Hyperbaric oxygen treatment increases killing of aggregating Pseudomonas aeruginosa isolates from cystic fibrosis patients

Background: Pseudomonas aeruginosa is a major pathogen of the chronic lung infections in cystic fibrosis (CF) patients. These persistent bacterial infections are characterized by bacterial aggregates with biofilm-like properties and are treated with nebulized or intravenous tobramycin in combination with other antibiotics. However, the chronic infections are close to impossible to eradicate due to reasons that are far from fully understood. Recent work has shown that re‑oxygenation of hypoxic aggregates by hyperbaric oxygen (O₂) treatment (HBOT: 100% O₂ at 2.8 bar) will increase killing of aggregating bacteria by antibiotics. This is relevant for treatment of infected CF patients where bacterial aggregates are found in the endobronchial secretions that are depleted of O₂ by the metabolism of polymorphonuclear leukocytes (PMNs). The main objective of this study was to investigate the effect of HBOT as an adjuvant to tobramycin treatment of aggregates formed by P. aeruginosa isolates from CF patients.Methods: The effect was tested using a model with bacterial aggregates embedded in agarose. O₂ profiling was used to confirm re‑oxygenation of aggregates.Results: We found that HBOT was able to significantly enhance the effect of tobramycin against aggregates of all the P. aeruginosa isolates in vitro. The effect was attributed to increased O₂ levels leading to increased growth and thus increased uptake of and killing by tobramycin.Conclusions: Re‑oxygenation may in the future be a clinical possibility as adjuvant to enhance killing by antibiotics in cystic fibrosis lung infections.     

Multiple antibiotic-resistant Pseudomonas aeruginosa and lung function decline in patients with cystic fibrosis

BackgroundThe goal of this study was to determine the association of multiple antibiotic-resistant Pseudomonas aeruginosa (MARPA) acquisition with lung function decline in patients with cystic fibrosis (CF).MethodsUsing data from Epidemiologic Study of Cystic Fibrosis (ESCF), we identified patients with spirometry data and MARPA, defined as PA (1) resistant to gentamicin and either tobramycin or amikacin, and (2) resistant to ≥ 1 antipseudomonal beta lactam. MARPA had to be detected in a respiratory culture after ≥ 2 years of PA-positive but MARPA-negative respiratory cultures. Multivariable piecewise linear regression was performed to model the annual rate of decline in forced expiratory volume in 1 second (FEV₁) % predicted 2 calendar years before and after the index year of MARPA detection, adjusting for patient characteristics and CF therapies.ResultsIn total, 4349 patients with chronic PA and adequate PFT data were identified; 1111 subsequently developed MARPA, while 3238 patients were PA positive but MARPA negative. Compared with patients who did not acquire MARPA, MARPA-positive patients had lower FEV₁ and received more oral (p < 0.013) and inhaled (p < 0.001) antibiotic therapy. Mean FEV₁ decline did not change significantly after MARPA detection (− 2.22% predicted/year before detection and − 2.43 after, p = 0.45). There was no relationship between persistent infection or FEV₁ quartile and FEV₁ decline.ConclusionsNewly detected MARPA was not associated with a significant change in the rate of FEV₁ decline. These results suggest that MARPA is more likely to be a marker of more severe disease and more intensive therapy, and less likely to be contributing independently to more rapid lung function decline.     

Comparison of real time diagnostic chemistries to detect Pseudomonas aeruginosa in respiratory samples from cystic fibrosis patients

BackgroundEarly eradication therapy is key to keeping the airways Pseudomonas aeruginosa infection-free and rapid identification is essential.MethodsWe used rapid DNA extraction and qPCR assays to detect bacterial, P. aeruginosa and strain-specific targets in samples using two qPCR chemistries. Using 459 respiratory samples from adult and children CF patients, we compared two qPCR methods to culture-based methods in terms of sensitivity and time to result.ResultsFor adult samples, there was 100% concordance between methods. There was no clear pattern in fluctuations in P. aeruginosa number during exacerbation. In child samples, qPCR methods identified additional P. aeruginosa positive samples. The time-to-result was reduced by over 24 h and copy number and colony forming unit could differ dramatically in some samples.ConclusionIf adopted, these methods could significantly improve early P. aeruginosa detection in diagnostic laboratories and therefore play a pivotal role in prolonging infection-free airways in CF patients.     

Survey of acute renal failure in patients with cystic fibrosis in the UK

BACKGROUND: There has been a recent increase in the number of reported cases of acute renal failure (ARF) in cystic fibrosis (CF). A study was undertaken to determine the incidence risk of ARF in patients with CF in the UK and to identify possible aetiological factors. METHODS: All doctors working at UK CF centres were asked if they had been involved with the management of a patient with CF who had developed ARF. Those responding positively were asked to request informed consent for entry into the study and the patient's case notes were then reviewed. The analysis was restricted to patients developing ARF between 1997 and 2004. A second questionnaire sought information on aminoglycoside prescribing practice. RESULTS: Responses were received from 55 of 56 centres with 64 reports, 9 of which were duplicates, leaving 55 cases. Consent was obtained for data extraction in 26 cases, of which 24 fitted the criteria for ARF (verified data). Median age at presentation with ARF was 9.7 years (range 0.4-31.8) and 12 cases were male. The incidence risk of ARF was 4.6 (verified data) to 10.5 cases (all data)/10,000 CF patients/year. In 21 cases (88%) an aminoglycoside was prescribed at onset of ARF or in the preceding week; 16 (76%) of those receiving an aminoglycoside had gentamicin. A renal biopsy was performed in 7 cases and histological examination revealed acute tubular necrosis in 6, all of whom had received gentamicin. Renal dialysis was required in 13 cases (54%). Complete recovery was seen in 22/24 patients (92%). CONCLUSIONS: ARF is increasingly being recognised in patients with CF. There is significant morbidity with most patients requiring dialysis. This study implicates intravenous aminoglycosides, particularly gentamicin, in the aetiology of ARF in CF.     

Transmission of Pseudomonas aeruginosa epidemic strain from a patient with cystic fibrosis to a pet cat

Chronic infection with Pseudomonas aeruginosa is common in cystic fibrosis (CF) and certain strains are more transmissible and virulent than others. Of these, the Liverpool Epidemic Strain (LES) is highly transmissible and cross infection has been reported between patients with CF and healthy non-CF relatives. However, the risk of transmission from humans to animals is unknown. The first report of interspecies transmission of the LES strain of P aeruginosa from an adult patient with CF to a pet cat is described. This development further complicates the issue of infection control policies required to prevent the spread of this organism.     

Extrapulmonary sites of Pseudomonas aeruginosa in adults with cystic fibrosis.

BACKGROUND: Pseudomonas aeruginosa infection is seldom eradicated in patients with cystic fibrosis despite intensive antipseudomonal treatment. Upper airway sites of infection may contribute to perpetuation of lower airways infection. This study was designed to find out which extrapulmonary sites are infected and whether the strains at these sites are identical to those in the lungs. METHODS: Sputum and upper airway samples from 42 patients were cultured for P aeruginosa and stool samples from 20 patients were also tested. Nineteen isolates from sputum and extrapulmonary sites from four patients were genotyped with the pCM tox probe. RESULTS: P aeruginosa was isolated from the sputum of 36 patients, 34 of whom had infection in the upper airways. Six of the 20 patients tested were positive for P aeruginosa in the stool. The nasopharynx was colonised in 30 patients, the oropharynx in 29, the middle meatus in 13, the external nares in six, and the inferior turbinate in four. Three of four patients tested had the same strain of P aeruginosa (a different one in each individual) in the sputum and the upper airways, and in two of the three the stool isolate was a different strain. CONCLUSION: Most adults with cystic fibrosis and P aeruginosa pulmonary infection have upper airway reservoirs of the organism and strains from these sites are identical to those in the lungs.     

Increased serum concentration of G-CSF in cystic fibrosis patients with chronic Pseudomonas aeruginosa pneumonia.

BACKGROUND: Chronic Pseudomonas aeruginosa lung infection is the major reason for premature death in patients with cystic fibrosis (CF). Infected patients experience a progressive deterioration of the lung tissue caused by a persistent accumulation of PMNs. We investigated if the pulmonary accumulation of PMNs is reflected as a migration of PMNs through the blood in chronically infected CF patients. METHODS: Blood and sputum samples from 37 stable, chronically (CF+P) and 6 non-infected (CF-P) CF patients without exacerbations were compared using FACS, leukocyte counting, and ELISA. Within the CF+P patients, the blood parameters were compared to the lung function (FEV1 and FVC) and to the sputum. Similar measurements were performed on 15 chronically infected CF patients before and after elective antibiotic treatment. RESULTS: In the CF+P patients the concentration of G-CSF in the sera and PMNs in the blood was increased and correlated to poor lung function. However, only the concentration of G-CSF in the sera was correlated to the concentration of TNF-alpha in the sputum. After the antibiotic treatment, the lung function was improved and the concentration of PMNs in the blood and G-CSF in the sera was reduced. CONCLUSION: G-CSF in the sera may contribute to the pulmonary inflammation in CF patients with chronic P. aeruginosa lung infection by regulating the number of PMNs available for migration and may be considered as an indicator of clinical status.     

A novel chromogenic medium for isolation of Pseudomonas aeruginosa from the sputa of cystic fibrosis patients

BackgroundA novel chromogenic medium for isolation and identification of Pseudomonas aeruginosa from sputa of cystic fibrosis (CF) patients was evaluated and compared with standard laboratory methods.MethodsOne hundred sputum samples from distinct CF patients were cultured onto blood agar (BA), Pseudomonas CN selective agar (CN) and a Pseudomonas chromogenic medium (PS-ID). All Gram-negative morphological variants from each medium were subjected to antimicrobial susceptibility testing, and identification using a combination of biochemical and molecular methods.ResultsP. aeruginosa was isolated from 62 samples after 72 h incubation. Blood agar recovered P. aeruginosa from 56 samples (90.3%) compared with 59 samples (95.2%) using either CN or PS-ID. The positive predictive value of PS-ID (98.3%) was significantly higher than growth on CN (88.5%) for identification of P. aeruginosa (P < 0.05).ConclusionsPS-ID is a promising medium allowing for the isolation and simultaneous identification of P. aeruginosa from sputa of CF patients.     

Genotype based evaluation of Pseudomonas aeruginosa eradication treatment success in cystic fibrosis patients

BackgroundLongitudinal data regarding the genotypes of Pseudomonas aeruginosa isolates after eradication treatment are limited. We followed cystic fibrosis patients after a first ever isolation of P. aeruginosa and evaluated the P. aeruginosa-free time period after eradication therapy.MethodsBetween January 2003 and December 2008 respiratory samples were cultured prospectively from 41 patients with a first ever P. aeruginosa isolate. Twenty five patients had at least one subsequent isolate. Treatment efficacy was assessed based on the time to a second isolation and on comparison of the RAPD genotypes of the P. aeruginosa isolates.ResultsEleven patients became chronically colonized during the study period. For ten of these the second isolate had the same genotype as the first isolate. Moreover, these patients had a significantly shorter P. aeruginosa-free time interval between the first ever and the second isolate compared to the 14 not chronically colonized patients (median 0 months versus 7.5 months, p < 0.05).ConclusionOur results indicate that the presence of a genotypically identical subsequent P. aeruginosa isolate and/or a short P. aeruginosa-free time interval after treatment are ominous signs and might be useful additional tools to predict impending chronic colonization. Current routine bacteriological methods for the detection of P. aeruginosa may lack the sensitivity to discriminate between true eradication and low bacterial persistence.     

Targeted exhaled breath analysis for detection of Pseudomonas aeruginosa in cystic fibrosis patients

BackgroundPseudomonas aeruginosa (PA) is an important respiratory pathogen for cystic fibrosis (CF) patients. Routine microbiology surveillance is time-consuming, and is best performed on expectorated sputum. As alternative, volatile organic compounds (VOCs) may be indicative of PA colonisation. In this study, we aimed to identify VOCs associated with PA in literature and perform targeted exhaled breath analysis to recognize PA positive CF patients non-invasively.MethodsThis study consisted of 1) a literature review to select VOCs of interest, and 2) a cross-sectional CF study. Definitions used: A) PA positive, PA culture at visit/chronically; B) PA free, no PA culture in ≥12 months. Exhaled VOCs were identified via quadrupole MS. The primary endpoint was the area under the receiver operating characteristics curve (AUROCC) of individual VOCs as well as combined VOCs against PA culture.Results241 VOCs were identified in literature, of which 56 were further evaluated, and 13 could be detected in exhaled breath in our cohort. Exhaled breath of 25 pediatric and 28 adult CF patients, PA positive (n=16) and free (n=28) was available. 3/13 VOCs were significantly (p<0.05) different between PA groups in children; none were in adults. Notably, a composite model based on 5 or 1 VOC(s) showed an AUROCC of 0.86 (CI 0.71–1.0) and 0.87 (CI 0.72–1.0) for adults and children, respectively.ConclusionsTargeted VOC analysis appears to discriminate children and adults with and without PA positive cultures with clinically acceptable sensitivity values.     

Spread of colistin resistant non-mucoid Pseudomonas aeruginosa among chronically infected Danish cystic fibrosis patients

BackgroundColistin resistant Pseudomonas aeruginosa have rarely been reported in cystic fibrosis (CF) patients.MethodsWe performed a 17-year prospective study on colistin susceptibility and compared our findings with clinical variables.ResultsThe first outbreak started in 1995 and lasted 5 years. It involved 27 CF patients who had inhaled colistin twice daily for a median of 10 years. Colistin resistant isolates persisted in individual patients for a median of 75 days after colistin was withdrawn.A second outbreak started in 2004. It involved 40 patients, 17 of whom were the same as in the first outbreak. Most resistant isolates belonged to two major clones that had similar genotypes in the two outbreaks. The P. aeruginosa isolates were all non-mucoid and they appeared in a group of chronically infected patients that had been admitted to the same ward for antibiotic treatment and had been followed at the same week-days in the outpatient clinic.Patients were individually isolated to avoid cross-infection and colistin inhalation was avoided in the CF outpatient clinic and in the ward after both outbreaks. Since 2004, no further spread has been observed.ConclusionIt is important that the colistin resistant clones do not spread to non-infected patients since colistin is an important antibiotic for eradication of initial and intermittent P. aeruginosa colonisation.     

Pseudomonas aeruginosa antimicrobial susceptibility test (AST) results and pulmonary exacerbation treatment responses in cystic fibrosis

Background Antimicrobial susceptibility testing (AST) of bacterial isolates is a time- and resource-intensive procedure recommended by cystic fibrosis (CF) treatment guidelines for antimicrobial selection for pulmonary exacerbation (PEx) treatment.Methods We studied relationships between Pseudomonas aeruginosa (Pa) isolate AST results, antipseudomonal PEx treatments, and treatment responses as change in weight and percent predicted forced expiratory volume in 1 s (ppFEV₁) as well as future antimicrobial treatment hazard for PEx occurring at a CF care center from 1999 through 2018. Treatments were categorized by “Pa coverage” as complete (all Pa isolates susceptible by AST to at least one administered agent), none (no isolates susceptible), incomplete (some, but not all isolates susceptible), and indeterminant (administered antipseudomonals not evaluated by AST). Weight and ppFEV₁ responses were compared across Pa coverage categories using unadjusted and adjusted general estimating equations; hazard of future treatment was assessed by Cox and logistic regression.Results Among 3820 antimicrobial PEx treatment events in 413 patients with Pa, 62.6% (2390) had complete Pa coverage; 8.9% (340), 2.4% (99), and 26.2% (1000), had no, incomplete, and indeterminant Pa coverage, respectively. Mean baseline to follow-up weight change was +0.74 kg [95% CI 0.63, 0.86]; ppFEV₁ change was +1.60 [1.29, 1.90]. Pa coverage category was not associated with significant differences in weight or ppFEV₁ change or with future antimicrobial treatment hazard.Conclusions We did not observe superior responses for AST-defined complete Pa coverage treatments versus lesser coverage treatments, suggesting that AST may be of little utility in choosing antimicrobials for CF PEx treatment.     

Effectiveness of a stepwise Pseudomonas aeruginosa eradication protocol in children with cystic fibrosis

Introduction

Antibiotic eradication therapy (AET) for initial Pseudomonas aeruginosa (Pa) infection is standard of care in children with cystic fibrosis (CF), but information is limited on treatment for patients who fail initial AET. The aim of this study was to evaluate the effectiveness of a multi-step protocol for AET for new-onset Pa infections in children with CF.