Spontaneous Mutations That Confer Antibiotic Resistance in Helicobacter pylori

Abstract

In this study, we systematically examined in vitro frequencies and spectra of the spontaneous mutations in Helicobacter pylori that confer resistance to clarithromycin (Cla r Antibiotic resistance is an increasing problem for the treatment of infectious diseases. Bacteria have evolved diverse mechanisms (pathways) of resistance to antimicrobial agents, including control of uptake and efflux of drugs, modification and detoxification of drugs, alteration and protection of the target sites, and acquisition of heterologous resistance genes from external sources. In Helicobacter pylori, the etiological agent of a wide range of gastric diseases, genetic determinants for resistance to several antibiotics, including clarithromycin, metronidazole, ciprofloxacin, and rifampin, have been determined. Remarkably, the known mechanisms of antibiotic resistance in H. pylori are all due to mutations in chromosomal genes. Clarithromycin resistance is associated with mutations in the 23S rRNA gene (22,25), which inhibit the binding of clarithromycin to the ribosome. Ciprofloxacin resistance is due to mutations in the gyrA gene, which encodes the A subunit of DNA gyrase (16), and rifampin resistance results from mutations in the rpoB gene, encoding the ␤ subunit of RNA polymerase (7). For metronidazole resistance, although several different mechanisms may exist, the predominant determinant has been shown to be the mutational inactivation of the rdxA gene that encodes an oxygen-insensitive NADPH nitroreductase (4, 9, 10, 13, 21).)The importance of de novo mutation in developing antibiotic resistance prompted us to ask how mutations occur in H. pylori (28). The first step in elucidating the mechanisms of mutagenesis is to define the background of frequency and specificity of spontaneous mutations. From the pioneering works of Luria and Delbruck (14) and recent developments in determining mutation rates of bacterial populations, it is known that determination of mutation rates is not simple (15,17), and determination of mutation spectra is particularly tedious. In this study we systematically examined the in vitro frequencies and spectra of spontaneous mutations in H. pylori that confer resistance to clarithromycin, metronidazole, amoxicillin, ciprofloxacin, and rifampin. MATERIALS AND METHODSH. pylori strains, growth medium, and antibiotics. H. pylori reference strains 26695, NCTC11639, and UA802 (26), as well as some isolates from University of Alberta Hospital, were used; all are susceptible to the antibiotics tested in this study. H. pylori strains were grown on BHI-YE broth (3.7% brain heart infusion with 0.3% yeast extract and 5% animal serum) or agar plates at 37°C under microaerobic conditions (5% CO 2 , 5% H 2 , and 90% N 2 ). Antibiotics used in this study include clarithromycin (Bayer), metronidazole (Sigma), ciprofloxacin (Bayer), rifampin (Sigma), and amoxicillin (Sigma).MIC test. H. pylori cells were grown for 2 days and suspended in sterile BHI-YE liquid medium, and the turbidity of the suspensions was adjusted to that...

Rifabutin-based 10-day and 14-day triple therapy as a third-line and fourth-line regimen forHelicobacter pylorieradication: A pilot study

“…Wang et al reported that the L525I point mutation induced strong resistance to rifampicin. 22 In our study, the strain harboring the L525I mutation showed resistance to rifabutin. The fact that the rifabutin-resistant strain was eradicated with a 14-day regimen as a fourth-line therapy suggested that there are two possibilities for successful eradication: first, the high dose of PPI may enforce the activity of amoxicillin and rifabutin, and second, rifabutin and amoxicillin may exhibit a synergistic effect on antimicrobial activity.…”

In vitro activity of fluoroquinolone and the gyrA gene mutation in Helicobacter pylori strains isolated from children

“…Moore et al (1995) reported four mutations of the A subunit of the DNA gyrase (GyrA) at amino acid 87 (Asn to Lys), 88 (Ala to Val), 91 (Asp to Gly, Asn or Tyr) and a double substitution at 91 and 97 (Ala to Val) in H. pylori strains with secondary resistance to ciprofloxacin. Moreover, in one in vitro study with the serial passage method, GyrA mutation at amino acid 91 (Asp to Ala, Gly, Asn or Tyr) was also reported in the ciprofloxacin-resistant strains (Wang et al, 2001). Our primary resistant strains showed three point mutations at amino acid 91 (Asp to Tyr, G271T; Asp to Asn, G271A; Asp to Gly, A272G).…”

“…5, 0 . 5 and 8 mg l À1 , respectively (Wang et al, 2001). H. pylori NCTC 49503 was studied as the control organism.…”

“…Oligonucleotide primers gyrAPF1 (59-ATGCATGAATTAGGTCTTACT-39) and gyrAP2 (59-TTCTTCAC TCGCCTTAGTCAT-39), flanking the gene fragment encoding the fluoroquinolone-resistance-determining region, were designed from the H. pylori gyrA gene. These primers were modified versions of the primer pair used by Wang et al (2001). PCR was performed with 30 cycles of denaturation at 94 8C for 30 s, annealing at 50 8C for 30 s, and extention at 72 8C for 2 min.…”

Differentiation of Metronidazole-Sensitive and -Resistant Clinical Isolates of Helicobacter pylori by Immunoblotting with Antisera to the RdxA Protein

“…Although a small number of metronidazole-resistant strains of H. pylori have been reported in which the nucleotide sequence of the rdxA gene has been unchanged there has been no further analysis of these mutants to confirm whether they have decreased RdxA activity or synthesis (1,14,31,35). We are now examining whether the RdxA enzymes produced by the two resistant strains are functionally inactive or whether other mechanisms are responsible for their resistant phenotype.…”