The burden of Shigella and Salmonella, Antibiotics Susceptibility Pattern and Associated Risk Factors among Diarrheic Children visited Alamura Health Center Southern Ethiopia: a cross-sectional study

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 paediatrics 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 paediatrics below the age of 14 years in which consecutive children with diarrhoea were included for the study. A structured questionnaire was used to collect sociodemographic and clinical data after assent and consent obtained from parents or caretaker. 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 guideline (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. Highly resistance to ampicillin (71.4%), augmentin and tetracycline (61.9%) each, whereas highly sensitive to ciprooxacin (95.2%), ceftriaxone & ceftazidime (85.9%) each, gentamycin (81%), chloramphenicol (76.2%), cefuroxime (66.7%) and cotrimoxazole (52.4%) identied. 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


Background
Diseases caused by the enteric pathogens are common public health problems in many parts of the 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 diarrhoea is in South Asia and sub-Saharan Africa (4). Studies in Ethiopia from different regions reported 4.3-17-45% (5-8) Shigella and 1-12.6% (6)(7)(8) Salmonella infection.
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 outbreaks in almost all over 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 diarrhoea and control the shedding of the organisms which otherwise might continue to spread among people and into 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 diarrhoea that visited Alamura health centre.

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 ve privates, one general and one comprehensive specialized Hospital and ten health centres. Alamura Health Centers was located in the Tabor sub-city and borderline between Fara and Hitata kebele near Alamura Mountain.

Study Design and population
A facility-based cross-sectional study conducted at Alamura Health Center. A convenient sampling technique was employed in which diarrheic paediatrics 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 paediatrics patients that were visited Alamura Health Center for the diarrheal case of illness.
Selected diarrheic paediatrics patient that visited Alamura Health Center during the study period was the study population. Paediatrics 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 is involuntary to sign consent and paediatrics refuse for assent excluded from the study.

Variable of the Study
The dependent variables were the presence of Salmonella and Shigella. The independent variables were sociodemographic factors that are age, sex, place of residence, educational status of the mothers, marital status, family size, monthly income, occupation of the family. Clinical and behavioural factors are history and type of diarrhoea, drinking water source, washing of child's hand after toilet, food/drink taken before illness, storage of cooked food for later use, the habit of handwashing before and after meal, washing habit of food containers, history of malnutrition and history of contact with domestic animals were assessed with a 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 diarrhoea 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 identi cation 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 identi cation. 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).
Antibiotics susceptibility testing A pure colony of isolated bacteria was mixed with normal saline to make a 0.

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 paediatrics 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 paediatrics' 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 diarrhoea positive was 12(57.1%) ( Table 2).
to CLSI guidelines antimicrobial susceptibility breaking points 2018 and recorded as sensitive (S), intermediate (I) or resistance (R) (20). 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 diarrhoea 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 predictor of Shigella and Salmonella. The strength of association was presented by odds ratio at 95% con dence interval and p-values ≤ 0.05 was considered as a statistically signi cant association.

Ethical Consideration
The study was conducted after formal permission was obtained from Southern Nation Nationality and People Regional Health O ce, Hawassa city administration health o ce, Alamura Health centre manager and laboratory head. The patients were included in the study if their parents or caretaker sign the consent. Culture results and antimicrobial susceptibility results were communicated to the concerned bodies of in health centre within 72 hrs and treatment accordingly.
Antimicrobial susceptibility pattern Salmonella typhi There was only one Salmonella typhi isolated. It was sensitive for cipro oxacin, gentamicin, ceftazidime, chloramphenicol, cefuroxime, ceftriaxone and co-trimoxazole and resistance for ampicillin and tetracycline.
Other Shigella species Shigella spp isolate was 100.0% sensitive to both ceftriaxone and cipro oxacin, 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 cipro oxacin, 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). Table 1 antimicrobial susceptibility pro le of Salmonella species, Shigella spp and Shigella dysentery isolated from diarrheic paediatrics patients in Alamura Health Center, South Ethiopia, 2019.  Table 2).  (Table 2). 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 signi cant association for Shigella and Salmonella infection with p-values ≤ .05. However, factors like the type of diarrhoea, history of contact with domestic animals, a habit of handwashing before meal and handwashing after a meal, and washing of food container were not statically signi cant associated factors (Table 2).

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 susceptibility pattern and associated risk factor among pediatric patient at Altamura Health Center.
In this study 20 (7.6%) 95% CI [4. [6][7][8][9][10][11].0] of Shigella spp was isolated which is comparable with a study conducted in Ethiopia 8.3% (6), 9.5% (26), in contrast to our nding a lower rate of Shigella species was reported from China 1.4% (27). Our study tried to identify Shigella dysenteriae from another Shigella app with available biochemical tests accordingly 11 (4.2%) 95% CI [1.9-6.8%] identi ed as Shigella dysenteriae. This rate is lower than a study reported from Nepal 14.5% (28) (49). Several factors may contribute to resistance by pathogens causing gastroenteritis in developing countries like Ethiopia. These include frequent overuse, misuse and factors related to the potency and quality of antimicrobials and the distribution of resistant strains (53).
Our nding in the multivariate analysis showed that who had a habit of washing the hands of a child after toilet sometimes as compared to those practice hand washing always 235.1 times at risk of infection. Similarly, those who store cooked food in an open container for later use was 34.44 times at risk of infection as compared to those who practice closing of the container with p-value ≤ 0.05 which is in agreement with a study conducted in Southern Ethiopia Arbaminch (8,25,54).

Conclusion
Our study indicated that there was a high rate of Shigellosis among diarrheic paediatrics patients that visited Alamura Health Center during the study period and single Salmonella was isolated. Ampicillin, augmentin and tetracycline are with high resistance and cipro oxacin, ceftriaxone, ceftazidime, gentamycin, chloramphenicol, cefuroxime and cotrimoxazole relatively sensitive. 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 focus on giving health education for hand wash after defecation and storing food in a closed container later use is mandatory.

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

Consent for publication: Not applicable
Availability of data and material: All the data supporting the ndings 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.