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Detection of the Mex Efflux Pumps in Pseudomonas aeruginosa by Using a Combined Resistance-Phenotypic Markers and Multiplex RT-PCR

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The aim of this study was to detect the expression of 4 clinically-important efflux pumps in the Resistance-Nodulation-Cell Division (RND) family including MexAB-OprM, MexXY, MexCD-OprJ and MexEF-OprN in Pseudomonas aeruginosa using a combination of resistance-phenotypic markers and multiplex RT-PCR (mRT-PCR). The antibiotic substrates specific for each Mex systems were used as phenotypic markers including carbenicillin, MexAB-OprM, erythromycin, MexCD-OprJ, norfloxacin and imipenem, MexEF-OprN and gentamicin, MexXY-OprM. The methods were validated with reference strains with known genotypes of the Mex systems and the potential applicability in clinical practice was tested with clinical isolates. The results for the reference strains support that the combination of resistance phenotype and mRT-PCR is a potential-attractive method for diagnosis of efflux-mediated resistance in P. aeruginosa. Further development to make it more practical for clinical use and study in a larger number of clinical isolates is required.
Cite this paper
Poonsuk, K. and Chuanchuen, R. (2014) Detection of the Mex Efflux Pumps in Pseudomonas aeruginosa by Using a Combined Resistance-Phenotypic Markers and Multiplex RT-PCR. Open Journal of Medical Microbiology, 4, 153-160. doi: 10.4236/ojmm.2014.43018
 

[1] Aksamit, T.R. (1993) Pseudomonas Pneumonia and Bacteremia in the Immunocompromised Patient. In: Fick, R.B.J., Ed., Pseudomonas aeruginosa: The Opportunist—Pathogenesis and Disease, CRC Press, Boca Raton, 177-188.
[2] Lister, P.D., Wolter, D.J. and Hanson, N.D. (2009) Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms. Clinical Microbiology Reviews, 22, 582-610.
http://dx.doi.org/10.1128/CMR.00040-09
[3] Poole, K. and Srikumar, R. (2001) Multidrug Efflux in Pseudomonas aeruginosa: Components, Mechanisms and Clinical Significance. Current Topics in Medicinal Chemistry, 1, 59-71.
http://dx.doi.org/10.2174/1568026013395605
[4] Morita, Y., Komori, Y., Mima, T., Kuroda, T., Mizushima, T. and Tsuchiya, T. (2001) Construction of a Series of Mutants Lacking All of the Four Major mex Operons for Multidrug Efflux Pumps or Possessing Each One of the Operons from Pseudomonas aeruginosa PAO1: MexCD-OprJ Is an Inducible Pump. FEMS Microbiology Letters, 202, 139-143.
http://dx.doi.org/10.1111/j.1574-6968.2001.tb10794.x
[5] Kohler, T., Michea-Hamzehpour, M., Henze, U., Gotoh, N., Curty, L.K. and Pechere, J.C. (1997) Characterization of MexE-MexF-OprN, a Positively Regulated Multidrug Efflux System of Pseudomonas aeruginosa. Molecular Microbiology, 23, 345-354.
http://dx.doi.org/10.1046/j.1365-2958.1997.2281594.x
[6] Aendekerk, S., Ghysels, B., Cornelis, P. and Baysse, C. (2002) Characterization of a New Efflux Pump, MexGHI-OpmD, from Pseudomonas aeruginosa That Confers Resistance to Vanadium. Microbiology, 148, 2371-2381.
[7] Wolter, D.J., Smith-Moland, E., Goering, R.V., Hanson, N.D. and Lister, P.D. (2004) Multidrug Resistance Associated with mexXY Expression in Clinical Isolates of Pseudomonas aeruginosa from a Texas Hospital. Diagn. Microbiol. Diagnostic Microbiology and Infectious Disease, 50, 43-50.
http://dx.doi.org/10.1016/j.diagmicrobio.2004.05.004
[8] Sevillano, E., Valderrey, C., Canduela, M.J., Umaran, A., Calvo, F. and Gallego, L. (2006) Resistance to Antibiotics in Clinical Isolates of Pseudomonas aeruginosa. Pathologie Biologie (Paris), 54, 493-497.
http://dx.doi.org/10.1016/j.patbio.2006.07.030
[9] Mesaros, N., Glupczynski, Y., Avrain, L., Caceres, N.E., Tulkens, P.M. and Van Bambeke, F. (2007) A Combined Phenotypic and Genotypic Method for the Detection of Mex Efflux Pumps in Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy, 59, 378-386.
http://dx.doi.org/10.1093/jac/dkl504
[10] Lomovskaya, O., Warren, M.S., Lee, A., Galazzo, J., Fronko, R., Lee, M., et al. (2001) Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents for Combination Therapy. Antimicrobial Agents and Chemotherape, 45, 105-116.
http://dx.doi.org/10.1128/AAC.45.1.105-116.2001
[11] Yoneda, K., Chikumi, H., Murata, T., Gotoh, N., Yamamoto, H., Fujiwara, H., et al. (2005) Measurement of Pseudomonas aeruginosa Multidrug Efflux Pumps by Quantitative Real-Time Polymerase Chain Reaction. FEMS Microbiology Letters, 243, 125-131.
http://dx.doi.org/10.1016/j.femsle.2004.11.048
[12] Watson, J.M. and Holloway, B.W. (1978) Chromosome Mapping in Pseudomonas aeruginosa. Journal of Bacteriology, 133, 1113-1125.
http://dx.doi.org/10.103/00006450-01000-00007
[13] Chuanchuen, R., Beinlich, K., Hoang, T.T., Becher, A., Karkhoff-Schweizer, R.R. and Schweizer, H.P. (2001) Cross-Resistance between Triclosan and Antibiotics in Pseudomonas aeruginosa Is Mediated by Multidrug Efflux Pumps: Exposure of a Susceptible Strain to Triclosan Selects nfxB Mutants Overexpressing MexCD-OprJ. Antimicrobial Agents and Chemotherapy, 45, 428-432.
http://dx.doi.org/10.1128/AAC.45.2.428-432.2001
[14] Chuanchuen, R., Narasaki, C.T. and Schweizer, H.P. (2002) The MexJK Efflux Pump of Pseudomonas aeruginosa Requires OprM for Antibiotic Efflux but Not for Efflux of Triclosan. Journal of Bacteriology, 184, 5036-5044.
http://dx.doi.org/10.1128/JB.184.18.5036-5044.2002
[15] Poonsuk, K., Tribuddharat, C. and Chuanchuen, R. (2013) Aminoglycoside Resistance Mechanisms in Pseudomonas aeruginosa Isolates from Non-Cystic Fibrosis Patients in Thailand. Canadian Journal of Microbiology, 59, 51-56.
http://dx.doi.org/10.1139/cjm-2012-0465
[16] Woods, C.R., Versalovic, J., Koeuth, T. and Lupski, J.R. (1993) Whole-Cell Repetitive Element Sequence-Based Polymerase Chain Reaction Allows Rapid Assessment of Clonal Relationships of Bacterial Isolates. Journal of Clinical Microbiology, 31, 1927-1931.
[17] CLSI. (2013) VET01-A4: Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard. 4th Edition. Clinical and Laboratory Standards Institute, Wayne, PA.
[18] Fang, X., Fang, Z., Zhao, J., Zou, Y., Li, T., Wang, J., et al. (2012) Draft Genome Sequence of Pseudomonas aeruginosa Strain ATCC 27853. Journal of Bacteriology, 194, 3755.
http://dx.doi.org/10.1128/JB.00690-12
[19] Winsor, G.L., Lam, D.K., Fleming, L., Lo, R., Whiteside, M.D., Yu, N.Y., et al. (2011) Pseudomonas Genome Database: Improved Comparative Analysis and Population Genomics Capability for Pseudomonas genomes. Nucleic Acids Research, 39, D596-D600.
http://dx.doi.org/10.1093/nar/gkq869
[20] Lamers, R.P., Cavallari, J.F. and Burrows, L.L. (2013) The Efflux Inhibitor Phenylalanine-Arginine Beta-Naphthylamide (PABetaN) Permeabilizes the Outer Membrane of Gram-Negative Bacteria. PLoS ONE, 8, e60666.
http://dx.doi.org/10.1371/journal.pone.0060666
[21] Kohler, T., Michea-Hamzehpour, M., Henze, U., Gotoh, N., Curty, L.K. and Pechere, J.C. (1997) Characterization of MexE-MexF-OprN, a Positively Regulated Multidrug Efflux System of Pseudomonas aeruginosa. Molecular Microbiology, 23, 345-354.
http://dx.doi.org/10.1046/j.1365-2958.1997.2281594.x
[22] Westbrock-Wadman, S., Sherman, D.R., Hickey, M.J., Coulter, S.N., Zhu, Y.Q., Warrener, P., et al. (1999) Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability. Antimicrobial Agents and Chemotherapy, 43, 2975-2983.
[23] Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Tsujimoto, H. and Nishino, T. (2000) Substrate Specificities of MexAB-OprM, MexCD-OprJ and MexXY-OprM Efflux Pumps in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 44, 3322-3327.
http://dx.doi.org/10.1128/AAC.44.12.3322-3327.2000
[24] Murata, T., Kuwagaki, M., Shin, T., Gotoh, N. and Nishino, T. (2002) The Substrate Specificity of Tripartite Efflux Systems of Pseudomonas aeruginosa Is Determined by the RND Component. Biochemical and Biophysical Research Communications, 299, 247-251.
http://dx.doi.org/10.1016/S0006-291X(02)02626-8
[25] Hocquet, D., Vogne, C., El Garch, F., Vejux, A., Gotoh, N., Lee, A., et al. (2003) MexXY-OprM Efflux Pump Is Necessary for Adaptive Resistance of Pseudomonas aeruginosa to Aminoglycosides. Antimicrobial Agents and Chemotherapy, 47, 1371-1375.
http://dx.doi.org/10.1128/AAC.47.4.1371-1375.2003
[26] Kohler, T., Michea-Hamzehpour, M., Plesiat, P., Kahr, A.L. and Pechere, J.C. (1997) Differential Selection of Multidrug Efflux Systems by Quinolones in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 41, 2540-2543.
[27] Ochs, M.M., McCusker, M.P., Bains, M. and Hancock, R.E. (1999) Negative Regulation of the Pseudomonas aeruginosa Outer Membrane Porin OprD Selective for Imipenem and Basic Amino Acids. Antimicrobial Agents and Chemotherapy, 43, 1085-1090.
[28] Wolter, D.J., Hanson, N.D. and Lister, P.D. (2008) Novel Mechanism of mexEF-oprN Efflux Pump Overexpression in Pseudomonas aeruginosa without Co-Regulation of oprD Expression. Abstracts. 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington DC, October 26 2008.
[29] Islam, S., Jalal, S. and Wretlind, B. (2004) Expression of the MexXY Efflux Pump in Amikacin-Resistant Isolates of Pseudomonas aeruginosa. Clinical Microbiology and Infection, 10, 877-883.
http://dx.doi.org/10.1111/j.1469-0691.2004.00991.x
[30] Poonsuk, K. and Chuanchuen, R. (2012) Contribution of the MexXY Multidrug Efflux Pump and Other Chromosomal Mechanisms on Aminoglycoside Resistance in Pseudomonas aeruginosa Isolates from Canine and Feline Infections. The Journal of Veterinary Medical Science, 74, 1575-1582.
http://dx.doi.org/10.1292/jvms.12-0239                       eww141223lx

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