'Silent' antibiotic resistance genes: an overlooked issue of considerable importance in antibacterial chemotherapy?

Antibiotics enable the treatment and cure of life-threatening bacterial infections, and represent one of the great successes of modern medicine. Unfortunately, the utility of these agents is being progressively eroded as bacteria evolve to resist their effects, and antibiotic resistance is now considered one of the three greatest threats to human health.

A key aspect of dealing with antibiotic resistance effectively in medical practice is strategic intelligence. Being in possession of up to date information about the proportion of bacterial strains in a given location that are resistant to particular antibiotics allows doctors to decide which would be the best antibiotics to use routinely to treat bacterial infection, and to avoid those which are probably not going to work because resistance is so commonplace. In the case of a life-threatening bacterial infection, knowing precisely which antibiotics the specific bacterium present in the patient is resistant or susceptible to enables the doctor to select the best antibiotic treatment to cure the patient.

This project is focussed on investigating a phenomenon that may be seriously undermining our strategic intelligence regarding antibiotic resistance. Recent work in the applicant's laboratory has established that some bacteria that are sensitive to antibiotics nonetheless carry genes that are normally associated with antibiotic resistance, but that these genes have become switched off ('silenced'). This phenomenon, which we have termed 'silencing of antibiotic resistance by mutation' (SARM) is of considerable concern, as bacteria with SARM would appear susceptible to an antibiotic when tested, but could then very quickly and easily become resistant to the antibiotic during treatment in a patient. Currently, we do not know how widespread SARM is amongst bacteria that cause disease, nor do we understand properly how SARM occurs. The present proposal aims to investigate both of these issues in the so-called 'superbug', Staphylococcus aureus.

To establish how common SARM is, a large collection of 1500 S. aureus isolates recovered from patients around the world will be tested. Each strain will undergo DNA sequencing of its genome to establish its complete genetic make-up, which will allow for the identification of genes that are known to be associated with antibiotic resistance. In addition, the susceptibility of each isolate to a wide range of commonly used antibiotics will be established to determine if the bacterium displays resistance to all the drugs that it has the genetic potential to display resistance to. Strains that carry antibiotic resistance genes, but do not exhibit resistance to the corresponding antibiotics, represent potential SARM strains. These strains will be studied in detail to establish how easily they can switch their 'silenced' resistance genes back on, and to understand the mechanism(s) by which SARM works.

A proportion of bacterial strains that are phenotypically sensitive to antibiotics carry antibiotic resistance genes that are not expressed as a consequence of genetic defects. This phenomenon, which we have termed 'silencing of antibiotic resistance by mutation' (SARM), appears to be reversible, and fully antibiotic-resistant revertants can therefore readily emerge from these populations upon antibiotic challenge. Consequently, SARM may represent a cause of unexpected therapeutic failure of antibacterial chemotherapy in patients, and could lead to considerable underestimation of the prevalence of resistance in surveillance studies conducted using susceptibility testing. The current state of knowledge regarding the molecular mechanisms of SARM is extremely limited, and systematic studies to establish the prevalence of this phenomenon in clinical isolates are lacking. This proposal seeks to undertake a detailed investigation of both of these aspects in the important human pathogen, Staphylococcus aureus. Thus, the major objectives of this study are (i) to establish the prevalence of SARM amongst clinical isolates of S. aureus, and determine the frequency with which SARM isolates revert to phenotypic antibiotic resistance, and (ii) to delineate the molecular basis for SARM. To identify staphylococcal isolates exhibiting SARM for both these objectives, a diverse collection of 1500 S. aureus strains will be screened for discordance between their phenotypic susceptibility to all clinically deployed antistaphylococal agents, and their resistance genotype as determined by whole genome sequencing. Strains exhibiting SARM, including a number that we have already identified, will undergo characterization to establish the location and identity of mutations that cause resistance gene silencing, and to determine exactly how SARM is mediated. Collectively, these studies will provide the first detailed insights into the SARM phenomenon.

The proposed study will support the regrowth of UK expertise in what has of late been an unrepresented area of research, but one that is of undeniable importance to human health - antibiotic resistance. The appointed PDRA will receive cutting-edge training from a well-established laboratory in the field, which, in combination with the state of the art training provided by the Sanger Institute, will deliver a highly-skilled researcher with excellent knowledge of antibiotic resistance and genomics, and with strong onward employment prospects in either academia or industry. In addition, the proposed project will foster a newly-forged collaboration between members of the antibiotic resistance research community and the Sanger Institute; this partnership has the potential to accelerate resistance research in the UK, both in this and in future projects in which high-throughput genomics can be brought to bear on the topic of antibiotic resistance.

The study will also provide detailed knowledge regarding the prevalence and mechanisms of SARM in S. aureus, revealing in the process the extent to which SARM may be negatively impacting our current ability to detect antibiotic resistance in patients and in surveillance studies employing susceptibility testing. Should it transpire that SARM is prevalent in staphylococci, this would undoubtedly transform the field, introducing an awareness of the potential failings of susceptibility testing results when choosing antibiotics for treatment, prompting development of approaches to better detect/ avoid overlooking SARM in the routine diagnostic laboratory, and thereby ultimately leading to reduced antibiotic treatment failure in patients. Collectively, the studies outlined here will serve to improve our fundamental understanding of antibiotic resistance in a way that may rapidly translate into important practical benefits in respect of our ability to treat bacterial infection in patients.