Uses of Staphylococcus aureus GeneChips in Genotyping and Genetic Composition Analysis

Abstract

Understanding the relatedness of strains within a bacterial species is essential for monitoring reservoirs of antimicrobial resistance and for epidemiological studies. Pulsed-field gel electrophoresis (PFGE), ribotyping, and multilocus sequence typing are commonly used for this purpose. However, these techniques are either nonquantitative or provide only a limited estimation of strain relatedness. Moreover, they cannot extensively define the genes that constitute an organism. In the present study, 21 oxacillin-resistant Staphylococcus aureus (ORSA) isolates, representing eight major ORSA lineages, and each of the seven strains for which the complete genomic sequence is publicly available were genotyped using a novel GeneChip-based approach. Strains were also subjected to PFGE and ribotyping analysis. GeneChip results provided a higher level of discrimination among isolates than either ribotyping or PFGE, although strain clustering was similar among the three techniques. In addition, GeneChip signal intensity cutoff values were empirically determined to provide extensive data on the genetic composition of each isolate analyzed. Using this technology it was shown that strains could be examined for each element represented on the GeneChip, including virulence factors, antimicrobial resistance determinants, and agr type. These results were validated by PCR, growth on selective media, and detailed in silico analysis of each of the sequenced genomes. Collectively, this work demonstrates that GeneChips provide extensive genotyping information for S. aureus strains and may play a major role in epidemiological studies in the future where correlating genes with particular disease phenotypes is critical.Monitoring the acquisition and maintenance of genes within bacterial populations is an essential component of understanding the epidemiology of emerging infectious diseases. Accordingly, much effort has been devoted toward developing methods to delineate the relatedness of Staphylococcus aureus strains that are circulating within both health care institutions and community settings.The techniques that are currently used for strain surveillance include spa typing, ribotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) (2,3,8,20). Although these methods have proven valuable in monitoring strain relatedness, none extensively defines the genes that constitute the organism(s) under investigation. Further evaluation of the genes of interest within an individual isolate can be accomplished by PCR amplification of the particular loci of interest followed by restriction enzyme analysis or sequencing. Vandenesch and colleagues recently combined PFGE, MLST, and PCR for 24 individual virulence genes in a study that was designed to examine the relatedness and genetic composition of 117 community-associated oxacillin-resistant S. aureus (CO-ORSA) isolates from several countries (22). The study demonstrated that despite geographic origin or relatedness of the strains, all CO-ORSA isolates contained the ...

Overexpression of Genes of the Cell Wall Stimulon in Clinical Isolates ofStaphylococcus aureusExhibiting Vancomycin-Intermediate-S. aureus-Type Resistance to Vancomycin

“…Custom-designed S. aureus GeneChips (Affymetrix, Inc., Santa Clara, Calif.) are described in detail by Dunman et al (7). Chromosomal DNA was labeled as described previously (7). For RNA samples, reverse transcription, cDNA fragmentation, and terminal labeling of cDNA fragments with biotin were carried out according to the manufacturer's instructions for antisense prokaryotic arrays (Affymetrix).…”

“…Custom-designed S. aureus GeneChips (Affymetrix, Inc., Santa Clara, Calif.) are described in detail by Dunman et al (7). Chromosomal DNA was labeled as described previously (7).…”

Characterization of a strain of community-associated methicillin-resistant Staphylococcus aureus widely disseminated in the United States

“…Briefly, chromosomal DNA was purified from each isolate, fragmented, and biotinylated at the 3Ј end. Then, 1.5 g of labeled DNA was hybridized to a GeneChip, and adjusted "present" and "absent" determinations were made for each locus represented on the array (21).…”

Characterization of a Strain of Community-AssociatedMethicillin-Resistant Staphylococcus aureus WidelyDisseminated in the UnitedStates

“…The genetic compositions of S. aureus pulsed-field types (PFTs) USA100-0022, USA300-0114, USA400-0051, and USA500-0004 isolates were determined using S. aureus Affymetrix GeneChips (Saur2a) (Affymetrix, Santa Clara, CA), as previously described (21); multiple isolates of the USA300 and USA400 PFTs were analyzed. Chromosomal DNA from each isolate was interrogated for the presence or absence of the 7,792 loci represented on the Saur2a chip (21). Briefly, chromosomal DNA was purified from each isolate, fragmented, and biotinylated at the 3Ј end.…”