Plates were covered with a Breathe-Easy® sealing membrane to avoid evaporation and incubated for 24 hours at 37°C. The lowest antibiotic concentration that inhibited visible bacterial growth was defined

the MIC. The determined MIC values are listed in Additional file 1: Table S1. Test for #Ipatasertib concentration randurls[1|1|,|CHEM1|]# persister cell formation Chemically defined RPMI 1640 medium was inoculated with 1 × 107 CFU of either exponential or stationary grown cryo-conserved bacteria. Freshly prepared antimicrobial substances were added at a final concentration of 100-fold MIC, if not stated otherwise. Suspensions were incubated with end-over-end rotation at 37°C. Samples were taken after 1, 2, 4, 6, and 8 hours for determination of CFU by serial dilution and plating. For this 100 μl of bacterial suspensions were immediately harvested by centrifugation, once washed in sterile 0.85% NaCl solution and spotted as 10 μl aliquots on sheep blood Columbia agar plates in serial dilutions. Plating of the aliquots

was performed in triplicates and all antibiotic killing experiments were performed at least with two biological replicates. Bacterial colonies were counted 24 and 48 hours after incubation at 37°C to ensure detection of slow growing bacteria. The results were analyzed with the GraphPad Prism 5 software and expressed in CFU/ml on a logarithmic scale. The limit of detection was defined as 100 CFU/ml and lower bacterial numbers were considered BB-94 ic50 not detectable (n. d.). If indicated statistical significance was determined by one-sided Student t test. Heritability of persistence An overnight culture was diluted

to an OD600 of 0.02 in fresh THB medium and further incubated until the early exponential growth phase was reached. Then bacteria were harvested by centrifugation, once washed with PBS, and inoculated in fresh RPMI medium containing 100-fold MIC of the respective antibiotic to a final Cyclic nucleotide phosphodiesterase bacterial concentration of 1 × 107 CFU/ml. The suspensions were incubated at 37°C with moderate end-over-end rotation. Samples were taken hourly as indicated and the CFUs were determined after removal of remaining antibiotics by washings as described above. After 3 hours of antibiotic treatment (surviving) bacteria were collected by centrifugation, once washed in PBS, inoculated in fresh THB medium and grown overnight. This culture was then used to start a new cycle of antibiotic treatment with exponential grown bacteria. This procedure was repeated with three consecutive cycles and the experiment performed at least with two biological replicates. Colonies were counted and CFUs calculated as described above. Test for persister cell elimination To dissect whether the antibiotic tolerant persister population of S. suis strain 10 comprises type I or type II persister cells, we performed a persister cell elimination test as described by Keren et al.[14], with some modifications. Briefly, an overnight culture of S. suis strain 10 was adjusted to OD600 = 0.