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Multi-Drug Resistant, Extended Spectrum β-Lactamase and Carbapenemase Producing Bacterial Isolates among Septicemia Suspected under Five Children in Tikur Anbesa Specialized Hospital, Addis Ababa Ethiopia

Background : Bloodstream infections due to bacterial pathogens are a major cause of morbidity and mortality among pediatric patients. Emergence of drug resistance in high classes of antibiotics among the bacterial pathogens is another issue of the public health concern. Objective : To determine Multi-drug resistant, extended spectrum β-lactamase and carbapenemase producing bacterial isolates among septicemia suspected under five Children in Tikur Anbesa Specialized Hospital, Addis Ababa Ethiopia. Methods : Across-sectional study was conducted from September 2017 to June 2018 among pediatric patients with febrile illness aged under five in Tikur Anbesa Specialized Hospital. 340 Blood samples were collected and processed following standard microbiological techniques and culture was performed using BacT/Alert machine in combination with conventional method. AST of the isolates was performed by Kirby-Bauer disc diffusion method and MIC technique Result: A total of 137(40.2%) bacterial pathogens were isolated from 340 pediatric patients suspected of BSI with febrile illness. Of these isolates 54% were Gram negative bacteria. Of the isolates 43 (31.4%) Klebsiella pneumonia Acinitobactor species were the most frequently isolated pathogens. Klebsiella pneumoniae isolates were 95.6% MDR, 23.7% ESBL, and 27.1% CRE in children. Conclusion : In this study, Klebsiella pneumoniae and S. aureus are common pathogens associated with BSI in pediatrics with high antimicrobial resistance. The prevalence of MDR 51.1%, CRE 30.5% and ESBL 25.4% were alarmingly high in bacterial isolates. ESBL producing organisms were common in Klebsiella species and Escherichia coli isolates. Since most of isolates exhibit multidrug resistant, invitro- susceptibility of antimicrobials is mandatory. Strengthing antimicrobial surveillance system and antimicrobial stewardship

are necessary for better management of antibiotics in addition to infection prevention practice in TASH settings.

Background
Blood stream infection (BSI) remains one of the most important causes of morbidity and mortality throughout the world. Approximately 200,000 cases of bacteremia occur annually with mortality rates ranging from 20-50% worldwide [1]. Blood stream infection (BSI) accounts for 10-20% of all nosocomial infections and is the eighth leading cause of mortality, in the United States some 17% of result in death [2]. In sub Saharan countries including Ethiopia septicemia is an important cause of illness and death in children, the mortality rate approaches 53% which makes it a significant health problem in developing countries [3].
In many studies a wide range of bacteria has been described in febrile patients including gram negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella species, Neisseria meningitidis, Haemophilus influenzae, and gram positive such as Coagulase negative staphylococci (CONS), Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and Enterococcus faecium [4]. The diagnosis of these infections can be confirmed by blood culture, which is routinely available in few Hospitals in developing countries [5].
Bacterial pathogens isolated from BSI are a leading cause of significant patient morbidity and mortality. The impact of specific etiologic agents on BSI patient outcome are tremendous; BSI increases the mortality rate, prolongs patient stay in an intensive care unit and in the Hospital, and leads to increased health care costs [6,7].
The timely and appropriate use of antibiotics is currently the only way to treat bacteremia. However, many bacterial pathogens have become resistant to antibiotic regimens and become a serious public health concern with economic and social implications throughout the world. Antibiotics resistance is a growing problem in developing countries such as Ethiopia. In Ethiopia the unregulated over-the-counter sale of these antimicrobials, mainly for self-treatment of suspected infection in humans, and to a lesser extent for use in animals without prescription, would inevitably lead to emergence and rapid dissemination of resistance [8]. Many studies have found that inadequate empirical therapy of bacteraemic infections is associated with adverse outcomes, including increased mortality and increased drug resistance emergence [9][10].
During the past few decades, antimicrobial resistance has increased worldwide, and the perspectives are alarming [11][12]. The nature, the magnitude and ways to cope with this problem are studied and described in the western world, while this base of knowledge is lacking in developing African countries [13][14][15]. We lack reports on mortality related to distribution of pathogens and their resistance patterns. Without such reports, guidelines for empiric treatment of severe bacterial diseases cannot be given. While updated studies on outcome in sepsis in Africa are almost non-existent, there are a few reports on bacterial culture results. The most alarming reports on antimicrobial resistance concern patients admitted to Hospitals [16], while community-acquired infections may have lower profiles of resistance [17]. In Ethiopia, the resource situation has not allowed antimicrobial resistance to be prioritized as major public health concern despite the obvious needs [18]. The aim of this study was to identify and determine multi-drug resistance bacteria among blood culture samples from under five patients attending to Tikur Anbesa Specialized Hospital by BacT/Alert and biochemical test.

Study area:
The study was conducted in Tikur Anbesa Specialized Hospital (TASH), the teaching Hospital of health Science College, Addis Ababa University. TASH is the largest specialized Hospital in Ethiopia, with over 700 beds, and serves as a training center for undergraduate and postgraduate medical students, dentists, nurses, midwives, pharmacists, medical laboratory technologists, radiology technologists, and others who shoulder the health problems of the community and the country at large. With more than 70 percent of childhood deaths attributable to communicable diseases and malnutrition, Ethiopia's healthcare resources have been directed primarily to treat and prevent diseases such as malaria and diarrhea [19].

Study design and period:
A cross-sectional study was conducted from September 2017 to June 2018 to identify the bacterial profiles and antimicrobial susceptibility pattern among septicemia under five patients with acute febrile illness in Tikur Anbesa Specialized Hospital in Addis Ababa.
Source population: All under 5 years pediatric patients who were suspected for septicemia and seek medical care at the study site during the study period Study population: All under 5 years pediatric patients who were requested for blood culture in the study site during the study period.

Inclusion and Exclusion criteria:
Children aged under five years including neonates with fever Patients who are diagnosed with sepsis, Sever sepsis and septic shock. In addition, all children who gave blood sample and their parents volunteer to give permission to participate on the study. However those participants clinically none febrile patients under five years. Patients who took antibiotics currently within the last 7 days were excluded.

Sample size calculation
The sample size for the study that infers the total population was determined using a single population proportion formula. The study considered the previous study of prevalence and antibiotic resistance of bacterial pathogens isolated from children under five in septicemia patients at Tikur Anbesa Specialized Hospital 27.9% bacterial isolation (20), at 95% level of confidence and 5% margin of error. n = (Zα)2 (pq)/ d2 Where: n = sample size Zα/2 = level of confidence P = diarrhea prevalence q = 1-p d2 = margin of error (0.05) n= z² * p * q, p=0.

Data collection procedure
Well standardized questionnaire was used to collect socio-demographic characteristics (sex, age, clinical presentation (fever, vomiting and household income). Patients visiting outpatient departments (pediatric and general medicine) and those admitted in the inpatient units were investigated for bloodstream infections by respective unit physicians.
At the onset of fever (>37∘C) or in the presence of any clinical symptoms compatible with infection.

Laboratory analysis
Blood sample collection: A venous blood culture specimen was taken with aseptic technique by cleansing of the collection site with 70% alcohol and subsequently followed by 10% povidone-iodine solution by trained laboratory personnel. About 2.5-5ml of blood specimen was collected and inoculated into aerobic 30ml BacT/ALERT PF Plus pediatric bottles at the blood to broth ratio of 1: 10-1:30. At least 2 sets of blood cultures were collected from a patient with suspected bacteremia prior to the initiation of antimicrobial therapy.
Culture Isolation and Identification: Venous blood to BacT/ALERT culture bottles were incubated in automatic BacT/ALERT® 3D at 37oC of 5% CO2 for 5 days for the primary isolation of the microorganism. Two aerobic blood culture bottles were used for each patient and growth in both bottles were considered positive. The microbial growth that could be detected by flag and audible sound of the instrument will subsequently be sub culture on 5% sheep blood Agar, chocolate, and MacConkey Agar plate (Oxoid Ltd, UK) and incubate at 37oC for 18-24 for bacterial isolation. The MacConkey agar plate was incubated aerobically while chocolate and blood agar were incubated in microaerophilic atmosphere (5-10% CO2) candle Jar. A negative result was checked by examining the flag and doing gram stain and a final subculture at the end of 5th day prior to discarding as negative. The significant growth colonies were examined morphologically for size, consistency, shape, hemolytic and ability to ferment lactose [21].  Descriptive statistics to analysis by using frequency, proportions graphs, crosstabs and odds ratio. Bivariate analysis was performed for each factors associated with enteric pathogens in pediatrics diarrhea. Regression analysis was conducted to identify associated factors and how they are associated with dependent variables .The strength of association was presented by odds ratio and 95% confidence interval and p-value of <0.05 was considered as statistical significant association between risk factors and enteric pathogens causing diarrhea and antimicrobial resistance of bacterial infection.

Ethical Considerations:
The study was conducted after ethical clearance was obtained from the research ethical committee of Department of Medical Laboratory sciences. An informed consent was obtained before collection of blood specimens and results were used in the management of patients. Those patients who clinically diagnosed as BSI in pediatric OPD and admitted willing to participate in the study and able to give blood sample during the study period were informed about the purpose of the study and written consent was sought for the study. Any information related with the patient result and clinical history was kept confidential.

Dissemination and Utilization of Results
After the completion of the study the research were disseminated to Department of Medical Laboratory Sciences, School of Allied Health Science, College of Health Science, and Addis Ababa University. It will also be submitted for scientific publication.

Operational Definitions
Antimicrobial resistance: occurs when microorganisms change in ways that render the medications used to cure the infections they cause ineffective.
Multidrug resistance (MDR): is antimicrobial resistance shown by a species of microorganism at least to one drug in three different classes of antibiotics.

Results
Among the study participants 122(35.9%) were males and 218 (64.1%) were females resulting in an overall female to male ratio of 1.7:1. The mean age of pediatrics participated in this study was 1.04±1.0 (SD) years. [ Table 1] Piperacillin-Tazobactam (58.1%). All Acinitobactor species were highly resistance to tested antimicrobials such as cefepime (100%), ceftazidime (90.9%), 72.7% for each meropenem and ciprofloxacillin. Pseudomonas spp also showed fifty percent (50%) resistance to antipseudomonal antibiotics gentamycin, ciprofloxacillin, cefepime, Amikacin and ceftazidime but it was susceptible 75% to meropenem and Piperacillin-Tazobactam. All Salmonella species completely susceptible to Ciprofloxacillin, ceftriaxone, and ampicillin and less susceptible to cotrimosazole (50%) as shown in Multi-drug resistant isolates : Antibiogram pattern of the isolates in this study showed that multidrug resistance among gram negative isolates the prevalence of multidrug resistance (MDR) in Pseudomonas aeruginosa showed that two (50%) of the isolates exhibit resistance to three antibiotics. In Klebsiella pneumoniae. majority of isolates 35(81.4%) were resistance to eight and more tested antibiotics even though 2(4.6%), 1(2.3%), 2(4.6%) and 2(4.6%) isolates were resistance consecutively to three ,four ,six and seven antibiotics respectively . Among eleven Acinitobacter spp 7(63.6%) isolates were resistance to eight and more antimicrobials and 1(9.1%) was resistance to seven antibiotics .the least isolate of gram negative bacteria Entrobacter cloacae 1(100%) was resistance to eight and more antibiotics. However there was no MDR in Citrobacter and Salmonella species.
Among the suspected 17 isolates 100% (n=17/17) were phenotypically confirmed for ESBL using combination disk method, K. pneumoniae 100% (n=16/16) and E. coli 100% (n= 1/1) were positive for ESBL ( figure). For result interpretation we use this result as the CLSI recommend this technique as reference for other phenotypic methods. We also use this test result to compare the findings of double disk method.

Double Disk Synergy Test (DDST):
All isolates (n=17) were further tested for ESBL production by double disk synergy procedure, another phenotypic confirmatory method.

Discussion
Blood stream infection (BSI) in pediatric patients associated with febrile illness is a major public health problem especially in developing countries where high child morbidity and mortality rate. So timely detection of bacteremia in blood culture set is a promising diagnostic tool established to rule out bacteremia and determination of its antimicrobial Susceptibility profile is necessary for clinicians to decide appropriate empirical therapy, which ultimately decreases the emergence of drug resistance [ The difference between studies might be due to differences patient condition in which our study includes more patients from ICU and impatient than outpatients in addition blood culture was performed by using more sensitive automated BacT/ALERT system. However we have isolated bacteria lower than the studies in Nigeria 47.6% [33], this was due to the patient condition in which others only include impatient and isolate anaerobic bacteria.  [48] frequently isolated pathogen in BSI was Pseudomonas other than Klebsiella pneumoniae in the same age group. The possible difference might be due the difference prescription of antibiotics for empirical treatment of patients before blood culture and difference of management in pathogens causing nosocomial infection across the counties in addition in our Hospital setting, nosocomial infections were not proper patient isolation system in the ward which further increase the survival of high drug resistant bacteria including Klebsiella pneumoniae.
A polymicrobial infection in our study was isolated in a single patient and etiologies both were from gram negative bacteria that tends to increase the severity of the diseases which is in agreement with previous study [49,50] even though some microbiologists consider polymicrobial growth as a contamination, but sepsis should be clinically correlated [50].
The trend of empirical treatment in our study 43.5% and the most prescribed antibiotics were ampicillin, gentamicin, ciprofloxacillin and third -generation cephalosporin (most common ceftriaxone) in which ampicillin and gentamicin were the most common combined drugs used. This was supported by the previous study in Tamale, Ghana [51].
The antimicrobial susceptibility of gram negative bacteria predominately Klebsiella pneumoniae isolates were high level of resistance to ampicillin(100%), cotrimosazole (90.7%) and gentamycin (88.4%),despite of sensitive to meropenem (62.8%), Piperacillin-Tazobactam (58.1%) was consistent with the studies by Zenebe et al [52] reported 100%resistance to ampicillin and Cotrimosazole, in Bahir dar ,Ethiopia by Hailu et al., [53] ampicillin 91.4%,cotrimosazole 77.1% and gentamicin 71% while in India the resistance of ampicillin, cotrimosazole and gentamycin done by Kumar et al.,[43] were 97%,88%,67% respectively. it was also comparable in Kaneti children Hospital, Nepal by kari et al [54] reported 100% resistance to ampicillin and least sensitive to Cotrimosazole and Gentamycin. The highest potent drugs 3 rd and 4 th generation cephalosporin, quinoles and carbapenem antibiotics also showed resistance which is a concern for treatment of BSI in pediatrics with septicemia.
The second most predominant GNB isolates in our study were Acinitobacter species resistance to most tested antimicrobials ceftazidime100%, cefepime 90.9% gentamycin 81.8%, torbomycin 81.8% ciprofloxacillin 72.7%, meropenem 72.7% was comparable with other previous studies where high resistance of Acinitobacter species was published [55,56]. However our result was high rate of resistance compared to the study conducted in South India by Zakariya et al., [57] in which meropenem 100% sensitive, while 67% were sensitive to gentamicin, ceftriaxone, ciprofloxacin, ceftazidime and Amikacin reported. This is the fact that we had relatively many isolates and might be due to inappropriate empirical use of meropenem as the first line treatment since most of isolates are from ICU patients in our Hospital.
The overall prevalence of multidrug resistance isolates MDR in our study was 51.1% of which most of them were Gram-negative bacteria with a very high resistance to betalactam antibiotics. This result is supported by the previous study in Ethiopia [20]. Among Gram negative bacterial isolates, Klebsiella 95.9% and Acinetobacter72.2% were dominant species. This was consistent with the study in north India [58].
The present study identified carbapenem resistance enterobacteriaceae (CRE) with the rate of 30.5% comparable with study conducted in Tanzania 35% [59]. The most carbapenem resistance was detected in 72.2% isolates of Acinetobacter spp. and in 62.8% of Klebsiella pneumoniae. This was inconsistent with the study in north India 64%, 92% [58] respectively.
The prevalence of ESBL-producing Enterobacteriaceae in our study is 25.4%. Among 43