Presenter: Jiyeon Jung, MD Research Attending: Sanjay Sethi, MD

1. 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 radiographyshows 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 COPDexacerbation.

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 withmolecular 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(+) forH. influenza in stable phase Sputum culture(+) for H. influenza inexacerbation 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. 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. Background

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

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 radiographyshows 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 inchronic bronchitis. NEJM 1975;292:563-71. • 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;176:356-61. • 4. Sethi S, Evans N, Grant BJB, Murphy TF. New strains of bacteria and exacerbations of COPDNEJM 2002:347:465-71.

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;177:853-60. • 8. Sethi S, Murphy TF. Infection in pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2355-2365.

32. Acknowledgement • Dr. Sanjay Sethi

33. Thank you.