Fig 1.
Growth curves of E. coli K-12.
(a) Growth curves of E. coli K-12 in three different media. Media: the blue plot indicates LB; red, NB and green, Ψ. (b) Growth curves of E. coli K-12 at four different pH values in the same LB medium. The blue plot indicates pH 3; red pH5; green pH 7 and purple pH 9. Six replicate growth curves were conducted and a typical growth curve for each condition is shown; the other five growth curves showed similar dynamics.
Fig 2.
The curve was built from 20 different gradient concentrations of trimethoprim; see Supporting S1 Text for information on the concentrations of trimethoprim used to construct the standard curve.
Fig 3.
Growth characteristics of E. coli.
(a) Growth curves of E. coli K-12 at pH 5 (dashed line) and pH 7 (solid line). For pH 5, the dashed blue line represents control samples, dashed red indicates samples challenged with 0.8 mg L-1 of trimethoprim added at the beginning of the lag phase (t = 0 h) and dashed green denotes samples challenged with 0.8 mg L-1 of trimethoprim and added at mid-exponential phase (t = 5 h). For pH 7, the solid purple line represents control samples, solid light blue indicates samples challenged with 0.8 mg L-1 of trimethoprim added at the beginning of the lag phase (t = 0 h) and solid orange denotes samples challenged with 0.8 mg L-1 of trimethoprim and added at the exponential phase (t = 5 h). (b) Column chart representing relative E. coli intracellular levels of trimethoprim after challenging with 0.8 mg L-1 of the drug at pH 5 (red columns) and pH 7 (blue columns) at different growth stages (time = 0 and 5 h) as detected by LC-MS analysis after cells were grown for a total of 18 h. Six replicate growth curves were conducted and a typical growth curve for each condition is shown; the other five growth curves showed similar dynamics.
Fig 4.
PCA and DFA on FT-IR spectra reveal pH and trimethoprim effects.
(a) PCA scores plot of PC1 vs. PC2 after CO2 removal around 2350 cm-1 and EMSC scaling. The total explained variance (TEV) of PC1 is 78.9% and for PC2 is 12.8%. (b) PC-DFA score plots of pH 5 and 7 samples. 20 PCs were extracted from PCA and used as inputs to DFA. These 20 PCs explain 99% of TEV; the legend in the plot shows the 95% confidence interval (CI) for the correct classification of the eight conditions. C, control.
Fig 5.
Multi-block PCA score plots from FT-IR spectra.
The plot shows the relationship between the effect of different concentrations of trimethoprim (0, 0.003, 0.03, 0.2 mg L-1) and that of different pH levels. Block scores plots showing the distribution of samples with different concentrations at (a) pH 5 and (b) pH 7.
Fig 6.
PC-DFA score plots of GC-MS profiles.
25 PCs were extracted from PCA and used as inputs to DFA, explaining 99% of the TEV. The legend in the figure shows the 95% CI for the correct classification of the 8 conditions. Significantly altered metabolites were mined through a combination of PC-DFA loadings and univariate significance testing (Student t-test). C, control.
Fig 7.
Metabolic effects of trimethoprim challenge on E. coli K-12 at pH 5 and pH 7.
When partially ionized at pH 7, trimethoprim is seen to impact on metabolism directly associated with the dihydrofolate pathway, as well as off-target effects upon nucleotide, sugar and amino acid metabolism, glycolysis, the TCA cycle, and up-regulation of osmoprotectants. When trimethoprim is in a poorly ionized state (pH 5), it appears to have a profound effect upon the up-regulation of amino acid metabolism.