Quinolone resistance mutations in topoisomerase IV: relationship to the flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus

Abstract

Mutations in the flqA (formerly ofx/cfx) resistance locus of Staphylococcus aureus were previously shown to be common after first-step selections for resistance to ciprofloxacin and ofloxacin and to map on the S. aureus chromosome distinctly from gyrA, gyrB, and norA.grlA and grlB, the genes for the topoisomerase IV of S. aureus, were identified from a genomic lambda library on a common KpnI fragment, and grlB hybridized specifically with the chromosomal SmaI A fragment, which contains the flqA locus. Amplification of grlA sequences (codons 1 to 251) by PCRs from nine independent single-step flqA mutants, one multistep mutant, and the parent strain identified mutations encoding a change from Ser to Phe at position 80 in four mutants, a novel change from Ala to either Glu or Pro at position 116 in three mutants, and no change in three mutants. In the multistep mutant, another resistance locus, flqC, was mapped by transformation to the chromosomal SmaI G fragment by linkage to omega(ch::Tn551)1051 (58%) and nov (97.9%), which encodes resistance to novobiocin. This fragment contains the gyrA gene, and flqC mutants had a mutation in gyrA encoding a change from Ser to Leu at position 84, a change previously found in resistant clinical isolates. In genetic outcrosses, the flqC (gyrA) mutation expressed resistance only in flqA mutants, including those with both types of grla mutations. The silent mutant allele of gyrA was present in a flqA background and expressed resistance only upon introduction of a grlA mutation. At fourfold the MIC of ciprofloxacin, the bactericidal activity of ciprofloxacin was reduced in a grlA mutant and was abolished in gyrA grlA double mutants. These findings provide direct genetic evidence that topoisomerase IV is the primary target of current fluoroquinolones in S. aureus and that this effect may result from the greater sensitivity of topoisomerase IV relative to that of DNA gyrase to these agents. Furthermore, resistance from an altered DNA gyrase requires resistant topoisomerase IV for its expression.

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).…”

Development of a rapid assay for screening point mutations associated with quinolone resistance in the Pseudomonas aeruginosa parC gene

“…In fluoroquinolone-resistant strains of other bacterial species, most quinolone resistance-associated mutations in the parC gene have been localized at the codons equivalent to Ser-80 and Glu-84 of P. aeruginosa ParC. [13][14][15][16][17][18][19] Therefore, our assay could screen the majority of mutations in the parC gene that are likely to occur commonly in quinolone-resistant P. aeruginosa strains.…”

Molecular Mechanisms of Antibiotic Resistance: The Need for Novel Antimicrobial Therapies

“…Compared to its parental strain, MT5224c9 showed an approximate 4-fold increase in MIC of ciprofloxacin yet an approximate 8-fold decrease in MIC of the courmarin novobiocin. The mutation is localized to SmaI chromosomal fragment A by a linkage to a Tn551 insertion in this fragment, which encodes topoisomerase IV grlB and grlA genes [166,167]. The mutation in question is Asn470Asp and is actually confined to the GrlB subunit of topoisomerase IV [162], which hydrolyzes ATP for the energydependent introduction of negative supercoils into the DNA during replication.…”