Study design and period
An institutional-based cross-sectional study was carried out among food handlers to evaluate the magnitude and antimicrobial resistance pattern of ESBL-producing E. coli and K. pneumoniae from stools of apparently health food handlers in Dilla University Student Cafeteria, Southern Ethiopia from November 2018 to September 2019. Three sample selection sites were identified prior to data collection. To select representative participants, the final sample size was proportionally allocated to each stratum, and food handlers were selected using a systematic random sampling technique. Finally, a total of 220 were included in the study based on the single population proportion formula.
Data collection and analysis
Data related to socio-demographic characteristics and personal hygiene practices were collected via face to face interviewing of the patient or guardian of the patient by using a well-structured questionnaire before laboratory sample collection. One environmental health and four medical laboratory professionals were recruited for data collection, supervision, and microbiological analysis. The data collectors were trained for 2 days by the principal investigator on observational data collection and specimen collection procedures.
Laboratory Data Collection and analysis
After interviewing, all respondents were asked to give a fresh stool specimen in a sterile, clean wide-mouthed plastic, and leak proof container by a clean wooden applicator stick for microbiological analysis. The isolation and characterization of E. coli and K. pneumoniae were performed based on the standard procedure. Briefly, a mixture of a stool sample (1 mL) was transferred to the selective media, Hicrome ESBL agar base, to assess the ESBL production in 2 hr (Oxoid, Ltd UK). An inoculum from Hicrome ESBL agar base was cultured onto MacConkey agar (Oxoid, Ltd UK). After overnight incubation at 37 °C the growth of E. coli and K. pneumoniae was differentiated by their colony characteristic, pigment production (pink to colorless flat or mucoid colonies), motility and Gram-staining techniques (Gram-negative rods, non-sporing, and non-capsulated).
A list of biochemical tests conducted for further identification of isolates were made by conformation of motility and other relevant biochemical tests. An isolate was considered as E. coli when it is indole positive(dark pink ring) and methyl-red positive, citrate negative (no change or remained green) and urea negative, gas and acid producer, and motile and wasconsidered as K. pneumoniae when it is indole and methyl-red negative, citrate positive, urea slow producing, and non-motile. In case of delay, the isolated bacteria were kept at 2–8 °C in the nutrient broth for not more than 24 hours until the antimicrobial sensitivity test was done.
Detection method of ESBL-producing E. coli and K. pneumonia
Disk-diffusion method was used to screen ESBL producer using ceftazidime (30 µg), cefotaxime (30 µg), and ceftriaxone (30 µg) (Oxoid, UK) as per CLSI criteria. A suspension of pure colony was wrapped on Mueller–Hinton agar (MHA) (Oxoid, UK) with a sterile cotton swab, and antibiotics discs were placed at a distance of 20 mm from each other and incubated at 35 ± 2 °C for 16–18 hours. The isolates with diminished susceptibility with breakpoint of ≤ 22 mm for ceftazidime, ≤ 25 mm for ceftriaxone, and ≤ 27 mm for cefotaxime were suspicion for ESBL production (15). Phenotypic Confirmations of ESBL producers was performed using double disc synergy between the indicator cephalosporin and clavulanic acid, according to EUCAST guidelines (23). A disc of amoxicillin/clavulanic acid (20/10 µg) was placed in the center of the MHA plate, and then cefotaxime (30 µg), ceftazidime (30 µg) and aztreonam (30 µg) were placed at a distance of 20 mm edge to edge from the amoxicillin/clavulanic acid disk. After 24hours incubation, the elevation of ≥ 5 mm zone inhibition between either of the cephalosporin disks and their respective amoxicillin/clavulanic acid disks was interpreted as ESBL producer (24) (Fig. 1)
Antimicrobial Susceptibility Testing
Antimicrobial susceptibility tests were performed for both Enterobacteriaceae by using the disc diffusion technique on Mueller–Hinton agar based on EUCAST guidelines (23) for the listed antibiotics: Ampicillin (10 µg), aztreonam (30 µg), cefotaxime (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), cefoxitin (30 µg) cefuroxime (30 µg), ciprofloxacin (5 µg), nitrofurantoin (300 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), tetracycline (30 µg) and imipenem (30 µg) (all from Oxoid; UK). The resistance and sensitivity results were interpreted according to the EUCAST guidelines (23). MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories. The quality and performance of culture media, biochemical tests, and antimicrobial susceptibility discs were checked using a control strain (E. coli ATCC 25922 and K. pneumoniae ATCC 700603) obtained from Ethiopian Public Health Institute, Addis Ababa.
The descriptive statistical analysis was performed by using SPSS version 24 software. The prevalence of ESBL producers and non- ESBL-producing isolates were categorized by personal hygiene related factors of food handlers and presented by calculating frequencies and percentages. Chi-square test was used with appropriate correction for the observation. Multivariate logistic regression analysis was used to identify risk factors for colonization by ESBL. P values < 0.05 were considered statistically significant.
Ethical clearance was obtained from the Ethical Review Committee of Dilla University medical and health Science College. Written informed consent was obtained from each study participant. Strict confidentiality was maintained during the interview process as well as anonymity was kept during data processing and report writing. Food handlers who have found to be positive for enteric pathogens (parasite and bacterial) were referred to their respective staff medical center for appropriate anti-parasitic and antimicrobial treatments.