Nucleotide sequence of the gyrA gene and characterization of ciprofloxacin-resistant mutants of Helicobacter pylori

Abstract

PCR was used to amplify a 238-bp region from Helicobacter pylori which corresponded to the quinolone resistance-determining region in Escherichia coli. The gyrA gene of H. pylori was cloned and sequenced. An open reading frame of 2,478 nucleotides coded for a polypeptide of 826 amino acids with a calculated molecular mass of 92,508 Da. The amino acid sequence showed an overall 52% identity with other bacterial gyrA genes but was most closely related to the gyrA subunit of Campylobacter jejuni (76.5% identity). Sequencing of the amplification product from ciprofloxacin-resistant mutants of H. pylori revealed four classes of mutations with substitutions at amino acid 87 (Asn3Lys), amino acid 88 (Ala3Val), and amino acid 91 (Asp3Gly, 3Asn, or 3Tyr) and a double substitution at amino acids 91 and 97 (Ala3Val). Ciprofloxacin-susceptible strains of H. pylori could be transformed to ciprofloxacin resistance by using the amplified fragment from resistant strains as donor DNA. Of the 11 ciprofloxacin-resistant mutants examined, only one did not have an alteration within the quinolone resistance-determining region, suggesting that, in H. pylori, resistance to quinolones is primarily a result of alterations in gyrA.Considerable evidence implicating Helicobacter pylori as an essential etiologic agent in type B chronic gastritis and peptic ulcer disease exists (2,19,20,37). Currently, H. pylori infections are treated with two or three antimicrobial agent combinations, as single therapy has proven ineffective (19, 21). As new approaches for antimicrobial agent treatment of H. pylori are developed, it is increasingly important to identify potential resistance problems. We have detected resistant H. pylori isolated from patients treated with ciprofloxacin, an antimicrobial agent which has potent in vitro activity, and therefore undertook an examination of such strains to determine their mechanism of resistance. Ciprofloxacin exerts antimicrobial activity by inhibiting the enzyme DNA gyrase (26). This enzyme, in addition to relaxing supercoiled DNA, is able to introduce negative supercoils into DNA and thus maintains the bacterial chromosome in a negatively supercoiled state (26). In addition, the enzyme is involved in DNA replication, recombination, and transcription (13, 32). The bacterial enzyme is a tetramer consisting of two A and two B subunits encoded by the gyrA and gyrB genes, respectively. In Bacillus subtilis (23), Staphylococcus aureus (12, 18), and Mycoplasma pneumoniae (4) these genes are contiguous on the chromosome, while in Escherichia coli (30), Klebsiella pneumoniae (6), Campylobacter jejuni (34), and Pseudomonas aeruginosa (14) the genes are located separately. As an enzyme essential for cell replication, DNA gyrase is an obvious target for antimicrobial agents. The fluoroquinolones exert their antimicrobial activity at the level of the A subunit of the DNA gyrase. This subunit, responsible for DNA cleavage and rejoining, is also the site of action for nalidixic acid. In E. coli, mutations in the gyrA gene wh...

Helicobacter pylori eradication in Western Australia using novel quadruple therapy combinations

“…mutations in the 23S rRNA, gyrA, rpoB and rdxA genes are associated with CLR, CIP, RIF and MTZ resistance respectively. [25][26][27][28][29][30] Nevertheless, the authors believe that the CLR/MTZ-based triple therapy is still effective as a first-line treatment, at least within Australia. Female patients are more likely to carry H. pylori strains resistant to both CLR and MTZ than male patients due to the higher incidence of previous treatment of gynaecological conditions with antibiotics.…”

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

“…Considering these facts, we believe that levofloxacin should be considered as one of the antibiotics used for second-or third-line H. pylori eradication therapy in 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).…”

Phenotypic and genotypic correlation as expressed in Helicobacter pylori resistance to clarithromycin and fluoroquinolones

“…This enzyme contains two A subunits and two B subunits, encoded by gyrA and gyrB, respectively [11]. Resistance to fluoroquinolones is mainly mediated by a mutation in the Quinolone Resistance Determining Region (QRDR) in the gyrA gene [12].…”