Published ahead of print on December 11, 2003, doi:10.1164/rccm.200309-1306OC
© 2004 American Thoracic Society
Adult Cystic Fibrosis Exacerbations and New Strains of Pseudomonas aeruginosaDepartments of Medicine and Pediatrics, University of Ottawa; Ottawa Health Research Institute, Ottawa, Ontario; Department of Medicine, University of Toronto, Toronto; and Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Correspondence and requests for reprints should be addressed to Shawn D. Aaron, M.D., The Ottawa Hospital, General Campus, Room 1812F, 501 Smyth Road, Ottawa, ON, K1H 8L6 Canada. E-mail: saaron{at}ottawahospital.on.ca
We hypothesized that in adults with cystic fibrosis, the acquisition of a new strain of Pseudomonas aeruginosa may be associated with a pulmonary exacerbation. Eighty-four patients who were chronically infected with P. aeruginosa were prospectively followed from eight centers over a 26-month period. Patients had sputum cultures performed every 3 months while clinically stable and at the time of an exacerbation. Forty patients (48%) had an exacerbation requiring intravenous antibiotics during the study period, and in 36 of these patients, their P. aeruginosa isolates were genetically typeable by pulsed-field gel electrophoresis. In 34 of the 36 patients (94%), P. aeruginosa recovered during clinical stability and at exacerbation were of the same genotype. In only two patients (6%; 95% confidence interval, 0–18%) was a new P. aeruginosa clone cultured during an exacerbation that had not been cultured during clinical stability. There were no significant differences in antibiotic susceptibilities, measured as mean minimal inhibitory concentrations, for isolates retrieved during clinically stable periods compared with isolates retrieved during exacerbations. We conclude that for the majority of adult patients with cystic fibrosis a new pulmonary exacerbation is not caused by the acquisition of a new strain of P. aeruginosa.
Key Words: cystic fibrosis • Pseudomonas aeruginosa • lung infection Pseudomonas aeruginosa is the most common respiratory pathogen cultured from adult patients with cystic fibrosis (CF), and chronic infection with P. aeruginosa is associated with reduced survival in this population (1, 2). Exacerbations of pulmonary symptoms are common in CF and are characterized by acute changes, such as increased cough, sputum, and dyspnea, in respiratory signs and symptoms (3, 4). Previous studies have demonstrated that the concentration of P. aeruginosa bacteria in sputum is increased in patients with CF during exacerbations and then falls with appropriate antibiotic treatment (5, 6). Initial antibiotic therapy of an exacerbation of CF is usually determined on the basis of the results of antibiotic susceptibility testing performed on isolates recovered from that patient's most recent sputum culture (7). Thus, clinical practice is based on the assumption that results of sputum cultures taken when the patient is clinically stable will reflect culture results during an exacerbation. Recently, prospective studies of exacerbations of chronic obstructive pulmonary disease have demonstrated that patients who are chronically infected with a single particular bacterial species may become infected with a new genetic strain of the same species during a chronic obstructive pulmonary disease exacerbation (8). We hypothesized that in patients with CF, the acquisition, or overgrowth, of a new strain of P. aeruginosa may be associated with a CF pulmonary exacerbation. To test this hypothesis, we prospectively collected sputum samples every 3 months from a cohort of patients with CF when they were clinically stable and also at the time of a pulmonary exacerbation, immediately before the administration of intravenous antibiotic therapy. Isolates of P. aeruginosa were subjected to pulsed-field gel electrophoresis (PFGE) typing and antibiotic susceptibility testing to determine whether the strains isolated during pulmonary exacerbations were genetically and phenotypically different compared with those isolated previously during periods of clinical stability.
Study Design The study was approved by the research ethics boards of each hospital, and all participants gave written informed consent. Adult patients with CF, who were chronically infected with P. aeruginosa, were recruited from eight Canadian CF centers from February 2001 to March 2003. All patients were at least 16 years old, were able to spontaneously produce sputum, and had a confirmed diagnosis of CF. Patients whose sputum chronically cultured other gram-negative bacteria in addition to P. aeruginosa were excluded. Patients were seen in the CF clinic every 3 months, and sputum samples were obtained at each visit. Patients were instructed to report to the clinic immediately when they had symptoms suggestive of an acute pulmonary exacerbation. An exacerbation was defined, as per the 1994 Cystic Fibrosis Foundation Microbiology Consensus Conference (4), as the presence of at least 3 of 11 new clinical findings (including increased cough, sputum production, fever, weight loss, school or work absenteeism, increased work of breathing, decreased exercise tolerance, or a deterioration in the chest exam, chest radiograph, FEV1, or hemoglobin saturation). To qualify, the exacerbation had to be judged by the CF clinician as being severe enough to warrant intravenous antibiotic therapy. Sputum was collected at the time of the exacerbation before antibiotics were administered.
Sputum Samples Minimum inhibitory concentrations to eight antipseudomonal antibiotics were determined for each isolate using e-test strips (AB Biodisk, Solna, Sweden). This methodology has been validated previously for use against CF-derived isolates (9).
Molecular Typing Restriction fragment profiles were visually compared and were interpreted based on guidelines recommended by Tenover and coworkers (11). Isolates with identical restriction fragment profiles were considered to represent a single strain. Isolates with restriction profiles that differed by one to three fragments (bands) were considered to be closely related strains that evolved from a single clone. Isolates with restriction profiles differing by four bands or more were considered to be different strains and therefore unrelated.
Statistical Analysis
Patients Eighty-four patients were entered into the study, and 40 patients (48%) had an exacerbation during the 26-month study period. However, four of these patients had baseline or exacerbation isolates of P. aeruginosa that were genetically untypeable by PFGE methods, and these four patients, therefore, were excluded from the analysis. The characteristics of the 36 study patients are shown in Table 1 . The mean age of the patients was 30 ± 9 years, and 21 of 36 (58%) patients were female. The patients' mean baseline FEV1 at the time of clinical stability was 1.81 ± 0.74 L (53% of predicted). The mean duration of follow-up from enrollment to pulmonary exacerbation was 135 ± 101 days (range 5–496 days). The median number of sputa collected (every 3 months) before exacerbation was two (range 1–5). At the time of an exacerbation, the mean FEV1 for the group was 1.51 ± 0.67 L, a drop of 17% compared with baseline.
Acute CF Exacerbations and New Strains of P. aeruginosa A total of 205 P. aeruginosa isolates were recovered from sputum specimens from 36 patients during clinically stable periods and during a pulmonary exacerbation. The mean number of P. aeruginosa morphotypes retrieved from sputum was 2.22 ± 0.92 during periods of stability and 2.25 ± 0.77 during an exacerbation. Quantitative cultures were performed for each isolate retrieved during exacerbations. At least 3 x 106 cfu of P. aeruginosa per milliliter were recovered from each isolate (range 3 x 106 to 1 x 1012 cfu/ml). The average density of P. aeruginosa in exacerbation sputums (i.e., the log10 value for the sum of all morphotypes) was 9.6 x 1010 cfu/ml of sputum. PFGE genotyping of the isolates revealed that 31 of 36 patients (86%) harbored only one strain of P. aeruginosa in their sputum during periods of clinical stability. Similarly, 32 of 36 (89%) patients harbored only one strain of P. aeruginosa in their sputum during an exacerbation (Table 2) .
In 34 of 36 patients (94%), P. aeruginosa recovered from baseline cultures and at exacerbation were of the same strain. In these cases, the P. aeruginosa recovered at exacerbations was an identical clone or a subclone of the P. aeruginosa recovered during a clinically stable period (Figure 1) .
In only 2 of 36 patients (6%; 95% confidence interval, 0–18%) was a new P. aeruginosa clone cultured during an exacerbation that had not been cultured during clinical stability. One of these two patients had replacement of their P. aeruginosa clone from baseline cultures with a completely different strain of P. aeruginosa at exacerbation. The second patient's sputum revealed a new unrelated P. aeruginosa clone that had not been recovered when the patient was clinically stable, in addition to the clone that he had grown previously (Figure 2) .
Table 2 summarizes the genotypes of the patients' isolates recovered at clinical stability and at exacerbation. Eleven patients, from three different cities in Ontario, were discovered to have a common ‘A’ strain of P. aeruginosa growing in sputum. The PFGE findings on these patients were confirmed by electrophoresis of random-amplified polymorphic DNA, using primer 272 (12).
Results from Post-treatment Isolates
Results from Patients Who Did Not Experience an Exacerbation during the Study Period
Acute CF Exacerbations and P. aeruginosa Susceptibility to Antibiotics
Antibiotic susceptibilities of the most resistant isolate cultured at exacerbation were compared with those of the most resistant isolate cultured during clinical stability for each patient. In 31 of 36 patients (86%), there were no significant shifts in MIC values for exacerbation isolates compared with clinical stability isolates. In 5 of 36 patients (14%), the MIC to at least two of eight antipseudomonal antibiotics was greater than two doubling dilutions higher in the most resistant exacerbation isolate compared with the most resistant isolate retrieved previously during clinical stability.
We used molecular typing techniques to prospectively test the hypothesis that the acquisition, or overgrowth, of a new strain of pathogenic P. aeruginosa may be associated with a CF pulmonary exacerbation. Our study found that the vast majority of adult patients with CF maintain the same strain of P. aeruginosa even when episodes of pulmonary exacerbations occur. Only 2 of 36 patients (6%) cultured a new strain of P. aeruginosa from sputum at exacerbation that was not cultured during clinically stable periods. The importance of our study is that we have demonstrated that for the majority of adult patients with CF, a new pulmonary exacerbation is not caused by the acquisition of a new strain of P. aeruginosa. This has implications for clinical care because initial antibiotic therapy of an exacerbation of CF is usually based on that patient's most recent sputum culture and sensitivity results obtained during a period of clinical stability. Clinical practice is based on the assumption that sputum cultures taken when the patient is clinically stable reflect culture results during an exacerbation, and our study suggests that this is indeed the case. The homogeneous genotype of the bacterial population during periods of clinical stability is not associated with uniform phenotype of P. aeruginosa in CF lungs, and colonies from the same strain from a single sputum often strongly differ in growth, morphology, or susceptibility to antimicrobial agents (13–16). Most changes in phenotype do not alter the global fragment fingerprint of the bacterial chromosome, probably because only a few loci are affected (17). However, given this, it is impossible to determine, on the basis of morphology alone, whether two P. aeruginosa colonies are of similar strain. Similarly, two distinct strains of P. aeruginosa can have identical morphology. This underlies a potential weakness of this study because it is theoretically possible that we failed to select for subculture from baseline and exacerbation sputa distinct strains of P. aeruginosa that were morphologically identical. However, we believe it is unlikely that we failed to identify relevant new strains because we used standard procedures used in CF clinical microbiology laboratories and we subcultured every distinct morphotype. Our criteria for selecting colonies for subculture were identical for baseline and exacerbation sputa, and the mean number of P. aeruginosa morphotypes retrieved from sputum for subculture was the same during periods of stability as during an exacerbation. If we had undersampled at times of clinical stability or sampled differentially for stable and exacerbation isolates, then the results of this study would be expected to be quite different and should have shown that new isolates appear at exacerbations that were not found during clinically stable periods. In fact, the results of our study reveal the opposite and argue against any possible bias posed by potential undersampling. Finally, the results of our study are directly applicable to the clinical setting because we used culturing procedures that are identical to those used in hospital microbiology laboratories. A similar study will need to be performed in pediatric patients to determine whether our results are generalizable to the pediatric population. Data from a longitudinal study by Burns and colleagues (18) suggest that infant patients with CF can be infected with more than one genotype of mostly nonmucoid P. aeruginosa and that in infants infection with different strain types can be acquired from environmental sources and may vary with time. Similar studies of deep-throat swabs taken from pediatric patients with CF by Grothues and coworkers (14) confirmed that 36% of their pediatric patients harbored two or three strain types of pseudomonas. Apparently, the microbiology of CF-associated P. aeruginosa infection differs in children compared with adults. Infants become infected with several strains of largely nonmucoid, environmental pseudomonas (18). In early life, no strain emerges as a dominant one, and the pseudomonas strains can be eradicated, at least temporarily, with antibiotic therapy (19–21). However, in adults, infection with pseudomonas becomes chronic (i.e., secretions cannot be sterilized with antibiotic therapy), and the bacteria are mucoid and form biofilms (22–24). In this environment, a single P. aeruginosa clone often predominates in the airway secretions of each patient. Longitudinal studies of stable adult patients with CF have reported that most patients with CF harbor a single clone or subclones of P. aeruginosa in their airways (17, 25–27). We have confirmed this observation in our present study, in which we have demonstrated that more than 80% of adult patients with chronic P. aeruginosa infection harbor only one strain of P. aeruginosa in their sputum even when longitudinally sampled over a 2-year period. We have also recently completed another study in 12 adults with stable CF that involved culturing and PFGE analysis of at least 15 P. aeruginosa isolates taken from each patient via sputum, bronchoalveolar lavage, and protected specimen brush bronchoscopy (Aaron and coworkers). This study found that 10 of 12 patients (83%) were each infected with a single strain of P. aeruginosa from all 15 isolates from all three sources. Thus, on the basis of our studies, and those of others (17), it can be concluded that the majority of adult patients with CF are each infected with a single clone of P. aeruginosa. A previous study by Boukadida and coworkers (28), of mostly pediatric patients, demonstrated that in three patients administration of antibiotics often led to changes to the strains isolated. Our study did not confirm this phenomenon; in 14 adult patients tested, postexacerbation sputum cultures did not recover any new strains of pseudomonas. Neither did intravenous antibiotic treatment eradicate the existing clone nor did it select out for growth of a new clone of P. aeruginosa. In summary, results from this study suggest that most adult patients with CF harbor a single clone of P. aeruginosa in their airways when they are clinically stable and that they maintain the same strain of P. aeruginosa even when episodes of pulmonary exacerbations occur. Thus, for the majority of adult patients with CF, reliance on sputum culture and sensitivity results taken during the patient's baseline state is appropriate to direct antibiotic therapy during exacerbations.
The authors thank Alain Plouffe for help with the PFGE analysis, Justin Tang for help with antibiotic susceptibility testing, and David Speert and Deborah Henry for help with random-amplified polymorphic DNA polymerase chain reaction analysis. The authors thank the following people who recruited patients into this study—Halifax, Nova Scotia: Pat Hazell; Montreal, Quebec: Dr. Yves Berthiaume, Dr. Alphonse Jeanneret, Nadia Beaudoin, Julie Langlois; Ottawa, Ontario: Kathleen Devecseri; Toronto, Ontario: Lesley Gaskin; Hamilton, Ontario: Dr. Andreas Freitag, Rosamund Hennessey; London, Ontario: Dr. Nigel Patterson, Patrice Keane; Edmonton, Alberta: Dr. Neil Brown, Josette Salgado; Vancouver, British Columbia: Dr. Pearce Wilcox, Holly Truchan.
Supported by the Canadian Institute of Health Research and the Canadian Cystic Fibrosis Foundation. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Conflict of Interest Statement: S.D.A. has no declared conflict of interest; K.R. has no declared conflict of interest; W.F. has no declared conflict of interest; K.V. has no declared conflict of interest; R.S. has no declared conflict of interest; E.T. has no declared conflict of interest; D.H. has no declared conflict of interest; D.K. has no declared conflict of interest; M.S.D. has no declared conflict of interest; F.C. has no declared conflict of interest. Received in original form September 23, 2003; accepted in final form December 9, 2003
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