Understanding mechanisms of antimicrobial resistance (AMR) in Streptococcus pneumoniae clinical isolates

The bacterium Streptococcus pneumoniae, also called pneumo, causes invasive diseases such as: pneumonia and meningitis, which lead to millions of deaths every year. To prevent and treat pneumo infections in hospitals and clinics, patients are given penicillin antibiotics, or drugs very similar to them. These drugs kill the bacteria growing inside the patient, combating the infection and curing the individual. Increasingly, strains resistant to penicillin are emerging across the world threatening our treatment strategies and jeopardising patient outcomes. Therefore, there is an urgent need to address penicillin resistance in pneumo to prevent patient deaths. This project will identify and characterise the biological underpinnings of resistance and aims to find way of re-sensitising strains to antibiotics we routinely use in clinics.
To address, and ultimately prevent, penicillin resistance in pneumo we need to understand how resistance occurs in the first place. This understanding is vital to end an all too familiar pattern of: a new drug discovery leading to resistant bacteria, resulting in a search for yet more drugs.

Penicillin resistance in pneumo does not occur in a single step, but emerges gradually as the bacterium acquires genetic alterations one at a time. Using pneumo strains isolated from patients, we have discovered a strain that is emerging on its journey towards antibiotic resistance and therefore contains a limited number of genetic changes. This strain is typical of the pattern of pneumo penicillin resistance in hospitals in the UK and already shows 'low-level' resistance to this drug. Importantly, strains of the same type are responsible for pneumo infections across the globe. Therefore cellular processes we discover in this study will be generally applicable to worldwide bacterial lineages (families of related strains). In preliminary work, we have already identified some of the genetic changes in this stain by genome sequencing, but we do not know which of these changes are important for underpinning the penicillin resistance. Our hypothesis is that one, or more, of these genetic changes is responsible for the 'low-level' resistance in this strain and these changes are an important step towards 'high-level' penicillin resistance in pneumo strains.

To test our hypothesis, we will carry out three parallel identification methods all powered by next-generation DNA sequencing: a whole genome profiling method (Tn-Seq) and two related whole-genome sequencing methods. These methods will identify the factors that are important for the strains to resist penicillin treatment and, once identified, we will carry out further work to understand how these new factors function. Importantly, our preliminary work has already identified a new resistance determinant and we will carry out experiments to understand its function in the pneumo cell.

Our primary aim is to re-sensitise the 'low-level' penicillin resistant clinical isolate to penicillin treatment and we will test our new understanding of resistance in a model infection system, which mimics treatment of patients in clinic. This research will help us tackle the global threat of antibiotic resistant infections, by finding ways to make sure drugs work properly when given to patients with deadly S. pneumoniae diseases.

The human pathogen S. pneumoniae is responsible for millions of cases of invasive disease annually with emerging penicillin resistant lineages threatening treatment outcomes for patients. Thus, there is an urgent need to understand the biology underpinning penicillin resistance for this pathogen to safeguard patient outcomes.

To achieve this, I have selected clinical isolates with low-level penicillin resistance for further study, reasoning these strains would have a limited number of resistance determinants and would offer a tractable platform for further investigations. I have discovered uncharacterised resistance determinants in these isolates that are essential for beta-lactam resistance in clinically important strain backgrounds. I hypothesise these unknown mechanisms are essential for beta-lactam resistance and support mosaic-pbp acquisition that drive higher levels of resistance.

This project will identify mutations in known and previously unknown S. pneumoniae genes linked to beta-lactam resistance in clinical strains with differing resistance profiles (Objective 1). I have made the exciting discovery of a new gene cluster linked to beta-lactam resistance and the function of target PBP enzymes. I will study the function of genes within this cluster alongside those identified in Aim 1 to understand the biological mechanisms underpinning resistance (Objective 2).

The ultimate aim of this study is to identify ways to 're-sensitise' strains to antibiotics, to a level where they now respond to treatment. I will use a zebrafish embryo model of infection to test how disruption of identified mechanisms impacts virulence and, importantly, if these disruptions have a favourable impact if infected zebrafish are treated with antibiotics (Objective 3). This unique approach will inform S. pneumoniae lineage surveillance and identify strategic points of weakness in beta-lactam resistance phenotypes that may be exploited for therapeutic gain.

The proposed project will develop an integrated, interdisciplinary platform to study S. pneumoniae antimicrobial resistant determinants. There will be a variety of impacts over a range of timescales and arenas.

- Impact on Research Staff (year 1 onwards): The PDRA linked to this proposal will be trained in the acquisition and analysis of next-generation sequencing datasets and will have experience of the zebrafish model of infection. The Research Imaging Specialist (Senior Experimental Officer) will have experience with S. pneumoniae quantitative live-cell microscopy, which has unique demands due to the autolytic property of these cells. We anticipate this experience will enable more complex microscopic experiments in future studies. Both staff members will have enhanced publication track records on the publication of the proposed research.

- Impact on Academia (year 1 onwards): Research scholars will benefit from the new information and models generated during the project, communicated both orally and via publication. This will open new lines of investigation for AKF and enhance an already present focus on S. pneumoniae research in Sheffield.

- Impact on Sheffield and the Florey Institute (year 1 onwards): This award will continue the upward trajectory of the Florey institute and build on the profile of Sheffield as a S. pneumoniae research hub.

- Impact on Healthcare (expected timescale year 2 onwards): We will maintain our clinical connections and regularly interact with clinicians and others associated with healthcare, exchanging information through seminars and organised Florey Institute events. This research will provide insight into the emergence of AMR lineages in the Yorkshire and Humber area which for S. pneumoniae are particularly important for high-risk groups (under 5s and over 60s). Longer-term impacts are covered in 'Policy Makers' below.

- Local communities (expected timescale year 2 onwards): via outreach activities will benefit from greater knowledge of this scientific area and a greater engagement with the scientific advances generated by their local university.

- Industry (expected timescale year 3 onwards): The identification of novel targets to combat antimicrobial resistance will be a major stimulus to the pharmaceutical industry. Direct links to Biotech companies such as Absynth and pharmaceutical companies such as GSK are in place through the Florey Institute to facilitate direct translation of key findings.

- Media (expected timescale year 3 onwards): On acceptance of a manuscript for publication, a press-release will be generated through the University of Sheffield media team to attract national coverage. AKF has liaised with the MRC Press Office for media engagement in the past and will approach this team again once any publication is accepted to maximise impact. Additional, supplementary posts will be publicised through Twitter, Facebook and on the Florey and University of Sheffield websites, which AKF administers, to raise awareness of the work locally.

- Policy makers (long term,>7 years): National and international clinical surveillance of emerging AMR lineages may be reappraised in the light of the data on identified resistance determinants (long term impact >10 years).

- Impact on Society: New approaches to combat infection would reduce morbidity, mortality and health costs (long term >10 years). This is particularly pertinent for the aging population demographics of the UK, as risk of S. pneumoniae infections cause deaths in localised outbreaks among the elderly living in close proximity. Increased understanding of the of AMR emergence in S. pneumoniae and of antimicrobial stewardship, facilitated by publicising this research, would remove some of the inappropriate demand for antimicrobials and encourage greater engagement between the public, health care professionals and industry to tackle infection related issues (short term,3-5 years).