FIGURE 1.

Persister character of clinical isolates. (A) Illustration of the persister assay in stationary phase cultures for ATCC 25923 (reference strain; black symbols and curve), one typical susceptible isolate with low-persister character (S-LP; 69687; red symbols and curve), and one typical resistant isolate with high-persister character (R-HP; 13890; blue symbols and curve). The graph shows the reduction in CFUs over time of incubation with moxifloxacin at 100 × MIC. The data were used to fit one phase exponential decay with plateau functions (R² = 0.99, 0.98, and 0.98). The dotted vertical line shows the time point (5 h) selected for performing the persister assay in the whole collection. (B) Relative persister fraction of each isolate stratified according to its susceptibility to moxifloxacin using EUCAST interpretive criteria (susceptible: MIC ≤ 0.25 mg/L). The graph shows the relative persister fraction [% persisters for the isolate/% persisters for ATCC 25923 (tick dotted line)]. A low-persister phenotype was attributed to isolates with a persister fraction ≤10 (thin dotted line) and a high-persister phenotype, to isolates with a persister fraction >10. On this basis, 3 subgroups were defined, namely susceptible isolates with low-persister fraction (S-LP, in red), susceptible isolates with high-persister fraction (S-HP, in green), and resistant isolates with high-persister fraction (R-HP, in blue), with dark and light colors corresponding to isolates from Vietnam (VN) or Belgium (BE), respectively. Individual data are shown together with the mean value and the 95% confidence interval. Persister tests were performed in 3 independent experiments each run in duplicate, the mean value for each isolate corresponding to one symbol. Statistical analysis: Mann-Withney test comparing (a) all moxifloxacin susceptible (MIC ≤ 0.25 mg/L) vs. all moxifloxacin-resistant (MIC ≥ 1 mg/L) isolates (no isolate with a MIC of 0.5 mg/L in this collection) or (b) S-LP vs. S-HP isolates: p < 0.0001. Kruskal–Wallis test comparing these three subgroups shows that the persister fraction is significantly different between S-LP and S-HP, but not between S-HP and R-HP (not shown on the graph).