Published ahead of print on April 29, 2004, doi:10.1164/rccm.200403-354OC
© 2004 American Thoracic Society
Persistent Colonization by Haemophilus influenzae in Chronic Obstructive Pulmonary DiseaseDivision of Infectious Diseases and Division of Pulmonary and Critical Care Medicine, Department of Medicine, and Department of Microbiology, University at Buffalo, State University of New York; and the Veterans Affairs Western New York Healthcare System, 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}acsu.buffalo.edu
Nontypeable Haemophilus influenzae colonizes the respiratory tract of adults with chronic obstructive pulmonary disease (COPD) and causes intermittent exacerbations. Isolates of H. influenzae collected monthly in a prospective study were subjected to molecular typing. During a 7-year study spanning 345 patient-months of observation, 122 episodes of negative cultures lasting 1 month or more, and that were preceded and followed by isolation of an apparently identical strain of H. influenzae, were found. Seventeen such episodes of negative cultures, lasting 6 months or more and spanning 203 patient-months, were studied in detail to test the hypothesis that these periods of negative cultures represented continuous colonization by the same strain of H. influenzae. Molecular typing by three independent methods established that the strains preceding and following the episodes of negative cultures were indeed identical. Strain-specific H. influenzae DNA was detected in some of the sputum samples that had yielded negative cultures. These results indicate that some patients with COPD are persistently colonized with H. influenzae and that sputum cultures underestimate the frequency of colonization of the respiratory tract by H. influenzae in COPD. This observation has a significant impact on understanding bacterial colonization in COPD.
Key Words: molecular epidemiology • outer membrane protein • pulsed-field gel electrophoresis • respiratory tract infection • sputum culture Nontypeable Haemophilus influenzae is present frequently in the airways of adults with chronic obstructive pulmonary disease (COPD) (1–4). In addition to colonizing during clinically stable periods, H. influenzae is an important cause of lower respiratory tract infections resulting in exacerbations of COPD (5–7). Acquisition of a new strain of H. influenzae is associated with the occurrence of an exacerbation of COPD (8). To begin to elucidate the dynamics of bacterial colonization of the respiratory tract in COPD, we are performing a prospective study of adults with COPD. Expectorated sputum is collected monthly and molecular typing is performed on bacteria isolated from cultures of sputum. H. influenzae is the most common potential pulmonary pathogen isolated, followed by Moraxella catarrhalis and Streptococcus pneumoniae (8). Isolates of H. influenzae are subjected to molecular typing and colonization patterns are being characterized. Isolates of H. influenzae are associated with a variety of colonization patterns (8–10). A common pattern observed in the case of H. influenzae is the isolation of a strain from sputum obtained at a clinic visit, followed by one or more clinic visits at which H. influenzae is absent in sputum culture, followed by isolation of the same original strain of H. influenzae at a subsequent clinic visit. This phenomenon of a period of negative sputum cultures preceded and followed by clinic visits at which an apparently identical strain was isolated, called "gaps," could have several possible explanations. First, one might question whether the typing methods are adequately sensitive to discriminate between strains. In other words, are the two apparently identical strains with intervening negative cultures truly identical? Second, patients may clear the strain and reacquire the same strain. Finally, the strain of H. influenzae may persist in the respiratory tract in the presence of negative sputum cultures. Elucidating the mechanism of this colonization pattern of H. influenzae has important implications in understanding the potential role of H. influenzae in the chronic airway inflammation observed in COPD, further defining the role of H. influenzae in causing exacerbations of COPD, characterizing the host response to H. influenzae in the respiratory tract, and interpreting the results of sputum cultures from adults with COPD. Over the course of the first 7 years of our prospective study of COPD involving monthly sputum cultures, 122 instances of such gaps of 1 month or more were observed over 345 patient-months of observation. Seventeen instances of the isolation of the apparent identical strain of H. influenzae with intervening negative cultures of 6 months or more were observed over 203 patient-months. The goal of the present study is to test the hypothesis that these prolonged periods of negative sputum cultures (gaps of 6 months or more) represent persistent colonization of the respiratory tract by H. influenzae in adults with COPD.
COPD Study Clinic One hundred four patients were enrolled in a prospective, longitudinal study from March 1994 through December 2000. Inclusion criteria were the presence of chronic bronchitis (11), absence of asthma and bronchiectasis based on clinical assessment, an ability to comply with a schedule of monthly clinic visits, and the absence of immunosuppressive or other life-threatening disorders. The patients were seen monthly at an outpatient clinic in the Buffalo Veterans Affairs Medical Center (Buffalo, NY), and whenever they had symptoms suggestive of an exacerbation. At each visit, clinical information and sputum and serum samples were obtained. A clinical evaluation was performed to determine whether the patient had stable disease or an exacerbation as previously described (8, 9).
Sputum Samples
Sputum Fibrinogen Measurement
Typing of H. influenzae Strains
Polymerase Chain Reaction
DNA was purified from pellets of sputum samples by standard phenol–chloroform extraction followed by ethanol precipitation (16). Each set of reactions included a negative control of sterile water in place of DNA. These controls consistently yielded negative results.
Data Analysis
Identification of Episodes of H. influenzae Colonization with Intervening Negative Cultures One hundred four patients were enrolled from March 1994 through December 2000 and these patients had 3,009 clinic visits. The frequency with which monthly cultures of sputum revealed the isolation of the apparently identical strain of H. influenzae with intervening negative cultures was determined by analysis of culture results and molecular typing data from the COPD Study Clinic. One hundred twenty-two instances of such gaps of 1 month or more were observed over 345 patient-months of observation. In all, 17 instances in 10 patients of periods of 6 months or more of negative cultures preceded and followed by an apparent identical strain of H. influenzae spanning 203 patient-months of observation were identified. Two patients (Patients 24 and 27) experienced two separate gaps of more than 6 months by different pairs of strains. One patient (Patient 3) experienced four separate gaps, three caused by the same strain and one caused by a different strain. One patient (Patient 73) experienced two gaps by the same strain (Visits 10 to 18 and Visits 19 to 30) and one long gap from Visit 4 to 34 by a different strain. Each of the gaps was considered individually. Characteristics of the 10 patients are shown in Table 2 . These 10 patients did not differ from the other 94 patients in age, smoking, lung function, frequency of exacerbation, or frequency of isolation of H. influenzae. These 17 episodes of prolonged gaps were studied in detail to test the hypothesis that H. influenzae continuously colonized the patients throughout the period in spite of negative cultures.
Assessment of the Quality of Sputum Samples To assess the adequacy of the sputum samples that yielded negative cultures, sputum supernatants were subjected to measurement of fibrinogen, which is a marker for lower respiratory tract secretions (12, 13). Sixteen sputum samples that were negative for H. influenzae in culture were compared with 15 samples that grew H. influenzae before and after the negative cultures from the same patients. To control for variability among patients, a paired comparison was performed with averaged values of fibrinogen obtained for each patient to determine whether there was a relationship between the presence of H. influenzae in cultures and sputum fibrinogen level. Fibrinogen was detected in all 31 samples. There was no difference in the level of fibrinogen in samples with positive cultures (median, 537.1 ng/ml; interquartile range, 1,214.5 ng/ml) compared with the level of fibrinogen in samples with negative cultures (median, 898.2 ng/ml; interquartile range, 2,148.8 ng/ml) (p = 0.89, Wilcoxon signed rank test). Therefore, the quality of the sputum samples is similar between samples that yielded positive and negative cultures.
Effect of Antibiotic Administration on Culture Results
Molecular Typing of H. influenzae Strains Isolates of H. influenzae recovered from sputum of patients monitored in the COPD Study Clinic were typed by subjecting whole bacterial cell lysates to SDS–PAGE (8, 14). SDS–PAGE patterns of all 17 pairs of isolates were identical to those of the corresponding isolates in the pair (Figure 1) .
To further assess the isolates of H. influenzae that preceded and followed negative culture periods of 6 months or more, genomic DNA was isolated and subjected to pulsed-field gel electrophoresis. In the example shown in Figure 2 , three sets are identical to each other in every band (lanes c and d, lanes d and e, and lanes f and g). The pair of isolates in lanes a and b differ by one band, indicating that a single genetic event, such as a point mutation or an insertion or deletion of DNA, accounts for this difference (17). Therefore, these are closely related isolates. Because this type of variation is observed when the same strain is cultured repeatedly over time or isolated multiple times from the same patient, isolates with one to three band differences are considered the same strain (17). Of the 17 episodes of colonization by H. influenzae with intervening negative cultures, 14 pairs of isolates were identical (all bands identical) and 3 pairs were closely related (1 to 3 band differences).
Analysis of P2 Sequences of H. influenzae Strains To further assess the 17 pairs of strains of H. influenzae that preceded and followed a period of negative monthly cultures, oligonucleotide primers corresponding to the gene that encodes outer membrane protein P2 were designed to amplify the gene from genomic DNA. Because the gene that encodes P2 contains regions with a high degree of sequence heterogeneity among strains, comparison of the sequence between two strains represents a "fingerprint" for assessing the identity of two strains (18–22). PCR with oligonucleotide primers corresponding to the conserved 5' and 3' ends of ompP2 yielded amplicons of the expected size for 16 strains (8 of 17 pairs). Numerous alterations in reaction conditions failed to yield PCR products for the remaining 9 pairs of isolates (see below). Determination of the sequences of the amplicons from the eight pairs of isolates confirmed that the primers amplified ompP2 and that, as expected, the genes of strains from different patients showed a high degree of sequence diversity among strains. Six of the eight pairs showed identical sequence in every nucleotide within the pair. One pair demonstrated a single point mutation (24P19H1 and 24P25H1). A second pair demonstrated that the second isolate of the pair had four point mutations and a nine-nucleotide insertion compared with the first isolate (48P38H1 and 48P45H1). These types of changes in ompP2 occur during persistent colonization by the same strain of the respiratory tract of adults with COPD (19, 23, 24). Therefore comparison of the ompP2 sequences within each of the pairs confirmed the results of typing by both SDS–PAGE and pulsed-field gel electrophoresis.
Identification of Isolates with Variant P2 Sequences
Analysis of P5 Sequences of H. influenzae Strains To summarize results of typing and sequencing, all 17 episodes of negative cultures were preceded and followed by the identical strain of H. influenzae as determined on the basis of 3 independent lines of evidence: (1) SDS–PAGE typing, (2) pulsed-field gel electrophoresis, and (3) matching nucleotide sequence of genes that encode P2 or P5 in the strain pair. The demonstration of the identity of the strain preceding and following the period of negative cultures provides evidence that these strains persistently colonized the respiratory tract. Although unlikely, an alternative explanation would be clearance and reacquisition of the same strain. Therefore, sputum samples with negative cultures during gaps were analyzed for the presence of H. influenzae DNA.
Analysis of Sputum Samples for H. influenzae DNA by PCR
Sputum pellets from positive cultures for strains of H. influenzae with "variant" P2 genes were studied by PCR to determine whether these P2 genes could be amplified from sputum samples. Primers that corresponded to the sequence of the variant P2 were designed and used in PCRs with sputum samples yielding positive cultures of H. influenzae strains with variant P2 genes. All failed to yield a PCR product. Positive controls with genomic DNA of H. influenzae strains with variant P2 consistently yielded a PCR product of the predicted size. Numerous adjustments in conditions of the reactions were attempted, but PCR products were not obtained from any sputum samples with these primers in spite of a perfect match in sequence for the P2 gene of the strain that was grown from the sputum sample. To determine whether the inability to amplify the P2 gene by PCR was specific for P2, primers corresponding to the conserved 5' and 3' ends of the genes that encode outer membrane proteins P5 and P6 were designed. DNA from pellets of sputum samples that were positive in culture for H. influenzae were subjected to PCR with P5 and P6 primers. All samples yielded negative results for PCR products that corresponded to the P5 and P6 genes in spite of a perfect match with genes whose sequences had been determined from the isolate in the culture. Controls with DNA from the corresponding strain as template yielded PCR products of the predicted size. We conclude that the DNA of these "variant" strains of H. influenzae is apparently less stable or less amenable to detection by PCR in sputum as revealed by the negative results with sputum samples that are positive in culture for strains that contain DNA with perfect homology to the oligonucleotide primers. This characteristic precluded study of the negative cultures preceded and followed by positive cultures for the presence of H. influenzae DNA.
In a prospective study to elucidate the dynamics of bacterial colonization of the respiratory tract in adults with COPD, we have observed several different colonization patterns in the case of H. influenzae. One of the patterns involves gaps in monthly cultures in the isolation of the same strain. Over the course of 7 years, 17 episodes of prolonged (greater than 6 months) periods of negative sputum cultures preceded and followed by apparently identical strains of H. influenzae, as determined by SDS–PAGE typing, were observed. The frequency of these prolonged gaps was a somewhat surprising result and led to concern as to whether the typing method was accurate. Therefore, strains were subjected to additional molecular typing methods to assess strain identity. Strains were subjected to analysis of large fragments of genomic DNA by pulsed-field gel electrophoresis, a highly reliable method that has been validated for several bacterial species, including H. influenzae (15, 17). Finally, as a third independent method of molecular typing, the sequences of genes that encode outer membrane proteins with a high degree of interstrain heterogeneity were determined and compared. A rigorous analysis of these strains by SDS–PAGE, pulsed-field gel electrophoresis of genomic DNA, and sequencing of genes that show heterogeneity among strains established that the same strain persists in the respiratory tract in spite of negative intervening sputum cultures. Measurement of fibrinogen levels confirmed that sputum samples that were culture positive and culture negative were comparable, excluding poor-quality sputum samples as an explanation for negative cultures. Analysis of detailed data on antibiotic administration revealed that antibiotic administration did not account for these periods of negative sputum cultures. An alternative explanation to consider for the periods of prolonged negative cultures might be clearing and reacquisition of the same strain. Strains of nontypeable H. influenzae demonstrate enormous genetic heterogeneity when compared with one another, making the reacquisition of the same strain circulating in the community a highly unlikely event. Furthermore, H. influenzae does not remain viable on inanimate objects in the environment for even short periods of time (minutes); therefore, reacquisition from the environment does not explain the prolonged gaps. Finally, analysis of sputum samples yielding negative cultures revealed the presence of strain-specific H. influenzae genes, further establishing persistent colonization as the mechanism of prolonged gaps. The homologous P2 gene was present in negative sputum samples from patients who were colonized by H. influenzae with a "conventional" P2 gene, providing further evidence of persistent colonization. Does the presence of H. influenzae DNA in these sputum samples reflect the presence of viable bacteria or, alternatively, could it represent persistent DNA in the absence of viable organisms? Studies of this phenomenon were performed in the chinchilla model of otitis media by Post and coworkers (27). The persistence of DNA in middle ear fluid was investigated after instillation of live H. influenzae, heat-killed H. influenzae, and purified DNA. No DNA was detected from middle ear fluid of animals given heat-killed bacteria or purified DNA after Day 3. However, DNA in middle ear fluid from animals given live bacteria persisted through Day 21, even though samples were culture negative after the administration of antimicrobial therapy. This study supports the notion that the presence of H. influenzae DNA in samples from the respiratory tract indicates the presence of viable organisms. The P2 genes of H. influenzae strains from 9 of these 17 episodes displayed a strikingly different sequence compared with other strains. In addition, these epidemiologically unrelated strains with different patterns in pulsed-field gels showed minimal diversity in ompP2 when compared with one another, a surprising observation based on the well-described diversity of P2 sequences among strains (18–22). These variant strains share a number of phenotypic differences with other strains of H. influenzae (our unpublished observations). We were unable to amplify the genes that encode outer membrane proteins P2, P5, or P6 from sputum samples that were positive in culture with isolates that contained all three of these genes. The DNA of these variant strains of H. influenzae is apparently less stable or less amenable to detection by PCR in sputum, as revealed by the negative results with sputum samples that are positive in culture for strains that contain DNA that is completely homologous to oligonucleotide primers. In spite of our inability to demonstrate H. influenzae DNA in this subset of sputum samples, the observation that the period of negative cultures were preceded and followed by the identical strain supports the conclusion of persistent colonization by H. influenzae. Although the frequency and duration of persistent colonization by H. influenzae in spite of negative sputum cultures was somewhat surprising, a similar observation has been made by other investigators using different methods. Groeneveld and coworkers (28) monitored 16 patients with COPD prospectively with sputum cultures. Inspection of their culture results and time lines reveals that patients were colonized by identical strains with intervening negative sputum cultures. Similarly, Bandi and coworkers (1) showed that intracellular H. influenzae was present in bronchial biopsies of 13 of 15 adults with acute exacerbations of COPD in spite of only 1 of the 15 having positive cultures in bronchial washes. Several mechanisms may account for these periods of negative cultures. Concentrations of bacteria at less than 103 colony-forming units/ml of sputum will yield negative cultures. H. influenzae is known to form biofilms in vitro and in vivo (29, 30). Bacteria that are present in the airway in the form of biofilms may yield negative cultures because routine culture methods are designed to detect planktonic growth and are less sensitive in detecting bacteria in biofilms. Finally, several lines of evidence indicate that H. influenzae is viable inside host cells, including macrophages and respiratory epithelial cells (31–34). One might hypothesize that bacteria enter host cells and "hide" from detection by culture, accounting for prolonged periods of negative cultures. It is tempting to speculate that H. influenzae may modulate its form of growth under different conditions in the human respiratory tract, accounting for periods of negative sputum cultures. For example, H. influenzae is known to grow as a biofilm and planktonically under different conditions (29). Could the cells be changing from planktonic growth to biofilms in the human respiratory tract as a survival strategy? Similarly, could H. influenzae move from extracellular to intracellular growth, driven by factors such as intermittent antibiotic exposure, mucosal immune response, inflammatory mediators, or other unknown factors? Further investigation will be necessary to test these hypotheses suggested by the observations in the present study. The observation of prolonged colonization of the respiratory tract in spite of negative sputum cultures has broad-ranging implications. From a practical standpoint, H. influenzae may be present in the respiratory tract even in the face of a negative sputum culture. It is estimated that approximately half of exacerbations of COPD are caused by bacteria (5, 6). These estimates are based on several lines of evidence, including results of sputum cultures. A patient with clinical evidence of an exacerbation with a negative sputum culture would cause most clinicians to conclude that the exacerbation had a nonbacterial etiology. The observation in the present study that H. influenzae is present in the respiratory tract even in the setting of a negative sputum culture raises the question that bacteria may cause a greater proportion of exacerbations than is revealed by sputum culture. This speculation requires further investigation. In view of the observations in the present study, it will be important to assess the role of persistent colonization by H. influenzae on the dynamics of respiratory tract colonization and infection. For example, does colonization by H. influenzae affect the likelihood of colonization or exacerbation caused by a strain of another bacterial species or another strain of H. influenzae? It is also interesting to speculate that colonization by H. influenzae might affect the incidence of viral infection in adults with COPD. Analysis of the 17 gaps studied in detail here revealed that this subset of 10 patients was colonized by H. influenzae for a total of 203 patient-months; sputum cultures predicted that colonization occurred for only 34 of those 203 patient-months. Thus, H. influenzae is present far more often in the respiratory tract of adults with COPD than is apparent on the basis of sputum culture. These results have important implications in understanding the role of bacteria in the course and pathogenesis of COPD. A hallmark of COPD is airway inflammation. Chronic bacterial colonization of the respiratory tract may contribute to airway inflammation in COPD (6). Because H. influenzae is known to induce airway inflammation, studies that have compared markers of airway inflammation in the presence and absence of bacteria on the basis of the results of sputum cultures require careful reinterpretation. The observation that H. influenzae is present far more often than is revealed by sputum culture should be considered in the design of future studies to elucidate the role of chronic bacterial colonization in airway inflammation and in studies of the role of bacteria in the course and pathogenesis of COPD.
The authors acknowledge the important contributions of Adeline Thurston for data management; study nurses Karen Eschberger and Nancy Evans; Phyllis Lobbins, Lori Grove, and Catherine Wrona for processing sputum samples; and Drs. Alan Lesse and Charles Berenson for their assistance in the COPD Study Clinic.
Supported by the Department of Veterans Affairs and by the National Institutes of Health (grants AI19641 and HL66549). 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: T.F.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.L.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.T.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 17, 2004; accepted in final form April 26, 2004
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ABSTRACT
METHODS
2 tests. Sputum fibrinogen values of culture-positive and culture-negative sputum samples were compared with the Wilcoxon signed rank test. A p value of less than 0.05 was considered significant. All statistical comparisons were done with StatView 5.0 (SAS Institute, Cary, NC). 
