Amoxicillin is often implemented in Helicobacter pylori treatment protocols. To date, amoxicillin-resistant H. pylori strains have rarely been detected, and only a total of 14 have been reported in the literature (1). Conspicuously, complete loss of the resistant phenotype was observed after these strains were stored at −80°C. Only one amoxicillin-resistant H. pylori strain has been isolated, in The Netherlands, in which in contrast, the amoxicillin resistance remained stable after repeated cycles of freezing and culture (5). The MIC for this strain was 8 μg/ml, which is relatively low.
Since 1996, we have isolated seven H. pylori strains exhibiting high-level amoxicillin resistance (MIC > 256 μg/ml) (Table 1). Four of these exhibited a stable resistance phenotype (strains ACR3, -4, -5, and -7). Strains ACR1, -2, and -4 were isolated from two 11-year-old girls and one 15-year-old girl with recurrent abdominal pain. They received triple therapy in accordance with the results of antibiotic susceptibility testing, and symptoms subsided. The H. pylori strains ACR3 (stable amoxicillin resistance) and ACR6 (unstable resistance) were isolated from an 8-year-old boy and an 11-year-old girl, respectively. Eradication therapy with amoxicillin, clarithromycin, and omeprazole was empirically initiated but remained without effect. Similarly, H. pylori strains ACR5 and ACR7 (stable amoxicillin resistance) were isolated from a 17-year-old girl and a 40-year-old woman with recurrent duodenal ulcers who had been unsuccessfully treated with triple combinations containing amoxicillin. Following treatment failure, the patients were examined by gastrointestinal endoscopy and H. pylori was isolated from both antral and corpus biopsy specimens. Identification was confirmed by 16S rRNA gene sequence analysis, and antibiotic resistance determinations revealed both strains to be highly resistant to amoxicillin.
TABLE 1.
MICs of antibiotics for seven H. pylori strains with stable and unstable amoxicillin resistance
| H. pylori strain and condition | MIC (μg/ml)a
|
||||||||
|---|---|---|---|---|---|---|---|---|---|
| Amoxicillin | Amoxicillin-clavulanic acid | Penicillin G | Cefuroxime | Cefotaxime | Azithromycin | Clarithromycin | Tetracycline | Metronidazole | |
| ACR1 | |||||||||
| Before storage | >256 | ND | ND | 0.016 | ND | 0.094 | <0.016 | 0.5 | >32 |
| After storage | <0.016b | <0.016 | 0.016 | 0.125 | 0.25 | 0.19 | <0.016 | 0.25 | 32 |
| ACR2 | |||||||||
| Before storage | >256 | ND | ND | 0.19 | ND | 0.25 | <0.016 | 0.064 | 0.094 |
| After storage | <0.016b | <0.016 | 0.125 | 0.016 | 0.016 | 0.064 | <0.016 | 0.094 | 0.094 |
| ACR3 | |||||||||
| Before storage | >256 | ND | ND | >256 | ND | >256 | >256 | 0.25 | 1.0 |
| After storage | >256c | >256 | >32 | >256 | >32 | >256 | >256 | 0.75 | >256 |
| ACR4 | |||||||||
| Before storage | >256 | ND | ND | 0.094 | ND | >256 | >256 | 0.25 | 0.125 |
| After storage | >256c | 0.38 | >32 | >256 | 0.023 | >256 | >256 | 0.047 | 0.047 |
| ACR5 | |||||||||
| Before storage | >256 | 0.064 | ND | >256 | ND | >256 | >256 | 0.38 | 0.064 |
| After storage | >256c | ND | ND | >256 | ND | >256 | >256 | <0.016 | <0.016 |
| ACR6 | |||||||||
| Before storage | >256 | 1.5 | ND | >256 | ND | >256 | >256 | >256 | 1.0 |
| After storage | <0.016b | <0.016 | ND | <0.016 | 0.002 | >256 | >256 | >256 | <0.016 |
| ACR7 | |||||||||
| Before storage | >256 | >256 | ND | >256 | ND | >256 | >256 | 0.75 | >256 |
| After storage | >256c | >256 | >256 | >256 | >256 | >256 | >256 | 0.50 | >256 |
As determined by the E-test method (AB BIODISK, Solna, Sweden). MICs were read after 3 to 4 days of incubation. ND, not determined.
Not stable.
Stable.
After storage at −70°C and reculture, strains ACR1, -2, and -6 lost their amoxicillin resistance, whereas strains ACR3, -4, -5, and -7 remained stable despite repetitive subculture or freeze-thaw cycles (Table 1). Although β-lactamase activity could not be detected by the nitrocefin assay, amoxicillin resistance was overcome by clavulanic acid in three strains (ACR4, -5, and -6). In contrast, strains ACR3 and -7 were also resistant to amoxicillin-clavulanic acid. All four strains with stable amoxicillin resistance were also resistant to cefuroxime.
The antibiotic resistance phenotypes suggest the existence of multiple resistance mechanisms in H. pylori. Antibiotic resistance testing of these bacteria may become increasingly necessary in patients experiencing treatment failures. In four of our cases, amoxicillin resistance correlated with treatment failure. There are general causes for concern. (i) The amoxicillin-resistant phenotype is transferable in vitro to amoxicillin-susceptible strains (5), presumably due to DNA exchange by transformation or a conjugation-like mechanism (2). Colonization of the stomach with other β-lactam-resistant bacteria may also lead to transfer of amoxicillin resistance to H. pylori. (ii) Amoxicillin MICs have been observed to increase upon repeated exposure of H. pylori to the antibiotic (3, 4). To date, MICs for stable amoxicillin-resistant bacteria have been in the range of 8 μg/ml. However, we report the emergence of high-level amoxicillin resistance, the MICs for our strains being >256 μg/ml, which may represent a major threat regarding an effective H. pylori eradication therapy.
In conclusion, it may be prudent to perform antibiotic susceptibility testing of H. pylori at an early stage whenever a treatment failure becomes apparent.
REFERENCES
- 1.Dore M P, Sepulveda A R, Mura I, Realdi G, Osato M S, Graham D Y. Explanation for variability of omeprazole amoxicillin therapy? Tolerance of H. pylori to amoxicillin. Gastroenterology. 1997;112:A105. [Google Scholar]
- 2.Kuipers E J, Israel D A, Kusters J G, Blaser M J. Evidence for a conjugation-like mechanism of DNA transfer in Helicobacter pylori. J Bacteriol. 1998;180:2901–2905. doi: 10.1128/jb.180.11.2901-2905.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Odenbreit S, Till M, Haas R. Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence. Mol Microbiol. 1996;20:361–373. doi: 10.1111/j.1365-2958.1996.tb02623.x. [DOI] [PubMed] [Google Scholar]
- 4.Sörberg M, Hanberger H, Nilsson M, Björkman A, Nilsson L E. Risk of development of in vitro resistance to amoxicillin, clarithromycin, and metronidazole in Helicobacter pylori. Antimicrob Agents Chemother. 1998;42:1222–1228. doi: 10.1128/aac.42.5.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Van Zwet A A, Vandenbrouke-Grauls C M J E, Thijs J C, van der Wouden E J, Gerrits M M, Kusters J G. Stable amoxicillin resistance in Helicobacter pylori. Lancet. 1998;352:1595. doi: 10.1016/s0140-6736(98)00064-6. [DOI] [PubMed] [Google Scholar]