Prevalence of Staphylococcus Aureus among Children Diagonosed with Acute Diarrhea in Kano, Nigeria

The burden of diarrheal disease is most critical in developing countries, facilitated by unsafe water supplies, poor sanitation, and nutritional deficiencies. The research was aimed to study the prevalence of Staphylococcus aureus among children diagnosed with acute diarrhea in Kura General Hospital Kano, Nigeria. Fecal specimens were collected in clean, dry and leak proof sterile bottle from 58 child patients (ranges from 1-5 years) admitted to Kura General Hospital and diagnosed with acute diarrhea from period of March to August 2017. The isolates were isolated and identified using Gram staining, Biochemical test (Catalase, Coagulase and DNase test), Mannitol fermentation and haemolysis test. The result showed that 34 samples out of 58 were positive for S. aureus. Higher incidence was found among males (20 subjects which accounted for 59%) than female with total of 14 subjects accounted for 41%. Highest frequency of diarrhea infection is found among subject with age between 1-2 years and more male (53%) were infected than female (47%). Statistical analysis of the result showed that there is no considerable statistical difference on prevalence of S. aureus among sex group and age categories of the subject at p<0.05. It is recommended that proper environmental sanitation, good personal hygiene and complete immunization against diarrhea disease are recommended.


Introduction
Over 1.7 billion cases of diarrheal infection are reported every year and these are associated with about 2.2 million deaths annually [1]. The burden of the disease is most severe and critical in a developing country which is facilitated by poor sanitation, unsafe water supply and nutritional deficiencies. Diarrhea may be infectious or non-infectious. It can be infectious when caused by bacteria, virus or parasite. Bacteria are one of the major causative agents of food illness such as diarrhea and accounted for about 60% of cases requiring hospitalization [2]. The global impact of foodborne illness is difficult to assess. However, it has been estimated that about 2.1 million children die due to diarrhea related in developing countries annually. It has been reported that water and food are the vehicle of diarrhea related illness [1]. Due Those having diarrhea for more than 30 days are said to have chronic diarrhea.
Staphylococcus aureus is gram positive bacteria, spherical in shape (cocci) mostly occur in singles, tetrads and irregular grape like cluster. It is the only strain that produce enterotoxins that can cause food poisoning. The food handler with lesion or carriage may initiate infection [4,5]. The species are host adapted with most of the known species inhibiting humans and other animals. These

Study area
The samples for the study were collected at Kura General

Sample Collection
Fecal specimens were collected in clean, dry and leak proof sterile bottle from 58 child patients (ranges from 1-5 years)

Isolation of S. aureus
A sterile wire loop was deep into the fecal sample of the patients and streaked onto the surface of Nutrient agar (Life save Biotech, USA), using standard method described by of Prescott et al. [9].
This procedure was applied for each of the sample and the plates were incubated at 370 C for 24 hours. The presumptive colonies of S. aureus on the plates were further sub-cultured to obtained pure culture. The pure isolates of S. aureus were preserved for further bacterial identification.

Bacterial Identification
The bacteria isolated were confirmed as S. aureus by conventional microbiological methods namely; Gram staining, biochemical test (such as catalase, coagulase and DNase test), mannitol and haemolysis test. Gram staining was done according to the methods described by Chessbrough [10]. Catalase, Coagulase and DNase test were done according to the method described by Holt et al. [11] and Cheesbrough [10]. Mannitol fermentation and haemolysis test was done according to the method described by Holt et al. [11].

Gram Staining
Gram staining was done according to method described by Cheesbrough [10]. A thin smear was made by emulsifying an overnight culture of the isolate in normal saline on a well labeled clean glass slide. The smear was air dried and fixed by heat. This is followed by flooding the slide with crystal violet as primary stain for 30 seconds and then rinsed the slide with distilled water. The smear was flooded with Lugol's iodine as a mordant to fix the primary stain and then rinsed with distilled water after 60 seconds.
The slide was decolorized using acetone and rinsed immediately.
Counter stain with safranin followed and left for 30 before being rinsed off. The stain smear was air dried and observed under microscope.

Catalase test
Catalase test was conducted using procedure described by Holt et al. [11]. A drop of 3% hydrogen peroxide was placed on a clean glass slide. An overnight culture of the isolate was picked using sterile wire loop and placed on the drop of the hydrogen peroxide; presence of bubbles observed indicated a positive catalase test.
loop from incubated Nutrient agar plate. The coony was emulsified in the blood plasma and observed for clot formation [11].

DNase test
Deoxyribonuclease agar medium was prepared according to manufacturer's instructions. An overnight over night broth culture of the isolate was spot inoculated on the surface of the medium and incubated at 370C for 24 hours. At the end of the incubation period, the agar surface was flooded with 1N hydrochloric acid and excess drained off [10].

Bacteriological analysis
The pure isolate obtained on Nutrient agar medium was picked using sterile wire loop and inoculate on the surface of Mannitol salt agar and blood agar (Lifesave Biotech, USA) and incubated at 370C for 24 hours. The changes in color of the medium from pink to yellow indicated positive results [11].

Statistical analysis
The prevalence of S. aureus isolates between sex and age categories was compared by using the Chi-square test (SPSS Version 19). Differences between the prevalence rates were considered significant when p<0.05.

Demographic distribution of patients
The demographic distribution of child patients diagnosed with acute diarrhea is presented in (Table 1). A total of 58 subjects (31 male and 27 female) were considered in the study. The age distribution of the subjects ranged from 1-5 years. Highest frequency is found among subject with age between 1-2 years.

Identification of S. aureus
The identification of S. aureus from the fecal samples of the subjects is presented in the table below (

Prevalence of S. aureus
The prevalence of S. aureus among fecal samples of the subjects is presented in ( Least prevalence was recoding among subjects with age category less than 1 with 6.6 %.

Discussion
Acute diarrhea remains as one of the most prevalent diseases affecting children in developing countries. The associated morbidity and mortality of the disease in children under five years of age is a major health problem in those countries. The present study was aimed at determining the prevalence of S. aureus in the fecal sample child patients diagnosed with acute diarrhea attending Kura General Hospital Kano. The finding of the study indicated that 34 out of the 58 samples were positive for S. aureus which accounted for 58% of the sample collected. Higher incidence was found among subjects with age category 2-3 years with prevalence of 17.2%.
Least prevalence was recoding among subjects with age category less than 1 with 6.6 %. Higher incidence of S. aureus among subject with age category 2-3 years is due to increased susceptibility to microbial infection, and this can be explained by the decline in the passive immunity because they no longer take breast feeding from their mothers due to weaning and this increase exposure to the diarrheal agents. Identification of S. aureus in the present study was based on Gram staining, cultural characteristics and biochemical characterization. All the 34 isolates were able to ferment Mannitol producing yellow colony, they also showed β-haemolysis on blood agar medium enriched with 5% sheep blood. Gram staining of the isolates exhibited a cluster of Gram-positive cocci. The isolates were positive for catalase, coagulase and DNase test. In catalase test, hydrogen peroxide was broken down into water and oxygen by enzyme catalase. The production of oxygen was indiated by bubble formation [12]. The positive result of coagulase test was confirmed by the formation of curd like clotting compared to negative control [13]. while older children in the group <48 months of age had the least prevalence (6.4%). The increased susceptibility of this age group to the diarrheal disease can be explained by the decline in the passive immunity and increased exposure to the diarrheal agents at this age. There is agreement that children less than 2 years of age are most affected by diarrhea [19] while some researchers have identified an increase incidence of the disease in children younger than one year [19]. It has been clear that weaning take place at this age category (1-2 years) which exposes the child to environmental contaminants in poor hygienic condition. Thus, early weaning is a putative factor to the onset of diarrhea in this age range. Therefore, early weaning and incomplete immunizations have a synergistic effect in enhancing disease.

Conclusion
Based on the finding of the study, S. aureus is one of the etiological agents of diarrhea. From the 58 fecal sample of examined, 34 samples which accounted for 58% were found to be positive for S. aureus. Higher incidence of the isolate was found among subjects with age category 2-3 with prevalence of 17.2%.
High prevalence of S. aureus among the subject was attributed to weaning, poor hygienic condition and lack of immunization. In the study, it is also found that higher incidence of diarrhea is more in male subjects than the female counter parts. It is recommended that proper environmental sanitation and delayed weaning is encouraged. Complete immunization against diarrhea disease is also recommended.