Mutations In gyrA Genes Research Articles

High rate of resistance to nalidixic acid in Salmonella enterica: its role as a marker of resistance to fluoroquinolones

High rate of resistance to nalidixic acid in Salmonella enterica: its role as a marker of resistance to fluoroquinolones

DEVELOPMENT OF FLUOROQUINOLONE RESISTANCE AND MUTATIONS INVOLVING GyrA AND ParC PROTEINS AMONG NEISSERIA GONORRHOEAE ISOLATES IN JAPAN

Purpose To investigate the development of fluoroquinolone resistance among Neisseria gonorrhoeae isolates in Japan and the frequency and patterns of mutations involving the GyrA and ParC proteins, which confer quinolone resistance to the bacteria, in isolates. Materials and Methods Antimicrobial susceptibilities of 145 gonococcal isolates, including 79 isolated from February 1992 through February 1993 and 66 isolated from February 1995 through February 1996, to six fluoroquinolones and several other antibiotics were compared with those of 27 isolates obtained from 1981 through 1984. To identify mutations in gyrA and parC genes of the isolates, the quinolone resistance-determining regions of the gyrA and parC genes were PCR-amplified and the PCR products were directly sequenced. Results The minimum inhibitory concentration for 90% of strains (MIC90) values of norfloxacin for the isolates from 1992 to 93 (4 micro g./ml.) and 1995 to 96 (8 micro g./ml.) were 16- and 32-fold, respectively, higher than those for isolates from 1981 to 84 (0.25 micro g./ml.). The MIC90 values of ciprofloxacin for isolates from 1992 to 93 (0.5 micro g./ml.) and 1995 to 96 (1 micro g./ml.) showed increase of 8- and 16-fold, respectively, in comparison with those from 1981 to 84 (0.063 micro g./ml.). The isolates from 1992 to 93 and 1995 to 96 were also less susceptible to newer fluoroquinolones including levofloxacin, sparfloxacin, DU-6859a and AM-1155, as compared with those from 1981 to 84. In 46 (67.6%) and 16 (23.5%) of the 68 gonococcal strains sequenced, GyrA and ParC mutations were identified, respectively. No ParC substitutions were identified in any isolates without co-existence of the GyrA mutation. A Ser-91 to Phe mutation, which was detected in 30 (65.2%) of the 46 isolates with GyrA mutations, was the most common GyrA mutation. Mutants with the single Ser-91 to Phe substitution in GyrA were 12-fold and at least 13-fold, respectively, less susceptible to norfloxacin and ciprofloxacin than the wild type. Conclusions The results obtained in this study suggest that a high prevalence of gonococcal isolates with the Ser-91 to Phe mutation in GyrA has reduced the susceptibility of this organism to fluoroquinolones in Japan.

Development of fluoroquinolone resistance in Enterococcus faecalis and role of mutations in the DNA gyrase gyrA gene.

We have analyzed the development of fluoroquinolone resistance between 1986 and 1993 among clinical isolates of Enterococcus faecalis from a French hospital. One hundred randomly selected isolates per year were screened for resistance to ciprofloxacin (MIC > 2 micrograms/ml) and for high-level resistance to gentamicin (MIC > 1,000 micrograms/ml). The percentages of ciprofloxacin-resistant strains for these years were as follows: 1986, 0; 1987, 1; 1988 to 1989, 2; 1990, 6; 1991, 16; 1992, 24; and 1993, 14. Eighty-three percent of the ciprofloxacin-resistant isolates were coresistant to high levels of gentamicin. Forty-eight high-level gentamicin-resistant E. faecalis strains, which were resistant (24 strains) or susceptible (24 strains) to ciprofloxacin, were examined by pulsed-field gel electrophoresis (PFGE) of SmaI-digested total DNA. Numerous PFGE types were observed among the ciprofloxacin-susceptible isolates, whereas one type was largely predominant among the ciprofloxacin-resistant strains, which suggests that the increase in fluoroquinolone resistance was due to the spread of a single clone. A 241-bp fragment of gyrA, corresponding to the quinolone resistance-determining region, was amplified and sequenced for seven ciprofloxacin-resistant isolates. Six strains had high levels of resistance (MICs, 32 to 64 micrograms/ml) and had a mutation at position 83 (Escherichia coli coordinates) from Ser to Arg (three strains) or to Ile (two strains) or at position 87 from Glu to Gly (one strain), whereas the low-level-resistant isolate (MIC, 8 micrograms/ml) had no mutations.

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.

Detection of gyrA and gyrB mutations in quinolone-resistant clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations.

Twelve quinolone-resistant clinical isolates of Escherichia coli (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.25 to 8 micrograms/ml) were transformed with plasmid pJSW101 carrying the gyrA+ gene and with plasmid pJB11 carrying the gyrB+ gene to examine the proportion of gyrA and gyrB mutations. Transformation with pJSW101 resulted in complementation (nalidixic acid MICs, 4 to 32 micrograms/ml; norfloxacin MICs, 0.06 to 0.25 micrograms/ml). In contrast, no change in MICs were observed after transformation with pJB11. A 418-bp fragment of gyrA from the 12 strains was amplified by PCR. Direct DNA sequencing of that fragment identified the causes of quinolone resistance in eight strains as a single point mutation leading to a substitution of the serine at position 83 (Ser-83) to Leu and in four strains as a single point mutation leading to a substitution of Asp-87 to Gly. Exchange of the fragment from one of these strains with that of gyrA+ and transformation of resistance with the hybrid gyrA plasmid indicated the contribution of Gly-87 to resistance and the stabilities of mutants containing GyrA (Gly-87). Thus, gyrA gene mutations are probably encountered more often than gyrB gene mutations in clinical isolates of E. coli. In addition, the substitution of Asp-87 to Gly can be encountered in such strains. On the basis of the level of resistance found in the fragment exchange experiment, the quinolone resistance attributable to Gly-87 appears to be comparable to that attributable to Leu-83. The levels of resistance found in the clinical isolates shown to have a Gly-87 mutation (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.5 to 4 micrograms/ml) suggest that the Gly-87 mutation causes resistance at the level of the nalidixic acid MIC (64 micrograms/ml) or the norfloxacin MIC (0.5 micrograms/ml or less) and that the additional increments in resistance seen in the other strains with higher levels of resistance may be attributable to additional mutations. The single-strand conformational polymorphism analysis with PCR products readily detected te Leu-83 and Gly-87 mutations.

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.

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.

Detection of 4-quinolone resistance mutation in gyrA gene of Shigella dysenteriae type 1 by PCR

To study 4-quinolone resistance, the N-terminal coding region of gyrA from nalidixic acid-susceptible and -resistant isolates of Shigella dysenteriae type 1 was amplified by PCR, cloned, and sequenced. DNA sequence analysis of gyrA from nalidixic acid-resistant isolates revealed a C-to-T transition at nucleotide position 248 leading to a Ser-83-to-Leu substitution which was absent in susceptible clinical isolates. Direct HinfI digestion of PCR-amplified DNA detected similar mutations. Thus, DNA gyrase A subunit mutation Ser-83 to Leu is implicated in 4-quinolone resistance in S. dysenteriae type 1.

Detection of gyrA gene mutations associated with ciprofloxacin resistance in methicillin-resistant Staphylococcus aureus: analysis by polymerase chain reaction and automated direct DNA sequencing.

A portion of the gyrA gene from amino acid codons 67 to 129 was sequenced in 34 methicillin-resistant Staphylococcus aureus strains (14 isolated in Minnesota, 10 isolated in Indiana, and 10 isolated in Tennessee). Twenty-eight of these strains were ciprofloxacin resistant. Sixteen of the strains contained a Ser----Leu mutation at codon 84; 3 contained strains a Ser----Ala mutation at codon 84; 3 strains contained two mutations, Ser----Leu at codon 84 and Ser----Pro at codon 85; and 6 strains contained a Glu----Lys mutation at codon 88. Six strains were wild type and ciprofloxacin susceptible. Several mutations from amino acid codons 84 through 88 can be associated with high-level quinolone resistance.