Exploiting targeted and untargeted approaches for the analysis of bacterial metabolites under altered growth conditions

Abstract

In the host, pathogenic microorganisms have developed stress responses to cope with constantly changing environments. Stress responses are directly related to changes in several metabolomic pathways, which could hamper microorganisms' unequivocal identification. We evaluated the effect of various in vitro stress conditions (acidic, basic, oxidative, ethanolic, and saline conditions) on the metabolism of Staphylococcus aureus, Bacillus cereus, and Pseudomonas aeruginosa, which are common lung pathogens. The metabolite profiles of the bacteria were analyzed using liquid chromatography coupled to triple quadrupole and quadrupole time-of-flight mass spectrometry. The advantages of targeted and untargeted analysis combined with univariate and multivariate statistical analysis (principal component analysis, hierarchical cluster analysis, partial least square discriminant analysis, random forest) were combined to unequivocally identify bacterial species. In normal in vitro conditions, the targeted methodology, based on the analysis of primary metabolites, enabled the rapid and efficient discrimination of the three bacteria. In changing in vitro conditions and specifically in presence of the various stressors, the untargeted methodology proved to be more valuable for the global and accurate differentiation of the three bacteria, also considering the type of stress environment within each species. In addition, species-specific metabolites (i.e., fatty acids, polysaccharides, peptides, and nucleotide bases derivatives) were putatively identified. Good intra-day repeatability and inter-day repeatability (< 10% RSD and < 15% RSD, respectively) were obtained for the targeted and the untargeted methods. This untargeted approach highlights its importance in unusual (and less known) bacterial growth environments, being a powerful tool for infectious disease diagnosis, where the accurate classification of microorganisms is sought.

Decreased Sphingosine Due to Down-Regulation of Acid Ceramidase Expression in Airway of Bronchiectasis Patients: A Potential Contributor to Pseudomonas aeruginosa Infection

“… 4 Likewise, figuring out the interaction between respiratory flora and host metabolites contributes to an explanation of the pathogenesis of respiratory diseases and leads to a breakthrough in treatment. 5 Analysis of respiratory microbial diversity, macrogenomics, and metabolomics in cystic fibrosis patients found unique combinations of bacterial genera and metabolites in different disease states and indicated that three additional exacerbations and treatments culminated in a new airway microbiome and metabolome. 6 However, the correlation between lower respiratory microbial diversity, P. aeruginosa infection, and host metabolites has not been thoroughly explained in bronchiectasis patients.…”