Antibiotic tolerance profile of starved planktonic and biofilm bacteria.

<p>Static cultures were grown for 24 h in M63B1_Gluc for wild-type prototroph TG1 (WT) and with the addition of 25 µg ml⁻¹ of leucine, lysine, or cysteine for the corresponding auxotrophs. Planktonic bacteria from the 24-h static culture were removed from each well and therefore separated from the attached-biofilm cells. (A, C) Planktonic bacteria were then collected, spun, washed and treated in parallel with the biofilm bacteria (B, D) with ticarcillin (100 µg ml⁻¹, 100× MIC) or ofloxacin (5 µg ml⁻¹, 80× MIC) in the absence of glucose for WT prototroph TG1 (M63B1 medium) or amino acids for the different auxotrophic mutant strains (M63B1_Gluc medium) for 24 h. Survivor cells were quantified by viable cell counts. Percent survival represents the tolerant population after 24 h of treatment compared to the total number of CFU prior to addition of antibiotics. Equivalent CFU were present in all compared planktonic populations before antibiotic treatment. Data represented are means ± SEM of at least three replicates. Asterisks indicate values significantly different from no starvation condition by the two-tailed unpaired t test: * <i>P</i>≤0.01, ** <i>P</i>≤0.001, *** <i>P</i>≤0.0001 and n.s. (not significant). The genotypes of the three amino acid auxotrophic mutants constructed in the WT prototroph TG1 genetic background are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003144#pgen.1003144.s007" target="_blank">Table S1</a> or as follows: leucine (Leu: Δ<i>leuC</i>::GB), lysine (Lys; Δ<i>lysA</i>::KmFRT), and cysteine (Cys; Δ<i>cysD</i>::KmFRT).