· web viewother underlying diseases (e.g. lung cancer, hydropneumothorax, dm, liver cirrohsis,...

Egyptian Journal of Medical Microbiology, October 2013 Vol. 22, No. 4

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Serotyping and Antimicrobial Resistance Pattern of Streptococcus Pneumoniae Strains in Patients with Community-acquired Pneumonia

Mona Sallam Embarek Mohamed1, Alaa Thabet Hassan2

1Department 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.

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

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


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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).

MATERIALS & METHODS

Study designA prospective study was carried out at

Assiut University Hospitals, Assiut, over a four- months 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 from60 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

collected into wide-mouthed sterile screw- capped 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 pneumoniaeand 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.


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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). Multidrug- resistant 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.

RESULTS

Study populationFrom 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%) hadbronchopneumonia, 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.

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 10- 20, 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 pneumonic- type 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).


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Characteristics of Streptococcus pneumoniaeserotypes

Of the 18 pneumococcal-isolates detected,16 (89%) strains showed seven different serotypes and 2 strains (11%) were non- typeable (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

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 un- typeable 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).

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)

60 patie

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Table 1: Demographic and clinical characteristics of patients (n=60)

Patients’ characteristics N (%) P-value Sex

Female 9 (15)Male 51 (85)

Geographical areaAssiut 52 (86.7)Qena 5 (8)New Valley 2 (3.3)Aswan 1 (1.7)

Site of admissionChest department 49(82)Chest intensive care unit 11(18)

Smoking index0 (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 findingsLobar 32 (53)Bronchopneumonia 19 (31.7)Interstitial 6 (10)

< 0.0001

< 0.0001

< 0.0005

<0.01

< 0.005Multilobar pneumonia 3 (5)Cavitation 2 (3)Pleural effusion 7 (11.7)


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Table 2: Clinical characteristics of pneumococcal pneumoniaPatient Age Gender Residence Site of

admissionSmoking index Underlying disease (bronchopulmonary

condition-immunosuppresion)Radiological findings

1 46 y M New Valley Chest Depart. 8 none Lt lower lobe pneumonia2 60 y M Assiut Chest ICU 32 Right lung cancer Bronchopneumonia3 55 y M Assiut Chest Depart. 25 none Interstitial pneumonia4 72 y M Assiut Chest Depart. 21 Lt sided consolidation collapse with Lt sided

pleural effusionLt lower lobe pneumonia

5 15 y M Qena Chest ICU 0 Lt hydropneumothorax Lt lower lobe pneumonia6 70 y M Assiut Chest Depart. 37 Rt pleural effusion with underlying collapse Rt lower lobe pnemonia7 47 y M Assiut Chest Depart. 16 none Interstitial pneumonia8 54 y F Assiut Chest Depart. 0 DM, Liver cirrohsis Lt lower lobe pneumonia9 55 y M Assiut Chest Depart. 22 none Lt lower lobe pneumonia10 63 y F Assiut Chest Depart. 0 none Rt upper lobe pneumonia11 35 y M Assiut Chest ICU Ex-smoker (previous index 18) Cardiac ischemia, massive hemoptysis Bronchopneumonia12 62 y M Assiut Chest Depart. 39 Respiratory failure Lt lower lobe pneumonia13 24 y M Assiut Chest Depart. 6 none Bronchopneumonia14 43 y M Assiut Chest Depart. 32 cardiomyopathy Lt sided pneumonia15 51 y M Assiut Chest ICU 30 Pulmonary embolism, Rt lower limb DVT,DM Multilobar pneumonia16 30y M Aswan Chest ICU 21 Respratory failure Bronchopneumonia17 56y F Assiut Chest Depart. 0 Pulmonary cavitation Rt lobar pneumonia18 60 y M Assiut Chest Depart. 35 Lt lower lobe collapse 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


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Table 3: Distribution of the detected serotypes of Streptococcus pneumoniae and their antimicrobial resistance against 10 antimicrobialsSerotype No. of isolates

(%)P value No. of isolates with indicated resistance Multiresistant

StrainsP AMP AMC E AZM B DA NA CRO LEV1 4 (22) <0.01 4 3 1 1 1 - - - - - 19V 4 (22) <0.01 4 4 1 1 1 - - - - - 16B 3 (16.7%) <0.05 3 3 1 1 1 - 1 - - - 119F 2 (11%) NS* 2 2 2 2 2 2 1 1 1 - 223F 1 (5.5%) NS 1 1 2 1 1 1 1 1 - - 114 1 (5.5%) NS - 1 - - - - - - - - -19A 1 (5.5%) NS 1 1 1 1 1 - - - - - 1NT** 2 (11%) NS 2 2 2 1 1 2 - 1 1 1 2Total 18 (100%) 17(94) 17(94) 10(55) 8(44) 8(44) 5(27.8) 3(16.7) 3(16.7) 2(11) 1(5.5) 9 (50%)P value <0.0001 <0.0001 <0.001 <0.005 <0.005 <0.01 <0.05 <0.05 - - <0.005

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


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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


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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 pneumococcal- serotypes in cases of CAP in Assiut. Other reports from Egypt described pneumococcal- serotypes 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 werethe 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


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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 multidrug- resistant 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.

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85

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ا ϧاالو ىϓات ϳوϟاالو Ϣأه .ةϣادϘϟة

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]

Accepted October 24th, 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. Non- typeable 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≥105 colony forming units (CFU)/ml [11]. Haemophilus influenzae isolates were identified based on colonial morphology, Gramstaining, 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, trimethoprim- sulfamethoxazole, 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 deviation or number and percentage as appropriate. The X2 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).


Table 1: Demographic and clinical characteristics of CAP patients (n=132)

Patients’ characteristics N (%)Sex

Female 43 (33)Male

Geographical area89 (67)

Assiut 98 (74)Qena 15 (11)Sohag 10 (7.6)

New Valley 5 (4)

Aswan 4 (3)Site of admission

Chest department 86 (65)Chest intensive care unit 46 (35)

Smoking index0 (non-smoker) 49 (37)Ex-smoker 19 (14)< 20 (mild smoker) 12 (9)20-30 (moderate smoker) 14 (11)>30 (heavy smoker) 38 (29)

Radiographic findingsLobar 75 (57)Bronchopneumonia 49 (37)Interstitial 1 (0.8)

132 patientsMultilobar pneumonia 3 (2.3)

Cavitation 1 (0.8)Pleural effusion 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 CAPAbbreviations: CAP= community-acquired pneumonia; Hib= H influenzae type b; Hif= H influenzae type f; NTHi= nontypable H

Embarek Mohamed et al 458influenzae


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 pneumonic- type 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

Underlying disease (bronchopulmonarycondition-immunosuppression)

Radiological findings

1 72y

M New Valley Chest Depart.

8 Right lung cancer Lt lower lobe pneumonia

2 66y

M Assiut Chest ICU 32 DCP, MV Bronchopneumonia

3 53y

M Assiut Chest Depart.

25 none Bronchopneumonia

4 41y


21 DM, hypertension Lt lower lobe pneumonia

5 35y

M Qena Chest ICU 0 RF, MV Bronchopneumonia

6 40y


37 Rt pleural effusion with underlying collapse

Rt lower lobe pnemonia

7 50y


16 none Interstitial pneumonia

8 54y

F Assiut Chest Depart.

0 DM, renal impairment Bronchopneumonia

9 60y



10 63y

F Sohag Chest Depart.

0 none Rt upper lobe pneumonia

11 47y

M Assiut Chest ICU Ex-smoker (previous index 27)

Cardiac ischemia, massive hemoptysis, MV

Bronchopneumonia

12 29y



13 43y


6 none Lt lower lobe pneumonia

14 55y


32 cardiomyopathy Bronchopneumonia

15 51y

M Assiut Chest ICU 30 Pulmonary embolism, Rt lower limb DVT,DM

Multilobar pneumonia

16 30y

M Aswan Chest ICU 21 RF, MV Bronchopneumonia

17 54y

F Assiut Chest Depart.

0 Pulmonary cavitation Bronchopneumonia

18 58y


35 Lt hydropneumothorax Lt lower lobe pneumonia

19 41y

M Sohag Chest Depart.

29 DM Bronchopneumonia

20 46y

M Qena Chest ICU 11 none Bronchopneumonia

21 33y

F Assiut Chest ICU 0 Lower limb DVT 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.


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

No (%) of isolates with indicated resistanceMultiresistant Strains (%)

P AX AMC SXT AZM CLR C CIP CRO LEV MER IMPNTHi 15 (71) 14(93) 2(13) 1(7) 10(67) 1(7) 0(0) 6(47) 1(7) 2(13) 0(0) 2(13) 1(7) 2(13)Hib 5 (24) 5(100) 1(20) 0(0) 3(60) 0(0) 0(0) 2(40) 0(0) 0(0) 0(0) 1(20) 0(0) 1(20)Hif 1 (5) 1(100) 1(100) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0)Total 21 (100) 20(95) 4(19) 1(5) 13(62) 1(5) 0(0) 8(38) 1(5) 2(9.5) 0(0) 3(14) 1(5) 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, β- lactam antimicrobials were the first therapeutic option for treating CAP due to H. influenzae [30]. Decreased susceptibilities to β- lactam 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.


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THE EGYPTIAN JOURNAL OF IMMUNOLOGY Vol. 22 (1), 2015Page: 85-91

Immunomodulatory Effects of Levofloxacin on Patients with Pneumonia in Assiut University Hospitals

1Mohamed S. Badari, 1Sherein G. Elgendy, 1Asmaa S. Mohamed, 2AlaaT. HassanDepartments 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 included40 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.

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 Gram- negative 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 MethodsEthics 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 87

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 hospital- acquired 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: 1- Patients 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 TNF- α and IL-10 ELISA kit, KOMA BIOTECH INC

(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.

ResultsForty 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


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) Patients Control *P1-value

Before levofloxacin administrationMean SE Median

36.43 4.1823.3

25.21 1.9627.8 NS

Range 8.2 98.0 16.7 33.5Mean SE 20.82 1.31 17.12 0.84

After levofloxacin administration (750mg once daily for 10 days) Median 18.5 17.9 NS

Range 11.5 46.2 10.7 20.0*P2-value 0.004 0.009

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.

IL-10 (pg/ml) Patients Control *P1-valueMean SE 42.54 2.83 51.48 1.76

Before levofloxacin administration Median 37.5 52.2 NSRange 16.4 79.2 40.2 60.5

After levofloxacin administration (750mg once daily for 10 days)

Mean SE Median

61.75 2.8561.3

28.57 1.3728.6 0.000

Range 20.0 100.1 20.0 33.8*P2-value 0.000 0.0051: Mann-Whitney Test.2: Wilcoxon Signed Ranks Test.* P > 0.05 is not significant (NS).

DiscussionSeveral 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


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 pro- inflammatory cytokines such as TNF-α, IL- 1ȕ, 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 pro- inflammatory 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 Gram- negative bacteria, LPS is the major pro- inflammatory 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

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 TNF- α, 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 pro- inflammatory 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 pro- inflammatory 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 pro- inflammatory cytokine mediators can induce systemic inflammatory response syndrome


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

human peripheral blood mononuclear cells. Antimicrob. Agents Chemother; 47:3704-3707.

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8. Guz K, Ploskonska G. (2007). Quinolones and eukaryotic topoisomerasis. In: Hooper DC, Wolfsonndfinal outcome of infection and clinical course JS, eds. Quinolones antimicrobial agents. 2 ed.

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.

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Original Research Article

British Microbiology Research Journal

7(6): 288-305, 2015, Article no.BMRJ.2015.121ISSN: 2231-0886

SCIENCEDOMAIN internationalwww.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

1Department of Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt.2Department of Chest Diseases, Assiut University Hospitals, Assiut, Egypt.

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 andapproved the final manuscript.

Article Information

DOI: 10.9734/BMRJ/2015/16317Editor(s):

Reviewers: (1) Hung-Jen Liu, Institute of Molecular Biology, National Chung Hsing University, Taiwan.

(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

Received 25th January 2015 Accepted 17th March 2015 Published 10th 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

289

were Haemophilus influenzae (19.4%), Escherichia coli (18%), Streptococcus pneumoniae (16.7%), Klebsiella pneumoniae (14%), Streptococcus pyogenes (10%), Pseudomonas aeruginosa (5.6%), methicillin resistant Staphylococcus aureus (5.6%), Acinetobacter baumannii (4.2%), and Moraxella catarrhalis (2.8%). The majority of the isolated strains showed high resistance rates to most groups of antibiotics where 91 (63%) of the isolated strains were multidrug resistant, 37 (26%) strains were extreme drug resistant and 16 (11%) bacterial strains were pandrug resistant. High resistance rates were observed against penicillins and cephalosporins. Moderate resistance rates were detected against the fluoroquinolones. High susceptibilities were detected to the carbapenem group. All the isolated Gram-positive bacteria were sensitive to linezolid.

Keywords: Chronic obstructive pulmonary disease; infection exacerbation; antimicrobial resistance; Upper Egypt.

ABBREVIATIONS

Acin. baumannii Acinetobacter baumanniiAECOPD acute exacerbation of Chronic obstructive pulmonary diseaseANOVA Analysis of varianceCLSI Clinical and Laboratory Standards InstituteCOPD Chronic obstructive pulmonary diseaseDCP decompensated Core-pulmonaleDVT deep venous thrombosisE coli Escherichia coliEMB Eosin Methylene BlueESR erythrocyte sedimentation rateFEV1 Forced expiratory volume in the first secondFVC forced vital capacityGOLD Global Initiative for Obstructive Lung DiseaseH. influenzae Haemophilus influenzaeI intermediateICU Intensive Care UnitIHD ischemic heart diseaseKl. pneumoniae Klebsiella pneumoniaeLTOT long term oxygen therapyM. catarrahlis Moraxella catarrahlisMDR Multidrug-resistantMRSA methicillin resistant Staphylococcus aureusMV mechanical ventilationPDR pandrug-resistantPs. aeruginosa Pseudomonas aeruginosaR resistantRF Respiratory failureS susceptibleSD Standard deviationSPSS Statistical package for social sciencesStaph. epidermidis Staphylococcus epidermidisStrep. pneumoniae Streptococcus pneumoniaeVTE Venous thromboembolismXDR 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


299

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 re- admission 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.

2. MATERIALS AND METHODS

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

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].

2.2 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

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.

2.5 Identification of the Causative Bacterial Strains

Samples were examined microscopically after staining with Gram´s stain and cultured directly on nutrient, blood, chocolate, mannitol salt, bile

291


esculin, CHROMagar, MacConkey´s, and Eosin Methylene Blue (EMB) agar plates. The cultured plates were incubated aerobically at 37ºC for 24- 48 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). Multidrug- resistant (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 was considered statistically significant.

3. RESULTS

3.1 Characteristics of COPD Patients

The study included 116 COPD patients consisted of 108 (93%) males and 8 (7%) females with agerange 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


292

(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 hadhypoalbuminemia, and 8 (7%) patients were hypertensive. Venous thromboembolism (VTE) was found in 13 (11%) patients with 6 (5%) patients suffered from pulmonary embolism and7 (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 (3attacks; 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 (16attacks; 11%). In 18 (24%) patients (24 attacks;

14%), no significant bacterial growth was found


293

(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. Optochin- sensitive 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


294

40

35 34

30 28

25

22

2016

15

1010 8 9

6

5 42 3

0JanFebMarApril MayJuneJulyAugSeptOctNovDec

bacteria, resistance rates were highest against the penicillin group and erythromycin. Resistance to bacitracin, vancomycin, and teicoplanin ranged from 58-64%. All the isolated Gram- positive 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

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.

Fig. 1. Monthly distribution of COPD infection exacerbations during January-December, 2013 (n=167)

Num

ber o

f infectio

n exacerba

tion att

acks

30

25

202016

stage IVstage III stage II stageI

16 1514 20

1012

4842

62

42

62

842

522 22

222

0

80

67 70

60

50MDR

XDR40

PDR 28 30

1815 20

4 6 101 1 2 2

0stage IV stage III stage II stage I


295

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

Fig. 3. Antibiotic resistance patterns in different COPD stages (n=144 attacks)


295

Table 1. Demographic and clinical characteristics of COPD patients (n=116)

Patients’ characteristics N (%)Sex

Female 8 (7)Male 108 (93)

GeographicalareaAssiut 80 (70)Sohag 4 (3)Qena 18 (15)Luxor 6 (5)

AdmissionAswan 8 (7)

ICU 84 (72)Chest Department 32 (28)

COPD stageI (mild) 2 (1.7)II (moderate) 8 (7)III (severe) 18 (15)

Number of attacks (no=167)/yearIV (very severe) 88 (76)

One attack 79 (68)Two attacks 24 (21)Three attacks 12 (10)Four attacks 1 (1)

Smokingindex0 (non-smokers) 12 (10)Ex-smokers 17 (15)Mild-smokers 2 (2) 87 (75)Moderate-smokers 33 (28)Heavy-smokers 52 (45)

Arterial blood gasesO2 tension Normal 12 (10)

Mild hypoxia 18 (16) 104 (90)Moderatehypoxia 70 (60)Severe hypoxia 16 (14)

CO2 tension Normal 36 (31)Hypercapnea 80 (69)


296

Patients’ characteristics N (%)

Chest X rayNormal chest x ray stage I: 2(2%); stage II: 1(0.9%) 3 (2.9)COPD changes * stage II:7(6%); stage III:18(15%); stage

IV:88(76%)113 (97)

Pneumonic infiltrates (co-morbid pneumonia) stage III: 2(2%) stage IV: 16(14%) 18 (15)Associated cardiopulmonary condition

Other systematic condition

Bacteriological diagnosis

DCP 58 (50)RF 86 (74)MV 8 (7)LTOT 18 (16)Pulmonary embolism 6 (5)Pleural effusion 4 (3)IHD 2 (2)Lungcarcinoma 2 (2)Alpha1-antitrypsin deficiency 3 (3)

Anaemia 44 (38)DM 30 (26)Hypoalbuminemia 12 (10)Leucocytosis 28 (24)Hypertension 8 (7)DVT 7 (6)Impaired liver function 8 (7)Impaired renal function 6 (5)Increased ESR 78 (67)Previous corticosteroid therapy 98 (84)

Significant bacterial growth during exacerbations (no of patients=116/ no of attacks=167) 88 patients (76%)/143 attacks (86%)Single etiological agent 72 patients (82%)/127 attacks (89%)Mixed infection 16 patients (18%)/ 16 attacks (11%)

No bacterial growth 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

5 1 6 7 8 9 10 11 12 MDR XDR PDR

doxycycline

penicillin

oxacillin

methicillin

carbencillin

bacitracin

erythromycin

vancomycin

teicoplanin

linezolid

neomycin

tobramycin

amikacin

gentamicin

meropenem

imipenem

aztreonam

4(14) ND** ND ND ND ND ND ND ND ND 14(50) 22(79) 5(18) 24(86) 2(7) 2(7) 18(64) 24(86) 2(7) 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)


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(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) 0(0) 0(0) 2(100) 2(100) 0(0) 0(0) 0(0) 0(0) ND ND ND ND ND ND ND 2(100) 0(0) 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) 2(100) 2(100) 2(100) 2(100) 0(0) 2(100) 2(100) 2(100) 0(0) ND ND ND ND ND ND ND 2(100) 0(0) 0(0)

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) 8(8.5) 76(81) 91(63) 47(33) 6(4)

Moham

ed et al.; BMR

J, 7(6): 288-305, 2015; Article no.BMR

J.2015.121

Table 2. Resistance patterns of isolated bacterial strains to antim

icrobial agents

297

*1-penicillins and penicillin combinations; 2-phenicols; 3-cephalosporines; 4-fluoroquinolones; 5-tetracyclines; 6-

polypeptides; 7-macrolides; 8-glycopeptides; 9-oxazolidinones; 10-am

inoglycosides; 11-carbapenems; 12-

monobactam

s. **ND

=not determined

Bacterial isolates Total 1* 2 3 4

No (%)

amoxicillin

amoxicillin

/ chloramphenicol

ceftriaxone

cefepime

ciprofloxacin

ceprodoxime

cefaclor

cefotaxime

levofloxacin

ofloxacin

lomefloxacin

H. influenzae 28 (19.4) 28(100) 16(57) 4(14) 12(43) 24(86) 10(36) 24(86) 18(64) 26(93) 8(29) 10(36) 14(50)

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) 8(100) 6(75) 6(75) 0(0) 2(25) 6(75)

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) 4(100) 2(50) 2(50) 0(0) 3(75) 2(50)

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) 2(100) 2(100) 0(0) 0(0) 0(0)

MRSA 8 (5.6) 8(100) 4(50) 4(50) 8(100) 8(100) 8(100) 6(75) 8(100) 8(100) 6(75) 8(100) 8(100)

Enterococci 2 (1.4) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 2(100) 2(100) 2(100)

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)

Moham

ed et al.; BMR

J, 7(6): 288-305, 2015; Article no.BMR

J.2015.121

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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 co- morbidities, 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. Gram- negative 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, Gram- negative bacteria were most commonly isolated.H. influenzae was the most common bacteria detected in our study. This is in correspondence

300


with previous works in Egypt [35] and other countries [36,37,38,39,40]. Strains ofH. 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-resistantS. 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 Gram- negative 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

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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 oxacillin- resistant 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].

5. CONCLUSION

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From our results we concluded that, Gram- negative 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

The authors have declared that no competing interests exist.

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Volume 7 • Issue 1 • 1000255

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.

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 BacteriaMona 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, Egypt2Department 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.

Keywords: Allovahlkampia spelaea; Amoeba-bacteria interactions; Co-culture experiments; Gram negative bacteria; Intracellular survival

IntroductionAllovahlkampia 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

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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.

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

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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 DiscussionAs 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.


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Co-culture of A. spelaea and bacteria

In our co-culture system, we followed the survival of bacteria insideA. 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. spelaeaFigure 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. aeruginosa- amoeba 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


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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.

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.


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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

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


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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.

Conlict of Interesthe authors declare that they have no conlict of interest.

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