A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria

Christian Strauss; Andrea Endimiani; Vincent Perreten

doi:10.1016/j.mimet.2014.11.006

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria

J Microbiol Methods. 2015 Jan:108:25-30. doi: 10.1016/j.mimet.2014.11.006. Epub 2014 Nov 20.

Authors

Christian Strauss¹, Andrea Endimiani¹, Vincent Perreten²

Affiliations

¹ Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
² Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland. Electronic address: vincent.perreten@vetsuisse.unibe.ch.

PMID: 25451460
DOI: 10.1016/j.mimet.2014.11.006

Abstract

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.

Keywords: DNA fragmentation; Detection; Drug resistance; Hybridization; Microarray; phi29 polymerase.

Publication types

Evaluation Study
Research Support, Non-U.S. Gov't

MeSH terms

Anti-Bacterial Agents / chemistry*
Bacterial Proteins / genetics*
DNA Primers / chemistry*
DNA Primers / genetics*
DNA, Bacterial / chemistry
DNA, Bacterial / genetics
Drug Resistance, Bacterial*
Gram-Positive Bacteria / drug effects*
Gram-Positive Bacteria / genetics*
Oligonucleotide Array Sequence Analysis / methods*
Polymerase Chain Reaction
Staining and Labeling

Substances

Anti-Bacterial Agents
Bacterial Proteins
DNA Primers
DNA, Bacterial