Quantitative real-time PCR assay for the rapid identification of the multidrug-resistant bacterial pathogen Stenotrophomonas maltophilia

Abstract

Stenotrophomonas maltophilia is emerging as an important cause of disease in nosocomial and community-acquired settings, including bloodstream, wound and catheter-associated infections. Cystic fibrosis airways also provide optimal growth conditions for various opportunistic pathogens with high antibiotic tolerance, including S. maltophilia. Currently, there is no rapid, cost-effective, and accurate molecular method for detecting this potentially life-threatening pathogen, particularly in polymicrobial specimens, suggesting that its true prevalence may be underestimated. Here, we used large-scale comparative genomics to identify a specific genetic target for S. maltophilia, with subsequent development and validation of a real-time PCR assay for its detection. Analysis of 165 Stenotrophomonas spp. genomes identified a 4kb region specific to S. maltophilia, which was targeted for Black Hole Quencher assay design. Our assay yielded the positive detection of 89 of 89 (100%) clinical S. maltophilia strains, and no amplification of 23 non-S. maltophilia clinical isolates. S. maltophilia was detected in 10/16 CF sputa, demonstrating the utility for direct detection in respiratory specimens. The assay demonstrated good sensitivity, with limits of detection and quantitation on pure culture of ~10 and ~100 genome equivalents, respectively. Our assay provides a highly specific, sensitive, and cost-effective method for the accurate identification of S. maltophilia, and will improve the diagnosis and treatment of this under-recognized pathogen by enabling its accurate and rapid detection from polymicrobial clinical and environmental samples.