PH plays a role in the mode of action of trimethoprim on Escherichia coli.

Haitham Alrabiah,J William Allwood,Royston Goodacre,Elon Correa,Yun Xu,Fernando Rodrigues-Lima

doi:10.1371/journal.pone.0200272

Haitham Alrabiah, J William Allwood + Show 4 more

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https://doi.org/10.1371/journal.pone.0200272

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Abstract

Metabolomics-based approaches were applied to understand interactions of trimethoprim with Escherichia coli K-12 at sub-minimum inhibitory concentrations (MIC≈0.2, 0.03 and 0.003 mg L-1). Trimethoprim inhibits dihydrofolate reductase and thereby is an indirect inhibitor of nucleic acid synthesis. Due to the basicity of trimethoprim, two pH levels (5 and 7) were selected which mimicked healthy urine pH. This also allowed investigation of the effect on bacterial metabolism when trimethoprim exists in different ionization states. UHPLC-MS was employed to detect trimethoprim molecules inside the bacterial cell and this showed that at pH 7 more of the drug was recovered compared to pH 5; this correlated with classical growth curve measurements. FT-IR spectroscopy was used to establish recovery of reproducible phenotypes under all 8 conditions (3 drug levels and control in 2 pH levels) and GC-MS was used to generate global metabolic profiles. In addition to finding direct mode-of-action effects where nucleotides were decreased at pH 7 with increasing trimethoprim levels, off-target pH-related effects were observed for many amino acids. Additionally, stress-related effects were observed where the osmoprotectant trehalose was higher at increased antibiotic levels at pH 7. This correlated with glucose and fructose consumption and increase in pyruvate-related products as well as lactate and alanine. Alanine is a known regulator of sugar metabolism and this increase may be to enhance sugar consumption and thus trehalose production. These results provide a wider view of the action of trimethoprim. Metabolomics indicated alternative metabolism areas to be investigated to further understand the off-target effects of trimethoprim.

Highlights

One of the most effective mechanisms of drug action is enzyme inhibition, often the mechanisms underlying the specific modes of action are not always fully understood [1,2]
For the experiments conducted in this study we chose to use E. coli K-12 strain MG1655 as the full genome sequence of this microorganism is available which has allowed construction of the metabolic pathways in this bacterium
[20] E. coli K-12 is a laboratory strain that has become adapted to life outside of the host and such adaptation may mean that this strain has lost its ability to survive in a human environment

Summary

Introduction

One of the most effective mechanisms of drug action is enzyme inhibition, often the mechanisms underlying the specific modes of action are not always fully understood [1,2]. This is typically because there is often an assumption that an antibiotic is an inhibitor of a specific enzyme (or another target), not realizing that this chemical can have other ‘off-target’ effects, such as binding to unidentified enzymes or indirect interactions with other.

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