Artificial Sputum Medium Research Articles

Pseudomonas aeruginosa AES-1 Exhibits Increased Virulence Gene Expression during Chronic Infection of Cystic Fibrosis Lung

Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients.

97 Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa

97 Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa

Characterization of JG024, a pseudomonas aeruginosa PB1-like broad host range phage under simulated infection conditions

BackgroundPseudomonas aeruginosa causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF). Once a chronic lung infection is established, P. aeruginosa cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic P. aeruginosa infections. However, little is known about the factors which influence phage infection of P. aeruginosa under infection conditions and suitable broad host range phages.ResultsWe isolated and characterized a phage, named JG024, which infects a broad range of clinical and environmental P. aeruginosa strains. Sequencing of the phage genome revealed that the phage JG024 is highly related to the ubiquitous and conserved PB1-like phages. The receptor of phage JG024 was determined as lipopolysaccharide. We used an artificial sputum medium to study phage infection under conditions similar to a chronic lung infection. Alginate production was identified as a factor reducing phage infectivity.ConclusionsPhage JG024 is a suitable broad host range phage which could be used in phage therapy. Phage infection experiments under simulated chronic lung infection conditions showed that alginate production reduces phage infection efficiency.

Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum

Pseudomonas aeruginosa airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Various in vitro models have been developed to study P. aeruginosa pathobiology in the CF lung. In this study we produced a modified artificial-sputum medium (ASMDM) more closely resembling CF sputum than previous models, and extended previous work by using strain PAO1 arrays to examine the global transcription profiles of P. aeruginosa strain UCBPP-PA14 under early exponential-phase and stationary-phase growth. In early exponential phase, 38/39 nutrition-related genes were upregulated in line with data from previous in vitro models using UCBPP-PA14. Additionally, 23 type III secretion system (T3SS) genes, several anaerobic respiration genes and 24 quorum-sensing (QS)-related genes were upregulated in ASMDM, suggesting enhanced virulence factor expression and priming for anaerobic growth and biofilm formation. Under stationary phase growth in ASMDM, macroscopic clumps resembling microcolonies were evident in UCBPP-PA14 and CF strains, and over 40 potentially important genes were differentially expressed relative to stationary-phase growth in Luria broth. Most notably, QS-related and T3SS genes were downregulated in ASMDM, and iron-acquisition and assimilatory nitrate reductase genes were upregulated, simulating the iron-depleted, microaerophilic/anaerobic environment of CF sputum. ASMDM thus appears to be highly suitable for gene expression studies of P. aeruginosa in CF.

Inhalable Antibiotic Delivery Using a Dry Powder Co-delivering Recombinant Deoxyribonuclease and Ciprofloxacin for Treatment of Cystic Fibrosis

To achieve efficient antibiotic delivery to the cystic fibrosis (CF) airway using a single inhalable powder co-encapsulating a mucolytic and an antibiotic. Inhalable dry powders containing deoxyribonuclease and/or ciprofloxacin (DNase, Cipro, and DNase/Cipro powders) were produced by spray-drying with dipalmitylphosphatidylcholine, albumin, and lactose as excipients, and their antibacterial effects were evaluated using the artificial sputum model. All powders showed mass median aerodynamic diameters below 5 microm. Both drugs were loaded in the dry powders without loss in quantity and activity. Dry powders containing DNase significantly decreased the storage modulus of the artificial sputum medium in less than 30 min. When applied to artificial sputum laden with Pseudomonas aeruginosa, Cipro/DNase powder showed better antibacterial activity than Cipro powder. The higher activity of the Cipro/DNase powder is attributable to the mucolytic activity of DNase, which promotes penetration of the dry powder into the artificial sputum and efficient dissolution and diffusion of ciprofloxacin. Inhalational delivery of antibiotics to the CF airway can be optimized when the sputum barrier is concomitantly addressed. Co-delivery of antibiotics and DNase using an inhalable particle system may be a promising strategy for local antipseudomonal therapy in the CF airway.

Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung

Pseudomonas aeruginosa colonizing the lung of cystic fibrosis patients is responsible for a decline in health and poor prognosis for these patients. Once established, growth of P. aeruginosa in microcolonies makes it very difficult to eradicate the organisms by antimicrobial treatment. An artificial sputum medium was developed to mimic growth of P. aeruginosa in the cystic fibrosis lung habitat and it was found that the organisms grew in tight microcolonies attached to sputum components. Several genes, such as algD, oprF and lasR but not fliC, were required for tight microcolony formation. Among the sputum components, amino acids, lecithin, DNA, salt and low iron were required for tight microcolony formation. Amino acids were also shown to be responsible for various other cystic-fibrosis-specific phenotypes of P. aeruginosa, such as diversification of colony morphology, alterations in LPS structure and hyperexpression of OprF. Since the amino acid content of sputum is elevated in severe lung disease, it is suggested that the tight microcolony biofilm is maintained in these conditions and that they contribute to the vicious cycle of disease severity and failure to eradicate the organism. Thus, growth of P. aeruginosa in artificial sputum medium is an appropriate model of chronic lung colonization and may be useful for evaluating therapeutic procedures and studying antibiotic-resistance mechanisms.