Microbiologic follow-up study in adult bronchiectasis
Paul T. Kinga,b,?, Stephen R. Holdsworthb, Nicholas J. Freezera,
Elmer Villanuevac, Peter W. Holmesa
aDepartment of Respiratory and Sleep Medicine, Monash Medical Centre, 246 Clayton Road,
Clayton, Melbourne 3168, Australia
bDepartment of Medicine, Monash Medical Centre, Monash University, 246 Clayton Road, Clayton, Melbourne 3168, Australia
cNational Breast Cancer Centre, Parramatta Road, Camperdown, Sydney 2050, Australia
Received 1 November 2006; accepted 17 March 2007
Available online 30 April 2007
There is minimal published longitudinal data about pathogenic microorganisms in adults
with bronchiectasis. Therefore a study was undertaken to assess the microbiologic profile
over time in bronchiectasis.
A prospective study of clinical and microbiologic outcomes was performed. Subjects were
assessed by a respiratory physician and sputum sample were collected for analysis.
Subjects were followed up and had repeat assessment performed.
Eighty-nine subjects were followed up for a period of 5.773.6 years. On initial assessment
the two most common pathogens isolated were Haemophilus influenzae (47%) and
Pseudomonas aeruginosa (12%) whilst 21% had no pathogens isolated. On follow-up review
results were similar (40% H. influenzae, 18% P. aeruginosa and 26% no pathogens). The
prevalence of antibiotic resistance of isolates increased from 13% to 30%. Analysis of a
series of H. influenzae isolates showed they were nearly all nontypeable and all were
different subtypes. Subjects with no pathogens isolated from their sputum had the mildest
disease, while subjects with P. aeruginosa had the most severe bronchiectasis.
Many subjects with bronchiectasis are colonized with the same bacterium over an average
follow-up of 5 years. Different pathogens are associated with different patterns of clinical
& 2007 Elsevier Ltd. All rights reserved.
Bronchiectasis remains a prevalent respiratory disease. It
has been estimated that there are more than 110,000 adults
in the US who have bronchiectasis.1In addition it overlaps
with chronic obstructive pulmonary disease (COPD) and two
recent studies have found that 29%2and 50%3of subjects
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0954-6111/$-see front matter & 2007 Elsevier Ltd. All rights reserved.
?Corresponding author. Department of Medicine, Monash Medical
Centre, 246 Clayton Rd, Clayton, Melbourne 3168, Australia.
Tel.: +6139546666; fax: +61395946495.
E-mail address: firstname.lastname@example.org (P.T. King).
Respiratory Medicine (2007) 101, 1633–1638
diagnosed with COPD have evidence on bronchiectasis on
high resolution computed tomography scanning (HRCT).
Bronchiectasis is characterized by chronic airway infec-
tion resulting in significant clinical disease and impaired
lung function. There have been a large number of studies
that have assessed factors involved in the aetiology of
bronchiectasis. However there is relative lack of information
available about the microbiology of this condition. In
particular, there is minimal longitudinal data about patho-
genic microorganisms in bronchiectasis. This information is
important as it has implications for the pathogenesis of this
condition as well as for appropriate antibiotic selection.
To examine the microbiologic profile in bronchiectasis a
prospective study of a cohort of adult patients with
bronchiectasis was performed.
The patient cohort consisted of adults who were referred by
their family doctors for assessment at Monash Medical
Centre, a University tertiary-referral hospital. Subjects
were seen between 1990 and 2004. Bronchiectasis was
diagnosed on high-resolution CT scanning by a consultant
radiologist using standard criteria.4
Subjects had a detailed clinical assessment performed by
a respiratory physician (PK/PH) and a sputum sample was
taken when the patient was clinically stable (i.e. patient
had not had an exacerbation and had not received
antibiotics for at least 1 month). Subjects were screened
for underlying causes of bronchiectasis with full blood
examinations, immunoglobulins, neutrophil and lymphocyte
function, aspergillus precipitins and skin testing, ciliary
function and cystic fibrosis mutation analysis. Patients were
followed up for a minimum period of 1 year when they were
reviewed again by a respiratory physician (PK/PH) and asked
to produce another sputum sample (when they were
clinically stable) for microbiological analysis. A total of
135 patients were screened of whom 89 subjects were able
to produce sputum samples suitable for microbiological
analysis on initial and follow-up review.
This project was approved by the Human Ethics Commit-
tee of Monash Medical Centre and informed consent was
obtained from all patients.
Collection of sputum samples
Patients were asked to perform chest physiotherapy for a
minimum of 5min prior to the expectoration of sputum.
Samples were processed in the Diagnostic Microbiology
laboratories of Monash Medical Centre (Southern Health
Pathology) if there were 425 leucocytes ando25 epithelial
cells per field using a low magnification lens. Specimens
were inoculated onto chocolate agar and horse blood agar
(50%)/McConcey agar plates and incubated at 351C in 5%CO2
for 48h. Specimens were then examined for growth.
Specimens for mycobacterial analysis were sent to the
Victorian Infectious Disease Reference Laboratory where
specimens had Ziehl-Nielsen staining performed and were
cultured on Lo ¨wenstein-Jensen medium. Negative bacterial
cultures were discarded after 7 days, negative cultures for
fungi after 4 weeks and Lowenstein-Jensen cultures after
6–8 weeks. Susceptibility testing was performed using
National Committee for Clinical Laboratory Standards.5
Sensitivity testing was performed using discs; VITEK (bio-
Merieux, Marcy-l’Etoile, France) and E-test (AB Biodisk,
Solna Sweden) for; Haemophilus influenzae and Moxarella.
catarrahalis (amoxicillin/erythromycin/tetracycline), Sta-
phylococcus aureus (methicillin), Streptococcus pneumoniae
(penicillin) and Pseudomonas/other Gram-negative species
Analysis for nontypeable H. influenzae
Samples of H. influenzae isolated from sputum samples were
collected in the Microbiology Laboratory at Monash Medical
Centre and frozen at ?701C in glycerol broth and stored for
further analysis. Isolates of H. influenzae were examined for
the presence of capsular serotypes (a–f), to assess whether
they were typeable or nontypeable. Samples were thawed,
inoculated in to agar and then taken to the Microbiology
Diagnostic Unit in the Department of Microbiology and
Immunology at Melbourne University for typing for poly-
saccharide capsules by using anti-sera a–f and biotyping.
NTHi samples were then taken to the Microbiology Research
Laboratory in the Department of Microbiology and Immunol-
ogy at Melbourne University, for outermembrane protein
analysis. This was performed by a standardized method
using sodium dodecyl sulfate-polyacrylamide gel electro-
Results were expressed as mean and standard deviation (SD)
or number of patients (n) and percentage (%). Statistical
analysis was performed to assess the association between
patient factors on initial review (age, sex, sputum volume,
hospitalisation in the last year for an exacerbation of
bronchiectasis, number of exacerbations per year, number
of lobes with bronchiectasis on HRCT, FEV1and FVC) with the
presence of the same microbial isolate on follow-up and
antibiotic resistance. Statistical analysis was performed
using Stata software (College Station, TX).
The patient cohort was predominantly female (70%) with an
average age of 57714 years. Most subjects (77%) had
idiopathic disease. None were current smokers and 18% had
had a history of previous smoking. Baseline characteristics of
the patients are summarised in Table 1.
Sputum isolates in subjects with bronchiectasis
On initial assessment the most common bacterium isolated
was H. influenzae, present in 42 (47%) of the cohort of 89
patients. The next most common bacteria were P. aerugi-
nosa isolated in 11 patients (12%), Moxarella catarrhalis in 7
patients (8%) and S. pneumoniae isolated in 6 (7%). Only 3
patients had Staphylococcus aureus isolated. Two patients
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P.T. King et al. 1634
had Aspergillus spp. isolated from their sputum and two had
Mycobacterium avium complex. A large number of patients
(21%) had no potential pathogenic microorganisms8(desig-
nated as no pathogens) isolated from their sputum. Results
are shown in Table 2.
Follow-up sputum samples were obtained an average
period of 5.773.6 years after initial review. Overall results
were fairly similar to the initial review, with the dominant
bacteria isolated being H. influenzae present in 36 patients
(40%) and P. aeruginosa isolated in 16 patients (18%). Again
there were a large proportion of subjects who had no
pathogen isolated from their sputum 23 patients (26%).
A comparison was performed between the results of
initial and follow-up sputum samples. The follow-up sputum
samples grew the same organism in 50 patients (56%). Of the
42 subjects who had H. influenzae isolated on initial
assessment, 27 of these patients (64%) had H. influenzae
isolated from their sputum on follow-up review. A similar
picture occurred in the 11 subjects with P. aeruginosa
isolated on their initial assessment, eight of whom (73%) had
Pseudomonas isolated again on follow-up. Logistic regres-
sion analysis showed that subjects who had the same isolate
on follow-up had a significantly higher number of exacerba-
tions (3.571.9 per year) compared with noncolonized
subjects (2.771.7) (p ¼ 0.04) with an odds ratio of 1.3
(95% confidence interval 1.0, 1.7).
Resistance to antibiotics
Antibiotic susceptibility testing was performed for all
isolates. On initial assessment 12 sputum isolates (13% of
patient group) showed antibiotic resistance. On follow-up
review there was a higher level of resistance with 27 isolates
(30%) demonstrating antibiotic resistance (Table 3). Resis-
tance to b-lactams in subjects with H. influenzae, Strepto-
coccus. pneumoniae or M. catarrhalis increased from 11% to
26%. Resistance to gentamicin in subjects with P. aeruginosa/
other Gram-negative pathogens increased from 14% to 39%.
The characteristics of patients with resistant and sensi-
tive pathogens are compared in Table 4. Patients with
resistant bacteria were significantly more likely to have
been hospitalised (52% versus 21%; p ¼ 0.006) and have had
a greater number of exacerbations (mean [SD] of 4.0 [1.8]
versus 2.8 [1.7]; p ¼ 0.007) compared to those with
sensitive bacteria. Logistic regression analysis resulted in a
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Baseline characteristics of patients (n ¼ 89).
CharacteristicsValues n (%)
Aetiology of bronchiectasis
Sputum isolates in 89 subjects with bronchiectasis.
Pathogen Initial assessment n (%) Follow-up review n (%) Same pathogen n (%)
Resistance to antibiotics.
(n ¼ 89)
(n ¼ 89)
Total12 (13%) 27 (30%)
Microbiologic outcome in bronchiectasis 1635
model relating presence of antibiotic resistance to hospita-
lisation (for an exacerbation of bronchiectasis) and the
number of exacerbations. On average, hospitalized patients
had an odds ratio (OR) of 3.5 (95% confidence interval [CI]
1.2, 9.6; p ¼ 0.017) for the presence of antibiotic resistance
compared to nonhospitalized patients, after controlling for
the number of exacerbations. Likewise, every exacerbation
event increased the odds of antibiotic resistance by 41%
(95% CI 6%, 88%) after controlling for hospitalisation status.
Analysis of H. influenzae samples
Typing of H. influenzae samples for the presence of capsular
polysaccharides demonstrated that 29 out of 30 sputum
isolates were nontypeable strains of H. influenzae and 1 was
an encapsulated type b strain. Analysis of outermembrane
proteins on 13 different NTHi isolates showed that they were
all different strains.
Features of the different microbiologic groups
The three most common findings on sputum analysis were H.
influenzae, P. aeruginosa and no pathogenic microorgan-
isms. Patients on the basis of the initial sputum samples
were divided into three different subsets (H. influenzae, P.
aeruginosa and no pathogens) and patient characteristics
(clinical, CT and spirometry features) were correlated with
the three different subsets. The no pathogen group had the
least severe clinical features, less extensive disease and
best lung function. The Pseudomonas group had the worst
clinical features and lung function and the most extensive
disease. Results are summarised in Table 5.
This study described the microbiologic profile of a series of
subjects with bronchiectasis followed up for an average
period of over 5.7 years. The spectrum of bacteria isolated
was similar to previous studies8–12with H. influenzae being
the most commonly isolated bacterium, followed by other
common pathogens including Streptococcus pneumoniae, M.
catarrhalis and P. aeruginosa.
Recent studies have assessed the microbiologic profile in
bronchiectasis8–14and the frequency of isolates often varies
significantly between different locations. The two main
pathogens isolated have been H. influenzae (mean of 42%
and a range of 29–70%) and P. aeruginosa (mean of 18% and
range of 12–33%). The behaviour of these two pathogens has
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Comparison between characteristics of patients with antibiotic resistant and sensitive pathogens.
CharacteristicsResistant (n ¼ 27)
Sensitive (n ¼ 62)
Females, n (%)
Hospitalization in past year, n (%)
Exacerbations in past year, n (%)
Number of lobes with bronchiectasis on HRCT
FVC (% predicted)
Table shows the patient characteristics on initial assessment and the association between the presence/absence of antibiotic
resistance on follow-up.
Features of different microbiologic groups.
No pathogens (n ¼ 19)
Haemophilus influenzae (n ¼ 43)
Pseudomonas aeruginosa (n ¼ 27)
Sputum volume (ml)
Hospitalizations (past year)
MRC dyspnoea score
Exacerbations (past year)
Lobes with bronchiectasis 2.271.0 2.370.8 2.971.2
FVC (% predicted)
MRC ¼ Medical Research Council.
P.T. King et al. 1636
not been well characterized in bronchiectasis but in other
forms of chronic bronchial disease such as cystic fibrosis and
COPD both have been shown to have many different
strains.15,16It has been recognised recently that both
are capable of forming biofilms.17,18There was a low
incidence of Staphylococcus aureus isolates in this cohort
and there is some evidence to suggest this pathogen is more
prevalent in cystic fibrosis.19Recent literature has empha-
sised the role of nontuberculosis mycobacterial (NTM)
infection in bronchiectasis.20Wickremasinghe et al.21found
that 2% of a United Kingdom cohort of subjects with
bronchiectasis had NTM, the same result as the current
study. One authority has suggested that NTM infection in the
United States may be associated with the relatively low hot-
As has been the case in previous studies a large proportion
of subjects in this study did not have pathogenic bacteria
isolated from purulent sputum. There are some factors that
need to be considered to explain these findings. The
specimens were obtained from standard sputum samples
and other sputum collection methods may have higher
yields. The gold standard for the collection of specimens
remains the protected bronchoscopy method. A recent study
showed that there was good correlation between broncho-
scopic procedures and standard sputum culture in patients
with bronchiectasis, (the yield of pathogenic bacteria from
sputum was 52%, 61% from protected specimen brush and
56% from bronchoalveolar lavage).8In subjects with nega-
tive cultures, pathogenic bacteria may only be found in low
numbers and as a consequence not be isolated using
standard cultures. The use of other techniques such as
polymerase chain reaction may increase the yield. Some
patients may have had infections with viruses, which have
not been assessed in this study. It has been suggested that up
to a third of exacerbations of COPD are due to viral
infections.23The role of viruses in bronchiectasis is not well
There do not appear to have been any previous studies
with long-term follow-up data on the sputum microbiology
of bronchiectasis. Over 50% of patients had the same
pathogen on follow-up review as on initial assessment. On
follow-up there was a higher incidence of P. aeruginosa but
the most common pathogen remained H. influenzae. Pasteur
et al.12studied a cohort of 150 adults with bronchiectasis
and as part of their assessment all subjects had multiple
sputum samples analysed over a 1 year period. This study
found that 66% of subjects were colonized with bacteria.
These two studies suggest that a large number of patients
become colonized with one pathogen and previous sputum
microbiologic analysis may be useful in guiding antibiotic
selection in bronchiectasis.
The incidence of antibiotic resistance increased between
the initial assessment and follow-up review, particularly in
H. influenzae and P. aeruginosa isolates. The frequency of
exacerbations on initial assessment was strongly associated
with the presence of antibiotic resistance on follow-up and
this presumably arose from the higher use of antibiotics.
Antibiotics were given as a course of 10 days with single drug
use in over 80% of cases. As such the regimen used was fairly
conservative and less than 15% of patients received long-
term antibiotic therapy (defined as more than 1 month of
continuous antibiotics). Previous hospitalisation was also a
major factor associated with antibiotic resistance. These
findings emphasise that more attention may be needed to
prevent the development of antibiotic resistance in such
patients. Potential strategies may include further reduction
in antibiotic usage, use of multiple antibiotics simulta-
neously to prevent resistance and avoidance of hospital
admission with more emphasis on outpatient parental
Despite the fact that H. influenzae is the most commonly
isolated pathogen in bronchiectasis there has been little
analysis of its characteristics in this condition. This study
confirmed that in bronchiectasis this bacterium is the
nontypeable form (NTHi) similar to other forms of bron-
chitis.24NTHi is a heterogeneous pathogen with over many
different strains25and there is considerable turnover with
new strains being acquired periodically.26It has been shown
that more than half of a cohort of subjects attending a cystic
fibrosis clinic in Melbourne had the same (epidemic) strain of
P. aeruginosa that appeared to arise as a consequence of
cross-infection.27The findings of 13 different types in 13
different patients studied, makes it unlikely that this cohort
has an epidemic strain of NTHi.
This cohort could be divided into 3 separate groups based
on results from sputum; no pathogenic microorganisms,
H. influenzae and P. aeruginosa. There was a spectrum
of severity in these 3 groups. The subjects in this study
form part of a cohort who had been previously studied
by the authors who found that the cohort had progressive
disease with worse lung function and symptoms over an
8-year follow-up period. It is possible that there may
be an evolution of disease from no pathogenic bacteria
to Haemophilus to Pseudomonas infection in bronchiec-
tasis. This appears to occur in CF.28,29This study did not
have a long enough follow-up period to make any clear
In conclusion, the most common isolates from sputum
analysis in this cohort of bronchiectasis subjects were H.
influenzae, no growth and P. aeruginosa. Over half of this
cohort was colonized with the same bacterium over a 5-year
follow-up period. Antibiotic resistance increased over time
and was associated with frequency of exacerbations and
We would like to thank the staff of the Diagnostic
Microbiology Laboratory at Monash Medical Centre for their
1. Weycker D, Edelsberg J, Oster G, et al. Prevalence and
2. O’Brien C, Guest PJ, Hill SL, et al. Physiological and radiological
characterisation of patients diagnosed with chronic obstructive
pulmonary disease in primary care. Thorax 2000;55:635–42.
3. Patel IS, Vlahos I, Wilkinson TM, et al. Bronchiectasis,
exacerbation indices, and inflammation in chronic obstructive
pulmonary disease. Am J Respir Crit Care Med 2004;170:
ARTICLE IN PRESS
Microbiologic outcome in bronchiectasis 1637
4. McGuinness G, Naidich DP. CT of airways disease and bronch- Download full-text
iectasis. Radiol Clin North Am 2002;40:1–19.
5. Standards NCCLS. Performance standards for antimicrobial
tests (M100-S8). Villanova, PA: National Committee for Clinical
Laboratory Standards. p. 18.
6. Barenkamp SJ, Munson Jr RS, Granoff DM. Subtyping isolates of
Haemophilus influenzae type b by outer-membrane protein
profiles. J Infect Dis 1981;143:668–76.
7. King PT, Hutchinson PE, Johnson PD, et al. Adaptive immunity to
nontypeable Haemophilus influenzae. Am J Respir Crit Care
8. Angrill J, Agusti C, de Celis R, et al. Bacterial colonisation in
patients with bronchiectasis: microbiological pattern and risk
factors. Thorax 2002;57:15–9.
9. Nicotra MB, Rivera M, Dale AM, et al. Clinical, pathophysiologic,
and microbiologic characterization of bronchiectasis in an aging
cohort. Chest 1995;108:955–61.
10. Roberts DE, Cole P. Use of selective media in bacteriological
investigation of patients with chronic suppurative respiratory
infection. Lancet 1980;1:796–7.
11. Pang JA, Cheng A, Chan HS, et al. The bacteriology of
bronchiectasis in Hong Kong investigated by protected catheter
12. Pasteur MC, Helliwell SM, Houghton SJ, et al. An investigation
into causative factors in patients with bronchiectasis. Am J
Respir Crit Care Med 2000;162:1277–84.
13. Ho PL, Chan KN, Ip MS, et al. The effect of Pseudomonas
aeruginosa infection on clinical parameters in steady-state
bronchiectasis. Chest 1998;114:1594–8.
14. King PT, Holdsworth SR, Freezer NJ, et al. Outcome in adult
bronchiectasis. COPD 2005;2:27–34.
15. Murphy TF, Sethi S, Klingman KL, et al. Simultaneous respiratory
tract colonizationby multiple
Haemophilus influenzae in chronic obstructive pulmonary
disease: implications for antibiotic therapy. J Infect Dis 1999;
16. Burns JL, Gibson RL, McNamara S, et al. Longitudinal assess-
ment of Pseudomonas aeruginosa in young children with cystic
fibrosis. J Infect Dis 2001;183:444–52.
17. Murphy TF, Kirkham C. Biofilm formation by nontypeable
Haemophilus influenzae: strain variability, outer membrane
antigen expression and role of pili. BMC Microbiol 2002;2:7.
18. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms:
from the natural environment to infectious diseases. Nat Rev
19. Shah PL, Mawdsley S, Nash K, et al. Determinants of chronic
infection with Staphylococcus aureus in patients with bronch-
iectasis. Eur Respir J 1999;14:1340–4.
20. Field SK, Fisher D, Cowie RL. Mycobacterium avium complex
pulmonary disease in patients without HIV infection. Chest
21. Wickremasinghe M, Ozerovitch LJ, Davies G, et al. Non-
tuberculous mycobacteria in patients with bronchiectasis.
22. Iseman M. Update in nontuberculosis mycobacteria. ATS: state
of the art. Chicago: American Thoracic Society; 2005.
23. Seemungal T, Harper-Owen R, Bhowmik A, et al. Respiratory
viruses, symptoms, and inflammatory markers in acute exacer-
bations and stable chronic obstructive pulmonary disease. Am J
Respir Crit Care Med 2001;164:1618–23.
24. Murphy TF. Haemophilus infections. In: Braunwald F, Kaspar H,
Longo J, editors. Harrisons principles of internal medicine. New
York: McGraw-Hill; 2001. p. 939–42.
25. Bruant G, Watt S, Quentin R, et al. Typing of nonencapsulated
haemophilus strains by repetitive-element sequence-based PCR
using intergenicdyad sequences.
26. Samuelson A, Freijd A, Jonasson J, et al. Turnover of none-
ncapsulated Haemophilus influenzae in the nasopharynges of
otitis-prone children. J Clin Microbiol 1995;33:2027–31.
27. Armstrong DS, Nixon GM, Carzino R, et al. Detection of a
widespread clone of Pseudomonas aeruginosa in a pediatric
cystic fibrosis clinic. Am J Respir Crit Care Med 2002;166:983–7.
28. Armstrong DS, Grimwood K, Carlin JB, et al. Lower airway
inflammation in infants and young children with cystic fibrosis.
Am J Respir Crit Care Med 1997;156:1197–204.
29. Gibson RL, Burns JL, Ramsey BW. Pathophysiology and manage-
ment of pulmonary infections in cystic fibrosis. Am J Respir Crit
Care Med 2003;168:918–51.
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P.T. King et al.1638