OVEREXPRESSION OF THE MARA OR SOXS REGULATORY GENE IN CLINICAL TOPOISOMERASE MUTANTS OF ESCHERICHIA-COLI

The contribution of regulatory genes to fluoroquinolone resistance was studied with clinical Escherichia coli strains bearing mutations in g yrA and parC and with different levels of fluoroquinolone resistance. Expression of marA and soxS was evaluated by Northern blot analysis of isolates that demonstrated increased organic solvent tolerance, a phe notype that has been linked to overexpression of marA, soxS, and rob. Among 25 cyclohexane-tolerant strains detected by a screen for increas ed organic solvent tolerance (M. Oethinger, W.V. Kern, J. D. Goldman, and S. B. Levy, J. Antimicrob, Chemother, 41:111-114, 1998), we found 5 Mar mutants and 4 Sox mutants. A further Mar mutant was detected amo ng 11 fluoroquinolone-resistant, cyclohexane-susceptible E. coli strai ns used as controls, Comparison of the marOR sequences of clinical Mar mutants with that of E. coli K-12 (GenBank accession no. M96235) reve aled point mutations in marR in all mutants which correlated with loss of repressor function as detected in a marO::lacZ transcriptional ass ay. We found four other amino acid changes in MarR that did not lead t o loss of function. Two of these changes, present in 20 of the 35 sequ enced marOR fragments, identified a variant genotype of marOR. Isolate s with the same gyrA and parC mutations showed increased fluoroquinolo ne resistance when the mutations were accompanied by overexpression of marA or soxS. These data support the hypothesis that high-level fluor oquinolone resistance involves mutations at several chromosomal loci, comprising structural and regulatory genes.