Shaken batch cultures of Pseudomonas aeruginosa contain biofilm-like heterogeneity

Two potential sources of differentially tolerant subpopulations of Pseudomonas aeruginosa in batch cultures are aggregation and growth rates. Here, we dissected these effects by treating batch cultures with tobramycin, colistin and a combination after various time intervals. Tobramycin was significantly more effective than colistin at early time points, while both showed similar efficacy at 24 and 48 h. Combination therapy was most effective at later stages, indicating the emergence of tolerant subpopulations. Reactivating growth of stationary-phase cultures by treating in fresh media altered antibiotic tolerance, further supporting the role of growth rate in tolerance. To examine the effect of aggregation, we separated batch cultures using filtration as well as examined the WT against a hyper-aggregating ΔwspF mutant. Aggregates demonstrated lower growth rates than single cells. However, microcalorimetry and colony enumeration after treatment revealed that aggregation only partially explained the variance associated with antibiotic treatment. Given that tobramycin and colistin target metabolically distinct subpopulations, this suggests that overall metabolic rate plays a larger role than aggregation regarding antibiotic tolerance in batch cultures.