Molecular cloning and functional analysis of a novel ATP-dependent multidrug efflux pump in Stenotrophomonas maltophilia
Abstract number: P2032
Al Hamad A., Burnie J., Upton M.
Objectives: To investigate the presence of a multidrug resistance encoding ABC-type efflux pump in Stenotrophomonas maltophilia, a nosocomial pathogen that shows significant degrees of intrinsic and acquired resistance to a wide variety of antimicrobial agents.
Methods: Open reading frames (ORFs) of a hypothetical ABC transporter that are composed of 500 to 800 aa with an N-terminal membrane spanning domain (MSD) and a C-terminal ATP-binding cassette (ABC) domain have been identified in the S. maltophilia genome. These were then aligned with experimentally proven antibiotic resistance encoding ABC transporters, and those with significant homology were cloned into a hypersusceptible acrAB mutant E. coli strain. MICs of several antimicrobial agents were determined and the post antibiotic effect (PAE) of ciprofloxacin was measured.
Results: The identified ABC transporter (ORF1) has been found to be similar to N-MDR1, C-MDR1, LmrA, and VcaM with amino acid homologies of 39.5%, 41.2%, 42.2%, and 67.5%, respectively. Overexpression of the identified gene led to an 8-fold increase in the MIC of ciprofloxacin, norfloxacin, and tetracycline; other antimicrobial agents have shown a 2-fold increase in the MIC. Heterologous expression of the ABC transporter gene in a supersusceptible E. coli strain led to shorter PAE when exposed to ciprofloxacin for 2 hours compared with the control strain expressing only the lacZ gene (0.9 versus 2.5 hours).
Conclusion: ORF1 is a novel ABC transporter in S. maltophilia, with a characteristic MSD-ABC domain organisation. Overexpression in a hypersusceptible E. coli strain was associated with a significant increase in MICs of several antimicrobial agents including ciprofloxacin, a drug that is commonly used in clinical practice. Additionally, data from the PAE experiment has shown a shorter effect in the strain containing the cloned ABC transporter when compared to its control, suggesting a lower intracellular concentration of the antibiotic in the test strain which may be attributed by a more efficient removal of the antibiotic by the identified ABC transporter.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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