Stable isotope profiles reveal active production of VOCs from human-associated microbes

Abstract

Volatile organic compounds (VOCs) measured from exhaled breath have great promise for the diagnosis of bacterial infections. However, determining human or microbial origin of VOCs detected in breath remains a great challenge. For example, the microbial fermentation product 2,3-butanedione was recently found in the breath of Cystic Fibrosis (CF) patients; parallel culture-independent metagenomic sequencing of the same samples revealed that Streptococcus and Rothia spp. have the genetic capacity to produce 2,3-butanedione. To investigate whether the genetic capacity found in metagenomes translates to bacterial production of a VOC of interest such as 2,3-butanedione, we fed stable isotopes to three bacterial strains isolated from patients: two gram-positive bacteria, Rothia mucilaginosa and Streptococcus salivarius, and a dominant opportunistic gram-negative pathogen, Pseudomonas aeruginosa. Culture headspaces were collected and analyzed using a gas chromatographic system to quantify the abundance of VOCs of interest; mass spectroscopy was used to determine whether the stable isotope label had been incorporated. Our results show that R. mucilaginosa and S. salivarius consumed D-Glucose-C to produce labeled 2,3-butanedione. R. mucilaginosa and S. salivarius also produced labeled acetaldehyde and ethanol when grown with HO. Additionally, we find that P. aeruginosa growth and dimethyl sulfide production are increased when exposed to lactic acid in culture. These results highlight the importance VOCs produced by P. aeruginosa, R. mucilaginosa, and S. salivarius as nutrients and signals in microbial communities, and as potential biomarkers in a CF infection.

Ethanol stimulates trehalose production through a SpoT-DksA-AlgU dependent pathway inPseudomonas aeruginosa

“…P. aeruginosa frequently causes chronic infections in individuals with the genetic disorder cystic fibrosis (CF). Diverse bacterial and fungal taxa often co-infect with P. aeruginosa in CF airways (2-6), and many of these taxa are robust fermenters capable of ethanol production (7, 8). Ethanol has also been identified as a volatile biomarker in exhaled breath condensates that discriminates between healthy individuals and those with CF (9).…”

Tracking Polymicrobial Metabolism in Cystic Fibrosis Airways: Pseudomonas aeruginosa Metabolism and Physiology Are Influenced by Rothia mucilaginosa-Derived Metabolites

“…Culture conditions and metabolomics. The bacterial strains chosen for this study were isolated from CF patients at the UCSD Adult CF Clinic: Pseudomonas aeruginosa PaFLR01 and Rothia mucilaginosa RmFLR01 (11,23). First, we took time points from R. mucilaginosa cultures to examine the kinetics of metabolites in glycolysis, the TCA cycle, amino acid biosynthesis, short-and long-chain fatty acid biosynthesis, and the pentose phosphate pathway in R. mucilaginosa, which was grown in triplicate in artificial-sputum medium (24) spiked with 100 mM [U- 13 C 6 ]D-glucose (Sigma-Aldrich and Cambridge Isotope Laboratory) under anaerobic and aerobic oxygen conditions (5% CO 2 ) at 37°C.…”

Multi-omics study of keystone species in the cystic fibrosis lung microbiome

“…The production of fermentation gas in the WinCF tubes and the increase in abundance of short-chain fatty acids in the metabolomes demonstrates the activity of fermenting bacteria and the release of metabolic by-products in sputum. Volatile fermentation products such as lactic acid and 2,3-butanediol have been previously detected in CF breath gas [30,36]. These molecules can induce pyocyanine production, dormancy and biofilm formation in P. aeruginosa, stimulating its growth and virulence [37,38] Bacteroides, Fusobacterium and Prevotella [39].…”