Helen L. Barr

Recombination is a key driver of genomic and phenotypic diversity in a Pseudomonas aeruginosa population during cystic fibrosis infection

The Cystic Fibrosis (CF) lung harbors a complex, polymicrobial ecosystem, in which Pseudomonas aeruginosa is capable of sustaining chronic infections, which are highly resistant to multiple antibiotics. Here, we investigate the phenotypic and genotypic diversity of 44 morphologically identical P. aeruginosa isolates taken from a single CF patient sputum sample. Comprehensive phenotypic analysis of isolates revealed large variances and trade-offs in growth, virulence factors and quorum sensing (QS) signals. Whole genome analysis of 22 isolates revealed high levels of intra-isolate diversity ranging from 5 to 64 SNPs and that recombination and not spontaneous mutation was the dominant driver of diversity in this population. Furthermore, phenotypic differences between isolates were not linked to mutations in known genes but were statistically associated with distinct recombination events. We also assessed antibiotic susceptibility of all isolates. Resistance to antibiotics significantly increased when multiple isolates were mixed together. Our results highlight the significant role of recombination in generating phenotypic and genetic diversification during in vivo chronic CF infection. We also discuss (i) how these findings could influence how patient-to-patient transmission studies are performed using whole genome sequencing, and (ii) the need to refine antibiotic susceptibility testing in sputum samples taken from patients with CF.

Association between socioeconomic status, sex, and age at death from cystic fibrosis in England and Wales (1959 to 2008): cross sectional study

Objective To determine the trend in the association between socioeconomic status and sex and median age at death from cystic fibrosis in England and Wales, over the past 50 years. Design Series of annual cross sectional studies of all registered deaths with a diagnosis of cystic fibrosis in England and Wales, from 1959 to 2008. Methods We obtained national mortality data for cystic fibrosis from the Office for National Statistics. From 1959 to 2000, the Registrar General’s Social Class coded socioeconomic status as manual or non-manual. From 2001 onwards, the National Statistics Socioeconomic Classification was implemented and socioeconomic status was split into three groups: professional and managerial, intermediate, and routine and manual. We calculated median age at death for every study year. We calculated the effects of sex and socioeconomic status on the odds of death above the median age at death for every study decade using logistic regression. Results From 1959 to 2008, 6750 deaths were attributed to cystic fibrosis in England and Wales. Males were more likely to die above the annual median age at death than females (from 1959 to 1999, adjusted odds ratio for socioeconomic status 1.28, 95% confidence intervals 1.13 to 1.45; from 2000 to 2008, 1.57, 1.18 to 2.08). Individuals in the highest socioeconomic class were also more likely to die above the median age of death than those in the lowest socioeconomic class (from 1959 to 2000, adjusted odds ratio for sex 2.50, 2.16 to 2.90; from 2001 to 2008, 1.89, 1.20 to 2.97). Conclusions Socioeconomic status and sex remain strong determinants of survival from cystic fibrosis in England and Wales, and the magnitude of these effects does not appear to have substantially reduced over time.

Pseudomonas aeruginosa quorum sensing molecules correlate with clinical status in cystic fibrosis

Pseudomonas aeruginosa produces quorum sensing signal molecules that are potential biomarkers for infection. A prospective study of 60 cystic fibrosis patients with chronic P. aeruginosa, who required intravenous antibiotics for pulmonary exacerbations, was undertaken. Clinical measurements and biological samples were obtained at the start and end of the treatment period. Additional data were available for 29 of these patients when they were clinically stable. Cross-sectionally, quorum sensing signal molecules were detectable in the sputum, plasma and urine of 86%, 75% and 83% patients, respectively. They were positively correlated between the three biofluids. Positive correlations were observed for most quorum sensing signal molecules in sputum, plasma and urine, with quantitative measures of pulmonary P. aeruginosa load at the start of a pulmonary exacerbation. Plasma concentrations of 2-nonyl-4-hydroxy-quinoline (NHQ) were significantly higher at the start of a pulmonary exacerbation compared to clinical stability (p<0.01). Following the administration of systemic antibiotics, plasma 2-heptyl-4-hydroxyquinoline (p=0.02) and NHQ concentrations (p<0.01) decreased significantly. In conclusion, quorum sensing signal molecules are detectable in cystic fibrosis patients with pulmonary P. aeruginosa infection and are positively correlated with quantitative measures of P. aeruginosa. NHQ correlates with clinical status and has potential as a novel biomarker for P. aeruginosa infection. P. aeruginosa QS molecules correlate with clinical status in cystic fibrosis and are biomarkers for infection http://ow.ly/MhzZp

Diagnostic and prognostic significance of systemic alkyl quinolones for P. aeruginosa in cystic fibrosis: a longitudinal study

Background Pulmonary P. aeruginosa infection is associated with poor outcomes in cystic fibrosis (CF) and early diagnosis is challenging, particularly in those who are unable to expectorate sputum. Specific P. aeruginosa 2-alkyl-4-quinolones are detectable in the sputum, plasma and urine of adults with CF, suggesting that they have potential as biomarkers for P. aeruginosa infection. Aim To investigate systemic 2-alkyl-4-quinolones as potential biomarkers for pulmonary P. aeruginosa infection. Methods A multicentre observational study of 176 adults and 68 children with CF. Cross-sectionally, comparisons were made between current P. aeruginosa infection using six 2-alkyl-4-quinolones detected in sputum, plasma and urine against hospital microbiological culture results. All participants without P. aeruginosa infection at baseline were followed up for one year to determine if 2-alkyl-4-quinolones were early biomarkers of pulmonary P. aeruginosa infection. Results Cross-sectional analysis: the most promising biomarker with the greatest diagnostic accuracy was 2-heptyl-4-hydroxyquinoline (HHQ). In adults, areas under the ROC curves (95% confidence intervals) for HHQ analyses were 0.82 (0.75–0.89) in sputum, 0.76 (0.69–0.82) in plasma and 0.82 (0.77–0.88) in urine. In children, the corresponding values for HHQ analyses were 0.88 (0.77–0.99) in plasma and 0.83 (0.68–0.97) in urine. Longitudinal analysis: Ten adults and six children had a new positive respiratory culture for P. aeruginosa in follow-up. A positive plasma HHQ test at baseline was significantly associated with a new positive culture for P. aeruginosa in both adults and children in follow-up (odds ratio (OR) = 6.67;-95% CI:-1.48–30.1;-p = 0.01 and OR = 70; 95% CI: 5–956;-p < 0.001 respectively). Conclusions AQs measured in sputum, plasma and urine may be used to diagnose current infection with P. aeruginosa in adults and children with CF. These preliminary data show that plasma HHQ may have potential as an early biomarker of pulmonary P. aeruginosa. Further studies are necessary to evaluate if HHQ could be used in clinical practice to aid early diagnosis of P. aeruginosa infection in the future.

Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages infected with P. aeruginosa: untreated < treated with GM-CSF < treated with IFN-γ < treated with GM-CSF and IFN-γ.

Glutamine supplementation in cystic fibrosis: A randomized placebo‐controlled trial

Pulmonary infection and malnutrition in cystic fibrosis are associated with decreased survival. Glutamine has a possible anti‐microbial effect, with a specific impact against Pseudomonas aeruginosa. We aimed to test the hypothesis that oral glutamine supplementation (21 g/day) for 8 weeks in adults with cystic fibrosis would decrease pulmonary inflammation and improve clinical status.

Weight gain during acute treatment of an initial pulmonary exacerbation is associated with a longer interval to the next exacerbation in adults with cystic fibrosis

Cystic fibrosis (CF) results in a variety of clinical phenotypes, including increased susceptibility to pulmonary infections [1] and malnutrition [2]. As a consequence, one of the priorities in the clinical care for individuals with a diagnosis of CF is to treat chest infections promptly [3], with the aim of minimising the decline in lung function that occurs in individuals who experience recurrent pulmonary exacerbations [4]. Optimising nutrition is another important consideration in the care of patients with CF [3]. It is well recognised that nutritional status and lung function are correlated [5] and that individuals with lower levels of nutrition have increased mortality [6] compared to those individuals with better nutrition. Weight gain during treatment for a cystic fibrosis exacerbation http://ow.ly/f1zl30dU9AO

P92 Systemic alkyl quinolones as novel biomarkers for pulmonary exacerbations in cystic fibrosis: a validation study

Introduction and objectives There is a clinical need to identify and validate biomarkers that are sensitive to treatment of infection in cystic fibrosis (CF). The aim of this study was to externally validate two novel biomarkers for pulmonary exacerbations in CF of the alkyl quinolone (AQ) class of quorum sensing molecules produced by Pseudomonas aeruginosa. Methods Retrospective analysis of 70 plasma samples from thirteen adults with CF obtained during treatment of fifteen discrete exacerbations treated with intravenous antibiotics. Plasma samples were obtained at the start, day five, day ten, at the end of treatment, and at clinical stability. Samples were analysed using liquid chromatography-mass spectrometry. Data were analysed using Spearman’s rank correlations and Wilcoxon matched pairs signed-rank tests using STATA 11 statistical software (Texas, USA). Graphs were produced in EXCEL 2011. Results Plasma 2-heptyl-4-hydroxyquinoline (HHQ) concentration significantly decreased by a median of 221 pmol/L (IQR: 158 to 258 pmol/L) or 73% (IQR 52 to 94%; p = 0.0007) during treatment for a pulmonary exacerbation (Figure 1). In the same interval, there was no significant change in plasma NHQ (median decrease of −3 pmol/L; IQR: −35 to 10 pmol/L; p = 0.65). During treatment for a pulmonary exacerbation, percent predicted FEV1 increased by 4% (IQR: 1 to 7%; p = 0.0086). Folloing systemic antimicrobial therapy, systemic IL6 concentration decreased by a median of 2.06 pg/mL (IQR: 1.02 to 3.55 pg/mL; p = 0.0022) and systemic calprotectin decreased by 1687 ng/mL (IQR: 291 to 3992 ng/mL; p = 0.0229). There was no significant association between change in plasma HHQ and change in FEV1 during treatment of a pulmonary exacerbation (Spearman’s correlation co-efficient, r = −0.42; p = 0.15). Conclusions Plasma HHQ declined significantly during treatment of a pulmonary exacerbation and merits further investigation as a biomarker for measuring treatment response in CF. There was no significant decline in plasma NHQ during systemic antimicrobial therapy. Abstract P92 Figure 1 Plasma HHQ concentrations at the start and end of 15 pulmonary exacerbations treated with systemic anti-pseudomonal antibiotics in 13 adults with CF HHQ = 2-heptyl-4-hydroxyquinoline pmol/L = picomoles per litre