Antibiotic regimen based on population analysis of residing persister cells eradicates Staphylococcus epidermidis biofilms

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.

Figure 1. Validation of a new method to isolate single biofilm-embedded cells for persister cell studies.

(A) CLSM of S. epidermidis RP62A biofilms treated with antibiotics at MBC_biofilm suggested isolation of single biofilm cells is necessary for persister cell quantification. (B) SEM of “single cells” isolated from mature biofilms with (left) or without filtration (right). (C) Quantitative comparison of densities (CFU/mL) of suspensions of single biofilm cells before and after filtration. (D) FACS of single biofilm cells before and after filtration.

Figure 2. Population analysis of single biofilm-embedded cells.

Population analysis profiling of single S. epidermidis biofilm cells with antibiotics at increasing concentrations identified three fractions of cells of different antibiotic tolerance. The first fraction represents the sensitive subpopulation; the majority of the population were killed following exposure to antibiotics at concentrations close to MBCs (4–8 mg/L for vancomycin and 0.5–1 mg/L for ciprofloxacin). The second fraction represents the persister cell population, which includes both TBK cells and dormant cells. This is a small population remaining tolerant to antibiotics at concentrations ranging from MBCs to sub-MBC_biofilm. The third fraction became visible on further increasing the antibiotic concentration (for ciprofloxacin) and treatment period (for vancomycin) to eradicateTBK cells and to select dormant cells.

Figure 3. Proportions of S. epidermidis persister cells in two different growth modes.

Total persister cells were isolated by exposing cell cultures to antibiotics at 8 X MIC for 3 h. Dormant cells were selected by exposing cell cultures to antibiotics at MBC_biofilm for 24 or 48 h. The proportion of TBK cells was calculated as the proportion of persister cells minus the proportion of dormant cells. Shown are the averages of three biological repeats in technical triplicates and the standard errors.

Figure 4. SEM of TBK cells and dormant cells isolated from S. epidermidis RP62A biofilms.

(A) TBK cells isolated with ciprofloxacin (3 h, 8 x MIC). (B) TBK cells isolated with oxacillin (24 h, MBC_biofilm). (C) Dormant cells isolated with vancomycin (48 h, MBC_biofilm). (D) Dormant cells isolated with oxacillin (48 h, MBC_biofilm). Scale bar = 5 μm.