Defining mechanisms of antiseptic tolerance against skin microbiota

Antiseptics are critical to prevention of infection in humans and animals as bacteria normally found on the skin are the major causes of infections when they gain access to the blood via breakage of the skin barrier or via catheters inserted for various medical reasons. In particular a group of staphylococci (related to MRSA) known as 'coagulase negative staphylococcus (CoNS)' live on skin and cause serious infections in animals and humans, particularly premature babies. Current measures to prevent infection rely on the use of antiseptics to remove pathogens from the skin and hence stop them causing infection. Two major antiseptics are used clinically:chlorhexidine and octenidine, with chlorhexidine also being widely used in animals. Together, these agents are crucial in preventing infections.

Recently there have been multiple reports documenting the identification of CoNS which are unusually tolerant to antiseptics prompting worries that they may become resistant to antisepsis which would result in increased infection risk to humans and animals.

Despite the widespread use of chlorhexidine and octenidine, little is known about how they work and how bacteria could develop resistance to them. The study proposed here will study how these agents work and explore whether antiseptic resistance can occur and if so whether mutant bacteria also become cross-resistant to antibiotics as a result.
We have recently assembled a collection of 1250 CoNS from the skin of babies from two neonatal intensive care units. One in the UK where chlorhexidine is used and one in Germany where octenidine is used. We have seen there are clear differences in antiseptic tolerance between the two units. In this study we will decode the genomes of all the strains we have collected and compare this information with antiseptic susceptibility. This will allow us to identify any genetic changes that cause antiseptic tolerance in the isolates. We will combine this approach with laboratory studies where we will expose selected isolates to each antiseptic and study how they become tolerant. Importantly, we have developed a human skin model we can use to do this which will be a realistic proxy for use in the real world. Again, we will sequence the genomes of any resistant mutants. Together this genetic analysis of resistant isolates and mutants will help us understand the genes involved in antiseptic resistance. We will also see if exposing isolates to the antiseptics makes them change their antibiotic resistance which could result in harder to treat infections.

The applicants have a strong background in studying disinfectants, bacterial genomics and skin responses to infection and we will use those skills here. The data we produce will be used and shared with clinical and veterinary colleagues to inform how best to use antiseptics in animals and humans with a focus on premature babies. We will be able to assess if there is a difference in the risk of bacteria becoming resistant to either of the two major antiseptics which can guide best practice. We will also generate useful information about the mechanisms by which both agents work which will help development of new antiseptics in future.

BACKGROUND AND OBJECTIVES:
The skin microbiome contains many different bacteria which can cause serious infections in humans and animals. Coagulase negative staphylococci are the major opportunistic pathogens present on skin. Antiseptics are crucial to prevention of infection although there is evidence for adaptation of staphylococci to these agents. We have assembled a large panel of coagulase negative staphylococci from babies in the UK where chlorhexidine is the antiseptic used and from Germany where octenidine is used. We have also developed an ex vivo human skin explant model and functional genomics tools to study bacterial adaptation to stress. The objectives of this proposal are to combine study of isolates with laboratory experiments to: understand the mechanisms of antiseptic tolerance and action and to study the potential impacts of antiseptics as drivers of antibiotic cross-resistance.

OVERVIEW OF EXPERIMENTAL DESIGN:
We will sequence all 1250 isolates and use this data with the susceptibility to antiseptics to identify genes and mutations which correlate with antiseptic tolerance. This will use computational approaches (a genome-wide association study) established by Co-I Page. We will also combine evolution experiments in the presence of both antiseptics with RNAseq and TraDIS transposon mutagenesis (using mutant libraries already made). Experiments will use a skin explant model to mimic antiseptic use in practice. We have already identified two chromosomal efflux pumps as putatively involved in antiseptic tolerance, NorA and NorB and this will be validated along with the role of key, novel candidate genes identified here.

APPLICATION AND EXPLOITATION
We are engaged with veterinarians, clinicians and industry and data will help develop best practices for antisepsis in animals and humans, in particular neonates. We will also uncover fundamental new biological insights into mechanisms of action and tolerance to two important antimicrobials.

Multiple groups will benefit from this research, including academics (see 'academic beneficiaries' section for details), veterinarians and clinicians involved in the management of infection, the public where antimicrobial resistance (AMR) is a topic of interest and industry where those developing antiseptics will benefit from the data generated in this project. The postdoctoral researchers to be appointed will also benefit in furthering their career and developing skills in functional genomics, molecular microbiology and host-pathogen interactions.

How will they benefit?
Clinical and veterinary beneficiaries:
A goal of this proposal is to provide quantitative and qualitative data to assess impacts of two important antiseptics in selection of tolerant mutants. This is likely to inform the development of neonatal specific guidelines for the use of antiseptics; Mark Webber is engaged with neonatologist Paul Clarke at the Norfolk and Norwich University Hospital (NNUH) and currently assisting in the analysis of microbiological outcomes from the 'ARCTIC' clinical trial comparing impacts of use of different chlorhexidine formulations on catheter colonisation and sepsis. With German colleagues at the Universitatsklinikum Schleswig-Holstein in Lubeck we aim to begin to formulate neonatal specific best practices for antisepsis combining data from this project with clinical data. We plan a stakeholder meeting to be held in Norwich to present our research to clinical and industrial colleagues in the final year of the project. This will allow us to identify how we can use information from this project to help improve clinical practice and determine the key next steps in this process. We will communicate both locally and internationally with clinical and veterinary colleagues. At the local level, regular meetings are already held (typically bi-monthly) between academics from the UEA Medical School and across the Norwich Research Park and clinical researchers from NNUH. For example, Webber has spoken twice in 2018 to NNUH including at the hospital wide 'Grand round' presentation. We will also visit colleagues at the Cambridge Veterinary School throughout the project.

The public and patients:
The work being undertaken in this proposal is obviously relevant to the public and patients and we are already engaged via the ARCTIC trial with a patient group, consisting of mothers who had premature babies which developed sepsis in the NNUH ICU. These interactions have proven hugely valuable in understanding how any changes to practice which may follow this study will be accepted as well as being an opportunity to communicate directly to a highly relevant sector of the public.
Antimicrobial resistance is undoubtedly of great interest to the general public. Our recent work with biocides and potential for roles in selection of AMR has been featured by the media including live interviews on television and print press reports around the world. The Quadram Institute Bioscience, NNUH and UEA all have press teams which are used to collaborating. These teams will handle media enquiries and write press releases: they are skilled at maximising exposure of interesting research developments to national and international media and we will work closely with them on presenting this topic to as wide an audience as possible.

Industry:
Development of novel biocidal formulations and understanding impacts of current products are major goals for industry. Webber currently supervises two iCASE students working on biocidal projects with another application submitted. There projects represent collaborations with three separate biocide manufacturers. We aim to disseminate our findings to industry and have initiated regular discussions with Schulke and Mayr (manufacturer of Octenidine), Procter and Gamble and GAMA Healthcare already which we will continue. Our stakeholder meeting in the final year will include industry representation (see letters of support).