The effect of three urease inhibitors on H. pylori viability, urease activity and urease gene expression

Abstract

Background: Treatment of Helicobacter pylori (H. pylori) infections is challenged by antibiotic resistance. The urease enzyme contributes to H. pylori colonization in the gastric acidic environment by producing a neutral microenvironment. We hypothesized that urease inhibition could affect H. pylori viability. This work aimed to assess the effects of acetohydroxamic acid (AHA), ebselen and baicalin on urease activity, bacterial viability and urease genes expression in H. pylori isolates.

Methods: Forty-nine H. pylori clinical isolates were collected. Urease activity was assessed using the phenol red method. The urease inhibition assay assessed inhibitors' effects on urease activity. Flow cytometry assessed the effect of inhibitors on bacterial viability. Real time PCR was used to compare urease genes expression levels following urease inhibition.

Results: Urease activity levels differed between isolates. Acetohydroxamic acid inhibited urease activity at a concentration of 2.5 mM. Although baicalin inhibited urease activity at lower concentrations, major effects were seen at 8 mM. Ebselen's major inhibition was demonstrated at 0.06 mM. Baicalin (8 mM) significantly reduced ATP production compared to untreated isolates. Baicalin, ebselen and acetohydroxamic acid significantly reduced H. pylori viability. Increased urease genes expression was detected after exposure to all urease inhibitors.

Discussion: In conclusion, higher concentrations of baicalin were needed to inhibit urease activity, compared to acetohydroxamic acid and ebselen. Baicalin, ebselen and acetohydroxamic acid reduced H. pylori viability. Therefore, these inhibitors should be further investigated as alternative treatments for H. pylori infection.

Keywords: Helicobacter pylori; bacterial viability; urease activity; urease genes; urease inhibitors.

Figure 1

Urease activity of study's isolates and relation to disease severity. Activity of H. pylori urease was measured as the difference between the optical density measured in 570 nm- OD₅₇₀ (t = 20 min) and initial OD₅₇₀ (t = 0). (A) The urease activity of the different isolates. (B) Urease activity in relation to infection severity. Each experiment was conducted in triplicates, n = 49.

Figure 2

Urease activity of acetohydroxamic acid (AHA)-treated. H. pylori isolates were treated with different concentration of AHA for 15 min, then urease activity was assessed over 20 min by measuring OD_570nm. (A) A representative graph of urease activity of one isolate, which was treated, with three different concentrations of AHA or untreated (Control). (B) Urease activity of 49 isolates treated for 10 min, with three different concentrations of AHA for 15 min was detected at t = 10 min and compared to the control (untreated). ***p < 0.001, n = 49. Each experiment was conducted in triplicates. AHA, acetohydroxamic acid.

Figure 3

Urease activity of isolates treated with baicalin and ebselen. H. pylori isolates were treated with different concentration of baicalin or ebselen for 15 min, and then urease activity was detected by measuring OD at 570 nm, during 20 min. (A) A representative graph of urease activity of one isolate, which was treated with baicalin at three different concentrations or untreated (Control). (B) Urease activity of the isolates treated with three different concentrations of baicalin for 15 min was detected at t = 10 min and compared to the control (untreated). (C) A representative graph of urease activity of one isolate which was treated with ebselen at three different concentrations for 15 min or untreated (Control). (D) Urease activity of the isolates treated with three different concentrations of ebselen for 15 min was detected at t = 10 min and compared to the control (untreated), ***p < 0.001, n = 49. Each experiment was conducted in triplicates. **p < 0.01.

Figure 4

ATP production in H. pylori isolates treated with urease inhibitors. Isolates were treated with the minimal urease inhibitory concentration of each inhibitor (AHA = 2.5 mM, baicalin = 8 mM and ebselen = 0.06 mM) for 24 h. ATP concentration was determined by luminescence. Untreated isolates served as control. ***p < 0.001, n = 47. AHA, Acetohydroxamic acid.

Figure 5

Viability of bacterial isolates treated with different urease inhibitors. Viability of bacterial isolates treated with different urease inhibitors. H. pylori representative isolates were incubated with urease inhibitors for 24 h (AHA = 2.5 mM, baicalin = 8 mM or ebselen = 0.06 mM) and then bacterial viability was measured by flow cytometry. The graphs in (A) present viable (blue) and dead (pink) cells of Control (untreated isolates), 2.5 mM AHA-treated isolates, 8 mM baicalin-treated isolates, and 0.06 mM-ebselen treated isolates. Propidium iodide, which stains dead bacteria, was detected by the FL4 channel, and SYTO9, which stains live bacteria, was detected by the FL1 channel. The graph in (B) present the percent of live bacteria in isolates treated with urease inhibitors, compared to control. *p < 0.05, ***p < 0.001, n = 10. AHA, acetohydroxamic acid.

Figure 6

Urease genes expression following exposure to urease inhibitors. Urease genes expression following exposure to urease inhibitors. H. pylori isolates were treated with urease inhibitors for 24 h, then RNA was extracted and real-time PCR was performed to detect the expression levels of ureA, ureB and the housekeeping gene 16s. *p < 0.05, ** p < 0.01, n = 13. AHA, acetohydroxamic acid; BAI, Baiclain; EBS, Ebselen.