Risk of development of in vitro resistance to amoxicillin, clarithromycin, and metronidazole in Helicobacter pylori

Abstract

We have studied initial killing, morphological alterations, the frequency of occurrence, and the selective growth of resistant subpopulations of Helicobacter pylori during exposure to amoxicillin, clarithromycin, or metronidazole by bioluminescence assay of intracellular ATP levels, microscopy, and a viable count assay. We found an induction of spheroplasts and a decrease in intracellular ATP levels after 21 h of exposure to high concentrations of amoxicillin. During clarithromycin exposure the onset of a decrease in intracellular ATP levels started after prolonged incubation, and with the highest concentration of clarithromycin an induction of coccoid forms was seen after 68 h. Metronidazole exposure resulted in the strongest initial decrease in intracellular ATP levels, and coccoid forms were seen after 21 h of exposure to high concentrations of metronidazole. Amoxicillin caused a low-level increase in resistant subpopulations, which indicates a need for surveillance of the amoxicillin susceptibility of H. pylori in order to detect decreasing susceptibility. No increase in the numbers of resistant subpopulations was demonstrated during clarithromycin exposure. Metronidazole selected resistant subpopulations, which caused high-level resistance in H. pylori.

FIG. 1

Monitoring of growth of H. pylori assayed by bioluminescence assay of ATP levels in bacteria in an unexposed culture (▪) and cultures exposed to amoxicillin at 0.008 (•), 0.015 (∗), 0.03 (○), and 0.25 (◊) μg/ml.

FIG. 2

Population analysis of H. pylori in unexposed cultures (▪ and □) and cultures exposed to amoxicillin at 0.008 (•), 0.015 (∗) and 0.03 (○) μg/ml.

FIG. 3

Monitoring of growth of H. pylori assayed by bioluminescence assay of ATP levels in bacteria in an unexposed culture (▪) and cultures exposed to clarithromycin at 0.008 to 0.25 μg/ml (◊, 0.250 μg/ml; ⧫, 0.125 μg/ml; ▴, 0.060 μg/ml; ○, 0.030 μg/ml; ∗, 0.015 μg/ml; •, 0.008 μg/ml).

FIG. 4

Population analysis of H. pylori in unexposed cultures (▪ and □) and cultures exposed to clarithromycin at 0.008 (•) and 0.015 (∗) μg/ml. 5E-04, 0.0005.

FIG. 5

Monitoring of growth of H. pylori assayed by bioluminescence assay of ATP levels in bacteria in an unexposed culture (▪) and cultures exposed to metronidazole at 0.5 to 4 and 32 μg/ml (⧫, 32 μg/ml; •, 4 μg/ml; ∗, 2 μg/ml; ○, 1 μg/ml; ◊, 0.5 μg/ml).

FIG. 6

Population analysis of H. pylori in an unexposed cultures (▪, □, and ▴) and cultures exposed to metronidazole at 1 to 4 μg/ml (○, 1 μg/ml; ∗, 2 μg/ml; •, 4 μg/ml).