Requirements for Pseudomonas aeruginosa Acute Burn and Chronic Surgical Wound Infection

Abstract

Opportunistic infections caused by Pseudomonas aeruginosa can be acute or chronic. While acute infections often spread rapidly and can cause tissue damage and sepsis with high mortality rates, chronic infections can persist for weeks, months, or years in the face of intensive clinical intervention. Remarkably, this diverse infectious capability is not accompanied by extensive variation in genomic content, suggesting that the genetic capacity to be an acute or a chronic pathogen is present in most P. aeruginosa strains. To investigate the genetic requirements for acute and chronic pathogenesis in P. aeruginosa infections, we combined high-throughput sequencing-mediated transcriptome profiling (RNA-seq) and genome-wide insertion mutant fitness profiling (Tn-seq) to characterize gene expression and fitness determinants in murine models of burn and non-diabetic chronic wound infection. Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes. By combining metabolic models generated from in vivo gene expression data with mutant fitness profiles, we determined the nutritional requirements for colonization and persistence in these infections. Specifically, we found that long-chain fatty acids represent a major carbon source in both chronic and acute wounds, and P. aeruginosa must biosynthesize purines, several amino acids, and most cofactors during infection. In addition, we determined that P. aeruginosa requires chemotactic flagellar motility for fitness and virulence in acute burn wound infections, but not in non-diabetic chronic wound infections. Our results provide novel insight into the genetic requirements for acute and chronic P. aeruginosa wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence.

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

“…Similarities have been observed in the active biosynthetic pathways of P. aeruginosa in the CF lung and murine surgical wound infections [127]. This suggests that (i) catabolite metabolism is shared between certain infection sites and (ii) other factors, such as host-inflammatory responses, may be the cause of infection chronicity.…”

“…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]. A follow up study rectified these issues, incorporating these components into SCFM.…”

Bacterial competition and quorum‐sensing signalling shape the eco‐evolutionary outcomes of model in vitro phage therapy

“…First, selection for phage resistance could be weaker in polymicrobial communities due to a competition‐mediated reduction in the focal pathogen density and relatively higher pleiotropic costs of adaptation. Competition could thus enhance the phage efficacy when treating acute CF and burn infections that are commonly co‐infected by QS signalling P. aeruginosa , S. aureus and S. maltophilia (Harrison, 2007; Turner et al., 2014). However, in contrary, P. aeruginosa resistance evolution to phages could be a more severe problem in chronic polymicrobial CF infections that are often dominated by P. aeruginosa mutants that have lost QS signalling ability during the long‐term adaptation (Andersen et al., 2015; Marvig et al., 2014; Smith et al., 2006).…”

“…The bacterium Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects many immunocompromised patients including cystic fibrosis (CF) and burn victim patients (Harrison, 2007; Turner, Everett, Trivedi, Rumbaugh, & Whiteley, 2014). P. aeruginosa is often characterized by multidrug resistance to conventional antibiotics (Strateva & Yordanov, 2009), and hence, the development of novel phage therapy treatments could potentially help a large number of patients (Harper & Enright, 2011).…”

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

“…DNA was extracted from each mutant pool largely as described previously (83). An aliquot (1-ml glycerol stock) was resuspended in 1 ml 1ϫ buffer A (84) plus 0.1% SDS and 0.1% sodium deoxycholate, transferred to a Lysing Matrix B 2-ml tube (MP Biomedicals), homogenized in a Mini-Beadbeater (BioSpec), and digested overnight at 55°C with 1 mg/ml proteinase K. DNA was then extracted with 1 ml 25:24:1 phenol-chloroform-isoamyl alcohol (pH 8.0), purified from the aqueous phase by isopropanol precipitation, washed with 75% ethanol, and dissolved in 500 l distilled water (diH 2 O).…”