Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR

Abstract

Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC beta-lactamases are limited because these organisms are usually resistant to all the beta-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 beta-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 beta-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 beta-lactamase expression in organisms with or without a chromosomal AmpC beta-lactamase gene.

FIG. 1.

AmpC dendrogram. Sequences were downloaded from the GenBank database, and structural genes were compared, as described in Material and Methods, by using the DNAsis program. Values in blue correspond to the percent similarity between the most distinct member of each cluster and the other members within that cluster. Primer pairs are correlated by the family of genes that they amplify.

FIG. 2.

Initial analysis of ampC multiplex PCR. Multiplex PCR products were separated in a 2% agarose gel. Lanes are labeled with the ampC gene used as template DNA; ACC, chromosomal ampC gene from H. alvei; M, 100-bp DNA ladder. The amplified product from each PCR is indicated on the left, and the size of the marker in base pairs is shown on the right.

FIG. 3.

Resolution of family-specific variation. Multiplex PCR products were separated in a 2% agarose gel. Lanes are labeled with the ampC gene used as template DNA; ACC, chromosomal ampC gene from H. alvei; M, 100-bp DNA ladder; (−), negative water control; C.o.(−), carryover negative control. The amplified product from each PCR is indicated on the left, and the size of the marker in base pairs is shown on the right.

FIG. 4.

Evaluation of chromosomal cross-hybridization. Multiplex PCR products were separated in a 2% agarose gel. Lanes are labeled with the name of the organism used as the source of template DNA (Table 1); M, 100-bp DNA ladder; (−), negative water control; C.o.(−), carryover negative control. The amplified product from each PCR is indicated on the left, and the size of the marker in base pairs is shown on the right.

FIG. 5.

Analysis of clinical isolates. Multiplex PCR products were separated in a 2% agarose gel. M, 100-bp DNA ladder; (−), negative water control; 4 Templates, MOX-1, LAT-1, DHA-1, and ACC; 2 Templates, FOX-1 and ACT-1; C.o.(−), carryover negative control. (A) Lanes 1 to 4, 6 to 8, and 12, E. coli isolates; lanes 5 and 9, K. pneumoniae isolates; lane 10, P. mirabilis isolate; lane 11, E. aerogenes isolate. (B) Lanes 1, 2, 4, and 10, E. coli isolates; lanes 3 and 5 to 9, K. pneumoniae isolates. The amplified product from each PCR is indicated on the right, and the size of the marker in base pairs is shown on the left.

FIG. 6.

WAVE analysis. (A) Chromatogram obtained by using multiplex PCR products amplified from the following DNA templates (bottom to top): FOX-1, ACT-1, ACC, DHA-1, LAT-1, MOX-1, combination of the six DNA templates listed above, and DNA marker pUC18. (B) Agarose gel electrophoresis of multiplex PCR products obtained by using three different combinations of DNA templates: 2 Templates, FOX-1 and ACT-1; 4 Templates, MOX-1, LAT-1, DHA-1, and ACC; and 6 Templates, combination of the six templates listed above. M, 100-bp ladder.The amplified product from each PCR is indicated on the left, and the size of the marker in base pairs is shown on the right.