Molecular and Proteomic Characterization of Invasive Staphylococcus aureus (ISA) Isolated from Pediatric Bloodstream Infections

Abstract

Staphyloccocus aureus is a Gram-positive cocci and a bacterial pathogen that is ubiquitous and genetically nimble. When untreated, invasive S. aureus infections (ISAs) have a mortality rate that exceeds 20% (1). S. aureus is equipped with a host of virulence determinants that mediate virulence mediated by cell-wall associated proteins and secreted toxins that are, for the most part, genetically regulated. Experiments to characterize the genetic determinants of pathogenicity in pediatric ISAs were undertaken because ISAs in this population remain largely uncharacterized in the current literature. A retrospective chart review identified ISA disease in pediatric patients (0-18 years) at Golisano Children’s Hospital in Syracuse, New York, and at the University of Arizona Medical Center, Diamond Children’s Hospital, Tucson, AZ. Genetic profiling of the bacteria from these pediatric cohorts used methods such as the polymerase chain reaction coupled to electrospray ionization mass spectrometry (PCR-ESI/MS), traditional PCR, and historic phenotypic methods. Isolates were characterized with respect to their genotype (USA strain type and clonal complex) and their common virulence determinants such as Accessory Gene Regulator (agr) type, leukocidins (lukD/E, pvl), hemolysins (hla, hlb, hld), superantigens (sea, tst), and staphylokinase (sak). Genetic profiles were compiled and compared to clinical disease attributes. In New York isolates, the only genes specifically found to be associated with virulence in pediatric ISAs were sea and luk-PV. The Tucson S. aureus isolates did not exhibit the same genetic virulence patterns, illustrating the premise that multiple strains cause invasive infection and that virulence gene expression differs between bacterial populations from geographic locations. Further study of the Arizona isolates focused on the identification of phenol soluble modulins (PSMs), a newly identified determinant of S. aureus virulence. Identification methods for PSMs use liquid chromatography coupled to multiple reaction monitoring mass spectrometry (MRM). Finally, the Bruker SepsiTyper extraction kid and BioTyper software system (MALDI-TOF mass spectrometry) was examined as a novel detection method for rapid, accurate identification of pathogens, including S. aureus, directly from blood culture samples.