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Published ahead of print on January 17, 2008, doi:10.1164/rccm.200709-1413OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 853-860, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200709-1413OC

Original Article

Pseudomonas aeruginosa in Chronic Obstructive Pulmonary Disease

Timothy F. Murphy1,2,3, Aimee L. Brauer1, Karen Eschberger1,3, Phyllis Lobbins1,3, Lori Grove1, Xueya Cai4 and Sanjay Sethi1,3

1 Department of Medicine and 2 Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo New York; 3 Veterans Affairs Western New York Healthcare System, Buffalo, New York; and 4 Department of Biostatistics, State University of New York at Buffalo, Buffalo, New York

Correspondence and requests for reprints should be addressed to Timothy F. Murphy, M.D., Medical Research 151, Buffalo Veterans Affairs Medical Center, 3495 Bailey Avenue, Buffalo, NY 14215. E-mail: murphyt{at}buffalo.edu


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Pseudomonas aeruginosa is isolated from adults with chronic obstructive pulmonary disease (COPD) in cross-sectional studies. However, patterns of carriage and the role of P. aeruginosa in COPD are unknown.

Objectives: To elucidate carriage patterns, phenotypes of strains, clinical manifestations, and the antibody response to P. aeruginosa in COPD.

Methods: A prospective study of adults with COPD was conducted. Isolates of P. aeruginosa were subjected to genotypic and phenotypic analysis. Sputum samples were studied for P. aeruginosa DNA, and immune responses were assayed.

Measurements and Main Results: We analyzed longitudinal clinical data, sputum cultures, pulsed-field gel electrophoresis of bacterial DNA, polymerase chain reaction of sputum, and immunoblot assays of serum. Fifty-seven episodes of acquisition of strains of P. aeruginosa were observed in 39 of 126 patients over 10 years. Acquisition of a new strain was associated with exacerbation. Thirty-one episodes of carriage were followed by clearance of the strain; 16 were of short (<1 mo) duration. Thirteen strains demonstrated persistence, and 13 strains were of indeterminate duration. Six strains were mucoid and were more likely to persist than nonmucoid strains (P = 0.005). Antibody responses developed in 53.8% of persistent carriage and in only 9.7% of short-term carriage episodes (P = 0.003). Antibiotics did not account for clearance.

Conclusions: Two distinct patterns of carriage by P. aeruginosa were observed: (1) short-term colonization followed by clearance and (2) long-term persistence. Mucoid strains showed persistence. Acquisition of P. aeruginosa is associated with the occurrence of an exacerbation. Serum antibody responses do not mediate clearance of P. aeruginosa.

Key Words: respiratory tract infection • exacerbation • immune response • sputum • polymerase chain reaction



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Pseudomonas aeruginosa is isolated from the sputum in chronic obstructive pulmonary disease, but carriage patterns, clinical manifestations, and immune responses have not been studied prospectively.

What This Study Adds to the Field
This study shows that (1) there are distinct patterns of carriage of P. aeruginosa in chronic obstructive pulmonary disease, (2) mucoid strains are uncommon but persistent, (3) acquisition of a new strain is associated with exacerbation, and (4) antibody responses are a marker for colonization.

 
Bacterial infection plays an important role in the course and pathogenesis of chronic obstructive pulmonary disease (COPD) (1–3). Several experimental approaches demonstrate that nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae cause intermittent or chronic respiratory tract infection in the setting of COPD (4–14). Pseudomonas aeruginosa is isolated from the sputum of 4–15% of adults with COPD in many cross-sectional studies (15–24). However, the dynamics of carriage over time, the host response, and the role of P. aeruginosa in the clinical course of COPD are not well characterized.

Infection with P. aeruginosa plays an important role in the course of other chronic lung diseases, including cystic fibrosis (CF) and bronchiectasis. Most patients with CF acquire P. aeruginosa, and many of these strains develop a mucoid phenotype. Chronic P. aeruginosa infection is difficult to eradicate and is responsible for much of the early mortality in CF (25–27).

P. aeruginosa colonizes the respiratory tract of people with bronchiectasis. Patients with bronchiectasis who are colonized by P. aeruginosa exhibit more advanced disease and more severe impairment of pulmonary function compared with those who remain free of colonization (28–31). P. aeruginosa causes intermittent exacerbations that characterize the course of bronchiectasis and that account for substantial morbidity in this clinical setting.

The role of P. aeruginosa in the course of COPD is less well characterized but has been the subject of increasing recent interest. P. aeruginosa is more likely to be isolated from patients with severe disease, particularly among patients who require mechanical ventilation for severe exacerbations (21, 32–37). The presence of P. aeruginosa in the lower airways using protected brush sampling is associated with symptoms of exacerbation (32). The presence of P. aeruginosa in a culture of sputum at the time of exacerbation is associated with an FEV1 of less than 35%, systemic steroid use, and prior antibiotic therapy within the preceding months (38). These and other observations indicate that P. aeruginosa is important in the setting of COPD. However, it is difficult to draw definitive conclusions regarding the extent to which P. aeruginosa contributes to adverse clinical outcomes because key questions regarding infection and colonization remain unanswered. To understand the role that P. aeruginosa plays in the course of the disease, it is critical to know the patterns of carriage, the clinical consequences of acquisition of and persistent colonization by P. aeruginosa, and whether immune responses can mediate clearance of P. aeruginosa from the respiratory tract of adults with COPD. Such information is important in interpreting the clinical significance of P. aeruginosa in the sputum and in designing studies to assess whether and when administration of antimicrobial therapy directed at P. aeruginosa in the setting of COPD is beneficial.

Carriage patterns of P. aeruginosa in COPD have not been examined in studies with a prospective design, and the frequency of mucoid strains in this setting is not known. Given the key role of mucoid strains in CF, it is important to know the frequency with which strains express the mucoid phenotype in COPD. The human immune response to P. aeruginosa in the setting of COPD has not been studied. The duration of carriage and whether different strains show different propensities for short-term or long-term carriage are not known.

The goals of the present study were as follows: (1) to elucidate carriage patterns of P. aeruginosa in a 10-year prospective study of adults with COPD, (2) to characterize the phenotypes of P. aeruginosa isolates in COPD, (3) to elucidate the clinical consequences of infection with P. aeruginosa, and (4) to characterize the human antibody response to P. aeruginosa in this clinical setting.


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
COPD Study Clinic
A prospective study of COPD at the Buffalo Veterans Affairs Medical Center has been described (8, 10). Details are in the online supplement. A total of 126 patients with COPD were enrolled between April 1994 and December 2004. Patients were seen monthly and whenever they had symptoms suggestive of an exacerbation. At clinic visits, clinical information and sputum and serum samples were obtained. A clinical evaluation was performed at each visit to determine whether the patient had stable disease or an exacerbation as described (8). This determination was made by one of two examiners (T.F.M., S.S.).

Sputum Samples
The processing of sputum samples is described in the online supplement. Briefly, spontaneously expectorated sputum was homogenized in 0.1% dithiothreitol, and serial dilutions were subjected to quantitative culture. Bacterial identification was performed using standard techniques. An isolate was identified as P. aeruginosa by colony morphology, the absence of lactose fermentation, and the presence of oxidase. The identity of all such isolates was confirmed by microscan analysis using the Negative Urine Combo Panel Type 33 (Dade Behring, Deerfield, IL). The presence or absence of a mucoid phenotype on Pseudomonas isolation agar plates was recorded for each isolate.

After aliquots of the homogenized sputum sample were removed for culture, the remainder of the sample was centrifuged at 27,000 x g for 30 minutes at 4°C. Supernatants and pellets were stored at –80°C.

Pulsed-Field Gel Electrophoresis
P. aeruginosa isolates were subjected to pulsed-field gel electrophoresis using previously described methods (8). Genomic DNA of the 230 isolates was separately cut with SpeI and NheI. Pulsed-field gel electrophoresis patterns were interpreted using the criteria of Tenover and colleagues (39) to determine when new strains were isolated from monthly sputum cultures. The online supplement includes details of the interpretation.

Analysis of Sputum Samples for P. aeruginosa DNA
DNA was extracted from pellets of sputum samples, and primers corresponding to the conserved oprf gene were used in polymerase chain reactions (PCRs) to identify P. aeruginosa DNA (40). Validation of the assay and details of the method are described in the online supplement.

Analysis of Antibody Response to P. aeruginosa
Serum antibody responses were detected by immunoblot assays with bacterial lysates of the patients' homologous strain using the method of Burns and colleagues (26). Sera from patients who had negative cultures for P. aeruginosa were assayed with strain PAO1. Bacterial lysates were subjected to immunoblot assays with serum samples to detect IgG.

Initially, one serum sample for each year the patient was followed in the study was assayed. An individual patient's serum samples were tested together on the same immunoblot assay. The development of new band(s) compared with a previous sample was determined to be the development of an antibody response. When a new antibody response was detected from yearly samples, monthly samples were tested to pinpoint the month at which the new antibody response occurred.

Statistical Analysis
The relative risk of an exacerbation when P. aeruginosa was present and upon acquisition of a new strain were calculated with generalized estimating equations to take into account the patients' repeated visits (8, 41). Details are provided in the online supplement.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
P. aeruginosa in Longitudinally Collected Samples
A total of 126 patients were enrolled and followed during the 10-year, 9-month period from April 1994 to the end of 2004. Patients made 5,100 clinic visits, at which 4,552 sputum samples were collected. A total of 230 (5.1%) sputum cultures grew P. aeruginosa. Figure 1 is a flow chart that shows the overall scheme of the source of cultures and distribution of strains following the analyses detailed in the following sections. Table 1 compares characteristics of the 39 patients who had a positive culture for P. aeruginosa at some time during follow-up with the 87 patients who never had a positive culture for P. aeruginosa. The groups were similar in several characteristics, including age, pack-years of smoking, and duration of underlying lung disease. Patients from whom P. aeruginosa was isolated were followed in the study for a longer period compared with those who had negative sputum cultures for P. aeruginosa. FEV1 showed a trend toward a lower value in the P. aeruginosa group, suggesting that longer follow-up of larger numbers of patients may reveal an association of severity of COPD with isolation of P. aeruginosa.


Figure 1
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  		Figure 1. Flow chart illustrating the distribution of sputum culture results among patients and the carriage patterns with regard to duration of carriage of strains of Pseudomonas aeruginosa in adults with chronic obstructive pulmonary disease. *Episodes of acquisition were determined by analysis of pulsed-field gel electrophoresis of 230 isolates. **Persistence and clearance of strains were determined by results of sputum culture, pulsed-field gel electrophoresis, and polymerase chain reaction analysis of sputum samples using criteria described in text.

 

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  		TABLE 1. CHARACTERISTICS OF PATIENTS WITH PSEUDOMONAS AERUGINOSA DURING FOLLOW-UP COMPARED WITH PATIENTS WITHOUT P. AERUGINOSA

 
Analysis of Culture-negative Sputum Samples for P. aeruginosa DNA
To assess the possibility that P. aeruginosa is present in sputum samples that are negative in culture, a PCR assay based on oligonucleotide primers corresponding to the oprf gene was developed to detect P. aeruginosa DNA in sputum. The online supplement includes a detailed description of the method using oligonucleotide primers corresponding to the oprf gene along with results of assays of various control samples establishing the specificity of the assay.

One pattern of carriage observed in 21 patients was the isolation of the identical strain of P. aeruginosa (determined by pulsed-field gel electrophoresis) with intervening negative cultures (Figure 2, top). To test the hypothesis that P. aeruginosa was present in sputum samples that were preceded and followed by identical strains (called gaps in culture), DNA was extracted from sputum pellets and used as a template in PCR with oprf primers. DNA extracted from a total of 102 sputum pellets obtained during these gaps from all 21 patients was studied in PCR. Seventy-two of the 102 samples yielded an amplicon that corresponded to the correct size of the fragment of oprf. At least one PCR reaction from "gaps" of each patient studied was positive for the oprf gene. Figure 2 shows an example of this analysis with one patient. Therefore, we concluded that sputum samples that are preceded and followed by the identical strain contain P. aeruginosa DNA and apparently represent persistent carriage of P. aeruginosa. This observation is similar to that observed with H. influenzae (42).


Figure 2
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  		Figure 2. (Top) Ethidium bromide stained pulsed-field gel of genomic DNA cut with SpeI from 11 isolates of Pseudomonas aeruginosa collected prospectively from a single patient. Clinic visits (approximately monthly) are noted. (Bottom) Ethidium bromide–stained agarose gel of polymerase chain reactions (PCRs) using DNA extracted from sputum samples that were negative in culture but preceded and were followed by positive cultures for P. aeruginosa as template. All samples are from the same patient whose strains are shown in the top gel. Primers corresponding to oprf that encodes a conserved outer membrane protein were used in PCR reactions. The arrow denotes the expected size of the oprf fragment amplified. Clinic visits (approximately monthly) are noted at the bottom. Molecular mass standards are noted on the right in kilobases.

 
Determination of Clearance and Persistence of P. aeruginosa
To assess the possible persistence of P. aeruginosa in sputum cultures that were negative in culture after the last positive culture before the patient left the study, pellets were studied by PCR to detect the presence of the oprf gene (192 sputum samples). On the basis of the results of cultures and PCR, each independent strain of P. aeruginosa was defined as "persistent," "cleared," or "indeterminate" as follows. On the basis of on previous observations in the case of H. influenzae (42), we required that the patient was followed for at least 6 months after the last positive culture and that a minimum of six sputum samples were available for testing to conclude that a strain was cleared. A strain was defined as cleared when this requirement was met and all sputum samples that followed the last positive culture were negative in PCR. A strain was defined as persistent when acquired at least 6 months before the end of the follow-up period and the strain was present in culture through the patients' last clinic visit or when at least one sputum sample after the last visit with a positive culture demonstrated the presence of the oprf gene by PCR. Episodes of colonization that did not meet the criteria for cleared or persistent strains were defined as indeterminate (i.e., strains acquired <6 mo before the last clinic visit or were followed for <6 mo after the last visit with a positive culture).

Patterns of Carriage
Figure 3 shows the distribution of duration of carriage for the 57 episodes of acquisition of new strains of P. aeruginosa among 39 patients. Distinct patterns of carriage were observed. Sixteen episodes were a single clinic visit in duration, and 13 episodes showed persistent colonization (see Figure 1). An additional 15 episodes were cleared after 2 to 22 months. The durations of 13 episodes of colonization were indeterminate. The duration of colonization by persistent strains (median, 35 mo; interquartile range [IQR], 40) was longer than that of strains that were cleared (median, 1 mo; IQR, 4; P < 0.0001, nonparametric Wilcoxon test).


Figure 3
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  		Figure 3. Duration of colonization of 57 episodes of acquisition of Pseudomonas aeruginosa in 36 adults with chronic obstructive pulmonary disease. x Axis: Duration of colonization in months. y Axis: Number of episodes of acquisition. The gray bars denote cleared strains, black bars denote persistent strains, and white bars denote strains of indeterminate duration based on criteria outlined in the text.

 
Mucoid Strains
Six strains of P. aeruginosa isolated from six different patients demonstrated a mucoid phenotype. Three strains were initially nonmucoid, with the phenotype changing to mucoid during carriage, and three were mucoid when initially cultured. Of the 44 strains whose duration of carriage could be determined, 4 of 4 mucoid strains persisted and were never cleared. Mucoid strains were more likely to persist than nonmucoid strains (4 of 4 [100%] vs. 9 of 40 [22.5%]; P = 0.005, Fisher's exact test). Furthermore, the duration of carriage by mucoid strains was longer than that of nonmucoid strains (49.5 mo; IQR, 25.5 vs. 3.0 mo; IQR, 9.5; P = 0.002, nonparametric Wilcoxon test).

Co-carriage with Other Bacteria
On the basis of combined analysis by culture and PCR, a total of 598 clinic visits in 39 patients were positive for P. aeruginosa. Sputum samples from 84 (14%) of these visits grew another bacterial pathogen in addition to P. aeruginosa (Table 2).


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  		TABLE 2. SPUTUM CULTURES POSITIVE FOR A SECOND PATHOGEN DURING 598 CLINIC VISITS IN WHICH PSEUDOMONAS AERUGINOSA WAS PRESENT

 
Relationship of Antibiotic Administration to Carriage Patterns
At each monthly clinic visit, medications were recorded. Patients in the COPD Study Clinic are veterans who receive all of their medical care at the Buffalo VA Medical Center. Therefore, accurate data on antibiotic administration are available. To assess the possibility that antibiotic therapy accounted for clearance of strains, episodes of colonization during which an antibiotic in a class with activity against P. aeruginosa was administered within 6 months of clearance of the strain were recorded. Of the 31 episodes of P. aeruginosa carriage that were followed by clearance of the strain, 10 (32.3%) received an antibiotic with potential activity against P. aeruginosa (fluoroquinolones, gentamicin, piperacillin-tazobactam) administered within 6 months of clearance of the strain. More details are provided in the online supplement. The remaining 21 (67.7%) cleared the strain without having received an active antibiotic. Of the 13 episodes of colonization by persistent strains, 11 (84.6%) received at least one course of antibiotic with activity against P. aeruginosa during colonization.

Clinical Manifestations of P. aeruginosa Carriage
The frequency of exacerbations in the 39 patients from whom P. aeruginosa was isolated was compared with that in the 87 patients who remained free of colonization by P. aeruginosa. Table 1 shows that, overall, no difference in the rate of exacerbations per year in patients with and without P. aeruginosa was observed (2.30 ± 0.25 vs. 2.45 ± 0.28; P = 0.67, t test). To explore this question further, the frequency of exacerbation during carriage of P. aeruginosa (based on culture and PCR analysis of sputum) was compared with the frequency of exacerbation during periods when P. aeruginosa was absent in the patients from whom P. aeruginosa was isolated. No difference in exacerbation frequency was observed during carriage of P. aeruginosa (2.98 ± 0.67 vs. 2.08 ± 0.28 exacerbations/yr; P = 0.23, t test).

We tested the hypothesis that a positive culture for P. aeruginosa was associated with an increased incidence of exacerbation. An exacerbation was present in 45 of 216 visits at which P. aeruginosa was isolated (20.8%) as compared with 706 of 3,883 visits at which P. aeruginosa was not isolated from sputum (18.2%; P = 0.33). Therefore, no association between a positive sputum culture for P. aeruginosa and frequency of exacerbation was observed (Table 3).


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  		TABLE 3. RELATIVE RISK OF EXACERBATION WITH PSEUDOMONAS AERUGINOSA COLONIZATION AND WITH ACQUISITION OF A NEW STRAIN OF P. AERUGINOSA

 
We tested the hypothesis that acquisition of a new strain of P. aeruginosa was associated with the occurrence of an exacerbation. Isolation of a new strain of P. aeruginosa was associated with a significant increase in the frequency of exacerbation. Twenty of 47 visits at which a new strain was isolated (42.6%) were associated with exacerbation, as compared with 731 of 4,002 visits at which no new strain was isolated (18.0%; P < 0.0001; relative risk, 3.36; 95% confidence interval, 1.88–6.03) (Table 3). We conclude that acquisition of a new strain of P. aeruginosa is associated with the occurrence of an exacerbation.

Immune Response to P. aeruginosa
Immunoblot assays with whole bacterial cell lysates of the homologous infecting strains were performed with one serum sample (1:8,000 dilution) from each year the patient was followed in the study (total of 516 serum samples). This dilution, based on a series of pilot assays with varying dilutions, was the highest dilution that yielded detectable bands in almost all adults with COPD. Of the 39 patients who acquired P. aeruginosa, 11 made new antibody responses (Figure 4). For each of these patients, selected serum samples obtained monthly were assayed to pinpoint the month when the new antibody response developed. Two patients developed two separate new immune responses during the course of their follow-up (Figure 4, left).


Figure 4
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  		Figure 4. Immunoblot assays of whole bacterial cell lysates of homologous strains of Pseudomonas aeruginosa from two patients with chronic obstructive pulmonary disease. Immunoblots were probed with serum samples (1:8,000) obtained at clinic visits (approximately monthly) as noted at the bottom. Serum IgG was detected with peroxidase-conjugated anti-human IgG. The arrows at the top indicate the development of immune responses.

 
To assess the specificity of the antibody responses to P. aeruginosa, immunoblot assays with annual serum samples obtained from 20 patients with COPD followed in the COPD Study Clinic who had never had a sputum culture positive for P. aeruginosa were tested in immunoblot assays with P. aeruginosa strain PAO1. None of these patients showed development of new antibodies. Serum from patients whose isolates changed to a mucoid phenotype during follow-up was tested in immunoblot assays with the mucoid and nonmucoid phenotypes, and the results were identical with both phenotypes. New antibody responses developed before the first positive culture for P. aeruginosa in 3 of 11 patients. The intervals between the development of antibody and the first positive cultures were 6, 9, and 23 months.

Immune Responses and Clearance of P. aeruginosa
Seven of the 13 episodes (53.8%) of persistent carriage were associated with the development of an antibody response. By contrast, only 3 of 31 episodes (9.7%) in which P. aeruginosa was cleared from the respiratory tract were associated with antibody responses (P = 0.003, Fisher's exact test).


   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study revealed several novel observations. Distinct patterns of carriage of P. aeruginosa were observed in adults with COPD, with some strains cleared quickly and others persisting. Acquisition of a new strain of P. aeruginosa is associated with the occurrence of an exacerbation, providing a new line of evidence that P. aeruginosa causes exacerbations. Antibody responses do not mediate clearance of P. aeruginosa in the setting of COPD. Rather, antibodies seem to be a marker for carriage in some patients. Finally, a small proportion of P. aeruginosa strains acquire a mucoid phenotype, and these mucoid strains persist.

Previously published work involving 56 months of follow-up of the same cohort of patients demonstrated that acquisition of a new strain of nontypeable H. influenzae, M. catarrhalis, or S. pneumoniae was associated with the occurrence of an exacerbation (8). The same study did not show an association of acquisition of a new strain of P. aeruginosa with exacerbation. However, the number of isolations of P. aeruginosa was small in the previous study. With longer follow-up and thus more episodes of acquisition and carriage of P. aeruginosa, the present study showed a strong association between acquisition of a new strain of P. aeruginosa and exacerbations. The rate of exacerbation at visits with a new strain of P. aeruginosa (42.6%) is similar to that seen with H. influenzae (44.5%) (43) and M. catarrhalis (48.8%). This finding provides evidence that P. aeruginosa causes some acute exacerbations of COPD.

To assess the potential role of immune responses to P. aeruginosa in mediating clearance from the respiratory tract, serum samples from all patients who acquired P. aeruginosa were studied in immunoblot assays with the homologous infecting strains. Of the 31 episodes of colonization in which the strain was cleared from the respiratory tract, three developed a new serum antibody response. These results suggest that clearance of P. aeruginosa is not mediated by antibody responses. However, one must be cautious in concluding the absence of an adaptive immune response. Although the method has the advantage of detecting conserved and strain-specific antibody responses because homologous strains were used, it is possible that mucosal antibody responses or cell-mediated immune responses have developed. In view of the rapidity with which many episodes of P. aeruginosa colonization were cleared and the absence of an antibody response in the vast majority of adults with COPD who clear P. aeruginosa, we speculate that innate immunity is important in clearance.

Of the 13 episodes in which strains persisted, seven were associated with the development of antibody responses to the colonizing strain. This observation is analogous to the situation of children with CF who are persistently colonized by P. aeruginosa and develop serum antibodies to P. aeruginosa antigens, suggesting that such antibodies are more a marker of persistent carriage by P. aeruginosa rather than functioning to mediate clearance (26, 27).

An additional line of evidence that the antibody responses detected are a marker for carriage is the observation that three patients in the present study developed new antibody responses to P. aeruginosa before their first positive culture. The most likely explanation for this observation is that sputum cultures remained negative despite the patient having acquired P. aeruginosa. The same phenomenon is observed in children with CF (26). This finding, together with the results of analysis of sputum by PCR, supports the conclusion that the absence of P. aeruginosa in a sputum culture does not exclude the possibility that the organism is present.

Several studies have demonstrated that prior exposure to antibiotics is associated with increased likelihood of isolating P. aeruginosa from the sputum of adults with COPD (21, 35, 38). Little is known about the role of antibiotics in determining clearance of P. aeruginosa from the respiratory tract in this setting. Because patients with COPD receive intermittent treatment with antibiotics, we investigated the possible effect of antibiotic administration in mediating clearance of P. aeruginosa. Antibiotics were administered orally for short durations for the treatment of exacerbations and were generally not administered in an effort to eradicate P. aeruginosa. In the majority of cases (21 of 31 [67.7%]), strains of P. aeruginosa were cleared in the absence of any antibiotic therapy. These results indicate that although antibiotics may play a role in selected episodes, factors other than antibiotics mediate clearance of P. aeruginosa from the respiratory tract of adults with COPD.

Analysis of sputum samples by PCR demonstrated that P. aeruginosa DNA is present in sputum samples that are negative in culture. Does the presence of P. aeruginosa DNA represent the presence of viable bacteria, or, alternatively, could it represent persistent DNA in the absence of viable bacteria? Three lines of evidence suggest that the presence of DNA indicates viable bacteria:

  1. All gaps in sputum cultures preceded and followed by the identical strain of P. aeruginosa contained at least one positive sample by PCR. This scenario is more likely explained by the persistence of P. aeruginosa rather than by clearance and reacquisition of the same strain because similar gaps in cultures of H. influenzae have been demonstrated to be caused by persistence (42).
  2. Several studies have demonstrated persistent colonization by P. aeruginosa despite intermittent negative cultures in CF (26, 27, 44).
  3. Studies in animal models demonstrate that purified DNA is cleared quickly from the respiratory tract, supporting the conclusion that bacterial DNA in respiratory tract samples indicates the presence of viable bacteria (45).

Although carriage patterns of P. aeruginosa in the course of CF are well characterized (26, 44, 46), little is known about such patterns in COPD. The present study demonstrates substantial differences between COPD and CF in carriage patterns with P. aeruginosa. Most patients with CF eventually acquire P. aeruginosa, and chronic P. aeruginosa infection is responsible for much of the early mortality in CF (25). By contrast, P. aeruginosa colonization does not seem to be an inevitability in COPD. Mucoid strains play a prominent role in the course of CF, whereas only a small proportion of strains are mucoid in COPD. In addition to these important differences, several parallels are seen. Once mucoid strains become established, they persist. Serum antibody responses to P. aeruginosa do not mediate clearance but rather are a marker for carriage in both settings. Finally, it may be difficult to eradicate P. aeruginosa with antibiotic therapy in COPD as in CF.

High-resolution computed tomography (HRCT) performed on cohorts of adults with COPD demonstrates the presence of bronchiectasis in up to 50% of patients, suggesting that subclinical bronchiectasis is part of the natural history of COPD (47, 48). The present study did not include HRCT on our cohort. However, because our patient population includes predominantly moderately severe and severe COPD, subclinical bronchiectasis is likely present in a proportion of our patients as well. In the study involving HRCT (48), P. aeruginosa was present in only 5 of 52 sputum cultures from adults with COPD, precluding conclusions regarding a possible association between bronchiectasis and P. aeruginosa. This will be an area of importance for study with larger numbers of patients.

In summary, this 10-year prospective study demonstrated several novel observations regarding P. aeruginosa in COPD. P. aeruginosa shows distinct patterns of carriage in adults with COPD, with some strains cleared quickly and others persisting. Acquisition of a new strain of P. aeruginosa is associated with exacerbations, providing a new line of evidence that P. aeruginosa causes some exacerbations. Antibody responses do not mediate clearance of P. aeruginosa in view of the observation that only 9.7% of episodes of clearance from the respiratory tract were associated with an antibody response. A small proportion of P. aeruginosa strains acquire a mucoid phenotype, and these mucoid strains persist. Future studies will be directed at investigating the clinical consequences and mechanisms of the various forms of colonization and infection of the respiratory tract of adults with COPD by P. aeruginosa.


   FOOTNOTES
 
Supported by a Merit Review grant from the Department of Veterans Affairs.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200709-1413OC on January 17, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 23, 2007; accepted in final form January 16, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sethi S, Murphy TF. Bacterial infection in chronic obstructive pulmonary disease in 2000: A state of the art review. Clin Microbiol Rev 2001;14:336–363.[Abstract/Free Full Text]
  2. Wilson R. The role of infection in COPD. Chest 1998;113:242S–248S.[CrossRef][Medline]
  3. White AJ, Gompertz S, Stockley RA. Chronic obstructive pulmonary disease. 6: The aetiology of exacerbations of chronic obstructive pulmonary disease. Thorax 2003;58:73–80.[Abstract/Free Full Text]
  4. Fagon J-Y, Chastre J, Trouillet J-L, Domart Y, Dombret M-C, Bornet M, Gibert C. Characterization of distal bronchial microflora during acute exacerbation of chronic bronchitis. Am Rev Respir Dis 1990;142:1004–1008.[Medline]
  5. Monso E, Ruiz J, Rosell A, Manterola J, Fiz J, Morera J, Ausina V. Bacterial infection in chronic obstructive pulmonary disease: a study of stable and exacerbated outpatients using the protected specimen brush. Am J Respir Crit Care Med 1995;152:1316–1320.[Abstract]
  6. Soler N, Torres A, Ewig S, Gonzalez J, Celis R, El-Ebiary M, Hernandez C, Rodriguez-Roisin R. Bronchial microbial patterns in severe exacerbations of chronic obstructive pulmonary disease (COPD) requiring mechanical ventilation. Am J Respir Crit Care Med 1998;157:1498–1505.[Medline]
  7. Pela R, Marchesani F, Agostinelli C, Staccioli D, Cecarini L, Bassotti C, Sanguinetti CM. Airways microbial flora in COPD patients in stable clinical conditions and during exacerbations: a bronchoscopic investigation. Monaldi Arch Chest Dis 1998;53:262–267.[Medline]
  8. Sethi S, Evans N, Grant BJB, Murphy TF. New strains of bacteria and exacerbations of chronic obstructive pulmonary disease. N Engl J Med 2002;347:465–471.[Abstract/Free Full Text]
  9. Sethi S, Wrona C, Grant BJ, Murphy TF. Strain-specific immune response to Haemophilus influenzae in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2004;169:448–453.[Abstract/Free Full Text]
  10. Murphy TF, Brauer AL, Grant BJ, Sethi S. Moraxella catarrhalis in chronic obstructive pulmonary disease: burden of disease and immune response. Am J Respir Crit Care Med 2005;172:195–199.[Abstract/Free Full Text]
  11. White AJ, Gompertz S, Bayley DL, Hill SL, O'Brien C, Unsal I, Stockley RA. Resolution of bronchial inflammation is related to bacterial eradication following treatment of exacerbations of chronic bronchitis. Thorax 2003;58:680–685.[Abstract/Free Full Text]
  12. Gompertz S, O'Brien C, Bayley DL, Hill SL, Stockley RA. Changes in bronchial inflammation during acute exacerbations of chronic bronchitis. Eur Respir J 2001;17:1112–1119.[Abstract/Free Full Text]
  13. Hurst JR, Perera WR, Wilkinson TM, Donaldson GC, Wedzicha JA. Systemic and upper and lower airway inflammation at exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2006;173:71–78.[Abstract/Free Full Text]
  14. Sethi S, Muscarella K, Evans N, Klingman KL, Grant BJB, Murphy TF. Airway inflammation and etiology of acute exacerbations of chronic bronchitis. Chest 2000;118:1557–1565.[CrossRef][Medline]
  15. Anzueto A, Niederman MS, Tillotson GS, Group BS. Etiology, susceptibility, and treatment of acute bacterial exacerbations of complicated chronic bronchitis in the primary care setting: ciprofloxacin 750 mg BID versus clarithromycin 500 mg BID. Clin Ther 1998;20:885–900.[CrossRef][Medline]
  16. Chodosh S, Schreurs A, Siami G, Barkman HW Jr, Anzueto A, Shan M, Moesker H, Stack T, Kowalsky S, Group BS. Efficacy of oral ciprofloxacin vs. clarithromycin for treatment of acute bacterial exacerbations of chronic bronchitis. Clin Infect Dis 1998;27:730–738.[Medline]
  17. Shah PM, Maesen FPV, Dolmann A, Vetter N, Fiss E, Wesch R. Levofloxacin versus cefuroxime axetil in the treatment of acute exacerbation of chronic bronchitis: results of a randomized, double-blind study. J Antimicrob Chemother 1999;43:529–539.[Abstract/Free Full Text]
  18. Davies BI, Maesen FPV. Clinical effectiveness of levofloxacin in patients with acute purulent exacerbations of chronic bronchitis: the relationship with in-vitro activity. J Antimicrob Chemother 1999;43:83–90.[Abstract]
  19. Nouira S, Marghli S, Belghith M, Besbes L, Elatrous S, Abroug F. Once daily oral ofloxacin in chronic obstructive pulmonary disease exacerbation requiring mechanical ventilation: a randomised placebo-controlled trial. Lancet 2001;358:2020–2025.[CrossRef][Medline]
  20. Patel IS, Seemungal TA, Wilks M, Lloyd-Owen SJ, Donaldson GC, Wedzicha JA. Relationship between bacterial colonisation and the frequency, character, and severity of COPD exacerbations. Thorax 2002;57:759–764.[Abstract/Free Full Text]
  21. Monso E, Garcia-Aymerich J, Soler N, Farrero E, Felez MA, Anto JM, Torres A. Bacterial infection in exacerbated COPD with changes in sputum characteristics. Epidemiol Infect 2003;131:799–804.[CrossRef][Medline]
  22. Groenewegen KH, Wouters EF. Bacterial infections in patients requiring admission for an acute exacerbation of COPD: a 1-year prospective study. Respir Med 2003;97:770–777.[CrossRef][Medline]
  23. Martinez FJ, Grossman RF, Zadeikis N, Fisher AC, Walker K, Ambruzs ME, Tennenberg AM. Patient stratification in the management of acute bacterial exacerbation of chronic bronchitis: the role of levofloxacin 750 mg. Eur Respir J 2005;25:1001–1010.[Abstract/Free Full Text]
  24. Papi A, Bellettato CM, Braccioni F, Romagnoli M, Casolari P, Caramori G, Fabbri LM, Johnston SL. Infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations. Am J Respir Crit Care Med 2006;173:1114–1121.[Abstract/Free Full Text]
  25. Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL. Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol 2002;34:91–100.[CrossRef][Medline]
  26. Burns JL, Gibson RL, McNamara S, Yim D, Emerson J, Rosenfeld M, Hiatt P, McCoy K, Castile R, Smith AL, et al. Longitudinal assessment of Pseudomonas aeruginosa in young children with cystic fibrosis. J Infect Dis 2001;183:444–452.[CrossRef][Medline]
  27. West SE, Zeng L, Lee BL, Kosorok MR, Laxova A, Rock MJ, Splaingard MJ, Farrell PM. Respiratory infections with Pseudomonas aeruginosa in children with cystic fibrosis: early detection by serology and assessment of risk factors. JAMA 2002;287:2958–2967.[Abstract/Free Full Text]
  28. Angrill J, Agusti C, Torres A. Bronchiectasis. Curr Opin Infect Dis 2001;14:193–197.[CrossRef][Medline]
  29. Angrill J, Agusti C, de Celis R, Rano A, Gonzalez J, Sole T, Xaubet A, Rodriguez-Roisin R, Torres A. Bacterial colonisation in patients with bronchiectasis: microbiological pattern and risk factors. Thorax 2002;57:15–19.[Abstract/Free Full Text]
  30. Ho PL, Chan KN, Ip MS, Lam WK, Ho CS, Yuen KY, Tsang KW. The effect of Pseudomonas aeruginosa infection on clinical parameters in steady-state bronchiectasis. Chest 1998;114:1594–1598.[CrossRef][Medline]
  31. Evans SA, Turner SM, Bosch BJ, Hardy CC, Woodhead MA. Lung function in bronchiectasis: the influence of Pseudomonas aeruginosa. Eur Respir J 1996;9:1601–1604.[Abstract]
  32. Rosell A, Monso E, Soler N, Torres F, Angrill J, Riise G, Zalacain R, Morera J, Torres A. Microbiologic determinants of exacerbation in chronic obstructive pulmonary disease. Arch Intern Med 2005;165:891–897.[Abstract/Free Full Text]
  33. Miravitlles M, Espinosa C, Fernandez-Laso E, Martos JA, Maldonado JA, Gallego M. Relationship between bacterial flora in sputum and functional impairment in patients with acute exacerbations of COPD. Study Group of Bacterial Infection in COPD. Chest 1999;116:40–46.[CrossRef][Medline]
  34. Nseir S, Di Pompeo C, Cavestri B, Jozefowicz E, Nyunga M, Soubrier S, Roussel-Delvallez M, Saulnier F, Mathieu D, Durocher A. Multiple-drug-resistant bacteria in patients with severe acute exacerbation of chronic obstructive pulmonary disease: prevalence, risk factors, and outcome. Crit Care Med 2006;34:2959–2966.[Medline]
  35. Ferrer M, Ioanas M, Arancibia F, Marco MA, de la Bellacasa JP, Torres A. Microbial airway colonization is associated with noninvasive ventilation failure in exacerbation of chronic obstructive pulmonary disease. Crit Care Med 2005;33:2003–2009.[CrossRef][Medline]
  36. Eller J, Ede A, Schaberg T, Niederman MS, Mauch H, Lode H. Infective exacerbations of chronic bronchitis: relation between bacteriologic etiology and lung function. Chest 1998;113:1542–1548.[CrossRef][Medline]
  37. Lin SH, Kuo PH, Hsueh PR, Yang PC, Kuo SH. Sputum bacteriology in hospitalized patients with acute exacerbation of chronic obstructive pulmonary disease in Taiwan with an emphasis on Klebsiella pneumoniae and Pseudomonas aeruginosa. Respirology 2007;12:81–87.[CrossRef][Medline]
  38. Lode H, Allewelt M, Balk S, De Roux A, Mauch H, Niederman M, Schmidt-Ioanas M. A prediction model for bacterial etiology in acute exacerbations of COPD. Infection 2007;35:143–149.[CrossRef][Medline]
  39. Tenover FC, Arbeit RD, Goering RV, Michelsen PA, Murray BE, Persing DH, Swaminathan B. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–2239.[Medline]
  40. Duchêne M, Schweizer A, Lottspeich F, Krauss G, Marget M, Vogel K, von Specht B, Domdey H. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene. J Bacteriol 1988;170:155–162.[Abstract/Free Full Text]
  41. Zeger SL, Liang KY. Longitudinal data analysis for discrete and continuous outcomes. Biometrics 1986;42:121–130.[CrossRef][Medline]
  42. Murphy TF, Brauer AL, Schiffmacher AT, Sethi S. Persistent colonization by Haemophilus influenzae in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2004;170:266–272.[Abstract/Free Full Text]
  43. Murphy TF, Brauer AL, Sethi S, Kilian M, Cai X, Lesse AJ. Haemophilus haemolyticus: a human respiratory tract commensal to be distinguished from Haemophilus influenzae. J Infect Dis 2007;195:81–89.[CrossRef][Medline]
  44. Frederiksen B, Koch C, Hoiby N. Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis. Pediatr Pulmonol 1997;23:330–335.[CrossRef][Medline]
  45. Post JC, Aul JJ, White GJ, Wadowsky RM, Zavoral T, Tabari R, Kerber B, Doyle WJ, Ehrlich GD. PCR-based detection of bacterial DNA after antimicrobial treatment is indicative of persistent, viable bacteria in the chinchilla model of otitis media. Am J Otolaryngol 1996;17:106–111.[CrossRef][Medline]
  46. Wood DM, Smyth AR. Antibiotic strategies for eradicating Pseudomonas aeruginosa in people with cystic fibrosis. Cochrane Database Syst Rev 2006;1:CD004197.[Medline]
  47. O'Brien C, Guest PJ, Hill SL, Stockley RA. Physiological and radiological characterisation of patients diagnosed with chronic obstructive pulmonary disease in primary care. Thorax 2000;55:635–642.[Abstract/Free Full Text]
  48. Patel IS, Vlahos I, Wilkinson TM, Lloyd-Owen SJ, Donaldson GC, Wilks M, Reznek RH, Wedzicha JA. Bronchiectasis, exacerbation indices, and inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2004;170:400–407.[Abstract/Free Full Text]



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