In Vivo Increase in Resistance to Ciprofloxacin inEscherichia coli Associated with Deletion of the C-Terminal Part of MarR

Bacterial strains. E. coli isolates EP1 and EP2 were isolated from the vagina and the urine of a patient with generalized follicular lymphoma who had received 750 mg of CIP twice daily for selective decontamination of the gut. E. coli ATCC 25922 was obtained from the American Type Culture Collection (ATCC). E. coli EP2 acrA::Tn10-Km was obtained from the clinical isolate EP2 by transposon mutagenesis and screening for susceptibility against FQ. E. coli S17 λpir harboring the plasmid pLOF/Km was kindly donated by V. de Lorenzo (6). E. coli S17 containing plasmid pBP591 with wild-type marR has been described previously (V. Hüllen, P. Heisig, and B. Wiedemann, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-64, p. 57, 1997).

PFGE.A rapid PFGE procedure was performed as described previously (8). Macrorestriction was performed withXbaI at 37°C for 3 h. Electrophoresis was performed with the contour-clamped homogeneous electric field (CHEF) mapper system (Bio-Rad, Hercules, Calif.). The run time was 14 h, with an initial switch time of 2.16 s and a final switch time of 35 s.

Susceptibility testing.MICs of CIP were determined according to NCCLS recommendations for microdilution assays (16). MICs of chloramphenicol and tetracycline were determined by Etest (AB BIODISK, Solna, Sweden). For testing of organic solvent tolerance (OST), isolates were inoculated on Mueller-Hinton agar at a concentration of 10⁵ CFU per spot and the plate was overlaid with hexane (H), cyclohexane (CH), and H-CH mixtures at ratios of 3:1, 1:1, and 1:3 to generate different levels of organic solvent toxicity. n-Hexane has a p_owof 3.9, and cyclohexane has a p_ow of 3.4 (3). The plates were checked for growth after 2 days.

DNA amplification and nucleotide sequence determination.Primers (Table 1) and PCR conditions for amplification of gyrA, gyrB, and parEwere used as previously described (7, 12). Primers and annealing temperatures for the amplification of parC, themar operon, marA and marB, andacrA are listed in Table 1. Complementary strands were sequenced in duplicate on a 310 DNA sequencer (Perkin-Elmer, Foster City, Calif.) using either PCR primer (6 μmol).

Plasmids and DNA manipulations.Strains and plasmids used in this study are listed in Table 2. Introduction of the marR frameshift mutation from isolate EP2 into the wild-type marR of isolate EP1 required subcloning of the EcoRI/PstI-digested PCR fragment (with primers marR-f₁₄₅₂ and marR-r₂₂₂₉) into the corresponding pUC18 site to yield plasmid pmarR-ΔC18. Probes for Northern blot analysis were generated by subcloning of marA and soxS into plasmid pcDNA3 (Invitrogen, Groningen, The Netherlands) and in vitro transcription from the SP6 and T7 promoters, respectively. The completemarA gene (1893 to 2282) was amplified in a DNA thermocycler using primers marA-f₁₈₉₃ and marA-r₂₂₈₁. For the amplification and cloning of soxS, primers soxS-f₃₃₁ and soxS-r₁₃₁₉were used. Genomic DNA from E. coli ATCC 25922 served as a template. The PCR fragments were cloned directionally into plasmid pcDNA3 to generate plasmids pc-marA and pc-soxS, respectively. Recombinant DNA techniques, transformation, and restriction enzyme digestions followed standard protocols (19).

Gene replacement.Introduction of the marRmutation into the wild-type marR gene of isolate EP1 was accomplished by homologous recombination between plasmid pmarR-ΔC18 and the bacterial chromosome, essentially as described previously (11). Plasmid pmarR-ΔC18 was introduced into isolate EP1 by electroporation (Genepulser; Bio-Rad). Resulting transformants were grown on Luria-Bertani (LB) agar plates supplemented with 50 mg of ampicillin/liter overnight at 37°C. Recombinant bacteria were then propagated in LB broth containing 32 mg of CIP/liter for selection ofE. coli carrying the desired recombination. Segregation of the plasmid after passaging of single colonies for 1 week was shown by growth inhibition of recombinant clones on LB agar plates containing ampicillin and by inability to amplify the specific marR DNA fragment by PCR with plasmid DNA preparations as templates using plasmid-specific primers.

Insertion mutagenesis.Transposon mutagenesis was performed using a suicide vector system as described previously (6, 10). The exconjugates were selected for reduced CIP MICs by transfer of single colonies to agar plates containing 64 or 2 μg of CIP/ml. Colonies growing selectively on plates with 2 μg of CIP/ml were further analyzed.

RNA extraction and Northern blot analysis.Overnight cultures were diluted 100-fold in LB broth and grown with shaking to mid-logarithmic phase at 37°C. Paraquat was added for the induction of the sox operon (45 min; final concentration, 1.3 mM; Sigma, Deisenhofen, Germany). For production of the marAprobe, plasmid pc-marA was linearized by HindIII digestion and in vitro RNA runoff transcription was performed with the RiboProbe Kit (Promega). Similarly, the soxS probe was obtained by XhoI digestion of plasmid pc-soxS and in vitro transcription. Digoxigenin (DIG)-labeled RNA probes were purified with the RNeasy Purification Kit (QIAGEN). The acrA probe was obtained by PCR. Northern blotting was performed using standard techniques (19).

Complementation assays.For the complementation of the mutant marR of isolate EP2, wild-type marR under the control of the bla promoter was introduced into isolate EP2 by mobilization with the filter mating technique. The donor strain,E. coli S17 carrying plasmid pBP591, and isolate EP2 were mixed in a 1:1 ratio and incubated at 37°C for 12 h on a minimal agar plate. Cells were resuspended in LB broth and plated on LB agar plates containing 50 μg of kanamycin/ml for selection.

Data analysis.SPSS 8.0 for Windows was used for calculation of Mann-Whitney U test results.