Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus

Abstract

In this study we describe a multiplex PCR assay for the detection of nine clinically relevant antibiotic resistance genes of Staphylococcus aureus. Conditions were optimized to amplify fragments of mecA (encoding methicillin resistance), aacA-aphD (aminoglycoside resistance), tetK, tetM (tetracycline resistance), erm(A), erm(C) (macrolide-lincosamide-streptogramin B resistance), vat(A), vat(B), and vat(C) (streptogramin A resistance) simultaneously in one PCR amplification. An additional primer pair for the amplification of a fragment of the staphylococcal 16S rDNA was included as a positive control. The multiplex PCR assay was evaluated on 30 different S. aureus isolates, and the PCR results correlated with the phenotypic antibiotic resistance data obtained by the broth microdilution assay. The multiplex PCR assay offers a rapid, simple, and accurate identification of antibiotic resistance profiles and could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.

FIG. 1.

Single and multiplex PCR amplification products for selected staphylococcal genes. Lanes: 1, mecA; 2, aacA-aphD; 3, tetK; 4, tetM; 5, erm(A); 6, erm(C); 7, vat(A); 8, vat(B); 9, vat(C); 10, 16S rRNA; 11, multiplex; M, molecular size standard. (A) DNA fragments were visualized on a 2.5% agarose gel and stained with ethidium bromide. (B) Fragment analysis was performed with the Agilent Bioanalyzer and the DNA 1000 LabChip kit.

FIG. 2.

Examples of multiplex PCR amplifications on template DNA of 14 different S. aureus isolates (lanes 1 to 14). Lanes: 1, 1719/00; 2, 3932/02; 3, 3940/02; 4, 3887/02; 5, 3211/02; 6, 3516/02; 7, 2513/02; 8, 2569/02; 9, 1450/93; 10, 3396/01; 11, 163/01; 12, 3980/01; 13, 1150/93; 14, 8325; 15, no template DNA; 16, DNA mixture with all targets.