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...

The Pseudomonas aeruginosa DNA gyrase gyrA gene was cloned and sequenced from strain PAO1. An open reading frame of 2,769 bp was found; it coded for a protein of 923 amino acids with an estimated molecular mass of 103 kDa. The derived amino acid sequence shared 67% identity with Escherichia coli GyrA and 54% identity with Bacillus subtUis GyrA, although conserved regions were present throughout the sequences, particularly toward the N terminus. Complementation of an E. coli mutant with a temperature-sensitive gyrA gene with the PAO1 gyrA gene showed that the gene is expressed in E. coli and is able to functionally complement the E. coli DNA gyrase B subunit. Expression of PAO1 gyrA in E. coli or P. aeruginosa with mutationally altered gyrA genes caused a reversion to wild-type quinolone susceptibility, indicating that the intrinsic susceptibility of the PAO1 GyrA to quinolones is comparable to that of the E. coli enzyme. PCR was used to amplify 360 bp of P. aeruginosa gyrA encompassing the so-called quinolone resistance-determining region from ciprofloxacin-resistant clinical isolates from patients with cystic fibrosis. Mutations were found in three of nine isolates tested; these mutations caused the following alterations in the sequence of GyrA Asp at position 87 (Asp-87) to Asn, Asp-87 to Tyr, and Thr-83 to Ile. The resistance mechanisms in the other six isolates are unknown. The results of the study suggested that mechanisms other than a mutational alteration in gyrA are the most common mechanism of ciprofloxacin resistance in P. aeruginosa from the lungs of patients with cystic fibrosis.The enzyme DNA gyrase is a type II DNA topoisomerase that is able to introduce negative superhelical coils into covalently closed bacterial DNA in an ATP-dependent process. Gyrase also plays a role in DNA replication, recombination, decatenation, and transcriptional regulation of some supercoiling-sensitive promoters (for a review, see reference 24). The gyrase holoenzyme is a tetramer of two A and two B subunits, the products of the gyrA and gyrB genes, respectively. The A subunits are responsible for DNA breakage and reunion, while the B subunits are the site of ATP hydrolysis. The enzyme has proven to be a good target for antimicrobial agents; the actions of the A subunits are inhibited by quinolones such as nalidixic acid and ciprofloxacin, while those of the B subunits are inhibited by coumarins such as coumermycin Al and novobiocin. The peptide antibiotic microcin B17 and the glycocinnamoylspermidine agent cinodine have also been shown to be inhibitors of DNA gyrase function (22,33). The contiguous gyrB-gyrA locus has been cloned and sequenced in Bacillus subtilis (19), Staphylococcus aureus (10), Mycoplasma pneumoniae (1), and Haloferax spp. (9). In Escherichia coli (32, 37), Klebsiella pneumoniae (3), Pseudomonas putida (23), Neisseria gonorrhoeae (31), and Campylobacter jejuni (34), the cloned gyrA and gyrB genes are not contiguous. The deduced amino acid sequences of the A and B subunits are very well conserved with...

HIV clinics in

An epidemiologically linked HIV-1-infected cohort, in which a nonprogressor donor infected two recipients who progressed to AIDS, was examined. Sequence analysis, over time, of HIV-1 vpr gene quasispecies from uncultured peripheral blood cells revealed an insertion of arginine at position 90 altering a highly conserved C-terminal motif, believed to play a role in Vpr nuclear targeting. Full genome analysis from each patient showed no gene defects in other gene regions, implying that the mutational selection was unique to the vpr gene. A detailed analysis of the vpr quasispecies showed very little amino acid diversity in the nonprogressing donor, whereas, following viral transmission, the amino acid diversity increased dramatically over time in tandem with disease progression in the two recipients. Although the R insertion at position 90 was present in all three individuals, the variable degree of additional amino acid changes over time may have influenced HIV disease in the nonprogressor donor and the two progressing recipients. These data provide the first evidence in favor of vpr gene evolution over time, which was host-driven. The status of the nonprogressing donor was consistent with a highly protective B-57 HLA type, which was absent in the two progressing recipients, implying a role for host HLA type and other immunologic selective pressures in vpr gene selection in vivo.