Targeting airborne bacterial infection: Studies on patient- and laboratory-generated mycobacterium tuberculosis aerosols.

Lead Research Organisation: University of Leicester
Department Name: Infection Immunity and Inflammation

Abstract

Globally, tuberculosis (TB) is the single most important cause of death and disease due to a bacterial agent. Like many other bacterial infections affecting the lungs, TB is transmitted by aerosols (tiny droplets expelled by coughing and other respiratory efforts) generated by infected individuals. Together TB and other airborne bacterial infections cause approximately 4 million deaths worldwide every year and remain a significant problem in the UK. In spite of the importance of aerosols in transmitting infection we have very little information on the biological properties that bacteria need to be transmitted by this route. Our previous research suggests both that the properties of bacteria expelled by TB cases are distinctive and that they are likely to change further during transit.
We recently established methods for capturing aerosols produced by TB patients and to determine the pattern of genes expressed by its bacterial cause, Mycobacterium tuberculosis (Mtb). These methods are built on the patient Cough Aerosol Sampling System (CASS) which has revealed that the risk of infection in household contacts of a TB can be predicted by the number bacteria the patient produces in a standard CASS sample. Our use of the CASS system here will enable us to relate our results to the infectiousness of the individual patients we sample.
Here we will characterise the CASS and a laboratory aerosol generation and sampling system, and use both to answer four questions: 1) is the TB aerosol more than just a simple sample of the mucus secretions (sputum) patients cough-up (sputum analysis has traditionally been used to assess infectiousness); 2) how does survival and gene expression of Mtb change with time and ambient conditions in laboratory aerosols; 3) which of Mtb's genes make important contributions to the bacterium's survival and transfer in aerosol; and 4) are the numbers of Mtb bacilli in aerosols underrepresented by standard agar culture methods?
By defining the Mtb genes and gene expression patterns important for aerosol transmission we aim to uncover new methods to control infection; these could include targeting critical Mtb gene products in patients to render them less infectious, altering ambient conditions to make them unfavourable for survival and transmission of Mtb and preventing the establishment of new infections. Reducing infectiousness could be particularly important in managing the increasing problem of drug resistant TB, where it may take a long time to render patients non-infectious. Simple alterations to ambient conditions could be valuable in the management of healthcare facilities, a particular problem in resource-limited settings. Finally, the potential for Mtb to change its properties in response to aerosol transfer raises the likelihood that we do not know what features the bacterium expresses when it is breathed in by a new potential victim and which of these features is essential to establishing infection. By identifying the way Mtb adapts to aerosol transfer, our work has clear potential to identify new Mtb targets for prophylactic therapy in recently exposed individuals and for vaccines that might prevent infection, a particularly weak feature of the current BCG vaccine and vaccine candidates in development.
Our study involves collaborations between clinicians (Pretoria), bacteriologists (Leicester), and specialists in the biology (Porton) and physics (Leeds) of aerosols. We will use exceptional facilities in South Africa, Leicester and Public Health England (Porton) to deliver the work. The Leeds group will develop computer models of our sampling systems to better define how completely the results reflect the aerosols sampled. These and our biological results will be used to develop new quantitative models of patient infectiousness.
We believe that understanding the TB aerosol will create new opportunities to control Mtb and provide important lessons applicable to other airborne infections.

Technical Summary

We recently established methods to obtain transcriptome profiles from TB patients' cough aerosol samples. These will be used here to elucidate the biological properties of Mtb that support its airborne transmission. As Mtb's survival depends on aerosol transfer from TB cases to contacts, interventions directed to this phase have major potential to impact on the global annual incidence of 9 million cases.
Our strategy is to sample cough aerosols produced by newly diagnosed TB patients in Pretoria and model these in vitro in contained aerosol chamber (Goldberg drum) studies. Use of CASS aerosol sampling, which has been shown to categorize patients associated with high and low risks of household transmission, will link our results to infectivity. We will test our hypothesis that the properties of Mtb in early aerosols (<30s post cough) are distinct from those in sputum and that this reflects a selective aerosolisation process. The drum studies will allow us to determine transcriptional adaptations of Mtb to the aerosol environment over extended times (up to 4h) and to screen for genes supporting aerosol transfer by use of an established Mtb transposon mutant library. These studies will be underpinned by nucleotide sequencing and will provide transcriptome, relative mutant frequencies and microbiomic profiles as required.
Both patient and lab studies will be used to evaluate the potential contribution to transmission of Mtb cells incapable of forming colonies on primary culture. High numbers of these growth factor dependent cells have been demonstrated by us in sputum and in preliminary aerosol studies.
The two sampling systems will be subjected to computational fluid dynamics modelling to assess internal airflows and the impact on sampling efficiency. Combining these methods our biological results will enable us to develop a case-centred model of infectivity and focus on identifying and exploiting vulnerabilities of Mtb transmission amenable to intervention.

Planned Impact

We aim to deliver new understanding of the biological factors that impact on the aerosol phase of Mycobacterium tuberculosis (Mtb) transmission. This most significant human pathogen, affecting one third of humankind, causes nearly 1.5 million deaths a year. The immediate benefits of this work will be to deliver a better understanding of the transmission process, providing numerous opportunities to develop strategies to target Mtb transmission and reduce the global burden of TB.
Our project is also designed to address generic issues in the airborne transmission of bacterial infections. We argue that our studies into the physiological responses of Mtb to the aerosol environment are likely to reveal features in common with other airborne bacterial pathogens of humans and animals (e.g. S. pneumoniae and M. bovis)
WHO WILL BENEFIT AND HOW? DEPARTMENT OF HEALTH; HEALTH PROTECTION ENGLAND; THE WORLD HEALTH ORGANISATION; GOVERNMENT; THE HEALTHCARE INDUSTRY. Primary beneficiaries will be organisations responsible for the management of TB and spread of infection. Better understanding of the limitations of sputum analysis as a means of predicting patient infectivity and how Mtb may change its expressed properties in transit will have implications for strategies to control transmission. Any measurable factors we discover that influence survival and adaptation in aerosol would have short term impact for guidance on patient isolation and management of room air and disinfection. In the medium term impact would arise from new innovation, regulation and guidance to control transmission, for example, in how best to ventilate hospital rooms.
MEDICAL COMMUNITY AND PATIENTS AND THOSE AT RISK OF EXPOSURE TO MTB. Practitioners concerned with TB control will benefit from the understanding gained in this project. In the short term simple procedures for reducing infectious aerosols may be developed and implemented. This will ultimately benefit the health and economic activity of those infected with, or at risk of exposure to Mtb. Our work also has the potential to identify features important for the establishment of new TB infections and vaccine strategies as well as principles applicable to other respiratory pathogens; these could lead to developments impacting their transmission.
PHARMACEUTICAL INDUSTRIES AND CHARITIES E.G. - GLOBAL ALLIANCE FOR TB DRUG DEVELOPMENT. In the longer term we aim to identify specific targets which have the potential to suppress patient infectivity. This is a particular concern for patients with highly drug resistant (MDR/XDR)-TB, who continue to produce infectious aerosols until such time as effective treatment regimens have been achieved. Organisations with interests in the development of new anti-TB drugs and vaccinations will benefit directly from our research outcomes; these include pharmaceutical companies in the UK, such as GSK and Astra Zeneca.
AGENCIES INVOLVED WITH THE CONTROL OF BACTERIAL AIRBORNE ANIMAL INFECTIONS INCLUDING BOVINE TUBERCULOSIS. Many of the members of the Mtb complex are spread by aerosol and the results of this project will also be of interest to those involved in the control of bovine TB and the spread of disease in other mammals.
PEOPLE. Beneficiaries include the research team and their network of collaborators, through the establishment of long term research partnerships. The UK trained workforce will benefit through the training of the research assistants who will acquire new skills in molecular biology, transcriptional analyses and aerosol science from the combined expertise of the applicants. Outcomes of this research will also be of interest to the media and public with the long term potential for the development of control measures to halt the spread of this scourge of mankind.
 
Description Porton Goldberg Drum 
Organisation Public Health England
Department Public Health England Porton Down
Country United Kingdom 
Sector Public 
PI Contribution We have provided bacterial inocula, nebuliser expertise and analytical expertise to experiments designed to determine survival of M. tuberculosis in aerosol and transcriptional adaptations to this environment
Collaborator Contribution Dr Katy-Anne Thompson and Mr Allan Bennett have co-designed the aerosol experiments and directly assisted in their delivery under containment level 2 and 3.
Impact In progress
Start Year 2017
 
Description Pretoria infectious diseases unit - Prof Anton Stoltz 
Organisation University of Pretoria
Country South Africa 
Sector Academic/University 
PI Contribution We have designed a patient sampling approach based on the Cough Aerosol Sampling System of Fennelly and colleagues. Leicester personnel have visited Pretoria to take and process samples for shipping back to Leicester
Collaborator Contribution Our Pretoria colleagues led by Prof Stoltz have identified and consented suitable patients and provided clinical support throughout. They have made material contributions to the sampling programme.
Impact In progress
Start Year 2017