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Journal of Clinical Microbiology, June 2002, p. 2153-2162, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.2153-2162.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection of Plasmid-Mediated AmpC ß-Lactamase Genes in Clinical Isolates by Using Multiplex PCR

F. Javier Pérez-Pérez1,2 and Nancy D. Hanson1*

Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, School of Medicine, Creighton University, Omaha, Nebraska,1 Department of Immunology, Microbiology, and Parasitology, Basque Country University, Vitoria-Gasteiz, Spain2

Received 11 December 2001/ Returned for modification 26 February 2002/ Accepted 14 March 2002

Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC ß-lactamases are limited because these organisms are usually resistant to all the ß-lactam antibiotics, except for cefepime, cefpirome, and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Six families of plasmid-mediated AmpC ß-lactamases have been identified, but no phenotypic test can differentiate among them, a fact which creates problems for surveillance and epidemiology studies. This report describes the development of a multiplex PCR for the purpose of identifying family-specific AmpC ß-lactamase genes within gram-negative pathogens. The PCR uses six sets of ampC-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis. ampC multiplex PCR differentiated the six plasmid-mediated ampC-specific families in organisms such as Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella enterica serovar Typhimurium. Family-specific primers did not amplify genes from the other families of ampC genes. Furthermore, this PCR-based assay differentiated multiple genes within one reaction. In addition, WAVE technology, a high-pressure liquid chromatography-based separation system, was used as a way of decreasing analysis time and increasing the sensitivity of multiple-gene assays. In conclusion, a multiplex PCR technique was developed for identifying family-specific ampC genes responsible for AmpC ß-lactamase expression in organisms with or without a chromosomal AmpC ß-lactamase gene.

* Corresponding author. Mailing address: Center for Research in Anti-Infectives and Biotechnology, Department of Microbiology and Immunology, Bldg. CRISS II, School of Medicine, Creighton University, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-5837. Fax: (402) 280-1875. E-mail: ndhanson{at}creighton.edu.

Journal of Clinical Microbiology, June 2002, p. 2153-2162, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.2153-2162.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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