Shigella and Salmonella, Antibiotics Susceptibility Pattern and Associated Risk Factors among Diarrheic Children in Southern Ethiopia: a cross sectional study CURRENT STATUS: POSTED

Background : Salmonella and Shigella is a major health problem worldwide, in developing countries like Ethiopia, it is responsible for high morbidity and mortality of children. This study aimed to determine the prevalence of Salmonella and Shigella infection, their antibiotic susceptibility pattern and associated risk factor among the diarrheic pediatrics patients that visited Alamura Health Center in southern Ethiopia. Method: A facility based cross-sectional study was conducted at Alamura Health Center from April 2018 – July 2019. The study was performed on pediatrics below the age of 14 years in which consecutive children with diarrhea were included for the study. A structured questionnaire was used to collect socio-demographic and clinical data after assent and consent obtained from parents or care taker. The stool sample cultured as per the standard operating procedure (SOP) of the microbiology laboratory. Antibiogram was performed by Kirby-Bauer disc diffusion method and was interpreted based on the Clinical and laboratory standard institute guidline (CLSI) version 2018. Results : Out of 263 children enrolled in the study, 50.5 % were females. The overall, 21/263 (8 %) 95% CI, (4.6 - 11.4%) Shigella and Salmonella was isolated. Shigella dysenteriae was dominantly isolated 11 (4.2%) followed by 9(3.42%) Shigella spp , and 1(0.38%) Salmonella typhi. Those with habit of washing the hands of children after toilet sometimes (AOR = 235.1, 95% CI, 20.9 - 2643.3, P = .000) and store cooked food in open container for later use (AOR = 36.44, 95% CI, 5.82 - 228.06, P = .000) showed statistically significant association. Conclusion: High level of Shigella spp and one Salmonella was isolated from diarrheic children at Alamura Health Center. Shigella dysentery was the most dominantly isolated. Those practised hands wash for their child after defecation for sometimes was 235.1-fold at risk of infection. Similarly, those store foods for later use in an open container was 36.44 times at risk of infection. Therefore, to alleviate this infection the concerned body should

world including Ethiopia [1,2]. Salmonella and Shigella are associated with a high burden of illness among children in the developing world [3]. Children are one of the victims to these infections accounting for approximately 8 per cent of all deaths among children under age 5 worldwide in 2017.
This interpreted to over 1,300 young children passed away each day, 480,000 children a year, regardless of the availability of humble active treatment. Most of these deaths due to diarrhea is in South Asia and sub-Saharan Africa [4]. Studies in Ethiopia from different regions reported that 4.3-17-45%[5-8] Shigella and 1-12.6 %[6-8] Salmonella infection was confirmed.
They are species of particular concerns as causes of enteric fevers, food poisoning and gastroenteritis [9]. They are Gram-negative rods which commonly inhabit intestinal tracts of humans and many animals [10]. It was estimated worldwide about 1.8 million cases of children died from diarrheal illness, a large proportion of which were attributed to Shigella and Salmonella spp. [11].
Different studies have reported that Shigella Spp. were associated with the majority of cases of bacillary dysentery which is prevalent mainly in developing nations. Whereas, Salmonella spp. were the most common cause of food-borne infection out breaks in almost allover the world [12]. In recent year the emergence and global dissemination of Salmonella and Shigella species resistance to ampicillin, chloramphenicol, tetracycline and co-trimoxazole increasingly documented in developing countries [13].
Infections of Shigella and Salmonella can be asymptomatic and can be treated with rehydration solutions unless the infection is by invasive strains [14]. Prescribing antibiotics might shorten the extent of diarrhea and control the shedding of the organisms which otherwise might continue to spread among people and in to the environment and further pose a risk of spread of infections [15].
Children are at high risk of these infections due to their weekend immune status and ease of contamination [16]. In developing countries, this increased due to poor sanitation, personal hygiene and lack of appropriate food supply that leads children to contaminate themselves [17]. Therefore, this study aimed to identify Shigella and Salmonella infections, their antibiotics susceptibility and associated risk factors among children with diarrhea that visited Alamura health center.

Study Area and Period
The study was conducted in the southern nation, nationalities and peoples region (SNNPR) at Hawassa Alamura Health Center. Hawassa is the capital city of Southern Nation Nationality People Region (SNNPR), located in the Southern part of Ethiopia, on the shores of Lake Hawassa which is one of the Great Rift Valley lakes and 270 km from the capital city of Ethiopia. Mean annual rainfall of about 950 mm, the temperature of 20 0 c and humidity of 70% -80%. The mean rainy season generally extends from June to october [18], gives the estimated population of Hawassa for 2015 as 351,469, with annual population growth, rate of just over 4%. The Hawassa city has seven sub cities with five privates, one general and one comprehensive specialized Hospital and ten health centers. Alamura Health Centers was located in the Tabor sub-city and borderline between Fara and Hitata kebele near Alamura Mountain.

Study Design and population
Facility based cross-sectional study was conducted at Alamura Health Center. A convenient sampling technique was employed in which diarrheic pediatrics patients that their family or guidance is volunteer to participate in the study were consecutively included until the calculated sample size was achieved. All diarrheic pediatrics patients that were visited Alamura Health Center for the diarrheal case of illness. Selected diarrheic pediatrics patient that visited Alamura Health Center during the study period was the study population. Pediatrics patients under 14 years of age whom his/her parents or guardians consented for the participation of the study included for the study. That parents/caretaker are involuntary to sign consent and pediatrics refuse for assent excluded from the study.

Variable of the Study
The dependent variables were the presence of Salmonella and Shigella and the independent variables were: sociodemographic factors that is age, sex, place of residence, educational status of the mothers, marital status, family size, monthly income, occupation of family and clinical and behavioral factors (previous history of diarrhea, type of diarrhea, source of drinking water, washing of child hand after toilet, food/drink taken before illness, storage of cooked food for later use, habit of hand washing before and after meal, washing habit of food containers, history of malnutrition and history of contact with domestic animals) were assessed with structured questionnaire.

Data Collection
The socio-demographic and clinical data collected after the parents/ caregiver informed about the aim of the study. Face-to-face interview conducted to collect the data with a structured questionnaire from parents or caretaker of the children who complained of diarrhea after they signed the consent and the child accepted the assent.

Laboratory diagnosis
The stool was collected using a screw cup container. The parents/caregiver instructed to bring a fresh stool sample before 30 minutes of collection and avoid contamination with urine and other materials.
All stool specimens placed into Carry Blair transport medium & transported to the microbiology laboratory of Hawassa University Comprehensive Specialized Hospital (HUCSH). The stool was inoculated on prepared culture media that is MacConkey, Xylose lysine deoxycholate (XLD) and selenite F-broth (Abtek, UK). The culture plates incubated aerobically at 37 o C for 24 hours.

Bacterial identification
The colonies examined morphologically for size, shape, and ability to ferment lactose. Those bacterial colonies with non-lactose fermenting characteristics with H 2 S for Salmonella and without H 2 S for Shigella picked for biochemical identification. Indole test, urease production, mannitol fermentation, hydrogen sulphide, gas production test, citrate utilization test, motility test, carbohydrate fermentation test, lysine decarboxylase test (LDC) and oxidase test were used to differentiate to genus and species level [19].

Data analysis
Data was entered to statistical package for the social science (SPSS) versions 20 and was analyzed to make inferences on the frequency of occurrence of enteric pathogens associated with diarrhea and to show bacterial resistance pattern to locally prescribe antibiotic substances. Descriptive statistics were performed to get the frequency of dependent and independent variables. Binary logistic regression analysis was conducted to identify real pridector of Shigella and Salmonella. The strength of association was presented by odds ratio at 95% confidence interval and p -value ≤ 0.05 was considered as a statistically significant association.

Ethical Consideration
The study was conducted after formal permission was obtained from Southern Nation Nationality and People Regional Health Office, Hawassa city administration health office, Alamura Health center manager and laboratory head. The patients were included in the study if their parents or care taker sign the consent. Culture results and antimicrobial susceptibility results were communicated to the concerned bodies of in health center within 72 hrs and treatmented accordingly.

Socio-demographic characteristics
A total of 263 diarrheic pediatric patients from Alamura Health Center was enrolled for the study with a mean and standard deviation of age 6.8 ±3.7 years. The frequency and percentage of pediatrics age range enrolled for the study were, 0-4, 88(33.5%), 5-9, 103(39.2%) and 10-14, 72 (27.4%). The almost equal ration of male to female enrolled for the study (130:133). Regarding the residence, most of the study subjects 155(58.9%) were from Urban area and 108(41.1) was from rural. Concerning the pediatrics' mother educational status most of them (81%) were educated which was included from reading and writing to university graduate level the rest 19 % are illiterates. The marital status of their mother 178 (67.7 %) was married, 43(16.3%) divorced and 41(15.6 %) widowed. The mean and standard deviation of the family size was 5.6± 1.9 persons. The average income of the family was 3743.3 ± 2568.1 Ethiopian birr. Most of the study participants have a large family size with relatively low income earned <1500 birr per month from this number diarrhea positive was 12(57.1%) ( Table   2).

Salmonella typhi
There was only one Salmonella typhi isolated. It was sensitive for ciprofloxacin, gentamicin, ceftazidime, chloramphenicol, cefuroxime, ceftriaxone and co-trimoxazole and resistance for ampicillin and tetracycline.

Other Shigella species
Shigellaspp isolate was 100.0% senstive to both ceftriaxone and ciprofloxacin, 77.8% for both ceftazidime and chloramphenicol, were as 66.7% for cefuroxime and 55.6% for gentamycin.
Resistance was seen 81.8% for ampicillin, 72.7% for tetracycline, and 55.6% for both co-trimoxazole and augmentin.

Shigella dysentery
Shigella dysentery isolate was 100% susceptible for gentamicin, 90.9 % for ciprofloxacin, 90% for ceftazidime, 72% for both ceftriaxone and chloramphenicol. Resistance was seen 45.5% for ampicillin, 55% for co-trimoxazole, 72.7% for tetracycline and 91% for augmentin ( Table 1).  However, in multivariate analysis, after adjustment, those who had a habit of washing the hands of children after toilet (AOR = 235.1, 95% CI, 20.9 -2643.3, P = .000) and store cooked food in open container (AOR = 36.44, 95% CI, 5.82 -228.06, P = .000) showed statistically significant association for Shigella and Salmonella infection with p -value ≤ .05. However, factors like the type of diarrhea, history of contact with domestic animals, a habit of hand washing before and after a meal, and washing of food container were not statically significant associated factors (Table 3).

Discussion
Our study does not indicate the total prevalence of Salmonella and Shigella infection in Hawassa town. were, it does not identify bacteria at species level this was due to lack of anti-sera in the market. The study determined the prevalence of Shigella and Salmonella, their antibiotics susceptablity patern and associated risk factor among pediatric patient at Altamura Health Center.

Declarations
Ethical Clearance: The study was conducted after formal permission was obtained from Southern Nation Nationality and People Regional Health Office, Hawassa city administration health office, Alamura Health center manager and laboratory head. The patients were included in the study if their parents or care taker sign the consent. Culture results and antimicrobial susceptibility results were communicated to the concerned bodies of in health center within 72 hrs and treatmented accordingly.

Consent for publication: Not applicable
Availability of data and material: All the data supporting the findings can be obtained from the corresponding author.

Competing of interest: The authors declare that they have no competing interests
Funding: this study was partially supported for data collection by Hawassa University.         Figure 1 Magnitude of Shigella Spp, Shigella dysentery and Salmonella typhi in diarrheic pediatrics patients from Alamura Health Center, southern Ethiopia, 2019.