Anne H. Neerincx

Identification of Pseudomonas aeruginosa and Aspergillus fumigatus mono- and co-cultures based on volatile biomarker combinations

Volatile organic compound (VOC) analysis in exhaled breath is proposed as a non-invasive method to detect respiratory infections in cystic fibrosis patients. Since polymicrobial infections are common, we assessed whether we could distinguish Pseudomonas aeruginosa and Aspergillus fumigatus mono- and co-cultures using the VOC emissions. We took headspace samples of P. aeruginosa, A. fumigatus and co-cultures at 16, 24 and 48 h after inoculation, in which VOCs were identified by thermal desorption combined with gas chromatography - mass spectrometry. Using multivariate analysis by Partial Least Squares Discriminant Analysis we found distinct VOC biomarker combinations for mono- and co-cultures at each sampling time point, showing that there is an interaction between the two pathogens, with P. aeruginosa dominating the co-culture at 48 h. Furthermore, time-independent VOC biomarker combinations were also obtained to predict correct identification of P. aeruginosa and A. fumigatus in mono-culture and in co-culture. This study shows that the VOC combinations in P. aeruginosa and A. fumigatus co-microbial environment are different from those released by these pathogens in mono-culture. Using advanced data analysis techniques such as PLS-DA, time-independent pathogen specific biomarker combinations can be generated that may help to detect mixed respiratory infections in exhaled breath of cystic fibrosis patients.

Optical parametric oscillator-based photoacoustic detection of hydrogen cyanide for biomedical applications

Abstract. A versatile, continuous wave, optical parametric oscillator is used in combination with photoacoustic spectroscopy for long-term trace gas experiments of volatile compounds emitted by biological samples. The optical parametric oscillator-based spectrometer (wavelength near 3 μm, 8-MHz linewidth, output power ∼1  W) is successfully tested for the detection of hydrogen cyanide (HCN) emission from clover leaves, and Pseudomonas bacteria; in addition, the presence of HCN in exhaled human breath is measured. For specific experiments, the spectrometer is operated continuously up to 10 days and has a detection limit of 0.4 parts-per-billion volume of HCN in air over 10 s, using the P8 rotational line in the ν3 vibrational band of HCN at 3287.25  cm−1. This results in an overall sensitivity of the system of 2.5×10−9  cm−1 Hz−1/2.

Real-time monitoring of hydrogen cyanide (HCN) and ammonia (NH3) emitted by Pseudomonas aeruginosa

We present the real-time monitoring of hydrogen cyanide (HCN) production from Pseudomonas aeruginosa (P. aeruginosa) strains in vitro, using laser-based photoacoustic spectroscopy. Simultaneously, the production of ammonia (NH3) was measured, and the influence of different factors (e.g. the medium, temperature and antibiotics treatment) was assessed. Both reference strains and clinical isolates of patients with CF were studied, and compared to other pathogens commonly present in lungs/airways of CF patients. Hydrogen cyanide production starts to rise as soon as P. aeruginosa bacteria reach the stationary phase ((9.0-9.5) × 10(9) colony forming units, CFUs), up to concentrations of 14.5 microliters per hour (µl h(-1)). Different strains of P. aeruginosa produced HCN to varying degrees, and addition of tobramycin strongly reduced HCN production within 2 h from application. Burkholderia cepacia also produced HCN (up to 0.35µl h(-1) in 9.0  ×  10(9) CFU) while other pathogens (Aspergillus fumigatus, Stenotrophomonas maltophilia, Mycobacterium abscessus) did not produce detectable levels. Our study reveals for the first time a broad overview of the dynamics of the HCN production in vitro.

Extended nitric oxide analysis may improve personalized anti-inflammatory treatment in asthmatic children with intermediate FENO50

Exhaled nitric oxide (F(E)NO) is elevated in asthma, and a clinical practice guideline has been published with recommendations for anti-inflammatory treatment. It summarizes that a F(E)NO at an expiratory flow rate of 50 ml s(-1) (F(E)NO50) above 35 ppb in children indicates eosinophilic inflammation, and the most likely response is to use inhaled corticosteroids. Intermediate F(E)NO50 between 20-35 ppb should be interpreted cautiously. The aim of the study was to investigate this guideline in a small group of asthmatic children. Thirty-seven asthmatic children; 23 boys and 14 girls, visited the outpatient clinic, and provided exhaled breath samples for offline NO measurement. These samples were analysed with chemiluminescence techniques. Three flow rates, namely 16, 90 and 230 ml s(-1) were used for the extended NO analysis (Högman-Meriläinen algorithm, HMA) to estimate the alveolar concentration (C(A)NO), diffusion rate of the airway wall (D(aw)NO) and airway wall content (C(aw)NO). For accuracy of the HMA, the estimated value of F(E)NO at 50 ml s(-1) (F(E)NO50) was compared with measured F(E)NO50. In nine children the difference was more than 5 ppb and the data were therefore excluded. Five children with F(E)NO50 <20 ppb had no known allergy and their F(E)NO50 geometrical mean (25th; 75th percentile) was 11 (10;14) and CawNO was 32 (20;43) ppb. Ten children with F(E)NO50  >  35 ppb had an allergy and had F(E)NO50 of 56 (47;60) ppb and C(aw)NO of 140 (121;172) ppb. Thirteen children with allergies, with intermediate F(E)NO50, had F(E)NO50 of 27 (25;30) ppb with a wide range of C(aw)NO. In five of these children, values were comparable to healthy children, 44 (43;50) ppb while eight children had elevated C(aw)NO values of 108 (95;129) ppb. Our data indicate the clinical potential use of extended NO analysis to determine the personal target value of F(E)NO50 for monitoring the treatment outcome. Furthermore, for children with intermediate F(E)NO50 more than half of them could possibly benefit from an adjustment of inhaled corticosteroids if the C(aw)NO value was considered.

Hydrogen cyanide emission in the lung by Staphylococcus aureus

Early detection of Pseudomonas aeruginosa in cystic fibrosis (CF) patients is crucial, since eradication in a later stage is extremely difficult [1]. Until now, it has been assumed that hydrogen cyanide (HCN) can be considered as a specific biomarker for P. aeruginosa in exhaled breath [2–5]. Only Burkholderia cepacia is also known to produce HCN in vitro [6, 7], but HCN is not a biomarker for B. cepacia complex infection in vivo [8]. To our knowledge, actual proof that HCN is not produced by other pathogens is lacking. Investigations into the emission of HCN by Staphylococcus aureus, a predominant CF pathogen throughout childhood, have been very limited. In a single report, five strains have been shown to produce low values in vitro [9], and in vivo studies have not been reported. Hydrogen cyanide is produced by S. aureus in vitro and in vivo and is not an exclusive biomarker for P. aeruginosa http://ow.ly/4nsh7y

Detection of Staphylococcus aureus in cystic fibrosis patients using breath VOC profiles

Staphylococcus aureus (S. aureus) is a common bacterium infecting children with cystic fibrosis (CF). Since current detection methods are difficult to perform in children, there is need for an alternative. This proof of concept study investigates whether breath profiles can discriminate between S. aureus infected and non-infected CF patients based on volatile organic compounds (VOCs). We collected exhaled breath of CF patients with and without S. aureus airways infections in which VOCs were identified using gas chromatography-mass spectrometry. We classified these VOC profiles with sparse partial least squares discriminant analysis. Multivariate breath VOC profiles discriminated infected from non-infected CF patients with high sensitivity (100%) and specificity (80%). We identified the nine compounds most important for this discrimination. We successfully detected S. aureus infection in CF patients, using breath VOC profiles. Nine highlighted compounds can be used as a focus point in further biomarker identification research. The results show considerable potential for non-invasive diagnosis of airway infections.

Ethylene, an early marker of systemic inflammation in humans

Ethylene is a major plant hormone mediating developmental processes and stress responses to stimuli such as infection. We show here that ethylene is also produced during systemic inflammation in humans and is released in exhaled breath. Traces of ethylene were detected by laser spectroscopy both in vitro in isolated blood leukocytes exposed to bacterial lipopolysaccharide (LPS) as well as in vivo following LPS administration in healthy volunteers. Exposure to LPS triggers formation of ethylene as a product of lipid peroxidation induced by the respiratory burst. In humans, ethylene was detected prior to the increase of blood levels of inflammatory cytokines and stress-related hormones. Our results highlight that ethylene release is an early and integral component of in vivo lipid peroxidation with important clinical implications as a breath biomarker of bacterial infection.

Combining ANOVA-PCA with POCHEMON to analyse micro-organism development in a polymicrobial environment

Revealing the biochemistry associated to micro-organismal interspecies interactions is highly relevant for many purposes. Each pathogen has a characteristic metabolic fingerprint that allows identification based on their unique multivariate biochemistry. When pathogen species come into mutual contact, their co-culture will display a chemistry that may be attributed both to mixing of the characteristic chemistries of the mono-cultures and to competition between the pathogens. Therefore, investigating pathogen development in a polymicrobial environment requires dedicated chemometric methods to untangle and focus upon these sources of variation. The multivariate data analysis method Projected Orthogonalised Chemical Encounter Monitoring (POCHEMON) is dedicated to highlight metabolites characteristic for the interaction of two micro-organisms in co-culture. However, this approach is currently limited to a single time-point, while development of polymicrobial interactions may be highly dynamic. A well-known multivariate implementation of Analysis of Variance (ANOVA) uses Principal Component Analysis (ANOVA-PCA). This allows the overall dynamics to be separated from the pathogen-specific chemistry to analyse the contributions of both aspects separately. For this reason, we propose to integrate ANOVA-PCA with the POCHEMON approach to disentangle the pathogen dynamics and the specific biochemistry in interspecies interactions. Two complementary case studies show great potential for both liquid and gas chromatography - mass spectrometry to reveal novel information on chemistry specific to interspecies interaction during pathogen development.