Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori

Abstract

Among the several factors that affect the appearance and spread of acquired antibiotic resistance, the mutation frequency and the biological cost of resistance are of special importance. Measurements of the mutation frequency to rifampicin resistance in Helicobacter pylori strains isolated from dyspeptic patients showed that approximately 1/4 of the isolates had higher mutation frequencies than Enterobacteriaceae mismatch-repair defective mutants. This high mutation frequency could explain why resistance is so frequently acquired during antibiotic treatment of H. pylori infections. Inactivation of the mutS gene had no substantial effect on the mutation frequency, suggesting that MutS-dependent mismatch repair is absent in this bacterium. Furthermore, clarithromycin resistance conferred a biological cost, as measured by a decreased competitive ability of the resistant mutants in mice. In clinical isolates this cost could be reduced, indicating that compensation is a clinically relevant phenomenon that could act to stabilize resistant bacteria in a population.

Figure 1

Mutation frequency to rifampicin resistance for H. pylori isolates. The dotted lines indicate the approximate mutation frequencies to rifampicin resistance for wild-type and mutator Enterobacteriaceae and P. aeruginosa (23, 24). Indicated are strains isolated from peptic ulcer patients (stippled), non-ulcer dyspepsia patients (open), and gastric cancer patients (vertical lines). The number of peptic ulcer and dyspepsia non-ulcer strains with mutation frequencies <10⁻⁶ were 3 and 11, respectively, and for strains with mutation frequencies >10⁻⁶ were 7 and 5, respectively. This difference is not statistically significant as calculated by Fischer's Exact test (P > 0.05).

Figure 2

Competition between the in vitro isolated ClaS (67:21) and ClaR (67:21, A₂₁₄₂ → G) pair in mice. Three different doses of bacteria were used: 10⁶ (diamond), 10⁷ (triangle), and 10⁸ (circle). Standard errors are indicated. For some time points an insufficient number of mice were examined to allow a statistical analysis.

Figure 3

Competition between clonally related pre- and posttreatment (ClaS and ClaR) pairs in mice. Pairs G34-G49, G142-G193, and G83-G162 were clinically isolated pairs, whereas the 67:21 ClaS-67:21 ClaR pair was isolated in vitro. One dose of bacteria (10⁸) was used. Standard errors are indicated.

Figure 4

Competition between clonally related pre- and posttreatment (ClaS/ClaR) pairs in laboratory medium. Pairs G34-G49, G142-G193, and G83-G162 were clinically isolated pairs, whereas the 67:21 ClaS-67:21 ClaR pair was isolated in vitro. Standard errors are indicated.

Figure 5

Probability of fixation as function of uN. Curves from left to right: s = −0.001, s = −0.01, s = −0.1, s = −0.2, s = −0.5, s = −1.

Figure 6

Take-over probability as a function of time (days) from Eq. 12. Data used: dash-dot, n₁⁰ = 10³, k₁ = 0.5, k₂=1.5; dashed, n₁⁰ = 10, k₁ = 0.5, k₂ = 1.5; dotted, n₁⁰ = 10³, k₁ = 0.2, k₂ = 1.2; solid, n₁⁰ = 10, k₁ = 0.2, k₂ = 1.2. For each pair of curves the left is with u₂ = 10⁻⁸ and the right one is with u₂ = 10⁻⁹.