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 2.3×10(7) cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3×10(6) 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.

Antimicrobial susceptibility and molecular typing of MRSA in cystic fibrosis.

OBJECTIVES: The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in cystic fibrosis (CF) patients in the United States is approximately 25%. Little is known about the relative proportion of hospital- versus community-associated strains or the antimicrobial susceptibility of MRSA in different CF centers. We hypothesized that the majority of MRSA isolates obtained from children with CF are those endemic in the hospital and that those associated with community acquisition (SCCmec IV) would be more resistant than typically seen in non-CF MRSA isolates. METHODS: We studied MRSA strains from seven pediatric CF centers to determine the clonal distribution based on DNA sequencing of the staphylococcal protein A gene (spa typing), the type of staphylococcal chromosomal cassette mec (SCCmec), and the proportion of strains with Panton-Valentine leukocidin (PVL). Antimicrobial susceptibility to systemic and topical antibiotics was compared between different MRSA types. RESULTS: We analyzed 277 MRSA isolates from unique patients (mean age 11.15 ± 4.77 years, 55% male). Seventy % of isolates were SCCmec II PVL negative and the remainder SCCmec IV. Overall 17% MRSA strains were PVL positive (all SCCmec IV). Spa typing of 118 isolates showed most of the SCCmec II strains being t002, while SCCmec IV PVL positive isolates were t008, and SCCmec IV PVL negative isolates represented a variety of spa-types. The proportions of SCCmec II strains and spa-types were similar among centers. Overall rates of resistance to trimethoprim-sulfamethoxazole (4%), tetracycline (7%), tigecycline (0.4%), linezolid (0.4%) as well as fosfomycin (0.4%), fusidic acid (3%), and mupirocin (1%) were low. No strains were resistant to vancomycin. SCCmec II strains had higher rates of resistance to ciprofloxacin and clindamycin (P < 0.001) than SCCmec IV strains. CONCLUSIONS: In this U.S. study, most MRSA isolates in the pediatric CF population were SCCmec II PVL negative. Rates of resistance were low, including to older and orally available antibiotics such as trimethoprim-sulfamethoxazole.

Association between mannan-binding lectin and impaired lung function in cystic fibrosis may be age-dependent.

An association between mannan-binding lectin (MBL) status and severity of lung function impairment in cystic fibrosis (CF) has been found in several studies, but not in others. To explore the possible basis for discrepancies in the literature, we related both MBL and L-ficolin concentrations to lung function and examined the results in relation to the age of the patients. For patients under 15 years of age, those with MBL < 200 ng/ml had better lung function than those with MBL > 200 ng/ml [median forced expiratory volume in 1 s (FEV(1)), 99% versus 83%; P = 0.05]. For patients over 15 years of age, those with MBL < 200 ng/ml had poorer lung function than those with MBL > 200 ng/ml (median FEV(1), 44% versus 55%; P = 0.1). Also, for the over 15-year-olds, the proportion of patients with FEV(1) values below the median was greater in the MBL-insufficient subgroup (P < 0.04). In other words, relative deficiency of MBL appears to accelerate the age-related decline in lung function in CF patients. No corresponding relationships could be found between L-ficolin concentration and lung function. These findings and interpretation lend support to the potential value of MBL replacement therapy in a small minority of cystic fibrosis patients.

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.

Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients.

Recent studies suggest that inflammation plays a role in the pathogenesis of lung disease in cystic fibrosis (CF). The goal of the present study was to quantitatively compare bronchoalveolar lavage fluid (BALF) inflammation and its relation to bacterial infection, between children with CF and children with other chronic respiratory problems. Differential cell counts, immunoreactive interleukin 8 (IL-8), and quantitative bacterial cultures were done in BALF from 54 CF (median age 1.8 yr) and 55 control patients (median age 1.0 yr) who underwent bronchoscopy for clinical indications. Among infected CF patients, those with Pseudomonas aeruginosa did not have more inflammation than those without P. aeruginosa. The ratio of neutrophils or of IL-8 to bacteria in BALF was significantly greater for CF patients compared with control subjects, regardless of pathogen. Calculation of linear regression for either neutrophils or IL-8, as a function of bacterial quantity, yielded positive slopes for both CF and control patients, but with significant elevations for CF. We conclude that the inflammatory response to bacterial infection is increased or prolonged in CF compared with control patients, and that this increase is not necessarily due to pathogens specific for CF (e.g., P. aeruginosa). These data may provide further rationale for anti-inflammatory therapy early in CF.