Is Hydrogen Cyanide a Marker of Burkholderia cepacia Complex? Francis J. Gilchrist, Hayley Sims, Alice Alcock, Andrew M. Jones, Rowland J. Bright-Thomas, David Smith, Patrik Spanel, A. Kevin Webb and Warren Lenney J. Clin. Microbiol. 2013, 51(11):3849. DOI: 10.1128/JCM.02157-13. Published Ahead of Print 21 August 2013.
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Is Hydrogen Cyanide a Marker of Burkholderia cepacia Complex? Francis J. Gilchrist,a,b,d Hayley Sims,c Alice Alcock,c Andrew M. Jones,b Rowland J. Bright-Thomas,b David Smith,d Patrik Špane˘l,d,e A. Kevin Webb,b Warren Lenneya,d Academic Department of Child Health, University Hospital of North Staffordshire, Stoke-on-Trent, United Kingdoma; Manchester Adult Cystic Fibrosis Centre, Wythenshawe Hospital, Manchester, United Kingdomb; Department of Microbiology, University Hospital of North Staffordshire, Stoke-on-Trent, United Kingdomc; Institute of Science and Technology in Medicine—Keele University, Guy Hilton Research Centre, Stoke-on-Trent, United Kingdomd; J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republice
Biofilm cultures of Burkholderia cepacia complex (BCC) infection have been found to generate the nonvolatile cyanide ion. We investigated if gaseous hydrogen cyanide (HCN) was a marker of BCC infection. Selected ion flow tube mass spectrometry analysis showed HCN was not elevated in the headspace of planktonic or biofilm cultures or in the exhaled breath of adult cystic fibrosis patients with chronic BCC infection. HCN is therefore not an in vitro or in vivo marker of BCC.
he volatile compound hydrogen cyanide (HCN) has been shown to be an in vitro marker of Pseudomonas aeruginosa, and the factors that affect its production have been established (1–3). HCN is also an in vivo marker of P. aeruginosa infection when measured in exhaled breath (4, 5). In children with cystic fibrosis (CF), mouth- or nose-exhaled breath can be used as healthy children have low or undetectable HCN levels (6). As healthy adults generate HCN in their mouth, only nose-exhaled breath should be used (7). It was thought that P. aeruginosa was the only cyanogenic organism in the CF lung, but a recent study demonstrated production of the nonvolatile cyanide ion by strains of Burkholderia cepacia complex (BCC) when cultured under biofilm but not planktonic conditions (8). We replicated the methodology of this in vitro experiment, but rather than using a selective electrode to measure cyanide ions trapped in sodium hydroxide, we used selected ion flow tube mass spectrometry (SIFT-MS) to measure the headspace concentration of HCN. In addition we investigated whether HCN is an in vivo marker of BCC infection by measuring its concentration in mouth- and nose-exhaled breath in adults with CF and chronic BCC infection. Ethical approval for the study was provided by the NRES Committee North West (11/NW/0102). Adult CF patients were recruited with chronic BCC infection but free from P. aeruginosa infection. Chronic BCC infection was defined as BCC in ⬎50% of sputum samples (minimum of 4 samples) over the previous 12 months. Freedom from P. aeruginosa infection was defined as the presence of no P. aeruginosa isolate in the previous 12 months (minimum of 4 samples). Patients provided mouth and nose exhalation breath samples directly into the SIFT-MS instrument for HCN analysis, as previously described (5). They also provided a sputum sample. The sputum was cultured and the isolated BCC
was recultured under planktonic and biofilm conditions. Biofilm culture conditions were developed by pipetting of 10 ml of BCCinoculated brain heart infusion (BHI) enrichment broth into a petri dish containing 4-mm-diameter sterile glass beads. Planktonic culture conditions were developed by pipetting inoculated BHI broth into a petri dish without glass beads. The dishes were individually sealed and incubated. Control cultures were prepared using the same methodology but with sterile BHI broth. Biofilm formation was assessed visually on a daily basis and quantitatively after 96 h of incubation. For the quantitative assessment, spectrophotometry was undertaken after crystal violet staining using a previously described methodology (8). For both planktonic and biofilm cultures, the headspace HCN concentration was measured at 24, 48, 72, and 96 h of incubation using a previously described methodology (2, 3). At 96 h, the headspace HCN concentrations were remeasured after acidification of the cultures using 1 ml HCl to promote the generation of gaseous HCN from cyanide ions. The mouth- and nose-exhaled breaths of patients free from both BCC and P. aeruginosa infection were analyzed as controls. Twelve CF patients (6 male) with chronic BCC infection but free from P. aeruginosa infection (BCC group) and 10 patients (6 male) free from both BCC and P. aeruginosa infection (control
Received 8 August 2013 Accepted 12 August 2013 Published ahead of print 21 August 2013 Address correspondence to Francis Gilchrist,
[email protected]. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02157-13
TABLE 1 Headspace HCN concentrations and spectroscopy results for biofilm and planktonic cultures after various durations of incubation Median HCN (IQR) concn, ppbv
Culture condition (no. of samples)
24 h
48 h
72 h
96 h
96 h ⫹ HCl
Mean (SD) absorbance of crystal violet, AU
BCC Biofilm (12) Planktonic (12)
0.5 (0.2–2.5) 1.6 (1.1–2.7)
2.2 (1.3–2.7) 2.3 (1.7–2.9)
2.1 (1.9–2.3) 3.5 (3.1–3.9.)
2.9 (1.5–3.5) 1.7 (1.4–4.2)
4.5 (4.1–5.5) 2.3 (1.6–3.4)
3.43 (0.31) 0.004 (0.002)
Control Biofilm (3) Planktonic (3)
0.3 (0.2–0.3) 0.3 (0.3–0.4)
1.8 (1.2–2.7) 1.2 (1.0–1.4)
2.6 (1.9–2.9) 2.2 (1.4–2.2)
0.9 (0.5–1.4) 3.1 (2.8–3.3)
1.2 (0.8–1.6) 2.6 (2.1–3.1)
0.006 (0.004) 0.005 (0.003)
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Gilchrist et al.
TABLE 2 Comparison of in vivo results between the BCC and control groups Median metabolite concn (IQR) in breath, ppbv Mouth exhaled
Nose exhaled
Group
Acetone
Ethanol
HCN
Acetone
Ethanol
HCN
BCC Controls
446 (387–526) 472 (437–558)
446 (387–526) 356 (322–846)
6.8 (0.8–19) 7.5 (2.3–20)
415 (362–527) 476 (356–563)
155 (133–257) 138 (121–303)
0 (0–0.3) 0 (0–3.2)
P value
0.55
0.26
0.74
0.79
0.37
0.53
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would be easily detected using SIFT-MS. The small increase in the gaseous HCN concentrations produced by acidification of the biofilm BCC cultures suggests some cyanide ions were present. Despite this, the HCN concentrations remained ⬍10 ppbv, meaning that the scale of the cyanogenesis was minimal compared to that seen by Ryall et al. The 34 BCC samples (from 9 species) used by Ryall et al. were a mixture of clinical and environmental, whereas we assessed 12 BCC clinical samples from 3 species. Ryall et al. found that cyanide concentrations varied between species, and so the different origins of the samples and the different species analyzed may have contributed to the discrepancy. Methodological issues also need to be considered. Both the cyanide ion-selective microelectrode used by Ryall et al. and our SIFT-MS instrument have previously been used successfully when investigating cyanide or HCN production by P. aeruginosa (2, 3, 10). The culture media were different in the 2 studies (Luria-Bertani and brain heart infusion [BHI]), which may have contributed to the discrepancy. It is also possible that the BCC cultures are producing a compound that chelates the HCN, preventing its release into the gas phase. In summary we did not identify elevated HCN concentrations in the headspace of BCC samples cultured under planktonic or biofilm condition or in the breath of patients chronically infected with BCC. We conclude that HCN is not an in vitro or in vivo marker of BCC infection. Further work is needed to investigate the discrepancy between the in vitro production of cyanide ions and gaseous HCN by BCC. REFERENCES 1. Carroll W, Lenney W, Wang T, Spanel P, Alcock A, Smith D. 2005. Detection of volatile compounds emitted by Pseudomonas aeruginosa using selected ion flow tube mass spectrometry. Pediatr. Pulmonol. 39:452– 456. 2. Gilchrist FJ, Alcock A, Belcher J, Brady M, Jones A, Smith D, Spane˘l P, Webb K, Lenney W. 2011. Variation in hydrogen cyanide production between different strains of Pseudomonas aeruginosa. Eur. Respir. J. 38:409 – 414. 3. Gilchrist FJ, Sims H, Alcock A, Belcher J, Jones AM, Smith D, Spanel P, Webb AK, Lenney W. 1 October 2012. Quantification of hydrogen cyanide and 2-aminoacetophenone in the headspace of Pseudomonas aeruginosa cultured under biofilm and planktonic conditions. Anal. Methods [Epub ahead of print.] doi:10.1039/C2AY25652E. 4. Enderby B, Smith D, Carroll W, Lenney W. 2009. Hydrogen cyanide as a biomarker for Pseudomonas aeruginosa in the breath of children with cystic fibrosis. Pediatr. Pulmonol. 44:142–147. 5. Gilchrist FJ, Bright-Thomas RJ, Jones AM, Smith D, Spane˘l P, Webb AK, Lenney W. 2013. Hydrogen cyanide concentrations in the breath of adult cystic fibrosis patients with and without Pseudomonas aeruginosa infection. J. Breath Res. 7:026010. doi:10.1088/1752-7155/7/2/026010. 6. Enderby B, Lenney W, Brady M, Emmett C, Špane˘l P, Smith,D. 2009. Concentrations of some metabolites in the breath of healthy children aged 7–18 years measured using selected ion flow tube mass spectrometry (SIFT-MS). J. Breath Res. 3:036001. doi:10.1088/1752 -7155/3/3/036001.
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group) were recruited. The median (interquartile range [IQR]) forced expiratory volume in 1 s (FEV1) was lower in the BCC group than in the control group: 1.7 (IQR, 1.4 to 2.0) versus 2.2 (IQR, 2.0 to 2.8) (P ⫽ 0.04). Age, body mass index (BMI), and forced vital capacity (FVC) were not significantly different. In the BCC group, there were 7 Burkholderia multivorans isolates (7 different strains), 3 Burkholderia cenocepacia isolates (all the ET-12 strain), and 2 Burkholderia latens isolates (2 different strains), as confirmed by the National Reference Laboratory within the last year using pulsed-field gel electrophoresis, recA sequencing, or species-specific PCR. Biofilms were identified visually on all biofilm cultures after 48 to 96 h of incubation. The mean (standard deviation [SD]) absorbance of crystal violet at 96 h was higher in the biofilm cultures than in the planktonic and control cultures: 3.43 (SD, 0.31) versus 0.005 (SD, 0.003) absorbance units (AU) (P ⬍ 0.001). The headspace HCN concentration measured using SIFT-MS remained at ⬍10 parts per billion by volume (ppbv) (equivalent to background concentrations) for both culture conditions at all time points. After acidification, there was a rise in the median headspace HCN concentration compared to the preacidification 96-h results (4.5 [IQR, 4.1 to 5.5] versus 2.9 [IQR, 1.5 to 3.5] ppbv) (P ⫽ 0.002), but all concentrations remained ⬍10 ppbv. Acidification did not produce a significant change in the median headspace HCN concentrations for the planktonic cultures: 2.3 (IQR, 1.6 to 3.4) versus 1.7 (IQR, 1.4 to 4.2) ppbv (P ⫽ 0.6) (Table 1). When data from all 22 patients (with and without BCC) were analyzed together, the systemic compound acetone had similar median breath concentrations for mouth- and nose-exhaled samples: 459 (IQR, 401 to 557) versus 445 (IQR, 354 to 567) ppbv (P ⫽ 0.60). In contrast, the median concentrations of ethanol and HCN, which are known to be generated in the mouth, were higher in mouth-exhaled breath samples than nose-exhaled breath samples: HCN, 6.8 (IQR, 1.2 to 20) versus 0 (IQR, 0 to 0.9) ppbv (P ⬍ 0.001); and ethanol, 410 (IQR, 346 to 555) versus 152 (IQR, 129 to 318) ppbv (P ⬍ 0.001). When the acetone, ethanol, and HCN concentrations were compared between the BCC and control groups, there were no significant differences for nose-exhaled or mouth-exhaled samples (Table 2). Using SIFT-MS, we did not identify elevated HCN concentrations in the headspace of biofilm or planktonic BCC cultures at any time point. This included analysis at a time when biofilm formation had been confirmed visually and with spectrophotometry. This is in contrast to Ryall et al., who observed cyanogenesis in 32 of 34 biofilm BCC cultures (8). The reason for this discrepancy is unclear. They found concentrations of trapped cyanide ranging from 60 M to 19 mM (equivalent to equilibrium headspace HCN gas concentrations of 7 parts per million by volume [ppmv] to 2,100 ppmv at 20°C) (9), which
Hydrogen Cyanide and Burkholderia cepacia Complex
7. Wang T, Pysanenko A, Dryahina K, Špane˘l P, Smith D. 2008. Analysis of breath, exhaled via the mouth and nose, and the air in the oral cavity. J. Breath Res. 2:037013. doi:10.1088/1752-7155/2/3/037013. 8. Ryall B, Lee X, Zlosnik JEA, Hoshino S, Williams HD. 2008. Bacteria of the Burkholderia cepacia complex are cyanogenic under biofilm and colonial growth conditions. BMC Microbiol. 8:108. doi:10.1186/1471-2180 -8-108.
9. Ma J, Dasgupta PK, Blackledge W, Boss GR. 2010. Temperature dependence of Henry’s law constant for hydrogen cyanide. Generation of trace standard gaseous hydrogen cyanide. Environ. Sci. Technol. 44:3028 – 3034. 10. Ryall B, Davies JC, Wilson R, Shoemark A, Williams HD. 2008. Pseudomonas aeruginosa, cyanide accumulation and lung function in CF and non-CF bronchiectasis patients. Eur. Respir. J. 32:740 –747.
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