Helicobacter pylori is a gram-negative microaerophilic bacterium that causes chronic gastritis and peptic ulcer disease in humans and is associated with gastric cancer. Antibiotic resistance in H. pylori is a major cause of therapy failure. In general, bacteria can acquire antibiotic resistance either by spontaneous mutation or by horizontal transfer of resistance genes. A possible mechanism for acquisition of antibiotic resistance by H. pylori is genetic transformation (7). Recently, the mechanism for transformation of H. pylori was suggested to be specific for DNA of members of the genus Helicobacter (6). This proposed genus-specific DNA uptake led us to investigate whether interspecies transfer of antibiotic resistance can take place in Helicobacter.
Previously, metronidazole resistance was reported for Helicobacter acinonychis isolates from captive tigers with ulcers (1). We observed that the H. acinonychis reference isolate (NCTC12686) and H. acinonychis isolate Sheeba (2) both consisted of a large metronidazole-sensitive subpopulation and a small metronidazole-resistant subpopulation. By repetitive subculture of colonies of each subpopulation, both homogeneous metronidazole-sensitive isolates (designated NCTC12686 MtzS and Sheeba MtzS) and fully metronidazole-resistant isolates (NCTC12686 MtzR and Sheeba MtzR; MICs for both were >256 μg/ml) were obtained. DNA was isolated from the metronidazole-resistant strains Sheeba MtzR and NCTC12686 MtzR and used for transformation of two metronidazole-sensitive H. pylori strains through natural transformation, essentially as described by Wang et al. (9). Metronidazole-resistant transformants were obtained for both H. pylori strains (Table 1). In contrast, no metronidazole-resistant colonies were obtained in a mock transformation with phosphate-buffered saline (Table 1).
TABLE 1.
Numbers of antibiotic-resistant colonies obtained upon transformationa
| Recipient strain and donor DNA | Selecting antibioticb | No. of colonies |
|---|---|---|
| H. pylori 26695 | ||
| H. acinonychis NCTC12686 MtzR | Metronidazole | >1,000 |
| H. acinonychis Sheeba MtzR | Metronidazole | >1,000 |
| Saline (no DNA) | Metronidazole | 0 |
| H. pylori J99 | ||
| H. acinonychis NCTC12686 MtzR | Metronidazole | 40 |
| H. acinonychis Sheeba MtzR | Metronidazole | 29 |
| Saline (no DNA) | Metronidazole | 0 |
| H. acinonychis Sheeba MtzS | ||
| H. pylori 1061 MtzR | Metronidazole | >1,000 |
| H. pylori NCTC11637 | Metronidazole | >1,000 |
| H. pylori pRdxA | Metronidazole | >1,000 |
| Saline (no DNA) | Metronidazole | 0 |
| H. pylori 1061 MtzR/ClaR | Clarithromycin | 140 |
| 23S rDNA PCR product of 1061 MtzR/ClaR | Clarithromycin | >1,000 |
| Saline (no DNA) | Clarithromycin | 0 |
| H. acinonychis NCTC12686 MtzS | ||
| H. pylori 1061 MtzR | Metronidazole | 500 |
| H. pylori NCTC11637 | Metronidazole | 57 |
| Saline (no DNA) | Metronidazole | 0 |
| H. pylori 1061 MtzR/ClaR | Clarithromycin | 374 |
| Saline (no DNA) | Clarithromycin | 0 |
Bacteria were transformed with approximately 1 μg of DNA as described previously (8).
Metronidazole, 20 μg/ml; clarithromycin, 2 μg/ml.
Conversely, the metronidazole-sensitive H. acinonychis strains Sheeba MtzS and NCTC12686 MtzS could be transformed to metronidazole resistance with chromosomal DNA isolated from two H. pylori strains which are metronidazole resistant due to known mutations that inactivate the rdxA nitroreductase gene (Table 1). Type strain NCTC11637 is resistant due to a transposon-induced deletion in rdxA (4); the metronidazole-resistant variant of strain 1061 (1061 MtzR) contains the null mutation containing rdxA of strain 439 (5). Transformation with this DNA resulted in high numbers of transformants, in contrast to control transformations (phosphate-buffered saline without DNA), which did not yield any metronidazole-resistant colonies. Additional evidence for interspecies DNA transfer comes from transformation with plasmid pRdxA, which contains a disrupted H. pylori-derived rdxA gene (8). Transformation of H. acynonychis with pRdxA resulted in similar numbers of transformants (Table 1).
Interspecies transformation was not limited to metronidazole resistance, as both H. acinonychis strains could be readily transformed to clarithromycin resistance. Both chromosomal DNA of the clarithromycin-resistant variant of strain 1061 (1061 MtzR/ClaR) (8), which is clarithromycin resistant due to a single base pair mutation (A2142-G) in the 23S ribosomal RNA gene (rDNA) (3), and an 850-bp PCR product of the 23S rDNA of this strain transformed both H. acinonychis strains to a clarithromycin-resistant phenotype (Table 1).
In conclusion, we provide evidence that (i) H. acinonychis is competent for natural transformation and (ii) H. pylori can acquire antibiotic resistance by uptake of DNA from other Helicobacter species and vice versa. To what extent interspecies gene transfer contributes to antibiotic resistance in the genus Helicobacter depends on the relative frequencies of intraspecies transfer, interspecies transfer, and mutation rate in vivo, which remain to be investigated.
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