New Strain Acquisition of Klebsiella spp, Enterobacter spp, and Escherichia coli in COPD is Not Associated with Exacerbati

ons.

Presenter: Jiyeon Jung, MD

Research Attending: Sanjay Sethi, MD

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.

A Case Vignette

• House staff ordered sputum culture in ED.

• Sputum culture came back positive for Klebsiella spp.

• How should this patient be treated?

History

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

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

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.

History

• Bacterial infections trigger COPD exacerbations.

• Four Major pathogens.

• A new bacterial strain plays a central role in the pathogenesis of an Exacerbation.

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.

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

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

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.

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

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.

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

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

Method/Bacterial Isolates

• Klebsiella spp, Enterobacter spp, and E. coli isolated from sputum were saved as frozen stocks at -70˚C.

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

Methods/Molecular typing

• Electrophoresis to see banding patterns.

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.

Result

• Baseline Characteristics of the PatientsAge (mean SD) 66.9 9.48

Sex (n) Male: 125 Female: 2

Race (n) Caucasian: 107African-American: 20

Years since diagnosis (mean SD) 10.5 11.2

Smoking status on enrollment (n) No: 85 Current smokers: 42

Smoking pack-years (mean SD) 79.8 41.1

FEV1 in liters (mean SD) 1.57 0.69

FEV1 % predicted (mean SD) 46.3 17.9 (15 - 99)

GOLD severity (number of subjects) 0 =5, 1 =1, 2 =45, 3 = 49, 4 = 27

Age (mean SD) 66.9 9.48

Sex (n) Male: 125 Female: 2

Race (n) Caucasian: 107African-American: 20

Years since diagnosis (mean SD) 10.5 11.2

Smoking status on enrollment (n) No: 85 Current smokers: 42

Smoking pack-years (mean SD) 79.8 41.1

FEV1 in liters (mean SD) 1.57 0.69

FEV1 % predicted (mean SD) 46.3 17.9 (15 - 99)

GOLD severity (number of subjects) 0 =5, 1 =1, 2 =45, 3 = 49, 4 = 27

Result

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

Result

• Sputum culture result

Pathogen Total isolates

(%)

New Strains/

Total isolates

(%)

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%)

Result

• REP-PCR of Klebsiella spp

Result

• Time Line and Molecular Typing

Result

• Association between new strain acquisition and exacerbation Pathogen Total

isolates (%)

New strains/ total isolates (%)

Proportion of exacerbations in visits with new strain (%)

Proportion of exacerbations in visits without new strain (%)

p value (GEE)

Klebsiella spp

115 (2.3)

51/115 (58.7)

8/51 (15.7) 912/5049 (18.1) 0.73

Enterobacter spp

114 (2.2)

53/114 (60.4)

12/53 (22.6) 919/5047 (18.2) 0.41

E. coli 89 (1.7)

19/89 (16.9)

3/19 (15.8) 928/5081 (18.3) 0.78

All 3 species 23/115( 26.5) 908/4985 (18.2) 0.62

Conclusion

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

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.

A Case Vignette

• House staff ordered sputum culture in ED.

• Sputum culture came back positive for Klebsiella spp.

• How should this patient be treated?

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

Reference

• 1. Tager I, Speizer FE. Role of infection in chronic 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.

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.

Acknowledgement

• Dr. Sanjay Sethi

Thank you.