1) Academic Department of Child Health, University Hospital of North Staffordshire,

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1 JCM Accepts, published online ahead of print on 21 August 2013 J. Clin. Microbiol. doi: /jcm Copyright 2013, American Society for Microbiology. All Rights Reserved Title: Is hydrogen cyanide a marker of Burkholderia cepacia complex? Running Title: HCN and BCC Authors: Francis J Gilchrist 1,2,4#, Hayley Sims 3, Alice Alcock 3, Andrew M Jones 2, Rowland J Bright-Thomas 2, David Smith 4, Patrik Špan l 4,5, A Kevin Webb 2, Warren Lenney 1,4 Institutions: 1) Academic Department of Child Health, University Hospital of North Staffordshire, Newcastle Road, Stoke-on-Trent, ST4 6QG, UK 2) Manchester Adult Cystic Fibrosis Centre, Wythenshawe Hospital, Southmoor Road, Manchester, M23 9LT, UK 3) Department of Microbiology, University Hospital of North Staffordshire, Newcastle Road, Stoke-on-Trent, ST4 6QG, UK 4) Institute of Science and Technology in Medicine - Keele University, Guy Hilton Research Centre, Thornburrow Drive, Stoke-on-Trent, ST4 7QB, UK 5) J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 3, Praha 8, Prague, Czech Republic # Correspondence to: Dr Francis Gilchrist, Academic Department of Child Health, University Hospital of North Staffordshire, Newcastle Road, Stoke-on-Trent, ST4 6QG, UK. francis.gilchrist@uhns.nhs.uk Keywords: Cystic fibrosis, microbiology, Burkholderia cepacia complex, hydrogen cyanide, SIFT-MS 1

2 Abstract Biofilm cultures of Burkholderia cepacia complex (BCC) infection have been found to generate the non-volatile 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. Downloaded from on September 10, 2018 by guest 2

3 The volatile compound hydrogen cyanide (HCN) has been shown to be an in-vitro marker of Pseudomonas aeruginosa (PA) and the factors that affect its production have been established (1 3). HCN is also an in-vivo marker of PA infection when measured in exhaled breath (4, 5). In CF children, 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 PA was the only cyanogenic organism in the CF lung but a recent study demonstrated production of the non-volatile cyanide ion by 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 if 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. Adult CF patients were recruited with chronic BCC infection but free from PA infection. Chronic BCC infection was defined as BCC in >50% of sputum samples (minimum of 4 samples) over the previous 12 months. Free from PA infection was defined as no PA 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 recultured under planktonic and biofilm conditions. Biofilm culture conditions were developed by pipetting 10ml of BCC inoculated Brain Heart Infusion (BHI) enrichment broth into a Petri dish 3

4 containing of 4mm 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 hours 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 hours incubation using a previously described methodology (2, 3). At 96 hours the headspace HCN concentrations were re-measured after acidification of the cultures using 1ml HCl to promote the generation of gaseous HCN from cyanide ions. The mouth and nose-exhaled breath of a patients free from both BCC and PA infection were analysed as controls. Twelve CF patients (6 male) with chronic BCC infection but free from PA infection (BCC Group) and 10 patients (6 male) free from both BCC and PA infection (control group) were recruited. The median (IQR) forced expiratory volume in one second (FEV1) was lower in the BCC group compared to the control group (1.7 ( ) vs 2.2 ( ) p=0.04). Age, body mass index (BMI) and forced vital capacity (FVC) were not significantly different. In the BCC group, 7 isolated Burkholderia multivorans (7 different strains), 3 Burkholderia cenocepacia (all ET-12 strain) and 2 Burkholderia latens (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 hours incubation. The mean (SD) absorbance of crystal violet at 96 hours was higher in the biofilm cultures compared 4

5 to the planktonic and control cultures (3.43 (0.31) vs (0.003) Absorbance Units (AU), p<0.001). The headspace HCN concentration measured using SIFT-MS remained <10ppbv (equivalent to background concentrations) for both culture conditions, at all time points. After acidification there was a rise in the median (IQR) headspace HCN concentration compared to the pre-acidification 96 hour results (4.5 ( ) vs 2.9 ( ) ppbv, p=0.002) but all concentrations remained <10ppbv. Acidification did not produce a significant change in the median (IQR) headspace HCN concentrations for the planktonic cultures (2.3 ( ) vs 1.7 ( ) ppbv, p=0.6). See Table 1. When data from all 22 patients (with and without BCC) were analysed together, the systemic compound acetone had similar median (IQR) breath concentrations for mouth and nose-exhaled samples (459 ( ) v 445 ( ) ppbv, p=0.60). In contrast, the median (IQR) 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 (1.2-20) v 0 (0-0.9) ppbv, p<0.001 and ethanol: 410 ( ) v 152 ( ) 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. See 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 60µM to 5

6 mM (equivalent to equilibrium headspace HCN gas concentrations of 7 ppmv to 2100 ppmv at 20 o C) (9) which 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 <10ppbv, 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 origin of the samples and the different species analysed may have contributed. Methodological issues also need to be considered. Both the cyanide ion-selective micro-electrode used by Ryall et al. and our SIFT-MS instrument have previously been successfully used when investigating the cyanide/hcn production by PA (2, 3, 10). The culture medium was different in the 2 studies (Luria-Bertani and BHI) which may have contributed. 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. 6

7 References 1. Carroll W, Lenney W, Wang T, Spanel P, Alcock A, Smith D Detection of volatile compounds emitted by Pseudomonas aeruginosa using selected ion flow tube mass spectrometry. Pediatr. Pulmonol 39: Gilchrist FJ, Alcock A, Belcher J, Brady M, Jones A, Smith D, Span l P, Webb K, Lenney W Variation in hydrogen cyanide production between different strains of Pseudomonas aeruginosa. Eur. Respir. J. 38: Gilchrist FJ, Sims H, Alcock A, Belcher J, Jones AM, Smith D, Spanel P, Webb AK, Lenney W. Quantification of hydrogen cyanide and 2-aminoacetophenone in the headspace of Pseudomonas aeruginosa cultured under biofilm and planktonic conditions. Anal. Methods, 2012 DOI: /C2AY25652E. 4. Enderby B, Smith D, Carroll W, Lenney W Hydrogen cyanide as a biomarker for Pseudomonas aeruginosa in the breath of children with cystic fibrosis. Pediatr. Pulmonol 44: Gilchrist FJ, Bright-Thomas RJ, Jones AM, Smith D, Span l P, Webb AK, Lenney W Hydrogen cyanide concentrations in the breath of adult cystic fibrosis patients with and without Pseudomonas aeruginosa infection. J Breath Res 7: Enderby B, Lenney W, Brady M, Emmett C, Špan l P, Smith D Concentrations of some metabolites in the breath of healthy children aged 7 18 years measured using selected ion flow tube mass spectrometry (SIFT-MS). Journal of Breath Research 3: Wang T, Pysanenko A, Dryahina K, Špan l P, Smith D Analysis of breath, exhaled via the mouth and nose, and the air in the oral cavity. J. Breath Res. 2: Ryall B, Lee X, Zlosnik JEA, Hoshino S, Williams HD Bacteria of the Burkholderia cepacia complex are cyanogenic under biofilm and colonial growth conditions. BMC Microbiol 8: Ma J, Dasgupta PK, Blackledge W, Boss GR Temperature dependence of Henry s law constant for hydrogen cyanide. Generation of trace standard gaseous hydrogen cyanide. Environ. Sci. Technol. 44: Ryall B, Davies JC, Wilson R, Shoemark A, Williams HD Pseudomonas aeruginosa, cyanide accumulation and lung function in CF and non-cf bronchiectasis patients. Eur. Respir. J 32:

8 Table 1: Headspace HCN concentrations and spectroscopy results for biofilm and planktonic cultures after various durations of incubation Culture type Culture conditions No. Median (IQR) HCN (ppbv) 24 hrs 48 hrs 72 hrs 96 hrs 96 hrs + HCl Mean (SD) absorbance of crystal violet (AU) BCC Biofilm ( ) ( ) ( ) ( ) ( ) 3.43 (0.31) BCC Planktonic ( ) ( ) ( ) ( ) ( ) (0.002) Control Biofilm ( ) ( ) ( ) ( ) ( ) (0.004) Control Planktonic ( ) ( ) ( ) ( ) ( ) (0.003) No.: number of samples, BCC: Burkholderia cepacia complex, IQR: inter-quartile range, ppbv: parts-per-billion by volume, HCN: hydrogen cyanide, HCl: hydrochloric acid, SD: standard deviation, AU: absorbance units Table 2: Comparison of in-vivo results between the BCC and control groups Mouth-exhaled Breath Nose-exhaled Breath Acetone Ethanol HCN Acetone Ethanol HCN BCC 446 ( ) 446 ( ) 6.8 (0.8-19) 415 ( ) 155 ( ) 0 (0-0.3) Controls 472 ( ) 356 ( ) 7.5 (2.3-20) 476 ( ) 138 ( ) 0 (0-3.2) p value BCC: Burkholderia cepacia complex. Concentration values are median (IQR) parts-per-billion by volume. 8