StaphyloSeq: Development of a next generation highly multiplexed amplicon sequencing tool for identification of various Staphylococcal species and strain types
Staphylococcal species are responsible for a multitude of infectious co-morbidities. Methicillin resistant Staphylococcus aureus (MRSA) continues to predominate the attention of the medical field, yet the scientific community progressively recognizes methicillin susceptible S. aureus (MSSA) as well as several coagulase-negative staphylococcal (CoNS) species for their pathogenic potential. Current methods of detection are focused on MRSA and involve time-consuming culturing or simple PCR-based amplification with little to no characterization of strain type. Strain identification allows for tracking of strains to understand risk factors for transmission and prediction of virulence, as strains are often associated with a particular phenotype. The need for a highly multiplexed, rapid tool for detection and characterization of various Staphylococcus species in a sample is increasingly significant. StaphyloSeq is a next generation amplicon sequencing assay developed to target various strain types of S. aureus and S. epidermidis, the two species largely responsible for septicemia and other infections. StaphyloSeq can be used directly on complex metagenomic samples, obviating culture methods, and is highly multiplexed, maximizing informative data in a single run. To design StaphyloSeq, we used whole genome sequencing and single nucleotide polymorphism (SNP) phylogenetic analysis to identify canonical SNPs (canSNPs) for each strain of interest and designed primers flanking the canSNPs for amplification. We validated StaphyloSeq on a panel of genomic DNA from known species and strain types, followed by a panel of metagenomic DNA from several specimen types, previously characterized by culture methods. Once fully developed the assay will target S. aureus, S. epi, S. hominis, S. haemolyticus, S. lugdunensis, as well as, antibiotic resistance genes and SCCmec typing regions. Use of this tool will allow rapid, multiplexed analysis of clinical samples obtaining information about species, strain typing, and virulence gene characterization.