Cystic Fibrosis Sputum Supports Growth and Cues Key Aspects of Pseudomonas aeruginosa Physiology

Abstract

The opportunistic human pathogen Pseudomonas aeruginosa causes persistent airway infections in patients with cystic fibrosis (CF). To establish these chronic infections, P. aeruginosa must grow and proliferate within the highly viscous sputum in the lungs of CF patients. In this study, we used Affymetrix GeneChip microarrays to investigate the physiology of P. aeruginosa grown using CF sputum as the sole source of carbon and energy. Our results indicate that CF sputum readily supports high-density P. aeruginosa growth. Furthermore, multiple signals, which reduce swimming motility and prematurely activate the Pseudomonas quinolone signal cell-to-cell signaling cascade in P. aeruginosa, are present in CF sputum. P. aeruginosa factors critical for lysis of the common CF lung inhabitant Staphylococcus aureus were also induced in CF sputum and increased the competitiveness of P. aeruginosa during polymicrobial growth in CF sputum.

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

“…Accordingly, these responses were particularly characteristic of P. aeruginosa and S. maltophilia pure cultures, a sputum sample dominated by P. aeruginosa (CF9) and an explant lung sample from a P. aeruginosa-dominated patient. The behavior of P. aeruginosa-and Climax Community-dominated samples matches much of what is recorded in the literature: that the organism grows anaerobically using denitrification in antibiotic-resistant biofilms (Worlitzsch et al, 2002;Palmer et al, 2005;Hogardt and Heesemann, 2010;Schobert and Jahn, 2010). The elevated pH in the media may be due to the production of ammonia from amino-acid breakdown, often observed with P. aeruginosa grown in a CF state (Verhoogt et al, 1992;Barth and Pitt, 1996;Thomas et al, 2000;Frimmersdorf et al, 2010), and previously demonstrated to accumulate in WinCF capillary tubes after the growth of CF microbes .…”

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

“…These differences become increasingly important when factors of clinical relevance, such as virulence and antimicrobial tolerance, are altered [13,118,119]. Nutritional cues similar to those of expectorated CF sputum have been incorporated into a synthetic CF sputum media (SCFM) that approximates P. aeruginosa gene expression to that observed in expectorated CF sputum [120]. Two noteworthy points of this study are (i) the lack of key components observed in CF sputum (notably DNA, fatty acids, N-acetyle glucosamine, and mucin) and (ii) the inability of the methods used (RNA-seq) to correctly predict fitness requirements [121][122][123][124][125][126][127].…”

Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum

“…Our laboratory recently used Tn-seq to reveal fitness requirements for P. aeruginosa during acute and chronic murine wound infection (12). A major finding of this study was that transcriptome-based approaches such as RNA sequencing cannot be used to predict fitness requirements (12,13), thus calling into question the utility of previous P. aeruginosa CF sputum transcriptome results (3,14) for elucidating fitness requirements in the CF lung.…”

“…The inflammatory components include large numbers of polymorphonuclear leukocytes, dead host cells, and serum components that enter the airway due to vascular leakage and pulmonary hemorrhage (1). The generation time of P. aeruginosa in CF sputum is variable but can be as short as 40 min, suggesting that sputum provides a robust growth environment for P. aeruginosa (3,4). Long-term colonization of the CF lung leads to the evolution of numerous presumed adaptive phenotypes including mucoidy, amino acid auxotrophy, loss of acute virulence factors, and antibiotic resistance (5)(6)(7).…”