Bacterial profile and antibiotic susceptibility patterns of acute exacerbation of chronic obstructive pulmonary disease in Assiut University Hospitals, Upper Egypt; a one-year prospective study.

The majority of chronic obstructive pulmonary disease exacerbations are caused by infections of the tracheobronchial tree. Previous data on bacterial exacerbations of COPD in Upper Egypt are limited. Hence, this study was conducted for the identification of the causative bacteria in exacerbations of COPD, and to illustrate their antimicrobial susceptibility patterns at Assiut University Hospitals, Upper Egypt. A total of 116 COPD patients who underwent 167 infection exacerbation attacks participated in this prospective study during 2013. Significant bacterial growth was found in 143 (86%) out of the 167 exacerbation attacks. The most common detected bacteria Original Research Article Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 289 were Haemophilus influenzae (19.4%), Escherichia coli (18%), Streptococcus pneumoniae (16.7%), Klebsiella pneumoniae (14%), Streptococcus pyogenes (10%), Pseudomonas aeruginosa (5.6%), methicillin resistant Staphylococcus aureus (5.6%), Acinetobacter baumannii (4.2%), and Moraxella catarrhalis (2.8%). The majority of the isolated strains showed high resistance rates to most groups of antibiotics where 91 (63%) of the isolated strains were multidrug resistant, 37 (26%) strains were extreme drug resistant and 16 (11%) bacterial strains were pandrug resistant. High resistance rates were observed against penicillins and cephalosporins. Moderate resistance rates were detected against the fluoroquinolones. High susceptibilities were detected to the carbapenem group. All the isolated Gram-positive bacteria were sensitive to linezolid.


INTRODUCTION
Exacerbation of chronic obstructive pulmonary disease (COPD) is defined as a sustained worsening of the patient's condition from the stable state and beyond normal day-to-day variations that is acute in onset and may warrant additional treatment in a patient with underlying COPD [1]. COPD exacerbations increase the rate of hospitalization and mortality and decrease the quality of life. The economic and social burden of AECOPD is extremely high. It is estimated that almost 35-45% of the total per capita health-care costs for COPD attributed to exacerbations alone [2]. Especially that more than half of the patients often require readmission in the subsequent period [3]. The majority of COPD exacerbations are caused by infections of the tracheobronchial tree [4]. A key characteristic of airway inflammation in COPD is the persistent presence of bacteria in the lower airways. The most commonly isolated bacteria in the lower respiratory tract of COPD patients were Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, with growing evidence of the significance of Pseudomonas aeruginosa infections in severe COPD disease [5]. Congestive heart failure, systemic infections, pulmonary embolism, pneumonia, air pollution, cold air, allergies, and smoking associated with 20-40% of COPD exacerbations [6]. People with moderate COPD have one exacerbation per year on average; those with severe COPD have two. However, these averages mask wide heterogeneity: many patients with COPD have exacerbations never or very infrequently; a few experience them almost every month [7]. Patients who experience frequent exacerbations may present an accelerating rate of lung function decline. Thus, the management of exacerbations by prompt diagnosis and effective treatment should be a major goal in COPD [8]. Previous data on infection exacerbations of COPD in Upper Egypt are limited. Hence, this study was conducted for the identification of the causative bacteria in acute exacerbation of COPD (AECOPD), and to illustrate their antimicrobial susceptibility patterns at Assiut University Hospitals, Upper Egypt.

Study Design and Population
COPD patients admitted at the Chest Department, Assiut University Hospitals, Upper Egypt who met the Global Initiative for Obstructive Lung Disease (GOLD) guidelines [9] and experienced one or more exacerbation attacks during the period between January 2013 and December 2013 were invited to participate in this prospective study. Tuberculous patients were excluded from the study. All participants signed the informed consent form that approved by the Institutional Ethics Committee. Questionnaires with demographic and clinical data were fulfilled. Smoking index was calculated as the product of tobacco use (in years) and the average number of cigarettes smoked per day/20 (1 pack has 20 cigarettes) [10].

Clinical Assessment
Patients underwent thorough clinical examination and pulmonary function tests that included spirometry, peripheral oxygen saturation, and chest X-ray. Forced expiratory volume in the first second (FEV 1 ) and forced vital capacity (FVC) were obtained from the flow-volume curve using a spirometer (Zan 300, Sensor Medics MGA USB, Germany). Static lung volumes were measured by closed-circuit helium dilution method. The reference values used were those of the American Thoracic Society standards before and 20 minutes after β-agonist (fenoterol 400 mcg) inhalation. The highest value of at least three measurements was selected and expressed as a percentage of reference values [9].

Laboratory Tests
Venous blood samples were obtained from patients for performing relevant chemical investigations; blood glucose level, liver function tests, kidney function tests, complete blood count, erythrocyte sedimentation rate (ESR). Arterial blood samples were obtained for measurement of blood gases.

Bacteriological Diagnosis
Valid early-morning sputum samples were collected into sterile cups from patients through effective coughing sometimes assisted by physiotherapy to obtain lung secretions as described previously [11]. Samples were transported directly to the Microbiology and Immunology Department, Faculty of Medicine, Assiut University where the bacteriological analyses were performed.

Antibiotic Susceptibility Testing
Susceptibilities of the isolated bacterial strains were determined to penicillins (amoxicillin and amoxicillin / clavulanic acid), phenicols (chloramphenicol), cephalosporins (ceftriaxone, cefepime, ciprofloxacin, cefaclor, ceprodoxime, and cefotaxime), fluoroquinolones (levofloxacin, ofloxacin, and lomefloxacin), and tetracyclines (doxycycline) (Bioanalyse, Turkey). In addition, susceptibilities of Gram-positive bacterial strains were tested against other penicillins (penicillin, oxacillin, methicillin, and carbencillin), polypeptides (bacitracin), macrolides (erythromycin), glycopeptides (vancomycin and teicoplanin), and oxazolidinones (linezolid). Susceptibilities of Gram-negative stains were tested also against aminoglycosides (gentamicin, amikacin, tobramycin, and neomycin), carbapenems (imipenem and meropenem), and monobactams (aztreonam). The test was performed using the disk diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines [13]. The results were interpreted as susceptible (S), intermediate (I), or resistant (R). Multidrugresistant (MDR) bacteria was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, extensively drug-resistant (XDR) bacteria was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories, and pandrug-resistant (PDR) bacteria was defined as non-susceptibility to all agents in all antimicrobial categories [14].

Bacteriological Analysis
Significant bacterial growth was found in 88 (76%) out of the 116 COPD patients during 143 (86%) out of the 167 exacerbation attacks either single (127 attacks; 89%) or mixed infections (16 attacks; 11%). In 18 (24%) patients (24 attacks; 14%), no significant bacterial growth was found (Table 1). A total of 144 bacterial strains were isolated in exacerbations of COPD either solely (113 strains) or mixed (31 strains). The distribution of bacterial isolates in different COPD stages is shown in Fig. 2 (Fig. 2). Thus, Gram-negative bacilli were detected in 82(49%) attacks. COPD patients infected with either M. catarrahlis or Staph. epidermidis were ≥ 60 years old with associated cardiopulmonary and/or systemic co-morbidities. Optochinsensitive Strep. pneumoniae strains were 17 (71%) in number, while seven (29%) strains were optochin-resistant. There was no significant association between the types of bacterial isolates or the optochin-sensitivity patterns of Strep. pneumoniae strains and the severity of COPD.

Antibiotic Susceptibility Patterns
High resistance rates were observed among the isolated bacterial strains against most groups of antibiotics where, 91 (63%) of the isolated strains were MDR, 47 (33%) strains were XDR and 6 (4%) bacterial strains were PDR (Table 2). Most isolates were resistant to amoxicillin, amoxicillin / clavulanic acid, cephalosporins (with exception to ciprofloxacin), ofloxacin, and lomefloxacin. About half the isolates were resistant to chloramphenicol, ciprofloxacin, levofloxacin, and doxycycline ( Table 2). Among the Gram-positive bacteria, resistance rates were highest against the penicillin group and erythromycin. Resistance to bacitracin, vancomycin, and teicoplanin ranged from 58-64%. All the isolated Grampositive bacteria were sensitive to linezolid ( Table 2). For Gram-negative isolates, the resistance rates to the aminoglycosides group ranged from high level to tobramycin and gentamicin to a slightly lower level (44%) to amikacin. Resistance rate was also high (81%) to aztreonam. Only few isolates (8.5%) showed resistance to the carbapenem group that belonged to H. influenzae, E. coli, Ps. aeruginosa, and M. catarrahlis strains (  (Table 2). No statistically significant difference was found between different antibiotic resistance patterns in the duration of hospital stay. The patterns of antibiotic resistance in different COPD stages are shown in Fig. 3. Death rates among MDR, XDR, and PDR infected patients were 2%, 6%, and 67%, respectively. The death rate was significantly higher for patients infected by PDR bacteria than those infected by XDR or MDR bacteria (Fisher's exact test; P=.000). On the other hand, there was no significant difference between different COPD stages regarding the antibiotic resistance patterns. For COPD cases (18 patients; 24%) with no significant bacterial growth, 17 patients had recovered and discharged while one patient died that had underlying pulmonary embolism.   Table 2. Resistance patterns of isolated bacterial strains to antimicrobial agents PDR 2(7) 4(15) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 6(4)

DISCUSSION
This study aimed to diagnose the spectrum of bacterial pathogens associated with AECOPD and their antimicrobial susceptibility patterns during the year 2013. The study included 116 AECOPD patients that admitted during the study period at Assiut University Hospitals, Upper Egypt. Most of the participated patients were old aged, suffered severe or very severe COPD, had associated cardiopulmonary or systemic comorbidities, and were critically ill that required admission at the ICU. Older age is a predictive factor for increased hospitalizations in COPD due to the higher degree of disability and co-morbidity in the older population [15]. The co-morbid conditions can trigger AECOPD and their presence is a predictor of poor clinical outcome [16]. The close association between COPD and cardiovascular diseases had been established during the last 15 years. It is estimated that the diagnosis of COPD increases the risk of cardiovascular disease by an OR of 2.7 [17]. Anemia and hypoalbuminemia that have been detected in our patients reflected the underlying nutritional status, and increased ESR and leucocytosis highlighted the underlying inflammatory process in those patients that are often observed during COPD exacerbations [18] and affect the clinical outcome of the disease [19]. Our patients were mostly smokers which reflected the effect of current smoking as a risk factor for severe exacerbations. Smoking perpetuates an ongoing inflammatory response that leads to airway narrowing and hyperactivity so patients become more prone to infection exacerbation attacks [20]. From a public health perspective, smoking cessation is the single most effective therapy for COPD and is associated with a decrease in symptoms, and improved health status [21]. Also we found that some ex-smokers had experienced exacerbation attacks which imply that smoking cessation was too late and the disease progression continued even after smoking cessation.
Almost all patients in this study had hypoxia and mostly had hypercapnea. Chronic hypoxia and hypercapnia were responsible for the pathogenesis of COPD [22] and hypercapnea is an independent risk factor for AECOPD and represents a marker of disease severity [23]. In our study, the duration of hospital stay was significantly longer in patients with severe and very severe COPD versus those with mild or moderate disease which corresponds to previous reports [24,25]. Shorter durations of exacerbations were a predictor of success of treatment while longer durations were a predictor of need for ventilatory support and poor outcome of the disease [3]. Most of our patients were under steroid therapy. Corticosteroids were routinely described in AECOPD patients as they reduce the airway inflammation [26]. However, they may be associated with adverse effects like fluid retention, hypertension, diabetes mellitus, and osteoporosis. Therefore, their use in AECOPD must be balanced against adverse effects [27]. In this work, the peak of exacerbation attacks occurred during January and a large proportion occurred during November. AECOPD attacks occurred mainly during winter months compared with summer [28]. A previous global study of exacerbation seasonability demonstrated that in the Northern Hemisphere, about 9% of patients had exacerbations between December and February compared to 5% in June to August with 80% winter excess in exacerbations, whereas in the southern hemisphere, 12% of patients had exacerbations in their winter compared to 7% in summer with 71% winter excess. A higher proportion of patients in the southern region reported an exacerbation in any seasonally adjusted month compared with the northern region [29]. In another study, it was found that the mean monthly exacerbation rates during winter were 2.16 fold higher than during summer [30]. In our work, a large proportion of AECOPD also occurred during April and May. This can be explained by the relatively constant temperatures in Egypt with a mean of ≥18ºC all year round.
Bacterial infections are generally considered to be the most common cause of AECOPD [31]. Previous studies have shown that approximately one third of COPD patients are colonized at any time [32]. In this work, Gram-negative bacilli were detected in about half of AECOPD. Gramnegative bacilli were also the predominant organisms in the study done by Siripataravanit et al. [33] in Thailand. Gram-negative bacilli and Enterobacteriaceae were the most common isolated bacteria in cases of AECOPD also in another study in China [34]. A change in the microbial pathogens seen during AECOPD from the usual pathogens to Gram-negative bacteria is in parallel with the deterioration of the patient´s lung function [31]. Our study population included admitted cases of AECOPD where there is deterioration of their lung function, hence, Gramnegative bacteria were most commonly isolated.
H. influenzae was the most common bacteria detected in our study. This is in correspondence . E. coli strains were the second common organism isolated in AECOPD in this study. In a previous study in Germany [42], E coli were the most common organism isolated in cases of AECOPD. Strep. pneumoniae strains were detected in 16.7% of AECOPD in this work. A previous documentation has shown that airway colonization with S. pneumoniae increases the risk of a first COPD exacerbation [43]. Also Sethi et al. [44], showed a significant increase in exacerbations when S. pneumoniae was isolated. About 29% of the isolated S. pneumoniae strains in this study were optochin-resistant. Optochin-resistant S. pneumoniae strains were first described in 1987 [45] and since then, their incidence in clinical sources increased steadily during the last decade [46]. Similar to our findings, H. influenzae and S. pneumoniae were the most prevalent organisms isolated in AECOPD previously in Pakistan [47], Germany [48], and the Netherlands [49]. Kl. pneumoniae were detected in 14% in AECOPD attacks in this study. This detection rate is higher than that detected previously [50,51]. A substantial number of our patients had P. aeruginosa with a percentage (5.6%) similar to previous report that was 6.3% [36]. On the other hand, our percentage was lower than other reports with a prevalence rate of 15% [49,52]. MRSA were detected in 5.6% of the participated patients. COPD was considered as an independent factor in the isolation of MRSA in ICU [53]. Acin. baumannii strains were found in 4.2% of AECOPD in our work. This isolation rate was comparable to previous studies in Bosnia and Herzegovina [50] and in Taiwan [54]. While Acin. baumannii is a major pathogen in nosocomial infections, community-acquired acinetobacter infections are of an increasingly concern because they mainly affect patients with certain co-morbidities such as COPD [55]. In our findings, the percent of detection of M. catarrhalis was 2.8%. Both of Acin. baumannii and M. catarrhalis were previously detected in cases of AECOPD in Bangladesh [56]. Our COPD patients that were infected by M. catarrhalis were old aged with associated co-morbidities. Wright et al. [57] found that the majority of respiratory isolates containing M. catarrhalis are from elderly patients with underlying cardiopulmonary diseases. Adherence of M. catarrhalis to epithelial cells increases in elderly patients [58]. M. catarrhalis has emerged as a main pathogen over the last two to three decades in patients with chronic obstructive pulmonary disease (COPD) [59]. In contrast to a previously reported data [60] that found a relationship between the severity of COPD and the type of isolated bacterial strains, our results found no significant association between the type of bacterial isolates and severity of COPD. This difference may be due to different demographic data and the small sample size of our study. A larger sample size is required to prove these findings.
To obtain high susceptibilities to antimicrobial agents, we tested the susceptibilities of the isolated bacterial strains to major groups of antibiotics that have effect against both Gramnegative and -positive bacteria. Our findings demonstrated high resistance rates among the isolated bacterial strains to different groups of antibiotics. Resistance was at the highest level to amoxicillin followed by the cephalosporins group (with exception to ciprofloxacin). This was similar to a previous study in Egypt [35]. Our data proved that the fluoroquinolones cannot be considered as the first option for treatment of AECOPD as recommended in previous reports [61,62] as their frequent usage can lead to the emergence of resistant strains that have been demonstrated in our results. About half of the bacterial isolates in our study were sensitive to chloramphenicol, ciprofloxacin, levofloxacin, and doxycycline. A similar rate of sensitivity to ciprofloxacin was also observed in a previous data from India [31]. Previous studies evidenced the high bacteriological eradication rate in AECOPD patients when treated by levofloxacin [63]. Levofloxacin appears to be only marginally affected in term of resistance rate [64]. In comparison to other members of the aminoglycoside group used in this study, resistance rate to amikacin was slightly lower among the Gram-negative isolates. This is similar to previous reports from Egypt [35,51]. Sensitivity of our Gram-negative bacteria was at the highest level to the carbapenem group which was similar to previous studies from Egypt [51] and China [65]. Nevertheless, some isolates (8.5%) showed resistance to that group. Resistance to imipenem was reported also previously in cases of AECOPD in coal workers [66]. In our study, as with previous findings [67,68,69], E. coli demonstrated a very high microbial resistance to antibiotics where 15% of the isolated E. coli strains were PDR. The majority of Gram-positive isolates in this study were resistant to penicillin which was similar to other findings [38]. A high resistance rate was also detected in Strep. pneumoniae strains in this study to penicillin. In recent years, resistance to penicillin increased rapidly among Strep. pneumoniae strains [60]. All Staph. aureus strains in our COPD patients were oxacillinresistant which was similar to a previous report in India [70], while methicillin-resistance was higher than the rates in previous reports [60,71,72]. All the isolated Gram-positive bacteria in this work were sensitive to linezolid, the first commercially available oxazolidinone antibiotic. Similarly, linezolid was active against Gram-positive isolates in previous studies in United Kingdom [73,74]. In this study, MDR bacteria were isolated in a rate of 63% which is higher than previous reports [51,75]. Isolation of MDR bacteria in cases of respiratory infections was recorded in previous studies from Vietnam [76], China [77,78], and Thailand [79]. Emergence of resistance to multiple antimicrobial agents in pathogenic bacteria has become a significant public health threat as there are fewer, or even sometimes no, effective antimicrobial agents available for infections caused by these bacteria. Gram-positive and -negative bacteria are both affected by the emergence and rise of antimicrobial resistance [14]. The situation is compounded by cross-resistance within and between classes of antibacterial agents, which further limits treatment options [80].