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Fernanda Paganelli

Thursday 26 February 2015

Biogenesis of Enterococcus faecium biofilms

Promotor: Prof. dr. M.J.M. Bonten
Defence: 26 February 2015

Nosocomial infections caused by Enteroccocus faecium have rapidly increased in the last three decades and treatment options become more limited. This is not only due to increasing resistance to antibiotics, but also because of biofilm-associated infections. Hospital-adapted pathogenic E. faecium is characterized by the presence of antibiotic resistance genes, as well as pathogenicity islands, capsule loci and other variable traits. These factors contribute to difficult-to-treat infections, frequently biofilm mediated, such as device-associated, urinary tract and surgical site infections. Biofilm is defined as a population of bacteria, encased in a matrix of exopolymeric substances, attached to various biotic and abiotic surfaces, usually at a solid-liquid interface. The transition from planktonic to the sessile state is triggered by environmental signals. In natural ecosystems, in general, nutrient availability is limited and, under these conditions, biofilm formation is an important adaptation for survival of microorganisms [9]. Biofilms can be formed on abiotic surfaces such as minerals, and on biotic surfaces, such as plants, animals, other microbes and also the human body.
Several processes have been described to be involved in specific steps of biofilm formation in different bacterial species; however, insights into E. faecium biofilm formation is limited. Therefore, the aim of the research described in this thesis was to identify new mechanisms involved in biofilm formation in E. Faecium and new potential therapeutic options to treat biofilm infections. Two distinct approaches were applied and described in this thesis, a targeted approach, based on molecular insights into biofilm biogenesis of other bacterial species, and an unbiased approach, based on Microarray-based Transposon Mapping (M-TraM), in combination with validation in in vitro and in vivo assays of targeted deletion mutants. Using these two approaches, we elucidated five new mechanisms involved in biogenesis of E. faecium biofilms, and a list of potential biofilm associated genes.
In chapter 2, the role of proteins and eDNA in the biofilm matrix is described in a set of community and hospital-associated E. faecium strains, in which we confirmed the role of the Secreted antigen A (SagA) protein in biofilm stability of hospital-associated E. faecium strains. In chapter 3, the major autolysin (AtlAEfm) was identified and its role in DNA release in the biofilm matrix and proper localization of surface proteins. Moreover, in this chapter we also identified a new autolysin-independent mechanism of DNA release. In chapter 4, the Enterococcus Biofilm Regulator B (ebrB) was identified as regulator of esp and its role in biofilm formation and gastrointestinal colonization was elucidated. In chapter 5 we used an unbiased approach based on Microarray-based Transposon Mapping (M-TraM) to identify genes essential for biofilm formation in vitro. M-TraM identified the yajC gene as most essential in this process. Deletion of yajC confirmed its role in biofilm formation in vitro but also in a rat endocarditis model. In addition, we described how yajC is involved in cell envelope stability in E. faecium. In chapter 6, we applied M-TraM to directly identify genes involved in infective endocarditis in an in vivo model, in rats. With this approach, we identified a putative phosphotransferase system (PTS), named fruA, which is highly enriched in E. faecium clinical isolates and absent in commensal isolates. By constructing a fruA deletion mutant we confirmed its role in biofilm formation in the presence of human serum. Finally, the findings and future perspectives of E. faecium biofilm treatments described in this thesis are discussed in chapter 7. This thesis increased our insights in the biofilm formation process and in E. faecium pathogenesis.