Forced oscillations and respiratory system modeling in adults with cystic fibrosis.

BACKGROUND: The Forced Oscillation Technique (FOT) has the potential to increase our knowledge about the biomechanical changes that occur in Cystic Fibrosis (CF). Thus, the aims of this study were to investigate changes in the resistive and reactive properties of the respiratory systems of adults with CF. METHODS: The study was conducted in a group of 27 adults with CF over 18 years old and a control group of 23 healthy individuals, both of which were assessed by the FOT, plethysmography and spirometry. An equivalent electrical circuit model was also used to quantify biomechanical changes and to gain physiological insight. RESULTS AND DISCUSSION: The CF adults presented an increased total respiratory resistance (p < 0.0001), increased resistance curve slope (p<0.0006) and reduced dynamic compliance (p<0.0001). In close agreement with the physiology of CF, the model analysis showed increased peripheral resistance (p<0.0005) and reduced compliance (p < 0.0004) and inertance (p<0.005). Significant reasonable to good correlations were observed between the resistive parameters and spirometric and plethysmographic indexes. Similar associations were observed for the reactive parameters. Peripheral resistance, obtained by the model analysis, presented reasonable (R=0.35) to good (R=0.64) relationships with plethysmographic parameters. CONCLUSIONS: The FOT adequately assessed the biomechanical changes associated with CF. The model used provides sensitive indicators of lung function and has the capacity to differentiate between obstructed and non-obstructed airway conditions. The FOT shows great potential for the clinical assessment of respiratory mechanics in adults with CF.

Burkholderia cepacia complex: clinical course in cystic fibrosis patients.

BACKGROUND: Pulmonary deterioration after B.cepacia complex (BCC) colonization has a heterogeneous pattern. The aim was to investigate the clinical outcome of BCC colonization in CF patients chronically colonized with P. aeruginosa. METHODS: CF patients chronically colonized with P. aeruginosa were divided into three groups: intermittent (I), chronic (II) and no colonization (III) with BCC. Body mass index (BMI) percentile and spirometric parameters were analyzed at three different times in each group. RESULTS: Fifty-six patients chronically colonized with P. aeruginosa were included. Of these, 27 also had evidence of BCC colonization (13 intermittent and 14 chronic). BMI percentile was significantly lower among patients chronically colonized by both P. aeruginosa and BCC. Mean values of FEV1 and FVC % were also significantly lower in these patients, both at the time of chronic BCC colonization and 24 months forward. CONCLUSIONS: Chronic BCC colonization is associated with significant loss of lung function. Lower BMI might be a risk factor for chronic BCC colonization, preceding these events.

Fatal Burkholderia gladioli infection misidentified as Empedobacter brevis in a lung transplant recipient with cystic fibrosis.

Data describing the risk of lung transplantation (LT), clinical features, and outcomes of patients with cystic fibrosis (CF) infected with Burkholderia gladioli are limited. Herein, we report a case of disseminated B. gladioli infection characterized by bacteremia, necrotizing pneumonia, lung abscess, and empyema in a lung transplant recipient with CF, highlight the importance of accurate microbiological identification, and review published outcomes of LT in CF patients infected with B. gladioli, which include cases of pneumonia, tracheobronchitis, bacteremia, and abscesses, and demonstrate an all-cause 1-year mortality of approximately 23%, often after combined medical and surgical treatment.

Mucoid and nonmucoid Burkholderia cepacia complex bacteria in cystic fibrosis infections.

RATIONALE: infection with Burkholderia cepacia complex (BCC) bacteria in cystic fibrosis (CF) is associated with an unpredictable rate of pulmonary decline. Some BCC, but not others, elaborate copious mucoid exopolysaccharide, endowing them with a gross mucoid phenotype, the clinical significance of which has not been described. OBJECTIVES: to determine whether there was a correlation between bacterial mucoid phenotype, as assessed in a semiquantitative manner from plate culture, and severity of disease as assessed by the rate of decline in lung function. METHODS: we performed a retrospective clinical review of 100 patients with CF attending the Vancouver clinics between 1981 and 2007 and analyzed the rate of lung function decline (% predicted FEV(1)). MEASUREMENTS AND MAIN RESULTS: patients infected exclusively with nonmucoid BCC had a more rapid decline in lung function (annual FEV(1) change, -8.51 ± 2.41%) than those infected with mucoid bacteria (-3.01 ± 1.09%; P < 0.05). Linear mixed-effects data modeling revealed a statistically significant inverse association between semiquantitative mucoid exopolysaccharide production and rate of decline of lung function. In vitro incubation of BCC with ceftazidime and ciprofloxacin but not meropenem caused conversion of BCC from mucoid to nonmucoid. CONCLUSIONS: our data suggest an inverse correlation between the quantity of mucoid exopolysaccharide production by BCC bacteria and rate of decline in CF lung function. Certain antibiotics may induce a change in bacterial morphology that enhances their virulence. A simple in vitro test of bacterial mucoidy may be useful in predicting the rate of decline of respiratory function in CF.

Quantitative proteomics (2-D DIGE) reveals molecular strategies employed by Burkholderia cenocepacia to adapt to the airways of cystic fibrosis patients under antimicrobial therapy.

Chronic respiratory infections caused by Burkholderia cenocepacia in patients with cystic fibrosis (CF) are characterized by low responsiveness to antibiotic therapy and, in general, to a more rapid decline of lung function. To get clues into the molecular mechanisms underlying the adaptive strategies employed to deal with the stressing conditions of the CF lung including antibiotic therapy, quantitative proteomics (2-D DIGE) was used to compare the expression programs of two clonal isolates retrieved from a chronically infected CF patient. Isolate IST439 was the first bacterium recovered while the clonal variant IST4113 was obtained after 3 years of persistent infection and intravenous therapy with ceftazidime/gentamicin. This isolate exhibits higher resistance levels towards different classes of antimicrobials. Proteins of the functional categories Energy metabolism, Translation, Nucleotide synthesis, Protein folding and stabilization are more abundant in IST4113, compared with IST439, suggesting an increased protein synthesis, DNA repair and stress resistance in IST4113. The level of proteins involved in peptidoglycan, membrane lipids and lipopolysaccharide synthesis is also altered and proteins involved in iron binding and transport are more abundant in IST4113. The quantitative comparison of the two proteomes suggests a genetic adaptation leading to increased antimicrobial resistance and bacterial persistence in the CF airways.

Burkholderia cenocepacia phenotypic clonal variation during a 3.5-year colonization in the lungs of a cystic fibrosis patient.

Chronic lung infection is the major cause of morbidity and premature mortality in cystic fibrosis (CF) patients. Bacteria of the Burkholderia cepacia complex are the most threatening pathogens in CF, and a better understanding of how these bacteria adapt to the CF airway environment and resist the host defense mechanisms and therapeutically administered antibiotics is crucial. To provide clues to the adaptive strategies adopted by Burkholderia cenocepacia during long-term colonization, we carried out a phenotypic assessment of 11 clonal variants obtained at the major Portuguese CF Center in Lisbon from sputa of the same CF patient during 3.5 years of colonization of the lungs, until the patient's death with cepacia syndrome. Phenotypic characterization included susceptibility assays against different classes of antimicrobials and characterization of cell motility, cell hydrophobicity and zeta potential, colony and cell morphology, fatty acid composition, growth under iron limitation/load conditions, exopolysaccharide production, and size of the biofilms formed. The results suggest the occurrence of clonal expansion during long-term colonization. For a number of the characteristics tested, no isolation time-dependent consistent alteration pattern could be identified. However, the values for antimicrobial susceptibility and swarming motility for the first B. cenocepacia isolate, thought to have initiated the infection, were consistently above those for the clonal variants obtained during the course of infection, and the opposite was found for the zeta potential. The adaptive strategy for long-term colonization, described here for the first time, involved the alteration of membrane fatty acid composition, in particular a reduction of the degree of fatty acid saturation, in the B. cenocepacia variants retrieved, along with the deterioration of pulmonary function and severe oxygen limitation.

The role of mucoidy in virulence of bacteria from the Burkholderia cepacia complex: a systematic proteomic and transcriptomic analysis.

Bacteria of the Burkholderia cepacia complex (BCC) are associated with severe infection in cystic fibrosis. Recent evidence shows that the mucoid phenotype is common in BCC bacteria; however, during chronic infection, transitions from the mucoid to nonmucoid morphology have been shown to take place. Here we use RNA microarray and proteomic isobaric tagging relative and absolute quantitation technologies to gain insight into a pair of mucoid and nonmucoid isolates of B. cenocepacia obtained from a chronically infected patient with cystic fibrosis in the year prior to her death. During chronic infection, the mucoid isolate lost the B. cepacia epidemic strain marker and acquired a mutation in the cepR gene. In the nonmucoid isolate, we observed overexpression at both the RNA and protein level of several described putative virulence factors, including a nematocidal protein AidA and the oxidative stress response protein AhpC. We show that this translates into increased resistance to oxidative stress in the nonmucoid isolate, a key microbial determinant for resistance against phagocytic cell killing. These data illuminate the biological differences between mucoid and nonmucoid BCC bacteria, provide targets for elucidating the genetic control of exopolysaccharide production in the BCC, and highlight that chronic infection can produce both genetically and phenotypically distinct microbial variants in the cystic fibrosis lung.

Potential mechanisms underlying the acute lung dysfunction and bacterial extrapulmonary dissemination during Burkholderia cenocepacia respiratory infection.

BACKGROUND: Burkholderia cenocepacia, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity of B. cenocepacia from the ET-12 lineage, that has been linked to the cepacia syndrome, and four clinical isolates recovered from CF patients with mild clinical courses were analysed in both in vitro and in vivo assays. METHODS: B. cenocepacia-infected BEAS-2B epithelial respiratory cells were used to investigate the bacterial cytotoxicity assessed by the flow cytometric detection of cell staining with propidium iodide. Bacteria-induced procoagulant activity in cell cultures was assessed by a colorimetric assay and by the flow cytometric detection of tissue factor (TF)-bearing microparticles in cell culture supernatants. Bronchoalveolar lavage fluids (BALF) from intratracheally infected mice were assessed for bacterial proinflammatory and procoagulant activities as well as for bacterial cytotoxicity, by the detection of released lactate dehydrogenase. RESULTS: ET-12 was significantly more cytotoxic to cell cultures but clinical isolates Cl-2, Cl-3 and Cl-4 exhibited also a cytotoxic profile. ET-12 and CI-2 were similarly able to generate a TF-dependent procoagulant environment in cell culture supernatant and to enhance the release of TF-bearing microparticles from infected cells. In the in vivo assay, all bacterial isolates disseminated from the mice lungs, but Cl-2 and Cl-4 exhibited the highest rates of recovery from mice livers. Interestingly, Cl-2 and Cl-4, together with ET-12, exhibited the highest cytotoxicity. All bacteria were similarly capable of generating a procoagulant and inflammatory environment in animal lungs. CONCLUSION: B. cenocepacia were shown to exhibit cytotoxic and procoagulant activities potentially implicated in bacterial dissemination into the circulation and acute pulmonary decline detected in susceptible CF patients. Improved understanding of the mechanisms accounting for B. cenocepacia-induced clinical decline has the potential to indicate novel therapeutic strategies to be included in the care B. cenocepacia-infected patients.

Burkholderia cepacia complex: impact on the cystic fibrosis lung lesion.

Burkholderia cepacia complex (Bcc) is a group of phenotypically similar nonfermenting, aerobic, gram-negative rods that infect 2 to 8% of patients with cystic fibrosis (CF). Bcc comprises several distinct species (formerly termed genomovars). Infection with Bcc has been associated with worse outcomes and survival in CF patients. Additionally, colonization with Bcc has been associated with heightened mortality following lung transplantation. Currently, the role of lung transplantation in Bcc-infected patients remains controversial. Antimicrobial therapy for Bcc is often ineffective because most strains are multidrug resistant. Combination therapy with two or three agents is typically administered, but optimal therapy has not been elucidated. Certain strains (particularly B. cenocepacia) have been associated with heightened transmissibility and virulence. Several genes or gene products have important roles in virulence, persistence of the organism(s), and transmissibility. The incidence of Bcc and specific species fluctuates over time and between centers. Bcc can be transmitted by social contact, and several major epidemic strains have disseminated globally. Strict infection control measures, including cohorting (segregation) of infected patients, dramatically curtail transmission and have become the standard of care, but such measures cause social isolation, stigmatism, and psychological impact among infected patients.

Head Tilt in Immunodeficient Mice Due to Contamination of Drinking Water by Burkholderia gladioli.

Immunodeficient mice in multiple holding rooms presented with head tilt, circling, spinning when picked up by the tail, dehydration, and lethargy. Burkholderia gladioli, a plant pathogen, was identified as the causative agent. Environmental testing revealed the presence of B. gladioli within the automatic watering system, water bottles, and sipper tubes. Here we describe steps taken to reduce the presence of this organism within the automatic watering system and water bottles. Facilities housing immunodeficient mice should take measures to minimize the accumulation of biofilm within their water-supply systems.

[The clinical course of Burkholderia cepacia complex bacteria respiratory infection in cystic fibrosis patients].

Bacteria of the Burkholderia cepacia complex (Bcc), a group of nine related species, are opportunistic pathogens in cystic fibrosis (CF) patients, associated with a poor prognosis and patient-to-patient transmissibility. The pulmonary deterioration in Bcc-colonised/ infected patients has a heterogeneous pattern leading, sometimes, to a fulminant development - the cepacia syndrome. To evaluate the relationship between colonisation/ infection by the different Bcc species and the clinical course, the authors carried out a retrospective study of 31 CF patients with Bcc bacteria isolations followed at Hospital de Santa Maria from January 1995 to March 2006. Patients were categorised into two groups: Group I, with intermittent isolations and Group II with chronic isolations. The prevalence of Bcc species was as follows: B. cepacia 57%, B. cenocepacia 43%, B. multivorans 7%, B. stabilis 13%. Three of the patients died of cepacia syndrome. The species B. cepacia and B. stabilis, usually less frequent in CF populations of Europe and America, were isolated in a considerable percentage of the patients examined. No correlation could be established between the species and the clinical outcome. Deteriorated but not stable patients from group II, whose lung function and pulmonary exacerbation caused hospitalisation could be retrospectively analysed, exhibited significant differences in the number of hospitalisations and pulmonary function (FEV1) in the year prior to and the years following Bcc isolation. Based on the available data, it is not currently possible to outline preventive measures through the molecular characterisation of Bcc isolates, reinforcing the notion that the recommended control measures must be followed.

Estimating in vivo airway surface liquid concentration in trials of inhaled antibiotics.

Antibiotic drugs exhibit concentration dependence in their efficacy. Therefore, ensuring appropriate concentration of these drugs in the relevant body fluid is important for obtaining the desired therapeutic and physiological action. Until recently there had been no suitable method available to measure or estimate concentration of drugs in the human airways resulting from inhaled aerosols or to determine the amount of inhaled antibiotics required to ensure minimum inhibitory concentration of a drug in the airway surface liquid (ASL). In this paper a numerical method is used for estimating local concentration of inhaled pharmaceutical aerosols in different generations of the human tracheobronchial airways. The method utilizes a mathematical lung deposition model to estimate amounts of aerosols depositing in different lung generations, and a recent ASL model along with deposition results to assess the concentration of deposited drugs immediately following inhalation. Examples of concentration estimates for two case studies: one for the antibiotic tobramycin against Pseudomonas aeruginosa, and another for taurolidine against Burkholderia cepacia are presented. The aerosol characteristics, breathing pattern and properties of nebulized solutions were adopted from two recent clinical studies on efficacy of these drugs in cystic fibrosis (CF) patients and from other sources in the literature. While the clinically effective tobramycin showed a concentration higher than the required in vivo concentration, that for the ineffective taurolidine was found to be below the speculated required in vivo concentration. Results of this study thus show that the mathematical ASL model combined with the lung deposition model can be an effective tool for helping decide the optimum dosage of inhaled antibiotic drugs delivered during human clinical trials.

Burkholderia cepacia complex: virulence characteristics, importance and relationship with cystic fibrosis.

BACKGROUND: Burkholderia cepacia has been described as a cause of opportunist infections in patients with immune deficiency because of the high transmission rates. Actually the B. cepacia is subdivided in nine different genomic species that show morphological similarity, called genomovars. High mortality rates have been associated with infections caused by genomovars in susceptible patients; antibiotics are not efficient because of the high resistance level and genomic mutability. Little is known about the epidemiological traits of this bacterium; therefore, their isolation remains a relevant technical problem. AIMS: The objective of this review is to describe Burkholderia cepacia as a bacterial complex with high pathogenicity and variability of habitats. MATERIALS AND METHODS: A systematic search was realized using the international bibliographic databanks SCIELO, HIGHWIRE, PUBMED, SCIRUS and LILACS to provide a useful and practical review for the health workers that do not know this microorganism. CONCLUSIONS: Today, B. cepacia complex is a very important problem for the acquired immunodeficiency syndrome and cystic fibrosis patients. The immunodeficiency caused by these diseases is a positive factor for this microorganism to infect and kill these patients. Therefore, this opportunistic pathogen should be pointed out as a risk to these patients and hospitals all over the world must be prepared to detect and combat this bacterium.

Comparison by electron microscopy of intracellular events and survival of Burkholderia pseudomallei in monocytes from normal subjects and patients with melioidosis.

INTRODUCTION: Burkholderia pseudomallei (B. pseudomallei) has been shown to persist intracellularly in patients with melioidosis, until reactivated by decreasing immunocompetence. We have previously demonstrated by transmission electron microscopy, the internalisation of B. pseudomallei by human macrophages and the occurrence of phagosome-lysosome fusion. METHODS: Phagocytosis and electron microscopy were used to compare the rate of phagosome-lysosome fusion and the intracellular survival of B. pseudomallei using monocytes obtained from five patients with melioidosis and five normal healthy adults. RESULTS: Ingested bacilli were seen in various stages of degradation, with a few remaining viable within phagolysosomes, and the proliferation of these viable bacteria was observed. Phagocytosis of B. pseudomallei by normal macrophages was two-fold higher than uptake by the melioidosis macrophages (p-value is less than 0.001). Three times more phagolysosomes were present in the normal macrophages, indicating that fusion occurred slowly and inefficiently in the melioidosis macrophages (p-value is less than 0.001), resulting in higher number of organisms within the melioidosis macrophages (p-value is less than 0.001). Both variables were inversely related to each other. CONCLUSION: Our observations suggest that phagolysosome fusion occurred slowly and inefficiently in monocytes of patients with melioidosis, leading to an increased number of intracellular organisms compared to monocytes obtained from healthy donors.

Virulence associated with outbreak-related strains of Burkholderia cepacia complex among a cohort of patients with bacteremia.

The Burkholderia cepacia complex includes 9 genomovars. The relative virulence of each is unknown. Host and pathogen features associated with mortality were evaluated among patients with B. cepacia complex bacteremia. Cases were ascertained through review of blood culture results for the period of May 1996 through May 2002. Isolates were identified to species level with 16S rDNA and recA-based species-specific polymerase chain reaction analyses and recA restriction fragment-length polymorphism. Strain typing was performed with pulsed-field gel electrophoresis. Fifty-three patients with B. cepacia complex bacteremia were identified; only 9 (17%) had cystic fibrosis. Twenty-five patients (47%) died within 14 days of bacteremia. After controlling for comorbid conditions and therapeutic interventions, 2 outbreak-related strains of Burkholderia cenocepacia (genomovar III) were associated with 14-day mortality (odds ratio, 5.5; 95% confidence interval, 1.20-25.02). B. cenocepacia is an emerging nosocomial pathogen. Certain strains are associated with an enhanced capacity for interpatient spread and poor outcome.

Clinical outcome of Burkholderia cepacia complex infection in cystic fibrosis adults.

BACKGROUND: The Burkholderia cepacia complex (BCC) is one of the most important groups of organisms infecting cystic fibrosis (CF) patients. The aim of the study was to examine how infection with BCC affects clinical outcome. METHODS: Nineteen CF adults infected with BCC and 19 controls infected with Pseudomonas aeruginosa were studied over a 4-year period. The best forced expiratory volume in 1 s (FEV(1)) and body mass index (BMI) for each year were recorded and annual rate of decline calculated. RESULTS: The BCC infected group displayed a significantly greater reduction of FEV(1) and BMI compared to the P. aeruginosa infected group (p=0.001 and p=0.009, respectively). Sixteen patients infected with a single Burkholderia cenocepacia strain had a significantly greater rate of FEV(1) decline compared to those infected with Burkholderia multivorans (n=3) or P. aeruginosa (p=0.01 and p<0.0001, respectively). The rate of BMI decline was significantly greater in patients infected with B. cenocepacia compared to those with P. aeruginosa (p=0.007), but not significantly different in those with B. multivorans (p=0.29). CONCLUSION: BCC infection is associated with an accelerated decline in pulmonary function and BMI. Infection with a single B. cenocepacia strain was associated with a more rapid decline in lung function than those infected with either B. multivorans or P. aeruginosa.

Pulmonary function, serum markers of inflammation, and IgG antibodies to core lipopolysaccharide of Burkholderia cepacia in adults with cystic fibrosis, following colonization with Burkholderia cepacia.

Eight patients with cystic fibrosis [CF] colonized with Pseudomonas aeruginosa (P. aeruginosa) had serial lung function, peripheral blood inflammatory markers, and serum IgG antibodies to Burkholderia cepacia (B. cepacia) lipopolysaccharide measured in the months preceding and following colonisation with B. cepacia. One patient experienced a fall in FEV(1) from 33% to 19% of predicted values, coinciding with the first sputum isolation of B. cepacia, and he died 12 weeks later. He had a rise in inflammatory markers preterminally, and this change was refractory to antibiotic therapy. There was no significant fall in FEV(1) % of predicted values in the remaining seven patients, and no significant changes in their serum markers of inflammation following colonization with B. cepacia over a median (range) period of 10.9 (7.3-12.0) months.

Infection of Burkholderia cepacia induces homeostatic responses in the host for their prolonged survival: the microarray perspective.

Burkholderia cepacia is an opportunistic human pathogen associated with life-threatening pulmonary infections in immunocompromised individuals. Pathogenesis of B. cepacia infection involves adherence, colonisation, invasion, survival and persistence in the host. In addition, B. cepacia are also known to secrete factors, which are associated with virulence in the pathogenesis of the infection. In this study, the host factor that may be the cause of the infection was elucidated in human epithelial cell line, A549, that was exposed to live B. cepacia (mid-log phase) and its secretory proteins (mid-log and early-stationary phases) using the Illumina Human Ref-8 microarray platform. The non-infection A549 cells were used as a control. Expression of the host genes that are related to apoptosis, inflammation and cell cycle as well as metabolic pathways were differentially regulated during the infection. Apoptosis of the host cells and secretion of pro-inflammatory cytokines were found to be inhibited by both live B. cepacia and its secretory proteins. In contrast, the host cell cycle and metabolic processes, particularly glycolysis/glycogenesis and fatty acid metabolism were transcriptionally up-regulated during the infection. Our microarray analysis provided preliminary insights into mechanisms of B. cepacia pathogenesis. The understanding of host response to an infection would provide novel therapeutic targets both for enhancing the host's defences and repressing detrimental responses induced by the invading pathogen.

Natural infection of Burkholderia pseudomallei in an imported pigtail macaque (Macaca nemestrina) and management of the exposed colony.

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.