Marian Garcia-Nuñez

Microbiome Diversity in the Bronchial Tracts of Patients with Chronic Obstructive Pulmonary Disease

ABSTRACT Culture of bacteria from bronchial secretions in respiratory patients has low sensitivity and does not allow for complete assessment of microbial diversity across different bronchial compartments. In addition, a significant number of clinical studies are based on sputum samples, and it is not known to what extent they describe the real diversity of the mucosa. In order to identify previously unrecognized lower airway bacteria and to investigate the complexity and distribution of microbiota in patients with chronic obstructive pulmonary disease (COPD), we performed PCR amplification and pyrosequencing of the 16S rRNA gene in patients not showing signs or symptoms of infection. Four types of respiratory samples (sputum, bronchial aspirate, bronchoalveolar lavage, and bronchial mucosa) were taken from each individual, obtaining on average >1,000 16S rRNA sequences per sample. The total number of genera per patient was >100, showing a high diversity, with Streptococcus, Prevotella, Moraxella, Haemophilus, Acinetobacter, Fusobacterium, and Neisseria being the most commonly identified. Sputum samples showed significantly lower diversity than the other three sample types. Lower-bronchial-tree samples, i.e., bronchoalveolar lavage and bronchial mucosa, showed a very similar bacterial compositions in contrast to sputum and bronchial aspirate samples. Thus, sputum and bronchial aspirate samples are upper bronchial tree samples that are not representative of the lower bronchial mucosa flora, and bronchoalveolar lavage samples showed the results closest to those for the bronchial mucosa. Our data confirm that the bronchial tree is not sterile in COPD patients and support the existence a different microbiota in the upper and lower compartments.

Severity-Related Changes of Bronchial Microbiome in Chronic Obstructive Pulmonary Disease

ABSTRACT Bronchial colonization by potentially pathogenic microorganisms (PPMs) is often demonstrated in chronic obstructive pulmonary disease (COPD), but culture-based techniques identify only a portion of the bacteria in mucosal surfaces. The aim of the study was to determine changes in the bronchial microbiome of COPD associated with the severity of the disease. The bronchial microbiome of COPD patients was analyzed by 16S rRNA gene amplification and pyrosequencing in sputum samples obtained during stable disease. Seventeen COPD patients were studied (forced expiratory volume in the first second expressed as a percentage of the forced vital capacity [FEV1%] median, 35.0%; interquartile range [IQR], 31.5 to 52.0), providing a mean of 4,493 (standard deviation [SD], 2,598) sequences corresponding to 47 operational taxonomic units (OTUs) (SD, 17) at a 97% identity level. Patients were dichotomized according to their lung function as moderate to severe when their FEV1% values were over the median and as advanced when FEV1% values were lower. The most prevalent phyla in sputum were Proteobacteria (44%) and Firmicutes (16%), followed by Actinobacteria (13%). A greater microbial diversity was found in patients with moderate-to-severe disease, and alpha diversity showed a statistically significant decrease in patients with advanced disease when assessed by Shannon (ρ = 0.528; P = 0.029, Spearman correlation coefficient) and Chao1 (ρ = 0.53; P = 0.028, Spearman correlation coefficient) alpha-diversity indexes. The higher severity that characterizes advanced COPD is paralleled by a decrease in the diversity of the bronchial microbiome, with a loss of part of the resident flora that is replaced by a more restricted microbiota that includes PPMs.

Variability and effects of bronchial colonisation in patients with moderate COPD

Sputum and lung function were periodically assessed in stable moderate chronic obstructive pulmonary disease (COPD) outpatients to determine relationships between bronchial colonisation and inflammation. Relationships between potentially pathogenic microorganism (PPM) typology, bronchial inflammation (neutrophilia, tumour necrosis factor-α, interleukin (IL)-1β, IL-6, IL-8, IL-10 and IL-12) and post-bronchodilator decline in forced expiratory volume in 1 s (FEV1) were analysed. PPMs periodically showing the same molecular profile using pulse field gel electrophoresis were considered long-term persistent. Bronchial colonisation was observed in 56 out of 79 follow-up examinations (70.9%) and was mainly due to Haemophilus influenzae, Pseudomonas aeruginosa and enterobacteria (n = 47). These PPMs were all related to sputum neutrophilia (p≤0.05, Chi-squared test), and H. influenzae was related to higher levels of IL-1β (p = 0.005) and IL-12 (p = 0.01), with a dose–response relationship (Spearman’s correlation coefficient of 0.38 for IL-1β (p = 0.001), and of 0.32 for IL-12 (p = 0.006)). Haemophilus parainfluenzae was not associated with an identifiable inflammatory response. Long-term persistence of the same strain was observed in 12 examinations (21.4%), mainly due to P. aeruginosa or enterobacteria. A neutrophilic bronchial inflammatory response was associated with a statistically significant decline in FEV1 during follow-up (OR 2.67, 95% CI 1.07–6.62). A load-related relationship to bronchial inflammation in moderate COPD was observed for colonisation by H. influenzae, but not for colonisation by H. parainfluenzae.

Presence and chromosomal subtyping of Legionella species in potable water systems in 20 hospitals of Catalonia, Spain.

OBJECTIVE To investigate the presence and clonal distribution of Legionella species in the water supply of 20 hospitals in Catalonia, Spain. SETTING 20 hospitals in Catalonia, an area of 32,000 km2, located in northeast Spain. METHODS Environmental cultures of 186 points of potable water supply and 10 cooling towers were performed for the presence of Legionella species. Following filtration and acid treatment, the samples were seeded in selective MWY (modified Wadowsky Yee)-buffered charcoal yeast extract-alpha agar. All isolates obtained were characterized microbiologically and genotyped by SfiI pulsed-field gel electrophoresis (PFGE). RESULTS 73 of 196 water samples, representing 17 of the 20 hospitals included in the study, were positive for Legionella pneumophila (serogroups 1, 2-14, or both). The degree of contamination ranged from 200 to 74,250 colony-forming units/L. Twenty-five chromosomal DNA subtypes were detected by PFGE. A single DNA subtype was identified in 10 hospitals, 2 DNA subtypes were observed in 6 hospitals, and 1 hospital exhibited 3 different DNA subtypes. Each hospital had its own Legionella DNA subtype, which was not shared with any other hospitals. CONCLUSIONS Legionella was present in the water of most of the hospitals studied; each such hospital had a unique, dominant chromosomal DNA subtype. The verification of several genomic DNA restriction profiles in such a small geographic area demonstrates the great genetic diversity of Legionella in the aquatic environment.

Factors related to persistence of Legionella urinary antigen excretion in patients with legionnaires' disease.

Abstract. The aim of this prospective study was to compare patient characteristics, clinical data, and evolution of Legionella pneumophila pneumonia according to the duration of Legionella urinary antigen excretion. Urine samples from 61 patients with Legionella pneumonia diagnosed by detection of urinary antigen were obtained periodically until urinary antigen could no longer be detected. Cases were divided into two groups based on the duration of urinary antigen excretion: group I (46 patients, <60 days) and group II (15 patients, ≥60 days). Groups were compared for patient characteristics, clinical data, and evolution of pneumonia. Antigen excretion ≥60 days was observed significantly more frequently in immunosuppressed patients (P=0.001) in whom the time to apyrexia was >72 h (P=0.002), although only the time to apyrexia remained significant on multivariate analysis (P=0.006). In conclusion, the duration of Legionella urinary antigen excretion was <60 days in most patients but was longer in immunosuppressed patients with a longer time to defervescence of fever.

Effect of Bronchial Colonisation on Airway and Systemic Inflammation in Stable COPD

Abstract The recovery of potentially pathogenic microorganisms (PPMs) from bronchial secretions is associated with a local inflammatory response in COPD patients. The objective of this study was to determine the relationships between bronchial colonisation and both bronchial and systemic inflammation in stable COPD. In COPD patients recruited on first admission for an exacerbation, bacterial sputum cultures, interleukin (IL)-1β, IL-6 and IL-8 levels, and blood C-reactive protein (CRP) were measured in stable condition. Bronchial colonisation was found in 39 of the 133 (29%) patients and was significantly related to higher sputum IL-1β (median [percentile 25–75]; 462 [121–993] vs. 154 [41–477] pg/ml, p = 0.002), IL-6 (147 [71–424] vs. 109 [50–197] pg/ml, p = 0.047) and IL-8 values (15 [9–19] vs. 8 [3–15] (×103) pg/ml, p = 0.002). Patients with positive cultures also showed significantly elevated levels of serum CRP (6.5 [2.5–8.5] vs. 3.5 [1.7–5.4] mg/l, p = 0.016). Bronchial colonisation by Haemophilus influenzae was associated with higher levels of IL-1β and IL-8 and clinically significant worse scores on the activity and impact domains of the St. Georges Respiratory Questionnaire. In conclusion, bronchial colonisation is associated with bronchial inflammation and high blood CRP levels in stable COPD patients, being Haemophilus influenzae related to a more severe inflammatory response and impairment in health-related quality of life.

Comparative study of community-acquired pneumonia caused by Streptococcus pneumoniae, Legionella pneumophila or Chlamydia pneumoniae.

The objective of this study was to compare epidemiological data and clinical presentation of community-acquired pneumonia (CAP) caused by Streptococcus pneumoniae, Legionella pneumophila or Chlamydia pneumoniae. From May 1994 to February 1996, 157 patients with S. pneumoniae (n=68), L. pneumophila (n=48) and C. pneumoniae (n=41) pneumonia with definitive diagnosis, were prospectively studied. The following comparisons showed differences at a level of at least p<0.05. Patients with S. pneumoniae pneumonia had more frequently underlying diseases (HIV infection and neoplasm) and those with C. pneumoniae pneumonia were older and had a higher frequency of chronic obstructive pulmonary disease (COPD), while L. pneumophila pneumonia prevailed in patients without comorbidity, but with alcohol intake. Presentation with cough and expectoration were significantly more frequent in patients with S. pneumoniae or C. pneumoniae pneumonia, while headache, diarrhoea and no response to betalactam antibiotics prevailed in L. pneumophila pneumonia. However, duration of symptoms ≥7 d was more frequent in C. pneumoniae pneumonia. Patients with CAP caused by L. pneumophila presented hyponatraemia and an increase in CK more frequently, while AST elevation prevailed in L. pneumophila and C. pneumoniae pneumonia. In conclusion, some risk factors and clinical characteristics of patients with CAP may help to broaden empirical therapy against atypical pathogens until rapid diagnostic tests are available.

Streptococcus pneumoniae and Legionella pneumophila pneumonia in HIV-infected patients

We compared the epidemiological data, clinical features and mortality of community-acquired pneumonia (CAP) by Streptococcus pneumoniae and Legionella in HIV-infected patients and determined discriminative features. An observational, comparative study was performed (January 1994 to December 2004) in 15 HIV patients with CAP by Legionella and 46 by S. pneumoniae. No significant differences were observed in delay until initiation of appropriate antibiotic therapy. Smoking, cancer and chemotherapy were more frequent in patients with Legionella pneumonia (p=0.03, p=0.00009 and p=0.01). Patients with Legionella pneumonia had a higher mean CD4 count (p=0.04), undetectable viral load (p=0.01) and received highly active antiretroviral therapy more frequently (p=0.004). AIDS was more frequent in patients with S. pneumoniae pneumonia (p=0.03). Legionella pneumonia was more severe (p=0.007). Extrarespiratory symptoms, hyponatraemia and increased creatine phosphokinase were more frequent in Legionella pneumonia (p=0.02, p=0.002 and p=0.006). Respiratory failure, need for ventilation and bilateral chest X-ray involvement were of note in the Legionella group (p=0.003, p=0.002 and p=0.002). Mortality tended to be higher in the Legionella group (6.7 vs 2.2%). In conclusion, CAP by Legionella has a higher morbimortality than CAP by S. pneumoniae in HIV-infected patients. Detailed analysis of CAP presentation features allows suspicion of Legionnaires’ disease in this subset.

Legionella pneumophila in cooling towers: fluctuations in counts, determination of genetic variability by pulsed-field gel electrophoresis (PFGE), and persistence of PFGE patterns.

ABSTRACT The concentrations of Legionella pneumophila in cooling towers may vary considerably over short periods of time, producing significant fluctuations throughout the year. Despite genetic variability, in small geographical areas the same indistinguishable pulsed-field gel electrophoresis patterns may be shared among different cooling towers and persist over time.

Functional Metagenomics of the Bronchial Microbiome in COPD

The course of chronic obstructive pulmonary disease (COPD) is frequently aggravated by exacerbations, and changes in the composition and activity of the microbiome may be implicated in their appearance. The aim of this study was to analyse the composition and the gene content of the microbial community in bronchial secretions of COPD patients in both stability and exacerbation. Taxonomic data were obtained by 16S rRNA gene amplification and pyrosequencing, and metabolic information through shotgun metagenomics, using the Metagenomics RAST server (MG-RAST), and the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) programme, which predict metagenomes from 16S data. Eight severe COPD patients provided good quality sputum samples, and no significant differences in the relative abundance of any phyla and genera were found between stability and exacerbation. Bacterial biodiversity (Chao1 and Shannon indexes) did not show statistical differences and beta-diversity analysis (Bray-Curtis dissimilarity index) showed a similar microbial composition in the two clinical situations. Four functional categories showed statistically significant differences with MG-RAST at KEGG level 2: in exacerbation, Cell growth and Death and Transport and Catabolism decreased in abundance [1.6 (0.2–2.3) vs 3.6 (3.3–6.9), p = 0.012; and 1.8 (0–3.3) vs 3.6 (1.8–5.1), p = 0.025 respectively], while Cancer and Carbohydrate Metabolism increased [0.8 (0–1.5) vs 0 (0–0.5), p = 0.043; and 7 (6.4–9) vs 5.9 (6.3–6.1), p = 0.012 respectively]. In conclusion, the bronchial microbiome as a whole is not significantly modified when exacerbation symptoms appear in severe COPD patients, but its functional metabolic capabilities show significant changes in several pathways.