Novel strategies to detect and eliminate persistent Mycobacterium tuberculosis - applications in a murine Cornell model

Tuberculosis is a curable disease but still remains one of the biggest killers in the world. It kills nearly 2 million people worldwide every year and 98% of tuberculosis deaths are in the developing world affecting mostly young adults in their productive years. A quarter of a million tuberculosis deaths are also infected with HIV which weakens human immune systems, most of these people are in Africa. Tuberculosis especially affects the most vulnerable populations including children, the poorest and malnourished. Tuberculosis is caused by the bacterium called Mycobacterium tuberculosis. One important characteristics of this disease is that the bacterium has an unusual ability to grow and survive for extended periods of time in human body. Therefore, it has been estimated that 2 billion people, equal to one third of the world's total population, are infected with the bacterium in whom it causes unnoticeable latent infection that gives rise to a 5-10% lifetime risk of active tuberculosis. These persistent bacteria cannot be cultured using standard microbiological methods and are not killed by the current tuberculosis drugs. Therefore, tuberculosis treatment needs at least 6 months with four drugs to cure the patients. This long-term treatment is extremely difficult to implement especially in developing countries because of lack of affordability in both patients and healthcare services and limited infrastructure. It is important that treatment duration is shortened using effective tuberculosis drugs as it will significantly reduce patient suffering, side effect and expenses incurred by both patients and their families. In addition, shorter TB treatment duration may allow an earlier return of patients to their productive activities.

The proposed research aims to identify and quantify those persistent bacteria in tuberculosis infected mice before and during treatment with new drugs to predict the outcome of tuberculosis treatment. The persistent bacteria will be "woken" by the addition of resuscitation promoting factors (RPFs) which are proteins produced by M. tuberculosis to restart growth. We have shown in our recent study that in mice there were persistent bacteria which depended on RPFs to grow. High-dose rifampicin which was added in the current treatment drug regimen was able to kill RPF-dependent persistent bacteria, as a result, treatment was cut short with no disease relapse. This meant that if we are able to remove RPF-dependent bacteria from patents, the treatment duration could be shortened with reduced relapse rate. In this proposal, we will apply the same principals and techniques which we have learnt from studying mice using a set of novel drug regimens to predict the outcome of human tuberculosis treatment, especially disease relapse. We will set up a model system in mice as a test bed to evaluate the potencies of new drug regimens before usage of them in much more expensive and time-consuming human testing.

1.Treatment with novel regimens in a modified Cornell model. The novel drug regimens containing rifapentine, bedaquiline, moxifloxacin will be tested in a modified Cornell mouse model in which treatment starts at 10 weeks after a low dose M. tuberculosis H37Rv infection. Treatment is given for 16 weeks by daily oral administration. Mouse organs will be harvested at two weeks interval to monitor CFU and broth counts (7H9 and culture filtrates). After treatment, the remaining mice are administered hydrocortisone for 8 week. CFU counts from lungs and spleens are performed to determine relapse.
2.Standardizing resuscitation promoting factor production and preparation of culture filtrates. M. tuberculosis is grown in 7H9 for 15 to 20 days until an optical density of 1 to 1.5 is reached. mRNA expression of 5 RPFs from the cultures is examined by semi-quantitative RT-PCR. The cultures are harvested by centrifugation and filtered with 0.2 um filter twice. The culture filtrates will be used immediately and RPF stability testing will be performed before and after storage at -20 or -70oC.
3.Resuscitation of M. tuberculosis in mouse organs. Broth counting is performed as serial 10-fold dilutions in triplicate in which 0.5 ml of organ homogenates are added to 4.5 ml of culture filtrates or 7H9. At 10-day intervals over a 2-month period of incubation, the broth cultures are examined for visible turbidity and confirmed by colonial morphology on 7H11 agar. The most probable numbers of viable bacilli are estimated from the patterns of positive and negative tubes.
4.Resuscitation of RPF-dependent persisters in Mycobacteria Growth Indicator Tube (MGIT). CFU count negative organ homogenates are incubated in MGITs, then culture filtrate is added into MGIT before and after 42 day incubation and continue to incubate for up to 2 months. Resuscitated persisters in MGIT tubes will be confirmed by observation of turbidity and fluorescence changes as well as plating on 7H11 agar plates.

This proposed research aligns with WHO "the end TB strategy" for rapid diagnosis, treatment and care and meets MRC's Strategic Priorities of 'Living a long and healthy life' and 'Research to people'. This outcomes of the project impact on

1. TB patients, Healthcare sector and general public. The outcomes of this project could lead to a novel clinical intervention and therefore, the ultimate beneficiaries will be TB patients. Tuberculosis is the single most important infectious disease in the world and leads to approximately 2 million deaths annually. Tuberculosis infects human populations of all age groups, but mostly affects adults in their most productive years. One million children fell ill with TB and 140000 children died from the disease in 2014. More than 95% of tuberculosis cases and deaths reside in the developing world. HIV co-infection contributes 20 to 30 times more risk for individuals to develop active tuberculosis. More than 20% of TB cases worldwide are attributable to smoking. Furthermore, about one third of the world's population harbor dormant M. tuberculosis and this provides a huge pool of potential disease (5 to 10% relapse). Effective TB control needs 6 months antibiotic therapy which is expensive (US $2000 per patient), challenging to implement, inevitably leads to poor patient compliance, high relapse rates and drug resistance. Shortening TB treatment duration with a low relapse rate will significantly reduce patient suffering, side effect and expenses incurred by both patients and their families. Our findings will have the potential to reduce the duration of TB treatment and therefore reduce burden of care and costs on both patients and healthcare systems.

2. Academic advance and education. The project will increase our understanding of fundamental problems of bacterial persistence to eradicate tuberculosis and how to identify persistent M. tuberculosis with aims to improve TB diagnosis and treatment. Therefore the most immediate beneficiary will be the academic community. The research has its novelty which will advance academic knowledge and UK competitiveness. In the public sector, the work will most directly impact on the academic research community and healthcare professionals who care for patients with tuberculosis. The outcome of the proposed research will be published in high impact peer-reviewed journals and presented in International and national conferences. This project will provide training for postdoctoral research assistant. There is an urgent need for researchers with expertise in Hazard Group 3 pathogens including working with M. tuberculosis infected animals in the UK. This is an excellent area for training future scientists such as PhD and master students. The training given in this project will be effectively transferable and of specific interest to pharmaceutical industry as well as academia.

3. Commercial exploitation: This project will potentially make new products for TB diagnosis and treatment. Essentially, to successfully eliminate these persisters, one must first detect them. One of the most intuitive and promising methods is to "wake up" the persistent bacteria from their dormant state, and induce them to recommence multiplication. Using RPF to wake up persistent bacteria may enable quantitative detection of the persistent population in patients with tuberculosis and holds promise for becoming a powerful risk stratification tool for clinical treatments. Accurate detection and quantification of persistent M. tuberculosis will make novel drug and drug regimen discovery feasible which may act as a persistent biomarker with predictive value to shortened tuberculosis treatment. Our persistent resuscitation systems have the potential to provide a fast but cheap screening for persistent tuberculosis at hospitals and clinical settings leading to fast diagnosis which potentially improves healthcare and reduced healthcare costs from early diagnosis.