Final Report Summary - BIOCHARGE (Investigation into the Biological Cost and Adaptation of the Host to Antibiotic Resistance on mobile Genetic Elements in Enterococcus species) Antibiotic resistance in bacterial populations currently represents one of the greatest challenges to modern medicine throughout the world. Enterococci are commensal bacteria that reside in humans, animals and the environment. They are also nosocomial pathogens responsible for a range of human diseases. One way of enterococci acquires antibiotic resistance genes is via horizontal gene transfer. Conjugative transfer of mobile genetic elements such as conjugative transposons (cTns) carrying resistance genes is one way antibiotic resistance genes are spread between different bacterial populations and is particularly common among the enterococci. The cTn Tn916 is the prototype of a large family of these highly promiscuous genetic elements and has been found in, or been transferred into, a wide range of bacteria.Long-term stability of cTns, carrying antibiotic resistance genes in bacterial populations is influenced by the biological cost that results from the carriage of these elements in a bacterial genome. It is generally accepted that the acquisition of antibiotic resistance genes leads to reduction in fitness of the host bacteria. The objectives of the project are to determine the genetic variability of Tn916 elements in a wide range of enterococci and to determine if these conjugative transposons affect the fitness of their host. The project also aims to determine if the host genome is able to evolve in order to compensate for any observed fitness loss. Description of the work performed in the project and main resultsA total of 100 tetracycline resistant Enterococcus strains were investigated in the project to determine the diversity of Tn916–like elements in these strains. The strains were obtained from Denmark, England, Portugal, Spain, Sweden, China and the USA. At least seven different enterococcal species were included in this study. These were Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus durans, Enterococcus gallinarum, Enterococcus casseliflavus, and unspeciated Enterococcus.Diversity of Tn916 –like elements were analysed by performing Polymerase Chain Reaction (PCRs) designed to amplify a large range of Tn916 –like cTns followed by restriction fragment length polymorphism analysis. Our data suggested a high prevalence of Tn916 in the diverse collection of enterococcal species investigated. Surprisingly there was little variation seen in between the isolated elements which is different from that seen in other genera e.g. the oral streptococci.In order to determine the biological cost of harbouring Tn916-like cTns in these bacteria, we transferred wild-type Tn916 and Tn6000 from suitable donor strains into suitable enterococcal lab strains and tested the growth in competitive fitness assays compared to the original host without the elements.Our results shown that the relative fitness cost of Tn6000 carriage is approximately 3% in all transconjugants tested, however, the cost of harbouring Tn916 varies among transconjugants and can be as high as 16.7%. To understand how Tn916 is maintained in wild-type Enterococcus spp. despite our finding of its high biological cost, a total of 42 E. faecium::Tn916 transconjugants were generated by filter-mating and analysed by competitive growth assays to determine their biological cost. We found that Tn916 could reduce the fitness of E. faecium as much as 46% immediately after acquisition. However, amelioration of such high fitness cost was observed after only 700 generations of growth in the absence of any selective pressure (tetracycline). This could explain, at least in part, the stability and the high prevalence of Tn916 in the enterococcal isolates worldwide.In order to investigate if the transcription of the tet(M) gene and downstream genes in Tn916 was involved in stability a mutation was made in the start codon of the tet(M) leader peptide. This mutation resulted in a permanent state of repression of transcription and therefore we expected the stability of the element to be affected. However after repeated sub-culturing of the mutant followed by replica plating on both tetracycline containing and antibiotic free agar plates we found that stability was still 100% in the colonies tested.Our study shows how bacteria are able to cope with the acquisition of foreign DNA and restore their fitness (reducing the biological cost) within a relatively short amount of time. Our data shows that once acquired the mobile genetic elements of the Tn916 family are inherently stable; at least in the enterococci and that cessation of use of tetracycline is not likely to lead to a decrease in the incidence of this resistance.