Study design, area and period
A Hospital-based cross-sectional study was conducted at the University of Gondar Comprehensive specialized Hospital in Gondar town; Northwest Ethiopia, from January-April 2019. The town is located 737 km far from Addis Ababa, the capital city of Ethiopia and 180 km far from Bahir Dar, the regional capital. According to the central and statistical agency of Ethiopia report in 2015, the town has twelve sub city, twenty-two urban and eleven rural kebeles with a total projected population of 323,900. There are 8 health centers, 21 private clinics and one primary hospital in the town. The hospital provides health care service for more than 5 million people living in North, South, West Gondar Zones, as well as urban and rural kebeles surrounding the town.
Sample size and sampling technique
The sample size was determined using the single population proportion formula. By taking the prevalence of ESBLs and CPE infection that was conducted at Tikur Ambesa Specialized Hospital which showed 0.52 (13, 20), a total of 384 study participants were enrolled by using convenient sampling technique.
Laboratory Methods
Specimen collection and processing: The study participants were instructed to collect approximately 2 gram of diarrheal stool in to a clean, leak-proof container. The specimen of the study participants were collected at the University of Gondar Comprehensive specialized Hospital laboratory. Each stool sample was immediately transported to school of Bio medical and Laboratory Sciences, Medical Microbiology laboratory section using Cary-Blair transport media. Following an aseptic technique, a loop full of diarrhoeal sample was inoculated onto MacConkey agar (oxoid, Code: CM0115) and incubated aerobically at 37°C for 16-24 hours.
Identification
Preliminary identification: Preliminary identification of bacteria was based on their colony characteristics of the organisms.
Biochemical tests: were performed on isolated colonies for identification of Enterobacteriaceae based on their biochemical reaction. Biochemical tests includes triple sugar iron agar, indole test, citrate utilization test, urease production test, lysine decarboxylase test and motility test (21).
Drug susceptibility testing: Modified Kirby-Bauer disk diffusion technique using Muller Hinton agar (MHA) (Oxoid, UK) was used for antimicrobial susceptibility testing. Bacterial s uspension of three to five isolated colonies was done using 0.85% normal saline and the turbidity was adjusted at 0.5 % MacFarland standard. Using sterile cotton applicator stick, the suspension had been inoculated on MHA and left at room temperature for 3-5 minutes until it becomes dry. Then, diifferent antibiotic discs including ceftazidime (30µg) and cefotaxime (30 µg) were applied on inoculated MHA and incubated for 24 hr at 37°C. Ceftazidime (30µg) and cefotaxime (30 µg) discs were used for presumptive identification of ESBL production. The zones of inhibition was measured by a ruler and the results were interpreted as susceptible, intermediate and resistant using CLSI 2019 performance Standards for antimicrobial susceptibility testing interpretation table. Zone of inhibition ≤ 22mm for ceftazidime and ≤27mm for cefotaxime, was considered as potential ESBL producers (22).
Laboratory test for detection of ESBL and CPE
Confirmatory test for ESBL producer: The potential ESBL-PE was confirmed by combined disk method.Colony suspension of suspected ESBL-PE was inoculated on to MHA, then Ceftazidime (30µg) and Ceftazidime-Clavunalic acid (30/10 µg), Cefotaxime (30 µg) and Cefotaxime-Clavunalic acid (30/10 µg) disks were placed at 20 mm distance apart. If a ≥5mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanate vs the zone diameter of the agent when tested alone, it was confirmed as ESBL-PE (22).
Screening test for CPE: Carbapenemase producing Enterobacteriaceae was screened by using Meropenem disks.Colony suspension of isolated bacteria was inoculated on to MHA, then Meropenem (10µg) disks were placed and incubated at 370c for 24 hrs. If the zone of inhibition is ≤ 19mm, it was considered as a potential CPE (22).
Confirmatory test for CPE: The suspected CPE is confirmed by Modified carbapenem inactivation method (mCIM). The isolated bacterial colony which was suspected for CPE was diluted with 2 ml of trypticase soya broth and meropenem (10µg) disk was immersed in the suspension; then incubate for 4 hours. A standard strain of meropenem susceptible E.coli ATCC 25922 was suspended in 0.85% normal saline and compared with MacFarland standard (1:10 dilution) then inoculated the whole plate of MHA. After 4 hrs incubation, meropenem disk was removed from the test tube and placed on the MHA plate which was inoculated by E.coli ATCC 25922 meropenem sensitive strain and incubated at 37oC for 18–24 hours. After incubation, if the zone of inhibition diameter between 6-15mm and 16-18mm with pinpoint colony, it was considered as carbapenem resistance Enterobacteriaceae (22).
Operational definitions
ESBL producers are bacteria that can produce the enzymes that confer resistance to most beta-lactam antibiotics (23).
MDR defined as resistance to three or more different classes of antibiotics (13).
Carbapenemases are beta-lactamase enzymes that inactivate almost all hydrolyzable beta-lactam antibiotics including the carbapenems (23).
Gastrointestinal tract complain is a discomfort in gastrointstinal tract with abdominal cramp, diarrhea, vomiting and distension of the abdomen (24).
Labolatory Quality control
All Medias were prepared according to the manufacturer’s instruction and following standard operational procedure. All materials, equipment, and procedures was adequately controlled based on pre-analytical, analytical and post-analytical stages of quality assurance that were incorporated in standard operating procedures at the School of Bio-medical and Labolatory Sciences of Bio-Medical complex of Medical Microbiology section. Culture media was checked for sterility by incubating 5% batch of the media at 37oc for 24 hours and performance test was checked by inoculating known control strains of Escherichia coli ATCC 25922 and K. pneumoniae ATCC® 700603 to confirm consistency of materials, methods and results. K. pneumoniae ATCCBAA1705 and ATCCBAA 1706 were used as a positive and negative quality control respectively for carbapenemase production.
Data analysis and interpretation
Data were collected, coded and entered in to EPI-Info version-7 to check completeness and clearance then transferred to SPSS version 20 for analysis.The characteristics of the study population were summarized using frequencies, percentages, mean and standard deviation and data was presented using tables.