(
A) Kill kinetics of
M. smegmatis mc
26 (left) and mc
2155(pUV3583cGFP) after exposure to 0.3 µg/ml ciprofloxacin in 384-well plates in the absence of hygromycin selection. Killing was assessed at each of the timepoints shown on the x-axis by harvesting eight wells, which were then serially diluted in four fold steps, after which 5 µl of each dilution were spotted onto rectangular petri dishes; colonies were counted after 2 and 3 days of growth. Error bars indicate the standard deviation. Outliers were not excluded from this analysis. The nearly identical kill kinetics of the two strains as assayed by cfu formation on non-selective growth medium confirms that the outgrowth of GFP positive wells when mc
2155(pUV3583cGFP) is exposed to ciprofloxacin in the absence of hygromycin selection is unrelated to differential plasmid retention by this highly transformable strain. (
B) Assessment of plasmid loss in mc
2155(pUV3583cGFP) during the time period shown in
A. Colonies were plated on both LB and LB containing 50 µg/ml hygromycin
B. The box-and whisker plot shows the ratio of hyg-resistant to hyg-sensitive colonies at each time point for the eight wells harvested. Open boxes are the untreated mc
2155(pUV3583cGFP), and shaded boxes are mc
2155(pUV3583cGFP) exposed to 0.3 µg/ml ciprofloxacin at t = 0. Whiskers indicate 5–95% CI. Plasmid stability appears to be only slightly affected in the presence of ciprofloxacin, contradicting the possibility that plasmid loss played a role in the pattern of outgrowth observed in the original selection depicted in
Figure 1. (
C) Outgrowth of
M. smegmatis mc
26 ciprofloxacin-resistant mutants under section in 0.3 µg/ml ciprofloxacin over 12 days. A total of 60 wells showed detectable fluorescence by day 12, yielding a mutation frequency of 1.86 × 10
−5 based on the peak population of approximately 4.4 × 10
3 bacteria per well observed 6 hr after initial exposure to ciprofloxacin in panel
A. (
D) Outgrowth, phenotypic, and genotypic characterization of selected wells. Outgrowth during the assay is indicated by increasing A600
nm and is heatmapped to highlight the timing and intensity of growth. After 12 days, representative wells were harvested and further expanded in 4 ml cultures with 0.3 µg/ml CIP prior to assaying for ciprofloxacin and INH sensitivity. PCR sequencing of mutants N12 and L07 revealed identical GG insertions in
rplO, resulting in a frameshift and premature truncation of the protein. The
lfrR open reading frame and
gyrA QRDR were unchanged in these mutants.