A cross sectional study design was employed from March 1 to April 30, 2021 in Addis Ababa which is the Capital City of the Federal Democratic Republic of Ethiopia, the diplomatic capital of Africa and the seat of different international and regional organizations. The city administration is divided in to ten sub city and 116 woreda. It hosts an estimated 3.2-38 million people, which is a 17% share of Ethiopia’s total urban population. Presently, Addis Ababa is experiencing an annual growth rate of 3.8% and is speculated to reach 4.7 million residents by 2030. The city covers a Landmass of 540 square kilo meters. The city is located at the heart of the country, at an altitude ranging from 2,100 meters at Akaki in the south to 3,000 meters at Entoto Hill in the North (21).
Within the city there are 13 government hospitals (five federal, six under Addis Ababa health bureau, one owned by police force and one armed force hospital) distributed throughout 10 sub cities. For this study, it was necessary to pick out major hospitals with a lot of instrumentation, a variety of medical services, higher bed and large run of patients. Hence, hospitals having 200 or more beds and those which provide 10 or more type of medical services are considered as major. Seven governmental hospitals in Addis Ababa meet these criteria. Out of these, 5 hospitals are selected randomly to making a sample size of 38.5%. Ten percent or more samples is considered as a good sample size for small populations (22). The selected hospitals included: Tikur Ambesa Specialized Hospital (TASH) having 543 beds and providing 26 different medical service, St. Paul’s Hospital Millennium Medical College (SPHMMC) with 337 beds and providing 25 different medical service, All African leprosy and tuberculosis rehabilitation training center (Alert hospital) with 241 beds and providing 16 different medical service, Yekatit 12 hospital medical college (Y12HMC) with 210 beds and 19 different medical service and Menilik ΙΙ referral hospital (MIIRH) with 203 beds and 15 different medical service(23).
Laboratory analysis was conducted at Ethiopian public health institute (EPHI) in clinical bacteriology and mycology national referral laboratory in collaboration with food microbiology laboratory. The laboratory has been accredited by Ethiopian National Accreditation Office.
Sampling Frequency and Sampling Technique
Hospitals that have higher bed and serving many patients were selected in ten sub- city of Addis Ababa. Based on these five governmental hospitals were included for this study. In each hospital, six sampling sites are employed to collect hospital effluent. These are a manhole used to collect a wastewater originate from adult ward, pediatric ward, labor ward, laboratory unit, laundry unit and a manhole of septic tank (which hold effluent from all source and we termed Mixed). In addition, one sampling unit viz. MDR TB ward was incorporated to collect hospital effluent only in alert hospital.
A total of 100 discrete (that is 50 at the morning and 50 at the afternoon) wastewater samples were collected from each ward/unit wastewater collecting manholes at the sampling site with four hour interval in the study period. This encompassed discrete effluent of 24 from each Y12HMC and MIIRH, 20 from each SPHMMC and ALERT, and 12 discrete effluents from TASH.
A “Grab sampling technique” was applied to collect the most representative samples according to guidelines of wastewater sampling techniques stated on Environment Protection Authority (EPA)(24) and American Public Health Association (APHA)(25). Discrete samples were collected in two rounds in each hospital for two months. The 1st and 2nd round samples were collected within 15 day interval. In each round, the discrete samples were collected two times a day with four hour interval from each sampling sites in each hospital in 150 ml cleaned and sterile microbiological glass bottles. Here 150 ml sterile glass container was used to collect 125 ml wastewater samples.
Hospitals’ effluent Samples were collected during their maximum activity period (usually 10:00 am- 2:30 pm) according to the method used by Nuñez and Moretton(10). In addition, samples were collected near the center of flow channel, at approximately 10-15 centimeter of the water depth, where the turbulence was at maximum and the possibility settling was minimized. Grazing (skimming) the water surface or slogging the bottle was avoided. The first sample was collected in the morning 10 -10:30 AM whereas the second sample was collected at 2-230 PM from each sampling site. After taking the sample, the neck of the bottle was wiped with 95% alcohol then the sample bottle labelled with date, code number, and time and sampling site. All samples were collected manually and transported instantly to Ethiopia Public Health Institute (EPHI) food microbiology and clinical laboratories with cold chin (4˚C) for bacteriological analysis within six hours of collection. A pair of new, non-powdered, disposable glove, a suitable gown and eye google were used each time while, collecting samples to avoid personal contamination. In the same token, heavy duty glove was used to clean and pick up the cover of manholes at time of sample collection.
In present study, there were different wards and units in the selected hospitals. Each ward/unit generated wastewater having different characteristics. So in order to locate in which sampling site the isolate found, the hospital effluents were collected at different manhole of each units/wards of hospital. The hospital effluents were collected at manhole of the adult ward, pediatric ward, delivery ward, laboratory unit, laundry service unit and at sampling site named MIXED (for this study purpose only). Here mixed sample indicates a hospital effluent in which its origin holding from different ward/unit and flow together hence it was difficult to identify at which specific unit/ward it came. In addition, wastewater was collected from MDR TB ward in case of Alert hospital only. The sampling site manholes located just at the outlet of wastewater of each ward/unit before discharged in to receiving water/ collecting septic tank and at the side of each ward/unit building. The geographical position of the sampling site/ unit was obtained and documented.
Data Collection Procedure
The important information’s were recorded using a pre-developed data collection form by asking the authorized body (about sampling unit/site, wastewater disinfection and disposal procedures), from record book/file (e.g. number of patients served during study period) and using google map application (for geographical location of the sample). After the sampling unit/site at each hospitals identified and its location as well as the source of wastewater, aseptically with care, the cover of each manholes are lifted up to collect the wastewater then covered immediately. The name of the hospital and sampling unit, time and round of collection, as well as its geographical location are recorded on pre-developed data collection format in addition to, labeling collecting bottle at time of sample collection. All of this information was collected by the principal investigator.
Isolation and Characterization of Pure Cultures
For isolation the bacteria, a loopful of a well-mixed sample suspension was inoculated using sterile inoculating loop on to MacConkey agar pate (Oxoid LTD, Basingstoke, Hampshire, England) and incubated aerobically at 37 oC for 18-24 hours.
After incubation for 24 hr. at 37oC, bacterial colonies with distinct coloration and morphology were randomly picked up and sub cultured on to another MacConkey Agar plate for further purification. Then purified colonies with distinct presumptive colonies of each suspected bacterial species and fermentation on MacConkey agar are sub cultured on tryptic soya agar (TSA)/nutrient agar (Oxoid LTD, Basingstoke, Hampshire, England) depending on the availability of media for biochemical test.
For identification pure colony from non-selective nutrient agar/TSA was sub cultured and identified based on the following biochemical tests: oxidase, indole, urea, motility, Lysine decarboxylase, citrate utilization and triple sugar iron tests as per the standards of microbiology procedure including E. coli ATCC 25922, as a control culture (26). Following purification and species identified, two –three purified colonies were preserved in Skimmy milk at -80oC for further characterization, after each isolate was assigned a unique identification number.
Screening isolates for ESBLs Producing
Those Enterobacteriaceae that was resistant or reduced susceptibility to the screening indicator cephalosporin (cefotaxime and/or ceftazidime) was considered as suspicious of ESBLs production. In other word, isolates that showed an inhibition zone size of ≤27 mm for cefotaxime (30μg) and/or ≤ 22 mm for ceftazidime (30μg) were considered as suspicious ESBL producers and selected for confirmation for ESBLs production.
Confirmation of ESBLs Producing Enterobacteriaceae
Confirmation of suspicious ESBL-producing isolate was verified by the combination disk method (CDT) as delineated by the 29th edition CLSI guide line (27). The test was performed using two cephalosporin antibiotics: ceftazidime (30μg), and cefotaxime (30μg) alone and in combination with beta-lactam inhibitor ((ceftazidime- clavulanic acid (30/10μg), and cefotaxime-clavulanic acid (30/10μg)) by dispensing on 0.5 McFarland turbidity bacterial suspension inoculated Muller Hinton agar (MHA) plate (Oxoid LTD, Basingstoke, Hampshire, England) and then incubated overnight (18-24 hours) at 37 oC as per 29th edition CLSI guideline. ESBL production was considered positive when ≥ 5mm increase in the zone diameter for the ceftazidime or cefotaxime tested in combination with clavulanic acid versus its zone when tested alone (27). E. coli ATCC 25922 was used as a negative control throughout the tests as a non-ESBL culture.
Antimicrobial Susceptibility Testing
Once the bacteria were isolated and identified from each sample collected, all Enterobacteriaceae isolates were assessed for non-susceptible pattern for 12 antibiotic agents by using the Kirby-Bauer disk diffusion method on MHA in line with 29th edition CLSI guideline (27). Bacterial inoculum was prepared by suspending the freshly grown bacteria in 4–5 ml sterile normal saline and the turbidity was adjusted to that of a 0.5 McFarland standard. Then a prepared bacterial inoculum suspension (0.5 McFarland standards) was streaked on MHA using sterile swap applicator stick and antimicrobial discs were placed. The antibiotic discs used for this study were: trimethoprim⁄ sulphametoxazole (SXT, 1.25/3.75ug), ciprofloxacin (CPR, 5ug), tazobactam+piperacillin (TZP, 30ug), cefoxitin (CXT, 30μg), chloramphenicol (CHL, 30μg), nitrofurantoin (F, 300 ug), amoxicillin/clavulanic acid (AMC, 20/10 μg), tobramycin (TOP, 10ug), meropenem (MER, 10μg), cefotaxime (CTX, 30ug), cefepime (CFP,30 μg), and ceftazidime (CAZ, 30 μg ). The antibiotic discs used were the product of Abtek Biologicals Ltd, Liverpool, United Kingdom. Inhibition zones were measured using ruler and isolates were categorized as: resistance, intermediate and susceptible for each antimicrobial agent using the break point as set in line with 29th edition CLSI guidelines (27). The isolates were going to be considered as MDR when they were non- susceptible for three or more classes of antibiotics (28). E. coli ATCC 25922 and P. aeruginosa ATCC 27853were used for quality control throughout the antimicrobial susceptibility tests as recommended by 29th edition CLSI.
Laboratory Data Quality Assurance
Sample collection, handling, transportation and microbiological analysis and interpretation of results were carried out using standard operating procedures. Before the tangible procedure; reagents, antimicrobial disks, and media were checked for damage, storage problems and expiry date. Laboratory equipment’s are appropriately cleaned and sterilized before use. Media’s was prepared according to the respective manufacturer’s instruction. Five percent of prepared media per batch was incubated overnight for sterility checkup. Quality control for new batch was performed using ATCC 25922 E. coli standard control to cross check the quality of antibiotics disks and culture media. For ESBLs confirmatory test E. coli ATCC 25922 (ESBLs negative) and K. pneumoniae ATCC 700603 (ESBLs positive) standard control strains are served at time of the procedure (27).
Data analysis and interpretation method
Data was entered and summarized using SPSS version 20 software (IBM Corporation, Armonk, NY, USA). Frequency and percentages of isolates, antibiotic susceptibility pattern of Enterobacteriaceae and ESBLs-pE were calculated. Tables and figures have been employed for data demonstration.
Statistical Data Quality Assurance
Before data entry, data from the data collection form was cross checked for its completeness and accuracy. Culture isolates and antibiotics susceptibility test results had been documented consciously ahead of entry to SPSS. Furthermore, data cleaning and double-data entry was implemented to assure quality of the data.