Staphylococcus aureus persisters tolerant to bactericidal antibiotics

Abstract

Bacterial persister cells are non- or slow-growing reversible phenotypic variants of the wild type, tolerant to bactericidal antibiotics. We analyzed here Staphylococcus aureus persister levels by monitoring colony-forming unit counts of planktonically grown cells treated with six different antimicrobials over time. The model laboratory strains HG001-HG003, SA113 and the small colony variant (SCV) strains hemB and menD were challenged by the compounds at different logs of minimal inhibitory concentration (MIC) in exponential or stationary growth phase. Antibiotic tolerance was usually elevated in SCV strains compared to normally growing cells and in stationary versus exponential phase cultures. Biphasic killing kinetics, typical for persister cell enrichment, were observed in both growth phases under different selective conditions. Treatment of exponential phase cultures of HG001-HG003 with 10-fold MIC of tobramycin resulted in the isolation of persisters which upon cultivation on plates formed either normal or phenotypically stable small colonies. Trajectories of different killing curves indicated physiological heterogeneity within persister subpopulations. Daptomycin added at 100-fold MIC to stationary phase SA113 cells rapidly isolated very robust persisters. Fractions of antibiotic-tolerant cells were observed with all S. aureus strains and mutants tested. Our results refute the hypothesis that S. aureus stationary phase cells are equivalent to persisters, as not all of these cells showed antibiotic tolerance. Isolation of S. aureus persisters of different robustness seems to depend on the kind and concentration of the antibiotic, as well as on the strain used.

Fig. 1. Time-dependent killing of exponential phase cultures

Strains were treated with (A) 10-fold MIC of ciprofloxacin, (B) 100-fold MIC of rifampin, (C) 10-fold MIC of tobramycin, (D) 100-fold MIC of tobramycin, or (E) 10-fold MIC of daptomycin. The limit of detection was 100 CFU/ml throughout all killing experiments. Error bars indicate standard deviations. The plate segment in Fig. 1C shows colonies of untreated HG001 cells (left) and SCV colonies obtained from the HG001 strain 1 h after tobramycin treatment (right). Note also the loss of pigmentation, typical for SCV strains.

Fig. 2. Time-dependent killing of stationary phase cultures

Strains were treated with (A) 100-fold MIC of tobramycin, (B) 100-fold MIC of daptomycin, or (C) 100-fold MIC of daptomycin/Ca²⁺.

Fig. 3. Time-dependent killing of stationary phase SA113 by combined daptomycin and tobramycin treatment

Both antibiotics were applied at 100-fold MIC.

Fig. 4. Long-term killing experiment

Stationary phase SA113 cultures were treated with 100-fold MIC each of daptomycin, tobramycin, ciprofloxacin, rifampin, or penicillin for 21 days. An identical SA113 culture without any antibiotic challenge served as a control.

Fig. 5. Test for heritability of persistence

Exponential phase SA113 cells were challenged with (A) 100-fold MIC of tobramycin or (B) 10-fold MIC of daptomycin/Ca²⁺ for 3 h in four consecutive cycles.