Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology

Abstract

Staphylococcus aureus is a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed an in silico typing scheme for the software SeqSphere ؉ (Ridom GmbH, Münster, Germany). The implemented target genes (n ‫؍‬ 182) correspond to those queried by the Identibac S. aureus Genotyping DNA microarray (Alere Technologies, Jena, Germany). The in silico scheme was evaluated by comparing the typing results of microarray and of WGS for 154 human S. aureus isolates. A total of 96.8% (n ‫؍‬ 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosome mec element types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences. Staphylococcus aureus is a Gram-positive facultative pathogenic bacterium that is responsible for a high percentage of hospitaland community-acquired infections worldwide. An infection with S. aureus may manifest itself in a broad variety of diseases, ranging from rather harmless local skin infections to severe bacteremia or intoxications (1). This extensive spectrum of virulence is owed, in part, to the bacterium's individual equipment with virulence factors. Analyzing these virulence factors is difficult because purified staphylococcal toxins do not essentially cause distinctive symptoms when administered in the absence of the bacterium, and the specific knockout of single virulence factors does not necessarily reduce the bacterial virulence (2). Thus, it seems that the combination of different virulence factors, their regulation and transcription, and their allelic variants play a crucial role in determining the eventually expressed virulence phenotype. Therefore, it is important to determine not only the presence or absence of single key factors, such as, e.g., Panton-Valentine leucocidin (PVL) or certain enterotoxins, but to obtain a comprehensive picture of the exact allelic variants of as many virulence-associated genes and their regulatory systems as possible. With regar...

Accuracy of Different Bioinformatics Methods in Detecting Antibiotic Resistance and Virulence Factors from Staphylococcus aureus Whole-Genome Sequences

“…Other previous studies have shown 100% concordance between the resistome and toxome in 14 MRSA isolates (24), 98.6% concordance across 5,288 susceptibility predictions in 308 S. aureus isolates (both MRSA and MSSA) (25), 100% concordance for selected resistance and toxin gene presence/absence in 18 MRSA strains (23), and 97% and 97% sensitivity and specificity, respectively, for Typewriter and 99.1% and 99.6% sensitivity and specificity, respectively, for Mykrobe for predicting phenotypic resistance in the Oxford validation samples used here (9,10). A comparison between microarray and WGS in 154 isolates reported 1.7% discordancy in detecting resistance and virulence genes (26), mainly due to the failure of WGS to detect enterotoxins and super antigens (similar to that for Typewriter in this study).…”

The Impact of the Staphylococcus aureus Virulome on Infection in a Developing Country: A Cohort Study

“…This might be due to true variations in the core genome or is related to technical issues. The latter, however, is unlikely, as the same bioinformatics algorithm has been applied and successfully validated with DNA microarray data ( Strauss et al, 2016 ).…”

“…Fourth, the non-detection of genes does not necessarily prove their biological absence, but may also be caused by technical limitations of shotgun sequencing and de novo assembly. However, the technical error rate of WGS was shown to be < 2% ( Strauss et al, 2016 ).…”

“…The S. aureus genomes were screened for the presence and allelic variants of 79 virulence factors (Supplementary Table S1 ). For each gene, a BLAST search was done against all reference alleles ( Strauss et al, 2016 ). All genes that were found with ≥ 95% nucleotide identity and ≥ 99% length overlap to any reference allele were included.…”

“…All genes that were found with ≥ 95% nucleotide identity and ≥ 99% length overlap to any reference allele were included. Targets that were partially identified on a cropped contig or “failed targets” (i.e., containing internal stop codons, frame shifts, or nucleotide ambiguities) were only included if they were found within operons/prophages and if the other genes of the respective operon/prophage were present (e.g., agr , ACME, hlg ) ( Strauss et al, 2016 ).…”