Article Figures & Data
Figures
- 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.
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Table S1 (Full microbiology results, including culture results, PCR results, and FISH results, for 69 clinical sputum samples)
Zipped PDF document, 9K.
- Supplemental file 1
-
Table S1 (Full microbiology results, including culture results, PCR results, and FISH results, for 69 clinical sputum samples)
