Imipenem/cilastatin treatment of multiresistant Pseudomonas aeruginosa lung infection in cystic fibrosis

Ten patients with cystic fibrosis and chronic broncho-pulmonary Pseudomonas aeruginosa infection received 45 mg imipenem/cilastatin per kg body weight/day, intravenously for two weeks. The treatment was safe with only minor side effects and clinical parameters improved considerably during therapy. In all patients resistance of Ps. aeruginosa to imipenem developed in the second week of treatment; in seven patients the therapy selected for a resistant strain and in three resistance developed in the original strain. The resistance persisted after cessation of treatment and thus the clinical usefulness of imipenem/cilastatin as monotherapy in CF-patients with Ps. aeruginosa seems to be limited.     

Serotyping of Pseudomonas aeruginosa from patients with cystic fibrosis of the pancreas.

Serotyping of 30 mucoid strains isolated from cystic fibrosis patients was carried out by slide agglutination tests with both live and heat-killed cells and by tube agglutination test with heat-killed cells. Comparison of the results obtained by these 2 methods revealed that tube agglutination with heat-killed cells was the superior method. More than half the strains were found to be Homma's serotype 15 (group M in the new schema [2]). Slide agglutination with live cells did not give clear results: some strains showed occasionally positive or negative agglutinations against the same serotype serum. Changes in serotypes (groups in the new schema [2]) were found in some strains, although the number was very small.     

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.     

beta-Lactam-resistant Pseudomonas aeruginosa with modified penicillin-binding proteins emerging during cystic fibrosis treatment.

The emergence of beta-lactam-resistant strains of Pseudomonas aeruginosa in a cystic fibrosis patient treated with high-dose tobramycin and piperacillin was studied. Two serotypes, M and K, were present before treatment and persisted, with changes in their beta-lactam resistance spectra, during treatment. The resistance was correlated with changes in the penicillin-binding proteins (PBPs) in both serotypes. In the low-level-resistant serotype K organism, PBP-3 either was absent or had lost the ability to bind [14C]penicillin G. Tow serotypes M strains, one with low- and one with high-level resistance to several antipseudomonal beta-lactam antibiotics, were isolated at progressively later stages of therapy. Several differences were noted between the PBP patterns of the resistant M and the susceptible M strains. The affinity for [14C]penicillin G was reduced in both resistant strains. PBP bands, with the exception of PBP-6 in the most resistant M type, were barely or not detectable at a [14C]penicillin G concentration of 39 microgram/ml. The graduated decrease in affinity for [14c]penicillin G was correlated with increasing beta-lactam resistance and with an increase in the quantity of the protein corresponding to PBP-6. The emergence of the low-level-resistant strains midway through, and of the highly resistant strain in the final stages of, the reported treatment strongly suggested that the resistance resulted from mutation in those strains present before treatment selected for by the high-dose piperacillin treatment.     

Priming of neutrophils for enhanced oxidative burst by sputum from cystic fibrosis patients with Pseudomonas aeruginosa infection

Neutrophils accumulate in the lungs of cystic fibrosis (CF) patients and inflict tissue damage by release of oxygen radicals and proteases. Here we report on the ability of sputum to prime neutrophils for enhanced release of oxygen radicals. Sol phase was prepared by ultracentrifugation of sputum obtained from CF patients attending the CF Clinic, Rigshospitalet, Copenhagen. The oxidative burst response of neutrophils from healthy individuals was measured by oxygen consumption, superoxide production and chemiluminescence. Neutrophils were preincubated with sputum or buffer and then stimulated with f-Met-Leu-Phe or zymosan. Appropriate controls were included in the experiments. It was shown that neutrophils preincubated with CF sol phase and stimulated with f-Met-Leu-Phe generated a three- to five-fold higher chemiluminescence response than those preincubated with buffer. There was no enhancement of the response when zymosan was used for stimulation of the cells. Neutrophils incubated with sol phase alone exhibited no response. Attempts were made to identify and partially characterize the priming factor(s). It was found that the sputum samples contained bacterial endotoxins. The priming activity was resistant to heating at 100 degrees C for 15 min, and was present only in fractions with molecules larger than 100 KD. It is suggested that the priming factor(s) consist of bacterial endotoxins and/or immune complexes. Activation and enhanced release of oxygen radicals from neutrophils may play an important role in the host defence as well as pathogenesis of tissue damage in the lungs of these patients.     

Mixed morphotype susceptibility testing of Pseudomonas aeruginosa from patients with cystic fibrosis

Disk diffusion antibiograms were determined for mixtures of Pseudomonas aeruginosa morphotypes isolated from the sputum of patients with cystic fibrosis (CF). The results were compared with the predicted susceptibility patterns derived from the antibiograms of individual morphotypes within the mixture. Fifty separate cultures, each yielding two, three, or four distinct morphotypes of Pseudomonas aeruginosa were evaluated. Overall, the correlation between observed and predicted results was 92.2% with only 2.9% of all observations leading to major disagreements in susceptibility. These data suggest that mixed morphotype susceptibility is potentially a useful method to monitor the collective resistance of colonizing strains of Pseudomonas aeruginosa from the respiratory tract of patients with CF.     

Aztreonam inhalation solution for suppressive treatment of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis

An aerosol form of aztreonam lysinate has recently been developed as a treatment for cystic fibrosis patients suffering from chronic Pseudomonas aeruginosa lung colonization. Local administration means the drug can reach mucus concentrations in the order of hundreds of times the MIC₅₀ of Pseudomonas associated with severe lung disease in cystic fibrosis, resulting in a significant reduction in airway bacterial density and a parallel improvement in lung function. These advantages are maintained over prolonged periods of treatments. Administration of the drug is optimized by the use of a specific eFlow^® system, resulting in considerable reductions in treatment times when compared with conventional nebulizers. The drug has been proven safe and no concomitant induction of resistance to Pseudomonas was found during the clinical trial period of 18 months. Aztreonam lysinate has been shown to ameliorate pulmonary function in cystic fibrosis patients with chronic airway Pseudomonas infection and this is paralleled by a reduction in bacterial density in the lungs. The increased availability of new aerosolized antibiotics for cystic fibrosis will lead to new scenarios in the treatment of the disease.     

Aztreonam inhalation solution for suppressive treatment of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis

An aerosol form of aztreonam lysinate has recently been developed as a treatment for cystic fibrosis patients suffering from chronic Pseudomonas aeruginosa lung colonization. Local administration means the drug can reach mucus concentrations in the order of hundreds of times the MIC(50) of Pseudomonas associated with severe lung disease in cystic fibrosis, resulting in a significant reduction in airway bacterial density and a parallel improvement in lung function. These advantages are maintained over prolonged periods of treatments. Administration of the drug is optimized by the use of a specific eFlow(?) system, resulting in considerable reductions in treatment times when compared with conventional nebulizers. The drug has been proven safe and no concomitant induction of resistance to Pseudomonas was found during the clinical trial period of 18 months. Aztreonam lysinate has been shown to ameliorate pulmonary function in cystic fibrosis patients with chronic airway Pseudomonas infection and this is paralleled by a reduction in bacterial density in the lungs. The increased availability of new aerosolized antibiotics for cystic fibrosis will lead to new scenarios in the treatment of the disease.     

Mercaptoethylguanidine inhibits the inflammatory response in a murine model of chronic infection with Pseudomonas aeruginosa

Chronic airway inflammation induced by Pseudomonas aeruginosa is the eventual cause of respiratory failure in most people affected by cystic fibrosis. Recent evidence implicates the involvement of free radical and oxidant stress in the pathogenesis of the inflammatory injury. Here we report the efficacy of a novel experimental therapeutic, mercaptoethylguanidine (MEG), which has combined actions as a selective inhibitor of the inducible nitric oxide synthase and as a scavenger of peroxynitrite, a potent oxidant formed in the reaction of nitric oxide and superoxide radical. Chronic pulmonary infection was established in FVB/N mice by intratracheal administration of 10(5) colony-forming units of P. aeruginosa in agar beads. Treatment with MEG (10 mg/kg/dose every 8 h i.p.) inhibited weight loss in the first 3 days and reduced histologic injury at 8 days postinfection. MEG also reduced myeloperoxidase activity, a marker of neutrophil infiltration, at 8 days and concentrations of the proinflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and macrophage inflammatory protein 2 in whole lung homogenates. MEG-treated animals and controls had similar perioperative mortality and comparable colony counts of P. aeruginosa at 8 days, indicating that MEG did not exacerbate infection. Our data suggest that MEG may be an effective immunomodulatory therapy of pulmonary inflammation induced by chronic infection.     

Diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by ELISA for anti-pseudomonas LPS IgG antibodies

An enzyme-linked immunosorbant assay (ELISA) with urease enzyme was developed with either a polyvalent pseudomonas smooth lipopolysaccharide (LPS) extract vaccine (PEV-02) or rough LPS (R-LPS) from P. aeruginosa rough mutant PAC605. Each ELISA was able to differentiate between sera from cystic fibrosis (CF) patients chronically colonized with P. aeruginosa and sera from non-colonized patients. Sera from non-colonized and intermittently colonized CF patients seldom reacted with any of the Pseudomonas LPS, whereas sera from chronically colonized CF patients reacted strongly with most of the sixteen smooth O-serotype vaccine components and with the PAC605 R-LPS, indicating the presence either of a number of different serotype specific IgG antibodies and/or IgG antibodies directed to a common antigenic component of LPS rough core. Absorption studies and immunoblot analysis demonstrated that in sera from CF patients who were chronically colonized with P. aeruginosa a significant component of the anti-P. aeruginosa antibodies is specific for the core of P. aeruginosa LPS and cross reactive with a number of serotypes of P. aeruginosa LPS.     

The efficacy and safety of ciprofloxacin and ofloxacin in chronic Pseudomonas aeruginosa infection in cystic fibrosis

The clinical efficacy and safety of ciprofloxacin and ofloxacin were compared in a prospective, randomized double blind, placebo combined cross-over study in 26 adult cystic fibrosis patients with chronic broncho-pulmonary Pseudomonas aeruginosa infection. Active treatment consisted of ciprofloxacin 750 mg orally twice daily or ofloxacin 400 mg orally twice daily; both treatments were given for 14 days, with three months between treatment periods; 21 patients completed both treatment periods. Treatment with both ciprofloxacin and ofloxacin was associated with a good clinical response as judged by clinical score, lung function tests and inflammatory parameters; no difference between ciprofloxacin and ofloxacin was found. Adverse reactions were seen in nine of 24 patients who received ciprofloxacin and in six of 23 who received ofloxacin. The majority were dyspeptic reactions or photosensitivity. No serious adverse reactions occurred. Three cases of treatment failure were found, one of which was associated with development of resistant P. aeruginosa during ofloxacin treatment. The mean MIC of both drugs increased during treatment but returned to pretreatment values within three months. Ciprofloxacin and ofloxacin seem to be valuable agents for intermittent treatment of chronic P. aeruginosa lung infection in adult cystic fibrosis patients.     

Mechanism of amikacin resistance in Pseudomonas aeruginosa isolates from patients with cystic fibrosis

We studied 27 amikacin-resistant isolates of Pseudomonas aeruginosa from patients with cystic fibrosis to determine the mechanism of antibiotic resistance. The absence of aminoglycoside-modifying enzymes (AMEs) in these isolates was inferred from the failure of DNA probes for 16 candidate AMEs to hybridize with DNA harvested from these isolates and, in addition, the uniform reduction in susceptibility to a panel of aminoglycosides. In eight of the 27 isolates that were resistant to amikacin at high levels (minimum inhibitory concentration ⩾ 250 μ/ml), plasmids were not detected. The ribosomes of these isolates were sensitive to amikacin in studies of protein synthesis by cell “ghosts.” These data suggest that impermeability is the mechanism of amikacin resistance in isolates of P. aeruginosa from patients with cystic fibrosis. Recognition of this mode of resistance may be difficult, as some isolates appeared to be borderline susceptible when tested against aminoglycosides other than amikacin, or had zone diameters that overlapped those obtained with amikacin-susceptible isolates.     

Emergence of ceftriaxone-resistant strains of Pseudomonas aeruginosa in cystic fibrosis patients

Cystic fibrosis patients with Pseudomonas aeruginosa chest infections were treated with ceftriaxone alone or ceftriaxone plus tobramycin. P. aeruginosa strains isolated before and after treatment were studied for changes in sensitivity to ceftriaxone. After therapy with either the single agent or the combination six strains from five patients were found to be resistant to ceftriaxone. No resistant strains were isolated before therapy. Resistance was mediated by excess production of Id beta-lactamase which in five of six strains was permanently derepressed. Bacterial resistance appearing during therapy reduces the value of this antibiotic in cystic fibrosis chest infections.     

Persistence mechanisms in Pseudomonas aeruginosa from cystic fibrosis patients undergoing ciprofloxacin therapy.

The mechanisms of persistence to ciprofloxacin in nine sets of Pseudomonas aeruginosa strains isolated during ciprofloxacin therapy of chronic lung infections in cystic fibrosis patients were studied. Low to moderate levels of ciprofloxacin resistance developed in each case. Each set of pretherapy ciprofloxacin-susceptible, during-therapy ciprofloxacin-resistant, and posttherapy ciprofloxacin-susceptible isolates were shown to be genotypically related by using a radiolabeled epidemiological gene probe. All ciprofloxacin-resistant isolates were found to have altered susceptibilities to both nalidixic acid and various chemically unrelated antibiotics. Analysis of possible resistance mechanisms showed that the strains had altered outer membrane protein or lipopolysaccharide profiles. Complementation of possible DNA gyrase mutations with a plasmid-borne, wild-type Escherichia coli gyrA gene indicated that altered DNA gyrase was at least partly responsible for ciprofloxacin resistance in all strains tested. Attempts to generate ciprofloxacin-susceptible revertants in vitro showed that in some strains reversion was rapid in the absence of ciprofloxacin, while in other strains it was not possible to generate revertants. These data indicate that persistence of Pseudomonas aeruginosa to ciprofloxacin involves changes in DNA gyrase and is associated with pleiotropic changes in outer membrane proteins and lipopolysaccharide.     

In vitro pharmacodynamics of ceftazidime against Pseudomonas aeruginosa isolates from cystic fibrosis patients.

The concentration/MIC (C/MIC) ratio maximizing the bactericidal activity of ceftazidime against 10 Pseudomonas aeruginosa isolates from cystic fibrosis patients was identified. Bactericidal activity was assessed by determining the percent difference in the area under the killing curve at each C/MIC ratio for all of the isolates from that of their growth control. The percent effect at each C/MIC ratio was fitted to a sigmoidal Emax model with maximum bactericidal activity defined as the C/MIC ratio that produced an effect that was 90% of the Emax. Our results suggest that at least some isolates may require higher C/MIC ratios than previously reported for maximal activity.     

Effect of ciprofloxacin in an in-vitro pharmacokinetic model against Pseudomonas aeruginosa isolated during cystic fibrosis lung infection

An in-vitro pharmacokinetic model was used to simulate ciprofloxacin concentrations in serum observed in vivo following oral doses of 250, 500 and 750 mg, in a culture of Pseudomonas aeruginosa isolated from the sputum of a cystic fibrosis patient. In the 12 h interval following each dose, a period of bactericidal activity was observed, succeeded by bacteriostasis and subsequent bacterial regrowth. In all cases bacterial regrowth was first observed when the ciprofloxacin concentration in the culture flask of the dynamic model fell below the MIC. For the three doses investigated, the surviving bacteria at the end of each 12-h dosing interval showed an increase in MIC compared with that of the culture before ciprofloxacin exposure. The post-antibiotic effect of ciprofloxacin against P. aeruginosa was observed for 1-5 h, and was dose related.