Dataset Information

Quantitative real-time PCR assay for the rapid identification of the intrinsically 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 (CF) 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 is 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 167 Stenotrophomonas spp. genomes identified a conserved 4?kb region in 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 of 16 CF sputa, demonstrating the assay's 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.

SUBMITTER: Fraser TA

PROVIDER: S-EPMC6861864 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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

Fraser Tamieka A TA Bell Mikaela G MG Harris Patrick N A PNA Bell Scott C SC Bergh Haakon H Nguyen Thuy-Khanh TK Kidd Timothy J TJ Nimmo Graeme R GR Sarovich Derek S DS Price Erin P EP

Microbial genomics 20191016 10

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 (CF) 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, parti ...[more]

PMID: 31617838

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Project description:Most studies on bacterial virulence focus on the pathogen itself. However, it is important to recall that the in-host behavior and the virulence of bacterial pathogens constitute a complex situation that depends on both the microorganisms and the infected host. While healthy people (the community) is infected by classical pathogenic microorganisms, able to cope with the anti-infection defenses of the host, in the case of people with basal diseases, debilitated or immunodepressed, the range of pathogens able to cause infection is wider and includes the so-named opportunistic pathogens, which lack the inherent ability to cause disease in healthy hosts and rarely produce infections in the community. Some of the most relevant opportunistic pathogens, as Stenotrophomonas maltophilia, have an environmental origin and, in occasions, present interesting biotechnological properties. Consequently, it is important knowing whether S. maltophilia isolates recovered from infections constitute a specific phylogenetic branch that has evolved toward acquiring a virulent phenotype as it happens in the case of classical pathogens or rather, any member of this bacterial species is capable of producing infection and its pathogenic behavior is mainly a consequence of the host situation. To address this question, we analyzed a set of environmental and clinical S. maltophilia strains. Our results indicate that this opportunistic pathogen presents a large core genome and that the distribution of genes in general, and of known virulence determinants in particular, is similar among environmental and clinical isolates. The majority of genes not belonging to the S. maltophilia core genome are present in just one or two of the analyzed strains. This indicates that, more than speciation into different lineages (virulent and environmental), the evolution of S. maltophilia is based in the strain-specific acquisition of genes, likely involved in the adaptation of this bacterial species to different microniches. In addition, both environmental and clinical isolates present low susceptibility to several antimicrobials. Altogether our results support that S. maltophilia does not present a specific evolutionary branch toward virulence and most likely infection is mainly the consequence of the impaired anti-infective response of the infected patients.

Project description:Clinical strains of Stenotrophomonas maltophilia are often highly resistant to multiple antibiotics, although the mechanisms of resistance are generally poorly understood. Multidrug resistant (MDR) strains were readily selected by plating a sensitive reference strain of the organism individually onto a variety of antibiotics, including tetracycline, chloramphenicol, ciprofloxacin, and norfloxacin. Tetracycline-selected MDR strains typically showed cross-resistance to erythromycin and fluoroquinolones and, in some instances, aminoglycosides. MDR mutants selected with the other agents generally displayed resistance to chloramphenicol and fluoroquinolones only, although two MDR strains (e.g., K1385) were also resistant to erythromycin and hypersusceptible to aminoglycosides. Many of the MDR strains expressed either moderate or high levels of a novel outer membrane protein (OMP) of ca. 50 kDa molecular mass, a phenotype typical of MDR strains of Pseudomonas aeruginosa hyperexpressing drug efflux systems. Indeed, the 50-kDa OMP of these S. maltophilia MDR strains reacted with antibody to OprM, the outer membrane component of the MexAB-OprM MDR efflux system of P. aeruginosa. Similarly, a ca. 110-kDa cytoplasmic membrane protein of these MDR strains also reacted with antibody to the MexB component of the P. aeruginosa pump. The outer and cytoplasmic membranes of several clinical S. maltophilia strains also reacted with the anti-OprM and anti-MexB antibodies. N-terminal amino acid sequencing of a cyanogen bromide-generated peptide of the 50-kDa OMP of MDR strain K1385, dubbed SmeM (Stenotrophomonas multidrug efflux), revealed it to be very similar to a number of outer membrane multidrug efflux components of P. aeruginosa and Pseudomonas putida. Deletion of the L1 and L2 beta-lactamase genes confirmed that these enzymes were responsible for the bulk of the beta-lactam resistance of K1385 and its parent. Still, overexpression of the MDR efflux mechanism in an L1- and L2-deficient derivative of K1385 did yield a modest increase in resistance to a few beta-lactams. These data are consistent with the MDR efflux mechanism(s) playing a role in the multidrug resistance of S. maltophilia.

Dataset Information

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

Publications

Quantitative real-time PCR assay for the rapid identification of the intrinsically multidrug-resistant bacterial pathogen <i>Stenotrophomonas maltophilia</i>.

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