scholarly journals Monitoring Tuberculosis Drug Activity in Live Animals by Using Near-Infrared Fluorescence Imaging

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Raphael Sommer ◽  
Stewart T. Cole
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Near Infrared ◽  
Imaging System ◽  
Limit Of Detection ◽  
Bacterial Load ◽  
Genetically Engineered ◽  
Fluorescent Imaging ◽  
Effective Vaccine ◽  
Content Type ◽  
Immunodeficient Mice

ABSTRACT Worldwide, tuberculosis (TB) is the leading cause of death due to infection with a single pathogenic agent, Mycobacterium tuberculosis. In the absence of an effective vaccine, new, more powerful antibiotics are required to halt the growing spread of multidrug-resistant strains and to shorten the duration of TB treatment. However, assessing drug efficacy at the preclinical stage remains a long and fastidious procedure that delays the progression of drugs down the pipeline and towards the clinic. In this investigation, we report the construction, optimization, and characterization of genetically engineered near-infrared (NIR) fluorescent reporter strains of the pathogens Mycobacterium marinum and Mycobacterium tuberculosis that enable the direct visualization of bacteria in infected zebrafish and mice, respectively. Fluorescence could be measured precisely in infected immunodeficient mice, while its intensity appeared to be below the limit of detection in immunocompetent mice, probably because of the lower bacterial load obtained in these animals. Furthermore, we show that the fluorescence level accurately reflects the bacterial load, as determined by CFU enumeration, thus enabling the efficacy of antibiotic treatment to be assessed in live animals in real time. The NIR fluorescent imaging system disclosed here is a valuable resource for TB research and can serve to accelerate drug development.

scholarly journals Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

2015 ◽  
Vol 22 (9) ◽  
pp. 1060-1069 ◽  
Author(s):  
Mariateresa Coppola ◽  
Susan J. F. van den Eeden ◽  
Louis Wilson ◽  
Kees L. M. C. Franken ◽  
Tom H. M. Ottenhoff ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Protective Efficacy ◽  
Bacterial Load ◽  
Effective Vaccine ◽  
Protective Capacity ◽  
Content Type ◽  
Ifn Γ ◽  
Vaccine Approach

ABSTRACTResponsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions.Mycobacterium bovisBCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity againstMycobacterium tuberculosislatency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a majorM. tuberculosislatency antigen which is highly expressed by “dormant”M. tuberculosisand well recognized by T cells from latentlyM. tuberculosis-infected individuals. In order to assess itsin vivoimmunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ+/TNF+) and IFN-γ+CD4+T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs ofM. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosischallenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential ofM. tuberculosislatency antigens to improve BCG efficacy. These data suggest a promising role forM. tuberculosislatency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.

scholarly journals Efficient 5-OP-RU-Induced Enrichment of Mucosa-Associated Invariant T Cells in the Murine Lung Does Not Enhance Control of Aerosol Mycobacterium tuberculosis Infection

2020 ◽  
Vol 89 (1) ◽  
pp. e00524-20 ◽  
Author(s):  
Charles Kyriakos Vorkas ◽  
Olivier Levy ◽  
Miroslav Skular ◽  
Kelin Li ◽  
Jeffrey Aubé ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Infections ◽  
Cell Activation ◽  
Bacterial Load ◽  
Cell Subset ◽  
Mycobacterium Tuberculosis Infection ◽  
Class I Mhc ◽  
Content Type ◽  
Mait Cells ◽  
Mait Cell

ABSTRACTMucosa-associated invariant T (MAIT) cells are an innate-like T cell subset in mammals that recognize microbial vitamin B metabolites presented by the evolutionarily conserved major histocompatibility complex class I (MHC I)-related molecule, MR1. Emerging data suggest that MAIT cells may be an attractive target for vaccine-induced protection against bacterial infections because of their rapid cytotoxic responses at mucosal services to a widely conserved bacterial ligand. In this study, we tested whether a MAIT cell priming strategy could protect against aerosol Mycobacterium tuberculosis infection in mice. Intranasal costimulation with the lipopeptide Toll-like receptor (TLR)2/6 agonist, Pam2Cys (P2C), and the synthetic MR1 ligand, 5-OP-RU, resulted in robust expansion of MAIT cells in the lung. Although MAIT cell priming significantly enhanced MAIT cell activation and expansion early after M. tuberculosis challenge, these MAIT cells did not restrict M. tuberculosis bacterial load. MAIT cells were depleted by the onset of the adaptive immune response, with decreased detection of granzyme B+ and gamma interferon (IFN-γ)+ MAIT cells relative to that in uninfected P2C/5-OP-RU-treated mice. Decreasing the infectious inoculum, varying the time between priming and aerosol infection, and testing MAIT cell priming in nitric oxide synthase 2 (NOS2)-deficient mice all failed to reveal an effect of P2C/5-OP-RU-induced MAIT cells on M. tuberculosis control. We conclude that intranasal MAIT cell priming in mice induces early MAIT cell activation and expansion after M. tuberculosis exposure, without attenuating M. tuberculosis growth, suggesting that MAIT cell enrichment in the lung is not sufficient to control M. tuberculosis infection.

scholarly journals Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis

2015 ◽  
Vol 59 (8) ◽  
pp. 4457-4463 ◽  
Author(s):  
Benoit Lechartier ◽  
Stewart T. Cole
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Load ◽  
Multidrug Resistant ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Drug Resistant Strains ◽  
Transfer Chain

ABSTRACTClofazimine (CZM) is an antileprosy drug that was recently repurposed for treatment of multidrug-resistant tuberculosis. InMycobacterium tuberculosis, CZM appears to act as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), to release reactive oxygen species upon reoxidation by O2. CZM presumably competes with menaquinone (MK-4), a key cofactor in the mycobacterial electron transfer chain, for its reduction by NDH-2. We studied the effect of MK-4 supplementation on the activity of CZM againstM. tuberculosisand found direct competition between CZM and MK-4 for the cidal effect of CZM, against nonreplicating and actively growing bacteria, as MK-4 supplementation blocked the drug's activity against nonreplicating bacteria. We demonstrated that CZM, like bedaquiline, is synergisticin vitrowith benzothiazinones such as 2-piperazino-benzothiazinone 169 (PBTZ169), and this synergy also occurs against nonreplicating bacteria. The synergy between CZM and PBTZ169 was lost in an MK-4-rich medium, indicating that MK-4 is the probable link between their activities. The efficacy of the dual combination of CZM and PBTZ169 was testedin vivo, where a great reduction in bacterial load was obtained in a murine model of chronic tuberculosis. Taken together, these data confirm the potential of CZM in association with PBTZ169 as the basis for a new regimen against drug-resistant strains ofM. tuberculosis.

scholarly journals Improving the Sensitivity of the Xpert MTB/RIF Assay on Sputum Pellets by Decreasing the Amount of Added Sample Reagent: a Laboratory and Clinical Evaluation

2015 ◽  
Vol 53 (4) ◽  
pp. 1258-1263 ◽  
Author(s):  
Nila J. Dharan ◽  
Danielle Amisano ◽  
Gerald Mboowa ◽  
Willy Ssengooba ◽  
Robert Blakemore ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Limit Of Detection ◽  
Smear Microscopy ◽  
Content Type ◽  
Clinical Specimens ◽  
Test Sensitivity ◽  
Smear Negative ◽  
Almost All ◽  
Culture Positive ◽  
Xpert Assay

The Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection ofMycobacterium tuberculosisin sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions ofM. tuberculosisinto sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained fromM. tuberculosisculture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in whichM. tuberculosiswas detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%;P= 0.03) and for smear-negative specimens (67% versus 58%;P= 0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increasedM. tuberculosisdetection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.

scholarly journals High Mycobacterium tuberculosis Bacillary Loads Detected by Tuberculosis Molecular Bacterial Load Assay in Patient Stool: a Potential Alternative for Nonsputum Diagnosis and Treatment Response Monitoring of Tuberculosis

2022 ◽  
Author(s):  
Emmanuel Musisi ◽  
Abdul Sessolo ◽  
Sylvia Kaswabuli ◽  
Josephine Zawedde ◽  
Patrick Byanyima ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Treatment Response ◽  
Bacterial Load ◽  
Diagnosis And Treatment ◽  
Response Monitoring ◽  
Sample Type ◽  
Content Type ◽  
Potential Alternative

This paper highlights the value of stool as a sample type for diagnosis of tuberculosis. While other studies have used DNA-based assays like the Xpert MTB/RIF and culture to detect Mycobacterium tuberculosis in stool, this is the first study that has applied TB-MBLA, an RNA-based assay, to quantify TB bacteria in stool.

scholarly journals Optimization of a Nucleic Acid-Based Reporter System To Detect Mycobacterium tuberculosis Antibiotic Sensitivity

2014 ◽  
Vol 59 (1) ◽  
pp. 407-413 ◽  
Author(s):  
Matthew C. Mulvey ◽  
Margaret Lemmon ◽  
Stephanie Rotter ◽  
Jonathan Lees ◽  
Leo Einck ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Limit Of Detection ◽  
Surrogate Marker ◽  
Antibiotic Sensitivity ◽  
Nucleic Acid Detection ◽  
Drug Resistant ◽  
Reporter System ◽  
Susceptible Population ◽  
Content Type

ABSTRACTWe previously reported the development of a prototype antibiotic sensitivity assay to detect drug-resistantMycobacterium tuberculosisusing infection by mycobacteriophage to create a novel nucleic acid transcript, a surrogate marker of mycobacterial viability, detected by reverse transcriptase PCR (M. C. Mulvey et al., mBio3:e00312-11, 2012). This assay detects antibiotic resistance to all drugs, even drugs for which the resistance mechanism is unknown or complex: it is a phenotypic readout using nucleic acid detection. In this report, we describe development and characteristics of an optimized reporter system that directed expression of the RNA cyclase ribozyme, which generated circular RNA through an intramolecular splicing reaction and led to accumulation of a new nucleic acid sequence in phage-infected bacteria. These modifications simplified the assay, increased the limit of detection from 104to <102M. tuberculosiscells, and correctly identified the susceptibility profile ofM. tuberculosisstrains exposed for 16 h to either first-line or second-line antitubercular drugs. In addition to phenotypic drug resistance or susceptibility, the assay reported streptomycin MICs and clearly detected 10% drug-resistant cells in an otherwise drug-susceptible population.

scholarly journals Real-Time In Vivo Detection and Monitoring of Bacterial Infection Based on NIR-II Imaging

2021 ◽  
Vol 9 ◽  
Author(s):  
Sijia Feng ◽  
Huizhu Li ◽  
Chang Liu ◽  
Mo Chen ◽  
Huaixuan Sheng ◽  
...  
Keyword(s):  
Bacterial Infection ◽  
Real Time ◽  
Bacterial Infections ◽  
Near Infrared ◽  
Imaging System ◽  
Bacterial Load ◽  
Dynamic Imaging ◽  
In Vivo Detection

Treatment according to the dynamic changes of bacterial load in vivo is critical for preventing progression of bacterial infections. Here, we present a lead sulfide quantum dots (PbS QDs) based second near-infrared (NIR-II) fluorescence imaging strategy for bacteria detection and real-time in vivo monitoring. Four strains of bacteria were labeled with synthesized PbS QDs which showed high bacteria labeling efficiency in vitro. Then bacteria at different concentrations were injected subcutaneously on the back of male nude mice for in vivo imaging. A series of NIR-II images taken at a predetermined time manner demonstrated changing patterns of photoluminescence (PL) intensity of infected sites, dynamically imaging a changing bacterial load in real-time. A detection limit around 102–104 CFU/ml was also achieved in vivo. Furthermore, analysis of pathology of infected sites were performed, which showed high biocompatibility of PbS QDs. Therefore, under the guidance of our developed NIR-II imaging system, real-time detection and spatiotemporal monitoring of bacterial infection in vivo can be achieved, thus facilitating anti-infection treatment under the guidance of the dynamic imaging of bacterial load in future.

scholarly journals Deficiency of Double-Strand DNA Break Repair Does Not Impair Mycobacterium tuberculosis Virulence in Multiple Animal Models of Infection

2014 ◽  
Vol 82 (8) ◽  
pp. 3177-3185 ◽  
Author(s):  
Brook E. Heaton ◽  
Daniel Barkan ◽  
Paola Bongiorno ◽  
Petros C. Karakousis ◽  
Michael S. Glickman
Keyword(s):  
Dna Damage ◽  
Mycobacterium Tuberculosis ◽  
Animal Models ◽  
Bacterial Load ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Content Type ◽  
Dna Break ◽  
Repair Systems ◽  
Double Strand Dna

ABSTRACTMycobacterium tuberculosispersistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, includingM. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed.M. tuberculosiselaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant toM. tuberculosislatency. However, very little information is available about the phenotype of DSB repair-deficientM. tuberculosisin animal models of infection. Here we testedM. tuberculosisstrains lacking NHEJ (a ΔkuΔligDstrain), HR (a ΔrecAstrain), or both (a ΔrecAΔkustrain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required forM. tuberculosispathogenesis.

scholarly journals Mechanistic Modeling of Mycobacterium tuberculosis Infection in Murine Models for Drug and Vaccine Efficacy Studies

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Nan Zhang ◽  
Natasha Strydom ◽  
Sandeep Tyagi ◽  
Heena Soni ◽  
Rokeya Tasneen ◽  
...  
Keyword(s):  
Immune Response ◽  
Mycobacterium Tuberculosis ◽  
Vaccine Efficacy ◽  
Vaccine Development ◽  
Bacterial Load ◽  
Mechanistic Modeling ◽  
Response Dynamics ◽  
Content Type ◽  
Immune Effect ◽  
Adaptive Immune

ABSTRACT Tuberculosis (TB) drug, regimen, and vaccine development rely heavily on preclinical animal experiments, and quantification of bacterial and immune response dynamics is essential for understanding drug and vaccine efficacy. A mechanism-based model was built to describe Mycobacterium tuberculosis H37Rv infection over time in BALB/c and athymic nude mice, which consisted of bacterial replication, bacterial death, and adaptive immune effects. The adaptive immune effect was best described by a sigmoidal function on both bacterial load and incubation time. Applications to demonstrate the utility of this baseline model showed (i) the important influence of the adaptive immune response on pyrazinamide (PZA) drug efficacy, (ii) a persistent adaptive immune effect in mice relapsing after chemotherapy cessation, and (iii) the protective effect of vaccines after M. tuberculosis challenge. These findings demonstrate the utility of our model for describing M. tuberculosis infection and corresponding adaptive immune dynamics for evaluating the efficacy of TB drugs, regimens, and vaccines.

scholarly journals Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility

2020 ◽  
Vol 202 (22) ◽  
Author(s):  
Paras Jain ◽  
Spencer Garing ◽  
Deepshikha Verma ◽  
Rajagopalan Saranathan ◽  
Nicholas Clute-Reinig ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Limit Of Detection ◽  
Reference Method ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Metabolic Inhibitors ◽  
Health Crisis ◽  
Content Type

ABSTRACT Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis. We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

Sign in / Sign up

Export Citation Format

Share Document