Impact of Artificial Sputum Medium Formulation on Pseudomonas aeruginosa Secondary Metabolite Production

Abstract

In vitro culture media are being developed to understand how host site-specific nutrient profiles influence microbial pathogenicity and ecology. To mimic the cystic fibrosis (CF) lung environment, a variety of artificial sputum media (ASM) have been created. However, the composition of these ASM vary in the concentration of key nutrients, including amino acids, lipids, DNA, and mucin. In this work, we used feature-based molecular networking (FBMN) to perform comparative metabolomics of Pseudomonas aeruginosa , the predominant opportunistic pathogen infecting the lungs of people with CF, cultured in nine different ASM. We found that the concentration of aromatic amino acids and iron from mucin added to the media contribute to differences in the production of P. aeruginosa virulence-associated secondary metabolites. IMPORTANCE Different media formulations aiming to replicate in vivo infection environments contain different nutrients, which affects interpretation of experimental results. Inclusion of undefined components, such as commercial porcine gastric mucin (PGM), in an otherwise chemically defined medium can alter the nutrient content of the medium in unexpected ways and influence experimental outcomes.

A library of promoter-gfpfusion reporters for studying systemic expression pattern of cyclic-di-GMP metabolism-related genes inPseudomonas aeruginosa

“…Bacterial strains and growth conditions P. aeruginosa strain PAO1 was cultured in Luria-Bertani (LB) medium (64), or Jensen's medium (65), or M63 medium (66), or Artificial Sputum Medium (ASM) (67).…”

The Use of Artificial Sputum Media to Enhance Investigation and Subsequent Treatment of Cystic Fibrosis Bacterial Infections

“…(Figure 3: Timeline of artificial sputum medium formulation). 2021) [33]. References in figure from top left-right [17,22,32,[34][35][36] and bottom left-right [15,37].…”

“…Formulations have been either derived from cited literature values (red) or are synthetically derived from CF sputum microanalysis (blue). Timeline adapted from Neve et al (2021) [ 33 ]. References in figure from top left-right [ 17 , 22 , 32 , 34 , 35 , 36 ] and bottom left-right [ 15 , 37 ].…”

“…The Ghani and Soothill formulation, however, lacked the second capacity. The cultures remained turbid, as in any growth medium, but lacked the conventional polymeric matrix seen in when growing P. aeruginosa biofilms [ 33 ]. This may be attributed to the absence of amino acids which contribute to secondary metabolite production and biofilm formation.…”

“…It became evident that a specific set of genes responsible for anabolic pathways for biosynthesis of thiamine, nicotinamide adenine dinucleotide (NAD), purines, folate, and branched amino acids and tryptophan metabolites was higher in CF sputum than in SCFM2. Therefore, the final iteration of the SCFM series (SCFM3) was modified by the addition of p -aminobenzoic acid, NAD+, adenine, guanine, xanthine, and hypoxanthine to compensate for their absence in the previous formulation and to address the abundant bioavailability of these metabolites in CF sputum [ 33 ].…”