Affiliation:
1. Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, School of Medicine, Creighton University, Omaha, Nebraska
2. Department of Immunology, Microbiology, and Parasitology, Basque Country University, Vitoria-Gasteiz, Spain
Abstract
ABSTRACT
Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC β-lactamases are limited because these organisms are usually resistant to all the β-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 β-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 β-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 β-lactamase expression in organisms with or without a chromosomal AmpC β-lactamase gene.
Publisher
American Society for Microbiology