Abstract
Biofilm formation is a major pathogenicity strategy of Staphylococcus epidermidis causing various medical-device infections. Persister cells have been implicated in treatment failure of such infections. We sought to profile bacterial subpopulations residing in S. epidermidis biofilms, and to establish persister-targeting treatment strategies to eradicate biofilms. Population analysis was performed by challenging single biofilm cells with antibiotics at increasing concentrations ranging from planktonic minimum bactericidal concentrations (MBCs) to biofilm MBCs (MBCbiofilm). Two populations of “persister cells” were observed: bacteria that survived antibiotics at MBCbiofilm for 24/48 hours were referred to as dormant cells; those selected with antibiotics at 8 X MICs for 3 hours (excluding dormant cells) were defined as tolerant-but-killable (TBK) cells. Antibiotic regimens targeting dormant cells were tested in vitro for their efficacies in eradicating persister cells and intact biofilms. This study confirmed that there are at least three subpopulations within a S. epidermidis biofilm: normal cells, dormant cells, and TBK cells. Biofilms comprise more TBK cells and dormant cells than their log-planktonic counterparts. Using antibiotic regimens targeting dormant cells, i.e. effective antibiotics at MBCbiofilm for an extended period, might eradicate S. epidermidis biofilms. Potential uses for this strategy are in antibiotic lock techniques and inhaled aerosolized antibiotics.
Highlights
Biofilm formation is a major pathogenicity strategy of Staphylococcus epidermidis causing various medical-device infections
We have previously proposed the existence of three cell subpopulations residing within S. epidermidis biofilms; normal cells that are rapidly killed by antibiotics, tolerant-but-killable (TBK) cells that only respond to high concentrations of antibiotics, and dormant cells that resist very high concentrations of antibiotics; the latter two comprise persister cells[35]
Three-dimensional reconstruction of confocal laser scanning microscopy (CLSM; see supplementary information for experimental details) images showed a large number of survivors residing in biofilms after exposure to vancomycin and oxacillin at MBCbiofilm for 24 h (Fig. 1A); the percentage of survivor cells is inconsistent with that of persister cells in biofilms proposed by other researchers and ourselves[3,34,35]
Summary
Biofilm formation is a major pathogenicity strategy of Staphylococcus epidermidis causing various medical-device infections. Many previous studies used antibiotics at 8–100 times minimum inhibitory concentration (MIC) and treatment periods between 2.5 hours (h) to 24 h to isolate persister cells[6,8,41] Though these methods have been validated to select the generally defined “persister cells”, insufficient killing of all vulnerable cells in a bacterial population has been reported[33,42,43]. To obtain comparable data on the prevalence of persister cells within bacterial biofilms, an ideal method would require the separation of individual cells from other biofilm-related factors that affect the performance of antibiotics Such a method should specify concentrations of antibiotics and exposure times needed to effectively eradicate non-tolerant cells. The specific aim was to accomplish two tasks in persister cell research recently raised by Lewis et al.[9], but related to S. epidermidis biofilms: 1) to detail the role of persister cells in antibiotic tolerance of biofilms; 2) to examine the possibility of eradication of persister cell and biofilms using conventional antibiotics in vitro
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