The effects of varying acidity on Helicobacter pylori growth and the bactericidal efficacy of ampicillin

Abstract

Background: Penicillins inhibit cell wall synthesis; therefore, Helicobacter pylori must be dividing for this class of antibiotics to be effective in eradication therapy. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens.

Aim: To determine the effects of acidity on bacterial growth and the bactericidal efficacy of ampicillin.

Methods: H. pylori were incubated in dialysis chambers suspended in 1.5-L of media at various pHs with 5 mM urea, with or without ampicillin, for 4, 8 or 16 h, thus mimicking unbuffered gastric juice. Changes in gene expression, viability and survival were determined.

Results: At pH 3.0, but not at pH 4.5 or 7.4, there was decreased expression of ~400 genes, including many cell envelope biosynthesis, cell division and penicillin-binding protein genes. Ampicillin was bactericidal at pH 4.5 and 7.4, but not at pH 3.0.

Conclusions: Ampicillin is bactericidal at pH 4.5 and 7.4, but not at pH 3.0, due to decreased expression of cell envelope and division genes with loss of cell division at pH 3.0. Therefore, at pH 3.0, the likely pH at the gastric surface, the bacteria are nondividing and persist with ampicillin treatment. A more effective inhibitor of acid secretion that maintains gastric pH near neutrality for 24 h/day should enhance the efficacy of amoxicillin, improving triple therapy and likely even allowing dual amoxicillin-based therapy for H. pylori eradication.

Figure 1

Box-whisker plots representing changes in mRNA from transcriptomal analysis. H. pylori were incubated for 4 hours at pH 3.0 and 7.4 in the presence of 5mM urea. At pH 3.0, there was a decrease in mRNA levels of cell wall biosynthesis (HP0159), cell division (HP0748, HP0749, HP1560), and penicillin binding (HP1565) genes. These data suggest that, at pH 3.0, the organisms are non-replicative, not synthesizing cell wall proteins, and have a reduced metabolism, rendering the bacteria ampicillin insensitive at pH 3.0.

Figure 2

H. pylori viability by the live/dead assay method in the absence or presence of ampicillin at neutral and acidic pH. H. pylori incubated for 8 (A) and 16 (B) hours at pH 3.0 in the absence of ampicillin were less viable then at pH 4.5 and 7.4. Ampicillin significantly decreased the viability of the bacteria at both pH 4.5 and 7.4 but had no effect at pH 3.0 at both incubation times.

Figure 3

H. pylori survival in the presence of ampicillin at neutral and acidic pH. After 8 hour exposure to acidity, there was no change in the number of CFUs at pH 4.5 or 7.4, and a marked decline with exposure to ampicillin at these pH levels, but no change with ampicillin at pH 3.0 (A). Similar data were obtained after 16 hour incubation (B).