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.     

Anti-Pseudomonas aeruginosa antibodies and lung disease in cystic fibrosis

The aim of our study was to diagnose and to control three aspects of the evolution of lung disease in CF: the absence of infection, the intermittent colonization and chronic infection by Pseudomonas aeruginosa. Therefore a study of anti-pseudomonas antibodies (Ab) (anti-protease, anti-elastin and antihexo-toxin A) for diagnosis and follow-up of CF patients was considered. Moreover, we related the presence of Ab to the sputum culture, to FEV1, to patient age and to genotype. Tbe Ab were dosed in 121 patients by quantitative ELISA method. Values < 1: 500 were considered negative, values> 1: 500 and < 1:1250 borderline, and > 1:1250 positive. 16.5% of patients did not have Ab, 17% had borderline values and 69.5% had positive values. All the patients with negative Ab had negative sputum culture; 47% of patients with borderline values had at least one positive culture while 53% were negative. 87% of patients with positive values had chronic colonization, 13% intermittent colonization. The increase in the Ab rate is statistically related to a more severe lung disease (p < 0.013). The presence of a severe mutation (?F 508) is related to positive values of Ab. Evaluation of anti-Pseudomonas aeruginosa is an important tool for diagnosis and follow-up of CF lung disease     

Immunoglobulin G antibodies toPseudomonas aeruginosa lipopolysaccharides and exotoxin A in patients with cystic fibrosis or bacteremia

IgG antibodies to ninePseudomonas aeruginosa lipopolysaccharides (LPS) and exotoxin A in sera from 11 patients with bacteremia and 51 patients with cystic fibrosis (CF) were analyzed. The methods used were enzyme immunoassay (EIA) and immunoblotting. Nine of the 11 bacteremic patients were infected with strains expressing an LPS serotype identical to one of the test antigens. In sera from six of these nine patients, antibody homologous to the serotype of the infecting strain was observed. An antibody response to heterologousPseudomonas aeruginosa LPS antigens was observed in nine patients. Eight of the bacteremic patients mounted an antibody response to exotoxin A. Thirty-five CF patients chronically colonized withPseudomonas aeruginosa possessed significantly higher levels of antibody to all of the test antigens than 16 patients with intermittent or no colonization (p<0.001). For exotoxin A and serotype 3 the sensitivity was 91 % and 94 %, and the specificity 94 % and 88 % respectively. When the results for exotoxin A and serotype 3 were combined, the sensitivity was 91 % while the specificity was 81 %. The pronounced antibody response to heterologous LPS antigens, as measured by the EIA and immunoblot, suggests expression of a common antigen determinant. A simplified serological assay utilizing exotoxin A and serotype 3 as test antigens may be useful for detectingPseudomonas aeruginosa infections in patients with CF and chronic colonization and in bacteremic patients from whom cultures are not available.     

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.     

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.     

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.     

Sera from adult patients with cystic fibrosis contain antibodies to Pseudomonas aeruginosa type III apparatus

Expression of type III proteins of Pseudomonas aeruginosa in patients with cystic fibrosis (CF) was investigated by measuring the immune response against components of the type III pathway. Twenty-three of the 33 sera contained antibodies against PcrV, a protein involved in translocation of type III cytotoxins into eukaryotic cells, and 11 of 33 had antibodies against ExoS, while most CF sera contained antibodies against PopB and PopD, components of the type III apparatus. These data indicate that P. aeruginosa commonly expresses components of the type III translocation apparatus in adult CF patients.     

Molecular epidemiological analysis ofPseudomonas aeruginosa strains causing failure of antibiotic therapy in cystic fibrosis patients

A combination of esterase electrophoretic typing and analysis of the restriction fragment length polymorphism of ribosomal DNA regions (ribotyping) was used to compare 27Pseudomonas aeruginosa strains isolated before and after two-week courses of anti-pseudomonal treatment in seven cystic fibrosis patients. A total of 12 courses of therapy were studied in which ciprofloxacin, ceftazidime, azlocillin or imipenem were used alone or in combination with tobramycin. Isolates at a count of 10⁶ cfu/ml of sputum were collected when there was evidence of therapeutic failure on the basis of persistence of isolates whether or not they were resistant to the antibiotic used for therapy. Emergence of resistance was observed in ten cases and failure to eradicate sensitive strains in five cases. Among the 27 isolates, eight zymotypes and five ribotypes were identified. With this typing approach, resistant post-therapy isolates were found to be identical to pre-therapy isolates in all cases but one. However, in one case an additional resistant strain was isolated after therapy besides that initially present. In all five cases in which susceptibility was still observed after treatment, pretherapy and post-therapy isolates were indistinguishable. Using this molecular typing approach, all the strains were typable. Thus combination of esterase typing and ribotyping should improve the analysis of therapeutic failure in cystic fibrosis patients.     

Typing of Pseudomonas aeruginosa strains in Norwegian cystic fibrosis patients

Objectives   Typing of Pseudomonas aeruginosa isolates from Norwegian cystic fibrosis (CF) patients with chronic Pseudomonas lung infection in order to see whether cross-infection might have occurred.
Methods   Isolates from 60 patients were collected during the years 1994–98, and typed by pulsed field gel electrophoresis.
Results   Seventy-one strains were identified. One large cluster of identical strains included 27 patients, and 13 smaller clusters of 2–4 patients were found (26 patients). Seven patients had a strain not shared by other patients (private strains). Harboring the main cluster strain was significantly associated with participation in summer camps and training courses ( P  = 0.004, chi-squared test). There were no associations with regular admissions to hospital (intravenous antibiotic courses) or smaller social gatherings of short duration. Small clusters and private strains were not associated with any of the risk factors. All strains were sensitive to colistin. The minimal inhibitory concentrations were generally lower in Norwegian P. aeruginosa strains compared with isolates from Danish patients.
Conclusions   Our results indicate that cross-infection with P. aeruginosa between cystic fibrosis patients has occurred.     

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.     

Aminoglycoside resistance in Pseudomonas aeruginosa isolated from cystic fibrosis patients

The authors studied 30 gentamicin-resistant and 17 gentamicin-sensitive strains of Pseudomonas aeruginosa isolated from respiratory cultures of patients with cystic fibrosis from five United States cities for the presence of plasmids, cross-resistance to other aminoglycosides, and the production of aminoglycoside-modifying enzymes. Four of 30 resistant strains and 3 of 17 sensitive strains contained one or more plasmids. Aminoglycoside cross-resistance to tobramycin, amikacin, and netilmicin was seen in 21 of 30 gentamicin-resistant strains. Seven strains that had low-level gentamicin resistance (minimum inhibitory concentrations [MIC] = 8-32 micrograms/mL) were sensitive to one or more of the other three aminoglycosides. Two strains with high-level gentamicin resistance (MIC greater than or equal to 128 micrograms/mL) were sensitive to amikacin. These two strains, each containing three plasmids, were the only isolates of nine tested that produced an aminoglycoside-modifying enzyme with activity against gentamicin. None of the plasmids was transferable by conjugation. Four strains, three of which contained one or more plasmids, produced an aminoglycoside 3'-0-phosphotransferase II. The authors propose that the mechanism of gentamicin resistance in P. aeruginosa from patients with cystic fibrosis is not commonly plasmid-mediated and likely is due to membrane impermeability to aminoglycosides.     

Antilipopolysaccharide antibodies and differential diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis

Chronic lung infection in cystic fibrosis is characteristically associated with polyagglutinable, serum-sensitive, mucoid strains of Pseudomonas aeruginosa. Enzyme-linked immunosorbent assay (ELISA) methods for standard-free quantitation of immunoglobulin G (IgG) and IgM antibodies to P. aeruginosa lipopolysaccharides (LPSs) have been developed. We now report the development of assays for quantitation of monomer and dimer total IgA and IgA anti-LPS antibodies. Use of these methods in diagnosis of early chronic P. aeruginosa lung infection was assessed. IgG and IgA anti-LPS levels increased significantly at the onset of chronic infection and continued to increase to very high levels in the later stages of infection. IgM anti-LPS levels also rose at the onset of chronic infection but did not increase further. The function of true- and false-positive rates was illustrated by using various concentrations of IgG, IgA, and IgM anti-LPS for discrimination of patients. Values that gave optimum separations were used for statistical evaluation of the diagnostic sensitivities and specificities of anti-LPS antibody concentrations. The results obtained in these assays were compared with a diagnosis, based on the number of precipitins in crossed immunoelectrophoresis, of serum samples from cystic fibrosis patients. In 64 paired serum samples taken before and immediately after the onset of chronic infection, as defined by crossed immunoelectrophoresis precipitins, the predictive values of a positive ELISA were 86% for IgG and 89% for IgA. The predictive values for a negative ELISA were 98% for IgG and 97% for IgA. Results of the IgM anti-LPS ELISA had a lower predictive value. Immunoblotting and absorption studies showed that IgG anti-LPS antibodies were directed specifically against LPS of P. aeuruginosa. ELISAs were developed to determine the specific IgG sublclasses involved. The increase in IgG anti-LPS involved all four subclasses. Highest anti-LPS titers were seen with IgG1 and IgG4, but the largest relative increases were seen with IgG2 and IgG3.     

Prospective study of serum antibodies to Pseudomonas aeruginosa exoproteins in cystic fibrosis.

Serum immunoglobulin G to four purified antigens from Pseudomonas aeruginosa, phospholipase C, alkaline protease, exotoxin A, and elastase, were determined in 62 patients with cystic fibrosis by enzyme-linked immunosorbent assay. The patients were followed for 12 to 24 months in a prospective study. Increased titers, i.e., titers more than 2 standard deviations above those of normal controls, were exclusively found in patients chronically colonized with P. aeruginosa and not in patients harboring only Staphylococcus aureus. The frequencies of elevated titers of antibody to the different antigens varied from 100% (phospholipase C) to 58% (alkaline protease and exotoxin A) to 15% (elastase) in the chronically colonized patients. Mean serum titer levels, expressed as multiples of the age-correlated upper normal limit (=1), were significantly higher to phospholipase C in patients with dual colonization with P. aeruginosa and S. aureus than in those colonized only with P. aeruginosa (P less than 0.001). Conversely, the other three antigens showed significantly higher serum antibody titer levels in patients harboring only P. aeruginosa (P less than 0.001). In five patients who became colonized with P. aeruginosa during the study period, serum antibodies to phospholipase C and exotoxin A increased first. Exceptions to the general pattern of antibody responses were found in three patients chronically colonized with Escherichia coli. They showed a delayed enhancement of anti-phospholipase C titers after the chronic P. aeruginosa colonization. Serum titers were not influenced by exacerbations of pulmonary infection or by antimicrobial therapy. The determination of titers of serum antibody to phospholipase C seems to be a valuable indicator of a chronic colonization with P. aeruginosa. The results further suggest that bacterial metabolism and interactions may influence the antibody response.     

Comparison of agar diffusion methodologies for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of patients with cystic fibrosis (CF). Improved antimicrobial chemotherapy has significantly increased the life expectancy of these patients. However, accurate susceptibility testing of P. aeruginosa isolates from CF sputum may be difficult because the organisms are often mucoid and slow growing. This study of 597 CF isolates of P. aeruginosa examined the correlation of disk diffusion and Etest (AB BIODISK, Solna, Sweden) results with a reference broth microdilution method. The rates of interpretive errors for 12 commonly used antipseudomonal antimicrobials were determined. The disk diffusion method correlated well (zone diameter versus MIC) for all of the agents tested. However, for mucoid isolates, correlation coefficients (r values) for piperacillin, piperacillin-tazobactam, and meropenem were <0.80. The Etest correlation with reference broth microdilution results (MIC versus MIC) was acceptable for all of the agents tested, for both mucoid and nonmucoid isolates. Category interpretation errors were similar for the disk diffusion and Etest methods with 0.4 and 0.1%, respectively, very major errors (false susceptibility) and 1.1 and 2.2% major errors (false resistance). Overall, both agar diffusion methods appear to be broadly acceptable for routine clinical use in susceptibility testing of CF isolates of P. aeruginosa.     

Evaluation of E-Test for determination of antimicrobial MICs for Pseudomonas aeruginosa isolates from cystic fibrosis patients.

We determined the E-Test and National Committee for Clinical Laboratory Standards standardized agar dilution MICs of ceftazidime, ciprofloxacin, piperacillin, and tobramycin for Pseudomonas aeruginosa during tests of 100 rough and mucoid P. aeruginosa isolates from cystic fibrosis patients. The levels of agreement (+/- 1 log2 dilution) between quantitative E-Test and agar dilution MIC results were 80, 97, 73, and 89% for ceftazidime, ciprofloxacin, piperacillin, and tobramycin, respectively. Comparison of the results after converting the MIC data to qualitative categories (susceptible, intermediate, and resistant) yielded levels of agreement of 84, 96, 88, and 93% for the same agents, respectively. Of the 39 qualitative discrepancies, 36 were minor and 3 were very major. We conclude that use of the E-Test is easier and more practical than use of the agar dilution method for most laboratories and that the E-Test furnishes results which are at least as accurate as those obtained by the agar dilution method. However, the higher cost of the E-Test method would likely discourage most laboratories from selecting it over disk diffusion for routine antimicrobial susceptibility testing of P. aeruginosa isolates from cystic fibrosis patients.     

Genetic determinants ofPseudomonas aeruginosa colonization in cystic fibrosis patients in Canada

The present study was aimed at analyzing whether the rate of colonization and the age at colonization withPseudomonas aeruginosa was genetically determined in cystic fibrosis (CF) patients. These two variables were calculated among 127 CF patients whose genotypes were known and who were monitored at the Clinique de Fibrose Kystique in Saguenay Lac-Saint-Jean. No statistically significant differences were found in the rate or the age at colonization when the patients were grouped by genotype; however, this result could be due to the small number of patients in each genotype group. The rate of colonization was significantly lower among CF patients carrying the A455E mutation, a mild allele with respect to exocrine pancreatic function, than among those carrying either the F508 or the 621+1G->T mutation, both of which are severe alleles. The results confirm previous reports that the rate of colonization withPseudomonas aeruginosa is, at least in part, genetically determined.     

Phenotypic and genotypic characterization of Pseudomonas aeruginosa from cystic fibrosis patients

Pseudomonas aeruginosa accounts for about one half of all pulmonary infections of cystic fibrosis (CF) patients. In this study, we analyzed 135 P. aeruginosa strains isolated from the expectorations of 55 CF adult patients attending a CF referral center over a period of five years. We assessed the genotype of the strains by pulsed-field gel electrophoresis (PFGE) and analyzed some phenotypic characteristics, such as O serotype, enzyme and mucous production, antibiotics susceptibility, and motility. PFGE allowed the typification of 97.1% of strains, revealing the presence of nine different genomic patterns. The pattern indicated as B was the most frequent, whereas patterns H and I were the most uncommon. Serotyping failed to identify 37.8% of strains and 29 out of 55 patients harbored almost one non-typable (NT) strain. During the five years of the study, we observed a progressive reduction of O6 and O10 types, but an increase of the O1 type and of NT strains. Most strains produced protease, hemolysin, and gelatinase, and were mobile. Several patients harbored the same serotype or genotype in sequential isolates, though characterized by a different susceptibility to antimicrobials. We did not observe a relationship between bacterial genotype and phenotype. This could be due to the fact that PFGE is not sensitive enough to detect subtle genotypic differences. The epidemiological importance of the genotypic characterization of bacteria-colonizing CF subjects and the surveillance measures to be adopted in CF centers are briefly discussed.     

A comparison of the efficiency in serotyping of Pseudomonas aeruginosa from cystic fibrosis patients using monoclonal and polyclonal antibodies

A well-known problem in serotyping Pseudomonas aeruginosa strains from cystic fibrosis patients using polyclonal sera is that more than half of the strains cannot be assigned to a single O-serogroup because of the high occurrence of polyagglutinable strains and nontypable strains. In the present study, the efficiency of a set of O-specific monoclonal antibodies in the simple slide agglutination test was compared with polyclonal sera for the serotyping of 243 isolates of P. aeruginosa from cystic fibrosis patients. Using the monoclonal antibodies, 213 (88%) strains were found to be typable and only 30 (12%) strains were nontypable. In contrast, when the polyclonal sera from Statens Seruminstitut were used, only 53 (22%) strains were typable. Similar results were obtained when polyclonal sera manufactured by Difco were used where only 61 (25%) strains were typable. We also investigated the consistency of each set of antibodies in typing of the same isolates on different days and found that the polyclonal sera showed higher reproducibility. The Statens Seruminstitut sera were found to have an 86% reproducibility while the Difco sera scored 81%; however, the percentage of strains that are nontypable remains below 25% despite the highly reproducible results obtained. The monoclonal antibodies were found to score a 75% reproducibility when the serotyping of the same strains was done both in the laboratories of Copenhagen and of Guelph. However, it should be noted that although the reproducibility was somewhat lower with the monoclonal antibodies, these highly specific antibodies are still clearly superior when compared with polyclonal sera used because more than 80% of the strains from cystic fibrosis can now be assigned to a single O-serogroup.     

Typing of polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients

The study assesses the reproducibility, typability and discriminatory power of several typing methods for Pseudomonas aeruginosa isolated from cystic fibrosis patients. 178 polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients were serotyped using polyclonal sera and monoclonal antibodies, phage typed, pyocin typed and reverse phage typed. 31 of these polyagglutinable isolates, six monoagglutinable isolates and three nontypable isolates were also typed by means of hybridization using a DNA probe. In a comparison of the methods used, on polyagglutinable isolates only, typability was 0% with polyclonal sera, 90% with monoclonal sera, 94% with phage typing, 85% with pyocin typing, 36% with reverse phage typing and 100% with DNA-prope typing. Using monoclonal antibodies, the reproducibility was 75%, while that of phage typing was 88%, pyocin typing 53% and reverse phage typing 62%. Typing with the DNA probe was not repeated. using polyclonal sera, repeated typing showed that 94% of the isolates were polyagglutinable. Using phage typing, 40% of the isolates belonged to phage type 31, while 60% were distributed amongst 32 phage types. Using monoclonal antibodies, 71% of the isolates belonged to 0-group 3, and these isolates showed 16 different phage types. Subdivision of the phage types was further achieved by both pyocin typing and reverse phage typing. The DNA probe typing made it possible in some cases to discriminate between isolates which were otherwise found identical with the conventional typing methods, while in other cases typing with the DNA probe recorded as identical isolates which conventional methods had typed as being different. These differences may be due to a high mutation rate caused by the selection pressure of antibiotics, and by the host immune response. According to our results, investigations of reproducibility and typability of old and new typing methods are essential when they are used in clinical situations. The low reproducibility of some of the typing methods in the present study affects the reliability of epidemiological investigations in cystic fibrosis patients. Usage of only one method may not be sufficient in cases of polyagglutinable strains from cystic fibrosis patients.