Figure 1.
Growth kinetics of H. pylori strain TK1402 with CLR.
Pre-cultured cells were grown in Brucella-FCS for 24 h with each concentration in a range of 0.5 µg/ml to 0.001 µg/ml or 0 µg/ml of CLR. After incubation for 24 h under microaerobic and shaking condition at 37°C, the optical densities of the cultures were determined. All of the results were expressed as the means ±1 standard deviation from at least three independent experiments.
Figure 2.
Effect of CLR on strain TK1402 biofilms.
The 2-day (a) and 3-day (b) biofilms were transferred into fresh Brucella-FCS with each concentration (0.5 µg/ml, 0.25 µg/ml, 0.125 µg/ml, 0.063 µg/ml, 0.031 µg/ml or 0 µg/ml) of CLR. After incubation for an additional 24 h under microaerobic and shaking conditions at 37°C, the biofilm biomass was measured with crystal violet. The biofilm biomass was calculated relative to starting biofilm biomass (0.53 and 1.51 for 2-day and 3-day biofilm, respectively), which was set at 1.0. All of the results are expressed as the means ±1 standard deviation from at least three independent experiments.*significantly different (p<0.05) relative to the level of starting biofilm biomass (starting biofilm biomass versus after biofilm biomass exposure to CLR).
Figure 3.
The effect of CLR on cell viability of the strain TK1402 biofilm.
After exposure of 2-day biofilm (closed squares) and planktonic cells (open circles) with each concentration of CLR, viable cells were measured using CFU counting. The initial CFU for 2-day biofilm and planktonic cells adjusted to an optical density at 600 nm of 0.14 were approximately 0.3×108 CFU. The CFU of CLR exposed biofilm or planktonic cells were measured. All of the results are expressed as the means ±1 standard deviation from at least three independent experiments. *significantly different (p<0.05) relative to CFU value (biofilm versus planktonic after treatment with the indicated concentrations of CLR concentration).
Figure 4.
SEM images of TK1402 biofilm after various concentrations of CLR treatment.
(a): the control cells (without treatment of CLR). (b): treated with 0.03 µg/ml of CLR. (c): treated with 0.06 µg/ml of CLR. (d): treated with 0.5 µg/ml of CLR. After treatment with the indicated concentrations of CLR, the biofilms were investigated using SEM. Scale bars are shown at the bottom of each electron microscope image.
Figure 5.
Expression of H. pylori efflux pump genes, HP605 (a), HP971 (b), HP1327 (c) and HP1489 (d).
The quantity of cDNA corresponding to these genes was determined by real-time PCR and was normalized to that of the 16S rRNA gene in each unique reaction. Each experiment was repeated three times with at least duplicate samples from each independently isolated RNA preparation. Data are expressed as the means of all of experiments ± standard deviations. *significantly different (p<0.05) relative to the mRNA expression level (planktonic versus biofilm).
Figure 6.
Induction of CLR resistance mutations in biofilm and planktonic cells.
2-day and 3-day biofilms (A and B) and planktonic cells (C and D) were exposed to each concentration of CLR. After an additional incubation for 24 h, cells were recovered in fresh Brucella-FCS agar, and the generation of CLR resistant mutants was assessed Brucella-FCS agar supplemented with 1.0 µg/ml CLR. When no CLR resistant cells were detected, exposure to CLR was repeated up to 5-times. The graph shows the accumulation ratio of the generated CLR resistant biofilm or planktonic cultures.