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1.
Figure 1

Figure 1. Antibiotic-tolerant populations of E. coli K-12 TG1 biofilms.. From: Starvation, Together with the SOS Response, Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin.

Twenty-four-hour biofilms of E. coli TG1 were preformed in M63B1Gluc and treated for a period of 24 h with concentrations up to 100- and 80-times the MIC values for ticarcillin and ofloxacin, respectively. Untreated controls represent 48 h biofilms in which fresh M63B1Gluc without antibiotic was added after 24 h, explaining their values above 100%, represented by a dotted line in each panel. Viable cells of the treated biofilm population were quantified by viable cell counts (A and B) and the XTT-reduction assay (C and D) and were compared to numbers obtained prior to antibiotic treatment. % survival (CFU) values are indicated on top of each bar and are means ± standard error of the means (SEM) of at least six replicates.

Steve P. Bernier, et al. PLoS Genet. 2013 January;9(1):e1003144.
2.
Figure 6

Figure 6. Ofloxacin tolerance in aging biofilms is SOS-response-dependent.. From: Starvation, Together with the SOS Response, Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin.

(A) The influence of the SOS response on the ofloxacin tolerance of aging biofilms was evaluated and compared between biofilms of wild-type TG1 (WT), ΔrecA, and lexAind3. (B) A close-up of the data obtained in panel A for the 24- and 48-h biofilms. Briefly, all biofilms were grown for 24, 48, 72 and 96 h in M63B1Gluc with medium renewal every 24 h and treated for 24 h with ofloxacin (5 µg ml−1, 80× MIC) in M63B1Gluc (no starvation). Survivors were quantified by viable cell counts. Percent survival represents the tolerant population after 24 h of treatment compared to untreated biofilm prior to addition of antibiotics. All compared biofilms had similar numbers of CFUs prior to antibiotic treatment (data not shown). Data represented are mean ± SEM of at least three replicates. Asterisks indicate values significantly different than WT (panel A). Statistics were performed by the two-tailed unpaired t-test: * P≤0.05, ** P≤0.01, *** P≤0.0001, and n.s. (not significant). The genotypes of the different strains used are detailed in Table S1 or as follows: WT (TG1), ΔrecA (TG1ΔrecA::KmFRT ), and lexAind3 (TG1lexAind3).

Steve P. Bernier, et al. PLoS Genet. 2013 January;9(1):e1003144.
3.
Figure 5

Figure 5. Induction of the SOS response and ofloxacin tolerance in aging biofilms.. From: Starvation, Together with the SOS Response, Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin.

(A) Induction of the SOS response was monitored by β-galactosidase assay in aging biofilms and planktonic cells using an E. coli K-12 derivative carrying a psulA::lacZ transcriptional fusion and the biofilm promoting factor F'tet. (B) The influence of biofilm age on ofloxacin tolerance was evaluated in biofilms of the reporter strain psulA::lacZ F'tet (black bars) and wild-type TG1 (white bars). Briefly, all biofilms were grown for 24, 48, 72 and 96 h in M63B1Gluc with medium renewal every 24 h. In (B) biofilms were treated for 24 h with ofloxacin (5 µg ml−1, 80× MIC) in M63B1Gluc, therefore in absence of starvation. Survivors were quantified by viable cell counts. Percent survival represents the tolerant population after 24 h of treatment compared to untreated biofilm prior to addition of antibiotics. Data represented are mean ± SEM of at least three replicates. Asterisks indicate values significantly different than 24-h biofilms. Statistics were performed by the two-tailed unpaired t-test: * P≤0.001 and ** P≤0.0001.

Steve P. Bernier, et al. PLoS Genet. 2013 January;9(1):e1003144.
4.
Figure 3

Figure 3. Antibiotic tolerance profile of starved planktonic and biofilm bacteria.. From: Starvation, Together with the SOS Response, Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin.

Static cultures were grown for 24 h in M63B1Gluc 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 (M63B1Gluc 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: * P≤0.01, ** P≤0.001, *** P≤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 Table S1 or as follows: leucine (Leu: ΔleuC::GB), lysine (Lys; ΔlysA::KmFRT), and cysteine (Cys; ΔcysD::KmFRT).

Steve P. Bernier, et al. PLoS Genet. 2013 January;9(1):e1003144.
5.
Figure 4

Figure 4. The high ofloxacin tolerance exhibited by starved biofilms is SOS-response-dependent.. From: Starvation, Together with the SOS Response, Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin.

The impact of SOS response loss-of-function mutations ΔrecA and lexAind3 on the ofloxacin tolerance of starved biofilms for glucose (A), leucine (B), lysine and tryptophan (C). Briefly, all biofilms were grown for 24 h in M63B1Gluc for all prototrophic strains and with the addition of 25 µg ml−1 of leucine, lysine or tryptophan for corresponding auxotrophic strains. All biofilms were treated for 24 h in M63B1Gluc containing ofloxacin (5 µg ml−1). For the glucose starvation environment, M63B1 without glucose was used instead of M63B1Gluc (Panel A - black bars). Ofloxacin hypertolerance of ΔleuCΔrecA was not restored when adding the vector control plasmid pAM34 (vector), but complete restoration of high ofloxacin tolerance upon leucine starvation was observed by in trans complementation using pAM34recA (precA). Complementation of ΔleuClexAind3 with pAM34recA (precA) failed to fully restore ofloxacin hypertolerance back to leucine auxotroph levels. Both pAM34 and pAM34recA were maintained by the presence of 0.5 mM IPTG. (D) The SOS response mutation ΔrecA (SOS-) did not affect ticarcillin hypertolerance of starved biofilms for leucine (Leu - ΔleuCΔrecA), lysine (Lys – ΔlysAΔrecA) or tryptophan (Trp - ΔtrpAΔrecA). Viable cells of the treated biofilm population were quantified by viable cell counts. Percent survival represents the tolerant population after 24 h of treatment compared to untreated biofilm prior to addition of antibiotics. All compared biofilms had similar numbers of CFUs prior to antibiotic treatment (data not shown). Data represented are means ± SEM of at least three replicates. Asterisks indicate values significantly different by the two-tailed unpaired t test: * P≤0.05, ** P≤0.01, *** P≤0.001, and n.s. (not significant). All strains used here were originally constructed in the WT TG1 genetic background and are detailed in Table S1. The different SOS response mutant strains made in amino acid auxotrophic background are derivatives of ΔleuC::ΔFRT (Leu - leucine), ΔlysA::ΔFRT (Lys - lysine), and ΔtrpA::ΔFRT (Trp – tryptophan).

Steve P. Bernier, et al. PLoS Genet. 2013 January;9(1):e1003144.
6.
Figure 2

Figure 2. Starvation leads to high antibiotic tolerance in biofilms of E. coli TG1.. From: Starvation, Together with the SOS Response, Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin.

The tolerance of non-starved biofilms (white bars; wild-type prototroph TG1 (WT)) to (A) ticarcillin (100 µg ml−1, 100× MIC) or (B) ofloxacin (5 µg ml−1, 80× MIC) was compared to biofilms starved for individual amino acids (black bars; auxotrophic mutants). (C) Addition of exogenous leucine (10 µg ml−1) or glucose (0.4%) restored ofloxacin sensitivity to biofilms. (D) Antibiotic tolerance profiles of biofilms grown and treated in LB-rich medium were indistinguishable from a leucine auxotroph and its WT prototroph. For panels A to C, biofilms of all tested amino acid auxotrophs were grown in M63B1Gluc for 24 h with the addition of 25 µg ml−1 of the required amino acid, while WT was grown in M63B1Gluc only. Antibiotic treatments were performed for a period of 24 h on 24-h biofilms. Conditions of starvation during treatment were achieved by removing the required amino acids (M63B1Gluc) for the respective auxotrophic strains or glucose for the WT prototroph (M63B1). In panel D, biofilms were grown for 24 h and treated for 24 h in LB-rich medium. Surviving cells were quantified by viable cell counts. Percent survival represents viable cells after 24 h of treatment compared to untreated biofilm prior to addition of antibiotics. All compared biofilms had similar numbers of CFU prior to antibiotic treatment (data not shown). Data represented are means ± SEM of at least three replicates. Asterisks indicate values significantly different from conditions of no starvation by the two-tailed unpaired t test: * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.001 and n.s. (not significant). The genotypes of all amino acid auxotrophic mutants constructed in the WT prototroph TG1 genetic background are described in Table S1 or as follows: tryptophan (Trp; ΔtrpA::KmFRT), histidine (His; ΔhisG::KmFRT), methionine (Met; ΔmetA::KmFRT), phenylalanine (Phe: ΔpheA::KmFRT), proline (Pro: ΔproC::KmFRT), tyrosine (Tyr; ΔtyrA::KmFRT), isoleucine (Ile; ΔilvA::KmFRT), arginine (Arg; ΔargH::KmFRT), cysteine (Cys; ΔcysD::KmFRT), lysine (Lys; ΔlysA::KmFRT), and leucine (Leu: ΔleuC::GB).

Steve P. Bernier, et al. PLoS Genet. 2013 January;9(1):e1003144.

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