Single or double mutational alterations of gyrA associated with fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli.

We looked for the presence of gyrA mutations in seven fluoroquinolone-resistant French clinical isolates of Campylobacter jejuni and Campylobacter coli. Three of the five isolates of C. jejuni and the two isolates of C. coli had high-level resistance to nalidixic acid (MICs 128-256 microg/ml) and ciprofloxacin (MICs 32 microg/ml). A gyrA mutation was found in all these isolates leading to the following substitutions: Thr86-Ile in four cases and Asp90-Tyr for one C. coli strain. One isolate had high-level resistance to nalidixic acid (MIC 64 microg/ml) but low-level resistance to ciprofloxacin (MIC 2 microg/ml) and also carried a gyrA mutation leading to a Thr86-Ala substitution. The last isolate of C. jejuni studied displayed an atypical resistance phenotype: It was resistant to high levels of ciprofloxacin (MIC 64 microg/ml) but remained fully susceptible to nalidixic acid (MIC 2 microg/ml). This phenotype was not explained by the presence of peculiar mutations in gyrA or gyrB. It carried a gyrA mutation leading to a Thr86-Ile substitution and was devoid of gyrB mutation. Despite numerous attempts with various degenerate oligonucleotide primers deduced from conserved regions of known parC genes, we were unable to amplify a corresponding sequence in C. jejuni or C. coli. First-step and second-step in vitro mutants, derived from reference strain C. coli ATCC 33559 with ciprofloxacin or moxifloxacin as selecting agents, were found to carry one and two mutations in gyrA, respectively. In contrast with the results obtained with clinical isolates, a variety of gyrA mutations were obtained in vitro.

Roles of gyrA mutations in resistance of clinical isolates and in vitro mutants of Bacteroides fragilis to the new fluoroquinolone trovafloxacin.

We determined whether gyrA mutations were present in fluoroquinolone-resistant laboratory mutants derived from the Bacteroides fragilis reference strain ATCC 25285 and in clinical isolates of B. fragilis. The two first-step mutants selected on ciprofloxacin (CIP) were devoid of gyrA mutations, whereas two of the three CIP-selected second-step mutants studied presented the same gyrA mutation leading to a Ser82Phe change. Unusual GyrA alterations, Asp81Asn or Ala118Val, were detected in two of the three first-step mutants selected on trovafloxacin (TRO), Mt3 and Mt1, respectively. The Ala118Val change had no effect on the susceptibility of Mt1 to CIP. No second-step mutant could be obtained with TRO as a selector. For the 12 clinical isolates studied, a Ser82Phe change in GyrA was found only in the 3 strains which showed the highest levels of TRO resistance (MIC, 4 microgram/ml). Thus, the resistance phenotypes and genotypes observed in fluoroquinolone-resistant clinical isolates of B. fragilis were similar to those found in CIP-selected laboratory mutants, whereas peculiar mutational events could be selected in vitro with TRO.

In-vitro activity of moxifloxacin against fluoroquinolone-resistant strains of aerobic gram-negative bacilli and Enterococcus faecalis.

MICs of the new fluoroquinolone, moxifloxacin, and those of ciprofloxacin, ofloxacin and sparfloxacin for 19 genetically characterized fluoroquinolone-resistant strains were determined by the agar dilution method. The MICs of moxifloxacin for Escherichia isolates with one mutation in gyrA (corresponding to Ser83-->Leu or Asp87-->Gly substitution) were 0.25-0.5 mg/L, while those of ciprofloxacin, ofloxacin and sparfloxacin were 0.06-0.25, 1 and 0.12-0.5 mg/L, respectively. These values were four- to 16-fold higher than those of the same antibiotics for the wild-type strain, E. coli KL16. Similar results were observed with clinical isolates of Salmonella spp. harbouring one mutation in gyrA leading to the substitution of Ser83 by Phe or Tyr. In the presence of two mutations in the E. coli gyrA gene, the MICs of moxifloxacin ciprofloxacin, ofloxacin and sparfloxacin were 2, 0.5, 4 and 1 mg/L, respectively; these were 32 times higher than the MICs of these agents for E. coli KL16. The MICs of the four quinolones for triple mutants with two mutations in gyrA and one in parC were even higher, i.e. 8, 8, 16 and 8-16 mg/L, respectively. The MICs of moxifloxacin for Campylobacter coli and Campylobacter jejuni strains with a gyrA mutation leading to Thr86-->Ile substitution ranged from 1 to 2 mg/L, while the MICs of ciprofloxacin, ofloxacin and sparfloxacin were 16-32 mg/L, 8-16 and 4-8 mg/L, respectively. For high-level ciprofloxacin-resistant (MICs of 32 mg/L) clinical isolates of Enterococcus faecalis with one substitution at position 83 in GyrA (E. coli coordinates), the MICs of moxifloxacin, ofloxacin and sparfloxacin were 8-16, > or = 128 and 32 mg/L respectively. In conclusion, moxifloxacin and other fluoroquinolones exhibit cross-resistance against aerobic gram-negative bacilli and enterococci. The in-vitro activity of moxifloxacin was greater than that of ofloxacin and slightly less than that of ciprofloxacin and sparfloxacin against Enterobacteriaceae, but greater than those of the three other compounds tested against Campylobacter spp and E. faecalis.

Analysis of the mutations involved in fluoroquinolone resistance of in vivo and in vitro mutants of Escherichia coli.

We searched for the mutations involved in high-level fluoroquinolone resistance (ciprofloxacin MIC > or = 8 microg/ml) of 11 clinical isolates of Escherichia coli. trans-Complementation tests with the wild-type gyrA and parC genes were positive for all strains whereas negative results were observed with the wild-type gyrB and parE genes. By PCR and sequencing, two mutations in gyrA, leading to Ser-83 --> Leu and Asp-87 --> Asn (7) or Gly (2) or Tyr (1) changes, were found in 10 strains, the eleventh presenting only the Ser-83 --> Leu change. In addition, all strains carried one change in ParC: Ser-80 --> Ile (8) or Arg (2); Glu-84 --> Lys (1). We described a novel and simple method permitting detection of the mutations in parC at codon 80, PCR-RFLP with HaeII. In vitro mutants, selected with ciprofloxacin in three successive steps were also studied. The first-step mutants were complemented by pJSW101 (gyrA+) but not by pEN260 (parC+), whereas the second-step and third-step mutants were complemented by both plasmids. Mutations occurred in the following order: (i) gyrA at codon 83 (Ser to Leu change), (ii) parC at codon 80 (Ser to Ile change), and (iii) gyrA at codon 87 (Asp to Asn change). Thus, these sequential mutations appear to be frequently involved in high-level fluoroquinolone resistance of E. coli.