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

Abstract

22Stenotrophomonas maltophilia is emerging as an important cause of disease in nosocomial 23 and community-acquired settings, including bloodstream, wound and catheter-associated 24 infections. Cystic fibrosis airways also provide optimal growth conditions for various 25 opportunistic pathogens with high antibiotic tolerance, including S. maltophilia. Currently, 26 there is no rapid, cost-effective, and accurate molecular method for detecting this potentially 27 life-threatening pathogen, particularly in polymicrobial specimens, suggesting that its true 28 prevalence may be underestimated. Here, we used large-scale comparative genomics to 29 identify a specific genetic target for S. maltophilia, with subsequent development and 30 validation of a real-time PCR assay for its detection. Analysis of 165 Stenotrophomonas spp. 31genomes identified a 4kb region specific to S. maltophilia, which was targeted for Black Hole 32 Quencher assay design. Our assay yielded the positive detection of 89 of 89 (100%) clinical 33 S. maltophilia strains, and no amplification of 23 non-S. maltophilia clinical isolates. S. 34 maltophilia was detected in 10/16 CF sputa, demonstrating the utility for direct detection in 35 respiratory specimens. The assay demonstrated good sensitivity, with limits of detection and 36 quantitation on pure culture of ~10 and ~100 genome equivalents, respectively. Our assay 37 provides a highly specific, sensitive, and cost-effective method for the accurate identification 38 of S. maltophilia, and will improve the diagnosis and treatment of this under-recognized 39 pathogen by enabling its accurate and rapid detection from polymicrobial clinical and 40 environmental samples.41 42Stenotrophomonas maltophilia is a Gram-negative, intrinsically multidrug-resistant bacterium 43 that is ubiquitous in aqueous environments such as soils, plant roots, water treatments and 44 distribution systems (1). Whilst conventionally overlooked as a laboratory contaminant, or as 45 a common commensal in hospitalized patients, S. maltophilia is increasingly being 46 recognized as an important nosocomial pathogen in its own right due to its ability to cause 47 life-threatening disease in immunocompromised individuals (2). This opportunistic pathogen 48 has been isolated from a variety of hospital settings including faucets, sinks, central venous 49 catheters, ice machines, tap water, and water fountains, reinforcing its nosocomial importance 50 (1, 3, 4). S. maltophilia most commonly infects people with meningitis, cancer, chronic 51 obstructive pulmonary disease (COPD), or cystic fibrosis (CF), with pneumonia, bacteraemia, 52 and wound and urinary infections being the most frequent clinical manifestations (5, 6). Risk 53 factors for S. maltophilia infection include prolonged hospitalization, neutropenia, 54 catheterization, and previous use of broad-spectrum antibiotics (7). The recommended 55 antibiotic treatment for S. maltophilia infections is co-trimoxazole; however, resistance 56 towards this antibiotic combina...