Effects of segregation on an epidemic Pseudomonas aeruginosa strain in a cystic fibrosis clinic

The detection of a clonal Pseudomonas aeruginosa strain in 21% of children attending a cystic fibrosis clinic during 1999, which may have led to a worse prognosis, prompted strict infection control measures, including cohort segregation. We determined whether these strategies interrupted cross-infection within the clinic. Patients from 1999 were observed and a cross-sectional study of the 2002 clinic was performed. By 2002, the epidemic strain prevalence had decreased from 21 to 14% (p = 0.03), whereas the proportion of patients with nonepidemic P. aeruginosa strains was unchanged. The age- and sex-adjusted relative risk for epidemic strains among sputum producers in 2002 compared with 1999 was 0.64 (95% confidence interval, 0.47, 0.87; p = 0.004). Increased mortality or transfer to another clinic did not explain this reduction. Although children with epidemic strains may have had increased mortality (adjusted odds ratio, 2.0; 95% confidence interval, 0.6-6.8), they did not demonstrate greater morbidity than those with other P. aeruginosa isolates. Successful infection control measures provided additional indirect evidence for person-to-person transmission of an epidemic strain within the clinic. Further studies are needed to resolve whether cohort segregation completely eliminates cross-infection and if acquisition of epidemic isolates is associated with worse outcomes.     

Pseudomonas aeruginosa empyema in an adult with cystic fibrosis

Pseudomonas aeruginosa is frequently grown from the sputum of adults with cystic fibrosis-related bronchiectasis. A rare case of pseudomonal empyema is reported in this clinical setting. Early diagnosis permitted successful treatment with closed needle aspiration and intravenous antibiotics.     

Detection of a widespread clone of Pseudomonas aeruginosa in a pediatric cystic fibrosis clinic

Cross-infection by Pseudomonas aeruginosa between unrelated patients with cystic fibrosis (CF) is believed to be uncommon. After detecting a genotypically identical strain of P. aeruginosa in five unrelated children with CF dying from severe lung disease, we determined its prevalence within a large CF clinic using pulsed-field gel electrophoresis and random amplified polymorphic DNA assays. The clinical status of P. aeruginosa-infected patients was also determined. Between September and December 1999, 152 patients, aged 3.9-20.7 years, provided sputum for culture. P. aeruginosa was detected in 118 children of mean (SD) age 13.5 (3.8) years. The genotyping techniques were concordant, showing that 65 (55%) infected patients carried an indistinguishable or closely related strain. No distinctive antibiogram or environmental reservoir was found. Patients with the clonal strain were more likely than those with unrelated isolates to have been hospitalized in the preceding 12 months for respiratory exacerbations. This study demonstrates extensive spread of a single, clonal strain of P. aeruginosa in a large pediatric CF clinic. Whether this strain is also more virulent than sporadic isolates remains to be determined. As transmissible strains could emerge elsewhere, other CF clinics may also need to consider molecular methods of surveillance for cross-infection.     

Factors associated with infection by Pseudomonas aeruginosa in adult cystic fibrosis.

Cross-infection with Pseudomonas aeruginosa is an emerging issue in the care of patients with cystic fibrosis (CF). This study sought to determine the extent of, and patient factors associated with, cross-infection in a tertiary referral adult CF centre. P. aeruginosa isolates were genotyped into two groups between November 2001 and February 2003, using pulsed-field gel electrophoresis after DNA digestion by the SpeI endonuclease, and identified as clustered if there was >80% homology in the macrorestriction profiles. Patient factors and measures of disease severity were identified a priori. In total, 157 out of 227 patients had a P. aeruginosa isolate genotyped. Of these, 94 patients (60%) were infected with clustered genotypes and 47 (30%) were infected with the newly described "Midlands 1" (Md1) genotype. A further 18 patients were infected with the previously identified "Liverpool" genotype and two with the "Manchester" genotype. Logistic regression analysis revealed that the predominant predictor of infection with Md1 was age at the time of referral to the centre, suggesting that infection may have occurred prior to referral in some patients. Md1 demonstrated a relatively benign anti-biogram and did not appear to be associated with more severe disease. In conclusion, the present study provides further evidence of the emerging importance of Pseudomonas aeruginosa cross-infection in cystic fibrosis.     

Superinfection with a transmissible strain of Pseudomonas aeruginosa in adults with cystic fibrosis chronically colonised by P aeruginosa

Infection with transmissible strains of Pseudomonas aeruginosa can occur in uncolonised patients, but cross infection (superinfection) of patients already colonised withP aeruginosa has not been reported. With genotypic identification, we found superinfection by a multiresistant transmissible strain of P aeruginosa in four patients with cystic fibrosis (CF) who were already colonised by unique strains of P aeruginosa. No evidence of environmental contamination was found, but all patients became superinfected after contact with colonised individuals during inpatient stays. Inpatients with CF who are colonised with P aeruginosa should be separated by strain type. Such strain typing can only be reliably done by genomic methods, but this has resource implications.     

Clonal strains of Pseudomonas aeruginosa in paediatric and adult cystic fibrosis units.

Despite recent reports of clonal strains of Pseudomonas aeruginosa in cystic fibrosis (CF) units, the need for routine microbiological surveillance remains contentious. Sputum was collected prospectively from productive patients attending the regional paediatric and adult CF units in Brisbane, Australia. All P. aeruginosa isolates were typed using pulsed-field gel electrophoresis. Spirometry, anthropometrics, hospitalisations and antibiotic sensitivity data were recorded. The first 100 sputum samples (first 50 patients at each clinic) harboured 163 isolates of P. aeruginosa. A total of 39 patients shared a common strain (pulsotype 2), 20 patients shared a strain with at least one other patient and 41 patients harboured unique strains. Eight patients shared a strain identical to a previously reported Australian transmissible strain (pulsotype 1). Compared with the unique strain group, patients harbouring pulsotype 2 were younger and had poorer lung function. Treatment requirements were similar in these two groups, as were the rates of multiresistance. In conclusion, 59% of patients harboured a clonal strain, supporting the need for routine microbiological surveillance. In contrast to previously described clonal strains, the dominant pulsotype was indistinguishable from nonclonal strains with respect to both colonial morphology and multiresistance. The clinical significance of clonal strains remains uncertain and requires longitudinal study.     

Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with multiresistant strains of Pseudomonas aeruginosa

We developed a rapid in vitro antibiotic susceptibility test to screen double- and triple-antibiotic combinations for bactericidal activity against 75 multiresistant Pseudomonas aeruginosa isolates referred from 44 cystic fibrosis (CF) patients. When used alone, the most effective intravenous antibiotic, meropenem, was bactericidal against only 44% of the isolates. High-dose tobramycin (200 microg/ml; concentrations achievable by aerosol administration) was bactericidal against 72% of isolates. Adding a second antibiotic significantly improved bactericidal activity. The most effective double-antibiotic combinations contained high-dose tobramycin plus meropenem, piperacillin/tazobactam, or ciprofloxacin, and were bactericidal against 88 to 94% of the isolates. Excluding high-dose tobramycin, the most effective intravenous double-antibiotic combinations contained meropenem plus ciprofloxacin, tobramycin (4 microg/ml), or cefipime, and were bactericidal against 85%, 71%, and 70% of isolates, respectively. Adding a third antibiotic did not significantly improve inhibition in vitro. We conclude that double-antibiotic combinations containing meropenem or high-dose tobramycin show the best bactericidal activity in vitro against multiresistant strains of P. aeruginosa. Addition of a third antibiotic to these double-antibiotic combinations may be unnecessary.     

Epidemiology of Pseudomonas aeruginosa in a cystic fibrosis rehabilitation centre.

Pseudomonas aeruginosa is the leading pathogen in cystic fibrosis (CF) lungs. Since there is great concern about clonal spread in CF centres, this study examined the P. aeruginosa genotypes of colonised residents of a CF rehabilitation centre. The isolates from the sputum of 76 P. aeruginosa-colonised patients were genotyped by fluorescent amplified fragment length polymorphism on arrival and departure. A total of 71 different P. aeruginosa genotypes were identified from 749 isolates. Forty-nine patients had one genotype, 20 had two genotypes and seven had three. Forty-four patients had one or more genotypes in common with other patients (i.e. cluster types). Thirty-two patients were colonised by a single genotype not shared by any other patient. Thirty-eight of the 44 patients with a cluster type already carried their cluster type strain(s) on arrival. Patient-to-patient transmission could not be excluded for eight patients. For five of these, this infection was transient. None of the environmental P. aeruginosa isolates had a genotype similar to the patients' genotypes. In summary, most patients were colonised by only one or two P. aeruginosa genotypes and the risk of persistent patient-to-patient transmission was low during the study period (4%). Most patients with a cluster-type strain carried this strain on arrival, indicating that transmission could have happened in the past. No environmental contamination could be established.     

Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis.

In the context of chronic lung infection due to Pseudomonas aeruginosa in cystic fibrosis (CF), attention has been focused on the presence of the most common mucoid phenotype. In this study, the presence of small-colony variants (SCVs) of P. aeruginosa in respiratory tract specimens from patients with CF was investigated, and the clinical conditions predisposing to SCVs were analyzed. P. aeruginosa SCVs were isolated from 33 of 86 P. aeruginosa-positive CF patients over a 2-year period. Fast-growing revertants with larger surface colonies could be isolated from SCV populations. Electron microscopy revealed no significant difference in cell size or morphology. MICs of a broad range of antipseudomonas agents for SCVs were two- to eightfold higher than values for revertants. Recovery of SCVs was correlated with parameters revealing poor lung function and was significantly associated with daily inhalation of tobramycin or colistin.     

IgG proteolytic activity of Pseudomonas aeruginosa in cystic fibrosis

To study how fragmented IgG antibodies might arise within the respiratory secretions of individuals with cystic fibrosis (CF), we screened protease extracts from CF polymorphonuclear leukocytes and mucoid and nonmucoid transformants of Pseudomonas aeruginosa from patients with CF for IgG proteolytic activity. All strains of P. aeruginosa tested exhibited IgG proteolytic activity. Incubation for 7 hr at 37 C was required to demonstrate generation of free Fc gamma immunoreactivity. Further analysis of these cleavage products of CF IgG demonstrated generation of Fc gamma polypeptides with 4S sedimentation coefficients and F(ab')2 fragments with 5S coefficients. Bacterial IgG proteolytic activity was inhibited by EDTA and was associated with levels of bacterial elastase exceeding 5 micrograms/mg of total protein. Pseudomonas elastase was significantly more active on IgG1 and IgG3; IgG2 and IgG4 were more resistant. This bacterial exoproduct appears to digest IgG molecules into Fab gamma, F(ab')2 fragments, and a free Fc gamma piece with a molecular weight of 40,000.     

Proteases of Pseudomonas aeruginosa in patients with cystic fibrosis

Radioimmunoassays were used to determine titers of antibody to alkaline protease (AP) and elastase (Ela) produced by Pseudomonas aeruginosa in sera and bronchial secretions, in vitro production of AP and Ela by P. aeruginosa isolates, and occurrence of these enzymes in bronchial secretions from patients with cystic fibrosis. Titers of serum antibodies to AP ranging from 1:10 to 1:545 and to Ela ranging from 1:10 to 1:725 were found in 83%-88% of patients with cystic fibrosis and chronic lung infections due to P. aeruginosa. Antibody titers in liquified bronchial secretions were approximately 10% of the serum titers. Thirty-one (93%) of 34 isolates produced both proteases in vitro in comparable amounts (concentration of protease in culture supernatant: AP, 0.01-480 micrograms/ml; Ela, 0.02-490 micrograms/ml). AP and Ela were detected in vivo when antibodies to the enzymes were absent. The results suggest that specific immune responses in patients with cystic fibrosis neutralize proteases of P. aeruginosa.     

Clinical profile of adult cystic fibrosis patients with frequent epidemic clones of Pseudomonas aeruginosa

Background and objective: Earlier reports suggested that Pseudomonas aeruginosa frequent epidemic clones circulating in cystic fibrosis (CF) centres had increased virulence. However, recent data show no consistent associations with virulence, and suggest attenuation of virulence in chronic infection. Changes to infection control programmes in relation to frequent epidemic clones should be based on their frequency, virulence across all age groups and mode of acquisition. The Australian epidemic strain‐1 (AES‐1) (or the Melbourne epidemic strain) and AES‐2 are common in CF clinics in mainland eastern Australia, but not in the environment. Both have shown increased virulence, but there are no data specifically in adults. This study examines the frequency and virulence of P. aeruginosa frequent epidemic clones in the adult CF clinic at Royal Prince Alfred Hospital, Sydney, Australia. Methods: Two hundred and fifty‐eight P. aeruginosa isolates from 112 participants were genotyped by pulsed field gel electrophoresis. Ninety‐eight patients were followed up for 1 year and associations sought between infection with a frequent epidemic clone, clinical outcome and antibiotic resistance. Results: Four frequent P. aeruginosa epidemic clones (AES‐1, AES‐2, S‐1, S‐2) affected almost 50% of participants. AES‐1 predominated (38%). AES‐1, AES‐2 and S‐1 were associated with increased exacerbations and hospital‐admission days. AES‐1 showed increased resistance to aminoglycosides and ticarcillin‐clavulanate. Conclusions: This study supports the potential threat of frequent P. aeruginosa epidemic clones in adult CF populations.     

Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway

The cystic fibrosis (CF) lung is chronically inflamed and infected by Pseudomonas aeruginosa, which is a major cause of morbidity and mortality in this genetic disease. Although aerosolization of Tobramycin into the airway of CF patients improves outcomes, the lungs of CF patients, even those receiving antibiotic therapy, are persistently colonized by P. aeruginosa. Recent studies suggest that the antibiotic resistance of P. aeruginosa in the CF lung is due to the formation of drug resistant biofilms, which are defined as communities of microbes associated with surfaces or interfaces, and whose growth is facilitated by thick and dehydrated mucus in the CF lung. In this review, we discuss some of the current models used to study biofilm formation in the context of biotic surfaces, such as airway cells, as well as the contribution of host-derived factors, including DNA, actin and mucus, to the formation of these microbial communities. We suggest that better in vitro models are required, both to understand the interaction of P. aeruginosa with the host airway, and as models to validate new therapeutics, whether targeted at bacteria or host.