1. Presenter: Jiyeon Jung, MD Research Attending: Sanjay Sethi, MD
New Strain Acquisition of Klebsiella spp, Enterobacter spp, and Escherichia coli in COPD is Not Associated with Exacerbations.
Presenter: Jiyeon Jung, MD
Research Attending: Sanjay Sethi, MD

2. A Case Vignette
A 68-year-old former heavy smoker with a history of chronic obstructive pulmonary disease (COPD) presents to the ED with a two-day history of worsened shortness of breath and increased sputum. Chest radiography shows hyperinflation and no acute infiltrates.

3. A Case Vignette House staff ordered sputum culture in ED.
Sputum culture came back positive for Klebsiella spp.
How should this patient be treated?

4. History
Since 1994, the study has been conducted in WNY VA to reveal a relationship between bacterial infection and COPD exacerbation.

5. History
The role of bacterial pathogens in COPD exacerbation was controversial.
The rate of bacterial isolation from sputum was found to be similar in stable COPD and during exacerbations.
On the basis of these observations, a seminal review in 1975 indicated that bacterial pathogens do not cause exacerbations and that their presence in sputum is due to chronic colonization.1

6. History
New molecular, cellular, immunologic techniques in the past two decades.
Possible to differentiate strains of bacterial pathogens over time from the patients with molecular typing.

7. History Bacterial infections trigger COPD exacerbations.
Four Major pathogens.
A new bacterial strain plays a central role in the pathogenesis of an Exacerbation.

8. History Example Sputum culture(+) for H. influenza in stable phase
Sputum culture(+) for H. influenza in exacerbation
New strain of H. influenza in molecular typing.

9. History
Sputum cultures are often not useful for identifying bacterial infection in patients with COPD exacerbations.
Gram stain and culture of sputum are similar during exacerbations and stable disease.
They do not distinguish between true pathogens and colonizing flora.
As per 2007 GOLD guideline, Sputum culture should Not be performed. 2
Molecular typing

10. History
Acquisition of a new strain of H. influenzae,Moraxella catarrhalis, Streptococcus pneumoniae, or Pseudomonas aeruginosa is strongly associated with the occurrence of an exacerbation. 2,4,5,6,7

11. Background
In severe COPD, Enterobacteriaceae (Gram negative bacilli) are also isolated from sputum during exacerbations and stable disease. 8
The dynamics of infection by these pathogens in COPD has not been examined.

12. Sethi S and Murphy T. N Engl J Med 2008;359:2355-2365

13. Hypothesis As four major pathogens.
Klebsiella spp, Enterobacter spp, and Escherichia coli
If they are causative, there should be an association between strain acquisition of these pathogens and exacerbation.

14. Methods/COPD Study Clinic
Prospective longitudinal study of COPD patients in an outpatient clinic
127 subjects enrolled from March 1994 to Dec 2004
Clinic visits monthly and suspected exacerbation
At each visit:
clinical information
expectorated sputum sample

15. Method
Exacerbation defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD)
Acute increase in symptoms beyond normal day-to-day variation. 2 This generally includes one or more of the following cardinal symptoms:
Cough increases in frequency and severity
Sputum production increases in volume and/or changes character
Dyspnea increases
Constitutional symptoms, a decrease in pulmonary function, and tachypnea are variably present during an exacerbation, but the chest radiograph is usually unchanged. 2

16. Method/Bacterial Isolates
Klebsiella spp, Enterobacter spp, and E. coli isolated from sputum were saved as frozen stocks at -70˚C.

17. Methods/Molecular typing
Isolates of Klebsiella spp, Enterobacter spp, and E. coli were typed by Repetitive extragenic palindromic-Polymerase chain reaction (Rep-PCR).
Primers
REP-1R-I: 5’-III ICG ICA TCI GGC-3’
REP-2-I: 5’-ICGICT TAT CIG GCC TAC-3’
PCR conditions (30 Cycles)
Denaturation: 94C for 1min
Annealing: 60C for 1 min
Extension, 72C for 2 min

18. Methods/Molecular typing
Electrophoresis to see banding patterns.

19. Methods/Molecular typing
Each strain was categorized as preexisting or new on the basis of molecular typing.
Strains not previously isolated from a subject were designated as new, whereas strains isolated previously were designated as preexisting.

20. Result
Baseline Characteristics of the Patients

21. Result
5100 clinic visits were completed by 127 patients, of which 931(18.2%) were exacerbation visits.

22. Result Sputum culture result Pathogen Total isolates (%) New Strains/
Klebsiella spp
115 (2.3%)
51/115 (58.7%)
Enterobacter spp
114 (2.2%)
53/114 (60.4%)
E. coli
89 (1.7%)
19/89 (16.9%)

23. Result
REP-PCR of Klebsiella spp

24. Result
Time Line and Molecular Typing

25. Result
Association between new strain acquisition and exacerbation

26. Conclusion
New strain acquisition does not appear to be a major mechanism of exacerbations of COPD for Klebsiella spp, Enterobacter spp, and Escherichia coli .

27. A Case Vignette
A 68-year-old former heavy smoker with a history of chronic obstructive pulmonary disease (COPD) presents to the ED with a two-day history of worsened shortness of breath and increased sputum. Chest radiography shows hyperinflation and no acute infiltrates.

28. A Case Vignette House staff ordered sputum culture in ED.
Sputum culture came back positive for Klebsiella spp.
How should this patient be treated?

29. Clinical implication Sputum culture should not have been done.
The initial antibiotics regimen should target likely bacterial pathogens (H. influenzae, M. catarrhalis, and S. pneumoniae in most patients). 8

30. Reference
1. Tager I, Speizer FE. Role of infection in chronic bronchitis. NEJM 1975;292:
2. Global strategy for the diagnosis, management and prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2007.
3. Sethi S, Eschberger K, et al. Airway bacterial concentrations and exacerbations of COPD. Am J Respir Crit Care Med 2007;176:
4. Sethi S, Evans N, Grant BJB, Murphy TF. New strains of bacteria and exacerbations of COPDNEJM 2002:347:

31. Reference
5. 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;195:81-9.
6. Murphy TF, Brauer AL, Grant BJ, Sethi S. Moraxella catarrhalis in COPD: burden of disease and immune response. Am J Respir Crit Care Med 2005;172:195-9.
7. Murphy TF, Brauer AL, Eschberger K, et al. Pseudomonas aeruginosa in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;177:
8. Sethi S, Murphy TF. Infection in pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:

32. Acknowledgement
Dr. Sanjay Sethi

33. Thank you.