Incidence of various gyrA mutants in 451 Staphylococcus aureus strains isolated in Japan and their susceptibilities to 10 fluoroquinolones.

Point mutations in the gyrA genes of 451 clinical strains of Staphylococcus aureus isolated in Japan were detected by a combination of nonradioisotopic single-strand conformation polymorphism analysis and restriction fragment length polymorphism analysis and by direct sequencing. Six types of gyrA mutations were observed in 149 of the 451 strains (33%), and ofloxacin MICs were greater than 6.25 micrograms/ml for 147 of the 149 strains (98.7%). These mutations were localized between codons 84 and 88, and they were associated with fluoroquinolone resistance. Two types of silent mutations were also found. Among these eight types of mutations, three types are novel, i.e., the serine at position 84 (Ser-84)-->Val (TCA-->GTA), Ser-84-->Leu (TCA-->TTA) plus Ile-86 (ATT-->ATC, silent), and Phe-110 (TTT-->TTC, silent). Among GyrA mutants, strains with a Ser-84-->Leu alteration and strains with a Glu-88-->Lys alteration were dominant. In contrast, few strains had Ser-84-->Ala and Glu-88-->Gly alterations. All fluoroquinolones tested showed greater than a fourfold decrease in their activities in terms of their MICs that inhibited 50% of strains tested for each GyrA mutant, in comparison with their MICs that inhibited 50% of strains tested for susceptible strains. Most of the currently available fluoroquinolones, such as norfloxacin, enoxacin, ofloxacin, ciprofloxacin, tosufloxacin, lomefloxacin, sparfloxacin, and fleroxacin, were ineffective against each mutant. Mutants containing a Ser-84-->Leu or Val alteration showed high-level resistance to fluoroquinolones, and one containing a Ser-84-->Ala alteration showed relatively low-level resistance. Double mutations were associated with a higher level of resistance than single mutations.     

Mutations in the gyrA gene of a highly fluoroquinolone-resistant clinical isolate of Escherichia coli.

We have determined the DNA sequence of the gyrA gene of the fluoroquinolone-resistant Escherichia coli isolate 205096 (MIC of ciprofloxacin, 128 micrograms/ml), which was recently demonstrated to be a gyrA mutant (P. Heisig and B. Wiedemann, Antimicrob. Agents Chemother. 35:2031-2036, 1991). Compared with the gyrA+ gene of E. coli K-12, 55 nucleotide changes were found. Three of these resulted in amino acid exchanges: Ser-83-->Leu, Asp-87-->Gly, and Asp-678-->Glu. A 0.7-kb DNA fragment containing two of these mutations (Ser-83-->Leu and Asp-87-->Gly) was isolated and fused in frame to the residual 3' coding region of gyrA+ in a plasmid to yield a chimeric gyrA gene (gyrA#). After introduction into E. coli 205096, this gyrA# gene does not increase the fluoroquinolone susceptibility of the resulting heterodiploid strain in a dominance test, while the gyrA+ gene does. The ciprofloxacin concentration necessary to inhibit by 90% (IC90) the supercoiling activity of gyrase isolated from E. coli 205096 is above 2,000 micrograms/ml. An identical result was found for gyrase reconstituted in vitro from the gyrB+ gene product and the chimeric gyrA# gene product. This is more than a 4,000-fold increase compared with the IC90 determined for gyrase from E. coli K-12 (gyrA+) (IC90, 0.5 microgram of ciprofloxacin per ml). No indications for the involvement of the gyrB gene or for alterations in quinolone permeation were found.     

Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro.

We have analyzed by gene amplification and sequencing mutations in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes of fluoroquinolone-resistant Streptococcus pneumoniae mutants obtained during therapy or in vitro. Mutations leading to substitutions in ParC were detected in the two mutants obtained in vivo, BM4203-R (substitution of a histidine for an aspartate at position 84 [Asp-84-->His]; Staphylococcus aureus coordinates) and BM4204-R (Ser-80-->Phe), and in two mutants obtained in vitro (Ser-80-->Tyr). An additional mutant obtained in vitro, BM4205-R3, displayed a higher level of fluoroquinolone resistance and had a mutation in gyrA leading to a Ser-84-->Phe change. We could not detect any mutation in the three remaining mutants obtained in vitro. Total DNA from BM4203-R, BM4204-R, and BM4205-R3 was used to transform S. pneumoniae CP1000 by selection on fluoroquinolones. For the parC mutants, transformants with phenotypes indistinguishable from those of the donors were obtained at frequencies (5 x 10(-3) to 8 x 10(-3)) compatible with monogenic transformation. By contrast, transformants were obtained at a low frequency (4 x 10(-5)), compatible with the transformation of two independent genes, for the gyrA mutant. Resistant transformants of CP1000 were also obtained with an amplified fragment of parC from BM4203-R and BM4204-R but not with a gyrA fragment from BM4205-R3. All transformants had mutations identical to those in the donors. These data strongly suggest that ParC is the primary target for fluoroquinolones in S. pneumoniae and that BM4205-R3 is resistant to higher levels of the drugs following the acquisition of two mutations, including one in gyrA.     

Ciprofloxacin-resistant Haemophilus influenzae strains possess mutations in analogous positions of GyrA and ParC.

The nucleotide sequences of the quinolone resistance-determining regions of the gyrA and parC genes from five ciprofloxacin-resistant strains of Haemophilus influenzae (MICs, 2 to 32 micrograms/ml) isolated from patients with cystic fibrosis and three ciprofloxacin-susceptible strains of H. influenzae (MICs, < or = 0.1 micrograms/ml) were determined. Four of the five resistant strains possessed at least one amino acid substitution in each of the GyrA and ParC fragments studied. The mutations identified in GyrA were a serine at residue 84 (Ser-84) to Leu or Tyr and Asp-88 to Asn or Tyr. ParC mutations were in positions exactly analogous to those identified in GyrA, namely, Ser-84 to Ile and Glu-88 to Lys. The Glu-88 to Lys ParC substitution was identified only in high-level ciprofloxacin-resistant strains. These mutations have been shown to be the origin of the observed resistance after transformation into ciprofloxacin-susceptible H. influenzae isolates. These results suggest that H. influenzae isolates require at least one amino acid substitution in both GyrA and ParC in order to attain significant levels of resistance to quinolones.     

Selection of moxifloxacin-resistant Staphylococcus aureus compared with five other fluoroquinolones

Fluoroquinolone-resistant mutants were selected from Staphylococcus aureus NCTC 8532 (F77), and two GrlA mutants of F77 (F193 and F194) with moxifloxacin, sparfloxacin, ofloxacin, grepafloxacin, levofloxacin and trovafloxacin. For mutants selected from F77, moxifloxacin, grepafloxacin and sparfloxacin selected preferentially for mutations in gyrA (Glu-88-->Lys). Ofloxacin and trovafloxacin selected most commonly for mutations in grlA, conferring substitutions for Ser-80. Three mutants of F77 were shown to have substitutions in both GrlA (Phe-80) and GyrA (Lys-88). Of the mutants selected from F193 (GrlA Phe-80), restriction fragment length polymorphism analysis of gyrA showed that 76/94 had a mutation at codon 84; those analysed in detail all had the substitution Ser-->Leu. Two mutants selected with grepafloxacin contained the substitution Lys-88. One mutant selected with trovafloxacin contained a novel mutation in gyrA substituting Gly-82-->Cys. Of the mutants selected from F194 (GrlA Tyr-80), 6/8 had a mutation in gyrA codon 84; of which three contained Leu. The MICs of most agents for mutants selected from F193 and F194 were similar, irrespective of the mutation selected. No mutants had any changes in grlB, and only one had a mutation in gyrB giving rise to the novel substitution Asp-437-->His. The mutations arising in first-step mutants were influenced by the fluoroquinolone used for selection. The phenotypes and genotypes of second-step mutants, derived from mutants with existing mutations in grlA, were similar, regardless of the selecting antibiotic.     

Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli.

The gene parC encodes the A subunit of topoisomerase IV of Escherichia coli. Mutations in the parC region analogous to those in the quinolone resistance-determining region of gyrA were investigated in 27 clinical isolates of E. coli for which ciprofloxacin MICs were 0.0007 to 128 micrograms/ml. Of 15 isolates for which ciprofloxacin MICs were > or = 1 microgram/ml, 8 showed a change in the serine residue at position 80 (Ser-80), 4 showed a change in Glu-84, and 3 showed changes in both amino acids. No mutations were detected in 12 clinical isolates for which ciprofloxacin MICs were < or = 0.25 micrograms/ml. These findings suggest that ParC from E. coli may be another target for quinolones and that mutations at residues Ser-80 and Glu-84 may contribute to decreased fluoroquinolone susceptibility.     

Mutations in gyrA gene of quinolone-resistant Salmonella serotypes isolated from humans and animals.

The quinolone resistance-determining regions (QRDRs) of the gyrA genes of quinolone-resistant clinical and veterinary salmonella isolates were sequenced. Substitutions analogous to a substitution of a Ser to a Phe at position 83 (Ser83-->Phe) and Asp87-->Gly or Tyr in Escherichia coli were found, as was a single novel mutation outside of the QRDR resulting in Ala119-->Glu. The data suggest that gyrA mutations are associated with quinolone resistance in veterinary and clinical salmonella isolates and that the limits of the QRDR may require revision.     

Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus.

Fluoroquinolone-resistant mutants were obtained in vitro from Staphylococcus aureus RN4220 by stepwise selection on increasing concentrations of ciprofloxacin. Results from sequence analysis of the quinolone resistance-determining region of GyrA and of the corresponding region of GrlA, the DNA topoisomerase IV subunit, showed an alteration of Ser-80 to Tyr (corresponding to Ser-83 of Escherichia coli GyrA) or Glu-84 to Lys in GrlA of both low- and high-level quinolone-resistant mutants. Second-step mutants were found to have, in addition to a mutation in grlA, reduced accumulation of norfloxacin or an alteration in GyrA at Ser-84 to Leu or Glu-88 to Lys. Third-step mutants derived from second-step mutants with reduced accumulation were found to have a mutation in gyrA. The results from this study demonstrated that mutations in gyrA or mutations leading to reduced drug accumulation occur after alteration of GrlA, supporting the previous findings (L. Ferrero, B. Cameron, B. Manse, D. Lagneaux, J. Crouzet, A. Famechon, and F. Blanche, Mol. Microbiol. 13:641-653, 1994) that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.     

High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA.

The mechanism of high-level fluoroquinolone resistance was studied in strains of Streptococcus pneumoniae, either selected in vitro or isolated from clinical samples. By using DNA from these high-level-resistant strains, low-level-resistant transformants (MIC of pefloxacin, > or = 32 micrograms/ml; MIC of ciprofloxacin, 4 micrograms/ml; MIC of sparfloxacin, 0.50 micrograms/ml) were obtained at high frequencies (ca.10(-2)), while high-level-resistant transformants (MIC of pefloxacin, > or = 64 micrograms/ml; MIC of ciprofloxacin, 16 to 64 micrograms/ml; MIC of sparfloxacin, > or = 8 micrograms/ml) were obtained only at low frequencies (ca.10(-4)). This suggested that mutations in at least two unlinked genes were necessary to obtain high-level resistance. Low-level resistance was associated with ParC mutations (change from Ser to Tyr at position 79 [Ser79Tyr], Ser79Phe, or Asp83Gly). ParC mutations were associated, in high-level-resistant strains and transformants, with alterations in the quinolone resistance-determining region of GyrA (Ser84Tyr, Ser84Phe, and/or Glu88Lys). Low-level resistance was shown to be necessary for expression of the gyrA mutations. No mutation in the region corresponding to the quinolone resistance-determining region of GyrB and no alteration of drug accumulation were found.     

In vitro antibacterial activities of new fluoroquinolones against clinical isolates of haemophilus influenzae with ciprofloxacin-resistance-associated alterations in GyrA and ParC

BACKGROUND: The in vitro antimicrobial activities of new fluoroquinolones were tested against quinolone-resistant Haemophilus influenzae of clinical isolates. METHODS: The nucleotide sequences of the gyrA and parC genes from three ciprofloxacin-resistant strains of Haemophilus influenzae (MIC, 1.56-6.25 microg/ml) were determined. The gyrase was purified from the clinical isolates, and the inhibitory activities of quinolones against the enzyme were tested. RESULTS: These strains possessed at least one amino acid substitution in each of the GyrA (asparagine at residue 88 (Asp-88) to Tyr, Ser-84 to Leu or Ser-84 to Leu and Asp-88 to Asn) and ParC (Glu-88 to Lys). The antibacterial activity of olamufloxacin against the resistant strains was most potent compared with other quinolones, and the inhibitory activities correlated with quinolone resistance of these strains. CONCLUSIONS: These results warrant the clinical effects of new types of fluoroquinolones, such as olamufloxacin, against respiratory tract and otolaryngology infections caused by ciprofloxacin-resistant H. influenzae.     

Mutations in gyrA and parC genes in nalidixic acid-resistant Escherichia coli strains from food products,humans and animals

Mutations in quinolone targets were analysed in 80 unrelated nalidixic acid-resistant (NALR) Escherichia coli strains whose nalidixic acid and ciprofloxacin MICs ranged from 32 to >256 mg/L and 0.03-64 mg/L, respectively. These strains were isolated from food products (23) and faecal samples from humans (15) and healthy animals (42). Thirteen nalidixic acid-susceptible (NALS) E. coli strains were also analysed. Mutations in gyrA and parC genes were studied by PCR and sequencing. No amino acid changes were detected in GyrA or ParC proteins of the 13 NALS strains. A single change in the GyrA protein was detected in all 61 NALR strains with ciprofloxacin MICs Leu (54), Ser-83-->Ala (one), Ser-83-->Val (one), Asp-87-->Asn (two), Asp-87-->Tyr (two) and Asp-87-->Gly (one). A double change in GyrA was found in 18 of 19 NALR strains with ciprofloxacin MICs >/= 4 mg/L. Amino acid substitutions were Ser-83-->Leu, with an additional change [Asp-87-->Asn (15), Asp-87-->Tyr (two) or Asp-87-->His (one)]. The remaining strain (ciprofloxacin MIC 4 mg/L) showed a single Ser-83-->Leu substitution. In respect of the ParC protein, a single change at Ser-80 or Glu-84 was found in 25 of 42 strains, with ciprofloxacin MICs ranging from 0.5 to 32 mg/L. A double substitution (Ser-80-->Ile and Glu-84-->Gly) was found in one strain (ciprofloxacin MIC 64 mg/L). No amino acid changes were detected in the GyrB protein of 18 NALR strains.     

Molecular characterization of nine different types of mutants among 107 inhibitor-resistant TEM beta-lactamases from clinical isolates of Escherichia coli.

DNA-DNA hybridization and sequencing were performed to determine the molecular basis of resistance to clavulanic acid in 107 inhibitor-resistant TEM (IRT) enzymes produced by Escherichia coli clinical isolates. These beta-lactamases derived from TEM-1 enzyme focused at pI 5.2 (n = 68) or 5.4 (n = 39) and were very poorly inhibited by clavulanic acid compared with TEM-1 enzyme. Results showed that the amino acid sequences of 84 of the 107 enzymes differ from TEM-1 by one or two substitutions previously described: Arg-244-->Ser (IRT-2) in 22 strains, Met-69-->Leu (TEM-33) in 17 strains, Met-69-->Val (TEM-34) in 14 strains, Met-69-->Ile (IRT-3) in 6 strains, Met-69-->Leu associated with Asn-276-->Asp (IRT-4) in 13 strains, and Met-69-->Val associated with Asn-276-->Asp (TEM-36) in 12 strains. A new combination, Met-69-->Ile with Asn-276-->Asp, was found in 20 strains and was called IRT-8. Two IRT enzymes not previously described were characterized. The substitution Met-69-->Val associated with a novel substitution Arg-275-->Leu occurred in one strain. The combination Met-69-->Leu and Asn-276-->Asp was associated with the novel substitution Trp-165-->Arg in two strains. These two novel enzymes were called IRT-9 and IRT-10, respectively. The implication of these novel mutated positions, 165 and 275, in resistance to inactivation by clavulanate was supported by crystallographic data on the TEM-1 enzyme and results of site-directed mutagenesis. Molecular characterization of these mutants showed great diversity among the genes coding for inhibitor-resistant TEM enzymes produced by clinical E. coli isolates.     

Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii.

The gyrA gene mutations associated with quinolone resistance were determined in 21 epidemiologically unrelated clinical isolates of Acinetobacter baumannii. Our studies highlight the conserved sequences in the quinolone resistance-determining region of the gyrA gene from A. baumannii and other bacteria. All 15 isolates for which the MIC of ciprofloxacin is > or = 4 micrograms/ml showed a change at Ser-83 to Leu. Six strains for which the MIC of ciprofloxacin is 1 microgram/ml did not show any change at Ser-83, although a strain for which the MIC of ciprofloxacin is 1 microgram/ml exhibited a change at Gly-81 to Val. Although it is possible that mutations in other locations of the gyrA gene, the gyrB gene, or in other genes may also contribute to the modulation of the MIC level, our results suggest that a gyrA mutation at Ser-83 is associated with quinolone resistance in A. baumannii.     

Examination of single and multiple mutations involved in resistance to quinolones in Staphylococcus aureus by a combination of PCR and denaturing high-performance liquid chromatography (DHPLC)

Detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that has been used to detect mutations in human DNA. We report on the first study to use this technique as a tool to detect mutations in genes encoding antibiotic resistance in bacteria. Three methicillin-sensitive and three methicillin-resistant clinical Staphylococcus aureus isolates, susceptible to ciprofloxacin (MIC Leu, Ser-112-->Pro, Glu-88-->Lys in GyrA, Glu-84-->Val, Ser-80-->Phe in GrlA, Pro-456-->Ser in GyrB and Glu-422-->Asp, Pro-451-->Ser, Asp-432-->Gly in GrlB. Mutations could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run) and reliable technique for characterizing antibiotic resistance in bacteria.     

Detection of grlA and gyrA Mutations in 344 Staphylococcus aureus Strains

Mutations in the grlA and gyrA genes of 344 clinical strains of Staphylococcus aureus isolated in 1994 in Japan were identified by combinations of single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing to identify possible relationships to fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA genes of 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA genes of 188 strains (54.7%). Among them, the grlA mutation of TCC→TTC or TAC (Ser-80→Phe or Tyr) and the gyrA mutation of TCA→TTA (Ser-84→Leu) were principal, being detected in 137 (39.8%) and 121 (35.9%) isolates, respectively. The grlA point mutations of CAT→CAC (His-77 [silent]), TCA→CCA (Ser-81→Pro), and ATA→ATT (Ile-100 [silent]) were novel, as was the GAC→GGC (Asp-73→Gly) change in gyrA. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC ≥ 3.13 μg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC at which 50% of the isolates are inhibited [MIC₅₀] = 50 μg/ml). Those with the Ser-80→Phe or Tyr alteration in grlA but wild-type gyrA showed a lower level of ciprofloxacin resistance (MIC₅₀ ≤ 12.5 μg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of a concomitant mutation at codon 73, 84, or 88 in gyrA (MIC ≤ 6.25 μg/ml). The new fluoroquinolone DU-6859a showed good activity with 186 of 193 isolates (96.4%) for which the MIC was ≤6.25 μg/ml.     

Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs.

The mutations in the quinolone resistance-determining region of the gyrA and gyrB genes from 27 clinical isolates of Escherichia coli with a range of MICs of ciprofloxacin from 0.007 to 128 micrograms/ml and of nalidixic acid from 2 to > 2,000 micrograms/ml were determined by DNA sequencing. All 15 isolates with ciprofloxacin MICs of > or = 1 micrograms/ml showed a change in Ser-83 to Leu of GyrA protein, whereas in clinical isolates with a MIC of > or = 8 micrograms/ml (11 strains), a double change in Ser-83 and Asp-87 was found. All isolates with a MIC of nalidixic acid of > or = 128 micrograms/ml showed a mutation at amino acid codon Ser-83. Only 1 of the 27 clinical isolates of E. coli analyzed showed a change in Lys-447 of the B subunit of DNA gyrase. A change in Ser-83 is sufficient to generate a high level of resistance to nalidixic acid, whereas a second mutation at Asp-87 in the A subunit of DNA gyrase may play a complementary role in developing the strain's high levels of ciprofloxacin resistance.     

Plasmid-mediated complementation of gyrA and gyrB in fluoroquinolone-resistant Bacteroides fragilis

OBJECTIVES: To identify whether mutations in gyrA and gyrB confer fluoroquinolone resistance in Bacteroides fragilis. METHODS: Eight fluoroquinolone-resistant (FQR) strains were complemented with plasmid-mediated B. fragilis wild-type gyrA (pMP1) and gyrB (pMP2), and MICs determined. Sequence analysis of the gyrA and gyrB quinolone resistance determining region (QRDR) was performed for all strains. RESULTS: MICs of fluoroquinolones were two- to 32-fold higher than wild-type for all mutants. Five mutants had a substitution in GyrA (Ser-82-->Phe), one mutant had a substitution in GyrA (Asp-81-->Gly), one mutant had a substitution in GyrB (Glu-478-->Lys), and one resistant strain did not contain mutations in the QRDR of gyrA or gyrB. Following complementation with pMP1 or pMP2, the MICs of fluoroquinolones were reduced two- to 32-fold for the mutants. CONCLUSION: These studies verify that substitutions in GyrA and GyrB confer resistance in B. fragilis. Other mechanisms are also responsible for resistance since not all resistant strains fully complemented to the wild-type phenotype.     

Characterization of fluoroquinolone-resistant mutants of escherichia coli selected in vitro.

Wild-type mutants highly resistant to fluoroquinolones were selected in vitro from a quinolone-susceptible Escherichia coli isolate by stepwise exposure to increasing concentrations of nalidixic acid and ciprofloxacin (CIP) either in liquid medium or on solid medium. Mutant R17 was selected by serial passage in liquid medium; the MIC of CIP for mutant R17 was 256 micrograms/ml. On solid medium, consecutive mutants MI, MII, MIII, MIVa, and MIVb were selected in four steps. The frequencies of mutations were between 10(-9) and 10(-11), and the MICs of CIP ranged from 0.5 microgram/ml (for mutant MI) to 256 micrograms/ml (for mutant MIVb). From the results of a dominance test with the gyrB+ plasmid (pBP547), no gyrB mutations were detectable. In the first step, mutant MI, a mutation from a Ser to a Leu residue at position 83 (a Ser-83-->Leu mutation), was detected in the quinolone resistance-determining region of the gyrA gene. In addition, the second-step mutation was associated with a reduced uptake of CIP and an altered outer membrane protein profile. The third mutation was identified as an Asp-87-->Gly mutation in the quinolone resistance-determining region of the gyrA gene. Concomitantly, a slight increase in the doubling time was detected. For two different four-step mutants, mutants MIVa and MIVb, the MICs of only some quinolones, including CIP, increased. The accumulation of CIP in the mutants was comparable to that in their parent MIII. The doubling time of mutant MIVa was similar to that of mutant MIII, but differed by a factor of 3 from that of the very slow growing mutant MIVb. In contrast, a clinical isolate of E.coli (isolate 205096) described previously (P. Heisig, H. Schedletzky, and H. Falkenstein-Paul, Antimicrob. Agents Chemother. 37:696-701, 1993) which has the same double mutation in gyrA had a doubling time comparable to that of the wild-type isolate.     

Molecular diversity of quinolone resistance in genetically related clinical isolates of Staphylococcus aureus and susceptibility to newer quinolones

The genes encoding topoisomerases (gyrA and grlA) and the norA promoter of 100 fluoroquinolone-susceptible and -resistant Staphylococcus aureus clinical isolates obtained in two geographically distant hospitals were analysed. The relationship between mutations found and the susceptibility to newer quinolones was determined. Thirty-nine strains were grouped in seven clones by pulsed-field gel electrophoresis (PFGE). The remaining 61 strains were classified as unrelated strains. In three clones, all strains showed the same grlA-gyrA-norA mutation profiles. Strains in the rest of the groups showed different mutation profiles, even though PFGE indicated that they possessed genetically similar populations. One cluster showed a high level of diversity; five different mutation profiles were detected in the six isolates belonging to this pattern. Two isolates had a Glu84 to Lys mutation in grlA and another isolate had this mutation combined with a Ser84 to Leu mutation in gyrA. Combination of a Ser80 to Phe mutation in grlA and a Ser84 to Leu in gyrA was found in the two other isolates. One of these also had a thymine to a guanine transversion at a position 89 nucleotides upstream of the norA start codon in the norA promoter. These results show that fluoroquinolone resistance in clinical S. aureus strains does not necessarily result from the spread of resistant clones. Fluoroquinolone resistance may develop independently in strains belonging to the same PFGE pattern by accumulation of different mutations over a quinolone-susceptible ancestor wild type or single grlA mutant.     

Identification of Novel Mutation Patterns in the parC Gene of Ciprofloxacin-Resistant Isolates of Neisseria gonorrhoeae

Of 65 ciprofloxacin-resistant, clinical isolates of Neisseria gonorrhoeae, 5 isolates exhibited ParC mutations previously undescribed in the gonococcus. For isolates containing two ParC mutations (the Ser-87→Ile and Glu-91→Gly mutations and the Gly-85→Cys and Arg116→Leu mutations) the MICs of ciprofloxacin (8.0 to 64.0 μg/ml) were higher than those for the isolate containing the single ParC mutation (Arg-116→Leu; MIC, 1.0 μg/ml).