Early eradication therapy against Pseudomonas aeruginosa in cystic fibrosis patients

In the hereditary disease cystic fibrosis (CF), chronic bacterial lung infections are linked to mutations in a gene encoding the epithelial chloride channel, the CF transmembrane conductance regulator 1. Decreased chloride secretion and increased sodium absorption leads to increased water absorption from the periciliary liquid layer on the respiratory epithelium, and the volume-depleted periciliary liquid layer impairs mucociliary clearance of bacterial organisms 1. The opportunistic bacterial pathogen Pseudomonas aeruginosa is the most prevalent pathogen and accounts for most of the morbidity and mortality in CF patients. Although antibiotic therapy has considerably improved the clinical condition of CF patients and their quality of life since the 1980s 2, it rarely leads to eradication of the pathogen from chronically infected CF airways. P. aeruginosa macrocolony formation in anaerobic mucus plugs 3, and the occurrence of hypermutable P. aeruginosa strains, characterised by a rapid development of antibiotic resistance, are thought to be responsible. Several studies have demonstrated that a delay of the onset of chronic P. aeruginosa infection and frequent eradication of the pathogen from CF airways is achieved when the antibiotic treatment is initiated shortly after bacterial lung colonisation 4–10. This may result from findings that early after airway colonisation, single P. aeruginosa cells prevail, which are more susceptible to antibiotic attack. However, the early eradication treatment regimen has not yet been accepted worldwide and several open questions remain concerning: 1) the optimal drug (combination); 2) the mode of delivery of the antibiotic(s); 3) the optimal duration of the regimen; 4) the length of the P. aeruginosa free-period after treatment; and 5) the impact of this treatment strategy on lung function, development of antibiotic resistant microorganisms, emergence of other pathogens and treatment costs.

Thus, the objectives of the present study were to assess the time period after early antibiotic therapy when P. aeruginosa was again cultured from the patients' respiratory tract, and to differentiate between re-growth of an identical P. aeruginosa strain in a subsequent infection episode and re-infection with a different organism. Furthermore, the current authors wanted to assess the impact of early antibiotic therapy on the decline of lung function in comparison with that of antibiotic maintenance therapy in age-matched and sex-matched CF patients suffering from chronic P. aeruginosa infection. Additionally, the authors wanted to know whether antimicrobial resistance patterns of P. aeruginosa isolates from CF patients undergoing early eradication therapy differ from the resistance patterns of P. aeruginosa strains isolated from chronically infected CF patients. Furthermore, the present authors sought to know whether P. aeruginosa eradication would result in the emergence of other bacterial or fungal pathogens in CF airways. Finally, the costs of the early antibiotic therapy regimen were calculated and compared with those of antibiotic maintenance therapy in chronically infected CF patients over a 7-yr period.

PATIENTS, MATERIALS AND METHODS

At the CF centre in Florence (Italy) 173 CF patients, including 42% of patients aged >18 yrs, were seen quarterly. Cultures for P. aeruginosa and other CF-related pathogens from respiratory secretions were performed at every visit. During 1992–2001, all P. aeruginosa-negative CF patients who became P. aeruginosa positive as evidenced by one positive respiratory culture (29 males, 29 females; mean age±sd at time of first colonisation 8.9±6.64 yrs) were immediately treated for 3 weeks with a combination of inhaled colistin (1×10⁻⁶ international units (IU) b.i.d. <6 yrs; 2×10⁻⁶ mL IU b.i.d. >6 yrs) and oral ciprofloxacin (30 mg·kg⁻¹·day⁻¹). Treatment was prolonged to 3 months and all patients received 2×10⁻⁶ mL IU b.i.d of colistin in cases when respiratory cultures did not become P. aeruginosa negative after the 3-week treatment 9.

Lung function, measured spirometrically (Renaissance PB100 apparatus; Nellcor Puritan Bennet, Pleasanton, CA, USA) as forced expiratory volume in one second (FEV₁) related to age-adjusted reference values, was determined for a period of 5 yrs in 18 patients submitted to P. aeruginosa eradication therapy and in 18 age-matched and sex-matched CF patients, chronically infected with P. aeruginosa. The CF patients of the control group had been chronically infected with P. aeruginosa in the period 1989–1992 before the treatment strategy was changed in the centre. Both patient groups did not differ statistically for CF genotype, pancreatic status, mean age, nutritional status, FEV₁, patient and parent compliance, environmental exposure to pathogens, physician practices or socioeconomic status at the beginning of early eradication treatment. A patient was selected when monitoring of lung function was possible for ≥5 yrs. Thus, particularly young patients who underwent early eradication therapy and in whom lung function data were not available were excluded. Patients were also excluded when a matched control individual was not available. The periods of lung function monitoring was comparable in the group of early treatment and the chronic infection group.

Antibiotics for treatment of pulmonary infections of CF patients are free in Italy according to law (law number 548, December 23, 1993). Antimicrobial susceptibility patterns of P. aeruginosa isolates from all patients were determined by the agar diffusion method. P. aeruginosa genotypes were assessed by pulsed field gel electrophoresis (PFGE) 11. Briefly, bacterial DNA was cut with the restriction enzyme SpeI overnight at 37°C and electrophoresis was run at 200 V for 24 h with 1–25 s pulse times. Ethidium bromide stained DNA bands were scanned (Cybertech, Berlin, Germany), digitalised and analysed using the Wincam software program (Cybertech). There were no systematic differences between the individuals whose strains were genotyped and those where strains for genotyping were not available. Serum anti-pseudomonal antibodies were determined by ELISA 12. Briefly, the purified P. aeruginosa antigens alkaline protease, elastase and exotoxin A, coated on albumin saturated microtiter plates were used to detect respective antibodies in CF serum samples. Additionally, serum samples were screened for anti-pseudomonal antibodies using crossed immunoelectrophoresis 13. To assess whether early eradication therapy of P. aeruginosa leads to a change of the respiratory flora in CF patients, bacterial and fungal pathogens from respiratory cultures of 46 CF patients were identified on agar plates before and after early eradication treatment. Between three to four cultures per patient, within periods of 8.2±3.4 months before treatment and 8.8±5.9 months after treatment, were assessed for: Staphylococcus aureus; methicillin-resistant S. aureus; Staphylococcus spp.; Burkholderia cepaci; Escherichia coli; Haemophilus influenzae; Klebsiella spp.; Stenotrophomonas maltophili; Acinetobacter spp.; Candida albicans; and Aspergillus fumigatus.

The cost of drugs used for early eradication treatment and for intravenous treatment of chronic P. aeruginosa lung infection was estimated for a 7-yr period 14.

RESULTS

Based on at least three consecutive negative respiratory P. aeruginosa cultures and negative serum antibody titers within a 6-month period after treatment, eradication of P. aeruginosa was achieved in 47 CF patients (81%) who had converted from P. aeruginosa negative to P. aeruginosa positive respiratory cultures. From these, 24 patients (51%) were recolonised with the pathogen one or more times during the study period. Genotyping of P. aeruginosa strains, which were available from 50 isolated episodes of P. aeruginosa recolonisation in 16 CF patients, revealed a different PFGE genotype in 73% of the subsequent episode (data not shown), further supporting the notion that eradication had been obtained in the majority of treated patients. There was no evidence for clonality of the P. aeruginosa CF isolates according to genotyping.

The mean±sd time period between two isolated episodes of P. aeruginosa colonisation in the early antibiotic treatment group of 47 CF patients was a median (range) of 18 (4–80) months, suggesting that this treatment regimen has a beneficial impact on the patients' clinical status. Indeed, the average lung function decline (fig. 1⇓) was significantly lower in early treated CF patients (mean±sd ΔFEV₁·yr⁻¹: -1.63±1.60%) than in chronically infected CF patients (ΔFEV₁·yr⁻¹: -4.69±2.95%; unpaired t-test: p<0.05). Lung function could not be assessed in all of the patients in the early antibiotic treatment group due to ages <5 yrs.

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Fig. 1—

The decrease of lung function (average forced expiratory volume in one second (ΔFEV₁)·yr⁻¹) determined in a 5-yr period in 18 cystic fibrosis (CF) patients treated early with inhaled colistin/oral ciprofloxacin (□) and in 18 age-matched CF patients chronically infected with Psuedomonas aeruginosa ().

P. aeruginosa isolates from early treated patients versus control patients were significantly more sensitive to the P. aeruginosa-specific antibiotics (Chi-squared test: p<0.05) ceftazidime, tobramycin and ciprofloxacin and revealed a nonmucoid phenotype (fig. 2⇓). The data show that P. aeruginosa is much more susceptible to antibiotics when therapy is administered early after lung colonisation.

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Fig. 2—

Antibiotic resistance patterns of 47 Pseudomonas aeruginosa isolates from 47 early treated patients (□) and 47 isolates from 47 chronically infected patients (). *: p<0.05.

Since eradication of P. aeruginosa may result in the emergence of other pathogens in the respiratory tract, bacterial and fungal pathogens from CF patients were identified before and after early eradication treatment (table 1⇓). No statistical difference was obtained for the investigated bacterial or fungal pathogens.

The 47 early-treated patients received a total of 104 antibiotic “maintainance” therapy courses over 7 yrs (0.32 courses·patient·yr⁻¹) costing in total \#8364;34,681 (\#8364;105.4·patient·yr⁻¹). The 47 age-matched, chronically P. aeruginosa-infected CF patients received 683 courses in 7 yrs (2.1 courses·patient·yr⁻¹) costing in total \#8364;1,767,025 (\#8364;5371·patient·yr⁻¹).

DISCUSSION

The present study answers several important questions. The period during which the patients remained free of P. aeruginosa after a 3 week to 3 month eradication therapy is encouragingly lengthy. In previous studies, the period in which the patients remained free of P. aeruginosa after early therapy was determined to be 3–25 months 6, and in one study >1 yr 7. Apparently, eradication is not immediately followed by re-infection of the patients with P. aeruginosa strains ubiquitously present in the environment. The treatment regimen also lead to true eradication as demonstrated by the diversity of genotyped sequential P. aeruginosa isolates from the majority of CF patients and by negative specific antibody titers against P. aeruginosa.

The Damocles sword of antibiotic resistance and adverse side effects is a common threat to conventional antibiotic therapy for chronically P. aeruginosa-infected CF patients, which has led to restrictive antibiotic treatment strategies in various CF centres. Early antibiotic therapy circumvents this problem efficiently. Since P. aeruginosa strains infecting CF airways are highly sensitive to many P. aeruginosa specific antibiotics, P. aeruginosa eradication is often achieved and the development of antibiotic resistance becomes marginal. The high success rate makes it conceivable that early antibiotic treatment has a beneficial impact on lung function compared with chronically infected CF patients and, thus, may well result in a higher life expectancy of early treated CF patients.

A further positive aspect of early antibiotic treatment of P. aeruginosa in CF concerns the costs of treatment. Compared with regular intravenous antibiotic therapy for chronically infected CF patients 2, the cost for early antibiotic treatment is ∼2%, since P. aeruginosa eradication leaves the patient for prolonged periods of time free of the pathogen. Besides the positive clinical aspects, the significantly reduced costs will undoubtedly make this therapy regimen attractive in many countries in which CF patients live. Reduced costs of early antibiotic treatment were also calculated by others 15. If widely applied it may substantially reduce the prevalence of chronic P. aeruginosa lung infections in CF patients. At present, only 39.3% of CF patients in the Cystic Fibrosis Centre of Tuscany (Meyer Pediatric Hospital, University of Florence, Italy) suffer from chronic P. aeruginosa lung infections as a result of early antibiotic therapy. Decreases of the prevalence of chronic P. aeruginosa lung infections in CF patients have also been reported from other centres 16. A larger cohort of patients needs to be studied to see if this development will lead to an increase of the prevalence of other pathogens in the respiratory tract. No increase in a number of CF-related bacterial and fungal pathogens were noticed after early treatment of P. aeruginosa, however, prolonged studies are needed to confirm this finding. An opposite development was observed in a group of young CF patients undergoing prophylactic treatment against S. aureus. The decrease of S. aureus was correlated with a significant increase in the number of patients harbouring P. aeruginosa.

In conclusion, taken together, the results of this study show that early antibiotic therapy for eradication of Pseusomonas aeruginosa is a powerful therapy regimen, which substantially reduces the prevalence of chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients.