Sequential genotyping of Pseudomonas aeruginosa from upper and lower airways of cystic fibrosis patients.

A controversy exists concerning the adequate specimen to characterise colonisation of cystic fibrosis (CF) airways by Pseudomonas aeruginosa. Oropharyngeal, sputum and bronchoalveolar lavage samples were evaluated from 38 stable CF patients for the detection of P. aeruginosa, genetically different isolates within the same host and longitudinal variations in the genotype during repeated examinations. Bacterial isolates were typed by pulsed-field gel electrophoresis of deoxyribonucleic acid macrorestriction fragments. Sensitivity, negative and positive predictive values and specificity to detect P. aeruginosa were 35.7, 73.5, 83.3 and 96.2% for oropharyngeal cultures in nonexpectorating patients and 91.7, 94.1, 100 and 100% for sputum cultures from expectorating patients, respectively. Genotypes of Pseudomonas isolates recovered from oropharyngeal swabs and sputum differed to the strains recovered by bronchoscopy in 55% and 40%, respectively. In 62% longitudinal variations in the genotype occurred. One-half of these alterations were detectable by bronchoscopy only. In conclusion, sputum samples were of equal value as specimens from bronchoalveolar lavage to detect Pseudomonas aeruginosa colonisation. Cultures from the oropharynx are not suitable for characterising bacterial conditions in the cystic fibrosis lung. Different genotypes within the same host and longitudinal genetic alterations are common and may be detectable in the bronchoalveolar lavage fluid exclusively.     

Outcomes of adults with cystic fibrosis infected with antibiotic-resistant Pseudomonas aeruginosa

BACKGROUND: Although Pseudomonas aeruginosa is the most common bacterial infection in adults with cystic fibrosis and frequently develops resistance to multiple classes of antibiotics, it has not been determined whether patients with multiple antibiotic-resistant Pseudomonas aeruginosa have worse clinical outcomes than patients with more susceptible strains. OBJECTIVES: This study assessed the impact of multiply-resistant P. aeruginosa on lung function, hospitalizations, antibiotic use, lung transplantation and survival in adults with cystic fibrosis. METHODS: In a cohort study at a university-based adult cystic fibrosis program, 75 consecutive adult cystic fibrosis patients who had P. aeruginosa isolated from sputum cultures were studied over a 4-year period. Outcomes included decline in FEV1, clinic visits, hospitalizations, courses and days of intravenous antibiotics, and lung transplantation. Multiple linear and Poisson regression for repeated measures were used to assess the outcomes. RESULTS: In comparison to patients with susceptible strains, patients with resistant P. aeruginosa had more severe baseline lung disease, more rapid decline in FEV1 (160 ml/year, p = 0.003) and were significantly more likely to undergo lung transplantation (17.6 vs. 0%, p = 0.005). CONCLUSIONS: Infection with multiple-antibiotic-resistant P. aeruginosa is associated with accelerated progression of cystic fibrosis, and has important implications for infection control strategies, antibiotic use and lung transplantation.     

Usefulness of parenteral colistin in treating of lower respiratory infection due to multidrug-resistant Pseudomonas aeruginosa

We report a case of cystic fibrosis in a 19-year-old woman who suffered from frequent exacerbations of lower respiratory infection due to multidrug-resistant Pseudomonas aeruginosa and who was successfully treated with parenteral colistin. Multidrug-resistant Pseudomonas aeruginosa isolated from sputum had become resistant to all parenteral antibiotics commercially available in Japan. She did not show clinical improvement despite treatment with several different combinations of available antibiotics. We therefore obtained parenteral colistin from a pharmacy outside Japan. She responded well to parenteral colistin without apparent side effects such as serious nephrotoxicity or neurotoxicity. Colistin is therefore an important alternative antibiotic for treating multidrug-resistant Pseudomonas aeruginosa and its use should be considered in severe infection. We hope that parenteral colistin will become available in Japan in the near future.     

Adult cystic fibrosis exacerbations and new strains of Pseudomonas aeruginosa

We hypothesized that in adults with cystic fibrosis, the acquisition of a new strain of Pseudomonas aeruginosa may be associated with a pulmonary exacerbation. Eighty-four patients who were chronically infected with P. aeruginosa were prospectively followed from eight centers over a 26-month period. Patients had sputum cultures performed every 3 months while clinically stable and at the time of an exacerbation. Forty patients (48%) had an exacerbation requiring intravenous antibiotics during the study period, and in 36 of these patients, their P. aeruginosa isolates were genetically typeable by pulsed-field gel electrophoresis. In 34 of the 36 patients (94%), P. aeruginosa recovered during clinical stability and at exacerbation were of the same genotype. In only two patients (6%; 95% confidence interval, 0-18%) was a new P. aeruginosa clone cultured during an exacerbation that had not been cultured during clinical stability. There were no significant differences in antibiotic susceptibilities, measured as mean minimal inhibitory concentrations, for isolates retrieved during clinically stable periods compared with isolates retrieved during exacerbations. We conclude that for the majority of adult patients with cystic fibrosis a new pulmonary exacerbation is not caused by the acquisition of a new strain of P. aeruginosa.     

Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis.

Iron availability is critical to many bacteria and increased iron has been described in airway secretions in cystic fibrosis (CF). The main aim of the present study was to assess the relationship between iron in CF sputum and the quantitative bacterial burden. Iron, ferritin and total cell counts (TCC) were assessed in sputum samples obtained from 15 clinically stable CF patients chronically infected with Pseudomonas aeruginosa. Sputum samples were also obtained at the commencement of episodes of acute exacerbation in 10 subjects and analyses were repeated in six of these exacerbation cases after i.v. antibiotic treatment. The relationship between iron indices and the presence of P. aeruginosa, as well as total anaerobic bacterial load, was determined. Sputum was also obtained from 10 CF patients with no evidence of infection with P. aeruginosa and 11 normal healthy controls. Sputum iron, ferritin and TCC were significantly elevated in all CF patients, even in those not infected with P. aeruginosa, compared with healthy controls. There was a strong positive relationship between sputum iron and P. aeruginosa in clinically stable patients, but not in samples obtained during an acute exacerbation. There was no relationship between sputum iron and anaerobic bacterial load. Antibiotic treatment significantly reduced sputum TCC and anaerobic bacterial load, but not iron, ferritin or the presence of P. aeruginosa during an exacerbation. In conclusion, the present study suggests that increased airway iron may be important to Pseudomonas aeruginosa persistence in cystic fibrosis.     

Approach to resistant gram-negative bacterial pulmonary infections in patients with cystic fibrosis

PURPOSE OF THE REVIEW: Patients with cystic fibrosis are living longer with chronic pulmonary bacterial infections. One consequence of antibiotic treatment of these chronic infections has been the increasing prevalence of antibiotic resistance seen in bacterial isolates recovered from patients with cystic fibrosis. RECENT FINDINGS: Bacteria such as Pseudomonas aeruginosa and Burkholderia cepacia are able to acquire antibiotic resistance by either spontaneous mutation or gene transfer via plasmids or integrins. In addition, bacteria survive by forming antibiotic-resistant biofilms within the airways of patients with cystic fibrosis. Therapeutic approaches to dealing with antibiotic-resistant bacterial pulmonary infections include the use of in vitro synergy testing to determine optimal double antibiotic combinations or multiple-combination bactericidal testing to determine bactericidal double and triple antibiotic combinations to use against the bacteria in the clinical setting of acute exacerbations. SUMMARY: Therapy for antibiotic-resistant bacterial infections in cystic fibrosis involves the use of new laboratory methods (synergy testing or multiple-combination bactericidal testing) to optimize antibiotic treatment strategies. Clinical trials are required to address whether treatment guided by susceptibility testing improves clinical outcomes. Future novel approaches will likely include drugs that can disrupt bacterial biofilm formation and the use of cationic peptide antimicrobial compounds.     

A randomised clinical trial of nebulised tobramycin or colistin in cystic fibrosis.

Chronic infection with Pseudomonas aeruginosa is associated with progressive deterioration in lung function in cystic fibrosis (CF) patients. The purpose of this trial was to assess the efficacy and safety of tobramycin nebuliser solution (TNS) and nebulised colistin in CF patients chronically infected with P. aeruginosa. One-hundred and fifteen patients, aged > or = 6 yrs, were randomised to receive either TNS or colistin, twice daily for 4 weeks. The primary end point was an evaluation of the relative change in lung function from baseline, as measured by forced expiratory volume in one second % predicted. Secondary end points included changes in sputum P. aeruginosa density, tobramycin/colistin minimum inhibitory concentrations and safety assessments. TNS produced a mean 6.7% improvement in lung function (p=0.006), whilst there was no significant improvement in the colistin-treated patients (mean change 0.37%). Both nebulised antibiotic regimens produced a significant decrease in the sputum P. aeruginosa density, and there was no development of highly resistant strains over the course of the study. The safety profile for both nebulised antibiotics was good. Tobramycin nebuliser solution significantly improved lung function of patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa, but colistin did not, in this study of 1-month's duration. Both treatments reduced the bacterial load.     

Clinical application of direct sputum sensitivity testing in a severe infective exacerbation of cystic fibrosis

A 16-year-old male with cystic fibrosis (CF) was admitted to hospital with a severe infective exacerbation. Despite standard management, including conventionally selected intravenous antibiotics for Pseudomonas aeruginosa, chest physiotherapy, and institution of noninvasive ventilation (NIV) for progressive hypercapneic respiratory failure, he continued to deteriorate. Direct sputum sensitivity testing (DSST) revealed a novel combination of antibiotics that resulted in a rapid and remarkable clinical improvement. DSST is a form of "whole" sputum sensitivity testing that provides information on antibiotic synergy, and may more accurately reflect in vivo antibiotic sensitivity patterns in cystic fibrosis.     

Concentration of tobramycin given by aerosol in the fluid obtained by bronchoalveolar lavage

Whereas previous studies have used only bronchial secretions and sputum, in the present study, bronchoalveolar (BAL) fluid was analysed for tobramycin levels after aerosolization of this antibiotic. In 20 adult patients with a variety of lung disorders, the concentration of tobramycin obtained in the first aliquot of the bronchoalveolar fluid varied from less than 0.1 to 9.2 micrograms ml-1 (mean 2 +/- 2.26 micrograms ml-1) with 18 samples above 0.4 micrograms ml-1. In most of the cases, the concentration of tobramycin achieved values of tobramycin in excess of the minimal inhibitory concentration for most of the microorganisms. Thus, sampling fluids by the bronchoalveolar technique offers a suitable method to study antibiotic levels at the site of broncho-pulmonary infection. These results may help explain why aerosol antibiotic treatment appears to be useful in selected patients, especially in cystic fibrosis patients chronically infected with Pseudomonas aeruginosa.     

Pseudomonas aeruginosa cross-infection among patients with cystic fibrosis during a winter camp

Twenty-seven patients with cystic fibrosis from our Danish Cystic Fibrosis Center went to a winter camp for 1 week in November of 1990. This study is based on 22 of these patients. Prior to attending camp, 17 out of 22 patients harbored Pseudomonas aeruginosa in their sputum, but 5 patients did not. After returning from camp, all 22 patients harbored P. aeruginosa in the sputum, including the 5 patients whose sputum was free of P. aeruginosa before they went. Epidemiological typing used pulsed-field gel electrophoresis of the P. aeruginosa isolates was performed. The typing results showed that the 5 cystic fibrosis patients who were free of P. aeruginosa in their sputum prior to the winter camp had acquired P. aeruginosa isolates identical to the P. aeruginosa strains isolated from the other 17 cystic fibrosis patients. This constitutes a cross-colonization rate of 100%, the highest rate ever detected among patients with cystic fibrosis. We conclude that separate holiday camps based on the infection status of the patients with cystic fibrosis are necessary to avoid cross-infection of patients not infected with P. aeruginosa.     

Case reports of the treatment with tobramycin solution for inhalation in severe bronchiectasis patients continuous infection of Pseudomonas aeruginosa]

Aerosolized tobramycin has been frequently used in cystic fibrosis patients in order to directly deliver the antibiotic to the endobronchial site of infection. Recently, we experienced three cases of severe chronic bronchial infection of Pseudomonas aeruginosa treated by aerosolized tobramycin inhalation (TOBI). For two cases, we succeeded in improving the patient's medical condition, quality of life, and reduced the amount of sputum. We consider that TOBI should be useful for chronic bronchial infection by Pseudomonas aeruginosa in the case of a patient who does not respond to usual antibiotics via other routes. But further investigation is necessary to reveal clinical efficacy and safety in non-CF patients with bronchiectasis.     

In vitro susceptibility of Pseudomonas aeruginosa from bacteremic and fibrocystic patients to four quinolones and five other antipseudomonal antibiotics

The in vitro antibacterial activities of 4 quinolones, 4 antipseudomonal beta-lactam antibiotics and tobramycin were determined by an agar dilution technique against 206 clinical isolates of Pseudomonas aeruginosa. 176 isolates derived from blood cultures (BC), the remaining 30 were piperacillin-resistant strains from sputum of cystic fibrosis (CF) patients. The CF strains were more resistant to all drugs tested and differed greatly in susceptibility pattern from the BC strains. Ciprofloxacin, ofloxacin and tobramycin were the most active drugs against the CF strains. More than 80% of the BC strains were susceptible to piperacillin and cefsulodin and more than 90% to ceftazidime, tobramycin, ciprofloxacin and ofloxacin. On a weight basis ciprofloxacin was the most active drug in both groups of strains. Our in vitro data suggest that several valuable drugs are now available in the treatment of infections caused by P. aeruginosa strains not previously exposed to antipseudomonal agents.     

Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients.

Recent studies suggest that inflammation plays a role in the pathogenesis of lung disease in cystic fibrosis (CF). The goal of the present study was to quantitatively compare bronchoalveolar lavage fluid (BALF) inflammation and its relation to bacterial infection, between children with CF and children with other chronic respiratory problems. Differential cell counts, immunoreactive interleukin 8 (IL-8), and quantitative bacterial cultures were done in BALF from 54 CF (median age 1.8 yr) and 55 control patients (median age 1.0 yr) who underwent bronchoscopy for clinical indications. Among infected CF patients, those with Pseudomonas aeruginosa did not have more inflammation than those without P. aeruginosa. The ratio of neutrophils or of IL-8 to bacteria in BALF was significantly greater for CF patients compared with control subjects, regardless of pathogen. Calculation of linear regression for either neutrophils or IL-8, as a function of bacterial quantity, yielded positive slopes for both CF and control patients, but with significant elevations for CF. We conclude that the inflammatory response to bacterial infection is increased or prolonged in CF compared with control patients, and that this increase is not necessarily due to pathogens specific for CF (e.g., P. aeruginosa). These data may provide further rationale for anti-inflammatory therapy early in CF.     

The role of piperacillin therapy in pulmonary exacerbations of cystic fibrosis: a controlled study

Piperacillin was evaluated as an antipseudomonas antibiotic in a double-blind controlled trial involving 18 pulmonary exacerbations of cystic fibrosis. Standard antibiotic treatment (flucloxacillin plus tobramycin) was compared with standard treatment plus intravenous piperacillin administered according to two regimens. No added benefit from piperacillin was demonstrable on the basis of improvement in symptoms, physical signs, weight gain, pulmonary function tests, radiologic signs, or sputum Pseudomonas bacterial counts. Some patients experienced sensitivity reactions to piperacillin. In vitro, piperacillin was a potent antibiotic against all beta-lactamase-producing mucoid strains of Pseudomonas aeruginosa; however, in spite of the fact that adequate serum antibiotic concentrations were achieved, sputum bacterial counts did not correlate with either the clinical status or the use of piperacillin therapy.     

RAPD-PCR characterization of Pseudomonas aeruginosa strains obtained from cystic fibrosis patients

OBJECTIVE: To characterize P. aeruginosa strains isolated from bronchoalveolar lavage fluid of cystic fibrosis (CF) patients over a 3 year period. MATERIAL AND METHODS: A prospective follow-up study was carried out in a population of cystic fibrosis patients. The random amplified polymorphic DNA (RAP.D) technique was used to amplify DNA of P. aeruginosa strains isolated from bronchoalveolar lavage fluid samples of five CF patients from the Servicio de Neumología y Cirugía del Tórax del Instituto Nacional de Pediatría (Mexico City Chest Clinic of the National Pediatrics Institute) in Mexico City, between June 1996 and June 2002. Amplification patterns were established for each isolate to accurately identify all strains and to carry out an epidemiological analysis of P. aeruginosa among the selected CF patients. RESULTS: Eighteen different DNA amplification patterns were defined and used to identify each P. aeruginosa strain isolated from the different bronchoalveolar lavage samples. No correlation was observed between the different P. aeruginosa strain genotypes and mucoid or non-mucoid phenotypes, as strains with different phenotypes showed similar amplification patterns. Several strains with different amplification patterns were identified in samples obtained from the same patient, suggesting coinfection with ore than one P. aeruginosa strain. Two siblings with CF shared similargenotypes, suggesting the occurrence of cross- contamination. Similar genotypes of P. aeruginosa strains were isolated throughout the study period. CONCLUSION: Genotypic characterization of P. aeruginosa strains in CF patients allows more accurate epidemiological analyses of this important host-agent relationship.     

Pseudomonas aeruginosa and Burkholderia cepacia cannot be detected by PCR in the breath condensate of patients with cystic fibrosis

The collection of sputum for microbiological examination in young cystic fibrosis patients can be very difficult. However, a knowledge of bacterial flora colonizing the patient's airways is of paramount importance for proper antimicrobial therapy. It is also known that cystic fibrosis patients colonized by Pseudomonas species have a poorer prognosis than Pseudomonas-negative patients. Noninvasive ways of diagnosing airway inflammation that require only minimal cooperation of the patient might yield new possibilities for early detection of airway colonisation. The breath condensate method as a noninvasive diagnostic technique seems especially appropriate for use in children. Therefore, the aim of this study was to evaluate whether the breath condensate method could be used for detection of Pseudomonas species in children with cystic fibrosis. In total, 32 breath condensate and seven sputum samples were obtained from 13 cystic fibrosis patients with Pseudomonas aeruginosa- or Burkholderia cepacia-positive sputum culture (20 samples were obtained during forced expiration). PCR for combined detection of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia was performed. PCR results of all breath condensate samples were negative for Pseudomonas aeruginosa, Stenotrophomonas maltophilia, or Burkholderia cepacia, while all sputum sample results were positive. A minimum DNA quantity of 10 fg could be detected in dilution series of the positive control group. We conclude that the breath condensate method cannot be used as a tool for detection of Pseudomonas species.     

493例支气管扩张急性感染的细菌学特点及药敏分析

目的分析支气管扩张症患者急性感染的细菌学特点及药敏情况。方法对本院呼吸科收治的493例支气管扩张急性感染患者痰标本的细菌培养及药敏试验进行分析。结果 493例痰标本分离出细菌200株,其中革兰阴性杆菌178株,占89.0%,革兰阳性球菌22株,占11.0%。根据五年统计结果,总数排在前6位的分别是铜绿假单胞菌(21.5%)、洛菲不动杆菌(15.5%)、肺炎克雷伯菌(8.5%),肠杆菌属(包括阴沟肠杆菌、产气肠杆菌、中间型肠杆菌及聚团肠杆菌)(8%)、鲍曼溶血不动杆菌(5.5%)和金黄色葡萄球菌(5%)。结论支气管扩张急性感染患者的细菌分布以革兰氏阴性菌为主,铜绿假单胞菌排在首位,提示合理选用抗菌药物对减少耐药菌的产生有重要作用。     

Aminoglycoside penetration, inactivation, and efficacy in cystic fibrosis sputum

We studied sputum tobramycin concentrations after intravenous administration in 10 cystic fibrosis patients. Tobramycin concentrations were determined by a bioassay and a radioenzymatic assay (REA). The bacterial density of Pseudomonas aeruginosa in sputum was examined serially during therapy. Bioactivity of tobramycin in the sputum was low and increased little during treatment. In contrast, tobramycin content (as assayed by REA) showed a progressive accumulation of the drug to high concentrations: a mean of 82 micrograms/g sputum after 3 wk of therapy in 4 patients. Pseudomonas aeruginosa was eradicated from the sputum in 3 of 4 patients receiving antibiotic therapy for 3 wk. Eradication correlated with tobramycin sputum concentrations measured by REA, which were 20-fold greater than the apparent tobramycin inhibitory concentration. A bactericidal effect of aminoglycosides in the presence of sputum in vitro could only be reliably produced with concentrations 25-fold the MIC. We conclude that tobramycin penetrates cystic fibrosis (CF) sputum and accumulates over time. Although CF sputum antagonized the bioactivity of aminoglycosides, 3 wk of intravenous therapy combined with an antipseudomonal beta-lactam antibiotic may be effective in eradication of P. aeruginosa from sputum of certain CF patients.     

Diagnosis of nosocomial bacterial pneumonia in intubated patients undergoing ventilation: comparison of the usefulness of bronchoalveolar lavage and the protected specimen brush

PURPOSE: To compare the usefulness of specimens recovered using a protected specimen brush and those recovered by bronchoalveolar lavage in the diagnosis of nosocomial pneumonia occurring in intubated patients undergoing ventilation, we performed both procedures in patients suspected of having pneumonia because of the presence of a new pulmonary infiltrate and purulent tracheal secretions. PATIENTS AND METHODS: Twenty-one patients (16 men and five women) with an average age of 57 +/- 12 years were studied. They had been receiving mechanical ventilation for 8 +/- 6 days before inclusion in the trial. The clinical suspicion for nosocomial bacterial pneumonia was high in these patients. Fiberoptic bronchoscopy was performed in each patient. Bronchoscopy specimens were obtained by a protected specimen brush and by bronchoalveolar lavage, and were then processed for quantitative bacterial and fungal culture using standard methods. Total cell counts were performed on an aliquot of resuspended original lavage fluid. Differential cell counts were made on at least 500 cells. In addition, 300 cells were examined at high-power magnification and the percentage of cells containing intracellular microorganisms and the average number of extracellular organisms per oil-immersion field were determined. RESULTS: Quantitative culture of specimens recovered using the protected specimen brush were positive (more than 10(3) colony-forming units [cfu]/ml) in five of five patients with subsequently confirmed pneumonia, and negative (less than 10(3) cfu/ml) in 13 of 13 patients without bacterial pneumonia, but results were not available until 24 to 48 hours after the procedure. Quantification of intracellular organisms in cells recovered by lavage was also useful in distinguishing patients with pneumonia (more than 25 percent of cells with intracellular organisms in five of five patients) from those without pneumonia (less than 15 percent of cells with intracellular organisms in all cases), and results were available immediately. In contrast, quantitative culture of lavage fluid and differential cell counts were of little value in identifying infected patients. CONCLUSION: The protected specimen brush and microscopic identification of intracellular organisms in cells recovered by lavage yield useful and complementary information, and together permit rapid and specific treatment of most patients with nosocomial pneumonia.     

Decision analysis of antibiotic and diagnostic strategies in ventilator-associated pneumonia

The optimal strategy for ventilator-associated pneumonia remains controversial. To clarify the tradeoffs involved, we performed a decision analysis. Strategies evaluated included antibiotic therapy with and without diagnostic testing. Tests that were explored included endotracheal aspirates, bronchoscopy with protected brush or bronchoalveolar lavage, and nonbronchoscopic mini-bronchoalveolar lavage (mini-BAL). Outcomes included dollar cost, antibiotic use, survival, cost-effectiveness, antibiotic use per survivor, and the outcome perspective of financial cost-antibiotic use per survivor. Initial coverage with three antibiotics was better than expectant management or one or two antibiotic approaches, leading to both improved survival (54% vs. 66%) and decreased cost (US dollars 55447 vs. US dollars 41483 per survivor). Testing with mini-BAL did not improve survival but did decrease costs (US dollars 41483 vs. US dollars 39967) and antibiotic use (63 vs. 39 antibiotic days per survivor). From the perspective of minimizing cost, minimizing antibiotic use, and maximizing survival, the best strategy was three antibiotics with mini-BAL.