This project will provide an ideal training for a veterinary graduate in in vitro molecular bacteriology, mathematical modelling and in vivo experimental skills. The PK/PD modelling techniques used will provide ideal training for a veterinary scientist to enter either academia or the pharmaceutical industry, where these skills are desperately needed in research which predicts drug dosing regimens which are both optimally efficacious and least likely to induce resistance in clinical use of antimicrobial drug products. Hypothesis The emergence and spread of resistance to antimicrobial drugs in porcine bacterial pathogens can be minimized by establishing dosage schedules that optimize bactericidal effects and by determining the mechanisms through which resistance arises. Aims Two key pathogens (A. pleuropneumonia, P. multocida) and the g.i.t. commensal potential pathogen (E. coli) and 4 drugs will be studied. The project will: (a) establish by PK-PD modeling methods, concentrations of each antimicrobial drug required to achieve defined levels of bacteriostatic and bactericidal effects and eradication of bacteria; (b) identify resistance and define the molecular mechanisms involved; and (c) design dosage schedules based on the derived PK-PD parameters and bacterial drug resistance findings. Investigational Plan In years 1 and 2, 6 porcine strains of each of the organisms, A. pleuropneumoniae, P. multocida and E. coli will be obtained from VLA centres. For each strain, in two matrices [Mueller Hinton Broth (MHB) and pig serum], MIC, MBC and 24-hour time-kill curves will be established in vitro. Serum will be used in addition to the conventional artificial medium (MHB), as it is a more therapeutically relevant matrix than MHB. Data will be obtained for 4 drugs, florfenicol, marbofloxacin, oxytetracycline and tiamulin, each representing a drug class with differing pharmacodynamic properties, using several multiples ranging from 0.5 to 8.0 x MIC. The time-kill data will be subjected to PK-PD modeling programmes to elucidate AUC/MIC values required for bacteriostatic, bactericidal and eradication responses. From these data dosages will be determined to provide bactericidal and eradication effects. Resistance of the selected bacteria to the antimicrobial drugs will be induced in vitro with 2-10 passages . In order to associate drug resistance with doses, a range of drug concentrations will be used for induction. The development of resistance will be identified by MIC tests and determination of the resistant genes. When the resistance is established, the in vitro experiments will be carried out using the drugs at MIC and MBC concentrations and the bacteria carrying or not carrying resistant genes. Bacteria will be isolated and their DNAs or total RNAs extracted for determination of resistant genes using PCR and qPCR technology. The proposed resistant genes are FlorA for florfenicol, a group of tet and otr (such as A-E, G and Y) for oxytetracycline; gyrA, parC and acrAB for marbofloxacin and cfr methyltransferase for tiamulin. In years 3 and 4 investigations will be conducted in vivo in a porcine model of enzootic pleuropneumonia currently in use in our laboratory. Based on the findings relating to PK/PD modelling and resistance pattern monitored in years 1 and 2, two drugs will be selected. Each will be administered in the pleuropneumonia model. Appropriate clinical, bacteriological, pathological and pharmacokinetic measurements will be made. These will include: (a) establishing plasma concentration-time profiles; and (b) harvesting organisms from the lung at post mortem and from faeces (E. coli) prior to induction of disease and at post mortem to identify any reduced sensitivity to the drugs and the molecular mechanism of the resistance, for comparison with in vitro studies. Data will be used to calculate optimal dosing regimens for clinical use.

Diseases caused by a range of parasites have a devastating impact on the health, welfare and productivity of sheep and cattle. Infections affect milk production, weight gain of young animals and in some cases, can cause acute disease and rapid death. To avoid the devastating consequences of parasitic infection, farmers use medicines to prevent disease. However resistance to many of these medicines is developing, meaning they are becoming less effective on many farms. To try and prevent further development and spread of resistance, through industry advisory bodies such as Control of Parasites Sustainably (COWS) and Sustainable Control of Parasites in Sheep (SCOPS) recommend diagnosing infection before animals are treated. However in many cases we lack diagnostic tests that can provide rapid results to guide treatment. The aim of this project is to develop and deliver pen-side tests, that farmers can use and that provide diagnostic information within 10 minutes. We will build on existing work, in which we have produced a lateral flow test to diagnose infection with the parasite, liver fluke. We will work with farmers, vets and animal health advisors, to ensure that pen-side tests are produced and delivered to meet farmers' needs and empower them to take decisions about treatment, supported by decision support systems. In addition to developing a lateral flow test for liver fluke, we will also work on a similar lateral flow test for bovine lungworm, a devastating and acute disease of calves and increasingly adult milking cows. We will evaluate the use of recombinant proteins as the basis for both lateral flow tests and working with a leading biotechnology we will produce proof of principal lateral flow tests, based on recombinant proteins. The architecture of both tests will be based on our existing, first generation liver fluke test. The project will deliver a better understanding of how to encourage the industry to adopt rapid diagnostic tests as they become available. It will demonstrate how results from those tests can be interpreted in a manner that suits most farmers and finally it will deliver a second generation liver fluke lateral flow test and a lungworm diagnostic test to a point where a commercial partner can consider taking them to full commercialisation. The results from the project will be delivered t project partticipants in the first instance and then rolled out to the industry through KE programmes supported by our project partners.

The 2019 World Health Organisation (WHO) report on Antimicrobial Resistance (AMR) identifies it as: "one of the greatest threats we face as a global community." The evolution of drug-resistant bacteria, our over-use of antibiotics and failure to develop new methods for tackling infection could leave us without viable treatments for even the most trivial infections within the next 3 decades. There have been significant efforts by the WHO and national agencies to raise awareness of AMR and reduce the use of antibiotics, but there is still an urgent need to intensify these efforts and, crucially, to develop alternatives. The aim of the programme is to address this need. The programme will consist of 4 interlinked work packages focussed on the core research objectives: (1) The development of human organoid models for studying interactions between bacteria and the tissue microenvironment and larger scale interactions with the host microbiome. (2) New microscopy methods to complement the organoid models and to facilitate rapid characterisation of bacteria for improved diagnosis. (3) New antimicrobial therapeutics and targeted delivery techniques to improve the use of existing antibiotics and provide viable alternatives. (4) "Drug-free" methods for treating infections and promoting immune function thereby further reducing the use of antibiotics and providing methods suitable for large scale environmental/industrial use. These will be supported by 2 parallel translational activities to enable development of the translational pathway and wider engagement with clinical and industrial stakeholders, policy-makers and the public.