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Approaches to control multi-resistant enterococci: studies on molecular ecology, horizontal gene transfer, fitness and prevention.

Summary

In European hospitals up to 60% of nosocomial infections are nowadays caused by antibiotic-resistant pathogens, including vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (VRE). VRE are among the most resistant opportunistic pathogens and have become the paradigm for the post-anti-microbial era. Genetic population analyses of Enterococcus faecium revealed the presence of a High-Risk Enterococcal Clonal Complex (HiRECC) resistant to multiple antibiotics and responsible for the vast majority of nosocomial VRE infections and hospital outbreaks worldwide. In this proposal, the evolutionary development of HiRECC in E. faecium and E. faecalis will be further unravelled and combined with new knowledge on intra- and inter-species gene transfer (including antibiotic resistance genes) and biological fitness costs of hospital adaptation.

Problem

Vancomycin-resistant enterococci (VRE) have become the paradigm of the post-antibiotic era. Nosocomial VRE-infections are rising in all European countries, with proportions >10% among enterococcal bloodstream infections in 9 countries in 2005 (Fig. 1). From a microbial perspective, emergence of VRE represents a dual-edged sword: they are amongst the most resistant opportunistic nosocomial pathogens with an increasing impact on patients’ health care. Moreover, and maybe even more important, their unprecedented capacity of genetic exchange make them perfect hubs for resistance genes facilitating horizontal gene transfer amongst bacterial species, most notably to MRSA. The emergence of VRE in European hospitals, where MRSA has been firmly established, creates a dooming perspective of large-scale horizontal transfer of resistance genes. In Europe different reservoirs of antibiotic-resistant enterococci (Enterococcus faecium and Enterococcus faecalis) exist: sewage, farm animals (pigs, calves and poultry), healthy humans and hospitalized patients. Interestingly, E. faecium strains colonizing different reservoirs have markedly different genetic characteristics (host-specificity) and a specific clonal complex (CC17), apparently well adapted to the hospital environment is associated with global spread within and between healthcare settings. In such settings, enterococci disseminate through high-efficient patient-to-patient transfer; via temporarily colonized staff or through contaminated medical instruments and surfaces. The abundance of antibiotic-resistant enterococci among farm animals in Europe has been linked to the massive use of antibiotics for therapy, prophylaxis and growth promotion. Importantly, E. faecium belonging to CC17 have never been isolated from animals. From the animal reservoir, enterococci spread to humans through the food chain and, once colonizing the human gut, animal-derived resistance determinants (such as vancomycin resistance genes) can be transferred to human-specific enterococci. Driven by the selective pressure in hospitals, acquisition of resistance determinants and other adaptive mechanisms like virulence genes, have favoured the emergence and spread of High-Risk Enterococcal Clonal Complexes (HiRECC) in which enterococci have progressively changed from innocent commensals to feared multi-resistant hospital-adapted pathogens.

Aim

The main project objectives are to determine the population structure of enterococci and the evolutionary development of HiRECC, to identify yet unknown HiRECC in E. faecalis and E. faecium, to increase our knowledge on transfer of resistance within Enterococcus species and between Enterococci and other bacterial species, and to improve our understanding of the biological fitness costs of hospital adaptation of Enterococci (Fig. 2). Furthermore, we aim to modulate human enteric colonisation with HiRECC by applying protective commensals and select viable candidates for immunotherapy. Elucidation of the population structure of both enterococcal species is a prerequisite for effective control of multi-resistant enterococci. Such knowledge also enables reconstruction of the evolutionary past of a species and prediction of its success in the future. Furthermore, essential insights in species adaptability and ecology (distribution, survival, and persistence (fitness) of bacteria and their genes in different environments) will be derived. For all these reasons identification of HiRECC is a fundamental aspect of our approach. Understanding processes that have lead to adaptation would provide fundamental insights in bacterial pathofysiology and will contribute to intervention strategies to reduce fitness c.q. adaptability of strains and identification of novel targets for antibiotics and immunotherapy. Knowledge on horizontal transfer of resistance is crucial to assess the risk of emergence of multiple antibiotic resistance among HiRECC and other pathogens.

Expected results

(1) Improved MLST scheme for E. faecium and E. faecalis with an improved resolution based on polymorphism in housekeeping genes and surface and extra cellular protein genes leading to the identification of HiRECC, community and animal associated CCs.

(2) A typing scheme for resistant plasmids to construct a catalogue of resistance determinants, transposons, and plasmids present in different host groups (hospital and non-hospital, animals, and the environment).

(3) In vitro and in vivo models to determine fitness costs related to different hospital environmental subspaces.

(4) Insight in ecological and bacterial determinants that promote hospital adaptation of enterococci in hospital environmental subspaces.

(5) A list of HiRECC associated genes, a collection of HiRECC mutants, and a redesigned E. faecium microarray, containing more genes from other E. faecium strains including plasmid encoding genes and the E. faecium PAI associated genes.

(6) A list of candidate molecules for immunotherapy and knowledge on the relations between virulence factor expression and growth conditions.

Potential applications

Antibiotic resistance clearly constitutes a major threat to public health. Drug resistance among bacteria is increasing, both in Europe and in North-America. Ironically, those persons admitted to hospitals are amongst the most vulnerable populations exposed to increasingly intractable Enterococcus infections, where VRE are causing deaths across the developed world each year. More and more European countries report hospital outbreaks of VRE, with nine European countries showing prevalence rates of VRE among nosocomial bloodstream infections above 10% (Fig. 1). Identification of HiRECC as distinct genetic subpopulations, improved knowledge on molecular ecology of mobile genetic elements and improved understanding of biological fitness costs of hospital adaptation of HiRECC will enable new strategies to reduce spread of resistance and infections among hospitalised patients. Furthermore, the identification of HiRECC-specific candidates for immunotherapy represents a unique opportunity to develop vaccination (passive and active immunization) as a new concept to prevent infection and colonization, respectively, with multi-resistant HiRECC.

Key words: Enterococcus faecium, Enterococcus faecalis, VRE, antibiotic drug resistance, horizontal gene transfer, population structure, genetic evolution, niche adaptation, colonisation modulation, Immunotherapy