Survey
* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project
Download Community-Acquired Pneumonia Caused By
Document related concepts
Transcript
Egyptian Journal of Medical Microbiology, October 2013 Vol. 22, No. 4 Serotyping and Antimicrobial Resistance Pattern of Streptococcus Pneumoniae Strains in Patients with Community-acquired Pneumonia Mona Sallam Embarek Mohamed1, Alaa Thabet Hassan2 1 Department of Microbiology and Immunology; 2 Department of Chest Diseases, Faculty of Medicine, Assiut University, Assiut, Egypt ABSTRACT Community-acquired pneumonia is a common disease and a frequent cause of morbidity and mortality worldwide. Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. The current study was conducted to determine the serotype distribution and antimicrobial susceptibility patterns of Streptococcus pneumoniae isolated from patients with community-acquired pneumonia at Assiut University Hospitals. From February 2013 to May 2013, sputum samples from 60 adult patients with community-acquired pneumonia were analyzed for bacterial etiology using conventional methods. Antimicrobial susceptibility and serotyping of Streptococcus pneumoniae was performed. bacterial agents were detected in 53 patients (88%). Streptococcus pneumoniae was the most common (30%) isolated bacteria. Eight co-infections were identified. The detected pneumococcal-serotypes were in decreasing order; 1, 9V, 6B, 19F, 23F, 14, and 19A. Pneumococcal-antibiotic resistance was highest for penicillin and ampicillin antibiotics. Streptococcus pneumoniae is the most common isolated bacteria in cases of community-acquired pneumonia which is associated with certain serotypes. Resistance to penicillin and other antimicrobial agents increased rapidly during the last years among pneumococcal strains worldwide. Keywords: Streptococcus pneumoniae, community-acquired pneumonia, serotyping, antimicrobial resistance. the cause of CAP in some patients (those who have had previous antimicrobial treatment or who have pulmonary co-morbidities)(7). Virulence of S. pneumoniae is mainly associated with the presence of capsular polysaccharides, which usually exhibit differences in size, composition, antiphagocytic properties and serotype specific immunogenicity. There are more than 90 pneumococcal serotypes, but less than a dozen are responsible for most of the infections(9). Many different serotypes of S. pneumoniae are capable of causing respiratory infection(10). The dominant serotypes associated with CAP worldwide include 14, 4, 1, 6A, 6B, 3, 8, 7F, 23F, 18C, 19F, and 9V. The distribution of serotypes differs among geographic regions. Serotypes 1 and 5 are common in developing countries, but are uncommon in the United States and Europe(11). A main problem is that CAP is caused by antimicrobial drug-resistant microbes(12). Although penicillin has long been the mainstay of treatment of pneumococcal infections, Streptococcus pneumoniae strains with decreased susceptibility to penicillin have become increasingly prevalent over the past 30 years and are now a serious problem worldwide. In addition, an increase in the prevalence of pneumococci resistant to macrolides has been INTRODUCTION Community-acquired pneumonia (CAP) is one of the most common acute infections requiring admission to hospital(1). The annual incidence of CAP varies from 5–11 per 1,000 population with the rates being higher in the elderly(2). The British Thoracic Society (BTS) guidelines for the management of CAP in Adults(3) has defined CAP as an acute illness with symptoms and signs of an acute lower respiratory tract infection associated with new radiographic shadowing. Typical symptoms of pneumonia include cough, pleuritic chest pain, and fever(4). The dominant risk factors for CAP are age, smoking and co-morbidities(1). A variety of pathogens are known to cause CAP that differ by region and country(5), yet, Streptococcus pneumoniae (pneumococcus) is the most common cause of CAP in adults(6) which accounts for about two-thirds of all cases of bacteraemic pneumonia(7). It is a major cause of morbidity and mortality among people all over the world(8). Other causative agents include Haemophilus influenzae, Mycoplasma pneumoniae, enteric gram-negative bacteria (enterobacteriaceae), Pseudomonas aeruginosa, Staphylococcus aureus, anaerobes, and respiratory viruses. Gram-negative bacilli (Enterobacteriaceae and pseudomonadas) are 69 Egyptian Journal of Medical Microbiology, October 2013 observed in Europe over recent years(13). Because infections caused by resistant pathogens are associated with higher morbidity and mortality than those caused by susceptible pathogens, the global impact of increasing resistance among CAP-organisms is a major concern(13). The incidence of CAP and its common complications, such as the requirement for intensive care and complicated para-pneumonic effusions, are increasing, making it essential for all physicians to have a good understanding of the management of CAP(1). Vol. 22, No. 4 collected into wide-mouthed sterile screwcapped cups that contained phosphate buffered saline (PBS) under complete aseptic conditions and transported directly to the laboratory at the Microbiology and Immunology Department, Faculty of Medicine, Assiut University. Identification of Streptococcus pneumoniae and other bacterial strains Samples were examined microscopically after staining with Gram´s stain and cultured directly on nutrient, blood, chocolate, mannitol salt, MacConkey´s, and Eosin Methylene Blue (EMB) agar plates. The chocolate and blood agar plates were incubated at 35–36°C with 5% CO2 for 24 hours for isolation of Streptococcus pneumoniae and Streptococcus pyogenes strains, respectively. Other plates were incubated aerobically at 37°C for 24-48 hours. The valid sputum culture defined as that had quantitative culture ≥105CFU/ml(16). Isolation of anaerobes was not considered. Bacterial isolates were identified based on colonial morphology, Gram staining, and standard biochemical reactions(17). For pneumococcal isolates, the catalase test, bile solubility test, and susceptibility to ethylhydrocupreine hydrochloride (optochin) were performed(17). Bile solubility testing The tube bile solubility test was performed by incubating the isolates overnight in tryptic soy broth and, after centrifugation, adding phosphate- buffered saline (pH 7.0) to the pellet to make a suspension with a final density of a McFarland standard of >1.5. Three to four drops of Na-taurocholate and Na-glycocholate (10%; Oxoid Ltd., United Kingdom) were then added to the suspension, which was carefully mixed. The tubes were incubated at 35°C for 3 h until analysis. Isolates were considered bile soluble if the suspension was clear and negative when opaque. Optochin susceptibility test The optochin susceptibility test was performed by the incubation of the isolate overnight on chocolate agar plates with optochin tablets (Rosco Diagnostica, Denmark) in CO2 and O2 atmospheres. Optochin susceptibility and resistance were defined as zones of inhibition of ≥18mm and <16mm (upon CO2 incubation) or ≥20mm and <18mm (upon O2 incubation), respectively. Serotyping Serotyping was performed by latex agglutination using Pneumotest kit containing 12 pool antisera (A-F + H, P-T) (Statens Serum Institut, Copenhagen, Denmark, Catalogue No. MATERIALS & METHODS Study design A prospective study was carried out at Assiut University Hospitals, Assiut, over a fourmonths period from February 2013 to May 2013; aiming to determine the antimicrobial susceptibility and serotype distributions of Streptococcus pneumoniae causing CAP among adult population. The study was approved by the medical ethical committee at the Faculty of Medicine, Assiut University, and oral consents were taken from all subjects prior to sample collection. Study population Adults with community-acquired pneumonia who attended the Chest Department were eligible for the study. Subjects who were receiving antibiotics were excluded. Pneumonia was defined by signs and symptoms suggestive of lower respiratory tract infection together with chest radiographic findings consistent with pneumonia as determined initially by the clinical physician. Questionnaires were fulfilled that included demographic and clinical data; age, gender, occupation, symptoms, admission, and associated risk factors (e.g. smoking, immunosuppressive condition). Smoking history was calculated as number of pack/year = number of cigarettes smoked per day × number of years smoked/20 (1 pack has 20 cigarettes)(14). Patients underwent thorough clinical examination, chest x-ray, and pulmonary function tests. Sample collection Valid sputum samples were collected from 60 patients with CAP through effective coughing sometimes assisted by physiotherapy to obtain lung secretions as described previously (15). Frothy saliva and secretions from pharynx were discarded and the patient was asked to produce another specimen. Samples were 70 Egyptian Journal of Medical Microbiology, October 2013 36158) according to manufacturer´s instructions. Equal quantities of antiserum and bacterial culture are mixed on a slide. A positive reaction is indicated when the capsule enclosing the pneumococcus swells and becomes visible (positive Quellung reaction). Antibiotic susceptibility testing Susceptibilities of pneumococcal isolates to amoxicillin/clavulanic acid, ampicillin, azithromycin, bacitracin, ceftriaxone, clindamycin, erythromycin, levofloxacin, nalidixic acid, and penicillin (Bioanalyse, Turkey) were determined. The test was performed using the disk diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines (18). The results were interpreted as susceptible (S), intermediate (I), or resistant (R). Multidrugresistant S pneumoniae (MDRSP) is defined as resistance to more than two different classes of antibiotics. 2.6. Statistical analysis The SPSS program version 16.0 was used for statistical analysis of data. Categorical variables were compared using Chi-square test and a P value <0.05 was considered statistically significant. Vol. 22, No. 4 Streptococcus pneumoniae was the most common isolated bacterial strain that was found in 18 (34%) patients of the study population (P=0.005) (Figure 2). Of the positive patients, 13 (24.5%) were infected by Klebsiella pneumoniae, 10 (19%) by Escherichia coli (E coli), six (11%) by Streptococcus pyogenes, three (5.7%) by Staphylococcus epidermidis, two (3.8%) by Staphylococcus aureus, and one patient (1.9%) infected by Pseudomonas aeruginosa (Figure 1). In total, eight co-infections were identified (13%): four co-infections with Streptococcus pneumoniae and Staphylococcus epidermidis, two co-infections with Streptococcus pneumoniae and Staphylococcus aureus, and two co-infections with Klebsiella pneumoniae and E coli. Most pneumococcal-cases (50%) were detected during February (P=0.005), four cases (22%) were detected during May, while three (17%) and two (11%) cases were detected during March and April, respectively (Figure 2). Characteristics of pneumococcal-positive patients Among the eighteen pneumococcal-cases, 15 (83%) were males and three (17%) were females (Table 2) with a mean age of about 50 years. Most (13) pneumococcal-cases were admitted at the Chest Department, while five cases admitted at the ICU. Five (27.8%) of pneumococcal-cases had the smoking index >30, five (27.8%) had the smoking index 20-30, one case (5.5%) had the smoking index of 1020, two cases (11%) had the smoking index <10, one case was ex-smoker that had a previous smoking index of 18, while four cases (22%) were non-smokers (Table 2). The most (61%) anatomical pneumonictype significantly associated with pneumococcal infection was lobar pneumonia that was detected in 11 cases (P<0.001). Left lower lobe pneumonia was found in eight cases while right sided pneumonia was found in three cases. Bronchopneumonia, interstitial pneumonia, and multilobar pneumonia were detected in four, two, and one cases respectively (Table 2). Twelve cases (66.7%) suffered from associated underlying diseases. Lung collapse was found in three cases with two of them had pleural effusion in addition. Respiratory failure was detected in two cases. Other underlying diseases (e.g. lung cancer, hydropneumothorax, DM, liver cirrohsis, cardiac ischemia, cardiomyopathy, and lung cavitation) were found in one case each (Table 2). RESULTS Study population From February 2013 to May 2013, a total of 60 patients (51 males and 9 females) with community-acquired pneumonia were prospectively enrolled in this study, most (49 patients) of them admitted at the Chest department. Almost all patients (86.7%) were residents of Assiut Province (Table 1). The mean age of patients was 46.2 years. Twenty three (about 38%) patients had the smoking index >30 (P<0.01), and 15 (25%) patients had the smoking index 20-30. All females enrolled in the study in addition to three males were non-smokers (Table 1). Of the 60 patients, lobar pneumonia was the most detected anatomical type in 32 cases (53%) (P< 0.005), 19 (31.7%) had bronchopneumonia, six (10%) had interstitial pneumonia, and three cases (5%) had multilobar pneumonia. Seven patients (11.7%) suffered from pleural effusion and two patients (3%) showed cavitations (Table 1). Detection of Streptococcus pneumoniae and other bacterial strains At least, a sole bacteriological agent was detected in 53 patients (88%). No bacterial isolates were detected in seven (12%) patients. 71 Egyptian Journal of Medical Microbiology, October 2013 Vol. 22, No. 4 pneumoniae were detected in nine (50%) cases (P<0.005) by being resistant to more than two classes of antibiotics. The most common types of multidrug-resistant S pneumoniae were serotypes 19F, 23F, 19A, and the two untypeable strains where all the detected strains were multidrug-resistant. For amoxicillin/clavulanic acid, ten (55%) pneumococcal-strains were resistant. Resistance to both erythromycin and azithromycin were in eight (44%) cases. Resistance to bacitracin, clindamycin, and nalidixic acid were found in five (27.8%), three (16.7%), and three (16.7%) cases, respectively (Table 3). Characteristics of Streptococcus pneumoniae serotypes Of the 18 pneumococcal-isolates detected, 16 (89%) strains showed seven different serotypes and 2 strains (11%) were nontypeable (Table 3). The detected serotypes were in decreasing order 1(22%), 9V (22%), 6B (16.7%), 19F(11%), 23F(5.5%), 14(5.5%), and 19A(5.5%). Antimicrobial susceptibility pattern Among the 18 pneumococcal-isolates, 17 (94%) were penicillin and ampicillin-resistant S pneumoniae (P<0.0001) (Table 3). Resistance to both ceftriaxone (two cases) and levofloxacin (one case) was the lowest. Multidrug-resistant S Figure 1: Bacterial isolates detected in cases of CAP. Each number of bacterial-positive samples is represented both with a bar and absolute values in the abscissa (* P=0.005). No bacterial isolates were detected in seven patients. Figure 2: Monthly distribution of pneumococcal pneumonia (* P=0.001) 72 Egyptian Journal of Medical Microbiology, October 2013 60 patients Table 1: Demographic and clinical characteristics of patients (n=60) Patients’ characteristics N (%) Sex Female 9 (15) Male 51 (85) Geographical area Assiut 52 (86.7) Qena 5 (8) New Valley 2 (3.3) Aswan 1 (1.7) Site of admission Chest department 49(82) Chest intensive care unit 11(18) Smoking index 0 (non-smoker) 12 (20) Ex-smoker 2 (3.3) 0-10 4 (6.7) 10-20 4 (6.7) 20-30 15 (25) >30 23 (38.3) Radiographic findings Lobar 32 (53) Bronchopneumonia 19 (31.7) Interstitial 6 (10) Multilobar pneumonia 3 (5) Cavitation 2 (3) Pleural effusion 7 (11.7) 73 Vol. 22, No. 4 P-value < 0.0001 < 0.0001 < 0.0005 <0.01 < 0.005 Egyptian Journal of Medical Microbiology, October 2013 Table 2: Clinical characteristics of pneumococcal pneumonia Patient Age Gender Residence Site of admission 1 46 y M New Valley Chest Depart. 2 60 y M Assiut Chest ICU 3 55 y M Assiut Chest Depart. 4 72 y M Assiut Chest Depart. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 15 y 70 y 47 y 54 y 55 y 63 y 35 y 62 y 24 y 43 y 51 y 30y 56y 60 y M M M F M F M M M M M M F M Qena Assiut Assiut Assiut Assiut Assiut Assiut Assiut Assiut Assiut Assiut Aswan Assiut Assiut Chest ICU Chest Depart. Chest Depart. Chest Depart. Chest Depart. Chest Depart. Chest ICU Chest Depart. Chest Depart. Chest Depart. Chest ICU Chest ICU Chest Depart. Chest Depart. Vol. 22, No. 4 Smoking index 8 32 25 21 0 37 16 0 22 0 Ex-smoker (previous index 18) 39 6 32 30 21 0 35 Underlying disease (bronchopulmonary condition-immunosuppresion) none Right lung cancer none Lt sided consolidation collapse with Lt sided pleural effusion Lt hydropneumothorax Rt pleural effusion with underlying collapse none DM, Liver cirrohsis none none Cardiac ischemia, massive hemoptysis Respiratory failure none cardiomyopathy Pulmonary embolism, Rt lower limb DVT,DM Respratory failure Pulmonary cavitation Lt lower lobe collapse Radiological findings Lt lower lobe pneumonia Bronchopneumonia Interstitial pneumonia Lt lower lobe pneumonia Lt lower lobe pneumonia Rt lower lobe pnemonia Interstitial pneumonia Lt lower lobe pneumonia Lt lower lobe pneumonia Rt upper lobe pneumonia Bronchopneumonia Lt lower lobe pneumonia Bronchopneumonia Lt sided pneumonia Multilobar pneumonia Bronchopneumonia Rt lobar pneumonia Lt lower lobe pneumonia Abbreviations: Depart.=department; DM=diabetes mellitus; DVT=deep venous thrombosis; F=female; ICU=Intensive care unit; Lt=left; M=male; Rt=right; y=year 74 Egyptian Journal of Medical Microbiology, October 2013 Vol. 22, No. 4 Table 3: Distribution of the detected serotypes of Streptococcus pneumoniae and their antimicrobial resistance against 10 antimicrobials Serotype No. of isolates with indicated resistance No. of isolates P value (%) P AMP AMC E AZM B DA NA 1 4 (22) <0.01 4 3 1 1 1 9V 4 (22) <0.01 4 4 1 1 1 6B 3 (16.7%) <0.05 3 3 1 1 1 1 19F 2 (11%) NS* 2 2 2 2 2 2 1 1 23F 1 (5.5%) NS 1 1 2 1 1 1 1 1 14 1 (5.5%) NS 1 19A 1 (5.5%) NS 1 1 1 1 1 - CRO 1 - LEV - Multiresistant Strains 1 1 1 2 1 1 NT** Total P value 1 2(11) - 1 1(5.5) - 2 9 (50%) <0.005 2 (11%) 18 (100%) NS 2 17(94) <0.0001 2 17(94) <0.0001 2 10(55) <0.001 1 8(44) <0.005 1 8(44) <0.005 2 5(27.8) <0.01 3(16.7) <0.05 1 3(16.7) <0.05 Abbreviations: AMC=amoxicillin/clavulanic acid; AMP=ampicillin; AZM=azithromycin; B=bacitracin; CRO=ceftriaxone; DA= clindamycin; E=erythromycin; LEV=levofloxacin; NA=nalidixic acid; P=penicillin; *NS=not significant; ** NT= non-typeable 75 Egyptian Journal of Medical Microbiology, October 2013 Vol. 22, No. 4 mostly affecting the lung tissue (e.g. lung collapse, pleural effusion, respiratory failure, and lung cancer). Presence of co-morbidities especially those involving reduced lung function are associated with higher risk of pneumonia(27, 31). In our study, lobar pneumonia was the most common anatomical type detected in enrolled cases. Lobar pneumonia is most commonly associated with community acquired pneumonia(32). About 61% of the pneumococcal infection in this study was lobar in nature. Among the clinical and radiological features, lobar distribution or alveolar consolidation were more frequent in pneumococcal-pneumonia than in other etiologies(33). Two of the involved pneumococcal-cases and one case suffered from pleural effusion and pulmonary cavitation, respectively. Cavity and pleural effusion were significantly frequent in cases of S. pneumoniae pneumonia(34). Up to date, there is no clear report that documented the prevalent pneumococcalserotypes in cases of CAP in Assiut. Other reports from Egypt described pneumococcalserotypes in cases of childhood meningitis where the major serotypes reported were 6B, 1, 19A, 23F, and 6A(35,36). Serogroup distribution of pneumococcal isolates varies between developing and developed countries as well as between different geographical regions(37). In the present study, seven different pneumococcal-serotypes were found. Serotypes 1, 9V, and 6B were the most frequently detected. These pneumococcal serotypes were previously detected in pneumococcal diseases in adult patients in Kuwait(21) and in children in Saudi Arabia(38). A study conducted in Egypt in the late 1970s used Quellung reaction, and identified type 1 as the most frequently observed capsular type(39). In a previous report from Algeria, the most common serotypes detected in cases of pneumococcal disease were 14 (19.5%), 23F (9.7%), 6B (9.3%), 19F (5.4%), and serotype 1 (5%)(40). In United Kingdom(41), serotypes 6B, 19F, and 23F were the top serotypes isolated in cases of respiratory infection. In the present study, as previously reported(10,40,42,43), a high rate of resistance to penicillin and other beta lactams was found. Among the 18 pneumococcal isolates, 94% were resistant to penicillin and ampicillin (P<0.0001). A strong correlation between serotypes and antimicrobial resistance patterns was observed in this study. The six serotypes 1, 9V, 6B, 19F, 23F and 19A were associated with DISCUSSION Community-acquired pneumonia (CAP) is a common disorder that is potentially life threatening, especially in older adults and those with co-morbid disease. Although many pathogens have been associated with CAP, it is a small range of key pathogens that cause most Cases(7). This study describes the epidemiologic characteristics, antibiotic susceptibility pattern, and serotype prevalence of S pneumoniae in patients with CAP at Assiut University Hospitals. So far, S pneumoniae is the most common pathogen in cases of CAP as reported previously in many researches either in Egypt(19) and the Arabian Peninsula like Tunisia(20) and Kuwait(21) or in other parts of the world in Chile(22), in a prospective multi-center study from 14 European centers(23), in USA(24) and in Japan(25). Prevalence of S pneumonia infection was mostly high during February and May. Infections with pneumococcal-pneumonia occur anytime but most often during the winter and early spring when respiratory illnesses are more common(26). In this study, incidence of pneumonia was higher in males (85%) than in females (15%). This is reported previously(27). The smoking habits in males make them more prone to the occurrence of pneumonia. The predisposition of cigarette smokers for development of respiratory infections caused by microbial pathogens is well recognized(28). Smoking cigarettes has a suppressive effect on the protective functions of airway epithelium, alveolar macrophages, dendritic cells, natural killer (NK) cells and adaptive immune mechanisms, in the setting of chronic systemic activation of neutrophils. Cigarette smoke also has a direct effect on microbial pathogens to promote the likelihood of infective disease, specifically promotion of microbial virulence and antibiotic resistance(28). About 38% of the pneumonia-cases in the study had the smoking index >30. A previous multivariate analysis was performed in USA(29) documented that both male sex and high smoking index are considerable risk factors for the occurrence of pneumonia. In a research conducted in Sweden(30), smoking and liver disease attributed to 14.9% and 8.0% of the mortality rate in adult patients with bacteraemic pneumococcal pneumonia. About 67% of pneumococcal-cases in this study were associated with co-morbidities 76 Egyptian Journal of Medical Microbiology, October 2013 high rates of resistance to penicillin, whereas serotype 14 was the least resistant serotype. Penicillin resistance was reported by several studies at different time intervals [36, 44, 45, 46, 47], with an increase in the pattern of resistance over time; in 1993, 71% S. pneumoniae were susceptible to penicillin(45), in 2000, 63% of isolates were susceptible to penicillin(46), in 2004 51% of isolates were susceptible to penicillin (36). Additionally, a surveillance report of the ARMed (Antibiotic Resistance Surveillance & Control in the Mediterranean Region) project which started in 2003 and continued for 2 years in the southeastern Mediterranean, reported 30% penicillin resistance and 25% erythromycin resistance among the S pneumoniae Egypt isolates(47,48). In this study, serotypes 19F, 23F, and 19A exhibited high rates of resistance to erythromycin and azithromycin and were significantly more likely to be multidrugresistant compared with other serotypes. In a study conducted from 1998-2004, 4% of S. pneumoniae isolates conferred multidrug resistance and 50% of these were characterized as serotypes 23F, 6B, and 6A(36). In a study in China (49), more than 75% of S pneumoniae strains were resistant to azithromycin and 20.3% were resistant to penicillin. Resistance to erythromycin, azithromycin and other macrolides were detected also in previous report from Saudi Arabia(50). Resistance to ceftriaxone and levofloxacin was the least among detected serotypes in our study. A retrospective multicenter study during 1999-2000 was conducted in 5 hospitals in Egypt revealed an increase in penicillin resistance, and little resistance to cefriaxone(46). Another study in Oman(43) detected 99% susceptibility rate to ceftriaxone among isolated pneumococcal strains. Therefore periodic monitoring of the patterns of antimicrobial resistance is necessary to guide effective treatment(51). Conclusion CAP has a disease burden in adult patients at Assiut University Hospitals. Streptococcus pneumoniae is the most common isolated bacteria in cases of CAP which is associated with certain serotypes. Resistance to penicillin macrolides and other antimicrobial agents increased rapidly during the last years among pneumococcal strains in Assiut and other Provinces in Egypt. Vol. 22, No. 4 REFERENCES 1. Brown JS. Community-acquired pneumonia. Clin Med. 2012 Dec; 12(6):538-43. 2. Khawaja A, Zubairi AB, Durrani FK, Zafar A. Etiology and outcome of severe community acquired pneumonia in immunocompetent adults. BMC Infect Dis. 2013 Feb 20;13:94. 3. Lim WS, Baudouin SV, George RC, Hill AT, Jamieson C, Le Jeune I, Macfarlane JT, Read RC, Roberts HJ, Levy ML, Wani M, Woodhead MA; Pneumonia Guidelines Committee of the BTS Standards of Care Committee. BTS guidelines for the management of community acquired pneumonia in adults: update 2009. Thorax. 2009 Oct;64 Suppl 3:iii1-55. 4. Liu YF, Gao Y, Chen MF, Cao B, Yang XH, Wei L. Etiological analysis and predictive diagnostic model building of community-acquired pneumonia in adult outpatients in Beijing, China. BMC Infect Dis. 2013 Jul 9;13:309. 5. Takahashi K, Suzuki M, Minh le N, Anh NH, Huong LT, Son TV, Long PT, Ai NT, Thole H, Morimoto K, Kilgore PE, Anh DD, Ariyoshi K, Yoshida LM. The incidence and aetiology of hospitalised community-acquired pneumonia among Vietnamese adults: a prospective surveillance in Central Vietnam. BMC Infect Dis. 2013 Jul 1;13:296 6. Yusuf J, Ahmad K, Mazumder S, Khouzam R. Streptococcus pneumoniae-associated pneumonia, meningitis, and endocarditis: a case of Austrian syndrome. J La State Med Soc. 2012 Nov-Dec;164(6):324-6. 7. File TM. Community-acquired pneumonia. Lancet. 2003 Dec 13;362(9400):1991-2001. 8. Maraki S, Christidou A, Tselentis Y. Antimicrobial resistance and serotype distribution of Streptococcus pneumoniae isolates from Crete, Greece. Int J Antimicrob Agents. 2001 Jun;17(6):465-9. 9. Ochoa TJ, Rupa R, Guerra H, Hernandez H, Chaparro E, Tamariz J, Wanger A, Mason EO Jr. Penicillin resistance and serotypes/serogroups of Streptococcus pneumoniae in nasopharyngeal carrier children younger than 2 years in Lima, Peru. Diagn Microbiol Infect Dis. 2005 May;52(1):59-64. 10. Noreddin AM, El-Khatib WF, Aolie J, Salem AH, Zhanel GG. Pharmacodynamic target attainment potential of azithromycin, clarithromycin, and telithromycin in serum 77 Egyptian Journal of Medical Microbiology, October 2013 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. and epithelial lining fluid of communityacquired pneumonia patients with penicillin-susceptible, intermediate, and resistant Streptococcus pneumoniae. Int J Infect Dis. 2009 Jul;13(4):483-7. Lynch JP 3rd, Zhanel GG. Streptococcus pneumoniae: epidemiology and risk factors, evolution of antimicrobial resistance, and impact of vaccines. Curr Opin Pulm Med. 2010 May;16(3):217-25. Wu CL, Ku SC, Yang KY, Fang WF, Tu CY, Chen CW, Hsu KH, Fan WC, Lin MC, Chen W, Ou CY, Yu CJ. Antimicrobial drug-resistant microbes associated with hospitalized community-acquired and healthcare-associated pneumonia: a multicenter study in Taiwan. J Formos Med Assoc. 2013 Jan;112(1):31-40. Lode HM. Managing community-acquired pneumonia: a European perspective. Respir Med. 2007 Sep;101(9):1864-73. Indrayan A, Kumar R, Dwivedi S. A simple index of smoking.2008 http://biostats.bepress.com/cobra/ps/art40 Henig NR, Tonelli MR, Pier MV, Burns JL, Aitken ML. Sputum induction as a research tool for sampling the airways of subjects with cystic fibrosis. Thorax. 2001 Apr;56(4):306-11. Koneman EW, Allen SD, Janda WM, Schreckenberger RC and Winn WC. Introduction to microbiology, part II: reporting of cultures from specific specimen sources. In Koneman EW, Allen SD, Janda WM, Schreckenberger RC, Winn WC, editors. Color Atlas and text book of diagnostic microbiology. Lippincott-Raven, Philadephia 121–171; 1997. Murray PR, Rosenthal KS, Pfaller MA. Streptococuss. page 241 inMedical Microbiology.6th edit.2009. Kumar S, Bandyopadhyay M, Mondal S, Pal N, Ghosh T, Bandyopadhyay M, Banerjee P. Tigecycline activity against metallo-β-lactamase-producing bacteria. Avicenna J Med. 2013 Oct;3(4):92-96. El Sayed Zaki M, Raafat D, El-Metaal AA, Ismail M. Study of human metapneumovirus-associated lower respiratory tract infections in Egyptian adults. Microbiol Immunol. 2009 Nov;53(11):603-8. Rachdi M, Boutiba-Ben Boubaker I, Mahjoubi-Rhimi F, Smaoui H, Hammami A, Kéchrid A, Slim A, Ben Redjeb S. Serotype distribution and antimicrobial resistance patterns of Streptococcus 21. 22. 23. 24. 25. 26. 27. 28. 29. 78 Vol. 22, No. 4 pneumoniae isolated in Tunisia. J Med Microbiol. 2011 Mar;60(Pt 3):391-3. Mokaddas EM, Rotimi VO, Albert MJ. Implications of Streptococcus pneumoniae penicillin resistance and serotype distribution in Kuwait for disease treatment and prevention. Clin Vaccine Immunol. 2008 Feb;15(2):203-7. Luchsinger V, Ruiz M, Zunino E, Martínez MA, Machado C, Piedra PA, Fasce R, Ulloa MT, Fink MC, Lara P, Gebauer M, Chávez F, Avendaño LF. Communityacquired pneumonia in Chile: the clinical relevance in the detection of viruses and atypical bacteria. Thorax. 2013;68(11):1000-6. Reissig A, Mempel C, Schumacher U, Copetti R, Gross F, Aliberti S. Microbiological diagnosis and antibiotic therapy in patients with communityacquired pneumonia and acute COPD exacerbation in daily clinical practice: comparison to current guidelines. Lung. 2013 Jun;191(3):239-46. Said MA, Johnson HL, Nonyane BA, Deloria-Knoll M, O'Brien KL; AGEDD Adult Pneumococcal Burden Study Team, Andreo F, Beovic B, et al. Estimating the burden of pneumococcal pneumonia among adults: a systematic review and metaanalysis of diagnostic techniques. PLoS One. 2013;8(4):e60273. Ishiguro T, Takayanagi N, Yamaguchi S, Yamakawa H, Nakamoto K, Takaku Y, Miyahara Y, Kagiyama N, Kurashima K, Yanagisawa T, Sugita Y. Etiology and factors contributing to the severity and mortality of community-acquired pneumonia. Intern Med. 2013;52(3):31724. Granados A, Podzamczer D, Gudiol F, Manresa F. Pneumonia due to Legionella pneumophila and pneumococcal pneumonia: similarities and differences on presentation. Eur Respir J. 1989 Feb;2(2):130-4. Torres A, Peetermans WE, Viegi G, Blasi F. Risk factors for community-acquired pneumonia in adults in Europe: a literature review. Thorax. 2013 Nov;68(11):1057-65. Feldman C, Anderson R. Cigarette smoking and mechanisms of susceptibility to infections of the respiratory tract and other organ systems. J Infect. 2013 Sep;67(3):169-84. Juthani-Mehta M, De Rekeneire N, Allore H, Chen S, O'Leary JR, Bauer DC, Harris TB, Newman AB, Yende S, Weyant RJ, Egyptian Journal of Medical Microbiology, October 2013 30. 31. 32. 33. 34. 35. 36. 37. Kritchevsky S, Quagliarello V; Health ABC Study. Modifiable risk factors for pneumonia requiring hospitalization of community-dwelling older adults: the Health, Aging, and Body Composition Study. J Am Geriatr Soc. 2013;61(7):11118. Naucler P, Darenberg J, Morfeldt E, Ortqvist A, Henriques Normark B. Contribution of host, bacterial factors and antibiotic treatment to mortality in adult patients with bacteraemic pneumococcal pneumonia. Thorax. 2013 Jun;68(6):571-9. Yende S, Alvarez K, Loehr L, Folsom AR, Newman AB, Weissfeld LA, Wunderink RG, Kritchevsky SB, Mukamal KJ, London SJ, Harris TB, Bauer DC, Angus DC. Epidemiology and Long-term Clinical and Biologic Risk Factors for Pneumonia in Community-Dwelling Older Americans: Analysis of Three Cohorts. Chest. 2013 Sep;144(3):1008-17. Reynolds JH, McDonald G, Alton H, Gordon SB. Pneumonia in the immunocompetent patient. Br J Radiol. 2010 Dec;83(996):998-1009. Moine P, Vercken JB, Chevret S, Gajdos P. Severe community-acquired pneumococcal pneumonia. The French Study Group of Community-Acquired Pneumonia in ICU. Scand J Infect Dis. 1995;27(3):201-6. Okada F, Ando Y, Matsushita S, Ishii R, Nakayama T, Morikawa K, Ono A, Maeda T, Mori H. Thin-section CT findings of patients with acute Streptococcus pneumoniae pneumonia with and without concurrent infection. Br J Radiol. 2012 Aug;85(1016):e357-64. Wasfy MO, Pimentel G, Abdel-Maksoud M, Russell KL, Barrozo CP, Klena JD, Earhart K, Hajjeh R: Antimicrobial susceptibility and serotype distribution of streptococcus pneumoniae causing meningitis in egypt, 1998-2003. J Antimicrob Chemother 2005, 55(6):958-64. Afifi S, Wasfy MO, Azab MA, Youssef FG, Pimentel G, Graham TW, Mansour H, Elsayed N, Earhart K, Hajjeh R, Mahoney F: Laboratory based surveillance of patients with bacterial meningitis in Egypt (19982004). Eur J Clin Microbiol Infect Dis 2007,26(5):331-40. Al-Mazrou A, Twum-Danso K, Al Zamil F, Kambal A. Streptococcus pneumoniae serotypes/serogroups causing invasive disease in Riyadh, Saudi Arabia: extent of coverage by pneumococcal vaccines. Ann Saudi Med. 2005 Mar-Apr;25(2):94-9. Vol. 22, No. 4 38. Shibl AM, Memish ZA, Al-Kattan KM. Antibiotic resistance and serotype distribution of invasive pneumococcal diseases before and after introduction of pneumococcal conjugate vaccine in the Kingdom of Saudi Arabia (KSA). Vaccine. 2012 Dec 31;30 Suppl 6:G32-6. 39. Guirguis N, Hafez K, El Kholy MA, Robbins JB, Gotschlich EC: Bacterial meningitis in Egypt: Analysis of CSF isolates from hospital patients in Cairo, 1977-78. Bull World Health Organ 1983,61(3):517-24. 40. Tali-Maamar H, Laliam R, Bentchouala C, Touati D, Sababou K, Azrou S, Azzam M, Amhis W, Oussadou L, Belouni R, Smati F, Rahal K. Serotyping and antibiotic susceptibility of Streptococcus pneumoniae strains isolated in Algeria from 2001 to 2010. Med Mal Infect. 2012 Feb;42(2):5965. 41. Farrell DJ, Felmingham D, Shackcloth J, Williams L, Maher K, Hope R, Livermore DM, George RC, Brick G, Martin S, Reynolds R; BSAC Working Parties on Resistance Surveillance. Non-susceptibility trends and serotype distributions among Streptococcus pneumoniae from community-acquired respiratory tract infections and from bacteraemias in the UK and Ireland, 1999 to 2007. J Antimicrob Chemother. 2008 Nov;62 Suppl 2:ii87-95. 42. Mahjoubi-Rhimi F, Kechrid A, Boutiba I, Mezghani S, Kamoun A, Smaoui H, Thabet L, Ben Redjeb S, Hammami A. [Antibiotic sensitivity of Streptococcus pneumoniae in Tunisia: results of a multicenter study (1998-1999)]. Tunis Med. 2003 Mar;81(3):167-71. 43. Al-Yaqoubi MM, Elhag KM. Serotype Prevalence and Penicillin-susceptibility of Streptococcus pneumoniae in Oman. Oman Med J. 2011 Jan;26(1):43-7. 44. Ministry of Health and Population, Egypt: Enhanced Surveillance 45. for Communicable Diseases, annual summary January-December 2000 report. http://www.geis.fhp.osd.mil/GEIS/Training /EgyptSurv2000.htm]. US Department of Defense Global Emerging Infections Surveillance and Response System. 46. Ostroff SM, Harrison LH, Khallaf N, Assaad MT, Guirguis NI, Harrington S, elAlamy M: Resistance patterns of streptococcus pneumoniae and haemophilus influenzae isolates recovered in Egypt from children with pneumonia. The antimicrobial 79 Egyptian Journal of Medical Microbiology, October 2013 47. 48. 49. 50. 51. resistance surveillance study group. Clin Infect Dis 1996,23(5):1069-74. El Kholy A, Baseem H, Hall GS, Procop GW, Longworth DL: Antimicrobial resistance in Cairo, Egypt 1999-2000: A survey of five hospitals. J Antimicrob Chemother 2003, 51(3):625-30. Borg MA, Tiemersma E, Scicluna E, Sande-Bruinsma N van de, de Kraker M, Monen J, Grundmann H, ARMed Project members and collaborators: Prevalence of penicillin and erythromycin resistance among invasive streptococcus pneumoniae isolates reported by laboratories in the southern and eastern Mediterranean region. Clin Microbiol Infect 2009, 15(3):232-7. Borg MA, Scicluna E, de Kraker M, SandeBruinsma N van de, Tiemersma E, Gur D, Redjeb S Ben, Rasslan O, Elnassar Z, Benbachir M, Pieridou Bagatzouni D, Rahal K, Dauod K, Grundmann H, Monen J: Antibiotic resistance in the southeastern Mediterranean -preliminary results from the Vol. 22, No. 4 ARMed project. Eurosurveillance 2006, 11(7):164-7. 52. Liu YN, Chen MJ, Zhao TM, Wang H, Wang R, Liu QF, Cai BQ, Cao B, Sun TY, Hu YJ, Xiu QY, Zhou X, Ding X, Yang L, Zhuo JS, Tang YC, Zhang KX, Liang DR, Lü XJ, Li SQ, Liu Y, Yu YS, Wei ZQ, Ying KJ, Zhao F, Chen P, Hou XN. [A multicentre study on the pathogenic agents in 665 adult patients with communityacquired pneumonia in cities of China]. Zhonghua Jie He He Hu Xi Za Zhi. 2006 Jan;29(1):3-8. 53. Fouda SI, Kadry AA, Shibl AM. Betalactam and macrolide resistance and serotype distribution among Streptococcus pneumoniae isolates from Saudi Arabia. J Chemother. 2004 Dec;16(6):517-23. 54. Youssef FG, El-Sakka H, Azab A, Eloun S, Chapman GD, Ismail T, Mansour H, Hallaj Z, Mahoney F: Etiology, antimicrobial susceptibility profiles, and mortality associated with bacterial meningitis among children in Egypt. Ann Epidemiol 2004,14(1):44-8. اﻟﺘ ﺼϒϴϨ اﻟϴδﺮوﻟﻮΟو ﻰϤϧ ﻂϣﻘﺎوϣاﻟ ﺔπϤاﻟ ﺎداتϴΤﻮϳﻟ ﺔϴϤﻜﺮوΑﺛ ﺎتϨﺎﺋϴا ﺎتϻاﻟ ﺮﺋ ﻮ ى ﻟﺘﻬﺎبϓﻰ ϣﺮοا ﻰϻاﻟاﻟﺮﺋﻮ ى ﻟﺘﻬﺎبϤﻜﺘΐδ Ϧϣ اﻟΠϤﺘϊϤ ϦδΣ ﺖΑء ﺛﺎϼϋ . **د،ﺪϤΤϣ ﺎركΒϣم اϼγ ﻰϨϣ .*د ﻮطϴγﺔ أόϣﺎΟ – ΐﻄϟﺔ اϴϠﺔ – آϋﺎϨϤϟﺎ واϴΟﻮϟﻮϴΑﻜﺮوϴϤϟ اϢδϗ* ﻮطϴγﺔ أόϣﺎΟ – ΐﻄϟﺔ اϴϠﻮط – آϴγﺔ اόϣﺎΟ ﺎتϴϔθΘδϣ – ﺔϳﺼﺪرϟﺮاض اϣ اﻷϢδϗ** ﺔϴοﺮϤϟﺎﻻت اΤϟ اϦϣ ﺮϴΜﻜϟ اΐΒδΗ وهﻰ.ﺎرϤϋ اﻷϒϠΘΨϣ ϦϴΑ ﺪوثΤϟﺔ اόﺎη ﺪوىϋ هﻰϊϤΘΠϤϟ اϦϣ ΐδΘﻜϤϟﻮى اﺮϟﻬﺎب اΘϟﺪوى اﻻϋ ان ﺎتϴﺎϨΛ ﺎتΑﻜﺮوϴϤϟ اϩﻰ رأس هﺬϠϋ ﻰΗﺄϳﻮى وﺮϟﻬﺎب اΘϟﻰ اﻻϓ ΐΒδΘΗ ﺎتΑﻜﺮوϴϤϟ اϦϣ ﺮاϴΜ ان آ.Ϣϟﺎόϟﻮى اΘδϣ ﻰϠϋ ﺎةϓﻮϟﺎﻻت اΣ ﻚϟوآﺬ .ﻮىﺮϟﻬﺎب اΘϟاﻻ ﺮاءΟﻬﺪف اΑ ﻚϟﻮط وذϴγﺔ أόϣﺎΟ ﺎتϴϔθΘδϤΑ ϊϤΘΠϤϟ اϦϣ ΐδΘﻜϤϟﻮى اﺮϟﻬﺎب اΘϟﺪى اﻻόΑ ﺎπϳﺮϣ ﻮنΘγ ﻰϠϋ ﺔγﺪراϟ اϩ هﺬΖϳﺮΟﺪ اϘϟو ﺎﻻتΣ ﻰϓ ﺪامΨΘγﺔ اﻻόﺎθϟﺔ اϳﻮϴΤϟﺎدات اπϤϟ اξόΒϟ ﻮىﺮϟﻬﺎب اΘϟﺎت اﻻϴﺎϨΛ ﺔϣﺎوϘϣ ﻂϤϧ ﺔϓﺮόϣ ﻚϟﻰ وآﺬΟﻮϟﺮوϴδϟ اϒϴϨﺼΘϟا ا ﻻΘϟا ﻬﺎبϟﺮﻮى. .ﺔϟﺰوόϤϟﺎت اΑﻜﺮوϴϤϟ اϦϣ ̃˼˹ ﺔΒδϨΑ ﺔ أىγﺪراϟﻰ اϓ ϦϴﺎرآθϤϟﻰ اοﺮϤϟ اϦϣ ﻮىﺮϟﻬﺎب اΘϟﺎت اﻻϴﺎϨΛ ﺎتΑﻜﺮوϴϣ Ϧϣ ˺́ ﺰلϋ ϢΗ ﺪϗو Ϧϣ ﻰ اىϟ اϢﻬϔϴϨﺼΗ ϢΘϳ Ϣϟ ﺰﻻتόϟ اϦϣ Ϧϴϋﻮϧ ﻰϟﺔ اϓﺎοﺎﻻΑ ﺔϔϠΘΨϣ ﻮاعϧ أϊΒγ ﻰϟﻰ اϤΘϨΗ ﺔϟﺰوόϤϟﺔ اϴΟﻮϟﺮوϴδϟﻮاع اϧ اﻷΖϧوآﺎ 1, 9V, 6B, 19F, : ﺔϴϟﺎΘϟﺎت اϋﻮϤΠϤϟﻰ اϟﻬﺎ اϟﺰϋ ϢΗ ﻰΘϟﺔ اϴΟﻮϟﺮوϴδϟﺎت اϋﻮϤΠϤϟ اΖϤΘϧﺪ اϗ و.ﺔϟﺰوόϤϟﺔ اϴΟﻮϟﺮوϴδϟﺎت اϋﻮϤΠϤϟا 23F, 14, 19A ﺔγﺪراϟ اϩﻰ هﺬϓ ﺔϟﺰόϤϟﻮى اﺮϟﻬﺎب اΘϟﺎت اﻻϴﺎϨΛ ﺪ أنΟﺪ وϘϠϓ ﺔϔϠΘΨϤϟﺔ اϳﻮϴΤϟﺎدات اπϤϠϟ ﻮىﺮϟﻬﺎب اΘϟﺎت اﻻϴﺎϨΛ ﺔϴγﺎδΣ ﻂϤϨϟ ﺔΒδϨϟﺎΑ Ζϧﺔ آﺎϟﺰوόϤϟﺎت اΑﻜﺮوϴϤϟ اϦϣ ̂ ˽̃ انΚϴΣ ﺪزϴϟﺎآﺮوϤϟﺔ اϋﻮϤΠϣ وϦϴϠϴδϨΒϟﺔ اϋﻮϤΠϤϟ ﺔϴϤΘϨϤϟﺔ اϳﻮϴΤϟﺎدات اπϤϠϟ ﺔϴϟﺎϋ ﺔϣﺎوϘϤΑ ﺰϴϤΘΗ ﺎآﺰونϳﺮΘϔϴδϟﺔ اϳﻮϴΤϟﺎدات اπϤϠϟ ﺔϠϴΌο ﺔϣﺎوϘϤΑ ﻮىﺮϟﻬﺎب اΘϟﺎت اﻻϴﺎϨΛ ﺰتϴϤΗ ﺎϤ آ.ﺔϳﻮϴΤϟﺎدات اπϤϟ اϦϣ ϦϴϋﻮϨϟ اϦϳﻬﺬϟ ﺔϣﺎوϘϣ .ϦϴγﺎδﻮآϠϓﻮϔϴϠϟوا ΚΤΒϟﻚ اϟ ذϦϣ و.19F, 23F, 19A ﺎتϋﻮϤΠϣ ϞΜϣ ﺮهﺎϴﺔ دون ﻏϳﻮϴΤϟﺎدات اπϤϠϟ ﺔϴϟﺎϋ ﺔϣﺎوϘϤΑ ﺰتϴϤΗ ﺔϴΟﻮϟﺮوϴδϟﻮاع اϧ اﻷξόΑ ﺎتΑﻜﺮوϴϤϟﺮ اΜﻮى هﻰ أآﺮϟﻬﺎب اΘϟﺎت اﻻϴﺎϨΛ ﻮط وأنϴγﺔ أόϣﺎΟ ﺎتϴϔθΘδϣ ﻰϓ ﺪوثΤϟﺔ اόﺎθϟﺮاض اϣ اﻷϦϣ ﻮىﺮϟﻬﺎب اΘϟ أن اﻻΞΘϨΘδϧ ξόΑ ﺰϴϤΗ ﺔϣﺎوϘϤϟ اϩﺮة وان هﺬϴΧﺔ اﻻϧﻰ اﻻوϓ ﺔϔϠΘΨϤϟﺔ اϳﻮϴΤϟﺎدات اπϤϠϟ ﻜﺮوبϴϤϟﺔ هﺬا اϣﺎوϘϣ ﺪ ازدادتϘϟ و.ﺪوىόϟ اϩﻬﺬϟ ﺔΒΒδϤϟا Ϧϣ ﻬﻮϟ ﺔϳﻮϴΤϟﺎدات اπϤϠϟ ϪΘϣﺎوϘϣ ﺔόΑﺎΘϣﻮى وﺮϟﻬﺎب اΘϟﺎﻻت اﻻΣ ﻰϓ ﻜﺮوبϴϤϟﺔ هﺬا اόΑﺎΘϣ ان.ﺮهﺎϴﺔ دون ﻏϴΟﻮϟﺮوϴδϟﺎت اϋﻮϤΠϤϟا أهϢ ﻻ وϟﻮ اϳ ﺎتϓ ﻻ و ﻰϧا ﺔ اϘϟﺎدϣﺔ. 80 Sci-Afric Journal of Scientific Issues, Research and Essays Vol. 2 (10), Pp. 456-461, October, 2014. (ISSN 2311-6188) http://www.sci-afric.org Research Paper Community-Acquired Pneumonia Caused By Haemophilus Influenzae in a Group of Non-Vaccinated Adult Population in Egypt. Mona Embarek Mohamed1, Mohamed A. El-Mokhtar Mahmoud1, Alaa Thabet Hassan2 1. Department of Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt. 2. Department of Chest Diseases, Faculty of Medicine, Assiut University, Assiut, Egypt. Author’s E-mail: [email protected] th Accepted October 24 , 2014 -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------ABSTRACT Community-acquired pneumonia is a common disease and a frequent cause of morbidity and mortality worldwide. Haemophilus influenzae is a leading cause of CAP. The current study was conducted to determine the serotype distribution and antimicrobial susceptibility patterns of Haemophilus influenzae isolated from unvaccinated adult patients with CAP at Assiut University Hospitals. Materials and Methods: From September 2013 to august 2014, sputum samples from 132 adult patients with CAP were analyzed for the detection of Haemophilus influenza using conventional methods. Antimicrobial susceptibility and serotyping of Haemophilus influenzae was performed. Results: Haemophilus influenzae were detected in 21(16%) CAP- patients. Nontypeable H influenzae were the most frequently isolated serotype that found in 15 (71%) of H influenzae-cases. H influenzae type b was found in 5 (24%) cases. While H influenzae type f was found in one (5%) case. Cases were detected mainly during January, February, and March. Resistance was highest for the B-lactam group of antibiotics. Conclusion: CAP has a disease burden in adult patients at Assiut University Hospitals, Egypt. H influenzae is a leading cause of CAP which was associated mostly with non-typeable serotypes. Resistance to penicillin and other antimicrobial agents increased rapidly during the last years among H influenzae strains. Key words: Haemophilus influenzae, community-acquired pneumonia, serotyping, antimicrobial resistance. INTRODUCTION Community-acquired pneumonia (CAP) was defined as pneumonia acquired outside the hospital setting [1]. It is one of the most common acute infections requiring admission to hospital. Risk factors for CAP include age, smoking, and co-morbidities [2]. The annual incidence of CAP varies from 5–11 per 1,000 population with higher rates in the elderly [3]. Haemophilus influenzae (H. influenzae) is one of the common causes of community-acquired lower respiratory tract (LRT) infections particularly CAP and invasive disease [4]. On the basis of the antigenic properties, six serotypes of encapsulated H. influenzae are distinguished (a, b, c, d, e, and f), and there are also non encapsulated or non-typeable H. influenzae (NTHi) [5]. Nowadays, non-typeable isolates (NTHi) account for the majority of LRTI after the introduction of Hib conjugate vaccines [6]. A main problem is that, CAP is caused by drug-resistant H influenzae strains. Although beta-lactams (as penicillin) has long been the mainstay of treatment of H. influenzae infections, strains with decreased susceptibility to penicillin have become increasingly prevalent and are now a serious problem worldwide [7]. Therefore, periodic monitoring of the patterns of antimicrobial resistance is necessary to guide effective treatment against H influenzae [8]. The incidence of CAP and its common complications, such as the requirement for intensive care and complicated para-pneumonic effusions, are increasing, making it essential for all physicians to have a good understanding of the management of CAP [1]. MATERIALS AND METHODS Study design This is a prospective study that carried out at Assiut University Hospitals, Assiut, Egypt over 12-months period from September 2013 to end August 2014; aiming to determine the serotype distribution and antimicrobial susceptibility profile of H. influenzae strains causing CAP among a group of adult population unvaccinated to H. influenzae. The study was approved by the medical ethical committee at the Faculty of Medicine, Assiut University, and oral consents were taken from all subjects prior to sample collection. Embarek Mohamed et al 456 Study population Un-vaccinated adults with community-acquired pneumonia who attended the Chest Department were eligible for the study. Proved tuberculosis patients and patients who were receiving antibiotics were excluded from the study. Pneumonia was defined by signs and symptoms suggestive of lower respiratory tract infection together with chest radiographic findings consistent with pneumonia as determined initially by the clinical physician. Questionnaires were fulfilled that included demographic and clinical data; age, gender, occupation, symptoms, admission, and associated risk factors (e.g. smoking, immunosuppressive condition, associated cardiopulmonary or systematic co-morbidities). Smoking history was calculated as number of pack/year = number of cigarettes smoked per day × number of years smoked/20 (1 pack has 20 cigarettes) [9]. Patients underwent thorough clinical examination, chest x-ray, and pulmonary function tests. Sample collection Samples were obtained within 24 hours after the patient´s admission to ensure community-acquired infection. Valid sputum samples were collected from 132 patients with CAP through effective coughing to obtain lung secretions as described previously [10]. Frothy saliva and secretions from pharynx were discarded and the patient was asked to produce another specimen. Samples were collected into wide-mouthed sterile screw-capped cups that contained brain heart infusion glycerol broth as a transport medium and transported to the laboratory at the Microbiology and Immunology Department, Faculty of Medicine, Assiut University where bacteriological diagnosis was performed. Identification of H. influenzae strains Samples were examined microscopically after staining with Gram´s stain and cultured on chocolate agar. The agar plates were incubated aerobically at 35–36°C with 5% CO2 for 24-48 hours. The valid sputum culture defined as that had quantitative culture 5 ≥10 colony forming units (CFU)/ml [11]. Haemophilus influenzae isolates were identified based on colonial morphology, Gram staining, and standard biochemical reactions according to the Bergey's Manual of Systematic Bacteriology [12]. Serotyping of H. influenzae strains Serotyping was performed by Haemophilus influenzae agglutination kit containing 6 pool antisera (a-f) (Difco, USA) according to manufacturer´s instructions. Briefly, a loopful of growth of the organism is mixed with a drop of the antiserum on an agglutination slide, mixed thoroughly and inspected for agglutination within one minute. Antibiotic susceptibility testing The susceptibility patterns of H. influenzae isolates to penicillin, amoxicillin, amoxicillin/clavulanic acid, trimethoprimsulfamethoxazole, clarithromycin, azithromycin, chloramphenicol, ceftriaxone, ciprofloxacin, levofloxacin, meropenem, and imipenem (Bioanalyse, Turkey) were determined. The test was performed using the disk diffusion method on Muller Hinton chocolate agar as recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines [13]. The results were interpreted as susceptible (S), intermediate (I), or resistant (R). Multidrug-resistant (MDR) H influenzae was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories [14]. Statistical analysis The SPSS program version 19.0 was used for the statistical analysis of data. Data were presented as mean and standard 2 deviation or number and percentage as appropriate. The X test was used to analyze categorical variables and a P value ˂0.05 was considered statistically significant. RESULTS Study population From September 2013 to August 2014, a total of 132 adult patients (89 males and 43 females) mostly (74%) were residents of Assiut Province with community-acquired pneumonia were prospectively enrolled in this study. Most (65%) patients were admitted at the Chest department (Table 1). The mean age of patients ranged from 28-72 years (mean± SD; 44.3 ± 27.5 years). Thirty eight (29%) patients were heavy smokers (P<0.01), 19 (14%) patients were ex-smokers, 14 (11%) patients were moderate smokers, and 12 (9%) patients were mild smokers. All females (43) enrolled in the study in addition to six males were non-smokers (Table 1). Of the 132 patients, lobar pneumonia was the most detected anatomical type in 75 (57%) patients (P<0.005), 49 (37%) patients had bronchpneumonia, 3 (2.3%) patients had multilobar pneumonia, and one patient (0.8%) had interstitial pneumonia. Three (2.3%) patients suffered from pleural effusion and one patient (0.8%) showed cavitation (Table 1). Embarek Mohamed et al 457 Table 1: Demographic and clinical characteristics of CAP patients (n=132) Patients’ characteristics Sex Female Male Geographical area Assiut Qena Sohag N (%) 43 (33) 89 (67) 98 (74) 15 (11) 10 (7.6) New Valley 5 (4) Aswan Site of admission Chest department Chest intensive care unit Smoking index 0 (non-smoker) Ex-smoker < 20 (mild smoker) 20-30 (moderate smoker) >30 (heavy smoker) 4 (3) 86 (65) 46 (35) 49 (37) 19 (14) 12 (9) 14 (11) 38 (29) Radiographic findings Lobar Bronchopneumonia Interstitial 75 (57) 49 (37) 1 (0.8) 132 patients Multilobar pneumonia 3 (2.3) Cavitation Pleural effusion 1 (0.8) 3 (2.3) Abbreviations: CAP= community-acquired pneumonia Characteristics of H influenzae serotypes A total of 21 H influenzae strains were detected. Non-typeable H influenzae (NTHi) were the most frequently isolated serotype that found in 15 (71%) of H influenzae-cases. H influenzae type b was found in 5 (24%) cases. While H influenzae type f (Hif) were found in one (5%) case (Fig 1). Characteristics of H influenzae cases H influenzae were detected in 21 (16%) CAP patients. Cases were detected mainly during January, February, and March (19% each), during December and May (14% each), November, April, and December (5% each) (Figure 1). Figure 1: Seasonal distribution of H influenzae strains in cases of CAP Abbreviations: CAP= community-acquired pneumonia; Hib= H influenzae type b; Hif= H influenzae type f; NTHi= nontypable H influenzae Embarek Mohamed et al 458 Seventeen (81%) patients were males and 4 (19%) were females with age ranged between 29-72 years (mean ± SD; 48.6 ± 11.8 years). Most (67%) H. influenzae-cases were admitted to the Chest department. Six (28.5%) of H influenzae-cases were moderate smokers, 5 cases (24%) were heavy smokers, 5 cases (24%) were non smokers, four cases (19%) were mild smokers, while one case (4.5%) was ex-smoker that had a previous smoking index of 27 (Table 2). The most (62%) anatomical pneumonictype significantly associated with H. influenzae infection was bronchopneumonia that was detected in 13 patients. Lobar pneumonia, were found in 6 (28%) patients, both of interstitial pneumonia and multilobar pneumonia were detected in one (5%) patient each (Table 2). Ten H. influenzae-cases (48%) suffered from associated cardiopulmonary conditions. Respiratory failure (RF) was found in 2 (9.5%) patients. Lung cancer, DCP, lung collapse, respiratory failure, pleural effusion, cardiac ischemia, cardiomyopathy, pulmonary embolism, pulmonary cavitation, and hydropneumothorax were found in one (5%) case each. Four (19%) patients were mechanically ventilated. Other systematic co-morbidities as diabetes mellitus (DM), hypertension, renal impairment, and deep venous thrombosis (DVT) were detected in 5 (24%) cases (Table 2). Table 2: Clinical characteristics of H. influenzae pneumonia (no=21) Patient Age Gender Residence Site of admission Smoking index 1 72 y 66 y 53 y 41 y 35 y 40 y 50 y 54 y 60 y 63 y 47 y M New Valley 8 M Assiut Chest Depart. Chest ICU Underlying disease (bronchopulmonary condition-immunosuppression) Right lung cancer 32 DCP, MV Lt lower lobe pneumonia Bronchopneumonia M Assiut 25 none Bronchopneumonia M Assiut 21 DM, hypertension M Qena Chest Depart. Chest Depart. Chest ICU 0 RF, MV Lt lower lobe pneumonia Bronchopneumonia M Assiut 37 M Assiut 16 Rt pleural effusion with underlying collapse none Rt lower lobe pnemonia Interstitial pneumonia F Assiut 0 DM, renal impairment Bronchopneumonia M Assiut 22 none Bronchopneumonia F Sohag 0 none M Assiut Chest Depart. Chest Depart. Chest Depart. Chest Depart. Chest Depart. Chest ICU Cardiac ischemia, massive hemoptysis, MV 29 y 43 y 55 y 51 y 30 y 54 y 58 y 41 y 46 y 33 y M Assiut none Bronchopneumonia M Assiut 6 none M Assiut 32 cardiomyopathy Lt lower lobe pneumonia Bronchopneumonia M Assiut Chest Depart. Chest Depart. Chest Depart. Chest ICU Ex-smoker (previous index 27) 39 Rt upper lobe pneumonia Bronchopneumonia 30 Multilobar pneumonia M Aswan Chest ICU 21 Pulmonary embolism, Rt lower limb DVT,DM RF, MV F Assiut 0 Pulmonary cavitation Bronchopneumonia M Assiut 35 Lt hydropneumothorax M Sohag 29 DM Lt lower lobe pneumonia Bronchopneumonia M Qena Chest Depart. Chest Depart. Chest Depart. Chest ICU 11 none Bronchopneumonia F Assiut Chest ICU 0 Lower limb DVT Bronchopneumonia 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Radiological findings Bronchopneumonia Abbreviations: DCP= decompensated Core-pulmonale; MV= mechanical ventilation; DM=diabetes mellitus; DVT=deep venous thrombosis; F=female; ICU=Intensive care unit; Lt=left; M=male; Rt=right; RF= respiratory failure. Embarek Mohamed et al 459 Antimicrobial susceptibility pattern Resistance to penicillin was the highest in all H. influenzae serotypes (P=0.000). Among the 21 H. influenzae isolates, 13(62%), 8(38%), 4(19%), 3(14%), 2(9.5%) strains were resistant to trimethoprim/sulfamethoxazole, chloramphenicol, amoxicillin, meropenem, and ceftriaxone, respectively. Resistance to amoxicillin/clavulanic acid, azithromycin, ciprofloxacin, and imipenem was found in one (5%) H. influenzae strain each. All H. influenzae strains in this study were found sensitive to clarithromycin and levofloxacin. NTHi showed the highest resistance to antibiotics versus Hib and Hif (P=0.01 and 0.001, respectively). Three H. influenzae strains were found to be MDR (Table 3). Table 3: Distribution of the detected serotypes of H. influenza and their antimicrobial resistance against 12 antimicrobials Serotype No (%) of isolates NTHi Hib Hif Total 15 (71) 5 (24) 1 (5) 21 (100) Multiresistant Strains (%) No (%) of isolates with indicated resistance P 14(93) 5(100) 1(100) 20(95) AX 2(13) 1(20) 1(100) 4(19) AMC 1(7) 0(0) 0(0) 1(5) SXT 10(67) 3(60) 0(0) 13(62) AZM 1(7) 0(0) 0(0) 1(5) CLR 0(0) 0(0) 0(0) 0(0) C 6(47) 2(40) 0(0) 8(38) CIP 1(7) 0(0) 0(0) 1(5) CRO 2(13) 0(0) 0(0) 2(9.5) LEV 0(0) 0(0) 0(0) 0(0) MER 2(13) 1(20) 0(0) 3(14) IMP 1(7) 0(0) 0(0) 1(5) 2(13) 1(20) 0(0) 3(14) Abbreviations: NTHi= non-typable H influanzae; Hib= H influenza type b; Hif= H influenza type f; P=penicillin; AX= amoxicillin; AMC= amoxicillin/ clavulanic acid;SXT= trimethoprim/sulfamethoxazole; AZM= azithromycin; CLR= clarithromycin; C= chloramphenicol; CIP= ciprofloxacin; CRO= ceftriaxone; LEV= levofloxacin; MER= meropenem; IMP= imipenem. DISCUSSION Up to date, there is no clear report that describes the prevalent H influenzae serotypes in cases of CAP in Assiut. This study describes the epidemiologic characteristics, antibiotic susceptibility patterns, and serotype prevalence of H. influenzae strains in unvaccinated patients with CAP at Assiut University Hospitals, Assiut, Egypt. H. influenzae play a crucial role in the etiology of CAP as evident in our study. This is reported previously either in Egypt [15], in the Arabian Peninsula like Saudia Arabia [16], or globally [17, 18, 19; 20]. Non-typeable H. influenzae (NTHi) were the most prominent serotype associated with CAP in this study in contrast to the capsulated strains Hib and Hif. This had been detected in other studies [21; 22]. This is explained by the introduction of Hib conjugate vaccines that increased the prevalence of non-capsulated strains of H. influenzae [6]. Prevalence of H. influenza pneumonia was mostly detected during December, January, February, March, and May. Infections with H. influenzae-pneumonia occur anytime but most often during the winter and early spring when respiratory illnesses are more common [23]. In this study, incidence of pneumonia was higher in males (67%) than in females (33%). This is reported previously [24]. The smoking habits in males make them more prone to the occurrence of pneumonia. The predisposition of cigarette smokers for development of respiratory infections caused by microbial pathogens is well recognized [25]. Smoking cigarettes has a suppressive effect on the protective functions of airway epithelium, alveolar macrophages, dendritic cells, natural killer (NK) cells and adaptive immune mechanisms, in the setting of chronic systemic activation of neutrophils. Cigarette smoke also has a direct effect on microbial pathogens to promote the likelihood of infective disease, specifically promotion of microbial virulence and antibiotic resistance [25]. About 29% of the pneumonia-cases in the study had the smoking index >30. A previous multivariate analyses were performed in USA [26] and Sweden [27] documented that the high smoking index are considerable risk factors for the occurrence of pneumonia. About 48% of H influenzae-cases in this study were associated with cardiopulmonary co-morbidities mostly affecting the lung tissue. Presence of co-morbidities especially those associated with reduced lung function are associated with higher risk of pneumonia [24; 28]. In our study, lobar pneumonia was the most common anatomical type detected in the 132 enrolled cases. Although lobar pneumonia is the most anatomical type associated with CAP [29], bronchopneumonia was reported to be the most anatomical type of CAP that associated with H. influenzae as evident from this study. For a long time, βlactam antimicrobials were the first therapeutic option for treating CAP due to H. influenzae [30]. Decreased susceptibilities to βlactam antibiotics among all H influenzae serotypes especially NTHi in this study could be explained by the frequent pulmonary co-morbidities found in the patients´ group. H influenzae strains in this study showed, in accordance with other studies [31;32], good response to amoxicillin/clavulanic acid, third-generation cephalosporins, oxazolidinones, quinolones, and carbapenems. Therefore, they are good therapeutic agents for treatment of CAP due to H. influenzae. All Hib strains in this study showed resistance to penicillin and 40% of Hib were resistant to chloramphenicol. This is consistent with previous studies from Africa where β-Lactamase production among Hib isolates is increasing, as recorded in these reports [33]. CONCLUSION CAP has a disease burden in adult patients at Assiut University Hospitals, Egypt. H influenzae is a leading cause of CAP which was associated mostly with non-typeable serotypes. Resistance to penicillin and other antimicrobial agents increased rapidly during the last years among H influenzae strains in Assiut and other Provinces in Egypt. Embarek Mohamed et al 460 References [1] Gordon RC. Community acquired pneumonia in adolescents. Adoles Med 2000; 11: 681-695. [2] Brown JS. Community-acquired pneumonia. Clin Med. 2012 Dec;12(6):538-43. [3] Khawaja A, Zubairi AB, Durrani FK, Zafar A. Etiology and outcome of severe community acquired pneumonia in immunocompetent adults. BMC Infect Dis. 2013 Feb 20;13:94. [4] Jordens JZ, Slack MPE: Haemophilus influenzae: Then and now. Eur J Clin Microbiol Infect Dis 1995, 14:935–948. [5] Wenger DJ and Ward J, “Haemophilus influenzae vaccine,” in Vaccines, S. A. Plotkin andW. A. Orenstein, Eds., pp. 229– 268, Saunders, Philadelphia, Pa, USA, 4th edition, 2004. [6] Resman F, Ristovski M, Forsgren A, Kaijser B, Kronvall G, Medstrand P, Melander E, Odenholt I, Riesbeck K: Increase of beta-lactam-resistant invasive Haemophilus influenzae in Sweden, 1997 to 2010. Antimicrob Agents Chemother 2012, 56:4408–4415 [7] Tristram S, Jacobs MR, Appelbaum PC: Antimicrobial resistance in Haemophilus influenzae. Clin Microbiol Rev 2007, 20:368–389. [8] Shaban L, Siam R. Prevalence and antimicrobial resistance pattern of bacterial meningitis in Egypt. Ann Clin Microbiol Antimicrob. 2009 Sep 24;8:26. [9] Indrayan A, Kumar R, Dwivedi S. A simple index of smoking. 2008 http://biostats.bepress.com/cobra/ps/art40 [10] Henig NR, Tonelli MR, Pier MV, Burns JL, Aitken ML. Sputum induction as a research tool for sampling the airways of subjects with cystic fibrosis. Thorax. 2001 Apr;56(4):306-11. [11] Koneman EW, Allen SD, Janda WM, Schreckenberger RC and Winn WC. Introduction to microbiology, part II: reporting of cultures from specific specimen sources. In Koneman EW, Allen SD, Janda WM, Schreckenberger RC, Winn WC, editors. Color Atlas and text book of diagnostic microbiology. Lippincott-Raven, Philadephia 121–171; 1997. [12] Holt JG., Krieg NR., Sneath PHA., Staley H., Williams ST. 1994. Bergey's manual of determinative bacteriology, 9th ed. Williams and Wilkins, Baltimore, MD [13] Kumar S, Bandyopadhyay M, Mondal S, Pal N, Ghosh T, Bandyopadhyay M, Banerjee P. Tigecycline activity against metallo-β-lactamase-producing bacteria. Avicenna J Med. 2013 Oct;3(4):92-96. [14] Pfeifer Y, Meisinger I, Brechtel K, Gröbner S. Emergence of a multidrug-resistant Haemophilus influenzae strain causing chronic pneumonia in a patient with common variable immunodeficiency. Microb Drug Resist. 2013 Feb;19(1):1-5. [15] El Sayed Zaki M, Goda T. Clinico-pathological study of atypical pathogens in community-acquired pneumonia: a prospective study. J Infect Dev Ctries. 2009 Apr 30;3(3):199-205. [16] Memish ZA, Almasri M, Turkestani A, Al-Shangiti AM, Yezli S. Etiology of severe community-acquired pneumonia during the 2013 Hajj-part of the MERS-CoV surveillance program. Int J Infect Dis. 2014 Aug;25:186-90. [17] Spoorenberg SM, Bos WJ, Heijligenberg R, Voorn PG, Grutters JC, Rijkers GT, van de Garde EM. Microbial aetiology, outcomes, and costs of hospitalisation for community-acquired pneumonia; an observational analysis. BMC Infect Dis. 2014 Jun 17;14:335. [18] Akter S, Shamsuzzaman SM, Jahan F. Community acquired bacterial pneumonia: aetiology, laboratory detection and antibiotic susceptibility pattern. Malays J Pathol. 2014 Aug;36(2):97-103. [19] Peto L, Nadjm B, Horby P, Ngan TT, van Doorn R, Van Kinh N, Wertheim HF. The bacterial aetiology of adult communityacquired pneumonia in Asia: a systematic review. Trans R Soc Trop Med Hyg. 2014 Jun;108(6):326-37. [20] Torres A, Blasi F, Peetermans WE, Viegi G, Welte T. The aetiology and antibiotic management of community-acquired pneumonia in adults in Europe: a literature review. Eur J Clin Microbiol Infect Dis. 2014 Jul;33(7):1065-79. [21] Efron AM, Moscoloni MA, Reijtman VR, Regueira M. [Surveillance of Haemophilus influenzae serotypes in Argentina from 2005 to 2010 during the Haemophilus influenzae type b conjugate vaccine era]. Rev Argent Microbiol. 2013 OctDec;45(4):240-7. [22] Puig C, Marti S, Hermans PW, de Jonge MI, Ardanuy C, Liñares J, Langereis JD. Incorporation of phosphorylcholine into the lipooligosaccharide of nontypeable Haemophilus influenzae does not correlate with the level of biofilm formation in vitro. Infect Immun. 2014 Apr;82(4):1591-9. [23] Prasad R. Community acquired pneumonia: clinical manifestations. J Assoc Physicians India. 2012 Jan;60 Suppl:10-2. [24] Torres A, Peetermans WE, Viegi G, Blasi F. Risk factors for community-acquired pneumonia in adults in Europe: a literature review. Thorax. 2013 Nov;68(11):1057-65. [25] Feldman C, Anderson R. Cigarette smoking and mechanisms of susceptibility to infections of the respiratory tract and other organ systems. J Infect. 2013 Sep;67(3):169-84. [26] Juthani-Mehta M, De Rekeneire N, Allore H, Chen S, O'Leary JR, Bauer DC, Harris TB, Newman AB, Yende S, Weyant RJ, Kritchevsky S, Quagliarello V; Health ABC Study. Modifiable risk factors for pneumonia requiring hospitalization of community-dwelling older adults: the Health, Aging, and Body Composition Study. J Am Geriatr Soc. 2013;61(7):1111-8. [27] Naucler P, Darenberg J, Morfeldt E, Ortqvist A, Henriques Normark B. Contribution of host, bacterial factors and antibiotic treatment to mortality in adult patients with bacteraemic pneumococcal pneumonia. Thorax. 2013 Jun;68(6):571-9. [28] Yende S, Alvarez K, Loehr L, Folsom AR, Newman AB, Weissfeld LA, Wunderink RG, Kritchevsky SB, Mukamal KJ, London SJ, Harris TB, Bauer DC, Angus DC. Epidemiology and Long-term Clinical and Biologic Risk Factors for Pneumonia in Community-Dwelling Older Americans: Analysis of Three Cohorts. Chest. 2013 Sep;144(3):1008-17. [29] Reynolds JH, McDonald G, Alton H, Gordon SB. Pneumonia in the immunocompetent patient. Br J Radiol. 2010 Dec;83(996):998-1009. Embarek Mohamed et al 461 [30] Mandell LA, Wunderink RG, Anzueto A, Bartlett JG, Campbell GD, et al. (2007) Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 44 Suppl. 2S27–S72 [31] Perez-Trallero E, Martin-Herrero JE, Mazon A, Garcia-Delafuente C, Robles P, et al. (2010) Antimicrobial resistance among respiratory pathogens in Spain: latest data and changes over 11 years (1996–1997 to 2006–2007). Antimicrob Agents Chemother 54: 2953–2959 [32] Blosser-Middleton R, Sahm DF, Thornsberry C, Jones ME, Hogan PA, et al. (2003) Antimicrobial susceptibility of 840 clinical isolates of Haemophilus influenzae collected in four European countries in 2000–2001. Clin Microbiol Infect 9: 431–436 [33] Ginsburg AS, Tinkham L, Riley K, Kay NA, Klugman KP, Gill CJ. Antibiotic non-susceptibility among Streptococcus pneumoniae and Haemophilus influenzae isolates identified in African cohorts: a meta-analysis of three decades of published studies. Int J Antimicrob Agents. 2013 Dec;42(6):482-91. THE EGYPTIAN JOURNAL OF IMMUNOLOGY Vol. 22 (1), 2015 Page: 85-91 Immunomodulatory Effects of Levofloxacin on Patients with Pneumonia in Assiut University Hospitals 1 Mohamed S. Badari, 1Sherein G. Elgendy, 1Asmaa S. Mohamed, 2Alaa T. Hassan Departments of 1Medical Microbiology & Immunology, Faculty of Medicine, and 2Chest Diseases, Assiut University hospitals, Assiut University, Assiut, Egypt The immunomodulatory effects of antibiotics could influence the degree of systemic and local responses to infection, so investigation of their intrinsic influence on the host’s inflammatory response appears to be essential. Fluoroquinolones are known to exert modulatory activity on immune responses to microbial infection. However the mechanism of this immunmodulation has not been well elucidated. The aim of the work, is to assess the immunomodulatory effects of a levofloxacin, through examining its effect on the concentrations of tumor necrosis factor α (TNF-α) and Interleukin – 10 (IL-10) in serum of pneumonic patients. After following local research ethics committee approval and informed consent. This study included 40 patients with different types of pneumonia, admitted to department of Chest Diseases, Faculty of Medicine, Assiut University Hospitals, Egypt. Also, 10 healthy volunteers served as randomized controls. Both patients and controls received levofloxacin (750 mg once daily for 10 days). Serum levels of TNF-α and IL-10 were measured in patients and control before and after levofloxacin administration (750 mg once daily for 10 days) using human TNF–α and IL-10 ELISA kits respectively. Levofloxacin caused a statistically significant decrease in the mean level of TNF- α in both patients (20.82±1.31 pg/ml) (P < 0.009) and control group (17.12 ±0.84 pg/ml) (P < 0.004). In contrast, there was statistically significant increase (P< 0.000) in the mean level of IL-10 in patients (61.75 ± 2.85 pg/ml) while statistically significant decrease (P< 0.005) in control group (28.57 ± 1.37pg/ml). In conclusion, our study demonstrates that treatment with levofloxacin affects production of TNF-α as a pro-inflammatory cytokine and IL-10 as an anti-inflammatory cytokines which may provide additional benefits in treatment of respiratory tract infections that are independent of its antibacterial properties. P neumonia is a leading cause of death in the world and the sixth most common cause of death in the United States. It is the number one cause of death from infectious diseases in the United States. In Europe, the overall incidence of community acquired lower respiratory tract infections (LRTIs) was found to be 44 cases per 1,000 populations per year in a single general practice. However, the incidence was two to four times higher in people aged over 60 years (Wei et al., 2009; Woodhead et al., 2005). There is growing evidence that certain antibiotics exert their beneficial effects not only by killing or inhibiting the growth of bacterial pathogens but also indirectly by their up regulatory effect on immune system. It has been noted that certain antibiotics (macrolides and fluoroquinolones) have immunomodulatory properties that improve the long term outcome of patients with inflammatory pulmonary diseases (Tauber & Nau, 2008). Levofloxacin is one of the newest third generation fluoroquinolones, it is the bacteriologically active L-isomer of ofloxacin (quinolone antibacterial agent). Levofloxacin has a broad spectrum of action, it diffuses through bacterial cell wall and acts by inhibiting and disrupting the function of DNA gyrase (bacterial type II topoisomerases) leading to blockage of bacterial cell growth. So levofloxacin acts as an efficient antibacterial agent by hijacking the natural ability 86 Immunomodulatory Effects of Levofloxacin on Patients with Pneumonia in Assiut University Hospitals of topoisomerase to create breaks in chromosomal DNA (Najma et al., 2009). Levofloxacin is a highly appropriate agent for treating respiratory tract infections due to its broad anti-bacterial spectrum of action for all of the most common respiratory tract pathogens, being effective against Gramnegative and Gram-Positive, as well as atypical organisms. Also its excellent pharmacokinetic and pharmacodynamic features which allows it to penetrate extremely well into lung tissue and bronchial secretions. In addition to its ability to penetrate into both phagocytic and epithelial cells which appears to be extremely important in inhibition of intracellular organisms. It achieves high concentrations in respiratory secretion and lung tissue and has a persistent activity in lung tissue “post antibiotic effect” (Carl, 2000). Fluoroquinolones have immunomodulatory effects that are independent on its antibacterial properties. The molecular mechanisms causing immunomodulatory effects are still under investigations. However activation of p38 mitogen-activated protein kinase (MAPK) pathway which is considered one of the major signal transduction pathways involved in inflammatory responses was proposed as the main effect of fluoroquinolones (Tauber and Nau, 2008). In the in vitro studies, fluoroquinolones exert their modulating effects only when used together with a co-stimulant. These studies generated heterogeneous data because of inhomogeneous effects triggered by different types of co-stimulants and differing responses of various cell lines on the stimuli. Studies in experimental animals showed significant clinical effects of flouroquinolones by attenuating cytokine responses in vivo (Dalhoff, 2005). The first study on the immunomodulatory activity of fluoroquinolones were independent on drug concentration and the analytical methods to quantitate cytokines were less sensitive, so the modulations of cytokines synthesis due to exposure to fluoroquinolones remained undetected. The older quinolone like nalidixic acid, enoxacin, fleroxacin, norfloxacin, and ofloxacin super induce cytokine synthesis at high concentration using human peripheral blood lymphocytes stimulated with phytohaemaglutinin, however, exposure to other stimulants led to inhibition of cytokine synthesis (Bailly et al., 1990) Ciprofloxacin inhibited I1-1α and I1-1 ȕ synthesis in lipopolysaccharide stimulated human peripheral blood lymphocytes but it augmented I1-1 synthesis in lipopolysaccharide stimulated Mono-Mac6 cells (Stunkel et al., 1991). Trovafloxacin significantly inhibited the secretion of I1-1α, I1- ȕ and GM-CSF and TNF-α by monocytes stimulated by LPS (Khan et al., 1998). Obviously, considerable study variations do exist due to differences in drug concentrations and stimulants used. In addition, the immunomodulatory effect of levofloxacin is not well studied and only few reports were done to evaluate immunomodulatory effect of levofloxacin on pro-inflammatory cytokine production however the experiments were carried out only in vitro. In addition, no data is available to describe the effects of levofloxacin on the anti-inflammatory cytokines. Therefore, this study aimed to evaluate the immunomodulatory effects of levofloxacin through examining its effect on the concentrations of TNF-α and IL-10 in serum of pneumonic patients and control group before and 10 days after its administration. Material and Methods Ethics Statement: Written informed consent was obtained from all patients and controls at the time of enrollment for their participation in the study. The study protocol was approved by the local Ethics Committee of the Faculty of Medicine, Assiut University. THE EGYPTIAN JOURNAL OF IMMUNOLOGY The study was conducted during the period from April 2011 to June 2013 as cooperation between Chest and Medical Microbiology & Immunology departments, Faculty of Medicine, Assiut University. Blood samples were withdrawn from 40 patients (28 males & 12 Females) including 15 patients with community acquired pneumonia (CAP), 12 patients with hospitalacquired pneumonia (HAP) and 13 patients with Ventilator-associated pneumonia (VAP). Patients with HAP and VAP were admitted due to reasons other than infection like pulmonary embolism, pneumothorax and bronchial asthma. In addition, 10 healthy volunteers served as randomized controls. Both patients and controls received levofloxacin (750 mg once daily for 10 days). Blood samples were taken before and 10 days after levofloxacin administration (750 mg once daily). Inclusion criteria: 1Patients with CAP provided by the BTS criteria for CAP (Wei et al., 2009) which is as follows: Symptoms of an acute lower respiratory tract illness (cough and at least one other lower respiratory tract symptom). New focal chest signs on examination. No other explanation for the illness, which is treated as CAP with antibiotics. Symptoms and signs consistent with an acute lower respiratory tract infection associated with new radiographic shadowing for which there is no other explanation (e.g. not pulmonary edema or infarction). Patients with HAP and VAP were diagnosed according to The American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) guidelines 2005 that distinguish the following types of pneumonia: Hospital-acquired pneumonia (HAP) is pneumonia that occurs 48 hours or more after admission and did not appear to be incubating at the time of admission. Ventilator-associated pneumonia (VAP) is a type of HAP that develops more than 48 to 72 hours after endotracheal intubation. Exclusion criteria: Patients with previous antibiotic history in < 15 days. Patients with diagnosed malignancies, collagen diseases, DM, hepatitis infection. All patients were subjected to complete clinical assessment. Routine investigations were performed including; complete blood pictures, erythrocyte sedimentation rates and bacteriological analysis of sputum. Blood samples were obtained under aseptic condition in sterile tubes without any anti-coagulant; each tube was labeled with the patient name, sex, age and the date of collection. Samples were spin down at 2000 r.p.m for 10 minutes; the serum was stored at 20ºC. Cytokine assay was performed by measuring TNF-α and IL-10 in serum samples using human TNFα and IL-10 ELISA kit, KOMA BIOTECH INC 87 (K0331123 and K0331131), respectively. All tests were done according to the manufacturer’s instructions:(a) 200 l of washing solution were added to each well and washed 3 times. (b) 100 l of standard (recombinant human TNF-α and IL-10) or samples were added to each well and incubated at room temperature for 2 hours. (c)The wells were aspirated and the plate washed 4 times. (d) 100 l of the diluted detection antibody (0.5 g/ml) were added per well, and incubated at room temperate for 2 hours. (e) 100 l of the diluted color development Enzyme (1: 20 dilute) were added per well, and incubated 30 minutes at room temperate. (f) The plate washed 4 times and 100 l of color development solution were added and incubated for (8 - 18 minutes). (g) Stop solution was added and the micro plate reader wavelength was set at 450 nm and the absorbance (OD) of each well was measured. (h) absorbance values of the strandard recombinant human TNF-α and IL-10 samples (supplied with the kit) were used to construct a standard curve from which the concentrations of the cytokines in the tested samples were calculated. Statistical Analysis All data were analyzed using the computerized statistical analysis (Statistical package for social science “SPSS version 16”). Concentrations of TNF-α and IL-10 were expressed as mean ± standard error of the mean (SEM). Differences in mean values of TNF-α and IL-10 concentrations before and after levofloxacin administration were calculated using Wilcoxon Signed Ranks Test. Mann–Whitney Test was used for comparison between mean values of patients and control. P-value is considered significant when less than 0.05. Results Forty patients with pneumonia including 15 patients with CAP, 12 patients with HAP and 13 patients with VAP were included in this study. Classical Bacteriological examinations showed that 42.5% of the samples showed Gram negative bacteria (Acinetobacter 5.0%, Klebsiella 25%, Pseudomonas 12.5%), while Gram positive bacteria were detected in 57.5% of the samples (MRSA 25.0%, Pneumococci 32.5%). This study showed that levofloxacin caused a statistically significant decrease in the mean 88 Immunomodulatory Effects of Levofloxacin on Patients with Pneumonia in Assiut University Hospitals level of TNF- α in both patients (P < 0.009) and control (P < 0.04) as shown in table (1). The mean value of TNF-α in the patients before levofloxacin administration was 36.43 ± 4.18 pg/ml while it was 20.82±1.31 pg/ml after levofloxacin administration. The mean value of TNF-α in control group before levofloxacin administration was 25.21 ± 1.96 while it was 17.12 ± 0.84 pg/ml after levofloxacin administration. Table 1. Serum TNF-(pg/ml) in patients with pneumonia and controls. TNF-(pg/ml) Before levofloxacin administration After levofloxacin administration (750mg once daily for 10 days) Mean SE Median Range Mean SE Median Range *P2-value Patients Control 36.43 4.18 23.3 8.2 98.0 20.82 1.31 25.21 1.96 27.8 16.7 33.5 17.12 0.84 18.5 17.9 11.5 46.2 0.004 10.7 20.0 0.009 *P1-value NS NS 1: Mann-Whitney Test. 2: Wilcoxon Signed Ranks Test. * P > 0.05 is not significant (NS) Regarding IL-10, levofloxacin caused a statistically significant increase in the mean level of IL-10 in patients and a statistically significant decrease in control group as shown in table (2). The mean value of IL-10 in patient before levofloxacin administration was 24.54 ± 2.83 pg/ml, while it was 61.75 ± 2.85 pg/ml after levofloxacin administration. The mean value of IL-10 in control group before levofloxacin administration was 51.48 ± 1.76 pg/ml, while after levofloxacin administration it was 28.57 ± 1.37pg/ml. Table 2. Serum IL-10 (pg/ml) in patients with pneumonia and controls. Patients Control *P1-value Before levofloxacin administration Mean SE Median Range 42.54 2.83 37.5 51.48 1.76 52.2 NS 16.4 79.2 40.2 60.5 After levofloxacin administration (750mg once daily for 10 days) Mean SE Median Range 61.75 2.85 61.3 20.0 100.1 0.000 28.57 1.37 28.6 20.0 33.8 0.005 IL-10 (pg/ml) *P2-value 0.000 1: Mann-Whitney Test. 2: Wilcoxon Signed Ranks Test. * P > 0.05 is not significant (NS). Discussion Several classes of antibiotics, including macrolides and quinolones exert modulatory effects on cytokine release by inflammatory cells (Parnham & Michael, 2005).It is important to define the immunomodulatory effects of these antibiotics which are commonly used in the therapy of respiratory tract infections, these effects seem to be related to the bacterial killing, as well as, the resolution of local inflammation. This may account for the therapeutic benefit of THE EGYPTIAN JOURNAL OF IMMUNOLOGY levofloxacin in these types of infections, even when bacterial eradication is not complete (Guz & Ploskonska, 2007). Some studies have demonstrated that in the early stages of infection, the local generation of proinflammatory cytokines such as TNF-α, IL1ȕ, IL-8, IL-12, gamma interferon (IFN-Ȗ), and possibly IL-6 are triggered by bacterial LPS (Pinsky, 2001). In the present study we evaluated the level TNF-α in vivo following the levofloxacin administration and concluded that levofloxacin led to statistically significant decrease in the mean level of TNF-α in both patients and control, which is consistent with the results obtained by Yoshimura & Kurita (1996), who found that levofloxacin suppressed tumor necrosis factor production by PBMC. Also, Choi et al. (2003) showed that the level of TNF-α as well as other proinflammatory cytokines reduced in pneumonic patients treated with levofloxacin. TNF is by far the best studied in pulmonary host defense, and has been shown to be of critical importance in a variety of animal models of pneumonia and a central mediator of the host’s response to infection. It is rapidly produced following either antigen specific or nonspecific stimulation and has, therefore, been designate an early response, or “alarm,” cytokine (Old, 1985).Lipopolysaccharide (LPS) is the best studied and most potent stimulus for TNF production. In Gramnegative bacteria, LPS is the major proinflammatory component of the cell walls, and the study of LPS-induced TNF expression by alveolar macrophages is, accordingly, very relevant to the role of TNF in the host defense response during Gram-negative pneumonia (Nelson et al., 2001). TNF is predominantly produced by cells of myeloid lineage, and serves as a major activator of both neutrophils and macrophages. Specifically, TNF enhances leukocyte microbial killing by augmenting 89 phagocytosis, oxidative burst, and release of proteases (Le & Butler, 1995). TNF also contributes to the accumulation of neutrophils in the area of inflammation by stimulating the expression of adhesion molecules on both vascular endothelial cells and phagocytic cells, and by inducing the production of chemotactic cytokines (Oswald & Huffinagle, 1996). Few mechanisms has been described to explain these findings First, the ability of levofloxacin to inhibit the production of TNFα, which occurs in very early stages of TNF-α synthesis, is probably due to its effect as a phosphodiesterase inhibitor, leading to cyclic AMP accumulation in the cells, resulting in enhanced cyclic AMP-protein kinase A activity, which in turn is known to inhibit TNF-α production (Blaine et al., 1997).Others have described the ability of fluoroquinolones to interfere with NF-κB activation by inhibiting the degradation of IκBα, thus reducing the levels of production of proinflammatory cytokines (Choi et al., 2003).So levofloxacin by its inhibitory effect on TNF-α may complement its direct antibacterial action by enhancing cellular defense mechanisms and facilitate the resolution of undesirably prolonged lung inflammation and improve outcome of infection. IL-10 is a cytokine with potent inhibitory effects on TH-l T cells and antigen presenting cells, such as monocyte/macrophages, causing down regulation of expression of major histocompatibility complex (MHC) class II molecules and attenuated release of proinflammatory cytokines, including TNF, TH-1 phenotype cytokines IFN- Ȗ and IL- 12 (Oswald et al., 1992). In our study, we found that serum levels of IL-10 were significantly elevated in all patients after taking levofloxacin. It is well known that excessive production of proinflammatory cytokine mediators can induce systemic inflammatory response syndrome 90 Immunomodulatory Effects of Levofloxacin on Patients with Pneumonia in Assiut University Hospitals and that these cytokines play an important role in the development of acute respiratory distress syndrome and multiple-organ dysfunction (Dinarello, 1997). On the other hand, IL-10 as an anti-inflammatory cytokine acts as specific inhibitor of this network (Opal & Depalo, 2000). Levofloxacin, by its effects on the production of TNF-α and IL-10, achieves the balance between pro and counter inflammatory agents which determine the final outcome of infection and clinical course of the disease. In conclusion, our study demonstrates that treatment with levofloxacin affects production of TNF-α as a pro-inflammatory cytokine and IL-10 as an anti-inflammatory cytokines which may provide additional benefits in treatment of respiratory tract infections and may lead to more efficient eradication of the offending pathogens. human peripheral blood mononuclear cells. Antimicrob. Agents Chemother; 47:3704-3707. 6. Dalhoff A. (2005). Immunomodulatory Activities of Fluoroquniolones, 33 (supple 2): 55-70. 7. Dinarello CA. (1997). Proinflammatory and antiinflammatory cytokines as mediators in the pathogenesis of septic shock. Chest; 112:321S329S. 8. Guz K, Ploskonska G. (2007). Quinolones and eukaryotic topoisomerasis. In: Hooper DC, Wolfson nd JS, eds. Quinolones antimicrobial agents. 2 ed. American Society for Microbiology, Washington DC; 139-160. 9. Khan AS, Slifer TR, Remington JS. (1998). Effect of trovafloxacin on production of cytokine by human monocytes. Antimicrob Agents Chemother; 42:1713-1717. 10. Le J, Butler. (1995). Tumor necrosis factor and interleukin-1: Cytokines with multiple overlapping biological activities. Lab Invest; 56:234-248. References 11. Nelson S, Mason CM, Kolls J, Boé D, Zhang M, Zhong P. (2001). Pathophysiology of pneumonia. Clin Chest Med; 16:1-12. 1. American Thoracic Society and the Infectious Diseases Society of America Guidelines. (2005). The Management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med; 171:388416. 12. Najma Sultana, Muhammad Saeed Areayne, Syeda Bushra Shakeb Rizvi, Muhammad Ahmed Mesaik. (2009). Synthesis, characterization and biological evaluation of a series of levofloxacin carboxamide analogues. Bull. Korean Chem. Soc; 30(10):22952298 2. Baily S, Fay M, Gougerot-Pocidalo JJ. (1990). Effects of quinolones on tumor necrosis factor production by human monocytes. Int J Immunopharmacol; 12:31-36. 13. Opal SM, DePalo VA. (2000). Anti-inflammatory cytokines. Chest; 117:1162-1172. 3. Blaine TA, Pollice PF, Rosier RN, Reynolds PR, Puzas JE, Keefe RJO. (1997). Modulation of the production of cytokines in titanium-stimulated human peripheral blood monocytes by pharmacological agents: the role of camp-mediated signaling mechanism. J. Bone Joint Surg; 79:15191528. 15. Oswald IP, Huffnagle GB. (1996). Afferent phase production of TNF-alpha is required for the development of protective T cell Immunity to Cryptococcus neuformans. J Immunol; 157:45294536. 4. Carl A DeAbate. (2000). MD Medical Director. Medical Research Center New Orleans, La USA. Respirotory Fluoroquinolone. Special interview, BIOMEDS international, Ltd. 5. Choi JH, Song MJ, Kim SH, Choi SM, Lee DG, Yoo JH, Shin WS. (2003). Effect of moxifloxacin on production of proinflammatory cytokines from 14. Old LJ. (1985). Tumor necrosis factor (TNF). Science; 230: 630-632. 16. Oswald IP, Wynn TA, Sher A. (1992). Interleukin 10 inhibits macrophage microbicidal activity by blocking the endogenous production of tumor necrosis factor alpha required as a costimulatory factor for interferon gamma–induced activation. Proc Nadt I Acad Sd USA; 89:8676-8680. 17. Parnham, Michael J. (2005). Immunomodulatory effects of antimicrobials in the therapy of respiratory tract infections. J Immunol; 18 (2): 125131. THE EGYPTIAN JOURNAL OF IMMUNOLOGY 18. Pinsky MR. (2001). Sepsis: A pro- and antiinflammatory disequilibrium syndrome. Contrib Nephrol; 132:354-366. 19. Stunkel KGE, Hewlett G, Zeler HJ. (1991). Cprofloxacin enhanced T cell function by modulating interleukin activities. Clin Exp Immunol; 86:525-531. 20. Tauber SC, Nau R. (2008). Immunomodulatory properties of antibiotics. Current Molecular Pharmacology; 1: 68-79. 21. Wei Shen L, Simon B, Robert G, Jamieson C, Jeune L, Macfarlane J, Read RC, Roberts HJ, Levy ML, 91 Wani M. (2009). The British Thoracic Society Guidelines for the management of community acquired pneumonia in adults Update 2009. Thorax. Vol 64 Supplement III. 22. Woodhead, Blasi F, Ewig S, Huchon G, Ieven M, Ortqvist A, Schaberg T, Torres A, van der Heijden G. (2005). Guidelines for the management of adult lower respiratory tract infections: Eur Respir J; 26:1138-1180. 23. Yoshimura T, Kurita C. (1996). Immunomodulatory action of Levofloxacin on cytokine production by human peripheral blood mononuclear cells. Chemotherapy; 42 (6):459-464. British Microbiology Research Journal 7(6): 288-305, 2015, Article no.BMRJ.2015.121 ISSN: 2231-0886 SCIENCEDOMAIN international www.sciencedomain.org 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 Mona Sallam Embarek Mohamed1*, Mohamed Ahmed El-Mokhtar1 and Alaa Thabet Hassan2 1 Department of Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt. Department of Chest Diseases, Assiut University Hospitals, Assiut, Egypt. 2 Authors’ contributions This work was carried out in collaboration between all authors. Authors MSEM and MAEM designed the study, performed the bacteriological and statistical analysis, wrote the protocol, and wrote the manuscript and managed literature searches. Author ATH performed the clinical assessment of patients, arranged the clinical data and participated in writing the manuscript. All authors read and approved the final manuscript. Article Information DOI: 10.9734/BMRJ/2015/16317 Editor(s): (1) Hung-Jen Liu, Institute of Molecular Biology, National Chung Hsing University, Taiwan. Reviewers: (1) Xiuhui Zhong, Institute of Traditional Chinese Veterinary Medicine, China. (2) Gulsen Meral, Kagithane State Hospital, Turkey. Complete Peer review History: http://www.sciencedomain.org/review-history.php?iid=993&id=8&aid=8767 th Original Research Article Received 25 January 2015 th Accepted 17 March 2015 th Published 10 April 2015 ABSTRACT 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 *Corresponding author: E-mail: [email protected]; Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 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. Keywords: Chronic obstructive pulmonary disease; infection exacerbation; antimicrobial resistance; Upper Egypt. ABBREVIATIONS Acin. baumannii AECOPD ANOVA CLSI COPD DCP DVT E coli EMB ESR FEV1 FVC GOLD H. influenzae I ICU IHD Kl. pneumoniae LTOT M. catarrahlis MDR MRSA MV PDR Ps. aeruginosa R RF S SD SPSS Staph. epidermidis Strep. pneumoniae VTE XDR Acinetobacter baumannii acute exacerbation of Chronic obstructive pulmonary disease Analysis of variance Clinical and Laboratory Standards Institute Chronic obstructive pulmonary disease decompensated Core-pulmonale deep venous thrombosis Escherichia coli Eosin Methylene Blue erythrocyte sedimentation rate Forced expiratory volume in the first second forced vital capacity Global Initiative for Obstructive Lung Disease Haemophilus influenzae intermediate Intensive Care Unit ischemic heart disease Klebsiella pneumoniae long term oxygen therapy Moraxella catarrahlis Multidrug-resistant methicillin resistant Staphylococcus aureus mechanical ventilation pandrug-resistant Pseudomonas aeruginosa resistant Respiratory failure susceptible Standard deviation Statistical package for social sciences Staphylococcus epidermidis Streptococcus pneumoniae Venous thromboembolism extensively drug-resistant 1. 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 289 Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 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. 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]. 2. MATERIALS AND METHODS 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. 2.1 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 2.2 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 (FEV1) 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]. 2.3 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. 2.4 Bacteriological Diagnosis 2.5 Identification of Bacterial Strains the Causative Samples were examined microscopically after staining with Gram´s stain and cultured directly on nutrient, blood, chocolate, mannitol salt, bile 299 Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 esculin, CHROMagar, MacConkey´s, and Eosin Methylene Blue (EMB) agar plates. The cultured plates were incubated aerobically at 37ºC for 2448 hours. Blood and chocolate agar plates were incubated at 35–36ºC with 5% CO2 for 48 hours for isolation of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis strains. Bacterial isolates were identified based on colonial morphology, Gram staining, and standard biochemical reactions according to the Bergey's Manual of Systematic Bacteriology [12]. 2.6 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]. 2.7 Statistical Analysis The SPSS program version 19.0 was used for the statistical analysis of data. Data were presented as mean and standard deviation or number and percentage as appropriate. A P value < 0.05 significant. was considered statistically 3. RESULTS 3.1 Characteristics of COPD Patients The study included 116 COPD patients consisted of 108 (93%) males and 8 (7%) females with age range 42-72 years (mean±SD, 57.6±8 years). Most (78; 67%) patients aged >55 years while, 38 (33%) patients aged 42-55 years. Eighty four (72%) patients were admitted to the Intensive Care Unit (ICU) while 32 (28%) patients were admitted to the Chest Department (Table 1). All patients were residents of Upper Egypt, primarily residents of Assiut (70%), followed by residents of the Governorates Qena (15%), Aswan (7%), Luxor (5%) and Sohag (3%). Of the 116 patients, 88 (76%) patients had very severe (stage IV) COPD, 18 (15%) patients had severe (stage III) COPD, 8 (7%) patients had moderate (stage II) COPD and 2 (1.7%) patients had mild (stage I) COPD. The duration of hospital stay ranged from 3-49 days (mean+/-SD, 14±9 days). It was significantly longer in patients with very severe versus those with severe, moderate, and mild COPD (ANOVA, P=0.024, P=0.001, and P=0.000, respectively) and in patients with severe COPD versus moderate and mild COPD (ANOVA, P=0.003 and P=0.000, respectively). The patients’ group (n=116) suffered from 167 infection exacerbations attacks during the study period from January to December 2013 with 79 (68%) patients experienced one attack/year, 24 (21%) patients experienced two attacks/year, 12 (10%) patients experienced three attacks/year, and one patient (~1%) experienced infection exacerbations four times / year (Table 1). The duration between attacks ranged from 3-225 days (mean±SD, 64.4±69.9 days). No significant difference was found regarding the frequency of exacerbation attacks / year or the duration between attacks with COPD stage. Eighty seven (75%) patients were smokers. Heavy-smokers were 52 (45%) patients, moderate-smokers were 33 (28%) patients, mild-smokers were 2 (2%) patients, ex-smokers were 17 (15%) patients, while 12 (10%) patients were non-smokers (Table 1). A significant positive correlation was observed between the COPD stage and the smoking index (r=0.438, P=0.000). A total of 104 (~90%) COPD patients had hypoxia. Mild, moderate, and severe hypoxia was detected in 18 (16%), 70 (60%), and 16 (14%) patients, respectively, while 12 (10%) patients had normal O2 tension. Hypercapnea was detected in 80 291 Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 (69%) COPD patients while, 36 (31%) patients had normal CO2 tension. Chest x ray was normal in stage I COPD patients and one patient in stage II, while characteristic COPD changes had been detected in all COPD stages III and IV cases. Chest x ray showed pneumonic infiltrates in 18 (15%) cases (Table 1). Cardiopulmonary co-morbidities were found in many COPD patients. Respiratory failure (RF) was found in 86 (74%) patients, decompensated Core-pulmonale (DCP) was detected in 58 (50%) of patients, 18 (16%) patients were managed with long term oxygen therapy (LTOT), 8 (7%) patients were under mechanical ventilation (MV). Other cardiopulmonary co-morbidities were detected in few patients; pleural effusion in 4 (3%) patients, alpha1-antitrypsin deficiency in 3 (~3%) patients, ischemic heart disease (IHD) and lung carcinoma were detected in 2 (~2%) patients each (Table 1). Laboratory tests showed that 78 (67%) COPD patients had high erythrocyte sedimentation rate (ESR), 44 (38%) patients had anemia, 30 (26%) patients were diabetic, 28 (24%) patients had leucocytosis, 12 (10%) patients had hypoalbuminemia, and 8 (7%) patients were hypertensive. Venous thromboembolism (VTE) was found in 13 (11%) patients with 6 (5%) patients suffered from pulmonary embolism and 7 (6%) patients had deep venous thrombosis (DVT). Impaired liver and renal functions were detected in 8 (7%) and 6 (5%) patients, respectively. Stage IV (very severe) COPD was significantly associated with the presence of DCP, RF, and high ESR (Fisher´s exact test, P=0.003, 0.003, and 0.001, respectively). Other co-morbidities had no significant association with COPD stage. Most (84%) of COPD patients were treated regularly with corticosteroids (Table 1). Thirty four (20%) exacerbation attacks detected in January, 28 (17%) of exacerbations detected in May, 25 (15%) in April, 22 (13%) during November, the least exacerbations were detected in September (4 attaks; 2%), August (3 attacks; 1.8%), and June (2 attacks; 1.2%) (Fig. 1). 3.2 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. The predominant bacterial strains were in decreasing order; Haemophilus influenzae (H. influenzae) (19.4%) that isolated in 22 attacks as a single pathogen and in 6 attacks combined with other pathogens. Escherichia coli (E. coli) were isolated in 18% of the attacks (singly in 18 attacks and mixed in 8 attacks), Streptococcus pneumoniae (Strep. pneumoniae) were found in 16.7% of attacks (singly in 20 attacks and mixed in 4 attacks). Other bacterial isolates were: Klebsiella pneumoniae (Kl. pneumoniae) (14%; singly in 14 attacks and mixed in 6 attacks), Streptococcus pyogenes (Strep. pyogenes) (10%; singly in 13 attacks and mixed in one attack), Each of Pseudomonas aeruginosa (Ps. aeruginosa) and, methicillin resistant Staphylococcus aureus (MRSA) were detected in 5.6% of the attacks (singly in 6 attacks and mixed in 2 attacks), Acinetobacter baumannii (Acin. baumannii) (4.2%; singly in 5 attacks and mixed in one attack), Moraxella catarrahlis (M. catarrahlis) (2.8%; singly in 4 attacks). Each of Staphylococcus epidermidis (Staph. epidermidis), Enterobacter, and Enterococci were detected in two (1.4%) attacks (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. 3.3 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 292 Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 strains. All (100%) M. Catarrahlis, Staph. epidermidis, and enterococcal strains were MDR. While 75%, 67%, 63%, 58%, and 57% of Ps. aeruginosa, Acin. baumannii, MRSA, Strep. pneumoniae, and Strep. pyogenes strains were MDR, respectively (Table 2). All Staph. aureus strains were methicillin and oxacillin-resistant. About 63% of the isolated Strep. pneumoniae strains were penicillin-resistant (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. 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). Most (84%) of H. influenzae strains were MDR, 2 (7%) strains were XDR, and another 2 (7%) strains were PDR. About 54% of detected E. coli strains were MDR, 31% of the isolates were XDR, and 15% of the isolates were PDR. About 40% of the isolated Kl. pneumoniae strains were MDR, while 60% were XDR and no isolates were PDR. XDR was found in all (100%) Enterobacter strains, 43% of Strep. pyogenes, 42% of Strep. pneumoniae, 37% of MRSA, 33% of Acin. baumannii, 25% of Ps. aeruginosa, 17% of Strep. pneumoniae, and 14% of Strep. pyogenes Number of infection exacerbation attacks 40 35 34 30 28 25 22 20 16 15 10 10 9 8 6 5 3 2 4 0 Jan Feb Mar April May June July Aug Sept Oct Nov Dec Fig. 1. Monthly distribution of COPD infection exacerbations during January-December, 2013 (n=167) 293 Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 30 25 20 20 stage IV stage III 16 16 15 20 14 stage II 10 12 stageI 4 8 4 2 6 2 4 2 2 8 5 4 6 2 2 2 2 2 2 2 2 0 Fig. 2. Bacterial strains detected during infection exacerbation of different COPD stages. Each number of bacterial-positive samples is represented both with a bar and absolute values in the abscissa. No bacterial isolates were detected in 24 attacks. Abbreviations: MRSA=methicillin resistant Staphylococcus aureus 80 67 70 60 50 MDR 40 XDR 28 PDR 30 15 4 18 20 10 6 1 1 2 2 stage II stage I 0 stage IV stage III Fig. 3. Antibiotic resistance patterns in different COPD stages (n=144 attacks) 294 Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 Table 1. Demographic and clinical characteristics of COPD patients (n=116) Patients’ characteristics Sex N (%) Female Male 8 (7) 108 (93) Assiut Sohag Qena Luxor Aswan 80 (70) 4 (3) 18 (15) 6 (5) 8 (7) ICU Chest Department 84 (72) 32 (28) I (mild) II (moderate) III (severe) IV (very severe) 2 (1.7) 8 (7) 18 (15) 88 (76) One attack Two attacks Three attacks Four attacks 79 (68) 24 (21) 12 (10) 1 (1) 0 (non-smokers) Ex-smokers Mild-smokers Moderate-smokers Heavy-smokers 12 (10) 17 (15) 2 (2) 33 (28) 52 (45) Normal Mild hypoxia Moderate hypoxia Severe hypoxia Normal Hypercapnea 12 (10) 18 (16) 70 (60) 16 (14) 36 (31) 80 (69) Geographicalarea Admission COPD stage Number of attacks (no=167)/year Smoking index Arterial blood gases O2 tension CO2 tension 295 87 (75) 104 (90) Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 Patients’ characteristics Chest X ray Normal chest x ray COPD changes * Pneumonic infiltrates (co-morbid pneumonia) Associated cardiopulmonary condition N (%) stage I: 2(2%); stage II: 1(0.9%) stage II:7(6%); stage III:18(15%); stage IV:88(76%) stage III: 2(2%) stage IV: 16(14%) 3 (2.9) 113 (97) DCP RF MV LTOT Pulmonary embolism Pleural effusion IHD Lung carcinoma Alpha1-antitrypsin deficiency 58 (50) 86 (74) 8 (7) 18 (16) 6 (5) 4 (3) 2 (2) 2 (2) 3 (3) Anaemia DM Hypoalbuminemia Leucocytosis Hypertension DVT Impaired liver function Impaired renal function Increased ESR Previous corticosteroid therapy 44 (38) 30 (26) 12 (10) 28 (24) 8 (7) 7 (6) 8 (7) 6 (5) 78 (67) 98 (84) 18 (15) Other systematic condition Bacteriological diagnosis Significant bacterial growth during exacerbations (no of patients=116/ no of attacks=167) Single etiological agent Mixed infection No bacterial growth 88 patients (76%)/143 attacks (86%) 72 patients (82%)/127 attacks (89%) 16 patients (18%)/ 16 attacks (11%) 18 patients (24%)/ 24 attacks (14%) Abbreviations: ICU=Intensive Care Unit; DCP=decompensated Core-pulmonale; RF=respiratory failure; MV=mechanical ventilation; LTOT=long term oxygen therapy; IHD=ischemic heart disease; DM=diabetes mellitus; DVT=deep venous thrombosis; ESR=erythrocyte sedimentation rate. * COPD changes include: hyperinflation of the lung, increase bronchovascular marking, low flattened diaphragm, ribbon-shaped heart, and cardiomegaly 296 5 1 6 7 8 9 10 11 12 MDR XDR PDR linezolid imipenem aztreonam ND ND ND ND ND ND 14(50) 22(79) 5(18) 24(86) 2(7) 2(7) 18(64) 24(86) 2(7) 18(69) ND ND ND ND ND ND ND ND ND 20(77) 22(85) 13(50) 18(69) 4(15) 4(15) 24(92) 14(54) 8(31) 4(15) 12(60) ND ND ND ND ND ND ND ND ND 14(70) 16(80) 10(50) 14(70) 0(0) 0(0) 14(70) 8(40) 12(60) 0(0) 4(50) ND ND ND ND ND ND ND ND ND 6(75) 6(75) 5(63) 6(75) 1(12.5) 0(0) 8(100) 6(75) 2(25) 0(0) 6(100) ND ND ND ND ND ND ND ND ND 0(0) 0(0) 0(0) 6(100) 4(67) 2(33) 0(0) 0(0) ND ND ND ND ND ND ND ND ND 4(100) 4(100) 4(100) 4(100) 0(0) 0(0) 4(100) 4(100) 0(0) 2(100) ND ND ND ND ND ND ND ND ND 2(100) 2(100) 2(100) 2(100) 1(50) 2(100) 2(100) 0(0) 2(7) 0(0) 2(100) 0(0) 6(25) 15(63) 22(92) 22(92) 17(71) 12(50) 16(67) 14(58) 16(67) 0(0) ND ND ND ND ND ND ND 14(58) 10(42) 0(0) 6(43) 10(71) 12(86) 12(86) 10(71) 10(71) 12(86) 10(71) 8(57) 0(0) ND ND ND ND ND ND ND 8(57) 6(43) 0(0) 0(0) 0(0) ND ND ND ND ND ND ND 2(100) 0(0) 6(75) 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) 4(50) 3(38) 0(0) ND ND ND ND ND ND ND 5(63) 3(37) 0(0) 0(0) ND ND ND ND ND ND 2(100) 0(0) 0(0) 0(0) 0(0) 2(100) 2(100) 0(0) 2(100) 2(100) 2(100) 2(100) 0(0) 0(0) 0(0) 2(100) 2(100) 2(100) 0(0) ND 64(44) 35(70) 44(88) 44(88) 39(78) 32(64) 38(76) 30(60) 29(58) 0(0) 60(64) 78(83) 41(44) 70(75) 8(8.5) 0(0) 0(0) 8(8.5) 76(81) 91(63) 47(33) 6(4) Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 teicoplanin ND Table 2. Resistance patterns of isolated bacterial strains to antimicrobial agents vancomycin ND 6(100) 2(33) 2(33) meropenem erythromycin gentamicin bacitracin amikacin carbencillin tobramycin methicillin neomycin oxacillin penicillin doxycycline 297 4(14) ND** Bacterial isolates 1* Total 2 3 4 lomefloxacin 24(86) 10(36) 24(86) 18(64) 26(93) E. coli 26 (18) 26(100) 22(85) 12(46) 22(85) 22(85) 20(77) 22(85) 26(100) 26(100) 16(62) 16(62) 22(85) Kl. pneumoniae 20 (14) 20(100) 16(80) 10(50) 14(70) 16(80) 12(60) 14(70) 16(80) 16(80) 13(65) 12(60) 12(60) Ps. aeruginosa 8 (5.6) 8(100) 6(75) 4(50) 8(100) 8(100) 0(0) 6(75) 0(0) Acin. baumannii 6(4.2) 6(100) 6(100) 6(100) 6(100) 6(100) 6(100) 6(100) 6(100) 6(100) 6(100) 6(100) 6(100) Mor.catarrahlis 4 (2.8) 4(100) 4(100) 0(0) 4(100) 4(100) 2(50) 2(50) 0(0) Enterobacter 2 (1.4) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) Str. pneumoniae 24 (16.7) 22(92) 14(58) 12(50) 20(83) 20(83) 14(58) 22(92) 18(75) 16(67) 14(58) 18(75) 20(83) Str.pyogenes 14 (10) 14(100) 8(57) 4(29) 12(86) 6(43) 6(43) 14(100) 12(86) 14(100) 4(29) 12(86) 14(100) Staph.epidermidis 2 (1.4) 2(100) 2(100) 0(0) 2(100) 2(100) 0(0) 2(100) MRSA 8 (5.6) 8(100) 4(50) 4(50) 8(100) 8(100) Enterococci 2 (1.4) 0(0) 0(0) 0(0) 0(0) 0(0) Total 144(100) 140(97) 100(69) 58(48) 110(76) 118(82) 80(56) 124(86) 116(81) 124(86) 71(49) 91(63) 108(75) 8(29) 10(36) 14(50) 2(25) 3(75) 2(50) 2(100) 2(100) 0(0) 8(100) 6(75) 8(100) 8(100) 6(75) 8(100) 8(100) 0(0) 0(0) 2(100) 2(100) 2(100) 0(0) 0(0) 0(0) 6(75) 0(0) Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 2(50) ofloxacin 12(43) 6(75) levofloxacin ciprofloxacin 4(14) 4(100) cefotaxime cefepime 28 (19.4) 28(100) 16(57) 8(100) cefaclor ceftriaxone ceprodoxime chloramphenicol amoxicillin / clavulanic amoxicillin * 1-penicillins and penicillin combinations; 2-phenicols; 3-cephalosporines; 4-fluoroquinolones; 5-tetracyclines; 6polypeptides; 7-macrolides; 8-glycopeptides; 9-oxazolidinones; 10-aminoglycosides; 11-carbapenems; 12monobactams.**ND=not determined 298 H. influenzae No (%) Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 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 299 Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 with previous works in Egypt [35] and other countries [36,37,38,39,40]. Strains of H. influenzae stimulate mucus hypersecretion and inhibit ciliary beat frequency. Furthermore, they can cause direct epithelial damage and their endotoxin increase epithelial expression of the pro-inflammatory cytokines thus providing potential mechanisms to upregulate the process of inflammation in COPD [41]. 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 300 Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 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]. 2. 3. 4. 5. 6. 7. 5. CONCLUSION From our results we concluded that, Gramnegative bacilli are the leading pathogens in patients with AECOPD in Upper Egypt with predominance of H. influenzae. Our bacteriological profiles highlighted the distribution of other pathogens, including E. coli, Strep. pneumoniae, and Kl. pneumoniae in AECOPD. The isolated bacterial strains characterized by high resistance rates to most groups of antimicrobials. Sensitivity was relatively high to the carbapenem group. COMPETING INTERESTS 8. 9. 10. 11. The authors have declared that no competing interests exist. REFERENCES 1. Burge S, exacerbations: 12. JA. Wedzicha Definitions COPD and 301 classifications. Eur Respir J Suppl. 2003; 41:46-53. Andersson F, Borg S, Jansson SA, Jonsson AC, Ericsson A, Prütz C, Rönmark E, Lundbäck B. The costs of exacerbations in chronic obstructive pulmonary disease (COPD). Respir Med. 2002;96(9):700-8. Mohan A, Premanand R, Reddy LN, Rao MH, Sharma SK, Kamity R, Bollineni S. Clinical presentation and predictors of outcome in patients with severe acute exacerbation of chronic obstructive pulmonary disease requiring admission to intensive care unit. BMC Pulm Med. 2006; 6:27. Donaldson GC, Seemungal TA, Bhowmik A, Wedzicha JA. Relationship between exacerbation frequency and lung function decline in chronic obstructive pulmonary disease. Thorax. 2002;57:847-52. Kyd JM, McGrath J, Krishnamurthy A. Mechanisms of bacterial resistance to antibiotics in infections of COPD patients. Curr Drug Targets. 2011;12(4):521-30. Qaseem A, Wilt TJ, Weinberger SE. Diagnosis and management of stable chronic obstructive pulmonary disease: A clinical practice guideline update from the American College of Physicians, American College of Chest Physicians, American Thoracic Society, and European Respiratory Society. Ann Intern Med. 2011; 155(3):179-91 Decramer M, Janssens W, Miravitlles M. Chronic obstructive pulmonary disease. The Lancet. 2012;379(9823):1341-1351. Makris D, Bouros D. COPD exacerbation: lost in translation. BMC Pulm Med. 2009; 29(9):6. Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease; 2014. Available:http://www.goldcopd.org Indrayan A, Kumar R, Dwivedi S. A simple index of smoking; 2009. Available:http://biostats.bepress.com/cobra /ps/art40 Henig NR, Tonelli MR, Pier MV, Burns JL, Aitken ML. Sputum induction as a research tool for sampling the airways of subjects with cystic fibrosis. Thorax. 2001;56(4): 306-11. Holt JG, Krieg NR, Sneath PHA, Staley H, Williams ST. Bergey's manual of Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 th 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. determinative bacteriology, 9 ed. Williams and Wilkins, Baltimore, MD; 1994. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests, 9th ed. Approved standard. M2-A9. CLSI, Wayne, Pennsylvania; 2006. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. Multidrug-resistant, extensively drug-resistant and pandrugresistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268-81. Soler-Cataluña JJ, Martínez-García MÁ, Román Sánchez P, Salcedo E, Navarro M, Ochando R. Severe acute exacerbations and mortality in patients with chronic obstructive pulmonary disease. Thorax. 2005;60:925–31. Rodriguez-Roisin R. Toward a consensus definition for COPD exacerbations. Chest. 2000;117(5 Suppl 2):398-401. Finkelstein J, Cha E, Scharf SM. Chronic obstructive pulmonary disease as an independent risk factor for cardiovascular morbidity. Int J Chron Obstruct Pulmon Dis. 2009;4:337-49. Donaldson GC, Seemungal TA, Patel IS, Bhowmik A, Wilkinson TM, Hurst JR, Maccallum PK, Wedzicha JA. Airway and systemic inflammation and decline in lung function in patients with COPD. Chest. 2005;128:1995–2004. Khilnani GC, Banga A, Sharma SK. Predictors of mortality of patients with acute respiratory failure secondary to chronic obstructive pulmonary disease admitted to an intensive care unit: A one year study. BMC Pulm Med. 2004;4:12. Hunter MH, King DE. COPD: management of acute exacerbations and chronic stable disease. Am Fam Physician. 2001; 64(4):603-12. Bolliger CT, Zellweger JP, Danielsson T, van Biljon X, Robidou A, Westin A, Perruchoud AP, Säwe U. Influence of longterm smoking reduction on health risk markers and quality of life. Nicotine Tob Res. 2002;4:433–9. Min JJ, Huo XL, Xiang LY, Qin YQ, Chai KQ, Wu B, Jin L, Wang XT. Protective effect of Dl-3n-butylphthalide on learning 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 302 and memory impairment induced by chronic intermittent hypoxia-hypercapnia exposure. Sci Rep. 2014;4:5555. Ringbaek TJ, Viskum K, Lange P. Does long-term oxygen therapy reduce hospitalisation in hypoxaemic chronic obstructive pulmonary disease? Euro Resp J. 2002;20:38-42. Kamei T, Yamamoto Y, Kajii F, Nakayama Y, Kawakami C. Systematic review and meta-analysis of studies involving telehome monitoring-based telenursing for patients with chronic obstructive pulmonary disease. Jpn J Nurs Sci. 2013;10(2):18092. Nowiński A, Kamiński D, Korzybski D, Stokłosa A, Górecka D. The impact of comorbidities on the length of hospital treatment in patients with chronic obstructive pulmonary disease. Pneumonol Alergol Pol. 2011;79(6):388-96. Wedzicha JA. Oral corticosteroids for exacerbations of chronic obstructive pulmonary disease. Thorax. 2000; 55(Suppl 1):23-7. McEvoy CE, Niewoehner DE. Adverse effects of corticosteroid therapy for COPD. Chest. 1997;111:732-43. de la Iglesia Martínez F, Pellicer Vázquez C, Ramos Polledo V, Nicolás Miguel R, Pita Fernández S, Diz-Lois Martínez F. Chronic obstructive pulmonary disease and the seasons of the year. Arch Bronconeumol. 2000;36(2):84-9. Jenkins CR, Celli B, Anderson JA, Ferguson GT, Jones PW, Vestbo J, Yates JC, Calverley PM. Seasonality and determinants of moderate and severe COPD exacerbations in the TORCH study. Eur Respir J. 2012;39(1):38-45. Rabe KF, Fabbri LM, Vogelmeier C, Kögler H, Schmidt H, Beeh KM, Glaab T. Seasonal distribution of COPD exacerbations in the Prevention of Exacerbations with Tiotropium in COPD trial. Chest. 2013;143(3):711-9. Madhavi S, Rama Rao MV, Janardhan Rao R. Bacterial etiology of acute exacerbations of chronic obstructive pulmonary. J. Microbiol. Biotech. Res. 2012;2(3):440-444. Sethi S, Murphy TF. Bacterial infection in chronic obstructive pulmonary disease in 2000: A state-of-the-art review. Clin Microbiol Rev. 2001;14(2):336-63. Siripataravanit S, Phaicharoen R, Termsetcharoen S, Klangprapun N. Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 34. 35. 36. 37. 38. 39. 40. 41. Bacteria associated with acute exacerbations of chronic obstructive pulmonary disease requiring mechanical ventilation and antimicrobial management in Respiratory Care Unit of Central Chest Institute of Thailand. J Med Assoc Thai. 2012;95(Suppl 8):11-8. Guo Z, Chang C, Chen Y, Zheng Y, Yao W. Bacteriology in acute exacerbation in patients hospitalized frequently for acute exacerbation of chronic obstructive pulmonary disease. Zhonghua Yi Xue Za Zhi. 2014;94(10):729-32. Agmy G, Mohamed S, Gad Y, Farghally E, Mohammedin H, Rashed H. Bacterial profile, antibiotic sensitivity and resistance of lower respiratory tract infections in upper egypt. Mediterr J Hematol Infect Dis. 2013;5(1):e2013056. Ko FW, Ng TK, Li TS, Fok JP, Chan MC, Wu AK, Hui DS. Sputum bacteriology in patients with acute exacerbations of COPD in Hong Kong. Respir Med. 2005; 99(4):454-60. Erkan L, Uzun O, Findik S, Katar D, Sanic A, Atici AG. Role of bacteria in acute exacerbations of chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis. 2008;3(3):463-7. Larsen MV, Janner JH, Nielsen SD, FriisMøller A, Ringbaek T, Lange P. Bacteriology in acute exacerbation of chronic obstructive pulmonary disease in patients admitted to hospital. Scand J Infect Dis. 2009;41(1):26-32. Molyneaux PL1, Mallia P, Cox MJ, Footitt J, Willis-Owen SA, Homola D, TrujilloTorralbo MB, Elkin S, Kon OM, Cookson WO, Moffatt MF, Johnston SL. Outgrowth of the bacterial airway microbiome after rhinovirus exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2013;188(10):122431. Barker BL, Haldar K, Patel H, Pavord ID, Barer MR, Brightling CE, Bafadhel M. Association between pathogens detected using quantitative polymerase chain reaction with airway inflammation in COPD at stable state and exacerbations. Chest; 2014. DOI: 10.1378/chest.14-0764. [Epub ahead of print] White AJ, Gompertz S, Stockley RA. Chronic obstructive pulmonary disease: The aetiology of exacerbations of chronic obstructive pulmonary disease. Thorax. 2003;58(1):73-80. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 303 Reissig A, Mempel C, Schumacher U, Copetti R, Gross F, Aliberti S. Microbiological diagnosis and antibiotic therapy in patients with communityacquired pneumonia and acute COPD exacerbation in daily clinical practice: comparison to current guidelines. Lung. 2013;191(3):239-46. Bogaert D, van der Valk P, Ramdin R, Sluijter M, Monninkhof E, Hendrix R, de Groot R, Hermans PW. Host-pathogen interaction during pneumococcal infection in patients with chronic obstructive pulmonary disease. Infect Immun. 2004; 72:818-23. Sethi S, Evans N, Grant BJ, Murphy TF. New strains of bacteria and exacerbations of chronic obstructive pulmonary disease. N Engl J Med. 2002;347:465-71. Kontiainen S, Sivonen A. Optochin resistance in Streptococcus pneumoniae strains isolated from blood and middle ear fluid. Eur. J. Clin. Microbiol. 1987;6:422424. Nagata M, Ueda O, Shobuike T, Muratani T, Aoki Y, Miyamoto H. Emergence of optochin resistance among Streptococcus pneumoniae in Japan. Open J. Med. Microbiol. 2012;2:8-15. Furqan S, Paracha SA. Frequency of Streptococcus pneumonia and Haemophilus influenza in acute exacerbation of chronic obstructive airway disease and their sensitivity to levofloxacin. J Pak Med Assoc. 2014;64(4):399-402. Klapdor B, Ewig S. Antimicrobial treatment of patients with severe acute exacerbation of COPD. Med Klin Intensivmed Notfmed. 2012;107(3):179-84. Groenewegen KH, Wouters EF. Bacterial infections in patients requiring admission for an acute exacerbation of COPD; a 1year prospective study. Respir Med. 2003; 97:770–7. Cukic V. The most common detected bacteria in sputum of patients with the acute exacerbation of COPD. Mater Sociomed. 2013;25(4):226-9. ElKorashy RI, El-Sherif RH. Gram negative organisms as a cause of acute exacerbation of COPD. Egyptian Journal of Chest Diseases and Tuberculosis. 2014; 63:345-349. Miravitlles M, Espinosa C, Fernandez-Laso E, Martos JA, Maldonado JA, Gallego M. Relationship between bacterial flora in sputum and functional impairment in Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. patients with acute exacerbations of COPD Patients Study Group. Levofloxacin COPD. Study Group of Bacterial Infection versus clarithromycin in COPD in COPD. Chest. 1990;116:40-6. exacerbation: Focus on exacerbation-free González-Castillo J, Cenci C, Rodriguezinterval. Eur Respir J. 2004;24(6):947-53. Adrada E, Candel FJ, de la Torre-Misiego 64. Blasi F. Therapeutic experience with F, Fernández C, Martín-Sánchez FJ. levofloxacin in pneumonia and COPD. Staphylococcus aureus infections and Infez Med. 2009;17(Suppl5):23-8. factors associated with resistance to 65. Ye F, He LX, Cai BQ, Wen FQ, Chen BY, methicillin in a hospital emergency Hadiarto M, Chen RC, Yuan JP, Sun HL. department. Rev Esp Quimioter. 2013; Spectrum and antimicrobial resistance of 26(4):337-45. common pathogenic bacteria isolated from Lin SH, Kuo PH, Hsueh PR, Yang PC, Kuo patients with acute exacerbation of chronic SH. Sputum bacteriology in hospitalized obstructive pulmonary disease in mainland patients with acute exacerbation of chronic of China. Chin Med J (Engl). 2013; obstructive pulmonary disease in Taiwan 126(12):2207-14. with an emphasis on Klebsiella 66. Liu PY, Sun YX, Gu DQ, Cheng JL. [Drug pneumoniae and Pseudomonas resistance of imipenem-resistant Gramaeruginosa. Respirology. 2007;12(1):81-7. negative bacilli in coal worker's Falagas ME, Karveli EA, Kelesidis I, pneumoconiosis chronic obstructive Kelesidis T. Community-acquired pulmonary disease patients with lower Community-acquired Acinetobacter respiratory tract infection]. Zhonghua Lao infections. Eur J Clin Microbiol Infect Dis. Dong Wei Sheng Zhi Ye Bing Za Zhi. 2007;26:857-868. 2013;31(9):700-2. Bari MR, Hiron MM, Zaman SM, Rahman 67. Anvari MS, Naderan M, Boroumand MA, MM, Ganguly KC. Microbes responsible for Shoar S, Bakhshi R, Naderan M. acute exacerbation of COPD. Mymensingh Microbiologic spectrum and antibiotic Med J. 2010;19(4):576-85. susceptibility pattern among patients with Wright PW, Wallace RJ, Shepherd JR. A urinary and respiratory tract infection. Int J descriptive study of 42 cases of Microbiol. 2014;682304. Branhamella catarrhalis pneumonia. Am J 68. Senbayrak Akcay S, Inan A, Cevan S, Med. 1990;88:2-8. Ozaydın AN, Cobanoglu N, Ozyurek SC, Catlin. Branhamella catarrhalisis: An Aksaray S. Gram-negative bacilli causing organism gaining respect as a pathogen. infections in an intensive care unit of a Clin Microbiol Rev. 1990;3:293-320. tertiary care hospital in Istanbul, Turkey. J Karalus R, Campagnari A. Moraxella Infect Dev Ctries. 2014;8(5):597-604. catarrhalis. A review of an important 69. Flamm RK, Sader HS, Jones RN. mucosal pathogen. Microbes Infect. 2000; Ceftaroline activity against organisms 2:547-9. isolated from respiratory tract infections in Li XJ, Li Q, Si LY, Yuan QY. USA hospitals: Results from the AWARE Bacteriological differences between COPD Program, 2009-2011. Diagn Microbiol exacerbation and community-acquired Infect Dis. 2014;78(4):437-42. pneumonia. Respir Care. 2011;56(11): 70. Rakesh G, Kasturi T, Yuvarajan S. 181824. Bacterial agents causing acute Canut A, Martín-Herrero JE, Labora A, exacerbations in Chronic Obstructive Maortua H. What are the most appropriate Pulmonary Disease (COPD) patients, their antibiotics for the treatment of acute antibiograms to Extended Spectrum Betaexacerbation of chronic obstructive Lactamases (ESBL) production in a tertiary pulmonary disease? A therapeutic care hospital, India. Int. J. Curr. Microbiol. outcomes model. J Antimicrob Chemother. App. Sci. 2013;2(11):273-282. 2007;60(3):605-12. 71. First report on antibiotic use and resistance Butorac-Petanjek B, Parnham MJ, in Viet Nam, ministry of health. Popovic-Grle S. Antibiotic therapy for Available;http://benhnhietdoi.vn/su-dungexacerbations of chronic obstructive khang-sinh/ pulmonary disease (COPD). J Chemother. 72. Thwaites GE. The management of 2010;22(5):291-7. Staphylococcus aureus bacteremia in the United Kingdom and Vietnam: A multiLode H, Eller J, Linnhoff A, Ioanas M. Evaluation of Therapy-Free Interval in 304 Mohamed et al.; BMRJ, 7(6): 288-305, 2015; Article no.BMRJ.2015.121 73. 74. 75. 76. centre evaluation. PLoS One. 2010; 5(12):14170 Wise R, Andrews JM, Boswell FJ, Ashby JP. The in-vitro activity of linezolid (U100766) and tentative breakpoints. J Antimicrob Chemother. 1998;42(6):721-8. Johnson AP, Warner M, Livermore DM. Activity of linezolid against multi-resistant gram-positive bacteria from diverse hospitals in the United Kingdom. J Antimicrob Chemother. 2000;45(2):225-30. Nseir S, Di Pompeo C, Cavestri B, Jozefowicz E, Nyunga M, Soubrier S, Roussel-Delvallez M, Saulnier F, Mathieu D, Durocher A. Multiple-drug-resistant bacteria in patients with severe acute exacerbation of chronic obstructive pulmonary disease: Prevalence, risk factors, and outcome. Crit Care Med. 2006;34(12):2959-66. Nguyen KV, Thi Do NT, Chandna A, Nguyen TV, Pham CV, Doan PM, Nguyen AQ, Thi Nguyen CK, Larsson M, Escalante S, Olowokure B, Laxminarayan R, Gelband H, Horby P, Thi Ngo HB, Hoang MT, Farrar J, Hien TT, Wertheim HF. Antibiotic use and resistance in emerging economies: A situation analysis for Viet Nam. BMC Public Health. 2013;13:1158. 77. Wnag L, Yang C, Zhang Q, Han B, Zhuang JJ, Chen M, Zou N, Li J, Duan MH, Zhang W, Zhu TN, Xu Y, Wang SJ, Zhou DB, Zhao YQ, Zhang H, Wang P, Xu YC. Prevalence and features of pathogenic bacteria in the department of hematology without bone marrow transplantation in Peking Union Medical College Hospital from 2010 to 2012. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2014;36(4):439-45. 78. Xia YL, Ge M, Wang Z. Pathogenic analysis of ventilator-associated pneumonia in the pediatric intensive care unit in high-altitude areas. Zhongguo Dang Dai Er Ke Za Zhi. 2014;16(8):787-90. Chaisathaphol T, Chayakulkeeree M. Epidemiology of infections caused by multidrug-resistant gram-negative bacteria in adult hospitalized patients at Siriraj Hospital. J Med Assoc Thai. 2014; 97(Suppl 3):35-45. Fogarty CM, Kohno S, Buchanan P, Aubier M, Baz M. Community-acquired respiratory tract infections caused by resistant pneumococci: Clinical and bacteriological efficacy of the ketolide telithromycin. J Antimicrob Chemother. 2003;51(4):947-55. 79. 80. © 2015 Mohamed et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history.php?iid=993&id=8&aid=8767 305 Bacteriology & Parasitology Mohamed et al., J Bacteriol Parasitol 2016, 7:1 http://dx.doi.org/10.4172/2155-9597.1000255 Research Article Open Access Allovahlkampfia spelaea is a Potential Environmental Host for Pathogenic Bacteria Mona Embarek Mohamed1*, Enas Abdelhameed Mahmoud Huseein2, Haiam Mohamed Farrag2, Fatma Abdel Aziz Mostafa3 and Alaa Thabet Hassan4 1Department of Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt 2Department of Parasitology, Faculty of Medicine, Assiut University, Assiut, Egypt 3Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza, Egypt 4Department of Chest Diseases, Assiut University Hospitals, Assiut, Egypt *Corresponding author: Mona Embarek Mohamed, Department of Microbiology and Immunology, Faculty of Medicine, Assiut University, 71515 Assiut, Egypt, Tel: +20-882413500/2411899; Fax: +20882333327, E-mail: [email protected] Received date: November 19, 2015; Accepted date: December 28, 2015; Published date: January 04, 2016 Copyright: © 2016 Mohamed ME, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Allovahlkampfia spelaea was identified for the first time in 2009. As a free living amoeba, it has been suggested to be a protective host for some bacterial pathogens against harsh environmental conditions and can transmit them to vulnerable hosts. We aimed in this study to test the interactions between Allovahlkampfia spelaea and some waterborne and foodborne bacteria and unravel if the tested bacteria can survive and multiply inside amoeba. We used a keratitis isolate of Allovahlkampfia spelaea grown in PYG medium containing proteose peptone, yeast extracts, and glucose. We examined amoeba interactions with Methicillin resistant Staphylococcus aureus, Escherichia coli 1, Klebsiella pneumoniae, Enterobacter aerogenes, Citrobacter cloaca, Proteus mirabilis, Raoultella terrigena, Raoultella ornitholytica, Aeromonas hydrophila and Pseudomonas aeruginosa using the co-culture assays. Amoebal survival rate with different bacterial strains were determined. With the exception of Proteus mirabilis that showed decreased survival rates inside amoebal cells, other bacterial isolates could survive and multiply inside Allovahlkampfia spelaea that was associated with decreased survival rates of the amoeba. Particularly, Pseudomonas aeruginosa, Aeromonas hydrophila and MRSA exhibited significantly increased multiplication rates inside amoeba. Our study demonstrated that Allovahlkampfia spelaea may act as a replicative host for pathogenic bacteria with environmental and clinical implications. Keywords: Allovahlkampia spelaea; Amoeba-bacteria interactions; Co-culture experiments; Gram negative bacteria; Intracellular survival Introduction Allovahlkampia spelaea (A. spelaea) belongs to the genus Allovahlkampia in the family Vahlkampiidae, class Heterolobosea, and Phylum Percolozoa [1] that was identiied for the irst time in 2009 [2]. A. spelaea is a free living amoeba (FLA) and has been found to be associated with keratitis as evident in a research conducted by Tolba et al. FLA is ubiquitous in nature [3] and some of them produce serious human infections [4]. Amoeba in nature may have contact with other microorganisms including bacteria. Amoebae are the dominant bacterial consumers, contributing to recycling of nutrients and maintaining the structure of the microbial community [5]. Most FLA genera are characterized by a biphasic life cycle consisting of a vegetative trophozoite stage and a physiologically static cyst stage [2]. Cysts are highly resistant and remain viable (and infective) for several years which facilitates spreading and colonization of new ecological niches [6]. On the other hand, bacteria have developed several antipredator strategies including cell size reduction, modiied cell morphology, modiication of cell wall characteristics, high-speed motility, bioilm or microcolony formation, and production of exopolymers or toxins [7]. In this case, amoebae may act as a protective host for some bacterial pathogens against harsh environmental conditions that normally kill. he role of FLA in survival and protection of pathogenic bacteria is increasingly J Bacteriol Parasitol ISSN:2155-9597 JBP, an open access journal recognized [8]. he amoebae aid in bacterial transmission to susceptible hosts thus constitutes a problem to the ecosystem health [4]. Additionally, bacteria become more resistant to disinfectants [5,8,9]. Many microorganisms are known to be hosted by FLA including; Acinetobacter spp., Aeromonas spp., Enterobacter spp., Escherichia coli (E. coli), Klebsiella pneumonia (Kl. pneumonia), Pseudomonas aeruginosa (Ps. aeruginosa), Salmonella spp., and Staphylococcus aureus (Staph. aureus) [10]. he hypothesis in our study was that A. spelaea may play a role for survival and multiplication of bacterial pathogens. Materials and Methods Molecular characterization of A. spelaea that was conducted at the Department of Medical Genomics, Graduate School of Frontier Sciences, he University of Tokyo, Japan. Culture and molecular characterization of A. spelaea A. spelaea used in this study was obtained from a patient with keratitis. A. spelaea was isolated on 1.5% non-nutrient agar made with Page’s saline (PAS) and seeded with E. coli kept at 30°C for 7 days. Cultures were examined using inverted microscope for presence of FLA and subcultured every 10 to 14 days by inverting a slice on a new agar plate as described previously [11]. he morphology of the trophozoite and cysts (non-stained and Giemsa’s Stained) were identiied using light microscope and inverted microscope according to Smirnov and Goodkov [12]. Molecular characterization of A. Volume 7 • Issue 1 • 1000255 Citation: Mohamed ME, Huseein EA, Farrag HM, Mostafa FAA, Hassan AT (2016) Allovahlkampfia spelaea is a Potential Environmental Host for Pathogenic Bacteria. J Bacteriol Parasitol 7: 255. doi:10.4172/2155-9597.1000255 Page 2 of 7 spelaea by polymerase chain reaction (PCR) of the 18S ribosomal RNA and sequencing was performed using primers described previously [11,13-15]. Bacterial cultures and antibiotic susceptibility tests Bacterial strains used in this study were isolated from cases with lower respiratory tract and urinary tract infections. he tested strains were Methicillin-resistant Staph. aureus (MRSA), Enterobacteriaceae [E. coli 1, Kl. pneumoniae, Enterobacter aerogenes (E. aerogenes), Citrobacter cloaca (C. cloacae), Proteus mirabilis (Pr mirabilis), Raoultella terrigena (R. terrigena), Raoultella ornitholytica (R. ornitholytica)], and other Gram negative bacteria; Aeromonas hydrophila (A. hydrophila) and Ps. aeruginosa. Gram negative bacteria were identiied up to the species level by API 20E system (BioMérieux, France) while detection of MRSA based on colonial morphology, Gram staining, and standard biochemical reactions according to the Bergey's Manual of Systematic Bacteriology [16]. Our Gram negative bacteria were all sensitive to imipenem and meropenem (Oxoid, England) while MRSA were sensitive to linezolid. Susceptibility tests were performed using the disk difusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines [17]. Bacteria were cultured in brain heart infusion broth overnight at 37°C without shaking prior to experimentation and were used at the stationary growth phase. Co-culture experiments A. spelaea was grown without shaking in 15 ml PYG medium (0.75 %, w/v, proteose peptone; 0.75 %, w/v, yeast extract; 1.5%, w/v, glucose) in tissue culture lasks at 30°C, as described previously [18]. he medium being refreshed 17-20h prior to all experimentation. his resulted in more than 95% of the amoebae in the trophozoite form. Supernatants from A. spelaea cultures were centrifuged at 2800×g for 30 min. New pellets were resuspended in PYG before being processed to recover cultivable bacteria. Co-culture experiments were performed with a slight modiication to a previous method [19]. A. spelaea was incubated in a concentration of 1×106 amoebae/mL PYG medium/well in 24 well plates until conluent. he cells were washed once with PAS. Next, Diferent bacterial strains were added in a concentration of 1×107 colony forming units (c.f.u)/well/mL PYG giving a multiplicity of infection (MOI) of 10. PH was adjusted to 7.2 and the plates were incubated for 1 h at 30°C to permit bacterial uptake. To kill residual extracellular bacteria, medium was replaced with PYG supplemented with 16 mg/L imipenem, a concentration greater than the highest MIC observed for all Gram-negative strains and linezolid in concentration of 1.5 µg/ml was used for MRSA. Plates were incubated for 1 h at 30°C. Antibiotics were removed by washing three-times with PAS. At the inal wash, the discarded supernatants were also plated onto nutrient agar plates to determine bacterial presence and 100 μl fresh PAS were added to wells. he microtiter plates were incubated again at 30°C (designated time 0 h). he wells were processed at 0 h, 8 h, 24 h, 48 h, and 72 h. To count the extracellular bacteria, PAS was carefully aspirated and sampled. To determine the number of intracellular bacteria, 100 μl of fresh PAS were added to the wells and the surface of each well bottom was scraped to remove adherent cells. Finally, amoebae were lysed by adding sodium dodecyl sulphate (SDS) in 0.5% inal concentration to each well for 20 min, and the number of bacteria was enumerated by plating on nutrient agar plates [20]. A. spelaea viability was monitored using the eosin dye exclusion assay using light and inverted microscope according to Wang and Ahearn [21]. Statistical analysis he SPSS program version 20.0 was used for the statistical analysis of data. Data were presented as number and percentage, or mean ± SD as appropriate. ANOVA test was used before data were transformed (Log10), p value <0.05 was considered statistically signiicant. Results and Discussion As newly discovered in 2009 in the karst caves of Slovenia, data on A. spelaea (Figure 1) and their interactions with bacteria are lacking. For identiication and characterization of A. spelaea, we depended upon 18S rRNA gene sequencing that revealed our strain to be A. spelaea strain SK1. Being a member of FLA, the role of A. spelaea in survival and multiplication of pathogenic bacteria should be considered. So, we aimed in this study to investigate survival and/or multiplication of the tested bacteria inside amoeba cells. Our bacterial strains were isolated from cases of urinary and respiratory tract infections and the majority of them are known to be natural contaminants of the water and food systems. Figure 1: Representative Micrograph of Allovahlkampia spelaea trophozoite (a) living, (b) dead stained with eosin stain, and (c) cyst with perinuclear ring (white arrow: living; black arrow: dead stained with eosin stain) (oil immersion x100 objective lens). J Bacteriol Parasitol ISSN:2155-9597 JBP, an open access journal Volume 7 • Issue 1 • 1000255 Citation: Mohamed ME, Huseein EA, Farrag HM, Mostafa FAA, Hassan AT (2016) Allovahlkampfia spelaea is a Potential Environmental Host for Pathogenic Bacteria. J Bacteriol Parasitol 7: 255. doi:10.4172/2155-9597.1000255 Page 3 of 7 Co-culture of A. spelaea and bacteria In our co-culture system, we followed the survival of bacteria inside A. spelaea at 30°C. Bacteria alone were incubated with various concentrations of SDS, and it was found that 0.5% SDS had no efect on bacterial viability. Bacterial survival and multiplication inside A. spelaea Figure 2 shows the intracellular bacterial counts in the presence of A. spelaea. With the exception of Pr. mirabilis that showed low counts throughout the experiment, our results showed that pathogenic bacteria survived and multiplied within the amoeba host. In particular, Ps. aeruginosa and A. hydrophila where the cell counts exceeded 5 log cycles at time 0h with highly signiicant diferences versus other bacterial strains (P <0.001 for both) and increased signiicantly in number to reach >8 and >7 log cycle at time 24h of co-incubation, respectively in comparison to other isolates (p <0.001 for both). he high multiplication rate of A. hydrophila inside FLA in our study has been demonstrated before [22,23]. Ps. aeruginosa is an environmental Gram-negative bacillus that colonizes hospital water systems and causes nosocomial infections [24]. Additionally, Ps. aeruginosaamoeba co-infections have been described in keratitis patients [25]. Our A. spelaea was isolated from a patient with keratitis, so the interactions between A. spelaea and Ps. aeruginosa are of special concern in those patients. he isolation of FLA naturally infected with Ps. aeruginosa [5,26-29] demonstrated the role of amoebae and their cysts as vectors for these intracellular bacteria [30]. Our results showed the intracellular multiplication of Ps. aeruginosa as supported previously [20,31,32]. In contrast to our indings, another report [33] supported the extracellular multiplication mode of Ps. aeruginosa with better growth outside than inside eukaryotic cells. In our work, MRSA intracellular counts were >4 log cycle at time 0h that signiicantly difered from other bacteria (p<0.001) and increased by >1 log cycle at time 24 h of co-incubation that difered from Ps. aeruginosa and A. hydrophila (p<0.001) but showed no signiicant diferences (p>0.05) against the Enterobacteriaceae group. Huws et al. [34] demonstrated the proliferation of epidemic strains of MRSA inside FLA. Our Enterobacteriaceae group started with ~ 1 log cycle growth at time 0 h that increased up to 4 log cycle for C. cloacae and E. coli 1, and >2 log cycle for Kl. pneumoniae, E. aerogenes, R. terrigena, and R. ornitholytica. Previous data demonstrated that Enterobacteriaceae can survive and multiply within amoeba host [23, 35-39] which is consistent with our indings. At 48h co-incubation, the intracellular counts for all bacteria in our study decreased onwards. he decrease ater 2-3 days of incubation, has been reported previously [19,40] which may be attributed to the limited intracellular life of bacteria, or the presence of viable not cultivable cells [41]. In our work, the intracellular viable count of Pr. mirabilis showed signiicantly the highest levels at time 0h (p=0.045, 0.021, 0.01, and 0.005 versus time 8h, 24h, 48h, and 72h, respectively). As the comparative counts of Pr. mirabilis in the presence of amoebae were lower, then, this was evidence of predation by A. spelaea. Extracellular bacterial counts he extracellular bacterial counts in our work, as shown in igure 2, occurred as a result of intracellular multiplication and subsequent release of vesicles containing live bacteria as reported [8,42]. hey were characterized by gradual increase from time 0h to 8h co-incubation (p≤ 0.001) until signiicantly maximum extracellular counts at time 24h co-incubation (p<0.001). hen bacterial counts decreased J Bacteriol Parasitol ISSN:2155-9597 JBP, an open access journal signiicantly to very low or undetected levels at 72h co-incubation (p>0.05 versus time 0h). Signiicant maximum extracellular viable counts at 24h co-incubation were detected for bacteria that showed the highest growth intracellulary; Ps. aeruginosa and A. hydrophila (p<0.001 for both versus other bacteria tested). Pr. mirabilis extracellular counts reached very low levels with insigniicant diferences between time 8h co-incubation until the end of experiment (p>0.05). here are varying accounts in the literature on the types of interaction between bacteria and FLA with diferent types of endocytosis and intracellular behaviors including intracellular lysis of bacteria, followed by its digestion by amoebae or intracellular survival and multiplication of bacteria leading to amoebal lysis which may be dependent on virulence of bacteria [43]. As shown in our work, the non-invasive bacteria are taken up by amoeba as a food source (in our case Pr. mirabilis), while the invasive bacteria are able to reside and multiply inside amoebae without being killed (in our case the other bacteria) [35], where they use amoebae as a transmission vehicle and develop resistance against other phagocytic cells in host tissues [4,44]. Nevertheless, the precise mechanisms of intracellular survival of our tested bacteria remain unclear and have to be determined. Although a previous report has demonstrated the ability of bacteria to inhibit the fusion of lysosomes with phagosomes as a critical step in the intracellular survival inside Acanthamoeba castellanii (A. castellanii) [45], so, our bacterial strains may use similar mechanisms to evade the amoeba-cell defenses. Other reports [34,40,46] that demonstrated the multiplication of Staph. aureus within FLA, suggested that Staph. aureus possess no speciic mechanism for evading digestion but have post-ingestion defenses such as a thicker cell wall, or an antioxidant yellow carotenoid. Pickup et al. [46] stated that Kl. pneumonae has the ability to resist phagocytosis and digestion as a result of polysaccharide capsule. he ability of many bacterial pathogens to survive intracellularly in A. spelaea may be a key step in the evolution of those bacteria to produce human and animal infections. he survival rate of A. spelaea in the presence of bacteria Number of viable A. spelaea in absence of bacteria increased from 1×106 cell/mL (100%) on time 0h to ~ 0.96×107 cells/mL (110%) and 1.88×107 (119%) cells/mL on 24h and 48h, respectively, and then survived to ~ 0.8×106 cell/mL (82%) at 72h (Figure 3). Growth of cocultivated A. spelaea, except with Pr. mirabilis, was inhibited with varying degrees. he statistical analysis showed highly signiicant diferences in survival rates of alone-cultivated compared to the cocultivated A. spelaea (p<0.001 for 24h and 48h, and p<0.002 for 72h except for R. ornitholytica and E. aerogenes where p=0.097 for both). A. spelaea growth at 24h co-incubation was highly signiicantly afected by Ps. aeruginosa and A. hydrophila where p were <0.008 and <0.01 versus other bacterial strains, respectively. In this context, it is well known that the invasive property of Ps. aeruginosa target the amoeba with their toxins that cause cell lysis [33,47]. A. spelaea growth was signiicantly enhanced by Pr. mirabilis (p<0.002, <0.003, and <0.003 for 24h, 48h, and 72h co-incubation versus other bacterial strains, respectively). he decrease in survival rates of A. spelaea when co-incubated with our bacterial strains suggests that amoebal lysis occurred as a result of bacterial multiplication. Nevertheless, bacterial growth did not result in total killing of the amoebal cells that survived until end of the experiment at 72 h, which suggests the adaptation of our bacteria to the intracellular environment without causing total protozoal lysis as reported previously [19]. Our results are in accordance with previously published data [5,38,48-50] that detected decreased amoebal survival in presence of pathogenic bacteria. Volume 7 • Issue 1 • 1000255 Citation: Mohamed ME, Huseein EA, Farrag HM, Mostafa FAA, Hassan AT (2016) Allovahlkampfia spelaea is a Potential Environmental Host for Pathogenic Bacteria. J Bacteriol Parasitol 7: 255. doi:10.4172/2155-9597.1000255 Page 4 of 7 Figure 2: Extracellular (▲) and intracellular (█) bacterial counts following co-incubation for 72h with Allovahlkampia spelaea (A. spelaea). Data are SE (bars) of the mean for three replicate experiments. he values of some error bars were too small to be presented. he predation/survival/intracellular replication data for our bacteria difered in some instances from some work on bacteria and FLA interactions already published [6,51] that revealed a dose-dependent proliferative response of FLA when co-incubated with bacteria like E. coli, Staph. aureus, C cloacae, and Ps. aeruginosa. We attribute this discrepancy between our results and previous data to the use of A. spelaea isolate that has not been investigated before, diferent bacterial J Bacteriol Parasitol ISSN:2155-9597 JBP, an open access journal strains used, diferent MOI, or diferent co-culture conditions as yet reported [19]. he predatory activity of FLA is known to be inluenced by several factors including the type and amount of surrounding bacteria [52,53]. As obvious in our indings, A. spelaea may act as a bacterial predator, or as a reservoir for bacteria, with environmental and clinical implications. Our results although may not relect all possible modality of interactions as a single amoebal host have been Volume 7 • Issue 1 • 1000255 Citation: Mohamed ME, Huseein EA, Farrag HM, Mostafa FAA, Hassan AT (2016) Allovahlkampfia spelaea is a Potential Environmental Host for Pathogenic Bacteria. J Bacteriol Parasitol 7: 255. doi:10.4172/2155-9597.1000255 Page 5 of 7 employed, demonstrated that many pathogenic bacteria are able to interact with A. spelaea, even species which were not expected to have an intracellular life cycle. A. spelaea efect on health ecosystem has two problems. First, A. spelaea serve as reservoirs for pathogenic bacteria. Second, A. spelaea species can themselves cause disease in humans or animals. In the environment, the interactions of bacteria and A. spelaea are expected to be much more complex than reported here, as diferent bacterial prey are present in diferent niches that can be colonized by competing bacterial and protozoan predators. Deciphering the mechanisms of bacteria-protozoa interaction will assist in a better understanding of A. spelaea and bacterial lifestyle. Figure 3: Viability of A. spelaea (106 cells/ml) following 72h incubation with diferent bacterial strains (107 CFU⁄ml). Results illustrate percentage survival relative to control (absence of bacteria) amoebal counts. Abbreviations; A. hydrophila: Aeromonas hydrophila, Ps. aeruginosa: Pseudomonas aeruginosa, C. cloacae: Citrobacter cloacae, E. aerogenes: Enterobacter aerogenes, E. coli 1: Escherichia coli 1, Kl. pneumoniae: Klebsiella pneumoniae, MRSA: Methicillin-resistant Staph. aureus, P mirabilis: Proteus mirabilis, R. ornitholytica: Raoultella ornitholytica, R. terrigena: Raoultella terrigena. Mean value of three replicate experiments are shown. he values of error bars were too small to be presented. Conlict of Interest he authors declare that they have no conlict of interest. References 1. 2. 3. 4. 5. Caliskan M (2012) Genetic diversity in Microorganisms. InTech, Croatia. Walochnik J, Mulec J (2009) Free-living amoebae in carbonate precipitating microhabitats of karst caves and a new vahlkampiid amoeba, Allovahlkampia spelaea gen. nov., sp. nov. Acta Protozool 48: 25-33. Laybourn-Parry J (1992) Protozoan plankton ecology. ChapmanHall, London. Siddiqui R, Khan NA (2012) War of the microbial worlds: who is the beneiciary in Acanthamoeba-bacterial interactions? Exp Parasitol 130: 311-313. Greub G, Raoult D (2004) Microorganisms resistant to free-living amoebae. Clin Microbiol Rev 17: 413-433. 6. J Bacteriol Parasitol ISSN:2155-9597 JBP, an open access journal 7. 8. 9. 10. 11. 12. de Moraes J, Alieri SC (2008) Growth, encystment and survival of Acanthamoeba castellanii grazing on diferent bacteria. FEMS Microbiol Ecol 66: 221-229. Hahn MW, Höle MG (2001) Grazing of protozoa and its efect on populations of aquatic bacteria. FEMS Microbiol Ecol 35: 113-121. Vaerewijck MJM, Baré J, Lambrecht E, Sabbe K, Houf K (2014) Interactions of foodborne pathogens with free-living protozoa: potential consequences for food safety. Compr Rev Food Sci Food Saf 13: 924-944. Schulz F, Lagkouvardos I, Wascher F, Aistleitner K, KostanjÅ¡ek R, et al. (2014) Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae. ISME J 8: 1634-1644. homas V, McDonnell G, Denyer SP, Maillard JY (2010) Free-living amoebae and their intracellular pathogenic microorganisms: risks for water quality. FEMS Microbiol Rev 34: 231-259. Edagawa A, Kimura A, Kawabuchi-Kurata T, Kusuhara Y, Karanis P (2009) Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan. Parasitol Res 105: 1109-1117. Smirnov AV, Goodkov AV (1999) An Illustrated list of basic morphotypes of Gymnamoebia (Rhizopoda, Lobosea). Protistology 1: 20-29. Volume 7 • Issue 1 • 1000255 Citation: Mohamed ME, Huseein EA, Farrag HM, Mostafa FAA, Hassan AT (2016) Allovahlkampfia spelaea is a Potential Environmental Host for Pathogenic Bacteria. J Bacteriol Parasitol 7: 255. doi:10.4172/2155-9597.1000255 Page 6 of 7 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. Schroeder JM, Booton GC, Hay J, Niszl IA, Seal DV, et al. (2001) Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identiication of acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol 39: 1903-1911. Pélandakis M, Pernin P (2002) Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment. Appl Environ Microbiol 68: 2061-2065. Hadziavdic K, Lekang K, Lanzen A, Jonassen I, hompson EM, et al. (2014) Characterization of the 18S rRNA gene for designing universal eukaryote speciic primers. PLoS One 9: e87624. Holt JG, Krieg NR, Sneath PHA, Staley H, Williams ST (1994) Bergey's manual of determinative bacteriology, 9th ed. Williams and Wilkins, Baltimore, MD. Clinical and Laboratory Standards Institute (2006) Performance Standards for Antimicrobial Disk Susceptibility Tests, 9th ed. Approved standard. M2-A9. CLSI, Wayne, Pennsylvania. Khan NA (2001) Pathogenicity, morphology, and diferentiation of Acanthamoeba. Curr Microbiol 43: 391-395. Anacarso I, de Niederhäusern S, Messi P, Guerrieri E, Iseppi R, et al. (2012) Acanthamoeba polyphaga, a potential environmental vector for the transmission of food-borne and opportunistic pathogens. Journal of Basic Microbiology 52: 261-268. Gao LY, Kwaik YA (2000) he mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila. Environ Microbiol 2: 79-90. Wang X, Ahearn DG (1997) Efect of bacteria on survival and growth of Acanthamoeba castellanii. Curr Microbiol 34: 212-215. Rahman M, Abd H, Romling U, Sandstrom G, Möllby R (2008) Aeromonas-Acanthamoeba interaction and early shit to a viable but nonculturable state of Aeromonas by Acanthamoeba. J Appl Microbiol 104: 1449-1457. Yousuf FA, Siddiqui R, Khan NA (2013) Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila. Parasit Vectors 6: 169. Lyczak JB, Cannon CL, Pier GB (2000) Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2: 1051-1060. Ziak P, Ondriska F, Mrva M (2003) Acanthamoeba keratitis ater use of sot contact lenses--case report. Cesk Slov Otalmol 59: 352-358. Pagnier I, Raoult D, La Scola B (2008) Isolation and identiication of amoeba- resisting bacteria from water in human environment by using an Acanthamoeba polyphaga co-culture procedure. Environ Microbiol 10: 1135-1144. Iovieno A, Ledee DR, Miller D, Alfonso EC (2010) Detection of bacterial endosymbionts in clinical acanthamoeba isolates. Ophthalmology 117: 445-452, 452. Siddiqui R, Khan NA (2012) Biology and pathogenesis of Acanthamoeba. Parasit Vectors 5: 6. Calvo L, Gregorio I, García A, Fernández MT, Goñi P, et al. (2013) A new pentaplex-nested PCR to detect ive pathogenic bacteria in free living amoebae. Water Res 47: 493-502. José Maschio V, Corção G, Rott MB (2015) identiication of Pseudomonas spp. as amoeba-resistant microorganisms in isolates of Acanthamoeba. Rev Inst Med Trop Sao Paulo 57: 81-83. Michel R, Burghardt H, Bergmann H (1995) Acanthamoeba, naturally intracellularly infected with Pseudomonas aeruginosa, ater their isolation from a microbiologically contaminated drinking water system in a hospital. Zentralbl Hyg Umweltmed 196: 532-544. Marciano-Cabral F, Cabral G (2003) Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 16: 273-307. Abd H, Wretlind B, Saeed A, Idsund E, Hultenby K, et al. (2008) Pseudomonas aeruginosa utilises its type III secretion system to kill the free-living amoeba Acanthamoeba castellanii. J Eukaryot Microbiol 55: 235-243. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. Huws SA, Smith AW, Enright MC, Wood PJ, Brown MR (2006) Amoebae promote persistence of epidemic strains of MRSA. Environ Microbiol 8: 1130-1133. Alsam S, Jeong SR, Sissons J, Dudley R, Kim KS, et al. (2006) Escherichia coli interactions with Acanthamoeba: a symbiosis with environmental and clinical implications. J Med Microbiol 55: 689-694. Snelling WJ, Moore JE, McKenna JP, Lecky DM, Dooley JS (2006) Bacterial-protozoa interactions; an update on the role these phenomena play towards human illness. Microbes Infect 8: 578-587. Jung SY, Alsam S, Kim KS, Khan NA (2008) Pathogen-pathogen interactions: a comparative study of Escherichia coli interactions with the clinical and environmental isolates of Acanthamoeba. World J Microbiol Biotechnol 24: 2339-2348. Chekabab SM, Daigle F, Charette SJ, Dozois CM, Harel J (2012) Survival of enterohemorrhagic Escherichia coli in the presence of Acanthamoeba castellanii and its dependence on Pho regulon. Microbiologyopen 1: 427-437. Denoncourt AM, Paquet VE, Charette SJ (2014) Potential role of bacteria packaging by protozoa in the persistence and transmission of pathogenic bacteria. Front Microbiol 5: 240. Huws SA, Morley RJ, Jones MV, Brown MR, Smith AW (2008) Interactions of some common pathogenic bacteria with Acanthamoeba polyphaga. FEMS Microbiol Lett 282: 258-265. Preston TM, Richards H, Wotton RS (2001) Locomotion and feeding of Acanthamoeba at the water-air interface of ponds. FEMS Microbiol Lett 194: 143-147. Gourabathini P, Brandl MT, Redding KS, Gunderson JH, Berk SG (2008) Interactions between food-borne pathogens and protozoa isolated from lettuce and spinach. Appl Environ Microbiol 74: 2518-2525. Siddiqui R, Malik H, Sagheer M, Jung SY, Khan NA (2011) he type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba. Exp Parasitol 128: 409-413. Salah IB, Ghigo E, Drancourt M (2009) Free-living amoebae, a training ield for macrophage resistance of mycobacteria. Clin Microbiol Infect 15: 894-905. Bozue JA, Johnson W (1996) Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome–lysosome fusion. Infect Immun 64: 668-673. Pickup ZL, Pickup R, Parry JD (2007) Efects of bacterial prey species and their concentration on growth of the amoebae Acanthamoeba castellanii and Hartmannella vermiformis. Appl Environ Microbiol 73: 2631-2634. Pukatzki S, Kessin RH, Mekalanos JJ (2002) he human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum. Proc Natl Acad Sci USA 99: 3159-3164. Bottone EJ, Madayag RM, Qureshi MN (1992) Acanthamoeba keratitis: synergy between amebic and bacterial cocontaminants in contact lens care systems as a prelude to infection. J Clin Microbiol 30: 2447-2450. Qureshi MN, Perez AA 2nd, Madayag RM, Bottone EJ (1993) Inhibition of Acanthamoeba species by Pseudomonas aeruginosa: rationale for their selective exclusion in corneal ulcers and contact lens care systems. J Clin Microbiol 31: 1908-1910. Matz C, Moreno AM, Alhede M, Maneield M, Hauser AR, et al. (2008) Pseudomonas aeruginosa uses type III secretion system to kill bioilmassociated amoebae. ISME J 2: 843-852. Weekers PH, Bodelier PLE, Wijen JPH, Vogels GD (1993) Efects of grazing by the free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis on various bacteria. Appl Environ Microbiol 59: 2317-2319. Rodríguez-Zaragoza S (1994) Ecology of free-living amoebae. Crit Rev Microbiol 20: 225-241. Khan NA (2006) Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol Rev 30: 564-595. 34. J Bacteriol Parasitol ISSN:2155-9597 JBP, an open access journal Volume 7 • Issue 1 • 1000255
