Analysis of volatile metabolites from in vitro biofilms of Pseudomonas aeruginosa with thin-film microextraction by thermal desorption gas chromatography-mass spectrometry

Abstract

Cystic fibrosis (CF) is an autosomal recessive inherited disease which leads to a production of thickened mucus in the airways. These conditions are conducive to poly-microbial infections, like chronic lung infection, in which Pseudomonas aeruginosa (P. aeruginosa) is the major pathogenic bacterium colonizing CF lungs at the end of the lifetime of CF patients. This in vitro study uses a P. aeruginosa biofilm model under partly cystic fibrosis conditions, with a sampling of volatile extracellular metabolites. The gas sampling was done with thin-film microextraction (TFME) and commercial polydimethylsiloxane (PDMS) films, whereas the analysis of loaded films was done by gas chromatography coupled to quadrupole mass spectrometry and thermodesorption (TD-GC-qMS). For this purpose, two commercially available films were characterized by means of thermogravimetry coupled to a qMS with atmospheric pressure photo ionization (TG-APPI-qMS), regarding homogeneity and temperature stability. The selected film was cleaned using a method developed in this study. The TD-GC-qMS method was successfully used for standards of volatile metabolites which were known to be produced by P. aeruginosa. Limits of detection and quantification of the method for middle and less polar compounds in low nanomolar range (0.5 nM and 1.5 nM) were achieved. The developed method was finally applied to investigate the extracellular volatile metabolites produced by biofilms of the strain P. aeruginosa DSM 50071 under aerobic and anaerobic conditions. In sum, eleven metabolites could be found under both conditions. Furthermore, it was shown in this study that different oxygen conditions (aerobic and anaerobic) resulted in emitting different extracellular volatile metabolites. Specific metabolites, like 1-undecene (aerobic) and 2-undecanone (anaerobic), could be identified. The results are promising, in that the biofilm model may be applicable for the identification of P. aeruginosa under clinical conditions. Furthermore, the model could be the basis for studying extracellular volatile metabolites from different mono-or co-cultures of various bacteria, as well as the implementation of pulmonary conditions, like these in CF lungs. This possibility allows the development of a non-invasive "at-bedside" breath analysis method for CF patients in focus of various bacterial infections.

Aspergillus Is Inhibited by Pseudomonas aeruginosa Volatiles

“…All these differences in findings are incompletely resolved, and require further study. An important observation in other prior studies is that there can be considerable variation in the production of different volatiles among various Pa strains [64,67,71,72]; culture conditions also affect the spectrum of Pa volatiles produced [61,67,72,76]. It is also possible that vapors from some of the Af media they used interacted with the Pa volatiles, which may explain the differences [57,62] from our results.…”

“…Many of the volatiles of interest to us as potential Pa inhibitors (Table 3) have been noted previously among Pa volatiles, including 2-undecanone [57,[63][64][65][66][67]71,76], 2-heptanone [64][65][66][67]71,76], and methyl thiocyanate [67]. 2-heptanone has been noted to be an inhibitor of micro-organisms (bacteria) [78].…”

“…Pa and Af are the most important bacterium and fungus in the microbiota in many clinical settings, particularly in the lungs of immunocompromised persons and those with CF. Advanced technology has been used to detect the volatiles of Pa and other microbes [ 57 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 ], and such technology is currently under study as methods for the noninvasive detection of these pathogens [ 66 , 72 , 73 , 74 , 75 , 76 ]. Nearly 400 volatiles have been described from Pa, of which ketones are the most highly represented [ 71 ].…”

Life Cycle Dominates the Volatilome Character of Dimorphic Fungus Coccidioides spp.

“…Second, our data show that the growth phase of the organism has a strong influence on the volatilome, and therefore, using in vitro culture conditions that can most closely replicate the in vivo environment and microbial growth phases may increase the likelihood of putative biomarker translation from bench to bedside. In addition to controlling the growth phase of dimorphic organisms, macronutrient, micronutrient, and oxygen availability should all be considered in the development of in vitro model systems that replicate the in vivo physiology of the pathogen ( 14 , 17 , 36 – 41 ). In this study, we observed 35 VOCs present in at least 67% of all spherule strains ( Fig.…”

Lifecycle dominates the volatilome character of the dimorphic fungusCoccidioidesspp

“…Second, our data show that the growth phase of the organism has a strong influence on the volatilome, and therefore using in vitro culture conditions that can most closely replicate the in vivo environment and microbial growth phases may increase the likelihood of putative biomarker translation from bench to bedside. In addition to controlling the growth phase of dimorphic organisms, macronutrient, micronutrient, and oxygen availability should all be considered in the development of in vitro model systems that replicate in vivo physiology of the pathogen (14, 17, 27, 30, 33, 36, 52, 53). Our next step in developing a Valley fever breath test is to translate in vitro biomarkers for use with an in vivo mouse model.…”