The antibiotics potentiator bicarbonate causes upregulation of tryptophanase and iron acquisition proteins in Escherichia coli.

We have reported that bicarbonate (NaHCO3 ) potentiates the activity of aminoglycosides in Escherichia coli, but the action mechanism was not identified. To eventually understand how NaHCO3 can potentiate antibiotics, we thought that a rational first step was to examine the effect of NaHCO3 separately and to inspect initial gene expression changes triggered by it. In this work, we started by confirming that NaHCO3 can reduce the number of viable E. coli bacteria. We then investigated, via RNAseq, gene expression changes induced by NaHCO3 . There were upregulated and downregulated genes, among the top upregulated genes c. 10-fold increase in expression) was tnaA, the gene encoding tryptophanase, the enzyme that degrades tryptophan to indole. Considering that higher expression of tnaA likely led to increases in indole, we tested the effect of indole and found both growth inhibition and synergy with NaHCO3 . We suggest that indole may participate in growth inhibition of E. coli. The RNAseq analysis also revealed upregulation (≥4-fold) of genes encoding proteins for the acquisition of iron and downregulation (≥16-fold) of genes encoding iron-sulphur-holding proteins; hence NaHCO3 apparently triggered also an iron-deficit response. We suggest that iron deficiency may also be involved in growth inhibition by NaHCO3 . SIGNIFICANCE AND IMPACT OF THE STUDY: Bicarbonate (NaHCO3 ) can enhance the activity of various antibiotics. This work investigated its action mechanism. We carried out a transcriptional analysis in Escherichia coli with the aim of defining initial bacterial changes potentially linked to the enhancing activity of NaHCO3 . Our approach differed from the longer term exposure to NaHCO3 recently used by other researchers, who noticed changes in the bacterial proton motive force. Based on our analysis, we propose two routes possibly linked to the effect of NaHCO3 . Conceivably, those routes are potential targets that could be manipulated by alternative means to augment the effect of antibiotics.

Bicarbonate enhances the in vitro antibiotic activity of kanamycin in Escherichia coli.

UNLABELLED: Growth of enteropathogenic Escherichia coli E2348/69 was inhibited by bicarbonate in a dose-dependent manner, showing approximately 5% growth reduction at 5 mmol l(-1) while kanamycin at 3·12 µg ml(-1) inhibited growth by 15%, yet when kanamycin and bicarbonate were combined at these concentrations, inhibition increased to 80%. Unexpectedly, at bicarbonate concentrations >20 mmol l(-1) enhancement of the antibiotic activity virtually disappeared, i.e. there was a paradoxical Eagle-like effect. How bicarbonate acts is unclear, but neutral or alkaline pH also enhanced the activity of kanamycin. However, several differences indicated a separate effect of bicarbonate. First, bicarbonate inhibited growth more than the corresponding increments in pH. Second, at low concentration, the antibiotic enhancing effect of bicarbonate was stronger than the effect of pH alone. Third, 5 mmol l(-1) bicarbonate significantly enhanced the activity of kanamycin while the corresponding pH had no effect. Fourth, the Eagle-like effect was exclusive of bicarbonate because changes in pH did not induce an analogous behaviour. Notwithstanding the mechanism, the enhancing effect of bicarbonate was indubitable. Consequently, it seems worthwhile to explore further its potential to improve the efficacy of aminoglycosides and maybe even other antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Bicarbonate at a low concentration enhanced the in vitro antibiotic activity of kanamycin and gentamicin. Even though the action mechanism of bicarbonate is hitherto unknown, it seems worthwhile to explore further its capacity to improve the efficacy of aminoglycosides. Clearly, the well-known harmful side-effects of aminoglycosides are a concern. However, it has recently been shown in a fish model that bicarbonate may protect ciliary cells against the damage caused by aminoglycosides. So, it seems possible that bicarbonate could help reduce aminoglycoside dosage at the same time that it might help lessen the damage to auditory ciliary cells in humans.