a Institute for Medical Microbiology and Hygiene, University of Regensburg, Regensburg; b Department for Pharmaceutical Biology, Institute of Pharmacy, University of Hamburg, Hamburg, Germany
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Abstract |
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Introduction |
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Materials and methods |
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E. cloacae strains Ecl#1 and Ecl#2 were isolated from the same patient on the same day. Ecl#1 was isolated as the sole organism from the conjunctiva, displaying clinical symptoms of conjunctivitis. Ecl#2 was recovered during surgical revision of peritonitis, after extensive resection of the pancreas and abscess formation. The patient had received a cumulative dose of CIP 1200 mg for the treatment of peritonitis until 6 days before the samples were taken. He was then switched to piperacillin/sulbactam, and finally to imipenem, when the microbiological results became available. E. cloacae DSMZ 3264 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). The E. coli strain S17 containing plasmid pBP591 with wild-type marR has been described previously.11 Enterobacter strains were identified by the VITEK system (bioMérieux Vitek, Hazelwood, MO, USA) and comparison of 16S rRNA sequences with GenBank sequences.
PFGE
A rapid pulsed-field gel electrophoresis (PFGE) procedure was carried out as described previously.12 Chromosomal DNA was digested with SpeI at 37°C for 3 h. Electrophoresis was carried out with the CHEF-mapper system (Bio-Rad, Hercules, CA, USA). 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, CAT and DOX were determined by Etest (AB Biodisk, Solna, Sweden). To test organic solvent tolerance, isolates were inoculated on to MuellerHinton agar at a concentration of 105 cfu/spot and the agar surface was overlaid with hexane and cyclohexane to generate different levels of organic solvent toxicity. Hexane and cyclohexane have distribution coefficients in octanol/water of 3.9 and 3.4, respectively.13 The plates were checked for visible bacterial growth after 2 days incubation at 37°C.
DNA amplification and nucleotide sequence determination
Primers and PCR conditions for amplification of partial regions of the gyrA, gyrB, parC, parE, marRA, acrB, soxS, robA, fis, yjcC, yjcD and ram genes are listed in Table 1. Complementary strands were sequenced in duplicate using PCR primers (6 pmol) on a 310 DNA sequencer (Perkin-Elmer, Foster City, CA, USA).
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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 (45 min, final concentration 1.3 mM; Sigma, Deisenhofen, Germany) was added to induce the sox operon or its equivalent. Digoxigenin-labelled probes were obtained by PCR. RNA was purified with the RNeasy Purification Kit (Qiagen, Hilden, Germany). Northern blotting was carried out using standard techniques,14 with glyceraldehyde phosphate dehydrogenase specific probes for control.
Complementation assays
For complementation tests, the marR wild-type gene of E. coli under the control of the bla promoter was introduced into Ecl#1 and Ecl#2 by mobilization, using the filter mating technique.15 The donor strain E. coli S17 containing plasmid pBP591 and the recipient strain, Ecl#1 or Ecl#2, were mixed in a 1:1 ratio and incubated at 37°C for 12 h on minimal agar. Cells were scraped from the surface of the agar, suspended in LB broth and plated on LB agar containing kanamycin 50 mg/L for selection.
Accumulation of norfloxacin
Accumulation of norfloxacin (NOR) was determined according to Martínez-Martínez et al.16 to demonstrate active efflux mechanism(s). Bacteria were incubated with NOR (10 mg/L, 10 min) in the absence and presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP, 0.1 mM). Fluorescence (proportional to the concentration of NOR) was determined on a Hitachi F 2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan; excitation 279 nm, emission 445 nm).
Promoter studies
PCR fragments of 252, 96 and 30 bp of marO of Ecl#1 immediately upstream of the transcription start of marR were cloned in-frame into the pUC18-derived reporter plasmid pIGJ containing the CAT and GFP genes using a BglII restriction site (Figure 2a). Plasmids pIGJ-1mar (252 bp), pIGJ-4mar (96 bp) and pIGJ-5mar (30 bp) were introduced into Ecl#1 and Ecl#2, and the expression of GFP was determined by fluorescence-activated cell sorting (FACS). The mean fluorescence intensity (MFI) was specified. FACS analysis was carried out on a FACSCalibur (Becton-Dickinson, San Jose, CA, USA) cell sorter using CellQuest (Becton Dickinson) analysis software. The graph was generated using WinMDI software.
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GenBank accession numbers of E. cloacae genes are listed in Table 1.
Data analysis
SPSS 8.0 for Windows was used for calculation of the MannWhitney U-test results.
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Results and discussion |
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Elevated MICs of the structurally unrelated antibiotics CAT and DOX, together with the increase in organic solvent tolerance displayed by Ecl#2 compared with Ecl#1, are consistent with an efflux phenotype. The role of an efflux mechanism in quinolone resistance of Ecl#2 was confirmed by its significantly lower NOR accumulation (P < 0.001). The metabolic uncoupler CCCP increased NOR accumulation in both strains; however, accumulation by Ecl#2 was affected more. Consistent with these findings, increased expression of marR and acrB in Ecl#2, compared with Ecl#1 and DSMZ 3264, was demonstrated by northern blotting (Figure 1). To investigate further the regulation of marRA expression in the E. cloacae strains and to evaluate the possible role of marOR, the promoter regions (marO) and the repressor genes (marR) of Ecl#1 and Ecl#2 were sequenced. The corresponding sequences in both strains were found to be identical. Both showed differences in MarR, compared with DSMZ 3264, at residue 6 (Tyr instead of Ile) and residue 80 (Val instead of Ala). Complementation of Ecl#2 with the marR wild-type sequence of E. coli had no effect on the MIC of CIP.
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In contrast to all other target genes, we were unable to identify a sox homologue by PCR using several sets of primers specific for the E. coli genes, under low-stringency conditions or by northern blotting with different probes. Although these results do not necessarily rule out the presence of a sox homologue in DSMZ 3264, Ecl#1 and Ecl#2, our results argue against it.
The hypothesis that the presence or absence of an unknown factor influences expression of the mar locus in Ecl#2 was challenged by the introduction of reporter plasmids into Ecl#1 and Ecl#2. When plasmids pIGJ-1mar (252 bp) and pIGJ-4mar (96 bp) were introduced into Ecl#1 and Ecl#2, expression of GFP was greater in Ecl#2 than in Ecl#1, as detected by FACS analysis (8547 MFI versus 1570 MFI; Figure 2b, data not shown for pIGJ-4mar). Introduction of pIGJ-5mar (30 bp) did not give a differential effect. This finding indicates that the region of bp 96 to 30 relative to the transcription start of marR (a region that in E. coli contains the Fis-binding site and the marbox for binding of MarA, SoxS and RobA)9 interacts with the putative factor. Experiments are in hand to identify the factor.
What is the basic level of activity of efflux pumps of Enterobacteriaceae under natural circumstances in the host?21 Our findings of constitutive overexpression of MarR (which is cotranscribed with MarA) in Ecl#2 in the present study and of MarA in E. coli2 indicate that physiological regulation of the AcrAB locus does not generate sufficient efflux pump activity under conditions in vivo. Two observations indicate that the constitutive overexpression of MarRA in Ecl#2 does not influence the biological fitness of the bacterium. First, the isolate was grown from a site of infection 6 days after the last dose of CIP and therefore has to be regarded as fully pathogenic. Second, growth curves (obtained by recording optical density over time until stationary growth; data not shown) of the two isolates Ecl#1 and Ecl#2 revealed no apparent difference compared with those of three other clinical wild-type E. cloacae isolates.
In conclusion, in this pair of closely related clinical isolates of E. cloacae, different levels of quinolone resistance can be explained by increased AcrAB efflux pump activity in the more resistant strain, in addition to a target mutation in GyrA in both strains. These data corroborate findings in vitro1 and in vivo2 concerning the sequential development of fluoroquinolone resistance.
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Acknowledgements |
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Notes |
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References |
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2
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Received 1 March 2001; returned 9 November 2001; revised 3 December 2001; accepted 11 January 2001