Original Article

J Nepal Health Res Counc 2012 Sep;10(22):218-23

Bacterial Etiology of Acute Diarrhea in Children Under Five Years of Age

Ansari S,1 Sherchand JB,1,2 Parajuli K,1 Mishra SK,1 Dahal RK,1 Shrestha S,1 Tandukar S,2 Pokhrel BM1
1

Department of Microbiology, Institute of Medicine, Kathmandu, Nepal, 2Public Health Research Laboratory, Institute of Medicine, Kathmandu, Nepal.

ABSTRACT Background: Diarrheal diseases are major problem of developing countries. Though precise data on childhood mortality associated with diarrheal diseases in Nepal is not available, it has been estimated that approximately 25% of child death are associated with diarrheal disease, particularly acute diarrhea. The purpose of this study was to assess the incidence of bacterial pathogens causing acute diarrhea in children under 5 years of age. Methods: A total of 525 children with acute diarrhea in a childrens hospital of Kathmandu, Nepal were enrolled between April 2011 to September 2011. Feacal specimens for culture were inoculated to the several media. The organisms were identified by different biochemical tests and serotyping. Their antibiotic sensitivity tests were performed by Kirby-Bauers disc diffusion method as recommended by CLSI. Results: Out of total 525 enrolled cases bacterial infection was found to be 46 (8.8%). Bacterial infection was found to be of highest, 36 (78.3%) in the age group between 6-24 months. Among the total enrolled cases the prevalence of Shigella species was 24 (4.6%) followed by Escherichia coli 12 (2.3%) and Salmonella species 10 (1.9%). Chloramphenicol and Tetracycline showed efficacy in 9 (90.0%) isolates of Salmonella species, Gentamycin showed efficacy in 22 (91.7%) isolates of Shigella species and Chloramphenicol showed 100% efficacy against Escherichia coli whereas 7 (70.0%) isolates of Salmonella species were resistant to ampicillin in vitro. MDR was highest 7 (70.0%) in Salmonella species. Conclusions: The bacterial pathogens were found to be a significant cause of acute diarrhea. The most common causative organism for acute diarrhea were Shigella spp. Awareness of improving hygiene and infectious diseases may reduce the burden of infection. Keywords: acute diarrhea; bacteria; pathogen. INTRODUCTION
Diarrhea is defined by World Health Organization (WHO) as having 3 or more loose or liquid stools per day or as having more stools than is normal for that person.1 Acute diarrhea, defined as an increased frequency of defecation (three or more times per day or at least 200 g of stool per day) lasting less than 14 days, may be accompanied by nausea, vomiting, abdominal cramping, clinically significant systemic symptoms, or malnutrition.2 In 2009 diarrhea was estimated to have caused 1.5 million deaths in children under the age of 5 years.3 Though precise data on childhood mortality associated with diarrheal diseases in Nepal is not available, it has been estimated that approximately 25% of child death are associated with diarrheal diseases, particularly acute diarrhea.4

Correspondence: Mr. Shamshul Ansari, Department of Microbiology, Institute of Medicine, Tribhuvan University Teaching Hospital, Kathmandu, Nepal. Email: [email protected], Phone: 9841455777.

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In developing countries 50-60% cases are of bacterial (Enteropathogenic Esherichia coli 25%, Campylobacter jejuni 10-18%, Salmonella spp. and Shigella spp. 5% each), 35% of viral (15-25% rotavirus) origin, and in many the cause is unidentified or mixed.5-9 Among the bacteriological pathogens Escherechia coli play an important role.10 Norfloxacin (10 g), Nalidixic acid (30 g), Cotrimoxazole (1.25/23.75 g), Gentamycin (10 g), Amikacin(30 g), Ciprofloxacin (5 g), Ofloxacin (5 g), Cefotaxime (30 g), and Ceftazidime (30 g), were used. In this study if the isolates were resistant to at least two or more than two groups of antimicrobial agents, they were regarded as MDR.11 Differences in proportions were assessed by Chi-square test. P values <0.05 were considered statistically significant.

METHODS
This study was a cross-sectional study conducted at Tribhuvan University Teaching Hospital, Department of Microbiology-Public Health Research Laboratory. Ethical approval was taken from the Institutional Review Board (IRB), Institute of Medicine, Tribhuvan University Teaching Hospital, Kathmandu, Nepal. Written informed consent was obtained from the childrens parents or guardian before enrollment. A total of 525 stool samples were collected from the children under 5 years of age visiting Kanti Childrens Hospital, Kathmandu, Nepal with acute diarrhea in the periods between April 2011 to September 2011. From each participating children, clinical data were obtained and stool sample was collected in a sterile container. Faecal specimens for culture were inoculated to several media for maximal yield, including solid agar and broth. Specimens received for detection of the most frequently isolated Enterobacteriaceae and Salmonella spp. and Shigella spp. were plated to a supportive medium, a slightly selective and differential medium. The specimen were inoculated to a MacConkey agar (Himedia) for the selection of Escherichia coli and Salmonella-Shigella agar (Himedia) for the selection of Shigella spp. and Salmonella spp. and thiosulfate citrate bile salt sucrose (TCBS) agar (Himedia) for the selection of Vibrio spp. Salmonell-Shegella broth (Himedia), Selenite F broth (Himedia) were used as the enrichment media for Salmonella spp. and Shigella spp. Alkaline peptone water was used as the enrichment medium for Vibrio spp. The aseptic condition was maintained through the experiment. Purity plate and quality control was maintained during the experiment. All culture plates and the enrichment media were incubated at 37 0C for overnight. All samples were tested for Escherichia coli, Shegella spp., Salmonella spp. and Vibrio cholerea by using grams stain, colony morphology, biochemical tests and agglutination with specific antisera. The antibiotic sensitivity tests of the pathogens isolated from the clinical specimen against different antibiotics were done using Mueller Hinton agar (MHA) (Himedia India) by the standard disk diffusion technique of modified Kirby-Bauer method as recommended by CLSI. For disk susceptibility testing, Ampicillin (10 g),

RESULTS
Out of total 525 enrolled cases 323 (61.5%) were from IPD and 202 (38.5%) were from OPD. Boys had higher diarrheal cases (64.2%) than girls (35.8%). The higher rate of diarrhea was in the age group of less than 2 years among which the rate was highest 367 (69.9%) in the age group of 6-24 months, 101 (19.2%) in the age group of less than 6 months and the least rate of 14 (2.7%) was found in the age group of 49-60 months (Table 1). The rate of diarrhea in less than 2 years of age was found to be statistically significant (P<0.01). Table 1. Age and gender wise distribution cases Age groups Male Female in months n (%) n (%) Less than 6 60 (17.8%) 41 (21.8%) 6 -24 238 (70.6%) 129 (68.6%) 25-36 18 (5.3%) 9 (4.8%) 37-48 10 (3.0%) 6 (3.2%) 49-60 11 (3.3%) 3 (1.6%) Total 337 (100%) 188 (100%) of diarrheal Total n (%) 101 (19.2%) 367 (69.9%) 27 (5.2%) 16 (3.0%) 14 (2.7%) 525 (100%)

Out of total enrolled cases bacterial infection was found to be 46 (8.8%). Bacterial infection was found to be of highest, 36 (78.3%) in the age group between 6-24 months (Figure 1), the lower age group of infants under 6 months was 7 (15.2%). The least infection (2.2%) was found in higher age group between 49-60 months. There were no detectable cases of bacterial infection among the age group of 37-48 months in this study. Occurrence of bacterial pathogens in children below 2 years of age was statistically significant than in those above 2 years of age (P<0.01). Bacterial pathogen infected cases were 22 (47.8%) among male while it was 24 (52.2%) among female. Among the total enrolled cases the Escherichia coli was 12 (2.3%), Shigella species was 24 (4.6%) and Salmonella species was 10 (1.9%) as shown in figure 2. The rate of Shigella spp. was found to be statistically significant (P<0.01).

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and 3rd generation cephalosporin showed better efficacy whereas 5 (41.7%) isolates were resistant to ampicillin followed by 4 (33.3%) resistant to nalidixic acid, cotrimoxazole and amikacin each in vitro (Table 3). Table 3. Antimicrobial sensitivity pattern of Escherichia coli (n=12) Antibiotics Sensitive Intermediate Resistant n (%) n (%) n (%) Ampicillin 7 (58.3) 0 (00) 5 (41.7) Nalidixic acid 8 (66.7) 0 (00) 4 (33.3) Norfloxacin 11 (91.7) 0 (00) 1 (8.3) Chloram12 (100) 0 (00) 0 (00) phenicol Cotrimoxazole 8 (66.7) 0 (00) 4 (33.3) Gentamycin 11 (91.7) 0 (00) 1 (8.3) Ciprofloxacin 11 (91.7) 0 (00) 1 (8.3) Tetracycline 9 (75.0) 0 (00) 3 (25.0) Ofloxacin 11 (91.7) 0 (00) 1 (8.3) Amikacin 8 (66.7) 0 (00) 4 (33.3) Ceftazidime 11 (91.7) 0 (00) 1 (8.3) Cefotaxime 11 (91.7) 0 (00) 1 (8.3) Among the total Shigella species, 22 (91.7%) isolates were susceptible to gentamycin followed by 21 (87.5%) isolates susceptible to fluoroquinolones and amikacin whereas 13 (54.2%) isolates were resistant to nalidixic acid followed by 12 (50.0%) isolates resistant to ampicillin and cotrimoxazole each in vitro (Table 4). Table 4. Antimicrobial sensitivity pattern of Shigella species (n=24) Antibiotics Sensitive Intermediate Resistant n (%) n (%) resistant n (%) Ampicillin 12 (50.0) 0 (0.0) 12 (50.0) Nalidixic acid 11 (45.8) 0 (0.0) 13 (54.2) Norfloxacin 21 (87.5) 1 (4.2) 2 (8.3) Chloram19 (79.2) 0 (0.0) 5 (20.8) phenicol Cotrimoxazole 12 (50.0) 0 (0.0) 12 (50.0) Gentamycin 22 (91.7) 0 (0.0) 2 (8.3) Ciprofloxacin 21 (87.5) 1 (4.2) 2 (8.3) Tetracyclin 14 (58.3) 0 (0.0) 10 (41.7) Ofloxacin 21 (87.5) 1 (4.2) 2 (8.3) Amikacin 21 (87.5) 0 (0.0) 3 (12.5) Ceftazidime 18 (75.0) 0 (0.0) 6 (25.0) Cefotaxime 16 (66.7) 0 (0.0) 8 (33.3) Among the Salmonella species, chloramphenicol and tetracycline showed efficacy in 9 (90.0%) isolates in vitro followed by amikacin, cotrimoxazole in 8 (80.0%) isolates and fluoroquinolones in 7 (70.0%) isolates whereas 7 (70.0%) isolates were resistant to ampicillin followed by 6 (60.0%) isolates resistant to nalidixic acid in vitro (Table 5).

Figure 1. Distribution of bacterial positive cases in different age groups

Figure 2. Distribution of different bacteria found in stool samples Table 2. Distribution of bacterial pathogen (n= 46) Type of organisms n (%) Escherichia coli 12 (26.1) Enteropathogenic Escherichia coli 8 (EPEC) 2 Enterotoxigenic Escherichia coli (ETEC) Enteroheamorrhagic Escherichia 2 coli(EHEC) EnteroinvasiveEscherichia coli 0 (EIEC) Shigella spp. 24 (52.2) Shigella boydii 12 Shigella sonnei 6 Shigella flexnerri 5 Shigella dysenteriae 1 Salmonella spp. 10 (21.7) Salmonella Typhi 2 Salmonella Paratyphi A 1 Other Salmonella spp. 7 Out of total bacterial enteropathogens positive cases, Shigella species were found to be highest constituting 24 (52.2%) followed by Escherichia coli constituting 12 (26.1%) and Salmonella species 10 (21.7%) as depicted in table 2. Among the Escherichia coli, chloramphenicol showed 12 (100%) efficacy while fluoroquinolones, gentamycin

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Table 5. Antimicrobial sensitivity pattern of Salmonella species (n=10) Sensitive Intermediate Resistant Antibiotics n (%) n (%) n (%) 3 (30.0) 0 (00) 7 (70.0) Ampicillin Nalidixic acid 4 (40.0) 0 (00) 6 (60.0) Norfloxacin 7 (70.0) 0 (00) 3 (30.0) Chloram9 (90.0) 0 (00) 1 (10.0) phenicol Cotrimoxazole 8 (80.0) 0 (00) 2 (20.0) Gentamycin 6 (60.0) 0 (00) 4 (40.0) Ciprofloxacin 7 (70.0) 0 (00) 3 (30.0) Tetracycline 9 (90.0) 0 (00) 1 (10.0) Ofloxacin 7 (70.0) 0 (00) 3 (30.0) Amikacin 8 (80.0) 0 (00) 2 (20.0) Ceftazidime 6 (60.0) 0 (00) 4 (40.0) Cefotaxime 6 (60.0) 0 (00) 4 (40.0) In present study 323 (61.5%) of cases were from IPD and 202 (38.5%) were from OPD. Bacterial infected cases showed non-significant difference between male (47.8%) and female (52.2%) (P>0.05). In our study bacterial pathogens were found in 46 (8.8%) of cases. Similar results were also found by Sherchand et al. in Nepal13 and Patel et al. in Oman.16 The prevalence of bacterial infection in cases of diarrhea among children in other countries varies between 5.3% and 54%.16 It appeared that infants below 6 months of age were initially protected to some extent by maternal antibodies against severe diarrhea, and they seem to have acquired adequate immunity between 12 and 16 months of age. The greater risk of infants and young children in the period between 6 to 12 months with declined levels of maternal antibodies have been documented.13 In this study too the age below 2 years were found to be the most infected by bacterial pathogens. Among 46 bacterial pathogen positive cases 36 (78.3%) were in the age between 6-24 months, followed by 7 (15.2%) in the age less than 6 months. Similar result was also found by Moyo et al.15 Escherichia coli were detected in 12 (2.3%) of cases with EPEC constituting 66.6% followed by ETEC and EHEC each 16.7%. Similar results were also reported by several other investigators.4,13,22,25,26 Figure 3. Distribution of MDR bacterial isolates MDR was highest 7 (70.0%) in Salmonella species followed by 14 (58.3%) in Shigella species and 7 (58.3%) in Escherichia coli (Figure 3). In this study, the Shigella species constituted 24 (4.6%) in cases of diarrhea (P<0.01). Similarly Moyo et al.15 in Tanzania found a prevalence of Shigella species to be 5.7% and other studies also have found the similar prevalence of 7.7% in Calcutta (India),17 3.2% in Mumbai (India)18 and 4.9% in Jordan.19 In present study among Shigella spp., Shigella boydi constituted 12 (50.0%), Shigella sonnei 6 (25%), Shigella flexneri 5 (20.8%), and Shigella dysenteriae 1 (4.2%) which was in consistent with study by Sherchand et al. in Nepal,13 Ghaemi et al. in Iran20 and Bodhidatta et al. in Thailand.21 The Salmonella spp. causing diarrhea in our study was reported in 10 (1.9%) cases. The finding of our study falls within the reported range of 1-5% of gastroenteritis cases in most developing countries.15 Our result concurs with different investigators from several places.18,19,22,23 Among Salmonella spp., Salmonella Typhi was reported in 2 (20.0%), Salmonella paratyphi A was reported in 1 (10.0%) and other Salmonella spp. were reported in 7 (70.0%) cases. No Vibrio spp. were isolated in this study. The spread pattern of Vibrio spp. suggested water borne infection in rainy seasons, although the quantity of water available is large in rainy season most water sources JNHRC Vol. 10 No. 3 Issue 22 September 2012

DISCUSSION
In this study out of 525 cases enrolled 337 were male and 188 were female with male to female ratio being 1.79:1. Children below 5 years of age were enrolled in this study. The maximum number of samples were from the age group of less than 2 years in which the age group of 6- 24 months constitutes maximum number 367 (69.9%) followed by the age group of less than 6 months 101 (19.2%). Among the total cases enrolled, the bacterial pathogen was found in 46 (8.8%) of cases. The rate of diarrhea was higher in male 337 (64.2%) than female 188 (35.8%) in this study. The higher positivity rate among boys was in agreement with the numerous studies.12-15 The most of the diarrheal cases were found in the age group less than 2 years of age among which the higher rate (69.9%) was found in the age group of 6-24 months than the age group of less than 6 months (19.2%). The prevalence of diarrhea in age less than 2 years was found to be statistically significant (P<0.01).

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are contaminated with excreted microorganisms from surface water runoff.24 While discussing about the antibiotic sensitivity profile, among Escherichia coli 100% isolates were found to be susceptible to Chloramphenicol while 11 (91.7%) isolates were susceptible to Gentamicin, Fluoroquinolones, and 3rd generation Cephalosporin and 9 (75.0%) isolates were found to be susceptible to Tetracycline whereas very few isolates were resistant to various antibiotics among them 5 (41.7%) isolates were resistant to Ampicillin. According to Kaminski et al,31 Ciprofloxacin was 100% effective against E. coli and 50% of isolates were resistant to Ampicillin and Al Gallas et al,10 found 71.4% isolates sensitive to Tetracycline and 20.4% isolates resistant to Ampicillin. Out of total Shigella species 22 (91.7%) were found to be sensitive to Gentamycin followed by 21 (87.5%) sensitive towards Fluoroquinolones and Amikacin whereas 13 (54.2%) isolates were resistant to Nalidixic acid. According to Das et al,27 from north Indian patients the strains of Shigella spp. isolated were highly susceptible to Norfloxacin, Gentamycin and increasing resistance to Nalidixic acid, according to Bodhidatta et al,21 Total 100% isolates of Shigella spp. were sensitive to Ciprofloxacin which concurs with our finding. Similar study by Sonawala et al,18 in India showed that Shigella spp. were 100% sensitive to Nalidixic acid which is much higher than our study and 98.8% of isolates were sensitive to Gentamycin which is consistent with our study. Chloramphenicol and Tetracycline were the most effective antimicrobials for Salmonella spp. with 9 (90.0%) isolates susceptible followed by Cotrimoxazole 8 (80.0%) and 7 (70%) of Salmonella isolates were susceptible to Fluoroquinolones whereas 7 (70.0%) isolates were resistant to Ampicillin followed by 6 (60.0%) isolates resistant to Nalidixic acid (NARS). Mandomando et al,22 reported 92% of Salmonella isolates were sensitive to Chloramphenicol, 85% isolates were sensitive to Tetracycline and 62% isolates of Salmonella spp. were resistant to Ampicillin. According to Patel et al,16 10% of the Salmonella isolates were resistant to Ampicillin and 7.1% were resistant to Nalidixic acid. This finding is much lower than our study. This may be due to empirical use of these antibiotics and development of resistance in our context. Dahifar et al from Tehran found 67% of Salmonella isolates were resistant to Nalidixic acid (NARS) which is consistent with our study.28 Patients infected with nalidixic acidresistant isolates (NARS) respond less well to fluoroquinolones, even when the isolates MICs are within the susceptible range for fluoroquinolones.29,30 According to our study Chloramphenicol, Fluoroquinolones, Gentamycin, and 3rd generation cephalosporin were the most effective antimicrobial for the bacterial pathogens. However, the use of Chloramphenicol and Fluoroquinolones are not recommended in younger children.22 Because of the microbial drugs, the antibiotic resistance abuse of antim of Salmonella strains from humans is expected to increase. It has been reported that there has been an increase in antibiotic resistance of Salmonella isolates from humans.32 In this study too, 7 (70.0%) of Salmonella spp. were MDR.

CONCLUSIONS
The study indicated that the frequency of diarrhea was higher in male children compared to female children. Chloramphenicol, Gentamicin and Fluoroquinolones were the most effective antibiotics while Ampicillin and Nalidixic acid were the least effective antibiotics in vitro against the bacterial isolates. Shigella species were common among the bacterial pathogen causing acute diarrhea in children under 5 years of age. These children can become a source of outbreaks. So the awareness on prevention of the infectious diseases, improving hygiene should be implicated to reduce the burden of infectious diseases.

ACKNOWLEDGEMENTS
We express our sincere thanks to Prof. Dr. Basista Prasad Rijal for his continuous support. The authors also express special thanks to all the working staffs, doctors, nursing in-charge and the subjects of this study of Kanti Childrens Hospital, Kathmandu, Nepal.

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