Pathogen Profiling: Rapid Molecular Characterization of Staphylococcus aureus by PCR/Electrospray Ionization-Mass Spectrometry and Correlation with Phenotype

There are few diagnostic methods that readily distinguish among community-acquired methicillin (meticillin)-resistant Staphylococcus aureus strains, now frequently transmitted within hospitals. We describe a rapid and high-throughput method for bacterial profiling of staphylococcal isolates. The method couples PCR to electrospray ionization-mass spectrometry (ESI-MS) and is performed on a platform suitable for use in a diagnostic laboratory. This profiling technology produces a high-resolution genetic signature indicative of the presence of specific genetic elements that represent distinctive phenotypic features. The PCR/ESI-MS signature accurately identified genotypic determinants consistent with phenotypic traits in well-characterized reference and clinical isolates of S. aureus. Molecular identification of the antibiotic resistance genes correlated strongly with phenotypic in vitro resistance. The identification of toxin genes correlated with independent PCR analyses for the toxin genes. Finally, isolates were correctly classified into genotypic groups that correlated with genetic clonal complexes, repetitive-element-based PCR patterns, or pulsed-field gel electrophoresis types. The high-throughput PCR/ESI-MS assay should improve clinical management of staphylococcal infections.Invasive infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) are among the most common complications of health care in the United States. Klevens et al. estimate that 94,360 invasive MRSA infections occur in the United States each year, with associated deaths in 18,650 cases (26). Infections are subcategorized as health care-associated MRSA (HA-MRSA) infections or as community-associated MRSA (CA-MRSA) infections; the latter occur in otherwise healthy people who have not experienced a hospital stay within the past 12 months (24, 35). CA-MRSA infections have largely been attributable to a few strains, designated pulsed-field types (PFTs) USA300 and USA400 (26, 29). Once introduced into a health care environment, CA-MRSA strains can be readily transmitted, blending with or replacing entrenched HA-MRSA strains (25, 35).Microbial genotyping analysis allows investigation into the clonality of an outbreak and risk factors associated with infection (5). Methods such as pulsed-field gel electrophoresis (PFGE) (39), repetitive-element-based PCR (rep-PCR) (42), and multilocus sequence typing (MLST) (10, 11) are used for microbial genotyping but are costly and labor-intensive and do not enable specific characterization of acquired genetic elements encoding virulence factors or toxins or of genes that mediate antibiotic resistance (40). A rapid technique for determining the MRSA strain genotype and its broader complement of genetic elements would enable a more comprehensive understanding of transmission dynamics and could lead to more effective actionable decisions related to bed management, prioritization of infection control resources, and treatment.Here, we describe the use of a rapid and high-throughput method to simultaneously genotype and characterize S. aureus specimens with respect to acquired genes encoding virulence factors, toxins, and antibiotic resistance determinants. The method is based on PCR coupled to electrospray ionization-mass spectrometry (ESI-MS) (8, 22, 37).The PCR/ESI-MS assay uses several novel strategies. First, broad-range primers, targeting sites that are highly conserved in all members of a microbe family, are used to amplify PCR products from groupings of microbes rather than single species. These primers are coupled with species- or strain-specific primers for the identification of specific pathogens or antibiotic targets. Second, PCR conditions are, by design, permissive and thus tolerant of mismatches, so that even sequences from novel strains can be amplified. Third, inosine and other “wild-card” nucleotides are used in primers to facilitate PCR analysis of mispaired sequences. Fourth, because MS simply measures the mass-to-charge ratio (m/z), the sequence of the amplicon need not be known in order to detect it. The technology offers advantages over routine single-target and multiplex PCR in that it is a full bioinformatics sequence analysis system.After amplification, MS is used to rapidly determine the precise mass-to-charge ratio for the amplified nucleic acid fragments present, and the A, C, T, and G contents (i.e., the base composition) of each amplicon are determined using proprietary software that creates a signature to allow organism identification and genotyping. This novel MS technology enables the rapid, sensitive, cost-effective, and simultaneous detection of a wide range of typical pathogenic organisms.We used the PCR/ESI-MS assay to analyze a well-characterized set of S. aureus strains from the CDC and geographically distinct clinical isolates from Maryland and Arizona. The PCR/ESI-MS technology effectively combines genotyping and characterization on a single high-throughput platform suitable for surveillance, infection control interventions, and patient management.