Bacterial biofilms are intrinsically resistant to antimicrobial treatment, which contributes to microbial persistence in clinical infections. Enterococcus faecalis is an opportunistic pathogen that readily forms biofilms and is the most prevalent enterococcal species identified in healthcare-associated infections. Since intrinsic resistance to multiple antibiotics is common for enterococci, and antibiotic resistance is elevated in biofilm populations, it is imperative to understand the mechanisms involved. Previously, we identified two glycosyltransferase genes whose disruption resulted in impaired nascent biofilm formation in the presence of antibiotic concentrations subinhibitory for parent growth and biofilm formation. The glycosyltransferases are involved in synthesis of the cell-wall-associated rhamnopolysaccharide Epa. Here we examined the effect of epa mutations on the temporal development of E. faecalis biofilms, and on the effects of antibiotics on pre-formed biofilms using scanning electron microscopy. We show that ΔepaOX mutant cells arrange into complex multidimensional biofilms independent of antibiotic exposure, while parent cells form biofilms that are monolayers in the absence of antibiotics. Remarkably, upon exposure to antibiotics parent biofilm cells restructure into complex three-dimensional biofilms resembling those of the ΔepaOX mutant without antibiotics. All biofilms exhibiting complex cellular architectures were less structurally stable than monolayer biofilms, with the biofilm cells exhibiting increased detachment. Our results indicate that E. faecalis biofilms restructure in response to cellular stress whether induced by antibiotics in the case of parent cells, or by deficiencies in Epa composition for the ΔepaOX strain. The data demonstrate a link between cellular architecture and antibiotic resistance of E. faecalis biofilms.
Bibliographical noteFunding Information:
This work was supported by PHS Grants AI58134 and AI122742 to G.M.D, and grant T90 DE0227232 awarded to J.L.D. from the National Institute of Dental and Craniofacial Research. Parts of this work were carried out in the Characterization Facility (CharFac) at the University of Minnesota, which receives partial support from the NSF through the Materials Research Science and Engineering Centers program. The Hitachi SU8230 SEM was provided by NSF MRI DMR-1229263. Glycosyl composition analysis was supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, U.S. Department of Energy grant (DE-FG02-93ER20097) to Parastoo Azadi at the Complex Carbohydrate Research Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Dental & Craniofacial Research, the NIH, NSF, or CCRC.