Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus

Abstract

A 4.2-kb DNA fragment conferring quinolone resistance was cloned from a quinolone-resistant clinical isolate of Staphylococcus aureus and was shown to possess a part of the grlB gene and a mutated grlA gene. S-80-->F and E-84-->K mutations in the grlA gene product were responsible for the quinolone resistance. The mutated grlA genes responsible for quinolone resistance were dominant over the wild-type allele, irrespective of gene dosage in a transformation experiment with the grlA gene alone. However, dominance by mutated grlA genes depended on gene dosage when bacteria were transformed with the grlA and grlB genes in combination. Quinolone-resistant gyrA mutants were easily isolated from a strain, S. aureus RN4220, carrying a plasmid with the mutated grlA gene, though this was not the case for other S. aureus strains lacking the plasmid. The elimination of this plasmid from such quinolone-resistant gyrA mutants resulted in marked increases in quinolone susceptibility. These results suggest that both DNA gyrase and DNA topoisomerase IV may be targets of quinolones and that the quinolone susceptibility of organisms may be determined by which of these enzymes is most quinolone sensitive.

Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus

“…Chromosomal DNA of RN4220, the parent strain, was digested with EcoRI, ligated to pND50 (Yamagishi et al 1996), a shuttle vector for S. aureus and E. coli, to construct a genomic library. TS0605 was transformed with the library, spread on LB agar plates, and then incubated at 43°C, the nonpermissive temperature.…”

Mechanism of quinolone resistance in Staphylococcus aureus

“…[12][13][14][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] However, the frequency of mutations in the grlA and gyrA of clinical isolates has been uncertain. Thus, we examined grlA and gyrA mutations in clinical isolates, and studied the relationship between mutation combinations in both these genes and the susceptibility of the various mutants to three fluoroquinolones.…”

“…11,18,19,26,33,36 In gyrA, ten types of mutations (Ser-84 AE Leu, Ser-84 AE Ala, Ser-84 AE Val, Ser-85 AE Pro, Glu-88 AE Lys, Glu-88 AE Gly, Ser-84 AE Leu and Ser-85 AE Pro, Ser-84 AE Leu and Ile-86 AE Ile, Ser-84 AE Leu and Glu-88 AE Lys, and Ser-84 AE Leu and Glu-88 AE Gly) were found in strains with high-level quinolone resistance. 9,13,23,28,30,31,36 From our data for the 344 clinical isolates of S. aureus, we found nine types of mutations in grlA and eight types in gyrA.…”

“…The finding that grlAgyrA double mutants exhibited higher-level ciprofloxacin resistance (MIC 50 ϭ50 µg/ml) confirmed previous genetic studies. 26,33 Combinations of mutations, including the Glu-84 AE Lys alteration in grlA and the Ser-84 AE Leu alteration in gyrA, conferred high-level quinolone resistance (MIC of ciprofloxacin м100 µg/ml).…”

Mechanisms of resistance to xenobiotics in human therapy

“…The Ser 80 residue of topoisomerase IV of S. aureus, which corresponds to Ser 83 of Gyr A, is substituted by Phe or Tyr in both high-and low-level quinolone-resistant isolates [163]. The Glu 84 Lys substitution also contributes to resistance [164].…”