Sputum Of CF Patients Research Articles

255 A Bacterial Serine Protease Inhibitor, Ecotin Inhibits Neutrophil Elastase in Cystic Fibrosis Airway Samples

OBJECTIVES/GOALS: Ecotin is a potent periplasmic serin protease inhibitor that was first described in Escherichia coli (E. coli). Ecotin is known to inhibit a broad range of serine proteases including NE. Therefore, we hypothesized that ecotin can inhibit the NE activity present in CF clinical sputum samples. METHODS/STUDY POPULATION: To investigate the function of ecotin on CF sputum, orthologues from Campylobacter rectus and Campylobacter showae were recombinantly expressed and purified from E. coli to generate C. rectus ecotin, C. showae ecotin, and E. coli ecotin. Ecotin was added to CF sputum supernatant samples at various concentrations and NE enzymatic activity was measured by a fluorometric assay kit. In addition, CF sputum and healthy sputum was added to PMNs isolated from healthy donors for 3.5 hours and NE was measured with immunofluorescence staining. The sputum was washed two times prior to measuring NE released and on the PMNs, and PBMCs was used as a control. RESULTS/ANTICIPATED RESULTS: Our results indicate a clear inhibition of NE activity in CF sputum supernatant. C. showae ecotin showed the greatest inhibitory effect on NE activity in CF sputum supernatant. We also saw that CF sputum supernatant causing healthy PMNs to release more NE suggesting that NE in the CF airway may trigger more NE release from newly incoming PMNs to the lung. Our next steps will be to determine if ecotin can inhibit NE on the PMN surface and released from PMNs. DISCUSSION/SIGNIFICANCE: Neutrophil elastases are produced by PMNs to kill microbial pathogens in the lung, however in CF, the pathogens are unable to be killed by NE and instead causes severe lung damage. PMNs and NE has been shown to be elevated in the sputum of CF patients and there is currently no NE therapeutic inhibitor that has been effective in these patients.

Citrullination of extracellular histone H3.1 reduces antibacterial activity and exacerbates its proteolytic degradation

BackgroundCystic fibrosis (CF), involves excessive airway accumulation of neutrophils, often in parallel with severe infection caused by Pseudomonas aeruginosa. Free histones are known to possess bactericidal properties, but the degree of antibacterial activity exerted on specific lung-based pathogens is largely unknown. Neutrophils have a high content of peptidyl deiminase 4 (PADI4), which citrullinate cationic peptidyl-arginines. In histone H3.1, several positions in the NH2-terminal tail are subject to citrullination. MethodsFull-length and segmented histone subunit H3.1 was investigated for bactericidal activity towards P. aeruginosa (strain PAO1). PADI4-induced citrullination of histone H3.1 was assessed for antibacterial activity towards P. aeruginosa. Next, the effect of neutrophil elastase (NE)-mediated proteolysis of histone H3.1 was investigated. Finally, PADI4, H3.1, and citrullinated H3.1 were examined in healthy control and CF patient lung tissues. ResultsFull-length histone H3.1 and sections of the histone H3.1 tail, displayed bactericidal activity towards P. aeruginosa. These antibacterial effects were reduced following citrullination by PADI4 or proteolysis by NE. Interestingly, citrullination of histone H3.1 exacerbated NE-mediated degradation. In CF lung tissue, citrullinated histone H3.1 and PADI4 immunoreactivity was abundant. Degraded histone H3.1 was detected in the sputum of CF patients but was absent in the sputum of healthy controls. ConclusionsCitrullination impairs the antibacterial activity of histone H3.1 and exacerbates its proteolytic degradation by NE. Citrullination is likely to play an important role during resolution of acute inflammation. However, in chronic inflammation akin to CF, citrullination may dampen host defense and promote pathogen survival, as exemplified by P. aeruginosa.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients.

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Bacteria in the airways of patients with cystic fibrosis are genetically capable of producing VOCs in breath

Breath contains hundreds of volatile organic compounds (VOCs), the composition of which is altered in a wide variety of diseases. Bacteria are implicated in the formation of VOCs, but the biochemical mechanisms that lead to the formation of breath VOCs remain largely hypothetical. We hypothesized that bacterial DNA fragments in sputum of CF patients could be sequenced to identify whether the bacteria present were capable of producing VOCs found in the breath of these patients.Breath from seven patients with cystic fibrosis was sampled and analyzed by gas-chromatography and mass-spectrometry. Sputum samples were also collected and microbial DNA was isolated. Metagenomic sequencing was performed and the DNA fragments were compared to a reference database with genes that are linked to the metabolism of acetaldehyde, ethanol and methanol in the KEGG database.Bacteria in the genera Escherichia, Lactococcus, Pseudomonas, Rothia and Streptococcus were found to have the genetic potential to produce acetaldehyde and ethanol. Only DNA sequences from Lactococcus were implicated in the formation of acetaldehyde from acetate through aldehyde dehydrogenase family 9 member A1 (K00149). Escherichia was found to be genetically capable of producing ethanol in all patients, whilst there was considerable heterogeneity between patients for the other genera. The ethanol concentration in breath positively correlated with the amount of Escherichia found in sputum (Spearman rho = 0.85, P = 0.015). Rothia showed the most versatile genetic potential for producing methanol.To conclude, bacterial DNA fragments in sputum of CF patients can be linked to enzymes implicated in the production of ethanol, acetaldehyde and methanol, which are VOCs that are predictive of respiratory tract colonization and/or infection. This supports that the lung microbiome can produce VOCs directly.

41 Taxonomic signatures of CF airway microbiota distinguish between patients with lower and higher pulmonary function decline

Objective To gain insight into the underlying causes of severe lung diseases, we compared the airway microbiota detected in sputum of CF patients with stable lung function (S) versus those with a substantial decline in lung function (SD). Methods Fifty-two patients belonging to three FEV1 groups (normal/mild, FEV1 >70%; moderate, 40 Results A sharp difference in the structure and composition of airway microbiota between S and SD patients was found, especially in patients with severe lung disease, where Pseudomonas represented the most abundant genus. A cooccurrence network analysis showed that microbial communities were less complex in SD patients than in S ones. Moreover, an inverse relationship between bacterial community diversity and disease severity was found. Indeed, the analysis of the main biodiversity indices on microbiota data has shown a significant drop passing from normal/mild FEV1 group to moderate lung disease FEV1 group suggesting that the patients with intermediate FEV1 values are experiencing changes in the airway assembly of taxa. Conclusion We can claim that bacterial lung microbiota is modified along with substantial decline in lung function. Higher levels of pulmonary deficiency can lead to a drastic decrement of bacterial community complexity leaving the way clear for the proliferation of opportunistic pathogens. Grants FFC #8/2012 and FFC #10/2014.

Neutrophils stain positive for IL-17A in sputum of CF patients

Neutrophils stain positive for IL-17A in sputum of CF patients

Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

AbstractDirect detection of Burkholderia cepacia complex (BCC) and its genomovars from sputum by molecular tests emerges as a method for rapid identification. In this study, four DNA extraction methods were evaluated for the identification for BCC from sputum of CF patients. Sputa from 28 CF patients were aliquoted and spiked with BCC reference strain. Boiling, phenol-chloroform, CTAB methods and a commercial spin column kit was used for DNA extraction. Total DNA yields were determined by spectrophotometry and single-round recA PCR was used for detection of BCC. No significant difference was observed in DNA yields from different extraction methods. Lower limit of detection for recA PCR was determined as 106 cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for CTAB and 13/16 (81.2%) of sputa for phenol-chloroform method and spin column kit in the assay sensitivity range determined in the study. Phenol-chloroform and commercial spin column kit were found to be better suited for DNA purification from sputum of CF patients for BCC identification. Diagnostic impact of single-round recA PCR directly from sputum was limited to chronically-infected patients.

99 Sensitivity of recA PCR for direct detection of Burkholderia cepacia complex (BCC) from sputum of CF patients

99 Sensitivity of recA PCR for direct detection of Burkholderia cepacia complex (BCC) from sputum of CF patients

Characterization of Small-Colony-Variant Stenotrophomonas maltophilia Isolated from the Sputum Specimens of Five Patients with Cystic Fibrosis

Cystic fibrosis (CF) patients are predisposed to chronic respiratory infection by nonfermentative gram-negative bacilli, including Stenotrophomonas maltophilia. S. maltophilia is highly resistant to most antibiotics, with the exception of sulfamethoxazole-trimethoprim (SXT). SXT-resistant S. maltophilia has been reported, but the mechanism of resistance is not well defined. Repeated findings of suspected small-colony-variant (SCV) S. maltophilia isolates from the sputa of five CF patients were confirmed by partial 16S rRNA gene sequencing. The SCV S. maltophilia isolates were the only S. maltophilia isolates in these cultures, and none were clonally related. DNA fingerprint analysis confirmed that once established, the SCV S. maltophilia strains persisted. Nutritional studies of SCV S. maltophilia have suggested auxotrophy in hemin, methionine, and thymidine associated with resistance to multiple antibiotics, including SXT. The phenotypic switch from wild-type to SCV S. maltophilia was reproducible in vitro by exposure to SXT, suggesting that prolonged exposure to antibiotics may select for both the SCV S. maltophilia phenotype and SXT resistance by interference with the dihydrofolate reductase pathway. Recovery of SCV S. maltophilia from the sputum of CF patients has implications for both laboratory testing and patient management.

Management of non-tuberculous mycobacteria in patients with cystic fibrosis.

Patients with CF are living longer with the present median survival close to 32 years of age. The increase in survival rates is attributed to better management of airway disease, including airway clearance techniques, better nutrition, new and innovative therapeutic approaches, and the development of broad-spectrum antibiotics. With improved survival, new pulmonary issues have developed. These include the emergence of multiresistant strains of Pseudomonas aeruginosa and as well as the emergence of organisms with increased virulence such as Burkholderia cepacia. Over the past few years several organisms have been identified with increasing frequency in the sputum of CF patients. These include Stenotrophomonas maltophilia, Achromobacter (Alcaligenes) xylosoxidans and nontuberculous mycobacteria (NTM). The clinical ramifications of these organisms are not entirely clear. The emergence of these organisms may be due to selection pressure from the increased use of antibiotics, improved culture techniques and/or the changing flora of an aging population. NTM have been recognised since the 1950s as potential pulmonary pathogens. NTM organisms are routinely found in the environment and are easily aerosolised. There have been documented cases of nosocomial outbreaks from medical equipment that utilises water. Person to person transmission is unlikely.

Infection control and the significance of sputum and other respiratory secretions from adult patients with cystic fibrosis

BackgroundThere is limited data available on the environmental and public health impact of the microbiological hazards associated with sputa from patients with cystic fibrosis [CF]. Pseudomonas aeruginosa, Burkholderia cenocepacia (formerly Burkholderia cepacia genomovar III), Staphylococcus aureus and Stenotrophomonas maltophilia are bacterial pathogens which are commonly found in the sputum of CF patients. A study was performed to ascertain the amount of sputum produced relating to microbial loading, as well as the diversity of bacteria present in a population of adult patients, with particular attention to pathogenic organisms.MethodsSputum from adult [>18 years old] CF patients [n = 20], chosen randomly from a population of 138 CF patients, was collected over a 24 h period on admission to the in-patient CF unit and enumerated quantitatively, as well as the sputa from 138 adult CF patients was examined qualitatively for the presence of infecting microflora. In addition, all different phenotypes from the sputum of each patient were identified phenotypically employing a combination of conventional identification methods [e.g. oxidase], as well as the API Identification schemes [API 20 NE, API 20 E].ResultsThis study demonstrated that patients with cystic fibrosis generate large numbers of bacteria in their sputum, approximating to 109 organisms per patient per day. Although these organisms are introduced to the environment from the respiratory tract mainly via sputum, relatively few represent true bacterial pathogens and therefore are not clinically important to the general public who are immunocompotent. The greatest risk of such environmental microbial loading is to other patients with CF and therefore CF patients should be made aware of the hazards of acquiring such organisms from the environment, as well as socializing with other CF patients with certain transmissible types, such as Pseudomonas aeruginosa and Burkholderia cenocepacia.ConclusionsEnvironmental health professionals should therefore be aware that CF patients are a greater risk to their peer grouping rather than to the general public or health care workers and that good personal hygiene practices with CF patients should be encouraged to minimize environmental contamination and potential acquistion.

Kinetic parameters of amikacin in cystic fibrosis children.

35 kinetic studies have been performed, in nine CF children three to 15 years old; six kinetic studies were performed in four non-CF children, one to 12 years old. The dosage was 5 to 12.5 mg/kg, i.v. during 0.5 to 1.0 h, three to four times per day. Amikacin concentrations were measured in the plasma of all children, and in the sputum of CF-patients, by fluorescent polarization (TDX Abbott). The pharmacokinetic parameters in the plasma did not differ significantly in both groups of patients. In CF children t1/2 = 0.94 h (SD = 0.25 h), Vd (area) = 0.257 l/kg (SD = 0.06 l/kg), total body clearance = 130.7 ml/min/1.73 m2 (SD = 32.4 ml/min/1.73 m2). In non-CF children t1/2 = 0.83 h (SD = 0.15 h), Vd (area) = 0.265 l/kg (SD = 0.04 l/kg) and clearance = 155 ml/min/1.73 m2 (SD = 17.4 ml/min/1.73 m2). The parameters were not affected by the dosage of amikacin. The peak plasma concentrations ranged from 19 to 43.8 mg/l. Amikacin peak level in the sputum of CF children never reached the average MIC (4 mg/l) of Pseudomonas aeruginosa strains isolated in these patients. Amikacin concentration in the sputum reached its highest value about 2 h after the completion of i.v. infusion and was directly related to the peak plasma concentration. According to these parameters, the best dosage regimen appeared to be 7.5 to 8 mg/kg or 225 to 240 mg/m2 administered intravenously in 1.0 h, three times per day.

Demonstration of neutrophil chemotactic activity in the sputum of cystic fibrosis patients with Pseudomonas aeruginosa infection.

The pathogenesis of tissue damage in the lungs of cystic fibrosis patients with chronic P. aeruginosa infection is quite complex and not well understood. Inflammatory cells, particularly neutrophils, are accumulated in the damaged tissues and in the sputum. This study demonstrates the presence of a heat-stable neutrophil chemotactic factor(s) in the sputum of CF patients. The chemotactic activity seems to be associated with the endotoxin present in the sputum. Chemotaxis of sol phase sputum was determined in a modified Boyden chamber assay. It was found that the sputum obtained from the patients showed strong chemotactic activity for peripheral blood neutrophils of normal individuals. The activity was greater than twice that of casein, a strong neutrophil chemo-attractant. Sputum at about dilution of 1:50 exhibited chemotactic activity equal to that of casein. Heat treatment of the sputum at 56 degrees C for 30 min, to inactivate complement, and at 100 degrees C for 15 min, to denature other proteins, somewhat reduced the chemotactic activity, but there was still strong chemotactic activity. The presence of endotoxin was demonstrated in both the non-heated and the heated samples by LAL and rocket immunoelectrophoresis. It is concluded that the sputum of CF patients contain chemotactic activity of heat-stable nature and most likely of bacterial origin endotoxin. These factors are involved in attraction of neutrophils to the lungs and sputum of these patients and contribute to the tissue damage of cystic fibrosis.