ANIHWA call2: Prevalence and optimised detection of resistance to antibiotics vital for animal and human health

The arsenal of antibiotics for use in medicine is ever decreasing, while the rates of resistance are ever increasing. There is a great need to identify and control all sources of antibiotic resistance, and minimise the transfer of resistance genes and/or bacteria within animals and between animals and humans. The aims of this collaborative project are to address resistance to three critically important classes of
antibiotic classes; polymyxins (colistin), aminoglycosides and carbapenems among gut microflora from pigs and cattle. Colistin is an important antibiotic in the treatment of animals with intestinal infections i.e. Escherichia coli and Salmonella species. Carbapenemases were thought to be restricted to human pathogens, since carbapenems are not used in veterinary medicine. However, they have recently been identified in food animals.

This project will evaluate the rates of resistance (or reduced susceptibility) to colistin, aminoglycosides and carbapenems and among Gram negative enteric bacteria from pigs and cattle, and compare these with the levels of prescribing in the different countries. We will identify the mechanisms leading to the resistance or reduced susceptibility to those antibiotics, and decipher their genetic environment. In cases where no known resistance mechanism can be identified we will utilise whole genome sequencing and functional metagenomics to decipher the novel resistance mechanism. Using this data we can then evaluate the relationship between mobile resistance elements within and between countries. Recently-developed rapid diagnostic techniques for cheap identification of carbapenemase-producing isolates will be applied to investigate carbapenem resistance. We will also develop further diagnostic tools for the identification of colistin and aminoglycoside resistances. The use of antibiotics in veterinary medicine, the emergence of antibiotic resistance and the potential transfer of resistance through the food chain to humans are topics of high priority at both the national and EU policy levels. Comprehensive EU-wide scientific data is required to guide future policy in this area and to ensure the maintenance of both animal and human health and welfare. Traceability and transparency within the food industry are vital to build on the reputation that Europe has built up over many years as an area of safe food production, with high standards in animal health and welfare.

Our research proposal will specifically address the following points:

1. Prevalence of resistance in gut (micro-)organisms

This project will focus on Escherichia coli, Salmonella, Klebsiella pneumoniae, Pseudomonas sp. and Acinetobacter sp. collected from pigs and cattle. The minimum inhibitory concentrations of colistin, aminoglycosides and carbapenems will be determined using sensititre broth dilution, agar dilution and E-test strips using the current guidelines.

2. Develop and adapt diagnostic tools

We will develop rapid diagnostic tests in order to identify and screen those isolates exhibiting reduced susceptibility to colistin, and high level resistance to aminoglycosides. We will also develop adequate phenotypic screening methods, which do not currently exist. Detection of carbapenemase production will be also performed by using rapid tests, i.e. Carba NP test.

3. Elucidate colistin, aminoglycoside, and carbapenem resistance mechanisms

In colistin-resistant isolates the pmrA/B and phoP/Q genes will be amplified by PCR and sequenced to identify mutations. Aminoglycoside resistant bacteria will be investigated for the presence of plasmid-mediated 16S rRNA methylase genes using microarrays, PCR and sequencing. Carbapenemase-producing isolates identified will be screened for the carbapenemase genes encoding class A (KPC) class B (metallo-beta-lactamases VIM, IMP, NDM) and class D (OXA) enzymes. Isolates will also be further analysed using whole genome sequencing (WGS) and functional metagenomics, in order to identify potentially new resistance mechanisms.

4. Identify the genetic environments of the mobile resistance mechanisms

Plasmids extracted from resistant bacteria will be sequenced to identify and characterize the genes conferring multi drug resistance. In the absence of plasmids, whole genome sequencing will be performed to determine the nature of the resistance and the presence of chromosomally integrated elements.

There will be numerous beneficiaries of the programme of research, which will lead to academic, economic and social impact. These include,
1. The academic research community through research outputs and scientific publications generated by the applicants, named researchers and project partners.
2. The BBSRC, by adding to the UK's capacity and research strength in antibiotic resistance research.
3. Industry (small medium enterprises and the pharmaceutical industry), who will have the opportunity to build upon new and novel data which will aid in the design of new targets for antibacterial drug discovery.
4. The public, through an effective programme of dissemination and engagement activities, will be told about our discoveries, which could impact on their health and wellbeing. Information about antibiotic resistance has appeal and interest to a broad lay audience.
5. Government departments across the EU who will be able to use the knowledge gained from the research to help formulate evidence based policy in relation to national and global infection challenges.

This project will deliver on the goals identified as priorities by ERA-Net ANIWHA.

1. Prevalence of antibiotic resistance in gut bacteria of pigs and cattle;
2. Genetic characterization and ecology of drug resistant bacteria and transfer of antimicrobial resistance in livestock production and to humans;
3. Use of newly-developed rapid diagnostic test and selective media affordable for diagnostic and surveillance laboratories;
4. Development of new selective media for colistin non-susceptible and aminoglycoside resistant isolates
5. Analysis and mitigation of resistance genesis with emphasis on resistance critical for human infections.
6. Large scale resistance plasmid database and identification of novel resistance mechanisms
7. Identification of novel resistance mechanisms

The development and validation of novel diagnostic tests will permit their implementation into national monitoring programs. The prevalence results can be exploited to develop political policies. Here we will evaluate to which extent the known and threatening resistance determinants for human health may also represent a threat for the animal health, and potentially if animals may serve as reservoirs for those resistance mechanisms. In addition, investigating novel and unknown resistance mechanisms within Europe among animal isolates will constitute a significant added-value. The development and adaptation of diagnostic tests will enable the first comparable detection of resistance to carbapenems in animal microflora. Given our expertise we expect to discover novel antibiotic resistance mechanisms and potentially mobile colistin resistance genes.

The results will be fed back to the specific national veterinary and public health departments and then fed into future policy making within each country via government reporting. The EU policies will be influenced by the transmission of the results directly to the ECDC and via the national veterinary research laboratories. The diagnostic tools developed could be utilized within the EU for rapid diagnostic testing of isolates. This project also has the potential to generate information on novel resistance mechanisms, which have not yet transferred from animals to humans, thus enabling identification and prevention strategies to be developed against the further transfer of these resistance genes.The direct impacts on animal health and welfare would be the provision of large databases of information, which can be used to minimise the emergence and spread of known and novel resistance mechanisms and rapid identification tests. This data is very important for the creation of risk assessment models to understand the risk of antibiotic use in animals on human health. The indirect benefits are to identify the mechanisms of resistance and potential modes of transfer between animals and humans.