M. Crowe

PCR-Based Detection and Identification of Burkholderia cepacia Complex Pathogens in Sputum from Cystic Fibrosis Patients

ABSTRACT PCR amplification of the recA gene followed by restriction fragment length polymorphism (RFLP) analysis was investigated for the rapid detection and identification ofBurkholderiacepacia complex genomovars directly from sputum. Successful amplification of the B.cepacia complex recA gene from cystic fibrosis (CF) patient sputum samples containing B.cepacia genomovar I, Burkholderiamultivorans, B. cepaciagenomovar III, Burkholderiastabilis, andBurkholderiavietnamiensis was demonstrated. In addition, the genomovar identifications determined directly from sputum were the same as those obtained after selective culturing. Sensitivity experiments revealed thatrecA-based PCR could reliably detect B.cepacia complex organisms to concentrations of 106 CFU g of sputum−1. To fully assess the diagnostic value of the method, sputum samples from 100 CF patients were screened for B. cepacia complex infection by selective culturing and recA-based PCR. Selective culturing identified 19 samples with presumptiveB. cepacia complex infection, which was corroborated by phenotypic analyses. Of the culture-positive sputum samples, 17 were also detected directly by recA-based PCR, while 2 samples were negative. The isolates cultured from bothrecA-negative sputum samples were subsequently identified as Burkholderiagladioli. RFLP analysis of the recA amplicons revealed 2 patients (12%) infected with B. multivorans, 11 patients (65%) infected with B. cepaciagenomovar III-A, and 4 patients (23%) infected with B.cepacia genomovar III-B. These results demonstrate the potential of recA-based PCR-RFLP analysis for the rapid detection and identification of B.cepacia complex genomovars directly from sputum. Where the sensitivity of the assay proves a limitation, sputum samples can be analyzed by selective culturing followed by recA-based analysis of the isolate.

A rapid molecular assay for the detection of antibiotic resistance determinants in causal agents of infective endocarditis

Aims: To develop and employ a PCR amplification system, directly from clinical specimens, for the rapid molecular detection of common antimicrobial resistance genes for streptococci, staphylococci and enterococci organisms causing infective endocarditis (IE).

Improved molecular detection ofBurkholderia cepacia genomovar III and Burkholderia multivorans directly from sputum of patients with cystic fibrosis

Optimum detection of the Burkholderia cepacia complex (BCC) from sputum of patients with cystic fibrosis (CF) is essential in preventing patient-to-patient transmission of this organism. The aim of this study was to develop an improved PCR assay with reference to sensitivity for the direct detection of BCC organisms from CF sputum employing the recA locus. The sensitivity results of three recA PCR assays were compared using various combinations of previously published primers. These included (i) a single-round approach using the primer set BCR1/BCR2, yielding a 1036-bp product, (ii) a single-round approach using the primer set BCR1/Mr, yielding a 465-bp product, and (iii) a semi-nested PCR (SN-PCR) approach using the primer set BCR1/BCR2 followed by BCR1/Mr. The sensitivity of these assays were determined by spiking B. cepacia-free sputum with known numbers of four strains of BCC, namely, genomovar II [B. multivorans] (C1576), genomovar IIIa (C5424, C6433) and genomovar IIIb (C1394). Following optimization, the chosen assay was performed on 14 patients. Employment of the single-round assay with BCR1/BCR2 was the least sensitive with a detection threshold of 10(7) cfu/g sputum for GIIIa and GIIIb, and 10(8) cfu/g sputum for GII. Sensitivity was improved by targeting the smaller amplification region of the recA locus (465 bp) employing the BCR1/Mr primer pair, in combination with a single-round approach, whereby the detection threshold was improved by 1 log for each genomovar. Employment of the semi-nested assay demonstrated optimum sensitivity, whereby the detection threshold increased to 10(1) and 10(2) cfu/g sputum for genomovar IIIa/IIIb and genomovar II, respectively. Subsequent genomovar characterisation can be performed by sequencing of the PCR amplicon without the need for culture which may be beneficial in patients in the initial stages of colonisation or who are transiently colonised and who may be culture-negative for BCC.

Culture-negative Bartonella endocarditis in a patient with renal failure: the value of molecular methods in diagnosis

Abstract Members of the genus Bartonella are increasingly recognised as a cause of culture-negative endocarditis, particularly in those patients with underlying risk factors (e.g., homelessness and alcoholism (B. quintana) or valvulopathy and cat ownership (B. henselae). The aortic and mitral valves are most commonly involved. Here, a case is reported of culture-negative right-sided endocarditis, without any of the above risk factors, due to Bartonella sp. in a 69-year-old man who presented with acute renal failure. The diagnosis was made using a broad-range 16S rRNA polymerase chain reaction (PCR) technique and direct automated sequencing on a peripheral blood sample, which was subsequently confirmed serologically. A review of the literature on Bartonella endocarditis is also presented. Molecular laboratory methods using peripheral blood or blood cultures may be very useful in the diagnosis of causal agents in culture-negative endocarditis and add further support to the recently inclusion of molecular (PCR) diagnosis, as a major Duke’s criterion, for the diagnosis of infective endocarditis.

Long‐term preservation of strains of Burkholderia cepacia, Pseudomonas spp. and Stenotrophomonas maltophilia isolated from patients with cystic fibrosis

Long‐term preservation methods are important in the maintenance of bacteria for downstream research applications. Most clinical laboratories have only limited resources for archiving isolates and therefore require cost‐effective and simple methods. An effective and cheap storage method using debrinated blood and maintenance at −80°C is described.

Molecular detection of an atypical, highly resistant, clonal Pseudomonas aeruginosa isolate in cystic fibrosis patients

BACKGROUND The identification of Pseudomonas aeruginosa (P. aeruginosa) isolates in sputum from cystic fibrosis (CF) patients can be challenging due to the multitude of phenotypic changes isolates undergo during adaptation to the microenvironment of the CF lung. METHODS We report the occurrence of shared P. aeruginosa isolates which failed identification by phenotypic methodologies and required species specific polymerase chain reaction. P. aeruginosa isolates were genotyped by macrorestriction analysis. RESULTS Analysis of atypical isolates revealed one clonal P. aeruginosa isolate and three smaller clusters. In contrast molecular typing of phenotypically characteristic P. aeruginosa isolates revealed only small clusters. Despite exhibiting higher levels of antimicrobial resistance, acquisition of atypical strains was not associated with significant changes in clinical decline. CONCLUSIONS Our experience highlights the importance of accurate identification of bacterial isolates in CF lung disease to detect clonal spread of atypical isolates.

Identification of a novel α-proteobacterium causing bacteraemia in an immunocompetent patient

Abstract An 89-year male with pyrexia and suspected bacteremia was admitted to hospital, where a Gram-negative rod was identified from blood culture. The organism was difficult to identify phenotypically and the resulting sequencing of a 559 bp section of the 16S rRNA gene did not have a high homology score (>97.0%) with any deposited GenBank accession numbers and hence was not able to be assigned to a species within any genus. Given that the isolate was a member of the alpha subclass of the Proteobacteria but did not fall into any of the known genera with more than 93.7% homology ( Brucella, Rhizobium, Ochrobactrum, Agrobacterium ), we believe this isolate to represent a novel α-proteobacterium, which was the cause of bacteraemia in this patient.