Increased Genome Instability in Escherichia coli lonMutants: Relation to Emergence of Multiple-Antibiotic-Resistant (Mar) Mutants Caused by Insertion Sequence Elements and Large Tandem Genomic Amplifications

ABSTRACTThirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coliAG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 μg/ml). The phenotype was linked to insertion sequence (IS) insertions in marRor acrRor unstable large tandem genomic amplifications which included acrABand which were bordered by IS3or IS5sequences. Five different lonmutations, 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 subpopulation of AG100 with lonmutations. That the lonlocus 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 × 10−4) of lon::IS186mutants, (ii) new lonmutants appeared during the selection on antibiotic-containing agar plates, (iii) lonmutants could slowly grow in the presence of low amounts (about 2× MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lonmutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lonmutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.