Assessment of fluorescent in situ hybridization and PCR-based methods for rapid identification of Burkholderia cepacia complex organisms directly from sputum samples

Abstract

Several species within the Burkholderia cepacia complex (BCC) have emerged as significant opportunistic pathogens of patients with cystic fibrosis (CF). BCC infection is typically associated with a poor clinical prognosis and decreased survival. These factors, combined with the existence of highly transmissible epidemic strains, have resulted in strict segregation of BCC- and non-BCC-infected patients to minimize cross infection. Accurate and rapid diagnosis of infections is essential to enable appropriate patient management. However, the rapidly evolving taxonomy of BCC poses a considerable challenge to diagnostics. In the present study, we assessed a commercially available fluorescent in situ hybridization (FISH) assay (seaFAST Cystic Fibrosis I kit) and a novel rRNA gene-based PCR assay for the rapid identification of BCC-positive sputa, irrespective of the BCC species. We report that, while the FISH assay fails to identify all BCC species, it does identify the majority of species, including the two most clinically relevant species, B. multivorans and B. cenocepacia. The sensitivity of the assay applied to sputum was limited by nonspecific background fluorescence. While sputum processing was optimized to minimize background, the resulting sensitivity for BCC detection was 8 x 10(5) CFU/ml. In contrast, the novel PCR assay reported herein exhibits 100% sensitivity and specificity for all BCC species and can detect 10(4) CFU/ml when applied to sputum. This novel rRNA gene-based assay is currently the most sensitive BCC-specific PCR assay for the detection of BCC direct from clinical samples and as such is a valuable addition to the field of BCC diagnostics.

FIG. 1.

Application of the BCC 16S rRNA gene-based PCR assay to BCC and closely related non-BCC species. A 196-bp product was obtained from all BCC species examined to date. B. cep., B. cepacia; B. mult., B. multivorans; B. ceno., B. cenocepacia; B. stab, B. stabilis; B. viet., B. vietnamiensis; B. dol., B. dolosa; B. amb., B. ambifaria; B. anth., B. anthina; B. pyrr., B. pyrrocinia; B. ubon., B. ubonensis; B. pseud., B. pseudomallei; B. caled., B. caledonica; Ralst., Ralstonia; Pand., Pandoraea. R-5630, R-15930, R-24201, R-24196, and R-16017 represent five presumptive novel species of the BCC (24a). Results shown are representatives of each species tested. Refer to Materials and Methods (“Bacterial strains and culture conditions”) for full details of the number of isolates of each species tested.

FIG. 2.

Application of the seaFAST Cystic Fibrosis I FISH kit to representative BCC and non-BCC strains. (A) B. cepacia; (B) B. multivorans; (C) B. cenocepacia; (D) B. stabilis; (E) B. vietnamiensis; (F) B. dolosa; (G-1 and G-2) B. ambifaria; (H) B. anthina; (I-1 and I-2) B. pyrrocinia; (J) B. ubonensis; (K) B. gladioli; (L) Ralstonia sp. The B. cepacia probe failed to identify four of four B. stabilis strains, one of three B. ambifaria strains, and two of four B. pyrrocinia strains tested. All other BCC species were reliably detected. No reactivity was observed with closely related species, including B. gladioli (K) and Ralstonia species (L). Results shown are representative of each species tested. Refer to Materials and Methods (“Bacterial strains and culture conditions”) for full details of the number of isolates of each species tested.

FIG. 3.

Application of the seaFAST Cystic Fibrosis I kit to sputa prior to optimization (A) and following optimization (B). (A) Sputum sample spiked with BCC organisms to an equivalent of >10¹⁰ CFU/ml. The arrow highlights two BCC cells showing weak fluorescence. (B) Clinical sputum sample containing 10⁷ CFU/ml BCC organisms, with strongly fluorescent BCC organisms clearly visible.

FIG. 4.

Assessing the impact of sputum dilution on the sensitivity of FISH analysis. Shown is a representative FISH analysis a clinical BCC-positive sputum sample (10⁷ CFU/ml) prepared according to the protocol in Materials and Methods. The sputum cell pellet was resuspended in either 0.1 ml (A), 0.5 ml (B), or 1 ml (C) saline. No BCC fluorescence was detectable in the 0.1-ml sputum suspension owing to the high background. While very weak fluorescence attributable to BCC organisms was occasionally evident when the pellet was resuspended in 0.5 ml saline (B, arrow), reliable detection of the BCC organism was achieved only when the pellet was resuspended in 1 ml.

FIG. 5.

Application of the 16S rRNA gene-based PCR assay (A) and the recA-based PCR assay (B) to DNA prepared from 10-fold serial dilutions of sputum spiked with known numbers of B. cenocepacia. (A) The rRNA gene-based PCR assay (196-bp product) detected the B. cenocepacia spike at 10⁴ CFU/ml. The assay is multiplexed with a human rRNA gene PCR assay to provide a positive control (270-bp product). Additional PCR bands evident between 300 and 400 bp are an artifact of multiplexing the BCC and human PCR primers and applying to a sputum DNA template. These bands are not evident when either primer pair is applied individually to sputum DNA or when primers are multiplexed and applied to bacterial DNA (data not shown). (B) The recA PCR (1,043-bp product) detected the B. cenocepacia spike at 10⁷ CFU/ml. The positive control (+ ve) is genomic DNA prepared from B. cenocepacia J2315. NTC, no-template control.