Prevention of Pseudomonas aeruginosa infection in cystic fibrosis patients

In patients with cystic fibrosis (CF) prevention of lung infections with Pseudomonas aeruginosa is of major importance. Principles to achieve this goal include vaccination, immediate use of antibiotics in patients newly colonized with the pathogen, and hygienic measures. The purpose of this review is to discuss recent developments in this context.     

Pseudomonas aeruginosa flagellar antibodies in patients with cystic fibrosis.

An enzyme-linked immunosorbent assay specific for flagellum type (a or b) of Pseudomonas aeruginosa was used to detect serum immunoglobulin antibodies in 98 random outpatients and 14 colonized cystic fibrosis patients. Antibodies were detected to both types of flagella in addition to M-2 lipopolysaccharide. Titers to both flagellar antigens (FlAg) were 10 to 100 times higher in cystic fibrosis patients than in random outpatients of a comparable age group. Mean antibody titers against b-type FlAg were 454 for outpatients (ages newborn to 21 years), whereas the mean titer for cystic fibrosis patients (ages 6 to 21 years) was 51,520. Titers against a-type FlAg were generally lower, with mean outpatient titers of 68 and mean cystic fibrosis patient titers of 34,323. Differences were also seen in antibody titer against M-2 lipopolysaccharide, but these differences did not correspond to M-2 FlAg titers. In 98 random outpatients (ages newborn to 86 years), FlAg titers generally increased with age. To demonstrate further specificity of the enzyme-linked immunosorbent assay for flagellum antibody, Western blots were performed with selected high-titer cystic fibrosis patient sera. Sera that had a high titer (greater than 25,600) for b- or a-type FlAg showed a corresponding reactive band. These results demonstrate that flagellum antibodies are produced in humans in response to P. aeruginosa infection.     

Mixed-morphotype broth microdilution susceptibility testing of Pseudomonas aeruginosa from cystic fibrosis patients.

Multiple morphotypes of Pseudomonas aeruginosa isolated from 50 respiratory specimens of cystic fibrosis patients were tested for correlation of broth microdilution susceptibility results of a mixed-morphotype inoculum with a predicted antibiogram of the individual isolates. The overall correlation was 96.0%, with only 1.6% very major or major errors.     

Ecology of Pseudomonas aeruginosa in patients with cystic fibrosis

The occurrence of various Pseudomonas aeruginosa strains in the sputum of 15 patients with cystic fibrosis (CF) was monitored over periods ranging from 2 to 60 months. Isolates of P. aeruginosa were typed by four different techniques, namely serotyping, active and passive pyocin typing, and phage typing. The maximum number of different serotypes found in the patients was three (one serotype in nine patients; two serotypes in five patients; three serotypes in one patient). Pyocin and phage typing showed no marked differences between strains of the same serotype in individual patients. Exacerbations of chronic respiratory infection were not associated with changes in the sputum flora, the composition of P. aeruginosa strains in which remains constant over long periods in patients with CF.     

Fecal isolation of Pseudomonas aeruginosa from patients with cystic fibrosis.

Fecal isolation of Pseudomonas aeruginosa was observed in 8 of 10 patients with cystic fibrosis who at the time of sampling also exhibited colonization of the respiratory tract. In contrast, P. aeruginosa cells were isolated at low frequency (9.1%) from the stools of 44 patients with cystic fibrosis with no previous history of chronic colonization. The results of this study suggest that the gastrointestinal tract is not a significant chronic reservoir of P. aeruginosa prior to pulmonary colonization.     

Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis

Pseudomonas aeruginosa is the predominant cause of chronic airway infection in cystic fibrosis (CF). CF airway isolates are often tested for antibiotic susceptibility but are rarely eradicated by the antibiotics identified as potentially effective. The growth state of P. aeruginosa in CF airways is probably different from that exhibited under conventional susceptibility testing conditions and may represent a bacterial biofilm. Biofilm susceptibility testing methods were adapted to create an assay for implementation in a clinical microbiology laboratory. This assay gave reproducible results when examined in 300 paired determinations with 12 antimicrobial agents, with a serious error rate of 5.7%. The biofilm assay was used retrospectively to test these 12 agents against 94 isolates from 41 CF patients. The biofilm inhibitory concentrations (BICs) were much higher than the corresponding conventionally determined MICs for the beta-lactam antibiotics (median values: aztreonam, >128 microg/ml versus 4 microg/ml; ceftazidime, 128 microg/ml versus 2 microg/ml; piperacillin-tazobactam, 256 microg/ml versus 4 microg/ml; and ticarcillin-clavulanate, 512 microg/ml versus 16 microg/ml, respectively) and doxycycline (>64 microg/ml versus 16 microg/ml); and similar for meropenem (4 micro g/ml versus < or = 1 microg/ml), ciprofloxacin (0.5 microg/ml versus 1 microg/ml), and the aminoglycosides amikacin (32 microg/ml versus 16 microg/ml), gentamicin (16 microg/ml versus 8 microg/ml), and tobramycin (4 microg/ml versus 2 microg/ml). The median BIC for azithromycin was 2 microg/ml, whereas isolates were uniformly resistant when tested by standard methods. This demonstrates the feasibility of adapting biofilm susceptibility methods to the clinical microbiology laboratory and opens the way to examining whether biofilm testing might be used to select more effective antibiotic combinations for CF airway infections than methods in current use.     

Influence of biofilm formation in the susceptibility of Pseudomonas aeruginosa from Brazilian patients with cystic fibrosis

Ferreira AG, Leão RS, Carvalho‐Assef APD, Folescu TW, Barth AL, Marques EA. Influence of biofilm formation in the susceptibility of Pseudomonas aeruginosa from Brazilian patients with cystic fibrosis. APMIS 2010; 118: 606–12. Biofilms play a key role in the occurrence of lung infections by Pseudomonas aeruginosa in patients with cystic fibrosis (CF). In this study, we examined 40 isolates of P. aeruginosa from CF patients according to their capacity to form biofilm. We also compared their in vitro response to antimicrobials according to different modes of growth (planktonic vs biofilm) and performed molecular typing. All isolates proved capable of forming biofilm. However, there was no difference in biofilm development according to the mucoid and nonmucoid phenotypes and among isolates obtained at different periods of the chronic infection. All isolates tested for antimicrobial susceptibility in the biofilm state (BIC) were consistently more resistant to antibiotics than the same isolate tested in the planktonic state. The molecular typing indicates a considerable clonal diversity among isolates. We identified five patients harboring the same strain over different periods. These strains, however, displayed different levels of biofilm formation and BIC values for antibiotics tested. The results of the present study demonstrate that there is a marked difference in the susceptibility profile according to the mode of growth of CF P. aeruginosa, as cells tested in the biofilm state proved consistently more resistant to antibiotics.     

Epidemiologic characterization of Pseudomonas aeruginosa in patients with cystic fibrosis

Objective   To determine persistence and variability of colonization with Pseudomonas aeruginosa in cystic fibrosis patients over long time periods, and to look for possible cross-colonization.
Methods   In total, 469 Pseudomonas aeruginosa isolates were obtained from 30 patients during the period from April 1994 to April 1996. The sources were mainly sputum and a few deep throat swabs. All grown strains dissimilar in macromorphology were processed separately. Typing with PFGE was carried out by contour-clamped homogeneous electric field electrophoresis. Genomic DNA was subjected to the rare-cutting restriction enzyme Spe I. For pyocin typing, the procedure described by Fyfe was applied.
Results   After typing with PFGE, we observed 40 restriction profiles. Eighteen different pyocin types were found. The most frequent pyocin type was type 3, followed by types 1 and 5. Twenty-two patients were persistently colonized by one clone specific and different for each patient, and four were co-colonized by a second clone also different for each of these patients. Cross-colonization had apparently been rare in the cystic fibrosis center of Leipzig.
Conclusions   Typing with PFGE is well suited for detailed investigations of colonization with Pseudomonas aeruginosa in cystic fibrosis patients. Pyocin typing can provide additional information for epidemiologic purposes.     

Antibodies to cell envelope proteins of Pseudomonas aeruginosa in cystic fibrosis patients.

Many vaccines containing somatic and secreted antigens of Pseudomonas aeruginosa have been reported. The vaccines containing lipopolysaccharide have been found to provide type-specific protection, but the endotoxin content of these vaccines does not make it feasible to use them in patients who are already debilitated. Outer membrane proteins could be effective as vaccines, as they can be purified free of lipopolysaccharide, and also because they are common to all serotypes of P. aeruginosa. To be effective as a vaccine, such proteins must be immunogenic and accessible from the outside of the intact bacterial cell. In this study, we showed that systemic antibodies were produced frequently to two cell envelope proteins with masses of 58,500 and 37,500 daltons and occasionally to 34,000-dalton protein of P. aeruginosa in cystic fibrosis patients with chronic lung infections. In rabbits immunized with whole, fixed cells of P. aeruginosa, antibodies were also produced against the 58,500-dalton proteins. Thus, the 58,500-dalton cell envelope protein of P. aeruginosa was the only immunogenic protein that was accessible to the immune system when whole, fixed cells were used for immunization. These serum antibodies did not protect the cystic fibrosis patients against further lung infection with P. aeruginosa.     

Risk of cross-colonization and infection by Pseudomonas aeruginosa in a holiday camp for cystic fibrosis patients.

The risk of cross-colonization and subsequent infection by Pseudomonas aeruginosa in holiday camps for cystic fibrosis patients was studied in 91 children by culturing sputum at their arrival, at their departure, 2 months later, and at regular intervals thereafter. The isolated strains were subjected to serotyping, phage typing, pyocin typing, and genotyping by random amplified polymorphic DNA fingerprinting-PCR. It was concluded from random amplified polymorphic DNA fingerprinting-PCR typing that the Pseudomonas flora was not constant in most children. Some children harbored one genotype, whereas some harbored two or more different genotypes simultaneously. Most culture-positive children easily acquired a strain of another genotype which replaced the former one or coexisted with the original one. The incidence of sputum conversion was 7.7% in previously negative children; the incidence of permanent colonization and infection was 1.9%. This risk was comparable with that observed in the community. We conclude that the risk of cross-infection is trivial compared with the obvious joy and social benefit derived from a holiday camp.     

Protease phenotypes of Pseudomonas aeruginosa isolated from patients with cystic fibrosis.

The protease phenotypes expressed by isolates of Pseudomonas aeruginosa from cystic fibrosis (CF) patients were evaluated. The majority of isolates tested produced elastase (65%) or alkaline protease (64%) or both. The mucoid phenotype expressed by many CF isolates of P. aeruginosa did not absolutely restrict the expression of protease activity, although a higher percentage of nonmucoid isolates was proteolytic. When isolates from CF patients chronically infected with P. aeruginosa were compared to isolates from CF patients colonized with this organism, both groups were found to contain comparable percentages of elastase-producing strains and mucoid strains. However, the group of isolates from colonized patients contained a higher percentage of strains producing alkaline protease and expressing general protease activity. In addition, the group of isolates from chronically infected patients contained more weakly proteolytic isolates than either the group from colonized CF patients or a group of isolates from pediatric patients without CF. These data suggest that protease production may be important in the initial colonization of the respiratory tract of CF patients by P. aeruginosa.     

Nosocomial acquisition of Pseudomonas aeruginosa by cystic fibrosis patients.

During a 4-year period, at least 12 of 40 patients with cystic fibrosis (CF) who were newly colonized with Pseudomonas aeruginosa had acquired it at CF recreation camps, clinics, or rehabilitation centers. After introduction of hygienic precautions at the CF clinic, only a single episode of nosocomial transmission of P. aeruginosa was detected at the CF ward during the subsequent 2 years.     

Transfer of a chromosomal locus responsible for mucoid colony morphology in Pseudomonas aeruginosa isolated from cystic fibrosis patients to P. aeruginosa PAO

The locus responsible for mucoid colony morphology in five independent clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients have been transferred by means of pM060-mediated conjugation to the genetically characterised strain P. aeruginosa PAO. Genetic mapping has shown that in all five strains the locus is on the chromosome between 89' and 94', although it is not possible to say that the same locus is involved in each case. The way is now open for a more detailed genetic analysis of the loci responsible for mucoid colony morphology.     

Mucosal immunity to Pseudomonas aeruginosa alginate in cystic fibrosis.

Patients with cystic fibrosis commonly acquire chronic pulmonary infection with alginate-producing Pseudomonas aeruginosa. The infection remains localized at the mucosal surfaces of the airways. Using enzyme-linked immunosorbent assays immunoglobulin concentrations and titers of specific antibodies to purified P. aeruginosa alginate and to P. aeruginosa sonicated antigens were measured in tears, saliva, sputum and serum. CF patients had significantly higher concentrations of IgG, IgA and SIgA in serum and saliva than controls. They also had significantly higher levels of specific antibodies to alginate and sonicated antigen in secretions and serum. Local production of IgA, IgG and IgM antibodies to P. aeruginosa was demonstrated. Only a minor proportion of specific IgA antibodies were present as secretory IgA in tears, saliva and sputum. The ratio of alginate-specific SIgA to specific monomeric IgA in sputum was significantly lower than the similar ratio in saliva, whereas the same ratio for specific P. aeruginosa sonicate antigens was found in saliva and sputum.     

Antimicrobial therapy for pulmonary pathogenic colonisation and infection by Pseudomonas aeruginosa in cystic fibrosis patients

Pseudomonas aeruginosa colonisation has a negative effect on pulmonary function in cystic fibrosis patients. The organism can only be eradicated in the early stage of colonisation, while reduction of bacterial density is desirable during chronic colonisation or exacerbations. Monthly, or at least 3-monthly, microbiological culture is advisable for patients without previous evidence of P. aeruginosa colonisation. Cultures should be performed at least every 2-3 months in patients with well-established colonisation, and always during exacerbations or hospitalisations. Treatment of patients following the first isolation of P. aeruginosa, but with no clinical signs of colonisation, should be with oral ciprofloxacin (15-20 mg/kg twice-daily for 3-4 weeks) plus inhaled tobramycin or colistin (intravenous treatment with or without inhaled treatment can be used as an alternative), while patients with acute infection should be treated for 14-21 days with high doses of two intravenous antimicrobial agents, with or without an inhaled treatment during or at the end of the intravenous treatment. Maintenance treatment after development of chronic P. aeruginosa infection/colonisation (pathogenic colonisation) in stable patients (aged>6 years) should be with inhaled tobramycin (300 mg twice-daily) in 28-day cycles (on-off) or, as an alternative, colistin (1-3 million units twice-daily). Colistin is also a possible choice for patients aged<6 years. Treatment can be completed with oral ciprofloxacin (3-4 weeks every 3-4 months) for patients with mild pulmonary symptoms, or intravenously (every 3-4 months) for those with severe symptoms or isolates with ciprofloxacin resistance. Moderate and serious exacerbations can be treated with intravenous ceftazidime (50-70 mg/kg three-times-daily) or cefepime (50 mg/kg three-times-daily) plus tobramycin (5-10 mg/kg every 24 h) or amikacin (20-30 mg/kg every 24 h) for 2-3 weeks. Oral ciprofloxacin is recommended for patients with mild pulmonary disease. If multiresistant P. aeruginosa is isolated, antimicrobial agents that retain activity are recommended and epidemiological control measures should be established.     

Acetamide broth for isolation of Pseudomonas aeruginosa from patients with cystic fibrosis.

The recovery of Pseudomonas aeruginosa was enhanced by incubating specimens in acetamide broth before subculture on cetrimide agar. This finding is of particular value in screening pediatric patients with cystic fibrosis for carriage of P. aeruginosa.     

Lipopolysaccharide is present in immune complexes isolated from sputum in patients with cystic fibrosis and chronic Pseudomonas aeruginosa lung infection.

Sputum samples from seven patients with cystic fibrosis and chronic P. aeruginosa lung infection were investigated for immune complexes by PEG precipitation and in two different complement binding assays. All seven patients were immune complex positive. The components involved in immune complex formation were identified by SDS-PAGE and immunoblotting. We found P. aeruginosa lipopolysaccharide as a major antigen. Both core and O-specific saccharide antigens could be demonstrated. IgG and IgA were the immunoglobulins involved, with IgG2 as the dominating IgG subclass. Lipopolysaccharide has a number of biological activities and its presence in sputum may have consequences for the pathogenesis of lung disease in cystic fibrosis.