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Antimicrob. Agents Chemother. doi:10.1128/AAC.01128-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Increased genome instability in Escherichia coli lon mutants: relation to emergence of multiple antibiotic resistant (Mar) mutants caused by insertion sequence elements and large tandem genomic amplifications

Center for Adaptation Genetics and Drug Resistance and the Departments of Molecular Biology and Microbiology; and of Medicine, Tufts University School of Medicine, Boston, Massachusetts 02111

^* To whom correspondence should be addressed. Email: stuart.levy{at}tufts.edu.

	Abstract

Thirteen spontaneous multiple antibiotic resistant (Mar) mutants of Escherichia coli AG100 were isolated on LB agar in the presence of tetracycline (4 µg/ml). The phenotype was linked to IS insertions in marR or acrR, or unstable large tandem genomic amplifications which included acrAB and were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a lon-mutated subpopulation of AG100. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7 x 10^-4) of lon::IS186 mutants; ii) new lon mutants appeared during the selection on antibiotic-containing agar plates; iii) lon mutants could slowly grow in the presence of low amounts (about 2 x MIC of the wild type) of chloramphenicol or tetracycline; iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB vs MacConkey) and the antibiotic used for the selection. A previously reported unstable amplifiable high level resistance observed after prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.