Prevalence, associated factors and antimicrobial susceptibility patterns of Salmonella and Shigella species among diarrheic under five children in Sultan Sheik Hassan Yabere referral Hospital, Jigjiga, Eastern Ethiopia

The study was conducted in Jigjiga town at Jigjiga University Sultan Sheik Hassan Yabere Referral Hospital from 05 August to 15 November 2022. Jigjiga town is the capital city of the Somali regional state, 630Km away from Addis-Ababa. Based on the 2007 Census conducted by the Central Statistical Agency of Ethiopia, this city has a total population of 277,560, of whom 149,292 are men and 128,268 women [7]. Jigjiga city has three governmental hospitals where two of them are general hospitals and one of them is specialized referral hospital. Jigjiga University Sheik Hassan Yabare referral hospital is comprehensive specialized referral [8].

An institutional based cross-sectional study design was used. All under-five children visited Sheik Hassan Yabere Referral Hospital during the study period were source population. All under-five children with diarrhea disease symptoms attended Sheik Hassan Yabere Referral Hospital during study period were the study population. All under-five children passing loose or watery diarrhea three or more times a day who attend the hospital during the study period were included. Children those who were critically ill and unable to give stool sample and those that were on antibiotic treatment with in the last two weeks were excluded.

The sample size was calculated using a single population proportion formula based on the assumption of 5% expected margins of error, considering 95% confidence interval, 10% non-response rate and by taking the prevalence of 17% of Shigella spp from previous study conducted in Arbaminch, southern Ethiopia [9]. The final total study subjects of this study were 239. Eligible study participants (children attending under five outpatients) were enrolled sequentially using convenient sampling technique.

The data were collected by two professional nurses and two trained medical laboratory professionals. Data on Socio-demographic, socio-economic characteristics, environmental factors and behavioral factors were collected by interview guided pretested structured questionnaire adopted from previous studies [10, 11]. The guardians were instructed to bring a freshly passed diarrhea stool of the study subjects in a sterile stool cup using clean applicator stick and to avoid contamination with urine and other materials. One teaspoonful of diarrhea stool sample was collected using a pre-labeled, clean and sterile screw cup container. The collected diarrhea stool was placed immediately in Cary Blair transport (Oxoid, UK) and transported to Jigjiga University Sultan Sheik Hassan referral hospital microbiology laboratory within 2 h of collection for investigation.

Prior to subjecting the sample to culture media, physical analysis of the stool sample was performed in order to check whether the sample was diarrheal or not, presence of blood, pus and mucus. Children with diarrhea, blood, mucus were included. Diarrhea stool specimen was inoculated using sterile wire loop on MacConkey Agar (Oxoid, Ltd), and Xylose lysine- Deoxycholate agar (XLD) (Oxoid Ltd). The inoculated plates ware incubated at 37 °C for 24 h (SOPs) [12]. After 24 h incubation, the plates were checked for colony characteristics of Salmonella and Shigella species. Furthermore, colorless to yellow colonies on MacConkey agar and pink to red colonies Xylose Lysine Deoxycholate (XLD) (Oxoid Ltd) [12] agar were typically considered as Non-lactose fomenters. In addition, the colonies considered as non-lactose fomenters were further identified by biochemical tests to confirm the identification to genus level of the pathogens Biochemical tests such as klingler iron agar (KIA), motility indole, lysine medium, simon’s citrate agar, and unease test were used [13].

The drug susceptibility test was done by a disk diffusion method by using Muller Hinton Agar (MHA) according the guidelines of the Clinical Laboratory Standard Institute [14]. Colony suspension was made using normal saline (0.85% NaCl) equivalent to 0.5% McFarland standard from grown overnight colonies [15]. A sterile cotton swab was dipped into the suspension in order to remove additional suspensions by gentle rotation of the swab against the surface of the tube and then spread evenly over the Muller Hinton agar (Oxoid, Ltd). The drugs were selected in accordance with the Ethiopian Drug Administration standard treatment guidelines and Control Authority’s and the Clinical Laboratory Standards Institute’s (CLSI). Based on the drugs those professionals use to treat bacteria diarrheal disease, each and every single of the isolates were tested for ampicilin (10 µg), tetracycline (30 µg), chloramphenicol (10 µg), norfloxacin, (5 µg), ciprofloxacin (5 µg), and ceftriaxone (30 µg) on the Muller Hinton agar, the plate were then incubated at 37^oC for 18 h and interpreted according Clinical Laboratory Standard Institute [14]. Finally, the diameter of the zone of inhibition was measured and interpreted as sensitive, intermediate, and resistant based on the Clinical and Laboratory Standards Institute [14].

Questionnaires was primarily written in English, translated to Somali and returned back to English to keep uniformity. Manufacturer instructions were followed during culture media preparation and sterilization. Sterility of culture media was checked by incubating 5% of each batch at 37^oC overnight and observed for growth. Culture media which show any growth were discarded and replaced by new batch. In addition, Quality control strains of the American Type Culture Collection (ATCC) like Escherichia coli ATCC 25,922, Shigella dysentery ATCC 13,313 and Salmonella typhi ATCC 13,311 [16] were used to check the performance of culture media, biochemical tests and antimicrobial susceptibility testing.

The laboratory analysis result was registered on the laboratory data collection pre-coded format sheet. Data were entered to Epi-data version 4.6 and was analyzed using SPSS version 22. Descriptive statistics and frequency table were used to summarize the data. Chi-square, bivariate and multi-variable analysis were used to predict the association between the dependent and independent variables. Variables with p-value ≤ 0.25 on bivariate logistic regression analysis were selected for multi-variable analysis. Adjusted odds ratio (AOR) with 95% confidence interval (CI) was used to measure the strength of the association. A P-value ≤ 0.05 was considered statistical significance.

Multi Drug Resistant (MDR) is a antimicrobial resistance shown by microorganism to at least one antimicrobial drug in three or more antimicrobial categories [18].

Improved drinking water is a source that, by the nature of construction, adequately protects from outside contamination, include piped water into a dwelling, yard of plot, public tap or standpipe, tube-well or borehole, protected spring, protected dug well, and rainwater collection [19].

Unimproved sources of drinking water include unprotected well (dug); unprotected spring, cart with small tank or drum; tanker truck-provided water, surface water [19].