Phenotyping of Pseudomonas aeruginosa biofilms in cystic fibrosis patients, biofilm biomarker identification, and understanding the nitric oxide mediated biofilm dispersal response.

Abstract

Colonisation of Pseudomonas aeruginosa (PA) in the airways is associated with persistent morbidity, and increased mortality in cystic fibrosis (CF) patients. Treatment strategies include aggressive antibiotic regimes aimed at eradicating or controlling the infection. However, structured biofilm aggregates offer an increased tolerance to antimicrobials, with eradication treatments failing in 10-40% of patients. Biofilms are largely unculturable, are unable to be diagnosed via culture-based methods. A lack of biomarkers for a biofilm infection escalates this diagnostic issue. Previous studies have explored the relationship of novel nitric oxide (NO) donors as biofilm dispersal agents for potential new antibiofilm therapy to be used in CF. Biofilms formed by CF isolates in vitro were found
to have varied responses to the NO donor sodium nitroprusside (SNP).

This study focuses both assessing the biofilm status of CF patients chronically infected with P. aeruginosa undergoing exacerbation and looks to combine the phenotypic biofilm status and proteomic data to identify potential biomarkers for biofilm infection. Secondly the study also aims to further explore the underlying mechanisms of the varied responses of clinical CF P. aeruginosa biofilms to the NO biofilm dispersal signal determining patient suitability for the therapeutic use of NO.

Using series of microbiological techniques including fluorescently labelled via in-situ hybridisation (FISH) microscopy, dispersal assays, biofilm culture models, and multi-omics methods, the biofilm status was determined for 62 patients and further assessed for alterations following antibiotic treatment. Sputum samples were processed for proteomic analysis to identify correlations between biofilm status and disease state to seek out potential human protein biomarkers for the presence of a biofilm infection. Histone H4 was observed positively correlate with the disease state, with a decreased abundance post antibiotics suggesting a potential infection marker. Despite showing no correlation to that of the biofilm biomass, histone H4 remains to be of interest for further analysis.
An extended panel of 10 PA isolates obtained from the sputum of Cystic fibrosis patients at the University hospital Southampton were assessed for their NO response. Interestingly, three distinct phenotypes were identified: dispersing, no change, and growth promotion. Isolates underwent whole genome sequencing, however, no obvious genetic adaptation to the NO signal was discovered suggesting deeper regulatory mechanisms to be responsible. Analysis of the transcriptomes of isolates with differing responses was unable to confidently identify any regulatory mechanism, but has suggested the roles of numerous elements, raising questions on the involvement of the two-component regulatory system, PhoPQ, reported to play a role in virulence and polymyxin resistance,
and flagella in the increased biofilm integrity in response to NO.

Overall, this study used biofilm phenotyping to characterise the patient biofilm status by identifying bacterial aggregates in expectorated sputum and highlighted the variability between individual patients, raising questions for personalised approaches to CF treatments. A potential biomarker,
histone H4, for the CF disease state and PA biofilm phenotype has been identified as being of interest for further proteomic analysis within an extended patient cohort. Further analysis of the bacterial isolates in the CF culture collection is required for full understanding of the varied NO dispersal response.

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Identifiers

ORCID for Declan Matthew Power: orcid.org/0000-0001-5049-713X

ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

ORCID for Gary Connett: orcid.org/0000-0003-1310-3239

ORCID for Saul Faust: orcid.org/0000-0003-3410-7642

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