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Multidrug-resistant Escherichia coli mutants selected with diazepam or lomefloxacin overexpressing soxS, sdiA, acrAB, TolC and PBP3

Abstract number: 1732_162

Tavío M.M., Aquili V., Fábrega A., González-Lama Z., Poveda J.B., Vila J.

Objectives: Multiple resistance of Escherichia coli to distinct classes of antimicrobials is usually attributed to simultaneous increased expression of AcrAB-TolC efflux system and loss of porin F generated by transcriptional activators marA, soxS or rob. AcrAB-TolC is also up-regulated by SdiA, an E. coli protein that regulates cell division.

In this study, multidrug resistant mutants selected in vitro with diazepam (DZ) and lomefloxacin (LOM) from two E. coli susceptible clinical isolates were characterised.

Methods: Mutants were selected with DZ or LOM at 2, 3, 4 or 6 × the MIC. Tolerance of strains to 10% cyclohexane was measured in liquid medium. Inner and outer membrane proteins (IMP and OMP) were analysed by electrophoresis on polyacrylamide gels. Penicillin-Binding-Proteins (PBPs) were detected with Bocillin FL and Imperial Protein Stain. Active efflux was evidenced by 0.05 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The expression of the acrA, acrB, marA, soxS, rob and sdiA genes was studied by reverse transcription of total RNA and PCR of cDNA (RT-PCR), using gapA gene as internal control of expression. PCR products were separated on polyacrylamide gels and detected using silver staining. Ag100 strain (induced or non-induced with 5 mM salicylate or 0.2 mM paraquat) was the control strain.

Results: Nine different clones were selected with DZ or LOM. Selection frequencies were around 10-9 to 10-8. The nine mutants increased resistance to quinolones, chloramphenicol and b-lactams (2–8 fold ceftazidime, cefpirome and aztreonam MICs). The presence of CCCP increased the susceptibility of mutants to antimicrobial agents. The OMP TolC increased in mutants, coinciding with their increased cyclohexane tolerance. Mutants and parent strains showed a high expression of acrA and acrB genes. Nevertheless, only mutants showed overexpression of sdiA and soxS and OmpF decrease. Neither parent strains nor mutants showed marA or rob overexpression. Increased PBP3 expression was detected in only the mutants selected with 6 × MIC of DZ or LOM.

Conclusions:

i. DZ (a non-antimicrobial drug) and LOM selected multiple antibiotic resistant mutants that overexpressed the same transcriptional regulators, soxS and sdiA, resulting in TolC increased expression in mutants.

ii. The OmpF decrease, which was generated by soxS overexpression, and increased TolC and PBP3 expression contributed to 2–8-fold increment in ceftazidime, aztreonam and cefpirome MICs in mutants.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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