Use of resequencing oligonucleotide microarrays for identification of Streptococcus pyogenes and associated antibiotic resistance determinants

Abstract

Group A streptococci (GAS) are responsible for a wide variety of human infections associated with considerable morbidity and mortality. Ever since the first systematic effort by Lancefield to group Streptococcus species by M protein variants, the detection and characterization of Streptococcus by different methods have been an evolving process. The ideal assay for GAS identification not only would provide quick and accurate diagnostic results but also would reveal antibiotic resistance patterns and genotype information, aiding not only in treatment but in epidemiologic assessment as well. The oligonucleotide microarray is a promising new technology which could potentially address this need. In this study, we evaluated the usefulness of oligonucleotide resequencing microarrays for identifying GAS and its associated antibiotic resistance markers. We demonstrated an assay platform that combines the use of resequencing DNA microarrays with either random nucleic acid amplification or multiplex PCR for GAS detection. When detecting Streptococcus pyogenes from coded clinical samples, this approach demonstrated an excellent concordance with a more established culture method. To this end, we showed the potential of resequencing microarrays for efficient and accurate detection of GAS and its associated antibiotic resistance markers with the benefit of sequencing information from microarray analysis.

FIG. 1.

Representative RPM v.1 chip image of GAS-positive clinical sample. (A) S. pyogenes-positive clinical sample hybridized to RPM v.1 chip following nucleic acid isolation and amplification using multiplex PCR primers. No significant cross-hybridization is observed. (B) Hybridization-based primary sequences generated through the chip image. GDAS software is used to apply an embedded version of the ABACUS (7) algorithm to produce an estimate of the correct base calls, comparing the respective intensities for the sense and antisense probe sets. (C) FASTA sequence output file and REPI analysis result of S. pyogenes showing the highest BLAST bit score across all respective tile regions where amplicons hybridized.