Assessment of Fluorescent In Situ Hybridization and PCR-Based Methods for Rapid Identification of Burkholderia cepacia Complex Organisms Directly from Sputum Samples

Bacterial strains and culture conditions.To determine the specificity and sensitivity of the diagnostic assays in the present study, a panel of relevant strains was assembled against which each assay was tested. This panel comprised the previously published BCC experimental strain panels (6, 16), together with B. gladioli (n = 3), B. caledonica (n = 3), Pseudomonas aeruginosa (mucoid and nonmucoid strains and representatives of major epidemic strains) (n = 34), other pseudomonads (n = 6), Stenotrophomonas maltophilia (n = 4), Ralstonia spp. (n = 6), Pandoraea spp. (n = 4), Achromobacter xylosoxidans (n = 5), and Acinetobacter baumannii (n = 2). The BCC experimental strain panels, obtained from the BCCM/LMG Bacteria Collection (Ghent, Belgium), comprise 8 B. multivorans isolates, 10 B. cenocepacia isolates, 3 B. ambifaria isolates, and 4 isolates each of B. cepacia, B. stabilis, B. vietnamiensis, B. dolosa, B. anthina, and B. pyrrocinia. All other strains were clinical isolates from the Edinburgh Strain Repository, identification of which was previously confirmed by relevant API and/or species-specific PCR. Subsequent to the analysis of the strain panel described above, the diagnostic assays were also applied to B. ubonensis LMG20358^T (BCCM/LMG Bacteria Collection, Ghent, Belgium), five presumptive novel species of the BCC (24a), and 37 clinical BCC isolates received by our laboratory in the course of this study (24 B. multivorans isolates, 11 B. cenocepacia isolates, and one isolate each of B. cepacia and B. vietnamiensis), bringing the total number of strains tested to 88 BCC isolates and 67 non-BCC isolates. All strains were cultured on nutrient agar with overnight incubation at 37°C.

Microbiological analysis of sputum.Sputum samples received by our laboratory were initially mixed with an equal volume of Sputolysin (Calbiochem) and vortexed thoroughly until liquefied. Tenfold serial dilutions of this sputum-Sputolysin mixture were prepared in sterile saline prior to plating on the following media: chocolate blood agar (10⁻² and 10⁻⁴ dilutions), blood agar (10⁻² and 10⁻⁴ dilutions), Pseudomonas isolation agar (Difco, Becton Dickinson) supplemented with glycerol (2% final concentration) (neat and 10⁻² and 10⁻⁴ dilutions), and Burkholderia cepacia medium supplemented with B. cepacia Selectatab (Mast Diagnostics) (neat and 10⁻² and 10⁻⁴ dilutions). By this protocol, the lower limit of detection by culture is 10² CFU/ml. Following incubation at 37°C for 48 to 72 h, preliminary identification of resulting cultures was performed, based on morphological examination of colony types and API20NE biochemical testing. Presumptive BCC organisms identified on the basis of colony phenotype and/or API20NE profile were confirmed as such by the BCC-specific recA PCR assay using the BCR1/BCR2 primer pair (15). Subsequent HaeIII RFLP analysis of the recA PCR product was followed by the relevant species-specific PCR.

FISH analysis of sputum.The 1:1 mixture of sputum-Sputolysin prepared previously (see “Microbiological analysis of sputum”) was further diluted in Sputolysin to an optical density at 600 nm (OD₆₀₀) of approximately 0.6 in a final volume of 1 ml. Typically this OD₆₀₀ equated to a final dilution factor ranging from 1:10 to 1:40, depending on the viscosity of the original sputum sample. This 1-ml sputum mixture was vortexed thoroughly and split into two equal aliquots. Both aliquots were centrifuged at 10,000 × g for 5 min, and the supernatant was removed. One cell pellet was retained for subsequent DNA extraction (see “Extraction of genomic DNA”), while the second cell pellet was resuspended in 1 ml sterile saline. Ten microliters of this saline suspension was applied per field on an eight-field microscope slide. FISH analysis was performed using the seaFAST Cystic Fibrosis I kit (IZINTA, Hungary; formerly SeaPro Theranostics International, The Netherlands) according to the manufacturer's instructions. The kit enables identification of four CF pathogens (B. cepacia, Haemophilus influenzae, P. aeruginosa, and S. maltophilia) through the use of two tandem probe pairs, the sequences of which are the same as the Burcep, Haeinf, PseaerA/PseaerB, and Stemal probes previously employed by Hogardt and colleagues (10). In brief, following application of samples to the slide, slides were dried at 55°C, fixed for 3 min in a SeaWAVE programmed microwave, and immersed in methanol for 10 min. After the slides were dried, 10 μl of reconstituted probe mixture was added to each field and slides were placed in a hybridization chamber containing filter paper soaked in 3 ml hybridization buffer. Hybridization at 46 to 48°C was performed in the SeaWAVE programmed microwave for 12 min according to the manufacturer's protocol, following which slides were washed for 16 min (46 to 48°C). Slides were rinsed briefly in methanol and allowed to dry prior to applying mounting medium and a coverslip. Fluorescence was read immediately using a Leica DM LB2 fluorescence microscope fitted with a Hamamatsu ORCA-ER digital camera.

Extraction of genomic DNA.To assess the specificity of the diagnostic PCR assay, crude genomic DNA was prepared from all isolates within the strain panel (see “Bacterial strains and culture conditions”). In brief, two bacterial colonies were resuspended in 20 μl lysis buffer (0.25% sodium dodecyl sulfate, 0.05 M NaOH) and incubated at 95°C for 15 min. After brief centrifugation, 180 μl sterile water was added and the mixture was centrifuged at 13,000 × g for 5 min. The supernatant containing genomic DNA was used directly in PCR assays.

For application of PCR assays to sputum, three alternative DNA extraction methods were compared to assess the impact on sensitivity of the PCR assays. Starting material for DNA extractions was the cell pellets obtained following centrifugation of the sputum-Sputolysin mixture (see “FISH analysis of sputum”). These cell pellets were either resuspended in 100 μl lysis buffer (described above) or 100 μl Chelex-100 (5%, wt/vol; Sigma) or processed using the QIAamp DNA kit (QIAGEN) according to the manufacturer's instructions (100-μl final elution). Pellet suspensions in lysis buffer were incubated at 95°C for 15 min prior to centrifugation at 13,000 × g for 5 min. Supernatant was diluted 1:10 prior to use in PCRs. Pellet suspensions in Chelex-100 were subjected to two cycles of heating (95°C, 5 min) and chilling on ice (5 min), prior to centrifugation at 13,000 × g (5 min). Supernatant from Chelex-100 preparations was used directly in PCRs.

PCR detection of BCC from sputum.Representative 16S rRNA sequences were aligned for all available BCC species, generating a consensus sequence which was then compared with alignments of relevant non-BCC 16S rRNA sequences. On the basis of these alignments, a BCC-specific 16S rRNA gene-based PCR assay was designed (forward, 5′-TCC GGA AAG AAA WCC TTG GY; reverse, 5′-AAT GCA GTT CCC AGG TTG AG). Additionally, a human 16S rRNA gene-based PCR assay was designed to act as a positive control in PCRs directly from sputum (forward, 5′-GCT CAG GGA GGA CAG AAA CC; reverse, 5′-AGT GGG TGA ACA ATC CAA CG). BCC PCRs were performed in a 50-μl volume containing 600 nM forward primer, 200 nM reverse primer, 3 mM MgCl₂, 260 μM of each deoxynucleoside triphosphate (dNTP), 4% (vol/vol) dimethyl sulfoxide, 1 U Taq polymerase (Invitrogen), and appropriate manufacturer's reaction buffer. For multiplex PCR assays with both human- and BCC-specific primers, 100 nM of each human primer was used, MgCl₂ was increased to 4 mM, dNTPs were increased to 400 μM, and Taq polymerase was increased to 1.5 U. Thermal cycling was performed on a GeneAmp PCR System 9700 (Applied Biosystems) with the following parameters: 95°C for 2 min; 40 cycles of 95°C (30 s), 55°C (20 s), and 72°C (30 s); and 72°C for 10 min. PCR products were electrophoresed on a 4% E-Gel (Invitrogen) and visualized by UV illumination.

Assessment of assay sensitivity using spiked sputum.To assess assay sensitivity, a known BCC-negative sputum sample was diluted with Sputolysin to an approximate OD₆₀₀ of 0.6. BCC organisms were grown overnight in Luria-Bertani broth or BM2-glucose minimal media (9) and viable counts performed. Aliquots of overnight culture were spun and cells resuspended in 1 ml of the sputum-Sputolysin mixture. Tenfold serial dilutions of this suspension were prepared using the sputum-Sputolysin mixture as the diluent. These suspensions were then processed for FISH and PCR as described above. Viable counts of the overnight culture enabled calculation of equivalent CFU/ml in the spiked sputum samples.

Specificity of BCC diagnostic assays applied to the strain panel.The BCC rRNA gene PCR assay exhibited 100% sensitivity and specificity for the identification of BCC when applied to the strains described above, correctly identifying all 88 BCC isolates tested. No reactivity was observed with any of the 67 non-BCC isolates tested, comprising pseudomonads (n = 40), non-BCC Burkholderia species (n = 6), S. maltophilia (n = 4), Ralstonia spp. (n = 6), Pandoraea spp. (n = 4), Achromobacter xylosoxidans (n = 5), and Acinetobacter baumannii (n = 2). Representative PCR results from BCC and non-BCC isolates are shown in Fig. 1. Inefficient PCR amplification was observed when the BCC PCR assay was applied to B. pseudomallei DNA, which occasionally resulted in a weak PCR band that could readily be distinguished from a true-positive result from BCC (Fig. 1).

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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.

In contrast to the PCR assay, the B. cepacia-specific probe within the seaFAST Cystic Fibrosis I FISH kit failed to identify all members of the BCC when applied to the strain panel (Fig. 2). No reactivity was observed with four of four B. stabilis strains, two of four B. pyrrocinia strains, and one of three B. ambifaria strains. Examination of available 16S rRNA sequences revealed a 3-nucleotide mismatch between the B. cepacia probe sequence and the published gene sequence for all B. stabilis strains sequenced and one-half of the B. pyrrocinia strains sequenced, thus explaining the observed lack of reactivity. The reason for the lack of reactivity observed with one B. ambifaria strain is unclear. The B. cepacia FISH probe did not cross-react with other closely related non-BCC species studied. Thus the specificity and sensitivity of the FISH kit for BCC when applied to the strain panel were 100% and 83%, respectively. Subsequent to the analysis of the strain panel, the FISH assay was shown to successfully identify B. ubonensis LMG20358^T (BCCM/LMG Bacteria Collection, Ghent, Belgium) (Fig. 2J) and five presumptive novel species of the BCC (24a) (data not shown).

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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.

Direct detection of BCC in sputum by FISH.Initially, FISH analysis was attempted on sputum that had been diluted 1:2 with Sputolysin, similar to the protocol described by Hogardt et al. (10), or 1:10 with Sputolysin (according to the manufacturer's instructions). These sputum-Sputolysin dilutions were applied directly to the microscope slide for FISH analysis. However in our hands, neither approach yielded satisfactory results. When sputum was diluted 1:2, a high level of nonspecific fluorescence prevented reliable identification of BCC organisms (Fig. 3A), even when present in high numbers (>10¹⁰ CFU/ml), while 1:10 dilutions with Sputolysin were associated with inefficient fixing of the sample to the slide. Optimization of the sputum preparation protocol focused on reducing the nonspecific fluorescence associated with the 1:2 Sputolysin dilution, thus enabling ready detection of the specific fluorescence attributable to BCC organisms (Fig. 3B). The optimized methodology (as given in Materials and Methods) essentially eradicated the background fluorescence by (i) performing dilution in Sputolysin (the extent of which varied according to sample viscosity), (ii) recovering the sputum cell pellet by centrifugation, and (iii) resuspending the pellet in sterile saline. When applied to serial dilutions of sputum spiked with B. cenocepacia or B. multivorans, this optimized methodology exhibited a lower limit of detection of 8 × 10⁵ CFU/ml (data not shown). The same sensitivity was obtained irrespective of whether the spike organism was grown in Luria-Bertani broth or BM2-glucose minimal media, despite the fact that the fluorescence signal was considerably weaker from cells grown in minimal media, presumably due to lower metabolic activity. When the seaFAST Cystic Fibrosis I FISH kit was used, a comparable lower limit of detection (≥7 × 10⁵ CFU/ml) was observed for the identification of P. aeruginosa within sputum (data not shown).

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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.

To reduce the nonspecific fluorescence and thus aid the rapid identification of BCC organisms within sputum, the sputum cell pellet was resuspended in a final volume of 1 ml saline, with only 10 μl of this being applied to the slide for FISH analysis. We anticipated that, while this dilution factor results in low background fluorescence, it would adversely affect the sensitivity of the assay. We assessed this by resuspending a sputum cell pellet in various volumes of saline (0.1, 0.5, and 1.0 ml) and applying 10 μl of each suspension to a slide for subsequent FISH analysis. Despite the sputum harboring 10⁷ CFU/ml BCC organisms (as determined by culture), no BCC-positive signal was detected when FISH was applied to the 0.1-ml pellet suspension due to high background fluorescence (Fig. 4A). Weak BCC-associated fluorescence was observed when FISH was applied to the 0.5-ml pellet suspension (Fig. 4B). However, strong fluorescence enabling reliable identification of BCC was observed only when the sputum cell pellet was resuspended in 1 ml with a resulting decrease in background fluorescence (Fig. 4C). Thus while the dilution factor results in fewer BCC organisms per field, the resulting low background effectively increases the sensitivity of the assay. Additionally, the low background fluorescence significantly reduces the time required for microscopy, thus increasing sample throughput. Attempts to improve sensitivity further by differential centrifugation of liquefied sputum (thus concentrating bacterial cells in the absence of material responsible for nonspecific fluorescence) were unsuccessful (data not shown).

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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.

The FISH results obtained using spiked sputum correlated fully with the results obtained when applied to 69 clinical sputum samples (see Table S1 in the supplemental material). With one exception, all BCC-positive sputum samples examined harbored at least 10⁶ CFU/ml BCC organisms (higher than the limit of detection by FISH) and thus were correctly identified as BCC positive by FISH analysis. FISH did not identify a B. vietnamiensis-positive sputum sample containing 5 × 10⁴ CFU/ml. No BCC-positive sputa examined in the course of this study harbored B. stabilis, B. pyrrocinia, or B. ambifaria, species which are associated with a false-negative FISH result (see above; Fig. 2). No FISH false positives were observed in any BCC culture-negative sputum samples studied.

Direct detection of BCC in sputum by PCR.Three different sputum DNA extraction protocols were tested in the present study to assess the impact that the quality and cleanliness of the DNA preparation has on PCR sensitivity. All three methods were successfully applied to the direct detection of BCC in sputum. However, the crude DNA extraction method (cell pellet resuspended in lysis buffer and heated at 95°C for 15 min) was associated with occasional PCR failures, as determined by the failure of the human 16S rRNA gene-based positive control. These PCR failures were presumed to arise from PCR inhibitors in the sputum that were not removed by the DNA extraction process. Consequently, the crude DNA extraction protocol was not investigated further. No PCR failures were observed with either of the other DNA extraction protocols tested (Chelex-100 supernatant or QIAamp DNA kit [QIAGEN]).

When the BCC-specific 16S rRNA gene-based PCR assay was applied to DNA prepared from serial dilutions of sputum spiked with B. cenocepacia, both the Chelex-100 and QIAamp DNA extraction protocols enabled reliable detection of 10⁴ CFU/ml when 10 μl of template DNA was used per 50-μl reaction mixture (Fig. 5A). A comparable limit of detection (between 10⁴ and 10⁵ CFU/ml) was observed for all other BCC species examined in the same way (genomovars I to X). In contrast, the recA-based PCR assay for the identification of BCC (15) was 100- to 1,000-fold less sensitive (limit of detection, 10⁷ CFU/ml) when applied directly to sputum (Fig. 5B), irrespective of the DNA extraction protocol.

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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.

When applied to 69 clinical sputum samples, the 16S rRNA gene-based PCR assay (applied to Chelex-100 supernatants) exhibited 100% sensitivity and specificity for detection of BCC-positive sputa compared with culture results (see Table S1 in the supplemental material). All BCC-positive sputum samples studied had bacterial counts in excess of the PCR limit of detection previously established using BCC-spiked sputum. No PCR false positives were observed in any BCC culture-negative sputum samples.