A biofilm is an assemblage of microbial cells associated with a surface and enclosed in a matrix of polysaccharide material[4]. Early detection of biofilm formation can be an essential step towards management of nosocomial infections[3].

Previous experiments have used a turbidostat environment to subject clinically isolated strains of E. faecalis to an increasing ramp of drug concentration with the antibiotic daptomycin (DAP). First approved in the United States in 2003, daptomycin (DAP) is considered a frontline antibiotic solely prescribed when other antibiotics, such as vancomycin, fail[2]. My current research project has utilized the biofilm formation assay adapted from the Murray group to compare biofilm formation ability of various strains of E. faecalis, and to determine if biofilm formation is correlated with increased drug resistance and specific genetic changes.

It was found that the strain R712 (DAP resistant) exhibited more biofilm formation than strain S613 (DAP sensitive). Furthermore, prior to addition of DAP, biofilm formation in the turbidostat population was increased relative to S613, suggesting growth in the vessel itself selected for biofilm formation independent of drug effects. However, most isolates from the end of the turbidostat experiment (78%) also showed increased biofilm formation compared to S613 (Fig 1). It was determined that specific genetic changes in LiaF, CLS (cardiolipin synthase), and other putative membrane proteins are linked to increased biofilm formation (Table 1). Colonies with an NFQ (74-76) deletion in CLS coupled with a mutation in a membrane protein exhibited the highest amount of biofilm formation, colonies with either the NFQ deletion or a mutation in a membrane protein exhibited an intermediate amount of biofilm formation, and those colonies with neither the NFQ deletion nor a mutation in membrane protein had the lowest amount of biofilm formation. This information may provide valuable drug targets in the future to reduce the virulence of E. faecalis infections.

Future research will focus on further characterization of the genes correlated to biofilm formation. For example, allelic replacement mutants may be used to determine exactly which mutations lead to increased biofilm formation. Also, more research will be conducted on how biofilm-linked mutants affect the activity of their proteins. Finally, microscopy may be used to further study biofilms.

REFERENCES

1. Arias, C.A., and B.E. Murray. (2012). The rise of the enterococcus: beyond vancomycin resistance. Nature Reviews Microbiology, 10, 266-278.

2. Baltz, R., V. Miao, and S. Wrigley. (2005). Natural Products to drugs: daptomycin and related lipopeptide antibiotics. Natural Product Reports, 22(6), 717-741.

3. Fonseca, A., Extremina, C., Costa, L. , Aguiar , A., & Peixe, L. (2010). Optimization of processing conditions for the quantification of enterococci biofilms using microtitre-plates. Journal of Microbiological Methods, 84, 167-173.

4. Heikens, E., Bonten, M., & Willems, R. . (2007). Enterococcal surface protein esp is important for biofilm formation of enterococcus faecium e1162. Journal of Bacteriology, 189(22), 8233-8240.

5. Toledo-Arana, A., Valle, J., Solano, C., Arrizubieta, M., & Lasa, I. (2001). The enterococcal surface protein, esp, is involved in enterococcus faecalis biofilm formation. Applied and Environmental Microbiology, 67(10), 4538-4545.