Investigating multi-drug resistant tuberculosis in the 3-dimensional bioelectrospray cell culture model

Tuberculosis is a bacterial disease that used to cause one third of all deaths in this country and is now is becoming progressively more difficult to treat. Indeed, some strains are now completely resistant to all antibiotics previously used to cure tuberculosis infection. One of the difficulties in identifying new antibiotic treatments is the lack of good systems to study how antibiotics work when the bacteria are inside human cells. We have developed a new way of studying tuberculosis and human blood cells by making tiny droplets that are impregnated with both cells and tuberculosis bugs. We will use this system to study how normal tuberculosis and drug-resistant tuberculosis are killed by antibiotics when the bacteria are infected into human cells. We have experiments showing that antibiotics that we know work in patients with tuberculosis also work in our new system but do not work in standard infection systems, and so this approach provides a new way of identifying antibiotics that will not be found by more traditional approaches.

Once we have developed the model further, we will investigate standard and newly discovered antibiotics in the droplets. We will then add a second level to the model, starving the cells of oxygen and nutrients to reflect conditions in patients with tuberculosis more accurately. We will study how this stress changes the behaviour of both the bacteria and the human cells. We can then integrate this system with modern fluidic systems to screen a large array of new treatments. Furthermore, once the system has been developed, we will be able to use this to investigate other bacterial infections which are also becoming more resistant to antibiotics. Therefore, this is a system of broad potential that can be used to address one of the major medical challenges of the modern era.

Mycobacterium tuberculosis is becoming progressively more resistant to antibiotics and totally drug-resistant strains have emerged. Current models to develop novel anti-mycobacterial agents have significant limitations. We have developed a 3-dimensional bioelectrospray cell culture model of TB incorporating primary human cells and extracellular matrix. Cell aggregates with cardinal features of human disease develop, and cytokines and MMPs elevated in patients are similarly secreted in this system. The extracellular matrix modulates the host-pathogen interaction. In antibacterial experiments, standard antibiotics work in both routine 2-D cell culture and also the 3-D system. However, pyrazinamide, a critical antibiotic in treatment of TB, only works in the 3-D cell culture system, clearly proving the potential of the system to screen for novel antibacterials that require an intact host immune response. This innovation award will develop the bioelectrospray model to study multi-drug resistant TB (MDR-TB) in the context of the host. We will characterise the model further, studying the up-regulation of mycobacterial stress genes in standard cell culture systems and the 3-D system. We will generate luminescent MDR-TB and study the effect of diverse antibiotics on mycobacterial proliferation and host cell survival. This will permit study of resistance in a 3-D system versus genotypic and standard resistance analysis. We will then model in vivo conditions further by studying hypoxic and nutrient-depleted granulomas, reflecting the complexity of in vivo conditions. Ultimately, the bioelectrospray model can be combined with a microfluidic system to deliver drugs in a pharmacologically relevant manner, modelling concentrations in patients and permitting high throughput assays. This is a potentially transformative system that is not only applicable to tuberculosis but also to all drug-resistant bacterial infections incorporating immune cells in a physiological matrix.

Impacts will be related both to the tuberculosis (TB) field and also more widely to the knowledge economy. The work will be of interest to pharmaceutical companies as it is directly translational and is addressing Cooksey's first gap in the translational pathway. The results will be relevant to a wide number of infections that require analysis of drug-resistant bacteria in the context of the host, other inflammatory diseases and conditions where advanced 3-dimenstional cell culture is required. Therefore, impacts will reach beyond the TB community.

Tuberculosis is a disease of global importance, infecting a third of the world's population and causing approximately 8 million new cases per year and killing approximately 1.4 million people per year. Despite the World Health Organisation highlighting that TB was a global health emergency in 1994, the disease incidence remains static and treatment has remained unchanged for over 30 years. Drug resistance is inexorably increasing, with the emergence of Multi-, Extensively- and now Totally-drug resistant strains. We will develop a better model to improve the understanding of the effects of antibacterials in TB and to identify new strategies to identify treatment for drug-resistant disease. We will investigate novel, innovative methodologies cutting across bioengineering and medical science which have wide applicability to other research fields. Furthermore, the bioelectrospraying of viable cells into 3-dimensional granulomas impregnated with components of the extracellular matrix will be relevant to all diseases where matrix remodelling is involved. We will train highly skilled researchers who will become competent in an extensive range of cutting-edge research techniques, including bioelectrospray methodology and multi-parameter readouts of both host and pathogen biology.

Our studies are of direct translational impact and so we anticipate pharmaceutical engagement and the potential to attract research and develop investment. We are already in discussion with GSK about testing emerging drugs in this model system. Tuberculosis can be considered the prototypic infectious disease to study in the 3-D bioelectrospray model, since a prolonged interaction between the pathogen and host occur, and so proving the efficacy of this approach for MDR-TB will not only identify novel approaches to TB, it will also prove the proof-of-concept for the efficacy of the model for diverse infectious diseases.

Ultimately, our studies will identify candidate compounds to go forward to experimentation in appropriate animal models and clinical trials to treat MDR- TB. The models developed will also have potential to identify antibacterials in diverse infections that require an intact host immune response for analysis and for the investigation of other inflammatory conditions.