Breathe easy membrane

The selected strains were tested by either serial dilution as describe above or measuring the growth curve in the selected medium. To measure the growth curve, the log phase cells were diluted to OD₆₀₀ = 0.1 in a final volume of 100 μl using fresh medium with or without stresses and cultured in the Costar™ Clear Polystyrene 96-well plates. Each strain was measured in triplicates. The 96-well plate was sealed with Breathe-Easy membrane (Sigma, Lot# MKBZ0331) and then cultivated in the Epoch 2 microplate spectrophotometer (Biotek) at 30 °C. OD₆₀₀ was measured every 10 min. Before each measurement, the plate was shaking at 282 cpm for 30 s. The data were analyzed using the on-board Gen5^TM software to generate growth curve, doubling time and specific growth rate.

For CLARIOStar© (BMG Labtech) plate reader assays, a 100 µL 2YT culture volume was grown in quadruplicate biological repeats (e.g. four single colonies) as an overnight pre-culture for 16 h. The OD₆₀₀ was recorded, before dilution into a fresh 96-well plate with 100 µL 2YT and antibiotics at a starting OD₆₀₀ of 0.02. Cell density was measured in a plate reader at 600 nm in a 96-well Greiner plate to a starting OD₆₀₀ of 0.02. Plates were sealed with a Breathe-Easy® membrane (Sigma) and grown at 30 °C for 6–12 h at 600 rpm. OD₆₀₀ and GFP measurements were recorded every 10 min. The experiment was repeated on two independent days to ensure reproducibility. Error bars represent standard deviation.

Two different lineages were founded from L. pneumophila strain Paris and propagated by serial passages in the presence of KKL-35 or norfloxacin, as previously described (7 (link), 8 (link)). Briefly, a suspension of L. pneumophila strain Paris in AYE was added to a concentration of 10⁸ CFU/ml in a 24-well polystyrene plate with 2-fold KKL-35 or norfloxacin concentrations ranging from 0.5 time to 8 times the MIC that was determined for the parental strain (norfloxacin, 0.25 mg/liter; KKL-35, 0.04 mg/liter). The plates were sealed with a Breathe-Easy membrane (Sigma-Aldrich) and incubated for 4 days at 37°C in air without agitation, after which the MIC was noted for each antibiotic. A 1:40 dilution of the bacteria from the well with the highest antibiotic concentration in which growth was observable was transferred to a new plate containing 2-fold KKL-35 or norfloxacin concentrations ranging from 0.5 time to 8 times the MIC of the previous cycle. Serial passages were repeated 10 times, and the experiment was performed twice independently.

For the time-kill assay, L. pneumophila strain Paris was resuspended in AYE medium at 3 × 10⁶ CFU/ml with a range of 2-fold dilutions of KKL-35. The tubes were then incubated at 37°C in air with shaking. Every 24 h, serial dilutions were plated on CYE agar and the numbers of CFU were counted. For MIC determination, no CLSI guidelines are available for testing the antibiotic susceptibility of Legionella strains. EUCAST guidelines were recently published but are based on the gradient strip test. KKL-35 strip tests are not commercially available, and we found the charcoal of CYE medium to seriously impede KKL-35 activity. Therefore, we used the previously described AYE broth microdilution method for MIC determination (44 (link)). Briefly, strains were resuspended in AYE medium and placed into the wells of a 96-well polystyrene plate, and a range of 2-fold dilutions of KKL-35 was added to the cultures. The inoculum (10⁶ CFU/ml) was verified by plating and counting of serial dilutions of the cultures at the beginning of the experiment. The 96-well plate was sealed with a Breathe-Easy membrane (Sigma-Aldrich) to prevent evaporation and was incubated for 48 h at 37°C in air with no agitation. At 48 h, the MICs were determined visually as the lowest concentrations inhibiting bacterial growth.

Biolog GENIII MicroPlates (Biolog, Hayward, CA, USA) was used to investigate the metabolism of evolved populations. Evolved populations stored in glycerol were inoculated onto the Inoculation Fluid-A (IF-A) provided by the manufacturer such that the OD₆₀₀ was between 0.02–0.05. After adjusting the OD₆₀₀, the inoculated IF-A was shaken well and distributed into a Biolog GENIII MicroPlate (100 μl in each well). The plate was then covered with a sterile Breathe-Easy membrane (Sigma-Aldrich, St. Louis, MO) and placed in a plate reader (SPARK, Tecan, Männedorf, Switzerland) at a preset temperature of 37°C. The plate reader was then run with the following parameters: temperature: 37°C, shaking: 250 rpm, OD₅₉₀ measurement: every 15 min, total running time: 36 h. From the OD₅₉₀ measures, area under the curve (AUC) was used as a proxy for metabolic activity. The Biolog assay was repeated for populations from fecal samples collected at weeks 1 and 12 from all 25 mice, with two replicates each.