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Applied and Environmental Microbiology, September 2007, p. 5507-5515, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.01013-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Monolaurin Resistance in Enterococcus faecalis{triangledown}

Muriel Dufour,1* Janet M. Manson,1,{dagger} Philip J. Bremer,2 Jean-Pierre Dufour,2 Gregory M. Cook,1 and Robin S. Simmonds1

Department of Microbiology and Immunology,1 Department of Food Science, University of Otago, P.O. Box 56, Dunedin, New Zealand2

Received 6 May 2007/ Accepted 30 June 2007

There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 µg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, P.O. Box 56, Dunedin, New Zealand. Phone: 64 3 479 5411. Fax: 64 3 479 8540. E-mail: muriel.dufour{at}stonebow.otago.ac.nz

{triangledown} Published ahead of print on 13 July 2007.

{dagger} Present address: Department of Ophthalmology, Harvard Medical School, and The Schepens Eye Research Institute, Boston, Massachusetts.

Applied and Environmental Microbiology, September 2007, p. 5507-5515, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.01013-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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