Marianne S. Muhlebach

Detection of Anaerobic Bacteria in High Numbers in Sputum from Patients with Cystic Fibrosis

RATIONALE Pulmonary infection in cystic fibrosis (CF) is polymicrobial and it is possible that anaerobic bacteria, not detected by routine aerobic culture methods, reside within infected anaerobic airway mucus. OBJECTIVES To determine whether anaerobic bacteria are present in the sputum of patients with CF. METHODS Sputum samples were collected from clinically stable adults with CF and bronchoalveolar lavage fluid (BALF) samples from children with CF. Induced sputum samples were collected from healthy volunteers who did not have CF. All samples were processed using anaerobic bacteriologic techniques and bacteria within the samples were quantified and identified. MEASUREMENTS AND MAIN RESULTS Anaerobic species primarily within the genera Prevotella, Veillonella, Propionibacterium, and Actinomyces were isolated in high numbers from 42 of 66 (64%) sputum samples from adult patients with CF. Colonization with Pseudomonas aeruginosa significantly increased the likelihood that anaerobic bacteria would be present in the sputum. Similar anaerobic species were identified in BALF from pediatric patients with CF. Although anaerobes were detected in induced sputum samples from 16 of 20 volunteers, they were present in much lower numbers and were generally different species compared with those detected in CF sputum. Species-dependent differences in the susceptibility of the anaerobes to antibiotics with known activity against anaerobes were apparent with all isolates susceptible to meropenem. CONCLUSIONS A range of anaerobic species are present in large numbers in the lungs of patients with CF. If these anaerobic bacteria are contributing significantly to infection and inflammation in the CF lung, informed alterations to antibiotic treatment to target anaerobes, in addition to the primary infecting pathogens, may improve management.

Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in patients with cystic fibrosis

Background Anaerobic bacteria are increasingly regarded as important in cystic fibrosis (CF) pulmonary infection. The aim of this study was to determine the effect of antibiotic treatment on aerobic and anaerobic microbial community diversity and abundance during exacerbations in patients with CF. Methods Sputum was collected at the start and completion of antibiotic treatment of exacerbations and when clinically stable. Bacteria were quantified and identified following culture, and community composition was also examined using culture-independent methods. Results Pseudomonas aeruginosa or Burkholderia cepacia complex were detected by culture in 24/26 samples at the start of treatment, 22/26 samples at completion of treatment and 11/13 stable samples. Anaerobic bacteria were detected in all start of treatment and stable samples and in 23/26 completion of treatment samples. Molecular analysis showed greater bacterial diversity within sputum samples than was detected by culture; there was reasonably good agreement between the methods for the presence or absence of aerobic bacteria such as P aeruginosa (κ=0.74) and B cepacia complex (κ=0.92), but agreement was poorer for anaerobes. Both methods showed that the composition of the bacterial community varied between patients but remained relatively stable in most individuals despite treatment. Bacterial abundance decreased transiently following treatment, with this effect more evident for aerobes (median decrease in total viable count 2.3×107 cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3×106 cfu/g, p=0.046). Conclusion Antibiotic treatment targeted against aerobes had a minimal effect on abundance of anaerobes and community composition, with both culture and molecular detection methods required for comprehensive characterisation of the microbial community in the CF lung. Further studies are required to determine the clinical significance of and optimal treatment for these newly identified bacteria.

Review: Staphylococcus aureus and MRSA in cystic fibrosis.

BACKGROUND Staphylococcus aureus (S. aureus) is one of the earliest bacteria detected in infants and children with cystic fibrosis (CF). The rise of methicillin resistant S. aureus (MRSA) in the last 10 years has caused a lot of attention to this organism. RESULTS The aim of this review is to provide a general overview of methicillin sensitive S. aureus (MSSA) and MRSA, discuss special aspects of S. aureus in cystic fibrosis, and to review treatment concepts. Microbiology of the organism will be reviewed along with data regarding the epidemiology of both MSSA and MRSA. Antibiotic treatments both in regards to acute management and eradication of MSSA and MRSA will be reviewed. Prophylaxis of MSSA in CF remains controversial. Treatment with anti-staphylococcal agents reduces the infection rate with MSSA but may lead to a higher rate of infection with P. aeruginosa. In regards to MRSA, there is a paucity of clinical data regarding approaches to eradication. CONCLUSIONS To advance the care of CF patients, controlled clinical trials are urgently needed to find the optimal approach to treating CF patients who are infected with either MSSA or MRSA.

Lipid-laden macrophage index and inflammation in bronchoalveolar lavage fluids in children

The presence of lipids in alveolar macrophages has been used clinically as an indicator of aspiration, a process associated with increased lung inflammation in animal models. The hypothesis is that the quantity of lipids in alveolar macrophages, measured as lipid-laden index (LLI), would correlate with lung inflammation in paediatric patients. Children with chronic respiratory symptoms (21 cystic fibrosis (CF), 24 non-CF) underwent flexible bronchoscopy with bronchoalveolar lavage (BAL) and 24-h intraoesophageal pH monitoring for clinical indications. Total cell counts, number and per cent of neutrophils and macrophages, and LLI were determined in the bronchoalveolar lavage fluids (BALF) from all children. BALF were also obtained from eight healthy, young nonsmoking adults for comparison. LLI in non-CF children were 6.9 +/- 3.5 (mean +/- SEM) which were higher than LLI in healthy adults (1.0 +/- 0.4), (p=0.045). Children with CF had very high LLIs (19.2 +/- 4.5) compared with both healthy adults (p=0.014) and children without CF (p=0.045). LLI did not correlate with airway inflammation in any group. LLI in children with abnormal pH probes had a tendency to be higher than in children with normal pH probes, but the difference was not significant (p=0.098). It is concluded that the lipid-laden index was significantly elevated in children with chronic respiratory symptoms compared with healthy adults, and in children with cystic fibrosis compared with those who have other chronic respiratory conditions. However, the lipid-laden index did not correlate with the quantity of bronchoalveolar lavage fluid inflammation. The lipid-laden index in children may, in part, reflect processes other than aspiration, such as airways obstruction.

Different expression ratio of S100A8/A9 and S100A12 in acute and chronic lung diseases

Calgranulins are a family of powerful chemoattractants, which have been implicated as biomarkers in inflammatory diseases. To determine how different respiratory diseases affect the expression of calgranulins, we measured the expression of S100A8/A9 and S100A12 in bronchoalveolar lavage fluid (BALF) of acute respiratory distress syndrome (ARDS) patients and healthy volunteers by ELISA. Analysis of calgranulin expression revealed a high level of S100A12 in the lavages of patients suffering from ARDS compared to controls (p<0.001). Based on the hypothesis that the increased expression of S100A12 relative to the S100A8/A9 heterodimer was a characteristic of respiratory diseases with neutrophilic inflammation, we measured calgranulin expression in BALF of cystic fibrosis (CF) patients. Despite similarly elevated levels of S100A8/A9, S100A12 was significantly higher in ARDS compared to CF BALF (p<0.001). The differential expression of calgranulins was unique for inflammatory markers, as an array of cytokines did not differ between CF and ARDS patients. Since ARDS is an acute event and CF a chronic inflammation with acute exacerbations, we compared calgranulin expression in sputum obtained from CF and patients with chronic obstructive lung disease (COPD). Levels of S100A12 and S100A8/9 were elevated in CF sputum compared to COPD sputum, but the ratio of S100A12 to S100A8/A9 was similar in COPD and CF and reflected more closely than seen in healthy controls. The results indicate that the regulation of human calgranulin expression and the ratio of S100A8/A9 to S100A12 may provide important insights in the mechanism of respiratory inflammation.

Respiratory manifestations in mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are a group of inherited, metabolic diseases caused by deficiency of lysosomal enzymes that degrade glycosaminoglycans (GAG). Loss of enzyme activity results in cellular accumulation of GAG fragments leading to the progressive multi-system manifestations. MPS are classified into seven clinical types based on eleven known lysosomal enzyme deficiencies of GAG metabolism. Respiratory involvement is seen in most MPS types with recurrent respiratory infections, upper and lower airway obstruction, tracheomalacia, restrictive lung disease, and sleep disturbances. Patients with airway obstruction are at high risk for anaesthetic complications. In this review, we present the respiratory manifestations in various MPS types and stages, evaluation of respiratory involvement, and treatment options for the progressive respiratory failure that occurs in MPS patients.

Plasma TGF-β1 in pediatric cystic fibrosis: Potential biomarker of lung disease and response to therapy

Transforming growth factor beta‐1 (TGF‐β1) is an important genetic modifier of lung disease severity in cystic fibrosis (CF), yet the mechanism behind this disease association remains unknown. Initial steps in the investigation of the relationship between TGF‐β1 and CF lung disease include determining the most appropriate available biospecimen for TGF‐β1 protein measurement.

The Role of Short-Chain Fatty Acids, Produced by Anaerobic Bacteria, in the Cystic Fibrosis Airway

RATIONALE Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune and inflammatory processes. OBJECTIVES To investigate the capacity of anaerobes to contribute to cystic fibrosis (CF) airway pathogenesis via SCFAs. METHODS Samples of 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFA levels in anaerobic supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of two SCFA receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings and 16HBE14o(-) and CFBE41o(-) cells were evaluated using reverse transcription polymerase chain reaction, Western blot analysis, laser scanning cytometry, and confocal microscopy. SCFA-induced IL-8 secretion was monitored by ELISA. MEASUREMENTS AND MAIN RESULTS Fifty-seven (52.3%) of 109 PWCF were anaerobe positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF younger (n = 24) and older (n = 85) than 6 years of age. All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic, and butyric acids. SCFA levels were higher in BAL samples of adults than in those of children. GPR41 levels were elevated in CFBE41o(-) versus 16HBE14o(-) cells; CF versus non-CF bronchial brushings; and 16HBE14o(-) cells after treatment with cystic fibrosis transmembrane conductance regulator inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells, with a higher production of IL-8 in CFBE41o(-) than in 16HBE14o(-) cells. CONCLUSIONS This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via up-regulated GPR41.

Iron accumulates in the lavage and explanted lungs of cystic fibrosis patients

Oxidative stress participates in the pathophysiology of cystic fibrosis (CF). An underlying disruption in iron homeostasis can frequently be demonstrated in injuries and diseases associated with an oxidative stress. We tested the hypothesis that iron accumulation and altered expression of iron-related proteins could be demonstrated in both the bronchoalveolar lavage (BAL) fluid and explanted lungs of patients with cystic fibrosis. BAL fluid collected from 10 children with CF showed elevated concentrations of protein, iron, ferritin, transferrin, heme, and hemoglobin relative to that obtained from 20 healthy volunteers. Using Perls Prussian blue staining, explanted lung from CF patients revealed increased iron in alveolar and interstitial macrophages. Similarly, there was an increased expression of ferritin, the iron importer DMT1, and the exporter ferroportin 1 in lung tissue from CF patients. We conclude that iron homeostasis is disrupted in CF patients with an accumulation of this metal and altered expression of iron-related proteins being evident in the lungs.

Pharmacokinetics of intravenous and oral linezolid in adults with cystic fibrosis.

ABSTRACT Linezolid is a treatment option for methicillin-resistant Staphylococcus aureus (MRSA) infections in cystic fibrosis (CF) patients. Little is known, however, about its pharmacokinetics in this population. Eight adults with CF were randomized to receive intravenous (i.v.) and oral linezolid at 600 mg twice daily for 9 doses in a crossover design with a 9-day washout. Plasma samples were collected after the first and ninth doses of each phase. Population pharmacokinetic analyses were performed by nonlinear mixed-effects modeling using a previously described 2-compartment model with time-dependent clearance inhibition. Monte Carlo simulation was performed to assess the activities of the linezolid dosing regimens against 42 contemporary MRSA isolates recovered from CF patients. The following pharmacokinetic parameter estimates were observed for the population: absorption rate constant, 1.91 h−1; clearance, 9.54 liters/h; volume of central compartment, 26.8 liters; volume of peripheral compartment, 17.3 liters; and intercompartmental clearance, 104 liters/h. Linezolid demonstrated nonlinear clearance after 9 doses, which was reduced by a mean of 38.9% (range, 28.8 to 59.9%). Mean bioavailability was 85% (range, 47 to 131%). At steady state, 600 mg given twice daily produced 93.0% and 87.2% probabilities of obtaining the target pharmacodynamic exposure against the MRSA isolates for the i.v. and oral formulations, respectively. Thrice-daily dosing increased the probabilities to 97.0% and 95.6%, respectively. Linezolid pharmacokinetics in these adults with CF were well described by a 2-compartment model with time-dependent clearance inhibition. Standard i.v. and oral dosing regimens should be sufficient to reliably attain pharmacodynamic targets against most MRSA isolates; however, more frequent dosing may be required for isolates with MICs of ≥2 μg/ml.