Most common bacterial agents in hospitalized patients with acute exacerbations of chronic obstructive pulmonary disease

Background/Aim. Infection is the major cause of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The aim of the study was to establish the most common bacterial agents of AE-COPD in patients hospitalized in a tertiary medical care institution. Methods. This retrospective study included the patients hospitalized due to infective AE-COPD in the Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica in a 12-month period. We evaluated clinical data, spirometry, pathogen etiology in the patients with positive sputum findings and disease outcome. Results. The study included a total of 81 patients, 47 (58.02%) males and 34 (41.97%) females of the mean age of 65.7 years. The mean history of the disease was 14.5 years. The median forced expiratory volume in one second (FEV1) was 30.12%. The most common isolated pathogens was Pseudomonas aeruginosa (n = 36; 38.29%), followed by Haemophilus influenzae (n = 25; 26.59%) and Streptococcus pneumoniae (n = 16; 17.02%). Coinfections were present in 9 (9.57%) of the patients. The median FEV1 values of 28.67%, 37.23%, and 42.26% were registered in the patients with Pseudomonas aeruginosa, Haemophilus influenza, and Streptococcus pneumonia induced infection, respectively. The case fatality rate (CFR) was 6.2%. Of the deceased, 100% had Pseudomonas aeruginosa-induced infection. A statistically significant difference in FEV1 values was registered between the patients with and without Pseudomonas aeruginosa in their sputum finding (p = 0.016). Conclusion. The most common pathogens in patients with AE-COPD were Pseudomonas aeruginosa, Haemophilus influenzae, and Streptococcus pneumonia. The CFR was 6.2%. All (100%) deceased patients had the infection induced by Pseudomonas aeruginosa.


Introduction
Chronic obstructive pulmonary disease (COPD) is a chronic progressive pulmonary disease which has a substantial morbidity and mortality 1,2 .Over three million people died of this disease in 2005.COPD was the sixth most frequent mortality cause in 1990.Furthermore, it was predicted to take the third top position among death causes by 2020 1 .With the disease progression, exacerbation occurs more frequently, inducing a faster lung function and life quality deterioration [3][4][5][6] .
The role of microorganisms, particularly bacteria, in COPD exacerbations has been in the focus of research for several decades.Over 70% of COPD exacerbations are due to respiratory infections, and bacterial infections are registered in more than 50% of the cases 2,4,[7][8][9] .Other exacerbation causes, including air pollution and temperature oscillations, are registered in 30% of the cases, 2, 10 while the causes of exacerbations remain obscure in even one third of the cases 4 .
The objective of this study was to identify the infectious agent of COPD exacerbations among the patients hospitalized in a tertiary medical care institution in one-year period.We analyzed if there existed a correlation between the bacterial agent and lung function (forced expiratory volume in one second -FEV 1 ), or the correlation between the bacterial agent and the treatment outcome, that is a fatal outcome of the disease.

Methods
This retrospective study, included the patients with acute exacerbation of COPD (AE-COPD) hospitalized in the Institute for Pulmonary Diseases of Vojvodina in 2012.The diagnosis was established in compliance with the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria, with postbronchodilation FEV1/FVC (FVC -forced vital capacity) of < 70%.Acute exacerbation was diagnosed if the usual daily symptoms changed in terms of deteriorated dyspnea, cough and sputum production, requiring the treatment change.
The following characteristics of all the patients included in the study were reviewed: demographic features, symptoms, smoking habits, disease outcome; they were all submitted to physical examination, spiroplethysmography, staging of the disease in compliance with the GOLD criteria, arterial blood gas analyses, complete blood count, fibrinogen measurement and chest radiography.It was also analyzed which patients required the non-invasive or invasive mechanical ventilation and who had a fatal outcome of their disease.The fatal outcome of the disease was analyzed in correlation to the FEV1 value and the presence of Pseudomonas aerugionsa in the sputum.
The patients with radiographycally verified pneumonia were excluded from the study.

Microbiology
Expectorated sputum specimens were collected into sterile containers and processed according to the standard procedures 11 .The microscopic sputum preparations were Gram stained, viewed under low magnification (100 ), and used to establish leucocyte and epithelial cell counts.The presence of ≥ 25 leucocytes and ≤ 10 epithelial cells in a single field of vision determined the specimen suitable for cultivation 12 .Sputum samples were sputolysine-homogenized, and then, using the calibrated 1 µL loop, cultivated in culture media: blood agar, chocolate agar, and MacConkey agar.Culture plates were then incubated at 37°C, the blood and chocolate agar in the presence of 5% CO 2 , and the MacConkey agar in aerobic conditions.The culture plates were examined 24 hours later in order to establish the clinically relevant pathogens.To isolate Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Staphylococcus aureus, blood and chocolate agar were used.MacConkey agar was used to isolate Pseudomonas species, Acinetobacter species and Enterobacteriaceae spp.Bacterial agents were identified by the conventional methods, in compliance with the standard procedures 13 .The bacterial growth was interpreted semiquantitatively, determining the number of Colony Forming Unit (CFU) in one milliliter of the sample.The growth of the isolate with ≥ 10 5 CFU/mL was considered diagnostically relevant 11 .

Statistical analysis
Statistical analysis was performed using descriptive statistics, absolute and relative frequencies, mean ± standard deviation (SD) values, χ2 test, Student t-test, and nonparametric test (Mann-Whitney test).

Results
During the examined 12-month period, the positive bacteriological sputum finding was registered in 81 patients treated in the Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica, with the total of 94 (27.6%) positive isolates of 340 examined sputum samples in total.Of these patients, there were 47 (58.02 %) males and 34 (41.97%)females.The mean age of the patients was 65.7 ± 8.48.The disease had a 14.5-year long mean history (SD = ± 8.75).Among the examined patients, 56 (69.1%) were active smokers, 17 (21.0%)were ex-smokers, and 8 (9.9%) were non-smokers.The patients' mean FEV 1 amounted to 30.12 ± 12.35%.Demographic features of the examined patients are presented in Table 1.
The patients' clinical features are presented in Table 2. Most patients (69; 85.2 %) had concurrent symptoms of dyspnea and productive cough, while in 12 (14.8%)patients the latter symptoms were accompanied with fever.The mean white blood count was 11.86 ± 5.02  10 9 /L.Four (4.9%) of the patients required non-invasive mechanical ventilation (NIV).Two (2.5%) of the patients were treated in the Semi-Intensive Care Unit and none in the Intensive Care Unit.The overall CFR amounted to 6.2%.
The most common pathogen was Pseudomonas aeruginosa isolated in 36 (38.29%) patients, followed by Haemophilus influenza isolated in 25 (26.59%) of the patients, and Streptococcus pneumoniae isolated in 16 (17.02%)patients.The mixed bacterial infection type was registered in 9 (9.57%) of the patients.The sputum bacteriology is presented in Table 3.    significant difference in FEV 1 values was registered between the patients who died and those who recovered (p = 0.307).A statistically significant difference in FEV 1 values was, however, registered between the patients with and without Pseudomonas aeruginosa isolated in their sputum samples (p = 0.016).

Disscusion
This retrospective one-year study, carried out in the Institute for Pulmonary Diseases of Vojvodina, analyzed demographic features, bacteriological sputum findings, clinical characteristics and disease outcome in the patients hospitalized for an acute exacerbation of the chronic obstructive pulmonary disease in 2012.
In our study, we found that 81 patients had positive sputum bacteriology, with the total of 94 positive isolates of 340 examined sputum samples in total, or 27.6% positive isolates of all the examined samples.In their report published in the Chest in 2007, Ko et al. 14 reported 32.3% positive sputum isolates of all the examined ones.In their article published in 2015, Ko et al. 15 also reported 37.8% of positive sputum findings, while Boixeda et al. 2 in their study in 2012 reported 24.1% of positive bacteriological sputum findings, correlating well to our results.
Pseudomonas aeruginosa was the most common bacterium isolated in our study, registered in 38.29% of the patients, followed by Haemophilus influenzae, isolated from 26.59% of the patients, and Streptococcus pnaumoniae, registered in 17.02% of the patients.In the study carried out by Ko et al. 15 in 2005, similar results were obtained for Haemophyllus influenzae, which was isolated in 23.1% of their cases, but not for Pseudomonas aeruginosa isolated in 6.3%, and Streprococcus pneumoniae isolated in only 4.0% of the cases, unlike our study results.Furthermore, Groenewegen and Wouters 16 reported Haemophilus influenzcae for the most common bacterium isolated in ther study (in 45% of the cases), followed by Streptococcus pneumoniae isolated in 27%, and Pseudomonas aeruginosa registered in 15% of their cases, deviating from the results obtained in our study.Our results also differ from those obtained in the study carried out in Taiwan by Lin et al. 5 in 2007, who isolated Klebsiela pneumoniae most frequently (19.6%), then Pseudomonas aeruginosa (16.8%), and Haemophilus influenzae (7.5%).
In our study, Haemophilus influenzae was more frequently isolated in the patients with FEV 1 > 30%, while Ko et al. 15 in their study carried out in 2005 isolated this bacterium more frequently in the patients with FEV 1 > 50%, correlating to the results reported by Lin et al. 5 In our study, Pseudomonas aeruginosa was more frequently isolated in the patients with FEV 1 < 30%, as it was the case in the study by Ko et al. 14 performed in 2007.In their study carried out in 2009, Larsen et al. 6 reported the patients with FEV 1 < 1.0 L had Pseudomonas aeruginosa-induced infections more frequently.Lin et.al. 5 also reported Pseudomonas aeruginosa was more frequently isolated in very severe COPD cases, while Groenewegen et al. 16 reported the positive sputum bacteriology was more frequently registered in the patients with lower FEV 1 in their study, but with no significant difference in the isolated bacterial species related to the severity of the disease.
All the deceased patients in our study had Pseudomonas aeruginosa-induced infection.Lin et al. 5 , applying the multivariate regression analysis in their study, have established the finding of Pseudomonas aeruginosa for one of independent case fatality risk factors, quite unlike Larsen et al. 6 who reported the presence of this bacterium in the sputum had no influence on the CFR, which was primarily affected by the patients' age and low FEV 1 .In our study, we registered no statistically significant difference in FEV 1 levels between the groups of deceased and recuperated patients (p = 0.307).However, the statistically significant difference in FEV 1 was established between the patients with and without the sputum finding of Pseudomonas aeruginosa (p = 0.016).
The major limitation of our investigation is that it included only bacteriological sputum findings, lacking the opportunity to perform virology analyses.Another limitation is that it provided no information on bacterial agents' sensitivity to antibiotics.In addition, our analysis included only hospital-treated exacerbations, lacking the insight into out-patient treated exacerbations.We will try to overcome these limitations in our additional future research work.

Conclusion
The most common bacterial agents isolated in our AE-COPD patients were Pseudomonas aeruginosa, Haemophilus influenzae, and Streptococcus pneumoniae.All the deceased patients had the infection induced by Pseudomonas aeruginosa.The patients with advanced disease stages had Pseudomonas aeruginosa sputum finding more frequently.

Table 3 Sputum bacteriology in AE-COPD patients, n = 81
FEV 1 findings were analyzed in correlation to the sputum bacteriology.The patients with isolated Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae had the mean FEV 1 values of 28.67%, 37.23%, and 42.26%, respectively.The correlation of sputum bacteriology and FEV 1 values are reviewed in Table4.The CFR was 6.2%.Of the deceased, 100% had the infection induced by Pseudomonas aeruginosa.No statistically Vol.74, No. 3