Direct PCR Detection of Burkholderia cepacia Complex and Identification of Its Genomovars by Using Sputum as Source of DNA

Abstract

We developed a nested PCR assay that detects the recA gene of the Burkholderia cepacia complex in sputum. The product of the first PCR round is also used to identify the genomovar of the pathogen. The protocol achieves high sensitivity and specificity with simple interpretation of genomovar status.Burkholderia cepacia is a multiresistant and transmissible opportunistic pathogen that frequently impairs the clinical state of patients with cystic fibrosis (CF) (1,3,9,10 (5). To reduce the probability of BCC spreading among patients with CF, a reliable early test that detects small quantities of the bacteria in clinical samples is needed. Conventional microbiological diagnostics of the BCC based on the results of culture and subsequent biochemical identification is insufficient for this purpose because of the potential risk of misidentification or false negativity (7). Moreover, these methods are not able to reliably distinguish the genomovars of the BCC (21).To increase the sensitivity and specificity of diagnostic routines and to identify the genomovars, use of molecular genetic methods is inevitable. Two target genes are commonly used for BCC analysis: the 16S rRNA gene (11,19,20) and the recA gene (13, 16). The recA gene polymorphisms enable both differentiation of the BCC from other closely related bacteria and its sorting into genomovars. Moreover, the differences in the recA sequences within genomovar III led to the establishment of two recA clusters designated III-A and III-B.The present study sought (i) to develop a rapid diagnostic method for early detection of BCC organisms and determination of their genomovars directly from sputum and (ii) to assess the occurrence of the BCC genomovars in Czech patients with CF.From May 2001 to April 2002, we collected 211 sputum samples from 134 consecutive CF patients attending the Prague CF center (55 males, 79 females; age range, 0 to 33 years). All samples were examined for the presence of the BCC by culture and our novel PCR protocol. For cultivation, specimens were liquefied with a homogenization solution (0.9% NaCl, 50 mM KH 2 PO 4 , 35 mM NaOH, 1% N-acetyl-L-cysteine), shaken for 20 min at 800 rpm, and cultured on blood, chocolate, Endo, Sabouraud, and MacConkey agars. The last 53 samples were also cultured on the selective B. cepacia agar (Oxoid, Basingstoke, United Kingdom) that came into use during the study. All of the BCC isolates were identified by the API 20 NE system (Biomerieux, Marcy l'Etoile, France) in accordance with the supplier's protocol. For PCR analysis, sputum was mixed with an equal volume of the homogenization solution and shaken for 1 h at 800 rpm. DNA was extracted from 100 l of liquefied sputum with the AMPLICOR Respiratory Specimen Preparation Kit (Roche, Indianapolis, Ind.) in accordance with the manufacturer's instructions. To check the DNA content in extraction aliquots, real-time PCR quantitation of the human albumin gene as an equivalent of the human DNA content was done on an ABI 7700 system (Applied Biosystems, Foster City, Calif....

Widespread clone of Burkholderia cenocepacia in cystic fibrosis patients in the Czech Republic

“…To check the accuracy of culture-based identification and to determine the genomovar status of the Bcc, extracted DNA from each isolate was subject to generic and genomovar-specific PCRs as described previously (Drevinek et al, 2002). All the isolates were assigned to B. cenocepacia, recA group IIIA.…”

Differentiation of Species Combined into the Burkholderia cepacia Complex and Related Taxa on the Basis of Their Fatty Acid Patterns

“…Identification of BCC isolates to the species level based on the results obtained by genotypic methods, such as restriction fragment length polymorphism analysis of the recA gene or 16S rRNA genes (17,18) and recA gene-based PCR (4), is mostly done with research tools provided in reference centers. Phenotypic testing is still frequently used in routine laboratories.…”

Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

“…For accomplishing a sensitivity of 1.2 x 10 1 to 1.3 x 10 2 cfu per gram of sputum, a semi-nested PCR is required, where consecutive genomovar identification must then be performed by direct sequencing, excluding RFLP analysis [5]. Drvinek et al reported a nested PCR method with a first round of recA PCR, followed by a genomovar-specific amplification step with a sensitivity of 10 3 cfu/ml [6]. FISH technique employed via a 16S rRNA probe was reported to have 4 x 10 5 cfu/ml detection sensitivity when applied directly on sputum samples, but genomovar identification was not possible [19].…”

“…It is reported that commercial systems lack sufficient sensitivity and specificity for accurate identification of bacteria belonging to BCC group [3,4]. Recently, PCR-based methods are emerging as useful diagnostic tools for rapid identification and typing of BCC, also enabling direct detection of bacterial DNA from patient's sputum [2,5,6]. The ability of these assays to detect BCC in sputum is dependent on both selected target sequence and efficiency of DNA extraction procedure.…”