Prevalence and antimicrobial susceptibility of Salmonella and Shigella species isolated from diarrheic children in Ambo town

Background Diarrhea, particularly of enteric bacterial pathogen, remains a major cause of morbidity and mortality in Ethiopia. Despite the high prevalence of diarrheal disease among under-five children, antibiotic resistance of bacterial pathogens test is not part of routine childcare in the study area. This study aimed to investigate the prevalence and antimicrobial susceptibility status of Salmonella and Shigella species among diarrheic children attending public health institutions in Ambo town, west Showa, Ethiopia. Methods Institutional based, cross-sectional study was carried out from January to July 2014 among 239 diarrheic children below five years of age in Ambo town, Ethiopia. Information about patient demographics, signs, and symptoms was obtained from the parents/guardians of each child using a questionnaire. Stool samples from diarrheic children were collected and processed for isolation of Salmonella and Shigella using conventional microbiology procedures. Suspected Salmonella isolates were confirmed by genus-specific PCR and serotyped using a slide agglutination test. Susceptibility to 10 commonly used antimicrobials was assessed using the Kirby Bauer disc diffusion method. Results From the 239 children screened, only nine (3.8%) of them were positive for either Salmonella (n = 3) or Shigella (n = 6) and 19 (7.9%) positive for the intestinal parasite. Three species of Shigella were identified: Shigella flexinari (n = 3), Shigella boydii (n = 2), and Shigella sonnei (n = 1). The three Salmonella isolates were S. chicago, S. caracas, and S. saintpaul. Salmonella and Shigella isolates were resistant to ampicillin (88.9%), followed by tetracycline (66.7%), cotrimoxazole (55.6%), chloramphenicol (44.4%), amoxicillin (33.3%), nalidixic acid (11.1%) and cefotaxime (11.1%). All isolates were sensitive to amikacin, ciprofloxacin, and gentamycin. Conclusion In this study, either Salmonella or Shigella species were detected only in 3.8% of diarrheic children in Ambo town, suggesting the dominance of other causes of diarrhea in the study area. A further study targeting other causes of diarrhea should be conducted to establish the major causes of childhood diarrhea in the study area.

antimicrobial resistance is the unrestricted use of over the counter drugs without medical supervision [6].
In Ethiopia, according to a study conducted in Jimma Health Center, 49.6% were positive for an intestinal parasite, Shigella, and Salmonella species. While Shigella species showed 100% resistance to ampicillin, amoxicillin, and cotrimoxazole, Salmonella isolates were resistant to amoxicillin. In contrast, all Shigella and Salmonella species were susceptible to ceftriaxone, ciprofloxacin, and gentamycin [7].
The level of antibiotic resistance gauges the clinical and the community malpractices related to the use of antibiotics and the associated risk of emerging infections. Despite this, Ethiopia's health facilities do not routinely perform the test for antibiotic resistance, at least among the most vulnerable segment of the population, the children affected by diarrhea. As a result, there is a poor understanding of antibiotic resistance on the most common etiologies of diarrhea, Salmonella, and Shigella species in central Ethiopia. Hence, this study intended to explore the magnitude and antimicrobial susceptibility of Salmonella and Shigella species isolated from diarrheic children in Ambo town.

Study design, area, and period
An institutional-based, cross-sectional study was carried out in Ambo Town Public Health Institutions (ATPHI)-at Ambo General Hospital, Ambo Health Center, and Awaro Health Center-from January to July 2014. These institutes provide health services for Ambo town and the surrounding districts. Ambo is one of the districts in the Western Shewa Zone, Oromia Region of Ethiopia. The 2007 national census of Ethiopia reported total populations for this district to be 108,406, of whom 54,186 were men and 54,220 were women [16].

Sample size
The sample size for the study was determined using a single population proportion formula. The prevalence and antimicrobial susceptibility of bacterial pathogens isolated from childhood diarrhea in Kenya were 17.7% [17]. It used as a reference and at a 95% level of confidence and a 10% non-response rate, the total of 250 children with diarrheal disease targeted to be included in the study as; Where: n = sample size. Zα/2 = level of confidence. P = diarrhea prevalence from previous study = 17.7%. q = 1-p. d = margin of error (0.05). The responded client size was 239 children less than five years of age presented with diarrhea to pediatric OPDs and Wards'. Diarrheic stool is defined as having loose or watery stools at least three times per day, or more frequently than usual for an individual (as per the WHO definition) [4]. A systematic random sampling method used to draw participants based pattern of previous patient flow. Accordingly, every other child whom the parents/guardians briefed on the aim of the study agreed and signed the consent to participate included in the study. Children who did not take an antibiotic for the current diarrheal attack included in the study.

Sample collection, handling, and transport
The collected clinical data includes body temperature, demographic data, and medical history. A single diarrheic stool specimen collected after a physical examination. At a hospital and health centers, the stool samples inoculated in Cary-Blair's transport medium (CA, USA). The samples transported in a cool box to Ambo University Microbiology Laboratory within four hours of collection, and it processed on the same day.

Microscopic examination
Stool examination performed at health institution laboratories (Ambo General Hospital, Ambo Health Center, and Awaro Health Center). Macroscopic and microscopic examination of the safeguarded specimens (formalin 10%) completed then focuses on formalin-ether sedimentation for intestinal parasites, WBC, and RBC conducted immediately upon sample collection before being inoculated into Cary-Blair's transport medium.

Culture and identification
All stool specimens cultured for isolation of Shigella and Salmonella species. The collected samples inoculated aerobically first in Selenite F broth (HIMEDIA, India) for the enrichment of Salmonella and Shigella species. Then, the samples inoculated to Xylose lysine desoxycholate agar (XLD) (OXOID, England) and incubated at 35-37°C for 14-16 h. A loopful of the fecal suspension directly inoculated onto MacConkey agar (SRL, India), and Salmonella-Shigella (SRL, India) agar, and incubated at 37°C for 18-24 h. MacConkey agar used to characterize most enteric bacteria toward their lactose utilization property, XLD (Shigella: red colonies, Salmonella: red with/without a black center), and SS agar used for the isolation of Shigella and Salmonella species. The presumptive colonies of each representative isolates, then characterized using standard biochemical tests.
Biochemical tests performed to characterize the enteric gram-negative bacteria include gram stain morphology, pigment production, motility, urease, citrate, hydrogen sulfide utilization, oxidase, indole, lysine, and sugar fermentation. The media used were nutrient broth ( Slide agglutination test was used to serotype the isolates of Salmonella and Shigella species using polyvalent/monovalent antisera. Presumptive Salmonella colonies were confirmed by genus-specific PCR [18]. The slide agglutination test for serotyping of Shigella species was carried out using antisera following the manufacturer instruction (Remel Europe Ltd). The Salmonella isolates were serotyped at the Public Health Agency of Canada, National Microbiology Laboratory at Guelph, OIE Salmonella Reference Laboratory, Guelph, Ontario [19].
The plates were incubated at 37°C for 24 h, and the zone of inhibition diameters were measured with a ruler and, interpreted according to CLSI guidelines, and the results recorded as sensitive (S), resistant (R), or intermediate (I) based on CLSI [20]. The study declared multidrug resistance (MDR) if the isolates were resistant to more than two (> 2) of the antimicrobial agents belonging to different classes [21,22]. Escherichia coli ATCC 25922 used as a quality control strain during the antimicrobial susceptibility test [20].

Statistical analysis
The data were entered into EpiData 3.02 and then transferred to SPSS version 17.0 statistical software for data processing and analysis. The descriptive statistics such as mean, standard deviation, and proportion used.

Results
A total of 239 children included in the study. Of these, more than three-quarters of them were from Ambo Hospital. Slightly more than half (52.3%) of the children were boys, and two-third of all the children were from urban areas. The children's age ranged from 5 to 60 months, with a mean of 27.96 (SD ± 17.09) months (Tables 1, and 2).
As shown in Table, four out of five children were living in non-crowded housing conditions, which is < 1.5 persons-per-room [23]. One out of six households was using water from unsafe sources such as rivers and domestic wells. Two out of five households with diarrheic children had at least one domestic animal in their household (Table 1). However, no statistically significant difference was observed in the proportion of identified enteric bacterial pathogens across socio-demographics characters.

Antimicrobial susceptibility of isolated Salmonella and Shigella isolates
The antimicrobial profile for isolated Salmonella and Shigella species (n = 9) to 10 antimicrobials drugs determined by the disk diffusion method following the recommendations of the CLSI [20]. The isolated Salmonella and Shigella species had variable degrees of resistance to all antimicrobial agents tested. The susceptibility of 31.1% of the isolates was below standard resistance breakpoints for ampicillin, amoxicillin, cotrimoxazole, chloramphenicol, nalidixic acid, tetracycline, and cefotaxime. The highest enteropathogenic bacteria resistance was observed against ampicillin (88.9%) followed by tetracycline (55.6%) and cotrimoxazole (55.6%), chloramphenicol (44.4%), amoxicillin (33.3%), nalidixic acid (11.1%), and cefotaxime (11.1%). All isolates were sensitive to amikacin, ciprofloxacin, and gentamycin. The resistance against tested ten antimicrobials was higher for Salmonella species (33.3%) than the Shigella species (30%). The highest rate of resistance observed was against ampicillin, which was 100% for Salmonella species and 83.3% for Shigella species. Conversely, the least antimicrobial resistance was to amikacin, ciprofloxacin, and gentamycin, where all isolates were sensitive. Shigella species showed no to a low level of resistance for cefotaxime and nalidixic acid (16.7%), respectively. Salmonella species showed no resistance against nalidixic acid. The resistance against the most commonly prescribed antibiotics, cotrimoxazole was 66.7% for Salmonella and 50% for Shigella isolates (Table 6).
Regarding multidrug resistance, five Shigella species and two Salmonella species were multidrug-resistant. Out of the resistant species while, two (22.2%) were resistant to one antimicrobial two (22.2%) were resistant to five antibiotic agents (Table 6).

Discussion
This study intended to examine the prevalence, and antimicrobial susceptibility status of Salmonella and Shigella strains isolated from under-five children with diarrhea presented to Ambo town public health institutions. The overall prevalence of Salmonella and Shigellainfection was 3.8%, and there was a high rate of multidrug resistance, especially for Salmonella species. While the highest resistance observed against ampicillin, the least was for amikacin, ciprofloxacin, and gentamycin.
Among Shigella isolates, Shigella flexneri (12.5%) was the most dominant, followed by Shigella boydii (8.3%) and Shigella sonnei (1.4%), which is comparable with a study done in Northern India [26]. On the contrary, study findings in Jimma, Southwest Ethiopia, and Salvador, Bahia, Brazil showed that the isolates were not comparable [8,27], respectively. The discrepancy might be due to a difference in the study population, study time, and illness due to other enteric pathogens.
There was a high rate of resistance to ampicillin for both Salmonella and Shigella species. Ampicillin is among the top dispensed drugs in Ethiopia for the last several years. The majority of such antibiotics (85%) are prescribed empirically [29]. That has made ampicillin familiar, popular, and accessible among the people, hence leading to a high rate of self-prescription. According to Mihrate et al., (2014), Ampicillin (11.1%) is the third most common self-prescribed drug only next to amoxicillin (61.1%) and, cotrimoxazole (27.8%). The selfprescription such antibiotics are common for gastrointestinal disorders [30,31]. Similarly, high levels of resistance were reported in previous studies [32][33][34]. The public health implication of such high resistance could be associated with the suboptimal water and sanitation conditions and inadequate sewage disposal systems. This could be further complicated by the hand hygiene practice of caregivers and/or mothers.
The study suggested that resistance shadowed a 'selection density'. In the assumption, ecology was the basis; i.e., in a particular geographic area as the more antibiotic used for individual persons, animals, or plants, there is a high probability of bacteria to develop antibiotic resistance. Antibiotic residues from human excreta stool can have an impact on the geographical ecosystem. They can easily pass to water, and soil thought manure and sewage. So, antibiotic-resistant bacterial strains may have been found in natural water [43].
This study has a small sample size and included only children with diarrhea and restricted to a public health facility, to this end, it will be difficult to generalize beyond the study settings. Besides, restricting the finding to Shigella and Salmonella species hindered the isolation of other common causes of diarrhea. Thus, a comprehensive study should be conducted to determine the common enteric pathogens that cause diarrhea along with sensitivity tests.

Conclusion
The low prevalence of Salmonella and Shigella species was identified from the diarrhea stool of children under five years in the study area. Among those isolates, antibiotic resistance was at a high rate against ampicillin. It was followed by cotrimoxazole and tetracycline while the isolates were sensitive to amikacin, ciprofloxacin, and gentamycin.

Acknowledgments
We would like to appreciate the staff of the microbiology laboratory at Ambo University and Addis Ababa University, Institute of Pathobiology, for their technical support of sample processing. We gratefully appreciate all parents/guardians who volunteered to provide data and guided their child for stool sample collection. We extend our thanks to Rasha Darghawth and Lauren Eby for the English language edition.
Authors' contributions WT, AM, and TA designed the research. WT & TE performed experiments. WT, AM, AA, TE, and TA contributed to data analysis and interpretation. WT and TA wrote the manuscript. WT, AM, AA, TE, and TA critically reviewed and approved the final version of the manuscript.

Funding
Addis Ababa University's financial funding support was for data collection, but not for study design, interpretation, and writing manuscript. Ambo University had support reagents, material, and laboratory facilities. Supporting institutions did not participate in the writing of this paper, nor did it read or approve it.

Availability of data and materials
The datasets used and analyzed in the study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Ethical clearance was obtained from the Ethics Review Committee of Department of Microbiology, Immunology, and Parasitology, College of Health Sciences, Addis Ababa University. Formal permission were letters obtained from the District Health Offices. Informed written consent obtained from the parents/ guardians of every child before taking the stool samples. Positive cases were reported to a physician or health professional. No fee was charged.