Identification and Detection of Stenotrophomonas maltophilia by rRNA-Directed PCR

Abstract

Stenotrophomonas maltophilia has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF). While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations. Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease, S. maltophiliacan be cultured from respiratory tract secretions. Identification ofS. maltophilia can be problematic, and analysis of isolates from the Burkholderia cepacia Research Laboratory and Repository showed that several isolates presumptively identified asB. cepacia by clinical microbiology laboratories were in fact S. maltophilia. To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify S. maltophilia directly from sputum. The SS-PCR was developed and tested against a panel of 112 S. maltophilia isolates collected from diverse geographic locations. To test for specificity, 43 isolates from 17 different species were analyzed. PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all S. maltophilia isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR. The utility of the SS-PCR to directly identify S. maltophilia in sputum was examined. Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR. Three samples were PCR positive, in complete concordance with the conventional laboratory culture. Thus, we have developed an SS-PCR protocol that can rapidly and accurately identifyS. maltophilia isolates and which can be used for the direct detection of this organism in CF patient sputum.

The Changing Microbial Epidemiology in Cystic Fibrosis

“…Although phylogenetically not as closely related to Burkholderia as the other species causing infection in CF, including Achromobacter and Ralstonia species, S. maltophilia can also be misidentified as a member of the B. cepacia complex (33,201). The use of rRNA-targeted PCR assays for species identification avoids this diagnostic pitfall (348).…”

Infection control recommendations for patients with cystic fibrosis: Microbiology, important pathogens, and infection control practices to prevent patient-to-patient transmission

“…247 The United States B. cepacia Reference Laboratory can provide genotypic confirmatory testing of S. maltophilia using PCR. 248…”

Identification of Bordetella spp. in respiratory specimens from individuals with cystic fibrosis

“…The 43 isolates were analysed at the BcRLR by using a range of species-specific 16S rDNA PCR assays for CF-related bacterial species, including species of Pseudomonas, Stenotrophomonas, Burkholderia, Ralstonia, Pandoraea and Achromobacter [2,3,[5][6][7][8][9][10][11]. All PCR assays were negative, except that 33 (77%) of the 43 isolates were positive according to a PCR assay designed to be specific for A. xylosoxidans [3].…”