Direct Assessment of Metabolite Utilization by Pseudomonas aeruginosa during Growth on Artificial Sputum Medium

Abstract

bWe grew Pseudomonas aeruginosa in LB and artificial sputum medium (ASM) (filtered and unfiltered) and quantified metabolite utilization and excretion by nuclear magnetic resonance (NMR) spectroscopy (metabolic footprinting or extracellular metabolomics). Utilization rates were similar between media, but there were differences in excretion-e.g., acetate was produced only in unfiltered ASM.T he opportunistic pathogen Pseudomonas aeruginosa is the major cause of morbidity and mortality for cystic fibrosis (CF) patients (1). In order to better understand P. aeruginosa infection, it is important to create model conditions that aim to mimic those in the CF lung, such as media designed to mimic the nutritional environment of sputum from CF patients. These include artificial sputum medium (ASM) (2), a complex medium made up of porcine mucin, DNA, and amino acids, and synthetic cystic fibrosis medium (SCFM) (3), a defined medium containing mainly amino acids and lactate. In addition, many studies use a more-conventional rich medium, like lysogeny broth (LB) (4), which is based on tryptone and yeast extract. All of these media support growth of P. aeruginosa to high cell numbers, but the question remains how the differences in composition alter the physiology and metabolism of the inoculated bacteria and, hence, the relevance to CF infections. Generally, approaches to validating this have depended on indirect means to assess metabolism-for instance, profiling gene transcription (3). However, transcriptional data are often insufficient to capture the actual metabolic changes that occur as a result of perturbing a bacterial cell. Because of this, we wanted to examine the actual changes in metabolite utilization by P. aeruginosa in a comparison of three different complex media, LB, ASM (oASM, prepared according to its original recipe [2]), and a filtered version of ASM (fASM, prepared according to the method of Kirchner et al. [5]). We have previously investigated metabolite utilization in SCFM in depth (6), so we did not include this medium again here.We grew wild-type P. aeruginosa PA14 under aerobic conditions in batch culture for 24 h (growth conditions described in detail in reference 6) and sampled 0.6 ml of culture at nine time points (before inoculation, hourly from 2 to 8 h, and then at 24 h). Of these samples, 0.1 ml was used for measuring the optical density (OD) for LB and fASM samples (oASM is turbid and the OD cannot be used to monitor cell growth). The supernatants were then analyzed by nuclear magnetic resonance (NMR) spectroscopy as detailed in Behrends et al. (7). The resulting spectra were assigned and integrated for each medium. In total, we were able to assign 19 compounds detectable across all media, of which 18 were able to be quantified (Table 1) (histidine was detected but not integrated here because of pH-induced resonance frequency shifts between spectra) and normalized to compound levels in the uninoculated media. Four further metabolites were able to be identified in at least two media. Finally...

Allelic polymorphism shapes community function in evolving Pseudomonas aeruginosa populations

“…Despite a number of recent studies focused on adaptive changes in evolved populations of P. aeruginosa in environments designed to mimic CF sputum (60)(61)(62)(63)(64)(65), there remain significant gaps in knowledge about how genomic and phenotypic diversity impacts upon on interactions within populations and on community-level phenotypes. In our current study, we focused on understanding how the evolution and coexistence of multiple lineages of P. aeruginosa shape community functions.…”

Effect of Media Modified To Mimic Cystic Fibrosis Sputum on the Susceptibility of Aspergillus fumigatus, and the Frequency of Resistance at One Center

“…It may prove to be the case that tests using such designed media prove more relevant to clinical outcome than those using standard methods, but this will require correlations of in vitro results, clinical courses, and posttreatment cultures. This has not yet even been done with P. aeruginosa infections, although studies of growth and metabolism of that bacterium in these media have been performed (30,31). Of note, the two media tested in which A. fumigatus could grow appeared to make the isolates generally more susceptible to drug inhibition, although digested sputum medium made the isolates more resistant to killing.…”

Antibacterial Effects of Bicarbonate in Media Modified to Mimic Cystic Fibrosis Sputum

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