Identification of Pseudomonas aeruginosa, Burkholderia cepacia complex, and Stenotrophomonas maltophilia in respiratory samples from cystic fibrosis patients using multiplex PCR

Abstract

A multiplex PCR method was developed to identify P. aeruginosa, B. cepacia complex, and S. maltophilia directly in sputum and oropharyngeal samples from CF patients. One hundred and six patients (53 male, and 53 female) attending our pulmonology clinic were studied from September 2000-April 2001. Two hundred and fifty-seven samples were cultured in selective media and submitted to multiplex PCR reactions, using three primer pairs targeting specific genomic sequences of each species, with an additional primer pair targeting a stretch of ribosomal 16S DNA, universal for bacteria, to act as a control. P. aeruginosa was isolated by culture in 56% of samples, B. cepacia complex in 4.3%, and S. maltophilia in 2.7%, while multiplex PCR identified P. aeruginosa in 78.7%, B. cepacia complex in 3.9%, and S. maltophilia in 3.1% of samples. Multiplex PCR results were verified by PCR reactions using different species-specific primers described in the literature and DNA sequencing of amplicons from a few samples. Comparing to culture results, the sensitivity and specificity values of multiplex PCR for bacterial identification were, respectively, 97.2% and 45.5% for P. aeruginosa, 45.5% and 97.9% for B. cepacia complex, and 40% and 97.6% for S. maltophilia. All 10 multiplex PCR-positive results for B. cepacia complex were confirmed using other species-specific primers described in the literature, while this approach confirmed results for S. maltophilia identification in 7/8 samples (87.5%). Sequencing of amplicons from samples culture-negative but multiplex PCR-positive for P. aeruginosa and B. cepacia complex confirmed their identity, while minor nucleotide differences among amplicons ruled out the hypothesis of PCR contamination.

Stenotrophomonas maltophilia: an Emerging Global Opportunistic Pathogen

“…PFGE, ERIC-PCR, gyrB restriction fragment length polymorphism (RFLP) analysis, and ribotyping have been used to analyze S. maltophilia isolates recovered from sputum samples (45,57,63,78,222,246,287,338,364). Multiplex PCR has been used to identify S. maltophilia, P. aeruginosa, and B. cepacia complex isolates in respiratory samples from CF patients (71). Primers designed to amplify a 149-bp fragment of the chitinase A gene of S. maltophilia were included in a multiplex procedure with universal primers to detect 16S rRNA genes and primers specific for P. aeruginosa and 16S rRNA genes of the B. cepacia complex.…”

“…This multiplex protocol demonstrated high negative predictive values (Ͼ90%) for the identification of the three pathogens, in contrast to the relatively low positive predictive values. It was proposed that the genomic heterogeneity observed among S. maltophilia strains may provide a reason for the low sensitivity of the method for the detection of S. maltophilia (71).…”

“…The genetic heterogeneity of S. maltophilia has been identified through the use of several molecular biology methods (71,246,308,339). The use of restriction fragment length polymorphism (RFLP) analysis of the gyrase B gene (gyrB) after HaeIII digestion demonstrated considerable diversity among S. maltophilia isolates (63).…”

Vitamin K 3 Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump

“…maltophilia mainly causes nosocomial infections (48), although communityacquired infections can also occur (49). The population at risk is mostly composed of immunocompromised hosts, including intensive care unit (ICU) patients, patients with dialysis catheters, hematological diseases, and cystic fibrosis, and those treated with a wide spectrum of antibiotics (50)(51)(52)(53). Although our results indicate that vitK3 might induce the expression of the smeVWX efflux pump even at low concentrations and antagonizes the effects of ofloxacin and chloramphenicol against S. maltophilia, both in the presence and in the absence of this efflux pump, the clinical significance of these findings remains to be clearly established.…”

Isolation of New Stenotrophomonas Bacteriophages and Genomic Characterization of Temperate Phage S1

“…Genotypic identification of the 26 bacterial strains used in this work was performed by multiplex PCR using primers specific for S. maltophilia, the Burkholderia cepacia complex, and Pseudomonas aeruginosa, plus a pair targeting a universal 16S RNA stretch as a positive control (6). The reactions were set up with 25-l aliquots using 2.5-l samples of exponential cultures without any processing as DNA sources.…”