.

Objectives: The rise of antibiotic-resistant Streptococcus pneumoniae (Sp) poses a significant global health threat, urging the quest for novel antimicrobial solutions. We have discovered that the human hormone l-thyroxine has antibacterial properties. In order to explore its drugability we perform here the characterization of a series of l-thyroxine analogues and describe the structural determinants influencing their antibacterial efficacy. Method: We performed a high-throughput screening of a library of compounds approved for use in humans, complemented with ITC assays on purified Sp-flavodoxin, to pinpoint molecules binding to this protein. Antimicrobial in vitro susceptibility assays of the hit compound (l-thyroxine) as well as of 13 l-thyroxine analogues were done against a panel of Gram-positive and Gram-negative bacteria. Toxicity of compounds on HepG2 cells was also assessed. A combined structure-activity and computational docking analysis was carried out to uncover functional groups crucial for the antimicrobial potency of these compounds. Results: Human l-thyroxine binds to Sp-flavodoxin, forming a 1:1 complex of low micromolar Kd. While l-thyroxine specifically inhibited Sp growth, some derivatives displayed activity against other Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, while remaining inactive against Gram-negative pathogens. Neither l-thyroxine nor some selected derivatives exhibited toxicity to HepG2 cells. Conclusions: l-thyroxine derivatives targeting bacterial flavodoxins represent a new and promising class of antimicrobials.

The Mla system and its role in maintaining outer membrane barrier function in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.

Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase.

OBJECTIVES: In vivo experimentation is costly and time-consuming, and presents a major bottleneck in anti-tuberculosis drug development. Conventional methods rely on the enumeration of bacterial colonies, and it can take up to 4 weeks for Mycobacterium tuberculosis to grow on agar plates. Light produced by recombinant bacteria expressing luciferase enzymes can be used as a marker of bacterial load, and disease progression can be easily followed non-invasively in live animals by using the appropriate imaging equipment. The objective of this work was to develop a bioluminescence-based mouse model of tuberculosis to assess antibiotic efficacy against M. tuberculosis in vivo. METHODS: We used an M. tuberculosis strain carrying a red-shifted derivative of the firefly luciferase gene (FFlucRT) to infect mice, and monitored disease progression in living animals by bioluminescence imaging before and after treatment with the frontline anti-tuberculosis drug isoniazid. The resulting images were analysed and the bioluminescence was correlated with bacterial counts. RESULTS: Using bioluminescence imaging we detected as few as 1.7 × 10(3) and 7.5 × 10(4) reporter bacteria ex vivo and in vivo, respectively, in the lungs of mice. A good correlation was found between bioluminescence and bacterial load in both cases. Furthermore, a marked reduction in luminescence was observed in living mice given isoniazid treatment. CONCLUSIONS: We have shown that an improved bioluminescent strain of M. tuberculosis can be visualized by non-invasive imaging in live mice during an acute, progressive infection and that this technique can be used to rapidly visualize and quantify the effect of antibiotic treatment. We believe that the model presented here will be of great benefit in early drug discovery as an easy and rapid way to identify active compounds in vivo.