Poor clinical outcomes associated with a multi-drug resistant clonal strain of Pseudomonas aeruginosa in the Tasmanian cystic fibrosis population

Background and objective: Clonal strains of Pseudomonas aeruginosa have been identified in large cystic fibrosis (CF) centres. Whether such strains are more virulent or whether cross‐infection between patients explains their widespread prevalence is unknown. This study described the epidemiology of P. aeruginosa infection in CF patients in Tasmania, Australia, an area with a high CF birth incidence. Patients in Tasmania are geographically dispersed and when this study was conducted (2003) there was no central CF clinic, with patients receiving treatment in regional hospitals. Methods: P. aeruginosa isolates from CF adults aged 15 years and over in Tasmania were genotyped using random amplified polymorphic DNA (RAPD)‐PCR and clonal strains confirmed with pulsed field gel electrophoresis. Results: Airway samples were obtained from 41 patients (82% of the adult CF population). P. aeruginosa was isolated from 34 patients and nine (26%) of these individuals harboured P. aeruginosa strains with identical RAPD‐PCR and pulsed field gel electrophoresis patterns (Australian Epidemic Strain III – AES III). AES III was isolated from patients in all regions of Tasmania and was distinct from the epidemic CF strains described on mainland Australia (AES I and II). The possible link between CF adults infected with AES III was attendance at family camps more than 12 years previously. Patients harbouring AES III had suffered significantly more exacerbations requiring hospitalisation during the 2 years prior to the study compared with patients infected with unique strains (P < 0.01). AES III displayed increased multi‐antibiotic resistance compared with other strains (P < 0.001). Conclusions: Clonal strains of P. aeruginosa may arise even in isolated CF populations. The increased exacerbation rate in patients infected with AES III and its antibiotic resistance profile strongly suggest increased virulence.     

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.     

The effect of alginate on the invasion of cystic fibrosis respiratory epithelial cells by clinical isolates of Pseudomonas aeruginosa

Chronic infection in the cystic fibrosis (CF) lung is characterized by Pseudomonas aeruginosa strains that overproduce the mucoid exopolysaccharide, alginate. Previous experiments have shown that long-term survival of P. aeruginosa in the CF lung may be facilitated by increased adherence and decreased invasion of respiratory epithelial cells. Therefore, mucoid and nonmucoid clinical isolates of P. aeruginosa were assayed for their ability to associate with and invade the CF respiratory epithelial cell line, CF/T43. Association assays and gentamicin exclusion assays demonstrated that mucoid P. aeruginosa associates with and invades CF/T43 cell monolayers significantly less than nonmucoid P. aeruginosa strains (P = .004, .02). Fluorescence microscopy invasion assays confirmed these results. The differences in association and invasion by the P. aeruginosa strains were not due to differences in lipopolysaccharide phenotype or cytotoxicity for CF/T43 respiratory epithelial cells. Exogenous bacterial alginate had no effect on the invasion of CF respiratory epithelia by a nonmucoid strain. Invasion assays with the wild-type P. aeruginosa strain PAO1 and isogenic algU and mucA mutant strains failed to show differences in invasion (P = .25). We conclude that (i) mucoid P. aeruginosa isolates associate with and invade CF/T43 respiratory epithelial cells with less efficiency than nonmucoid P. aeruginosa, (ii) these differences are not due to variations in lipopolysaccharide phenotype between strains, (iii) neither exogenous nor endogenous alginate affects the ability of P. aeruginosa to invade CF/T43 respiratory epithelial cells, and (iv) invasion of CF/T43 respiratory epithelial cells by a laboratory reference strain of P. aeruginosa does not appear to be regulated by AlgU.     

Distribution and transmission of Pseudomonas aeruginosa and Burkholderia cepacia in a hospital ward

Genotyping and antibiotic susceptibility testing were used to analyze Pseudomonas aeruginosa and Burkholderia cepacia strains from sink drain from 14 pediatric patients with cystic fibrosis (CF) and from hospital personnel as part of a 4 week prospective study of strain transmission in a pediatric ward. A total of 87.5% of all washbasin drains were contaminated with P. aeruginosa [10² to 10⁵ colony forming units (CFU)/ml sink fluid], whereas B. cepacia was found only once in a sink drain. From the eight CF patients already infected with P. aeruginosa upon entering the ward, we isolated six genotypes that were identical with strains found in sink drains of the ward. Four of the 16 members of the personnel had one positive P. aeruginosa hand culture. B. cepacia was never found in patients or on personnel hands. Hand washing in contaminated sinks (≥ 10³ CFU/ml) led to positive P. aeruginosa or B. cepacia hand cultures. P. aeruginosa or B. cepacia embedded in sputum were transmissable by hand shaking for up to 180 min, whereas both pathogens suspended in physiological saline were transmissable to other hands only up to 30 min. Genotyping of P. aeruginosa revealed strain transmission from CF patients or the environment to other patients or the personnel, as well as one transmission from the environment to a CF patient. The ability of CF sputum to prolong survival of P. aeruginosa and B. cepacia may be important for strain transmission. The results suggest that improved hygienic measures are required to prevent routes of bacterial transmission via the hands and sink drains. Pediatr Pulmonol. 1996; 21:90–100. © 1996 Wiley-Liss, Inc.     

Epidemiology of Pseudomonas cepacia colonization among patients with cystic fibrosis

Colonization with Pseudmonas cepacia in patients with cystic fibrosis (CF) has been associated with increased morbidity and early death, compared with colonization by P. aeruginosa. The mode of acquisition of P. cepacia is not fully understood, although person-to-person spread appears likely. Recent epidemiologic studies support the importance of social contact in the spread of P. cepacia among patients with CF. This study was undertaken to investigate the epidemiology of P. cepacia colonization among patients with CF attending the CF clinic at our center. Isolates of P. cepacia were collected from patients at two CF treatment centers, including ours. Additional isolates were collected from patients without CF in the hospital ICU, from other teaching hospitals, and from the environment. Profiles of enzymes were obtained by ultrathin polyacrylamide gel electrophoresis of P. cepacia extracts. A predominant electromorphic type (ET) was found among P. cepacia isolates from patients at both centers, suggesting a common source or person-to-person transmission. The majority of hospital isolates fell into a single, different ET. Surveillance swabs of respiratory equipment at our CF clinic did not grow P. cepacia. Attendance of patients at CF summer camp correlated strongly with P. cepacia colonization (P < 0.0001). Pediatr Pulmonol. 1994;18:108–113 . © 1994 Wiley-Liss, Inc.     

Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms

The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilm-mode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.     

Longitudinal assessment of Pseudomonas aeruginosa in young children with cystic fibrosis

Pseudomonas aeruginosa lung infection is an important cause of morbidity and mortality in cystic fibrosis (CF). Longitudinal assessment of the phenotypic changes in P. aeruginosa isolated from young children with CF is lacking. This study investigated genotypic and phenotypic changes in P. aeruginosa from oropharynx (OP) and bronchoalveolar lavage fluid (BALF) in a cohort of 40 CF patients during the first 3 years of life; antibody response was also examined. A high degree of genotypic variability was identified, and each patient had unique genotypes. Early isolates had a phenotype distinct from those of usual CF isolates: generally nonmucoid and antibiotic susceptible. Genotype and phenotype correlated between OP and BALF isolates. As determined by culture, 72.5% of patients demonstrated P. aeruginosa during their first 3 years. On the basis of combined culture and serologic results, 97.5% of patients had evidence of infection by age 3 years, which suggests that P. aeruginosa infection occurs early in CF and may be intermittent or undetectable by culture.     

Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients

The present study was undertaken to investigate the appearance and location of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung and in sputum. Samples include preserved tissues of CF patients who died due to chronic P. aeruginosa lung infection prior to the advent of intensive antibiotic therapy, explanted lungs from 3 intensively treated chronically P. aeruginosa infected CF patients and routine sputum from 77 chronically P. aeruginosa infected CF patients. All samples were investigated microscopically using hematoxylin–eosin (HE), Gram and alcian‐blue stain, PNA FISH and immunofluorescence for alginate. Investigation of the preserved tissues revealed that prior to aggressive antibiotic therapy, P. aeruginosa infection and destruction of the CF lung correlated with the occurrence of mucoid (alginate) bacteria present in aggregating structures surrounded by pronounced polymorphonuclear‐leukocyte (PMN) inflammation in the respiratory zone (9/9). Non‐mucoid bacteria were not observed here, and rarely in the conductive zone (1/9). However, in the explanted lungs, the P. aeruginosa aggregates were also mucoid but in contrast to the autopsies, they were very rare in the respiratory zone but abundant in the sputum of the conductive zone (3/3), which also contained abundances of PMNs (3/3). Non‐mucoid and planktonic P. aeruginosa were also observed here (3/3). In conclusion, the present intensive antibiotic therapy of chronic P. aeruginosa infections, at the Copenhagen CF Centre, seems to restrain but not eradicate the bacteria from the conductive zone, whereas the remaining healthy respiratory zone appears to be protected, for a long period, from massive biofilm infection. This strongly suggests that the conductive zone serves as a bacterial reservoir where the bacteria are organized in mucoid biofilms within the mucus, protected against antibiotics and host defenses. Pediatr Pulmonol. 2009; 44:547–558. © 2009 Wiley‐Liss, Inc.     

Biofilm production using distinct media and antimicrobial susceptibility profile of Pseudomonas aeruginosa

Biofilm production is an important mechanism for bacterial survival and its occurrence together with antimicrobial resistance represents a challenge for clinical management. Here, we evaluated the ability for biofilm production among P. aeruginosa isolates from patients with or without cystic fibrosis (CF) using two distinct media, besides determining the antimicrobial susceptibility profile of these isolates for eight antimicrobial agents. The ability for biofilm production when TSB medium was used was higher than when used CF sputum media (p = 0.0198). However, P. aeruginosa isolates from CF have demonstrated similar performance for biofilm production, independently of the medium used. Besides, among the biofilm-producing isolates, those recovered from CF were more resistant to the carbapenems (meropenem and imipenem) agents than those isolates from non-CF isolates.     

Detection of viable but non-culturable <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in cystic fibrosis by qPCR: a validation study

Background

Routine culture-based diagnosis of Pseudomonas aeruginosa lung infection in Cystic Fibrosis (CF) patients can be hampered by the phenotypic variability of the microorganism, including its transition to a Viable But Non-Culturable (VBNC) state. The aim of this study was to validate an ecfX-targeting qPCR protocol developed to detect all viable P. aeruginosa bacteria and to identify VBNC forms in CF sputum samples.

Methods

The study involved 115 P. aeruginosa strains of different origins and 10 non-P. aeruginosa strains and 88 CF sputum samples, 41 Culture-Positive (CP) and 47 Culture-Negative (CN). Spiking assays were performed using scalar dilutions of a mixture of live and dead P. aeruginosa ATCC 9027 and a pooled P. aeruginosa-free sputum batch. Total DNA from sputum samples was extracted by a commercial kit, whereas a crude extract was obtained from the broth cultures. Extracellular DNA (eDNA) interference was evaluated by comparing the qPCR counts obtained from DNase-treated and untreated aliquots of the same samples. The statistical significance of the results was assessed by the Wilcoxon test and Student’s t test.

Results

The newly-developed qPCR protocol identified 96.6% of the P. aeruginosa isolates; no amplification was obtained with strains belonging to different species. Spiking assays supported protocol reliability, since counts always matched the amount of live bacteria, thus excluding the interference of dead cells and eDNA. The protocol sensitivity threshold was 70 cells/ml of the original sample. Moreover, qPCR detected P. aeruginosa in 9/47 CN samples and showed higher bacterial counts compared with the culture method in 10/41 CP samples.

Conclusions

Our findings demonstrate the reliability of the newly-developed qPCR protocol and further highlight the need for harnessing a non-culture approach to achieve an accurate microbiological diagnosis of P. aeruginosa CF lung infection and a greater understanding of its evolution.

     

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.     

Correlation between specific IgG subclass antibodies to Pseudomonas aeruginosa and opsonic activity in serum from patients with cystic fibrosis

Heat-stable opsonins from sera of patients with cystic fibrosis (CF) non-CF patients with chronic Pseudornonas aeruginosa infection, healthy children, and adults were investigated for their ability to promote phagocytosis of ³⁵S-labeled P. aeruginosa by human polymorphonuclear neutrophils. Healthy children had significantly lower levels of opsonic activity than adults. Sera from patients with CF without chronic P. aeruginosa lung infection showed significantly higher levels of opsonic activity compared to healthy children. Sera from patients with CF in the early stage of chronic infection had similar opsonic activity as non-CF patients with chronic infection. Sera from patients with CF in the late stage of chronic infection had higher opsonic activity than other infected patients, but not different from adult controls. An inverse correlation was found between levels of specific antibodies to P. aeruginosa and opsonic activity in the group of patients in a late stage of infection. An inverse correlation was also found between levels of IgG1 and lgG3 to P. aeruginosa St-Ag and opsonic activity during the late stage of infection. Infection with P. aeruginosa in CF did not induce significantly increased opsonic activity. It seems that antibodies to P. aeruginosa may have inhibitory opsonic activity. Pediatr Pulmonol. 1994; 17:31–40 . © 1994 Wiley-Liss, Inc.     

Detection of Pseudomonas (Burkholderia) cepacia using PCR

Pseudomonas cepacia colonization of the lung is associated with increased morbidity and mortality for cystic fibrosis (CF) patients. The lack of a sensitive detection method for Pseudomonas cepacia in CF sputum has resulted in controversy regarding its epidemiology. We designed a PCR method to detect P. cepacia using P. cepacia 16 S rRNA sequences as the amplification target region. The PCR amplification with purified DNA as template yielded the expected 209-bp products from P. cepacia, but not from related Pseudomonas species of medical importance or other bacteria which have been reported to colonize CF patients. In serial dilution experiments as few as 10² P. cepacia CFU were detectable. When sputum samples from three CF patients chronically colonized with P. cepacia and P. aeruginosa were analyzed, P. cepacia was detected in all three specimens by PCR, but only in two when selective culture was performed. Our data support the potential role of PCR technology in the rapid, sensitive, and definitive detection of P. cepacia in CF sputum samples, even in the context of concomitant P. aeruginosa colonization. Pediatr Pulmonol. 1995; 20:44–49 . © 1995 Wiley-Liss, Inc.     

Sequential genotyping of Pseudomonas aeruginosa from upper and lower airways of cystic fibrosis patients.

A controversy exists concerning the adequate specimen to characterise colonisation of cystic fibrosis (CF) airways by Pseudomonas aeruginosa. Oropharyngeal, sputum and bronchoalveolar lavage samples were evaluated from 38 stable CF patients for the detection of P. aeruginosa, genetically different isolates within the same host and longitudinal variations in the genotype during repeated examinations. Bacterial isolates were typed by pulsed-field gel electrophoresis of deoxyribonucleic acid macrorestriction fragments. Sensitivity, negative and positive predictive values and specificity to detect P. aeruginosa were 35.7, 73.5, 83.3 and 96.2% for oropharyngeal cultures in nonexpectorating patients and 91.7, 94.1, 100 and 100% for sputum cultures from expectorating patients, respectively. Genotypes of Pseudomonas isolates recovered from oropharyngeal swabs and sputum differed to the strains recovered by bronchoscopy in 55% and 40%, respectively. In 62% longitudinal variations in the genotype occurred. One-half of these alterations were detectable by bronchoscopy only. In conclusion, sputum samples were of equal value as specimens from bronchoalveolar lavage to detect Pseudomonas aeruginosa colonisation. Cultures from the oropharynx are not suitable for characterising bacterial conditions in the cystic fibrosis lung. Different genotypes within the same host and longitudinal genetic alterations are common and may be detectable in the bronchoalveolar lavage fluid exclusively.     

Molecular typing and biological characteristics of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Brazil

The present study had as objective to evaluate the genotypic diversity and biological characteristics, such as hemolysin, protease, elastase of 56 clinical strains of Pseudomonas aeruginosa isolated from 13 cystic fibrosis (CF) patients attending at the School Hospital of Campinas State University (UNICAMP), Brazil. Genotypic diversity has been determined by Ribotyping (RT) and the pattern of the enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) of each strain. The production of elastase was significantly different only among mucoid and nonmucoid isolates. Joint results obtained by (RT) and ERIC-PCR methods were able to discriminate all strains isolated from both the same and different patients. Additionally, we observed four strain clusters with low diversity. The most infective strains were located in just two clusters. These results suggest that either there is a strong selection towards a specific genotype or that specific isolates could be responsible for the initial and subsequent colonization processes. More studies are necessary to know if these conclusions can be generalized for the general CF population.     

In vitro susceptibility of Pseudomonas aeruginosa from bacteremic and fibrocystic patients to four quinolones and five other antipseudomonal antibiotics

The in vitro antibacterial activities of 4 quinolones, 4 antipseudomonal beta-lactam antibiotics and tobramycin were determined by an agar dilution technique against 206 clinical isolates of Pseudomonas aeruginosa. 176 isolates derived from blood cultures (BC), the remaining 30 were piperacillin-resistant strains from sputum of cystic fibrosis (CF) patients. The CF strains were more resistant to all drugs tested and differed greatly in susceptibility pattern from the BC strains. Ciprofloxacin, ofloxacin and tobramycin were the most active drugs against the CF strains. More than 80% of the BC strains were susceptible to piperacillin and cefsulodin and more than 90% to ceftazidime, tobramycin, ciprofloxacin and ofloxacin. On a weight basis ciprofloxacin was the most active drug in both groups of strains. Our in vitro data suggest that several valuable drugs are now available in the treatment of infections caused by P. aeruginosa strains not previously exposed to antipseudomonal agents.     

Placebo-controlled,double-blind,randomized study of aerosolized tobramycin for early treatment of Pseudomonas aeruginosa colonization in cystic fibrosis

In chronic Pseudomonas aeruginosa pulmonary infection of patients with cystic fibrosis (CF), antibiotic therapy generally fails to eradicate the bacterial pathogen. The mucoid bacterial phenotype, high sputum production by the host, and low airway levels of antibiotics seem to be responsible for the observed decrease in antibiotic efficacy. We hypothesized that early antibiotic treatment by inhalation in CF patients may be able to prevent or at least delay airway infection. In a prospective placebo-controlled, double-blind, randomized multicenter study, 22 CF patients received either 80 mg b.i.d. of aerosolized tobramycin or placebo for a period of 12 months shortly after the onset of P. aeruginosa pulmonary colonization. Two patients in the tobramycin and six patients in the placebo group stopped inhalation before the 12 month treatment period. Using life table analysis, the time to conversion from a P. aeruginosa-positive to a P. aeruginosa-negative respiratory culture was significantly shorter in the tobramycin-treated group than in the placebo group (P < 0.05, log rank test). Lung function parameters and markers of inflammation did not change in either group during treatment. The results of this study suggest that early tobramycin inhalation may prevent and/or delay P. aeruginosa pulmonary infection in CF patients. Pediatr. Pulmonol. 1998; 25:88–92. © 1998 Wiley-Liss, Inc.     

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.     

Multiple of isolates of Pseudomonas aeruginosa with differing antimicrobial susceptibility patterns from patients with cystic fibrosis.

Clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis were studied in an effort to determine the unique characteristics of the infecting strains and to elucidate the pattern of colonization. Of 413 patients studied, 81% were chronically infected with P. aeruginosa. Patients from whom P. aeruginosa was never or only occasionally isolated were in better clinical condition than the chronically infected patients. Isolates were classified into six morphologic varieties: classic, rough, mucoid, gelatinous, dwarf, and enterobacter. Most patients had two or more of these varieties. Such multiple varieties from the same individual were of the same serotype but often differed in antibiotic susceptibility as determined by both the disk and the minimal inhibitory concentration methods. These differences were apparent when mucoid strains were compared with nonmucoid strains and when nonmucoid strains were compared with one another. Studies of antibiotic susceptibility should be performed on each morphologically different type of P. aeruginosa obtained from patients with cystic fibrosis.