Isolation of extended spectrum beta-lactamase producing Enterobacteriaceae among children with urinary tract infection: A cross sectional study.

Background: Resistance of Gram-negative bacteria to antibiotics had increased at an alarming pace over the last two decades. Empirical antibiotic treatment of UTI practice created opportunity for frequent use of antibiotics that resulted in emergence of resistant strains. Extended spectrum beta-lactamase producing Enterobacteriaceae are emerging worldwide and are usually multi-drug resistant; thus, limiting treatment options for these organisms. Hence, it is mandatory to scrutinize the extent of the problem in the study area. Objective: We investigated the prevalence and correlates of extended spectrum beta-lactamase producing Enterobacteriaceae and their antibacterial resistance profile among children with urinary tract infection (UTI) at Hawassa University Comprehensive Specialized Hospital. Method: A hospital-based cross sectional study was conducted from February 1-July 30/ 2018. A semi-structured questionnaire was used to collect data from 284 children suspected of UTI. Bacterial species were isolated using standard bacteriological techniques. Antibiotic susceptibility testing was done using Kirby-Bauer disk diffusion method and ESBL production was detected by double disc synergy test. Data entry, cleaning and statistical analysis was done using SPSS version 20. Results: Overall, Enterobacteriaceae accounted for 75% (72/96) of UTI infections. The most frequent Enterobacteriaceae were E. coli , 44.4% (32/72) and K. pneumoniae , 27.8% (20/72). Overall, ESBL-producers within Enterobacteriaceae accounted for 41.7% (30/72) and magnitude of multi-drug resistance level was 86.1% (62/72). ESBL producing K. pneumoniae and E. coli accounted for 70% (14/20) and 37.5% (12/32), respectively of all the respective isolates. Previous UTI within the past 12 month (AOR= 0.08, 95% CI (0.01-0.57) and medium family wealth index (AOR = 0.03, 95% CI (0.00-0.27) were associated with infection of ESBL-producing Enterobacteriaceae . Conclusion: ESBL producing Enterobacteriaceae were prevalent among UTI cases; where E. prescribed drugs.

coli and K. pneumoniae were the major isolated resistant strains that contributed mainly to the problem. Majority of Enterobacteriaceae isolated were resistant to commonly prescribed drugs. Key words : ESBL, Antimicrobial Resistance, UTI, Children.

Introduction
Gram negative bacteria, especially the family Enterobacteriaceae are the common causes of both community and hospital acquired urinary tract infection (UTI). UTI ranks second next to upper respiratory tract infection where Escherichia coli ( E. coli) and Klebsiella species are the most common pathogens [1,2].
Over the past two decades, there has been wide use of extended broad-spectrum antibiotics to counter the increasing rates of ESBL-producing bacteria in patients with UTI [3]. ESBL-producing bacteria have been described as a pandemic [4]. Usually initial antibiotic treatment for UTI is empirical until the culture and sensitivity results are obtained. The improper and frequent use of antibiotics resulted in emergence of resistant strains. Resistance of Gram-negative bacteria to antibiotics had increased at an alarming pace over the last two decades [1]. Extended-Spectrum Beta-Lactamase (ESBL) producing bacteria, mostly E. coli and Klebsiella species are complicating treatment of UTI worldwide [5].
The resistance of Gram-negative bacteria is usually due to plasmid-mediated enzymes called ESBLs [6]. Bacterial resistance to β-lactam antibiotics occurs by three mechanisms: failure of the β-lactam to reach the penicillin-binding proteins (PBPs), low binding affinity to the PBPs and inactivation of the drug by β-lactamases. Among these β-Lactamases are the commonest cause of bacterial resistance to β-lactam antimicrobials [7].
ESBLs are β-lactamases containing serine in their active site and are capable of hydrolyzing penicillin group of antibiotics, broad-and extended-spectrum cephalosporins, monobactam, and aztreonam, thereby causing resistance to them. However, they do not hydrolyze cephamycin antibiotics such as cefoxitin and cefotetan. They are generally inactive against the carbapenem antibiotics such as imipenem, meropenem, and ertapenem. Currently, carbapenems are regarded as the drugs of choice for treatment of infections caused by ESBL-producing organisms [8].
Antimicrobial resistance (AMR) in healthcare facilities is a global public health problem.
Worldwide, the increasing rate of antibiotic resistance over time is leading to serious difficulty of antimicrobial treatment [9]. Although AMR is a global problem, the impact is higher in Sub-Saharan Africa (SSA) due to limited available resources for healthcare infrastructure and wide irrational use of antimicrobial agents. Of all those who take antibiotic, more than one-third do not get prescriptions from a doctor, and about a quarter obtain antibiotics from an informal dispenser [7,10].
The risk of ESBL-producing Enterobacteriaceae was investigated in different part of the nation on clinical samples. The extent of these problems was not investigated in the study area previously. The study tried to explore prevalence and antimicrobial resistance profile of ESBL-producing Enterobacteriaceae isolated from UTI suspected children. Findings of the study will help improve local empiric drug choice for the treatment of UTI in children.

Study area, design & period Hawassa University College of Medicine and Health Sciences Comprehensive Specialized
Hospital is a tertiary level teaching hospital found in Hawassa city, Southern Ethiopia.
Hawassa is the capital city of Southern Nations Nationalities and Peoples Regional State (SNNPRs) and is located 273 km to South of Addis Ababa. HUCSH has an estimated catchment of more than 5 million. According to information from hospital HMIS unit, the pediatric outpatient department (OPD) receives about 9,000 cases annually. The Pediatric Department of HUCSH has a total of 65 beds with average monthly admission rate of 75 children. We employed a cross sectional study design from February 1/ 2018 to July 30/ 2018 to enroll children with suspected UTI at the Pediatrics Department of HUCSH.

Study Participants
Children below the age of 15 years who were clinically suspected to have UTI and presenting to Hawassa University Comprehensive Specialized Hospital (HUCSH) were enrolled. Written informed assent was obtained from guardians or patients before data collection.

Inclusion criteria:
Patients who had at least one of the signs and symptoms of urinary tract infection like; frequency, urgency, dysuria, abdominal pain, back pain, fever (>38.0°C).

Exclusion criteria:
Patients who received antibiotics within two weeks before presentation to the hospital were excluded.

Sample size determination and Sampling techniques
Sample size calculated using single-population proportion formulae with assumptions based on a previous study done at Tikur Anbessa Specialized Hospital, which showed prevalence of extended spectrum β-lactamase (ESBL) of 78.6% [8] gave the largest sample size. The final sample size for a population survey considering a 10% non-response rate gave 284 subjects suspected of UTI.

Formulae:
(Sample size) n = Z 2 α/ 2 p (1-p)/ d 2 Where: n = the number of study subjects from which samples will be taken P = anticipated population proportion. Prevalence of ESBL taken as 0.786 Z = Standard normal distribution value at 95% CI, which is 1.96 d = Degree of precision taken as 5%.

Socio-demographic and clinical data:
Socio-demographic characteristics like; age, sex, residence, paternal and maternal educational and occupational status, family living standard and clinical history of participants such as hospital admission and history of UTI within the past 12 months were recorded.

Laboratory data collection:
A total of 284 early morning mid-stream (MSU) and catheterized urine samples was collected using sterile, clean, transparent, screw-capped, wide-mouth plastic cups. All urine samples were properly labeled with unique code, age, sex, and time of collection and the sample was transported to microbiology laboratory within two hours of collection.
By using the standard wire loop which has 1µl diameter approximately 0.001 ml urine was inoculated on 5% sheep Blood agar (OXOID Ltd. England) and MacConkey agar (OXOID Ltd., Basingstoke, United Kingdom) plate and the plates were incubated aerobically at 37°C for 24hrs.
A significant bacterium was determined on Blood agar plate (BAP) if single midstream and catheterized urine culture yields at least 10 5 CFU/mL [9]. All types of colonies on the primary plates were examined macroscopically for hemolysis, changes in physical appearance of differential media, and the colony characteristics were recorded. Cefotaxime (CTX: 30μg), ceftazidime (CAZ: 30μg) and ceftriaxone (CTR: 30μg) were placed on the MHA using an automated dispenser and lightly pressed on to make sure they are firmly placed to the media. These plates were incubated for 18-24 hours at 37 0 C. Isolates showing inhibition zone size below the CLSI stated break points were considered a potential ESBL-producer when the inhibition zone size for: ceftazidime < 22 mm, cefotaxime < 27 mm and ceftriaxone < 25 mm.

Confirmatory test for ESBL producing Enterobacteriaceae:
The ESBLs detection was carried out by double disc synergy test using third generation cephalosporins and modified double disc synergy test using cefepime along with the third generation cephalosporins. All the strains which were positive for ESBL screening test was selected for checking for ESBLs production.

Double disk synergy test (DDST)
The double disc synergy test (DDST) is used as primary isolation method to identify the ESBL producing organisms. Antibiotic discs of amoxicillin / clavulanic acid (20/10μg) at the center; cefotaxime (30μg) and ceftazidime were placed at a distance of 15 mm apart center to center and incubated at 37˚C for 18-24 hours. A clear extension of cefotaxime and ceftazidime inhibition zones towards the disc containing clavulanic acid was considered as ESBL producer. Isolates which were screened and found positive for ESBL production may be negative for confirmatory test using DDST due to the coproduction of other beta lactamases like AMPC lactamase. In such cases, modified double disk synergy test were used using Cefepime antibiotics for inhibiting other beta lactamases (AMPC lactamase).

Modified Double disk synergy test (MDDST)
The test is performed by using a disc of amoxicillin-clavulanate (20/10 μg) along with four cephalosporins (cefotaxime, ceftriaxone, ceftazidime and cefepime). A disc which contained amoxicillin clavulanate (20/10 μg) was placed in the centre of the plate. The discs of third generation cephalosporin (cefotaxime, ceftriaxone and ceftazidime) and fourth generation cephalosporin (cefepime) were placed 15 mm and 20 mm apart respectively, centre to centre to that of the amoxicillin clavulanate disc on the surface of MHA plate, as recommend by CLSI, 2017. The plates were incubated at 37˚C for 24 hours.
Any distortion or increases in the zone toward the disc of amoxicillin-clavulanate were considered as positive for ESBLs production.

Antimicrobial Susceptibility Testing
Antibiotic susceptibility testing was done by Kirby Bauer disk diffusion method on MHA as recommended by the CLSI guidelines [9, 10] for the following antimicrobial discs:

Data Analysis
The data were analyzed by using SPSS version 20 software. All variables with a P-value <0.25 in bivariate analysis were taken to multivariable logistic regression analysis. On multivariable analysis, variables with P-value <0.05 at 95% confidence interval were considered as statistically significant. Wealth status of the family used as a variables using principal component analysis (PCA).

Ethical Considerations
Written informed assent were obtained from study participants or caretakers of children.
Patient privacy was protected by de-identification of records. Names of patients were coded. All data obtained in the course of the study were kept confidential, and was used solely for the purpose of the study. Positive laboratory result from the study participant was communicated to their physicians for appropriate treatment or management.

Antibiotic resistance profile of isolated Enterobacteriaceae
The antibiotics resistance profile of Enterobacteriaceae isolated in urine specimen against 12 antibiotics is presented in  (Table 6).

Discussion
In this study; 284 urine samples were collected from study participants. Among these; 90 of the samples were culture positive and a total of 96 bacterial species were isolated.
The ESBLs prevalence rate in the present study was much lower than those reported from Tikur Anbessa Specialized Hospital (78.6%) conducted variably on urinary and septicemia isolates [8]. The decline observed in our study might be attributed to the inclusion of only urine specimens; additionally, the high prevalence (78.6%) might be because of small number of Enterobacteriaceae isolated compared to the current study. The ESBL prevalence was also lower than those reported in Burkina Faso 58%, from clinical samples [18] and Nepal 62.31% from urinary isolates [19], these might be due to the inclusion of all age groups in the former study.
On the other hand, prevalence of ESBL observed in this study was higher than other studies conducted in Adama 25% [20], Morocco 25.5% [21], Iran 28.4% [22] [17]. and also comparable with study in Addis Ababa, Ethiopia (Amoxacillin/clavulanic acid 91.5%, trimethoprim-sulfamethoxazole 89.8%) [15]. This may suggest that the production of ESBLs by the family of Enterobacteriaceae may limit the treatment option available for the management of the infections caused by this group of bacteria. Thus the members of the family of Enterobacteriaceae that were proven to be produce ESBL pose a serious problem in the clinical management of UTI which trigger to put strong effort for the prevention of ESBL occurrence.

Conclusion
Our research finding evidenced that ESBL producing bacteria was prevalent among children with UTI. From the total bacteria species isolated, Enterobacteriaceae covered majority of the percent. Most of these Enterobacteriaceae were isolated from patients who attended inpatient department. Regarding ESBL producing specific bacteria species, ESBL producing Klebsiella species were the most frequent isolates followed by E. coli among Enterobacteriaceae. Majority of Enterobacteriaceae had resistance to commonly prescribed antibiotics. In the study, the most effective drug for ESBL producers and non-ESBL producers identified was nitrofurantoin.

Limitations of the Study
Certain clinical features such as ICU admission and circumcision (only for boy) did not assess as potential associated factor for ESBL production due to few numbers of cases.
Molecular epidemiological study and characterization of ESBL types were not conducted.

Data Availability
Raw data can be presented by the principal investigator upon reasonable request.

Ethical Approval and consent to participate
Ethical clearance was granted from Hawassa University College of Medicine and Health Sciences Institutional Review Board with a reference number IRB/156/10. Written informed assent was obtained from all study participant parents/guardians by telling them about the objective of the study before collecting the laboratory data. All data obtained in the course of the study were kept confidential, and was used solely for the purpose of the study. Moreover, all investigators were assured to guarantee safety and proper care of the study participants.

Consent for publication
Not applicable

Competing Interest
The authors declare that they have no competing interests.        Figure 1 The graph displays percent of ESBL production of individual bacteria out of the total ESBL producing Enterobacteriaceae isolates (n=30).