Common Pathogens and Their Resistance to Antimicrobials in Community Acquired Pneumonia (CAP): A Single Center Study in Bangladesh

Pneumonia is a worldwide, serious threat to health and an enormous socio-economic burden for health care system. Community-acquired pneumonia (CAP) is associated with a signicant mortality and morbidity. Knowledge of predominant microbial patterns in CAP constitutes the basis for initial decisions about empirical antimicrobial treatment. The aim of this study was to identify the bacterial etiology of CAP in adult hospitalized patients and to see their antibiotic sensitivity pattern as well as to observe their clinical prole and short term outcome. It was a hospital based prospective observational study. A total of 87 hospitalized patients diagnosed with CAP were enrolled consecutively from the medicine ward of Chittagong Medical College Hospital (CMCH). Sputum for Gram staining, Z N staining, culture sensitivity, blood culture and sensitivity and PCR for Legionella pneumophila, Mycoplasma pneumoniae, Chlamydophila pneumonia and Streptococcus pneumonia were done. Patients were followed up for in-hospital outcome and 30-day mortality. The mean (±SD) age was 49.59±16.97 years and male female ratio was 1.56:1. Fever, chest pain and cough were the most common clinical features. Sputum culture, blood culture and PCR were positive in 60.9%, 1.1% and 4.6% of the samples respectively. Klebsiella pneumoniae was identied in the sputum culture of the majority of the patients (39.1%), followed by Pseudomonas aeruginosa (10.3%), Staphylococcus aureus and Escherichia coli (5.7%). The only one sample which was positive in blood culture and it was Staphylococcus aureus. Streptococcus pneumoniae was identied in all the 4 PCR positive cases. The highly sensitive drugs were meropenem, levooxacin and amikacin. The mean (±SD) duration of hospital stay was 6.34±2.37. In hospital mortality and 30-day mortality was 6.9% and 16.1% respectively. Gram-negative bacteria pre-dominate in the bacteriologic prole of CAP using conventional sputum and blood culture. There is need for further conventional serologic tests for atypical and viral pathogens in all patients admitted with CAP.


Introduction
Pneumonia is de ned as an acute respiratory illness associated with recently developed radiological pulmonary shadowing which may be segmental, lobar or multi-lobar 1 . Community acquired pneumonia (CAP) is de ned as pneumonia acquired outside the hospital by an immune-competent individual. It is to be distinguished, on the basis of a wider spectrum of pathogens, from nosocomial pneumonia (which arises more than 48 hours after hospital admission or within 3 months of discharge) and from pneumonia in an immune-compromised host (e.g., in the setting of neutropenia, iatrogenic immunesuppression with drugs, status post organ or stem-cell transplantation, HIV infection, or a congenital immune de ciency 2 . Underlying diseases (Chronic obstructive pulmonary disease, compromised immune system, dementia, gastro esophageal re ux disease, etc.) increase susceptibility of the patients for pneumonia 3 .
Alternatively, habitual pathogens could show particular patterns of antimicrobial resistance 4 .
Undoubtedly the knowledge of these microbiological characteristics is critical and represents the basis for empirical treatments. Serious co-existing illness has been identi ed as modifying factors of severity of pneumonia 5 . On the basis of these appreciation, published guidelines on pneumonia advocate speci c criteria for antibiotic selection and the management of patients in the presence of co-morbid diseases 6 .
As the etiology of CAP varies geographically the understanding of local epidemiology may play an important role in making proper empirical treatment choices before laboratory test results are available. This is especially true for Bangladesh and other developing countries where healthcare systems operate on poor hygiene system and lack proper facilities to contain infections. In these countries, early treatment is usually based on the patient's clinical symptoms rather than diagnostic results. Therefore, patient's early prognosis to nal outcome might be much improved by available epidemiologic data for the most frequently isolated pathogenic organisms 7 . However, such complete data is scarce in Chittagong, Bangladesh.
There is a need to conduct regular prevalence and antibiogram studies to develop empirical guidelines for treatment of CAP in that particular region. The hospital antibiogram is a periodic summary of antimicrobial susceptibilities of local bacterial isolates submitted to the hospital's clinical microbiology laboratory. Antibiograms are often used by clinicians to assess local susceptibility rates, as an aid in selecting empiric antibiotic therapy, and in monitoring resistance trends over time within an institution.
Antibiograms can also used to compare susceptibility rates across institutions and track resistance trends 8 .
On the other hand, antimicrobial resistance is a well-known important emerging clinical and public health problem. Controlling antimicrobial resistance is a major issue confronting organized health care today.
The irrational use of antibiotics has an in uence in the spread of antimicrobial resistance among bacteria 9 . Both under-use and over-use of broad spectrum antibiotics for an infection as common as CAP could be harmful, particularly in Asia where mortality is high, resources scarce and antibiotic resistance an increasing problem 10 .
National data on incidence, etiology and mortality of CAP is not available in Bangladesh. Pneumonia's mortality is highly correlated with socio-economic factors and is a far less frequent cause of death in high-income countries. It occurs about ve times more frequently in the developing world than the developed world. The incidence of CAP ranged from 4 million to 5 million cases per year, with about 25% requiring hospitalization 11 . Death rates in hospital admitted patients are between 5-10% but may be as high as 50% in severe illness 1 . The annual incidence of CAP in Europe is 1.6-10.6/1,000 adults 12 . In Asia, CAP causes an estimated one million adult deaths per year (160,000 cases per year among those aged 15-59 years) 7 .
CAP is a frequent cause of antibiotic prescription, either in hospital or in the ambulatory setting.
Antibiotics prescribed for CAP exert a selection pressure favoring the emergence of multidrug resistant bacteria, not only on lung pathogens (like S. pneumoniae or M. pneumoniae), but also on bacteria colonizing the skin or the gastrointestinal tract, like Staphylococcus aureus or enterobacteriaceae. The emergence of methicillin-resistant S. aureus, and lately of extended spectrum betalactamase (ESBL)producing enterobacteriaceae, are worrying consequences of widespread antibiotic use. A high correlation is present between the prevalence of S. pneumoniae resistance towards diverse antibiotic classes and the density of prescription of the same antibiotic classes. On an individual level, one-time antibiotic administration enhances the prevalence of colonization by resistant bacteria up to one year 13 .
In CAP, in approximately 6% of the cases a MDR pathogen is involved, the most frequent described being S. aureus and P. aeruginosa. A study by Aliberti et al. reported that MDR microorganisms were involved in 3.3% to 7.6% CAP cases in which the most commonly identi ed MDR pathogen was methicillin-resistant S. aureus (MRSA). Community-acquired methicillin-resistant S. aureus (CA-MRSA) has become an important CAP pathogen in endemic areas in Europe 14 . Since the recommendation of current international guidelines for severe CAP is empiric therapy with β-lactam with macrolide or uoroquinolone, which may not provide adequate protection against MRSA, microbiological diagnosis of these cases is very important 11 .
To get updated information for proper therapeutic interventions, periodic evaluation and regional surveillance programs is necessary for the development local data about the antimicrobial sensitivity to respiratory pathogens for CAP. In this study, we aimed to identify the common bacterial pathogens in hospitalized patients with CAP from Chittagong, Bangladesh. We also determined pathogens' antibiotic susceptibility patterns to evaluate the changing trend of antimicrobial susceptibility in this region. Meanwhile the short term clinical outcomeof antibiotic therapy for CAP during hospital stay was also observed.
Though hospitalization of adult patients with CAP are increasing in Bangladesh, information regarding their clinical presentation, microbiological characteristics, antimicrobial susceptibility pattern that is required for choosing empiric antibiotic treatment and outcome of patients are lacking. Microorganisms causing CAP vary in their susceptibility to antimicrobials from place to place and time to time. CMCH is the 2 nd largest tertiary care hospital in Bangladesh and a referral centre to the hospitals in the surrounding districts. Up to date knowledge of the microbial organisms and antibiotic susceptibility pattern of patients with CAP is essential for de ning empirical treatment. There is a paucity of recent study regarding these issues in Bangladesh especially in peripheral hospital settings, where a large number of CAP patients with different comorbidities are managed routinely.

Methodology
The hospital based prospective observational study was conducted from August 2018 to July 2019 in the Department of Medicine at Chittagong Medical College Hospital. Patients of both sexes age above 18 years who were diagnosed as CAP admitted in the Department of Medicine was included in the study as consecutive sampling method. Total 85 admitted patients were included after screening of exclusion criteria like chemical pneumonitis, malignancy, radiological evidence of brosis, collapse, bronchiectasis, lung abscess and tuberculosis, suspicion of immunosuppression or known immunosuppressive status like HIV, Hematological or lymphoid malignancy. Patient on immunosuppressive drugs-steroids and chemotherapy and getting antibiotic for more than 48 hours were also excluded.
Prior approval for the study was taken from the institutional ethical review committee of Chittagong Medical College. Patients diagnosed clinically as CAP were enrolled in the study. Investigations like hematologic measurements (TC of WBC, Hb%, ESR), blood culture, chest X-ray P/A view, sputum for Gram staining and culture sensitivity, sputum for AFB for 3 consecutive samples, blood urea and random blood sugar were done. For scanty production of cough, patient's sputum was collected after nebulization by hypertonic normal saline. Patient with positive radiological ndings of consolidation was enrolled in the study. During treatment, oral temperature was recorded and frequently physical examinations were performed up to discharge. Patients were asked to report 30 days after discharge for follow up.

Results
The mean (±SD) age was 49.59±16.97years with ranged from 18 to 76 years and maximum number (35.6%) of patients was found in the age group of 40-59 years. There was male predominance with a male to female ration of 1.56:1 (Table I). Different laboratory ndings of the enrolled CAP patients show that, sputum gram stain was positive in 55 (63.2%) patients while Z-N stain was negative in entire sample. Sputum culture yield growth in 53 (60.9%) sample while blood culture only in 1 (1.1%) sample.
PCR was positive in 4 (4.6%) sample.   In hospital mortality rate of the CAP patients in this study was 6.9% and 30-day mortality rate was 14.1%.
Average length of hospital stay was 6 days. About one tenth of the total patients develop sepsis and need ICU support. Patients who died within 30 days, majority had either Klebsiella pneumonia or Pseudomonas aeruginosa. Among survivors in addition of these two organisms Staphylococcus aureus, Escherichia coli and Streptococcus pneumonia were identi ed. There was some variation in the distributions of the isolated organisms in different age groups. Pseudomonas aeruginosa were more prevalent in CAP patient's age ≥60 years. In contrast no Escherichia coli was identi ed among this age group. Overall the isolated organisms in the study were found to be highly sensitive for Meropenem (96.2%), Amikacin (96.2%), Levo oxacin (88.5%) and Vancomycin (80.0%).
In hospital mortality rate of the CAP patients in this study was 6.9% and 30-day mortality rate was 14.1%. Average length of hospital stay was 6 days. About one tenth of the total patients develop sepsis and need ICU support. Patients who died within 30 days, majority had either Klebsiella pneumonia or Pseudomonas aeruginosa. Among survivors in addition of these two organisms Staphylococcus aureus, Escherichia coli and Streptococcus pneumonia were identi ed. There was some variation in the distributions of the isolated organisms in different age groups. Pseudomonas aeruginosa were more prevalent in CAP patient's age ≥60 years. In contrast no Escherichia coli was identi ed among this age group.

Discussion
This hospital based prospective observational study was conducted to determine the clinicobacteriological pro le and in-hospital outcome of hospitalized adult patients with CAP. For this purpose, 87 patients of CAP who had clinical and radiological features of pneumonia and were admitted in different medicine wards of CMCH were enrolled in the study.
The microbial diagnosis of CAP was con rmed in 65.5% of patients with standard sputum culture, blood culture and PCR test (53/87 were sputum culture positive and 4/87 were PCR positive). However, this rate varies in different studies. With different laboratory testing the etiological diagnosis could be con rmed in 29%, 49% and 75.6% in the studies of Naik et al, Salam et al, and Bansal et al, respectively 15,16,17 .
Comparatively high incidence of the etiological diagnosis in the present study is probably explained by the strict inclusion criteria. Patients with a history of getting antibiotic for more than 48 hours were excluded from the present study. Studies like Naik et al have comparatively relaxed inclusion criteria and included patients irrespective of their duration of antibiotic history and consequently a lower yield of organisms was found. However, no causative organisms were identi ed in a signi cant proportion of patients (34.5%) in the present study. The possible causes for the inability to determine etiology in these patients were lack of sensitivity of laboratory investigations, prior antibiotic treatment and lack of more sophisticated investigations which are expensive and require highly trained personnel. Other prospective studies for evaluating the causes of CAP in adults have failed to establish an etiologic diagnosis in 40 -60% of cases even with extensive diagnostic testing 18,19 .
The most common organism isolated from sputum culture was Klebsiella pneumoniae followed by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. However, globally, Streptococcus pneumoniae (pneumococcus) is widely accepted as being the most common pathogen in CAP, usually presented with acute symptoms of lower respiratory tract infection. An estimated prevalence of 19.3% to 34% was reported for S. pneumoniae in Europe 20,21 . A previous study conducted in another tertiary care hospital of Bangladesh (Salam et al., 2016) Streptococcus pneumoniae was the most frequent organism isolated from the sputum culture from the hospitalized patients with CAP. Though S. pneumonia is commonest organism reported globally, but some studies, conducted in last two decades from neighboring countries in South Asia have reported higher incidence of gram-negative organisms among culture-positive pneumonias. Studies from Malaysia reported that, gram-negative bacteria, Klebsiella pneumoniae and Pseudomonas aeruginosa, were the most common causes of CAP in their series 18 .
Another study from India reported that, Klebsiella pneumoniae (42.85%) was the most common followed by Pseudomonas aeruginosa (28.57%), Staphylococcus aureus (21.43%) and Streptococcus pneumoniae (7.14%) 22 . Mythri et al. reported that the most common pathogen was Klebsiella spp followed by S. pneumoniae and P. aeruginosa. These ndings indicate that, CAP by Gram-negative organisms constitute a signi cant burden in our locale and the spectrum of organisms is subject to geographical alternations 23 . However, compared with western studies, S. pneumonia was of less relative importance in Asian countries. Gram-negative bacilli and Mycobacterium tuberculosis were more important in this continent.
To increase the microbiological diagnosis in the present series PCR was done to detect Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila and Streptococcus pneumoniae. However, only 4 samples yield positive result and all of them were Streptococcus pneumoniae. Intracellular pathogens are frequent causes of pneumonia, in these cases the clinical presentation is 'atypical', characterized by sub-acute symptoms, non-productive cough, low fever, normal white blood cells count and with frequency associated extrapulmonary manifestations. However, the incidence is variable; depending in part on the di culties with microbiological cultures they grow poorly in standard culture media and culture requires expertise 24 .
It was observed from this study that isolated Klebsiella strain was mostly resistant to commonly used antibiotics for CAP like Amoxicillin-Clavulanate, Ce xime, Cefuroxime, clarithromycin and Ceftazidime. P. aeruginosa were highly resistant to Amoxicillin-Clavulanate, Ce xime, Cefuroxime and clarithromycin. S. aureus were highly resistant to clarithromycin, azytyhromycin, Amoxicillin-Clavulanate, Ceftazidime and Ce xime. Other isolated organisms like Pseudomonas, Escherichia coli, were also resistant to Blactamase inhibitor, Macrolides and third generation cephalosporin. This study also revealed Meropenem, Amikacin and levo oxacin were the most sensitive antibiotics for the organisms identi ed form the CAP patients. However, Meropenem is costly and not recommended by the guideline published by American thoracic society (2004) and Infectious disease society of America (IDSA 2004) 25 .
Frequent use beta-lactam antiobiotic and Macrolides for the treatment CAP are rst line regimens but emerging strain are more resistant to these conventional antibiotics. Multi drug resistant to betalactamase, Macrolides and Fluroquinolone is an emerging problem and complicating the management of CAP 26 . Alarming outcome of resistant bacteria was also observed in a study conducted by Salaam et al in Dhaka Medical College Hospital.
The present study was conducted over a short period of just over nine months and it is possible that less common pathogens were not detected during the study. A larger multi-center study is needed to obtain accurate information on the epidemiology of CAP in the country. Moreover, the etiology remained undetermined in 21.4% of patients who died during hospitalization. This emphasizes the need of further investigations in patients in whom the bad prognostic factors are present at the time of admission so as to establish the etiology, start early treatment and thereby reducing mortality.

Conclusion
In conclusion, we found that the Gram-negative bacilli like Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli were common organism for CAP identi ed by sputum culture. Staphylococcus aureus was found by PCR test. For CAP that required hospitalization sensitivity results were in favor of Meropenem, Amikacin and Levo oxacin. Overall mortality in hospital and 30-day were high.