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

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. Therefore this study aimed 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, 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. Three hundred forty 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 pneumoniae and Acinitiobactor 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. Strengthen antimicrobial surveillance prevention

system and antimicrobial stewardship are necessary for better management of antibiotics in addition to infection prevention practice in TASH settings. Key words: Blood stream infection, BacT/Alert, multi-drug resistance, Extended spectrum betalactamase, Carbapenem resistance entrobacteriaciae. Background Blood circulatory system infection stays one of the most significant reasons for horribleness and mortality all through the world. Roughly 200,000 instances of bacteremia happen every year with death rates running from 20-half overall [1].
Blood circulatory system infection represents 10-20% of every single nosocomial disease and is the eighth driving reason for mortality, in the United States some 17% of result in death [2]. In sub Saharan nations including Ethiopia septicemia is a significant reason for sickness and passing in youngsters, the death rate approaches 53% which makes it a noteworthy medical issue in under developed countries [3].
In numerous investigations a wide scope of microscopic organisms has been portrayed in febrile patients including gram negative microorganisms, for example,  [4].
The determination of these microorganisms can be affirmed by blood culture, which is routinely accessible in couple of Hospitals in developing nations [5].
Bacterial pathogens isolated from blood stream infection are a main source of critical patient morbidity and mortality. The effect of explicit etiologic specialists on BSI persistent result are enormous; BSI builds the death rate, drags outpatient remain in an emergency unit in the Hospital, and prompts expanded human services costs [6,7].
The auspicious and suitable utilization of anti-microbial is as of now the best way to treat bacteremia. In any case, numerous bacterial pathogens have turned out to be impervious to anti-infection regimens and become a genuine general wellbeing worry with financial and social ramifications all through the world. Anti-microbial resistance is a developing issue in developing nations, for example, Ethiopia. In Ethiopia the unregulated over-the-counter closeout of these antimicrobials, for the most part for self-treatment of suspected disease in people, and to a lesser degree for use in creatures without remedy, would unavoidably prompt rise and quick dispersal of obstruction [8]. Numerous examinations have discovered that lacking experimental treatment of bacteraemic infection is related with antagonistic results, including expanded mortality and expanded medication obstruction development [9][10].
During the previous couple of decades, antimicrobial opposition has expanded around the world, and the points of view are disturbing [11]. The nature, the greatness and approaches to adapt to this issue are contemplated and depicted in the western world, while this base of learning is deficient in creating African nations [12][13][14].
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 [15], while community-acquired infections may have lower profiles of resistance [16]. In Ethiopia, the resource situation has not allowed antimicrobial resistance to be prioritized as major public health concern despite the obvious needs [17]. The aim of this study was to identify and determine Multi-drug resistant, extended spectrum β-lactamase and carbapenemase producing bacterial isolates among blood culture samples from under five patients attending to Tikur Anbesa Specialized Hospital by automated BacT/Alert machine.

Study setting
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 [18].

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.

Inclusion and Exclusion criteria
Children aged under five years including neonates with fever and those 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.

Sampling technique
The study subjects were selected using convenient sampling technique from all patients attending Tikur Anbesa specialized Hospital among under five children with febrile illness clinically diagnosed at pediatric OPD, ICU and impatient pediatric ward admitted during the study period. Sampling technique was employed for those children fulfill the inclusion criteria.

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.

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% povidoneiodine 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. For gram negative bacteria convectional biochemical test was performed [20].

Antibiotic Susceptibility Test
Pure Colony of isolated bacterial organism was mixed with 0.85% normal saline and measured at 0.5 McFarland standards for susceptibility testing. The bacterial isolates were tested against the following drugs commonly used; for gram negative bacteria Tobramycin (10μg), Amoxicilin-Clavulanate (20/10μg), Amikacin

Detection of Extended spectrum beta-lactamase
Initial screening for ESBL was done by the diameters of zones of inhibition produced by Ceftazidime (30µg), Ceftriaxone (30µg) and Cefotaxime (30µg) found to be within the CLSI screening criteria. These breakpoints indicative of thought for ESBL production are: for CAZ≤22mm, CRO, ≤ 25 mm and for CTX≤ 27mm.
Phenotypic detection of ESBL production was confirmed by double disk synergy test and combined disk test according to CLSI(2017) guideline.

Combined disk (double disk potentiate) Test (CDT)
A Ceftazidime (30 µg) disk and Cefotaxime (30µg) dick were used alone and their combination with Clavulanic acid (30 µg/10 µg) for phenotypic confirmation of the presence of ESBLs. A ≥5 mm increase in zone diameter for either of the Cephalosporin disks and their respective Cephalosporin/Clavulanate disk were interpreted as ESBL producer. This method (according to CLSI) is used as reference phenotypic method for comparing double disk synergy method.

Double Disk Synergy Test (DDST)
The organism to be tested was spread onto a Mueller-Hinton agar plate. The antibiotic disks used are Ceftriaxone (30 µg), Cefotaxime (30 µg), Ceftazidime (30µg), Aztreonam (30µg) and Amoxicillin/ Clavulanic acid (20/10 µg). The four antibiotics were placed at distances of 20 mm (edge to edge) from the Amoxicillin/Clavulanic acid disk placed in the middle of the plate. After 24-h incubation, if an enhanced zone of inhibition between either of the cephalosporin antibiotics and the Amoxicillin/Clavulanic acid disk occurred, the test was considered positive.

Media preparation as per manufacturer instructions and laboratory Standard
Operating Procedures (SOP) was strictly followed. Verify that media meet expiration date and quality control parameters per CLSI. Labeling container, media, filling the forms were carried out.
Visual inspections of cracks in media or plastic petri-dishes, unequal fill, hemolysis, evidence of freezing, bubbles, and contamination were performed. Use ATCC control strain for each isolated bacterium including E. coli 25922, S. aureus 25923, Pseudomonas areuginosa 27853, H.influnzae 10479. Report the results on log sheet and stored for further data. Samples were stored at -80 0c in skim milk.

Data analysis and interpretation
SPSS versions 20.0 was employed to analyze the work and to make inferences on the frequency of occurrence of the bacterial pathogens associated with febrile illness and to show the resistance pattern to antibiotic substances. Descriptive statistics to analysis by using frequency, proportions graphs, crosstabs and odds ratio. Bivariate analysis was performed for each factors associated with blood stream infection. 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.

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.

Socio-demographic characteristics
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 high positive blood culture were identified as shown (Figure1).

Bacteria pathogens among blood stream infection
Of 340 paired blood sample bottles, a total of 137(40.2%) bacterial pathogens were isolated from pediatric patients suspected of BSI with febrile illness. Among positive blood culture results about 54% of them were Gram negative bacteria with.Klebsiellapneumoniae was the highest incidence 31.4% followed by Acinitiobactor species (8.7%). Double infection from pseudomonas species and Klebsiellaoxytoca were identified in one patient as shown (Figure2).

Antimicrobial susceptibility Testing
Trends of antibiotics prescribed were assessed prior to blood sample collection before 7days and about 148(43.5%) participants have taken antibiotics empirically, of these 49(33.1%) were culture positive during the study. Ampicillin and Gentamicin were among the most common empirical prescribed antibiotics. After collection of positive blood culture results about 20 antibiotics were applied in 137(40.2%) isolates and it revealed that the most prescribed antibiotics cotrimosazole, gentamycin, and ciprofloxacin showed high resistance.

Antimicrobial susceptibility pattern of Gram negative bacterial isolates
In 74 gram negative isolates with exception of Salmonella species, susceptibilities of beta-lactam antibiotics, fluoroquinolones, aminoglycosides, and carbapenems were applied for isolates from pediatric patients. The predominant gram negative isolates from BSI were Klebsiellapneumoniae species showed resistance to ampicillin (100%) and cotrimosazole (90.7%). On the other hand, the isolates susceptible to meropenem (62.8%) and 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 ciprofloxacin. Pseudomonas species also showed fifty percent (50%) resistance to anti-pseudomonal antibiotics gentamycin, ciprofloxacin, cefepime, Amikacin and ceftazidime but it was susceptible 75% to meropenem and Piperacillin-Tazobactam. All Salmonella species completely susceptible to Ciprofloxacin, ceftriaxone, and ampicillin and less susceptible to cotrimosazole (50%) ( Table 2).

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 Klebsiellapneumoniae, 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, sixand seven antibiotics respectively. Among eleven Acinitobacter species 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 ( Table 3).

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 However it was inconsistent that the predominant GNB isolation rate varies from country to country where in India by Kante et al., [45], Indonesia by Murni et al., [46] frequently isolated pathogen in BSI was Pseudomonas other than 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 even though some microbiologists consider polymicrobial growth as a contamination, but sepsis should be clinically correlated [47].
The trend of empirical treatment in our study 43.5% and the most prescribed antibiotics were ampicillin, gentamicin, ciprofloxacin 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 [48].

The antimicrobial susceptibility of gram negative bacteria predominately
Klebsiellapneumoniae isolates were high level of resistance to ampicillin(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% ciprofloxacin 72.7%, meropenem 72.7% was comparable with other previous studies where high resistance of Acinitobacterspecies was published [52,53]. However our result was high rate of resistance compared to the study conducted in South India by Zakariya et al., [54] 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 86.5% of which most of them were Gram-negative bacteria with a very high resistance to beta-lactam antibiotics. This result is supported by the previous study in Ethiopia [

Declarations
The author's declare that the study is their original work

Ethics approval and consent to participate
The study was conducted after it was approved by the department of Medical ) . An informed consent was obtained from mother /guardian before collection of blood specimens and results were used in the management of patients.
Written consent was sought for the study and any information related with the patient result and clinical history was kept confidential.

Availability of data and material
The data is available in first author and can provide when necessary

Competing interests
We declare the is no competing interest