RESUMO
INTRODUCTION: Vitamin-D deficiency has been linked to an increased risk of respiratory infections. The objective of this study was to determine the frequency and clinical importance of vitamin-D deficiency in patients with bronchiectasis. METHODS: 25-hydroxyvitamin-D was measured by immunoassay in 402 stable patients with bronchiectasis. Patients were classified as vitamin-D deficient (serum 25-hydroxyvitamin-D <25 nmol/l), insufficient (25 nmol/l-74 nmol/l) or sufficient (≥ 75 nmol/l). Disease severity was assessed, including exacerbation frequency, measurement of airway inflammatory markers, sputum bacteriology and lung function over 3 years follow-up. RESULTS: 50% of bronchiectasis patients were vitamin-D deficient, 43% insufficient and only 7% sufficient. This compared to only 12% of age and sex matched controls with vitamin-D deficiency (p<0.0001). Vitamin-D deficient patients were more frequently chronically colonised with bacteria (p<0.0001), 21.4% of vitamin-D deficient subjects were colonised with Pseudomonas aeruginosa compared to 10.4% of insufficient patients and 3.6% of sufficient patients, p=0.003. Vitamin-D deficient patients had lower FEV(1)% predicted (p=0.002), and more frequent pulmonary exacerbations (p=0.04). Vitamin-D deficient patients had higher sputum levels of inflammatory markers and demonstrated a more rapid decline in lung function over 3 years follow-up. Defects in neutrophil function and assessment of airway LL-37 levels did not provide a mechanistic explanation for these findings. Vitamin-D deficient patients had, however, higher levels of Vitamin-D Binding Protein in sputum sol. CONCLUSIONS: Vitamin-D deficiency is common in bronchiectasis and correlates with markers of disease severity. The mechanism of this association is unclear.
Assuntos
Bronquiectasia/epidemiologia , Bronquiectasia/microbiologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Deficiência de Vitamina D/epidemiologia , Idoso , Bronquiectasia/fisiopatologia , Estudos de Casos e Controles , Doença Crônica , Estudos de Coortes , Contagem de Colônia Microbiana , Comorbidade , Feminino , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Prognóstico , Valores de Referência , Testes de Função Respiratória , Infecções Respiratórias/fisiopatologia , Medição de Risco , Índice de Gravidade de Doença , Escarro/microbiologia , Reino Unido/epidemiologia , Vitamina D/análogos & derivados , Vitamina D/sangue , Vitamina D/uso terapêutico , Deficiência de Vitamina D/diagnóstico , Deficiência de Vitamina D/tratamento farmacológicoRESUMO
RATIONALE: The vicious cycle hypothesis of bronchiectasis argues that bacterial colonization leads to airway inflammation and progressive lung damage. The logical extension of this hypothesis is that acute or chronic antibiotic therapy should improve airway inflammation and clinical outcome. There are little data to support this hypothesis in patients with non-cystic fibrosis (CF) bronchiectasis. OBJECTIVES: To determine whether acute or chronic antibiotic therapy improves airway inflammation and clinical outcome in non-CF bronchiectasis. METHODS: The relationship between bacterial load and airway and systemic inflammation was investigated in 385 stable patients, 15 stable patients treated with intravenous antibiotics, and 34 patients with an exacerbation of bronchiectasis treated with intravenous antibiotics. Long-term antibiotic therapy was investigated using samples from a 12-month controlled trial of nebulized gentamicin. MEASUREMENTS AND MAIN RESULTS: In stable patients, there was a direct relationship between airway bacterial load and markers of airway inflammation (P < 0.0001 for all analyses). High bacterial loads were associated with higher serum intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 (P < 0.05 above bacterial load ≥1 × 10(7) cfu/ml). In stable patients, there was a direct relationship between bacterial load and the risk of subsequent exacerbations (odds ratio, 1.20; 95% confidence interval, 1.11-1.29; P < 0.0001) and severe exacerbations (odds ratio, 1.11; 95% confidence interval, 1.01-1.21; P = 0.02). Short- and long-term antibiotic treatments were associated with reductions in bacterial load, airways, and systemic inflammation. CONCLUSIONS: High airway bacterial loads in non-CF bronchiectasis are associated with airway and systemic inflammation and a greater risk of exacerbations. Short- and long-term antibiotic therapy reduce markers of airways and systemic inflammation.
Assuntos
Antibacterianos/administração & dosagem , Bronquiectasia/tratamento farmacológico , Bronquiectasia/patologia , Adulto , Idoso , Carga Bacteriana , Biomarcadores/metabolismo , Bronquiectasia/microbiologia , Moléculas de Adesão Celular/metabolismo , Estudos de Coortes , Esquema de Medicação , Feminino , Nível de Saúde , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Resultado do TratamentoRESUMO
RATIONALE: Bronchiectasis is a chronic debilitating disease with few evidence-based long-term treatments. OBJECTIVES: A randomized controlled trial assessing the efficacy of nebulized gentamicin therapy over 1 year in patients with non-cystic fibrosis bronchiectasis. METHODS: Sixty-five patients were randomized to either twice-daily nebulized gentamicin, 80 mg, or nebulized 0.9% saline, for 12 months. All were reviewed at three-monthly intervals during treatment and at 3 months' follow-up. MEASUREMENTS AND MAIN RESULTS: At each review the following were assessed: quantitative and qualitative sputum bacteriology; sputum purulence and 24-hour volume; FEV(1), FVC, and forced expiratory flow, midexpiratory phase; exercise capacity; Leicester Cough Questionnaire and St. George's Respiratory Questionnaire; and exacerbation frequency. Fifty-seven patients completed the study. At the end of 12 months' treatment, compared with the saline group, in the gentamicin group there was reduced sputum bacterial density with 30.8% eradication in those infected with Pseudomonas aeruginosa and 92.8% eradication in those infected with other pathogens; less sputum purulence (8.7% vs. 38.5%; P < 0.0001); greater exercise capacity (510 [350-690] m vs. 415 [267.5-530] m; P = 0.03); and fewer exacerbations (0 [0-1] vs. 1.5 [1-2]; P < 0.0001) with increased time to first exacerbation (120 [87-161.5] d vs. 61.5 [20.7-122.7] d; P = 0.02). The gentamicin group had greater improvements in Leicester Cough Questionnaire (81.4% vs. 20%; P < 0.01) and St. George's Respiratory Questionnaire (87.5% vs. 19.2%; P < 0.004) score. No differences were seen in 24-hour sputum volume, FEV(1), FVC, or forced expiratory flow, midexpiratory phase. No P. aeruginosa isolates developed resistance to gentamicin. At follow-up, all outcome measures were similar to baseline. CONCLUSIONS: Regular, long-term nebulized gentamicin is of significant benefit in non-cystic fibrosis bronchiectasis but treatment needs to be continuous for its ongoing efficacy. Clinical trial registered with www.clinicaltrials.gov (NCT 00749866).
Assuntos
Antibacterianos/administração & dosagem , Bronquiectasia/tratamento farmacológico , Gentamicinas/administração & dosagem , Administração por Inalação , Aerossóis , Idoso , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Função Respiratória/métodos , Testes de Função Respiratória/estatística & dados numéricos , Cloreto de Sódio/administração & dosagem , Inquéritos e Questionários , Resultado do TratamentoRESUMO
Cationic host defense peptides are key, evolutionarily conserved components of the innate immune system. The human cathelicidin LL-37 is an important cationic host defense peptide up-regulated in infection and inflammation, specifically in the human lung, and was shown to enhance the pulmonary clearance of the opportunistic pathogen Pseudomonas aeruginosa in vivo by as yet undefined mechanisms. In addition to its direct microbicidal potential, LL-37 can modulate inflammation and immune mechanisms in host defense against infection, including the capacity to modulate cell death pathways. We demonstrate that at physiologically relevant concentrations of LL-37, this peptide preferentially promoted the apoptosis of infected airway epithelium, via enhanced LL-37-induced mitochondrial membrane depolarization and release of cytochrome c, with activation of caspase-9 and caspase-3 and induction of apoptosis, which only occurred in the presence of both peptide and bacteria, but not with either stimulus alone. This synergistic induction of apoptosis in infected cells was caspase-dependent, contrasting with the caspase-independent cell death induced by supraphysiologic levels of peptide alone. We demonstrate that the synergistic induction of apoptosis by LL-37 and Pseudomonas aeruginosa required specific bacteria-epithelial cell interactions with whole, live bacteria, and bacterial invasion of the epithelial cell. We propose that the LL-37-mediated apoptosis of infected, compromised airway epithelial cells may represent a novel inflammomodulatory role for this peptide in innate host defense, promoting the clearance of respiratory pathogens.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Brônquios/microbiologia , Brônquios/patologia , Epitélio/microbiologia , Epitélio/patologia , Infecções por Pseudomonas/patologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Brônquios/efeitos dos fármacos , Caspases/metabolismo , Comunicação Celular/efeitos dos fármacos , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Fímbrias Bacterianas/efeitos dos fármacos , Fímbrias Bacterianas/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Proteína X Associada a bcl-2/metabolismo , CatelicidinasRESUMO
Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Using high-resolution mass spectrometry, we recently reclassified one such peroxiredoxin, bacterioferritin comigratory protein (BCP) of Escherichia coli, as an atypical 2-Cys peroxiredoxin that functions through the formation of an intramolecular disulfide bond between the active and resolving cysteine. An engineered E. coli BCP, which lacked the resolving cysteine, retained enzyme activity through a novel catalytic pathway. Unlike the active cysteine, the resolving cysteine of BCP peroxiredoxins is not conserved across all members of the family. To clarify the catalytic mechanism of native BCP enzymes that lack the resolving cysteine, we have investigated the BCP homologue of Burkholderia cenocepacia. We demonstrate that the B. cenocepacia BCP (BcBCP) homologue functions through a 1-Cys catalytic pathway. During catalysis, BcBCP can utilize thioredoxin as a reductant for the sulfenic acid intermediate. However, significantly higher peroxidase activity is observed utilizing glutathione as a resolving cysteine and glutaredoxin as a redox partner. Introduction of a resolving cysteine into BcBCP changes the activity from a 1-Cys pathway to an atypical 2-Cys pathway, analogous to the E. coli enzyme. In contrast to the native B. cenocepacia enzyme, thioredoxin is the preferred redox partner for this atypical 2-Cys variant. BCP-deficient B. cenocepacia exhibit a growth-phase-dependent hypersensitivity to oxidative killing. On the basis of sequence alignments, we believe that BcBCP described herein is representative of the major class of bacterial BCP peroxiredoxins. To our knowledge, this is the first detailed characterization of their catalytic activity. These studies support the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia/química , Peroxirredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Catálise , Linhagem Celular , Escherichia coli/enzimologia , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NADP/metabolismo , Oxirredução , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Transporte Proteico , Compostos de Sulfidrila/química , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.
Assuntos
Antibacterianos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Fragmentos de Peptídeos/química , beta-Defensinas/química , Animais , Antibacterianos/farmacologia , Dimerização , Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , beta-Defensinas/farmacologiaRESUMO
BACKGROUND: Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP. METHODS: A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age- and sex-matched volunteers. Growth of pathogens at >10(4) colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from "non-VAP". Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves. RESULTS: Seventy-two patients had recoverable lavage-24% had VAP. BALF interleukin-1beta (IL-1beta), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1alpha were significantly higher in the VAP group (all p<0.005). Using a cut-off of 10 pg/ml, BALF IL-1beta generated negative likelihood ratios for VAP of 0.09. In patients with BALF IL-1beta <10 pg/ml the post-test probability of VAP was 2.8%. Using a cut-off value for IL-8 of 2 ng/ml, the positive likelihood ratio was 5.03. There was no difference in cytokine levels between patients with sterile BALF and those with growth of <10(4) cfu/ml. CONCLUSIONS: BALF IL-1beta and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations.
Assuntos
Interleucina-1beta/análise , Interleucina-8/análise , Pneumonia Associada à Ventilação Mecânica/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/microbiologia , Métodos Epidemiológicos , Feminino , Humanos , Mediadores da Inflamação/análise , Masculino , Pessoa de Meia-Idade , Pneumonia Associada à Ventilação Mecânica/microbiologia , Adulto JovemRESUMO
Microaspiration of Pseudomonas aeruginosa contributes to the pathogenesis of nosocomial pneumonia. Trappin-2 is a host defense peptide that assists with the clearance of P. aeruginosa through undefined mechanisms. A model of macrophage interactions with replicating P. aeruginosa (strain PA01) in serum-free conditions was developed, and the influence of subantimicrobial concentrations of trappin-2 was subsequently studied. PA01 that was pre-incubated with trappin-2 (at concentrations that have no direct antimicrobial effects), but not control PA01, was cleared by alveolar and bone marrow-derived macrophages. However, trappin-2-enhanced clearance of PA01 was completely abrogated by CD14- null macrophages. Fluorescence microscopy demonstrated the presence of trappin-2 on the bacterial cell surface of trappin-2-treated PA01. In a murine model of early lung infection, trappin-2-treated PA01 was cleared more efficiently than control PA01 2 hours of intratracheal instillation. Furthermore, trappin-2-treated PA01 up-regulated the murine chemokine CXCL1/KC after 2 hours with a corresponding increase in neutrophil recruitment 1 hour later. These in vivo trappin-2-treated PA01 effects were absent in CD14-deficient mice. Trappin-2 appears to opsonize P. aeruginosa for more efficient, CD14-dependent clearance by macrophages and contributes to the induction of chemokines that promote neutrophil recruitment. Trappin-2 may therefore play an important role in innate recognition and clearance of pathogens during the very earliest stages of pulmonary infection.
Assuntos
Elafina/imunologia , Receptores de Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Infiltração de Neutrófilos/imunologia , Infecções por Pseudomonas/imunologia , Animais , Células Cultivadas , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Pneumopatias/imunologia , Pneumopatias/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fagocitose , Pseudomonas aeruginosa/imunologia , Proteínas Recombinantes/imunologiaRESUMO
Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.
Assuntos
Complexo Burkholderia cepacia/genética , Complexo Burkholderia cepacia/patogenicidade , Burkholderia/genética , Burkholderia/patogenicidade , Fibrose Cística/microbiologia , Genoma Bacteriano , Complexo Burkholderia cepacia/efeitos dos fármacos , Complexo Burkholderia cepacia/isolamento & purificação , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , Primers do DNA , DNA Bacteriano/genética , DNA Circular/genética , Resistência Microbiana a Medicamentos , Amplificação de Genes , Humanos , Plantas/microbiologia , Plasmídeos , Reação em Cadeia da Polimerase , Escarro/microbiologiaRESUMO
BACKGROUND: This study reviews the impact of changing infection control practices at the Manchester Adult Cystic Fibrosis Centre (MACFC) upon the epidemiology of Burkholderia species infections. METHODS: We reviewed strain and genomovar typing of all available Burkholderia isolates at our centre between 1983-2006. RESULTS: The incidence/prevalence of infection with Burkholderia species between 1983-1990 was below 5%/9% each year. There was a rise in incidence/prevalence of Burkholderia species between 1991 and 1994 with a peak of 16.3%/31.2% in 1992. Following complete cohort segregation, the incidence has fallen to below 3% for all but one year and the prevalence has gradually reduced to 9.3% in 2005. Currently, there is an increase in the prevalence to 10.6% for the first time since 1994, predominantly due to patients with unique infections transferring into the unit from referring centres. The presence of unique strains now exceeds transmissible strains for the first time since 1991. CONCLUSIONS: Infection control measures including patient segregation have controlled spread of transmissible B. cenocepacia strains, but not the acquisition of unique strains. Unique strains of Burkholderia species now account for the majority of new infections at the Manchester Adult Cystic Fibrosis Centre.
Assuntos
Infecções por Burkholderia/epidemiologia , Infecções por Burkholderia/microbiologia , Burkholderia/isolamento & purificação , Fibrose Cística/complicações , Técnicas de Tipagem Bacteriana , Infecções por Burkholderia/transmissão , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Inglaterra/epidemiologia , Humanos , Incidência , Controle de Infecções/métodos , Epidemiologia Molecular , Prevalência , Estudos Prospectivos , Escarro/microbiologiaRESUMO
Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.
Assuntos
Corantes Fluorescentes/metabolismo , Bactérias Gram-Negativas/isolamento & purificação , Lipídeo A/metabolismo , Pulmão/microbiologia , Peptídeos/metabolismo , Animais , Bronquiectasia/microbiologia , Bronquiectasia/patologia , Humanos , Unidades de Terapia Intensiva , Pulmão/patologia , Macrófagos Alveolares/metabolismo , Polimixinas/farmacologia , Ovinos , Razão Sinal-Ruído , Relação Estrutura-AtividadeRESUMO
BACKGROUND: There are no randomized controlled trials of statin therapy in patients with severe bronchiectasis who are chronically infected with Pseudomonas aeruginosa. METHODS: Thirty-two patients chronically infected with P aeruginosa were recruited in this double-blind cross-over randomized controlled trial. Sixteen patients were recruited in each arm, were given atorvastatin 80 mg or placebo for 3 months followed by a washout period for 6 weeks, and then crossed over and administered the alternative therapy for 3 months. RESULTS: Twenty-seven patients completed the study. Atorvastatin did not significantly improve the primary end point of cough as measured by the Leicester Cough Questionnaire (mean difference, 1.92; 95% CI for difference, -0.57-4.41; P = .12). However, atorvastatin treatment resulted in an improved St. Georges Respiratory Questionnaire (-5.62 points; P = .016) and reduced serum levels of CXCL8 (P = .04), tumor necrosis factor (P = .01), and intercellular adhesion molecule 1 (P = .04). There was a trend toward improvement in serum C-reactive protein and serum neutrophil counts (P = .07 and P = .06, respectively). We demonstrated in vitro that atorvastatin 10 µM reduced formyl-methionyl-leucyl phenylalanine-induced upregulation of CD11b expression and changes in calcium flux, reflecting an ability to decrease neutrophil activation. CONCLUSIONS: We demonstrated that atorvastatin reduced systemic inflammation and improved quality of life in patients with bronchiectasis who were infected with P aeruginosa. These effects may be due to an ability of atorvastatin to modulate neutrophil activation. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01299194; URL: www.clinicaltrials.gov.
Assuntos
Anti-Inflamatórios/administração & dosagem , Atorvastatina/administração & dosagem , Bronquiectasia/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Infecções por Pseudomonas/complicações , Adolescente , Adulto , Idoso , Antibacterianos/uso terapêutico , Bronquiectasia/complicações , Cálcio/metabolismo , Tosse/etiologia , Estudos Cross-Over , Citocinas , Método Duplo-Cego , Esquema de Medicação , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Ativação de Neutrófilo/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/isolamento & purificação , Qualidade de Vida , Escarro/microbiologia , Resultado do Tratamento , Capacidade Vital/efeitos dos fármacos , Adulto JovemRESUMO
This study aimed firstly to establish the distribution and copy number within the Burkholderia cepacia complex of three insertion sequences (IS402, IS407 and IS1416) that possess the ability to activate transcription and hence influence gene expression. A second aim was to map the genomic insertion sites of one of the active insertion sequences (IS407) to establish putative links between insertion site and downstream gene activation. The resulting data revealed that all three insertion sequences were present in one-third of the 66 isolates tested. The three insertion sequences were prevalent across the nine B. cepacia complex species, although IS402 was absent from the 16 Burkholderia anthina strains tested and IS407 was absent from all 10 Burkholderia pyrrocinia strains. IS407 copies from six strains (two Burkholderia cenocepacia strains and one strain each of Burkholderia multivorans, Burkholderia stabilis, Burkholderia vietnamiensis and B. anthina) were mapped to the genome using hemi-nested inverse PCR. Insertions were found upstream of genes with wide-ranging functions. This study suggests that the abundance and distribution of these active insertion sequences is likely to affect genomic plasticity, and potentially gene transcription and pathogenicity.
Assuntos
Complexo Burkholderia cepacia/genética , Mapeamento Cromossômico , Elementos de DNA Transponíveis , Infecções por Burkholderia/microbiologia , Complexo Burkholderia cepacia/classificação , Complexo Burkholderia cepacia/patogenicidade , Fibrose Cística/microbiologia , Microbiologia Ambiental , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Humanos , Transcrição Gênica , Ativação TranscricionalRESUMO
Ninety-one percent of Burkholderia cepacia complex reference strains (66 out of 72) displayed a yellow slope-green butt colour reaction after growth in Stewart's medium indicating the oxidation of glucose and the absence of an arginine dihydrolase system. This same colour reaction was observed for Burkholderia gladioli and several Ralstonia species, but not for Pseudomonas aeruginosa, Stenotrophomonas, Achromobacter, Pandoraea and several other Gram-negative non-fermenting bacilli. We therefore consider growth in Stewart's medium a useful, simple, rapid and inexpensive screening test to reduce the number of false positive isolates from B. cepacia complex selective media.
Assuntos
Técnicas de Tipagem Bacteriana , Infecções por Burkholderia/microbiologia , Complexo Burkholderia cepacia/classificação , Meios de Cultura , Fibrose Cística/microbiologia , Indicadores e Reagentes , Complexo Burkholderia cepacia/crescimento & desenvolvimento , HumanosAssuntos
Amidoidrolases/antagonistas & inibidores , Complexo Burkholderia cepacia/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Treonina/análogos & derivados , Infecções por Burkholderia/microbiologia , Complexo Burkholderia cepacia/enzimologia , Humanos , Ácidos Hidroxâmicos/química , Testes de Sensibilidade Microbiana , Treonina/química , Treonina/farmacologiaRESUMO
The purpose of this study was to develop a novel strategy for the isolation and identification of Burkholderia cepacia complex bacteria from the home environment of cystic fibrosis (CF) patients. Water and soil samples were enriched in a broth containing 0.1% l-arabinose, 0.1% l-threonine, and a mixture of selective agents including 1 microgml(-1) C-390, 600U ml(-1) polymyxin B sulfate, 10 microgml(-1) gentamycin, 2 microgml(-1) vancomycin and 10 microgml(-1) cycloheximide. On selective media (consisting of the same components as above plus 1.8% agar), several dilutions of the enrichment broth were inoculated and incubated for 5 days at 28 degrees C. Isolates with different randomly amplified polymorphic DNA patterns were inoculated in Stewart's medium. Putative B. cepacia complex bacteria were confirmed by means of recA PCR and further identified by HaeIII-recA restriction fragment length polymorphism analysis. Our results suggest that these organisms may be more widespread in the home environment than previously assumed and that plant associated soil and pond water may be reservoirs of B. cepacia complex infection in CF patients.
Assuntos
Burkholderia cepacia/classificação , Burkholderia cepacia/isolamento & purificação , Infecções por Burkholderia/etiologia , Infecções por Burkholderia/microbiologia , Burkholderia cepacia/genética , Fibrose Cística/microbiologia , Humanos , Indicadores e Reagentes , Polimorfismo de Fragmento de Restrição , Polimixina B , Microbiologia do SoloRESUMO
BACKGROUND: Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density. This potentially suggests a role for the latter in the pathogenesis of exacerbations. However, other data suggesting that changes in P. aeruginosa sputum culture status may not reliably predict an improvement in clinical status, and data indicating no significant changes in either total bacterial counts or in P. aeruginosa numbers in sputum samples collected prior to pulmonary exacerbation sheds doubt on this assumption. We used our recently developed lung segmental model of chronic Pseudomonas infection in sheep to investigate the lung microbiota changes associated with chronic P. aeruginosa lung infection and the impact of systemic therapy with colistimethate sodium (CMS). METHODOLOGY/PRINCIPAL FINDINGS: We collected protected specimen brush (PSB) samples from sheep (n = 8) both prior to and 14 days after establishment of chronic local lung infection with P aeruginosa. Samples were taken from both directly infected lung segments (direct) and segments spatially remote to such sites (remote). Four sheep were treated with daily intravenous injections of CMS between days 7 and 14, and four were treated with a placebo. Necropsy examination at d14 confirmed the presence of chronic local lung infection and lung pathology in every direct lung segment. The predominant orders in lung microbiota communities before infection were Bacillales, Actinomycetales and Clostridiales. While lung microbiota samples were more likely to share similarities with other samples derived from the same lung, considerable within- and between-animal heterogeneity could be appreciated. Pseudomonadales joined the aforementioned list of predominant orders in lung microbiota communities after infection. Whilst treatment with CMS appeared to have little impact on microbial community composition after infection, or the change undergone by communities in reaching that state, when Gram negative organisms (excluding Pseudomonadales) were considered together as a group there was a significant decrease in their relative proportion that was only observed in the sheep treated with CMS. With only one exception the reduction was seen in both direct and remote lung segments. This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment. CONCLUSIONS/SIGNIFICANCE: The proportional increase in Pseudomonadales relative to other Gram negative bacteria in the lungs of sheep treated with systemic CMS highlights the potential for such therapies to inadvertently select or create a niche for bacteria seeding from a persistent source of chronic infection.
Assuntos
Antibacterianos/administração & dosagem , Colistina/análogos & derivados , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/microbiologia , Animais , Carga Bacteriana , Líquido da Lavagem Broncoalveolar/microbiologia , Doença Crônica , Colistina/administração & dosagem , Modelos Animais de Doenças , Feminino , Injeções Intravenosas , Pulmão/microbiologia , Pulmão/patologia , Masculino , Microbiota , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Infecções Respiratórias/patologia , Carneiro DomésticoRESUMO
DNA-DNA hybridisation experiments between isolates representing Burkholderia cepacia genomovar III recA lineages IIIA and IIIB reinforced the classification of both phylogenetic subgroups as a single genospecies, distinct from B. cepacia (genomovar I). A formal classification of B. cepacia genomovar III encompassing the recA lineages IIIA and IIIB, and the new recA lineages IIIC and IIID, as B. cenocepacia sp. nov., with LMG 16656 as the type strain, is proposed.
Assuntos
Burkholderia/classificação , Burkholderia/genética , Infecções por Burkholderia/microbiologia , Burkholderia cepacia/genética , Fibrose Cística/microbiologia , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Filogenia , Recombinases Rec A/genética , Análise de Sequência de DNARESUMO
Burkholderia cepacia is an important pathogen in patients with cystic fibrosis (CF) and much is now known of its epidemiology. In contrast, its virulence mechanisms are poorly understood. The lipopolysaccharide (LPS) of B. cepacia, a well-recognised virulence factor of other gram-negative bacteria, is known to be strongly endotoxic in vitro. The aim of this study was to observe if there were any links between the structure of B. cepacia LPS and virulence. This has been investigated by polyacrylamide gel electrophoresis and immunoblotting to define the chemotype and antigenic cross reactivity of B. cepacia LPS. Strains (16) belonging to different genomovars of the B. cepacia complex were selected to represent epidemic and non-epidemic clinical isolates and environmental strains. All strains belonging to genomovars I and II (the latter now renamed B. multivorans) had smooth LPS. However, isolates belonging to genomovar III, the group to which most of the epidemic CF isolates belong - including the highly transmissible strain (ET 12) which has been found in both the UK and North America - were of either rough or smooth LPS chemotype. In this study, B. cepacia J2315 represents the ET 12 lineage, and has a rough chemotype. Rabbit antiserum raised to strain J2315 revealed that the LPS core of this strain was antigenically related to some but not all other genomovar III strains, but it also cross-reacted strongly with all B. multivorans (genomovar II) and most genomovar I strains. Intra-strain phenotypic variation was demonstrated between bacteria grown in broth or on solid agar with a concomitant variation in antigenic cross reactivity. There was no clear evidence to associate any particular LPS phenotype with epidemic or non-epidemic strains, but changes in phenotype in vitro may provide clues to the survival and adaptability of B. cepacia in hostile environments and possibly to its ability to produce an inflammatory response in vivo.