Growth Condition-Dependent Esp Expression by Enterococcus faecium Affects Initial Adherence and Biofilm Formation

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Abstract
A genetic subpopulation of Enterococcus faecium , called clonal complex 17 (CC-17), is strongly associated with hospital outbreaks and invasive infections. Most CC-17 strains contain a putative pathogenicity island encoding the E. faecium variant of enterococcal surface protein (Esp). Western blotting, flow cytometric analyses, and electron microscopy showed that Esp is expressed and exposed on the surface of E. faecium , though Esp expression and surface exposure are highly varied among different strains. Furthermore, Esp expression depends on growth conditions like temperature and anaerobioses. When grown at 37°C, five of six esp -positive E. faecium strains showed significantly increased levels of surface-exposed Esp compared to bacteria grown at 21°C, which was confirmed at the transcriptional level by real-time PCR. In addition, a significant increase in surface-exposed Esp was found in half of these strains when grown at 37°C under anaerobic conditions compared to the level in bacteria grown under aerobic conditions. Finally, amounts of surface-exposed Esp correlated with initial adherence to polystyrene ( R 2 = 0.7146) and biofilm formation ( R 2 = 0.7535). Polystyrene adherence was competitively inhibited by soluble recombinant N-terminal Esp. This study demonstrates that Esp expression on the surface of E. faecium (i) varies consistently between strains, (ii) is growth condition dependent, and (iii) is quantitatively correlated with initial adherence and biofilm formation. These data indicate that E. faecium senses and responds to changing environmental conditions, which might play a role in the early stages of infection when bacteria transit from oxygen-rich conditions at room temperature to anaerobic conditions at body temperature. In addition, variation of surface exposure may explain the contrasting findings reported on the role of Esp in biofilm formation.

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