Studies on the antibacterial effects of statins--in vitro and in vivo

Background

Statin treatment has been associated with a beneficial outcome on respiratory tract infections. In addition, previous in vitro and in vivo experiments have indicated favorable effects of statins in bacterial infections.

Aim

The aim of the present study was to elucidate possible antibacterial effects of statins against primary pathogens of the respiratory tract.

Methods

MIC-values for simvastatin, fluvastatin and pravastatin against S. pneumoniae, M. catarrhalis and H. influenzae were determined by traditional antibacterial assays. A BioScreen instrument was used to monitor effects of statins on bacterial growth and to assess possible synergistic effects with penicillin. Bacterial growth in whole blood and serum from healthy volunteers before and after a single dose of simvastatin, fluvastatin and penicillin (positive control) was determined using a blood culture system (BactAlert).

Findings

The MIC-value for simvastatin against S pneumoniae and M catarrhalis was 15 µg/mL (36 mmol/L). Fluvastatin and Pravastatin showed no antibacterial effect in concentrations up to 100 µg/mL (230 µmol/L). Statins did not affect growth or viability of H influenzae. Single doses of statins given to healthy volunteers did not affect growth of pneumococci, whereas penicillin efficiently killed all bacteria.

Conclusions

Simvastatin at high concentrations 15 µg/mL (36 µmol/L) rapidly kills S pneumoniae and M catarrhalis. However, these concentrations by far exceed the concentrations detected in human blood during simvastatin therapy (1–15 nmol/L) and single doses of statins given to healthy volunteers did not improve antibacterial effects of whole blood. Thus, a direct bactericidal effect of statins in vivo is probably not the mechanism behind the observed beneficial effect of statins against various infections.     

Elevated expression of both mRNA and protein levels of IL-17A in sputum of stable Cystic Fibrosis patients

Background

T helper 17 (Th17) cells can recruit neutrophils to inflammatory sites through production of IL-17, which induces chemokine release. IL-23 is an important inducer of IL-17 and IL-22 production. Our aim was to study the role of Th17 cells in cystic fibrosis (CF) lung disease by measuring IL-17 protein and mRNA levels and IL-22 and IL-23 mRNA in sputum of clinically stable CF patients and by comparing these levels with healthy controls.

Methods

Sputum induction was performed in adult CF patients outside of an exacerbation and healthy control subjects. IL-17A protein levels were measured in supernatants with cytometric bead array (CBA) and RNA was isolated and quantitative RT-PCR was performed for IL-17A, IL-22 and IL-23.

Results

We found significantly higher levels of IL-17A protein and mRNA levels (both: p < 0.0001) and IL-23 mRNA levels (p < 0.0001) in the sputum of CF group as compared to controls. We found very low levels of IL-22 mRNA in the CF group. The levels of IL-17 and IL-23 mRNA were higher in patients chronically infected with Pseudomonas aeruginosa (P. aeruginosa) as compared to those who were not chronically infected with P. aeruginosa. The presence of Staphylococcus aureus (S. aureus) on sputum did not affect the IL-17 or IL-23 levels. There was no correlation between IL-17 or IL-23 levels and FEV₁ nor sputum neutrophilia.

Conclusion

The elevated levels of IL-17 and IL-23 might indicate that Th17 cells are implicated in the persistent neutrophil infiltration in CF lung disease and chronic infection with P. aeruginosa.     

Effect of temperature on cystic fibrosis lung disease and infections: a replicated cohort study

Background

Progressive lung disease accounts for the majority of morbidity and mortality observed in cystic fibrosis (CF). Beyond secondhand smoke exposure and socio-economic status, the effect of specific environmental factors on CF lung function is largely unknown.

Methods

Multivariate regression was used to assess correlation between specific environmental factors, the presence of pulmonary pathogens, and variation in lung function using subjects enrolled in the U.S. CF Twin and Sibling Study (CFTSS: n = 1378). Significant associations were tested for replication in the U.S. CF Foundation Patient Registry (CFF: n = 16439), the Australian CF Data Registry (ACFDR: n = 1801), and prospectively ascertained subjects from Australia/New Zealand (ACFBAL: n = 167).

Results

In CFTSS subjects, the presence of Pseudomonas aeruginosa (OR = 1.06 per °F; p<0.001) was associated with warmer annual ambient temperatures. This finding was independently replicated in the CFF (1.02; p<0.001), ACFDR (1.05; p = 0.002), and ACFBAL (1.09; p = 0.003) subjects. Warmer temperatures (−0.34 points per °F; p = 0.005) and public insurance (−6.43 points; p<0.001) were associated with lower lung function in the CFTSS subjects. These findings were replicated in the CFF subjects (temperature: −0.31; p<0.001; insurance: −9.11; p<0.001) and similar in the ACFDR subjects (temperature: −0.23; p = 0.057). The association between temperature and lung function was minimally influenced by P. aeruginosa. Similarly, the association between temperature and P. aeruginosa was largely independent of lung function.

Conclusions

Ambient temperature is associated with prevalence of P. aeruginosa and lung function in four independent samples of CF patients from two continents.     

The population genetics of Pseudomonas aeruginosa isolates from different patient populations exhibits high-level host specificity

Objective

To determine whether highly prevalent P. aeruginosa sequence types (ST) in Dutch cystic fibrosis (CF) patients are specifically linked to CF patients we investigated the population structure of P. aeruginosa from different clinical backgrounds. We first selected the optimal genotyping method by comparing pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and multilocus variable number tandem-repeat analysis (MLVA).

Methods

Selected P. aeruginosa isolates (n = 60) were genotyped with PFGE, MLST and MLVA to determine the diversity index (DI) and congruence (adjusted Rand and Wallace coefficients). Subsequently, isolates from patients admitted to two different ICUs (n = 205), from CF patients (n = 100) and from non-ICU, non-CF patients (n = 58, of which 19 were community acquired) were genotyped with MLVA to determine distribution of genotypes and genetic diversity.

Results

Congruence between the typing methods was >79% and DIs were similar and all >0.963. Based on costs, ease, speed and possibilities to compare results between labs an adapted MLVA scheme called MLVA9-Utrecht was selected as the preferred typing method. In 363 clinical isolates 252 different MLVA types (MTs) were identified, indicating a highly diverse population (DI  = 0.995; CI  = 0.993–0.997). DI levels were similarly high in the diverse clinical sources (all >0.981) and only eight genotypes were shared. MTs were highly specific (>80%) for the different patient populations, even for similar patient groups (ICU patients) in two distinct geographic regions, with only three of 142 ICU genotypes detected in both ICUs. The two major CF clones were unique to CF patients.

Conclusion

The population structure of P. aeruginosa isolates is highly diverse and population specific without evidence for a core lineage in which major CF, hospital or community clones co-cluster. The two genotypes highly prevalent among Dutch CF patients appeared unique to CF patients, suggesting specific adaptation of these clones to the CF lung.     

The airway microbiota in cystic fibrosis: a complex fungal and bacterial community--implications for therapeutic management

Background

Given the polymicrobial nature of pulmonary infections in patients with cystic fibrosis (CF), it is essential to enhance our knowledge on the composition of the microbial community to improve patient management. In this study, we developed a pyrosequencing approach to extensively explore the diversity and dynamics of fungal and prokaryotic populations in CF lower airways.

Methodology and Principal Findings

Fungi and bacteria diversity in eight sputum samples collected from four adult CF patients was investigated using conventional microbiological culturing and high-throughput pyrosequencing approach targeting the ITS2 locus and the 16S rDNA gene. The unveiled microbial community structure was compared to the clinical profile of the CF patients. Pyrosequencing confirmed recently reported bacterial diversity and observed complex fungal communities, in which more than 60% of the species or genera were not detected by cultures. Strikingly, the diversity and species richness of fungal and bacterial communities was significantly lower in patients with decreased lung function and poor clinical status. Values of Chao1 richness estimator were statistically correlated with values of the Shwachman-Kulczycki score, body mass index, forced vital capacity, and forced expiratory volume in 1 s (p = 0.046, 0.047, 0.004, and 0.001, respectively for fungal Chao1 indices, and p = 0.010, 0.047, 0.002, and 0.0003, respectively for bacterial Chao1 values). Phylogenetic analysis showed high molecular diversities at the sub-species level for the main fungal and bacterial taxa identified in the present study. Anaerobes were isolated with Pseudomonas aeruginosa, which was more likely to be observed in association with Candida albicans than with Aspergillus fumigatus.

Conclusions

In light of the recent concept of CF lung microbiota, we viewed the microbial community as a unique pathogenic entity. We thus interpreted our results to highlight the potential interactions between microorganisms and the role of fungi in the context of improving survival in CF.     

Treatment of Aspergillus fumigatus in patients with cystic fibrosis: a randomized, placebo-controlled pilot study

Background

Many patients with cystic fibrosis develop persistent airway infection/colonization with Aspergillus fumigatus, however the impact of A. fumigatus on clinical outcomes remains unclear. The objective of this study was to determine whether treatment directed against Aspergillus fumigatus improves pulmonary function and clinical outcomes in patients with cystic fibrosis (CF).

Methods

We performed a double-blind randomized placebo-controlled pilot clinical trial involving 35 patients with CF whose sputum cultures were chronically positive for A. fumigatus. Participants were centrally randomized to receive either oral itraconazole 5 mg/kg/d (N = 18) or placebo (N = 17) for 24 weeks. The primary outcome was the proportion of patients who experienced a respiratory exacerbation requiring intravenous antibiotics over the 24 week treatment period. Secondary outcomes included changes in FEV₁ and quality of life.

Results

Over the 24 week treatment period, 4 of 18 (22%) patients randomized to itraconazole experienced a respiratory exacerbation requiring intravenous antibiotics, compared to 5 of 16 (31%) placebo treated patients, P = 0.70. FEV₁ declined by 4.62% over 24 weeks in the patients randomized to itraconazole, compared to a 0.32% improvement in the placebo group (between group difference = −4.94%, 95% CI: −15.33 to 5.45, P = 0.34). Quality of life did not differ between the 2 treatment groups throughout the study. Therapeutic itraconazole blood levels were not achieved in 43% of patients randomized to itraconazole.

Conclusion

We did not identify clinical benefit from itraconazole treatment for CF patients whose sputum was chronically colonized with A. fumigatus. Limitations of this pilot study were its small sample size, and failure to achieve therapeutic levels of itraconazole in many patients.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00528190","term_id":"NCT00528190"}}NCT00528190     

Chemical analysis of cellular and extracellular carbohydrates of a biofilm-forming strain Pseudomonas aeruginosa PA14

Background

Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A–L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the “scaffolding” polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A–L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated.

Principal Findings

In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1–3)-α-D-QuiNAc-(1–3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic β-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ∼20% of dry weight) of LPS-like material.

Conclusions

We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of P. aeruginosa.     

Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

Background

To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA).

Methodology

Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated.

Results

The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations - DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold.

Conclusions

Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections.     

Whole blood-derived microRNA signatures in mice exposed to lipopolysaccharides

Background

Lipopolysaccharide (LPS) is recognized as the most potent microbial mediator presaging the threat of invasion of Gram-negative bacteria that implicated in the pathogenesis of sepsis and septic shock. This study was designed to examine the microRNA (miRNA) expression in whole blood from mice injected with intraperitoneal LPS.

Methods

C57BL/6 mice received intraperitoneal injections of varying concentrations (range, 10–1000 μg) of LPS from different bacteria, including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enterica, and Serratia marcescens and were killed 2, 6, 24, and 72 h after LPS injection. Whole blood samples were obtained and tissues, including lung, brain, liver, and spleen, were harvested for miRNA expression analysis using an miRNA array (Phalanx miRNA OneArray® 1.0). Upregulated expression of miRNA targets in the whole blood of C57BL/6 and Tlr4^−/− mice injected with LPS was quantified using real-time RT-PCR and compared with that in the whole blood of C57BL/6 mice injected with lipoteichoic acid (LTA) from Staphylococcus aureus.

Results

Following LPS injection, a significant increase of 15 miRNAs was observed in the whole blood. Among them, only 3 miRNAs showed up-regulated expression in the lung, but no miRNAs showed a high expression level in the other examined tissues. Upregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection on real-time RT-PCR was dose- and time-dependent. miRNA induction occurred after 2 h and persisted for at least 6 h. Exposure to LPS from different bacteria did not induce significantly different expression of these miRNA targets. Additionally, significantly lower expression levels of let-7d, miR-25, miR-92a, miR-103, and miR-107 were observed in whole blood of Tlr4^−/− mice. In contrast, LTA exposure induced moderate expression of miR-451 but not of the other 7 miRNA targets.

Conclusions

We identified a specific whole blood–derived miRNA signature in mice exposed to LPS, but not to LTA, from different gram-negative bacteria. These whole blood-derived miRNAs are promising as biomarkers for LPS exposure.     

Cytosolic phospholipase A2α mediates Pseudomonas aeruginosa LPS-induced airway constriction of CFTR -/- mice

Background

Lungs of cystic fibrosis (CF) patients are chronically infected with Pseudomonas aeruginosa. Increased airway constriction has been reported in CF patients but underplaying mechanisms have not been elucidated. Aim: to examine the effect of P. aeruginosa LPS on airway constriction in CF mice and the implication in this process of cytosolic phospholipase A2α (cPLA2α), an enzyme involved in arachidonic acid (AA) release.

Methods

Mice were instilled intra-nasally with LPS. Airway constriction was assessed using barometric plethysmograph. MIP-2, prostaglandin E2 (PGE2), leukotrienes and AA concentrations were measured in BALF using standard kits and gas chromatography.

Results

LPS induced enhanced airway constriction and AA release in BALF of CF compared to littermate mice. This was accompanied by increased levels of PGE2, but not those of leukotrienes. However, airway neutrophil influx and MIP-2 production remained similar in both mouse strains. The cPLA2α inhibitor arachidonyl trifluoro-methyl-ketone (ATK), but not aspirin which inhibit PGE2 synthesis, reduced LPS-induced airway constriction. LPS induced lower airway constriction and PGE2 production in cPLA2α -/- mice compared to corresponding littermates. Neither aspirin nor ATK interfered with LPS-induced airway neutrophil influx or MIP-2 production.

Conclusions

CF mice develop enhanced airway constriction through a cPLA2α-dependent mechanism. Airway inflammation is dissociated from airway constriction in this model. cPLA2α may represent a suitable target for therapeutic intervention in CF. Attenuation of airway constriction by cPLA2α inhibitors may help to ameliorate the clinical status of CF patients.     

Reduced Mucosal Associated Invariant T-Cells Are Associated with Increased Disease Severity and Pseudomonas aeruginosa Infection in Cystic Fibrosis

Background

Primary defects in host immune responses have been hypothesised to contribute towards an inability of subjects with cystic fibrosis (CF) to effectively clear pulmonary infections. Innate T-lymphocytes provide rapid pathogen-specific responses prior to the development of classical MHC class I and II restricted T-cell responses and are essential to the initial control of pulmonary infection. We aimed to examine the relationship between peripheral blood lymphocyte phenotype and clinical outcomes in adults with CF.

Methods

We studied 41 subjects with CF and 22, age matched, non-smoking healthy control subjects. Lymphocytes were extracted from peripheral blood samples and phenotyped by flow-cytometry. Lymphocyte phenotype was correlated with sputum microbiology and clinical parameters.

Results

In comparison to healthy control subjects, mucosal associated invariant T (MAIT)-lymphocytes were significantly reduced in the peripheral blood of subjects with CF (1.1% versus 2.0% of T-lymphocytes, P = 0.002). MAIT cell concentration was lowest in CF subjects infected with P. aeruginosa and in subjects receiving treatment for a pulmonary exacerbation. Furthermore a reduced MAIT cell concentration correlated with severity of lung disease.

Conclusion

Reduced numbers of MAIT cells in subjects with CF were associated with P. aeruginosa pulmonary infection, pulmonary exacerbations and more severe lung disease. These findings provide the impetus for future studies examining the utility of MAIT cells in immunotherapies and vaccine development. Longitudinal studies of MAIT cells as biomarkers of CF pulmonary infection are awaited.     

Enhanced susceptibility to lipopolysaccharide-induced arthritis and endotoxin shock in interleukin-32 alpha transgenic mice through induction of tumor necrosis factor alpha

Introduction

The present study assessed the potential functions of interleukin (IL)-32α on inflammatory arthritis and endotoxin shock models using IL-32α transgenic (Tg) mice. The potential signaling pathway for the IL-32-tumor necrosis factor (TNF)α axis was analyzed in vitro.

Methods

IL-32α Tg mice were generated under control of a ubiquitous promoter. Two disease models were used to examine in vivo effects of overexpressed IL-32α: Toll-like receptor (TLR) ligand-induced arthritis developed using a single injection of lipopolysaccharide (LPS) or zymosan into the knee joints; and endotoxin shock induced with intraperitoneal injection of LPS and D-galactosamine. TNFα antagonist etanercept was administered simultaneously with LPS in some mice. Using RAW264.7 cells, in vitro effects of exogenous IL-32α on TNFα, IL-6 or macrophage inflammatory protein 2 (MIP-2) production were assessed with or without inhibitors for nuclear factor kappa B (NFκB) or mitogen-activated protein kinase (MAPK).

Results

Single injection of LPS, but not zymosan, resulted in development of severe synovitis with substantial articular cartilage degradation in knees of the Tg mice. The expression of TNFα mRNA in inflamed synovia was highly upregulated in the LPS-injected Tg mice. Moreover, the Tg mice were more susceptive to endotoxin-induced lethality than the wild-type control mice 48 hours after LPS challenge; but blockade of TNFα by etanercept protected from endotoxin lethality. In cultured bone marrow cells derived from the Tg mice, overexpressed IL-32α accelerated production of TNFα upon stimulation with LPS. Of note, exogenously added IL-32α alone stimulated RAW264.7 cells to express TNFα, IL-6, and MIP-2 mRNAs. Particularly, IL-32α -induced TNFα, but not IL-6 or MIP-2, was inhibited by dehydroxymethylepoxyquinomicin (DHMEQ) and U0126, which are specific inhibitors of nuclear factor kappa B (NFκB) and extracellular signal regulated kinase1/2 (ERK1/2), respectively.

Conclusions

These results show that IL-32α contributed to the development of inflammatory arthritis and endotoxin lethality. Stimulation of TLR signaling with LPS appeared indispensable for activating the IL-32α-TNFα axis in vivo. However, IL-32α alone induced TNFα production in RAW264.7 cells through phosphorylation of inhibitor kappa B (IκB) and ERK1/2 MAPK. Further studies on the potential involvement of IL-32α-TNFα axis will be beneficial in better understanding the pathology of autoimmune-related arthritis and infectious immunity.     

In vivo fluorescence imaging of bacteriogenic cyanide in the lungs of live mice infected with cystic fibrosis pathogens

Background

Pseudomonas aeruginosa (PA) and Burkholderia cepacia complex (Bcc), commonly found in the lungs of cystic fibrosis (CF) patients, often produce cyanide (CN), which inhibits cellular respiration. CN in sputa is a potential biomarker for lung infection by CF pathogens. However, its actual concentration in the infected lungs is unknown.

Methods and Findings

This work reports observation of CN in the lungs of mice infected with cyanogenic PA or Bcc strains using a CN fluorescent chemosensor (4′,5′-fluorescein dicarboxaldehyde) with a whole animal imaging system. When the CN chemosensor was injected into the lungs of mice intratracheally infected with either PA or B. cepacia strains embedded in agar beads, CN was detected in the millimolar range (1.8 to 4 mM) in the infected lungs. CN concentration in PA-infected lungs rapidly increased within 24 hours but gradually decreased over the following days, while CN concentration in B. cepacia-infected lungs slowly increased, reaching a maximum at 5 days. CN concentrations correlated with the bacterial loads in the lungs. In vivo efficacy of antimicrobial treatments was tested in live mice by monitoring bacteriogenic CN in the lungs.

Conclusions

The in vivo imaging method was also found suitable for minimally invasive testing the efficacy of antibiotic compounds as well as for aiding the understanding of bacterial cyanogenesis in CF lungs.     

Is the Improvement of CF Patients,Hospitalized for Pulmonary Exacerbation,Correlated to a Decrease in Bacterial Load?

Background

Cystic Fibrosis (CF) patients are vulnerable to airway colonization with Pseudomonas aeruginosa. In case eradication fails after antibiotic treatment, patients become chronically colonized with P. aeruginosa, with recurrent pulmonary exacerbation, for which patients typically are hospitalized for 2 weeks and receive intravenous antibiotic treatment. Normally, improvement of the patients'' health is established.

Aim

Determination of the correspondence between patient improvement and changes of the P. aeruginosa and total bacterial load in the sputum.

Methods

Eighteen CF patients with exacerbation were included for a total of 27 hospitalization episodes. At day 1, 8 and 15, inflammation and lung function parameters were determined, together with the P. aeruginosa load in the sputum using culture, quantitative PCR (qPCR) and propidium monoazide qPCR.

Results

Patients improved during hospitalization (decrease in levels of C-reactive protein, white blood cell counts and erythrocyte sedimentation rate, increase of FEV₁), reaching normal values already after one week. Also the P. aeruginosa load and the total bacterial load decreased during the first week of antibiotic treatment (p<0.05), except for patients with a low lung function (FEV₁≤39.4%), for whom no significant decrease of P. aeruginosa was established. Comparison of culture-based and propidium monoazide qPCR-based quantification of P. aeruginosa showed that at the end of the treatment on average 62% of the P. aeruginosa cells are not cultivable, indicating that many cells are alive but dormant, or dead but still structurally intact.

Conclusion

Improvement of the clinical status is accompanied with a decrease of the P. aeruginosa load, whereby both occur mainly during the first week of antibiotic treatment. However, for patients with a low lung function, no decrease of the P. aeruginosa load is observed. Comparison of detection techniques shows that a large amount of noncultivable or dead bacteria are present in the samples.     

Molecular Epidemiology of Chronic Pseudomonas aeruginosa Airway Infections in Cystic Fibrosis

Background/Methods

The molecular epidemiology of the chronic airway infections with Pseudomonas aeruginosa in individuals with cystic fibrosis (CF) was investigated by cross-sectional analysis of bacterial isolates from 51 CF centers and by longitudinal analysis of serial isolates which had been collected at the CF centers Hanover and Copenhagen since the onset of airway colonization over 30 years.

Results

Genotyping revealed that the P. aeruginosa population in CF is dominated by a few ubiquitous clones. The five most common clones retrieved from the CF host also belonged to the twenty most frequent clones in the environment and in other human disease habitats. Turnover of clones in CF airways was rare. At the Hanover clinic more than half of the patient cohort was still harbouring the initially acquired clone after twenty years of airway colonization. At the Copenhagen clinic, however, two rare clones replaced the initially acquired individual clones in all but one patient.

Conclusion

The divergent epidemiology at the two sites is explained by their differential management of hygiene and antipseudomonal chemotherapy. Hygienic measures to prohibit patient-to-patient transmission and the modalities of antipseudomonal chemotherapy modify the epidemiology of the chronic P. aeruginosa infections in CF.     

The B Lymphocyte Differentiation Factor (BAFF) Is Expressed in the Airways of Children with CF and in Lungs of Mice Infected with Pseudomonas aeruginosa

Background

Chronic lung infection with Pseudomonas aeruginosa remains a major cause of mortality and morbidity among individuals with CF. Expression of mediators promoting recruitment and differentiation of B cells, or supporting antibody production is poorly understood yet could be key to controlling infection.

Methods

BAFF was measured in BAL from children with CF, both with and without P. aeruginosa, and controls. Mice were intra-nasally infected with P. aeruginosa strain LESB65 for up to 7 days. Cellular infiltration and expression of B cell chemoattractants and B cell differentiation factor, BAFF were measured in lung tissue.

Results

BAFF expression was elevated in both P. aeruginosa negative and positive CF patients and in P. aeruginosa infected mice post infection. Expression of the B cell chemoattractants CXCL13, CCL19 and CCL21 increased progressively post infection.

Conclusions

In a mouse model, infection with P. aeruginosa was associated with elevated expression of BAFF and other B cell chemoattractants suggesting a role for airway B cell recruitment and differentiation in the local adaptive immune response to P. aeruginosa. The paediatric CF airway, irrespective of pseudomonal infection, was found to be associated with an elevated level of BAFF implying that BAFF expression is not specific to pseudomonas infection and may be a feature of the CF airway. Despite the observed presence of a potent B cell activator, chronic colonisation is common suggesting that this response is ineffective.