Submitted: November 20, 2020 | Approved: December 14, 2020 | Published: December 15, 2020
How to cite this article: Ali M, Diso SU, Abdullah MS, Mu’azu L, Nas FS. Determination of antibiotics susceptibility profile of Shigella species isolated from children with acute diarrhea. Int J Clin Microbiol Biochem Technol. 2020; 3: 054-058.
Copyright: © 2020 Ali M, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Antibacterial activity; Diarrhea; Shigella; Susceptibility profile
Diarrheal diseases continue to be the major cause of morbidity and mortality among children under 5 years. This study aimed to isolate, identify and determining the prevalence, antimicrobial susceptibility profile of Shigella sp associated with acute diarrhea among children in Kano, Northern Nigeria. A cross sectional study was conducted among children less than 5 years diagnosed with acute diarrhea and admitted to paediatric ward of Murtala Muhammad Specialist Hospital Kano. Stool samples from a total of 37 (20 male and 17 female) subjects were used to isolate and identified the pathogen. Antimicrobial susceptibility test was conducted using disc diffusion method. The result showed 12 out of 37 samples were positive for Shigella sp which accounted for 32.4%. Higher incidence of Shigella sp was found among subjects of age between 2 – 3 years. The isolates were 100% resistant to Ampicillin. High resistance was also observed in Amoxicillin (83.33%), Chloramphenicol (58.33%) and Tetracycline (25%). The isolates are 100% sensitive to ciprofloxacin, 66.7% to Levofloxacin and Gentamicin each and 58.33% to Erythromycin. Three (3) isolates were resistance to Ampicillin and Amoxicillin, 5 isolates were resistance to Ampicillin, Chloramphenicol and Amoxicillin while 2 isolates were resistance to Ampicillin, Chloramphenicol, Tetracycline and Amoxicillin. It is concluded that Shigella sp is one of the etiological agent of diarrhea in children. Ciprofloxacin, levofloxacin and Gentamicin are drugs of choice for treating diarrhea caused by Shigella sp.
Globally, Diarrheal diseases remain one of the leading causes of morbidity and mortality among children < 5 years of age . About 21% of all deaths in children under the age of five (years) are estimated to be due to diarrhoea and related infections. In Sub-Saharan Africa, it is the second leading cause of death in children under 5 years of age [2,3]. About 37% of all paediatric/childhood deaths in Nigeria are caused by acute diarrhoea, with most of the deaths occurring during the first year of life . Diarrhoea is defined as three or more episodes of watery loose stools in the last 24 hours [4,5]. Diarrhoea can be caused by different agents such as bacteria, parasites and virus . The main etiology of the diarrhoea is related to a wide range of bacteria (such as Campylobacter jejuni, Escherichia coli, Salmonella species, Shigella species, Vibrio cholera, Yersinia enterocolitica, and Aeromonas species), enteroparasites (Giardia lamblia, Cryptosporidium species and Entamoeba histolytica), and viruses (adenovirus, Norwalk virus, and rotavirus) . Among the bacterial causative agents, Salmonella and Shigella remain the major contributors to acute enteric infections and diarrhoea. The common route of infection by these pathogens is the ingestion of contaminated foods and drinks. The problem of antimicrobial resistance in bacterial pathogens causing diarrheal diseases continues to be alarming .
Shigellosis is caused by Shigella sp and it is a worldwide problem although more prevalent in developing countries . Shigellosis is common in areas where living standards are very low and access to safe and adequate drinking water and proper waste disposal systems are often very limited, or even absent . Shigella sp are limited to the intestinal tract of humans and cause bacillary dysentery leading to watery or bloody diarrhea . A variety of raw vegetables, salads, meat, milk, and other dairy products can serve as vehicles for the transmission of Shigella sp. Therefore, the most common causes of contamination are unsanitary practices of food handlers and fecally contaminated water . All age groups are affected by Shigella, but the age group under 5 years is most susceptible, because of low personal cleanliness and partially developed immunity and absence of past exposures .
Microbial resistance to antibiotics is one of the most serious health problems threatening human well-being today. Antibiotic resistance is a type of drug resistance where a microorganism is able to survive exposure to an antibiotic . The widespread use of antibiotics both inside and outside of medicine is playing a significant role in the emergence of resistant bacteria . Infections from resistant bacteria are now too common and some pathogens have even become resistant to multiple types or classes of antibiotics (antimicrobials used to treat bacterial infections) . This resistance has complicated the selection of antibiotics for the treatment of enteric bacterial pathogens, particularly to commonly used antimicrobial agents such as ampicillin, tetracycline and trimethoprim–sulfamethoxazole . Since most diarrheal diseases are treated empirically, it is important to know the susceptibility pattern of the prevalent pathogens. There is a great need to establish the identity and antibiotic susceptibility patterns of different bacterial agents which cause enteric infections in order to introduce effective treatment for diarrheal illness . Hence, this study was aimed to isolate, identify and determine the antibiotic susceptibility profile of Shigella sp associated with acute diarrhea among children in Kano, Northern Nigeria.
The study was conducted at paediatric ward of Murtala Muhammad Specialist Hospital Kano. Kano State is one of the states located in Northern Nigeria. It is geographically coordinated at 110 3’ N and 80 3’ E latitude and longitude respectively. It shares borders with Kaduna state to the west, Bauchi state to the South, Jigawa state to the East, Katsina state to the North. It has a total area of 20,131 km2 (7,777 sqm) and population of 13,405,300 .
The inclusion criteria for the study participants were being a child of five years of age or less and diagnosed with acute diarrhea. Those having a history of antibiotics use two weeks before enrolment were excluded from the study. A simple structured questionnaire was used to collect participants’ demographic information such as sex, age, residential area and socio-economic of the parents.
Fecal samples collection
Thirty-seven (37) Fecal samples from children (ranges from 1 – 5 years) diagnosed with acute diarrhea admitted to paediatric ward of Murtala Muhammad Specialist Hospital Kano were collected in clean, dry and leak proof sterile bottle from period of January to April 2017. The specimens were immediately transported to Laboratory of Science Laboratory Technology in School of Technology, Kano State Polytechnics for bacterial isolation and identification.
Isolation and identification of Shigella sp
Isolation of Shigella sp was conducted according to the method describe by Prescott, et al. . During the process, a sterile wire loop was deep into the fecal sample of the patients and streaked onto the surface of Nutrient agar and MacConkey agar (Life save Biotech, USA). The procedure was repeated for sample and the plates were incubated 370 C for 24 hours. The presumptive colony of Shigella sp from each plate was further sub-cultured to obtained pure culture. The pure isolates of Shigella sp were preserved in peptone water for further use. The preserved isolates were confirmed as Shigella sp using conventional microbiological methods which include Gram staining, lactose fermentation and biochemical (Indole, methyl orange, voges proskauer, nitrate reduction and citrate utilization) tests according to the methods described by Cheesbrough .
Antibiotic susceptibility testing
The Shigella sp isolates were subjected to antibiotic susceptibility testing using the agar disc diffusion method as described by Bauer, et al. . Mueller Hinton agar (MHA) plates were inoculated with overnight culture of each isolate by streak plating. The standard antibiotic sensitivity discs were then aseptically placed at equidistance on the plates and allowed to stand for 1 hour. The plates were then incubated at 37 °C for 24 hours. Sensitivity pattern of the isolates to Ampicillin (30 μg), Tetracycline (30 μg), Amoxicillin (30 μg), Erythromycin (10 μg), Chloramphenicol (30 μg), Levofloxacin (10 μg), Gentamicin (30 μg) and Ciprofloxacin (30 μg), produced by Abtek pharmaceutical limited, were determined. Isolates were divided into two groups based on the zone of inhibition produced by the antibiotic disc; susceptible and resistant according to the European committee on antimicrobial susceptibility testing (EUCAST) breakpoint for interpretation of MICs and zone diameters . The experiment was conducted in triplicate and the average zone of inhibition was recorded.
Ethical clearance (MOH/off/797/T.I/49) for the study was obtained from Kano State Ministry of Health through Health Service Management Board Kano with the consent of Murtala Muhammad Specialist Hospital Kano ethical committee.
Age and sex distribution of the subjects
Table 1 represents the frequency, sex and age of the subjects. A total of 37 participated in the study with 20 males accounted for 54% and 17 females (46%). Subjects within age category 1 – 2 years has the highest frequency with 11 subjects accounted for 29.7%, followed closely by subjects within 2 – 3 years 9 (24.4%). Least frequency was recorded among subjects within age category 4 – 5 years with total frequency of 4 (10.8%).
Identification of Shigella sp
The result for identification of Shigella sp is presented in table 2. The isolate was identified based on Gram staining, cultural characteristics and biochemical test. The result showed that the isolate is Gram negative bacteria, positive for MR, nitrate reduction and indole tests but negative for VP, urease and citrate utilization test. The isolate cannot ferment lactose and produce transparent colourless colony in both MacConkey and Salmonella-Shigella agar.
Prevalence of Shigella sp
The Prevalence of Shigella sp in the fecal sample of the subjects with acute diarrhoea is presented in table 3. The result showed 12 out of 37 samples were positive for Shigella sp which accounted for 32.4%. Higher incidence of Shigella sp was found among subjects of age between 2 – 3 years.
Antibiotic sensitivity testing
The result for antibiotic susceptibility testing is presented in table 4. From the result, the percentage resistance of the isolates to the tested antibiotics is 37.5%. The result showed that all Shigella sp were resistant to Ampicillin. High resistance was also observed in Amoxicillin (83.33%), Chloramphenicol (58.33%) and Tetracycline (25%). The isolates are 100% sensitive to ciprofloxacin. Other antibiotics that are effective against the isolate include Levofloxacin and Gentamicin with sensitivity of 66.7% each and Erythromycin (58.33%).
Multidrug resistance pattern of the isolates
The multi-drug resistance pattern of the isolates is presented in table 5. The result showed that 3 isolates were resistance to Ampicillin and Amoxicillin, 5 isolates were resistance to Ampicillin, Chloramphenicol and Amoxicillin while 2 isolates were resistance to Ampicillin, Chloramphenicol, Tetracycline and Amoxicillin.
Acute diarrhoea might strike at any age, but it is mostly a children disease affecting those younger than 5 years. The period of weaning is a major factor to favor the occurrence of diarrhoea, while many other risk factors such as low socioeconomic level, poor hygiene habits, unsavory dwelling, elevated environmental exposure to entero-pathogenesis and poor nutritional status may be considered . In table 2, the isolate was tested for gram staining and biochemical test. The isolate produces colourless and transparent colony on MacConkey and Salmonella-Shigella agar medium; hence cannot ferment lactose. In the present study, Shigella sp was found as one of the bacteria associated with acute diarrhoea in children. Out of 37 samples examined, 12 were positive for Shigella sp which accounted for 32.4%. Higher incidence of Shigella sp was found among subjects of age between 2–3 years. Several studies were conducted to characterize bacteria associated with gastro-intestinal infection and found Shigella sp as one of the bacteria associated with diarrhea. The finding of this study was in conformity with that of Elmanama and Abdelateef  in Palestine and Sheikh, et al.  in Iran who both found Shigella in stool sample of Children diagnosed with diarrhea as 16.7% and 13.2% respectively. Similar study was conducted by Obi, et al.  on enteric bacterial pathogen in stools of residents of urban and rural regions of Nigeria, the results shows the most frequently encountered pathogens in rural area are E. coli, followed by Salmonella, Shigella, Enterobacter and Campylobacter. A study conducted by Lopez, et al.  on Entero-pathogenic agents isolated in persistent diarrhoea, the result shows that Salmonella, E. coli and Shigella as the major enteric bacteria responsible for gastric disorder. This finding supported the present study.
On the susceptibility of the isolates against the antibiotics used, some of the antibiotics were active against the isolates while resistance to others. The isolates were 100% resistant to Ampicillin. High resistance was also observed in Amoxicillin (83.33%), Chloramphenicol (58.33%) and Tetracycline (25%). The isolates are 100% sensitive to ciprofloxacin, 66.7% to Levofloxacin and Gentamicin each and 58.33% to Erythromycin. This finding showed Shigella is sensitive to β - lactam drugs (Amoxicillin and ampicillin) while sensitive to Fluroquinolones such as ciprofloxacin and Levofloxacin. Isolates were also sensitive to Gentamicin. This finding correlate with that of Gebreegziabher, et al.  and Ameye, et al.  who both found resistivity of Shigella to Amoxicillin and Ampicillin but sensitive to Fluroquinolones such as Ciprofloxacin. High resistance to Amoxicillin and Ampicillin may be attributed to the fact that these antibiotics have been used for long in the country ampicillin and because of their easy availability and potential for misuse. Shigella isolates produce R plasmids that code for several resistance genes and can confer multiple antibiotic resistances . Similarly, analyses of antibiotics resistance genes of Shigella isolates causing diarrhea in children under age of 5 years in South eastern Africa revealed the presence of oxa-1-like β-lactamases for ampicillin, dfrA1 genes for trimethoprim-sulfamethoxazole/co-trimoxazole, tetB genes for tetracycline and Chlacetyltransferase (CAT) activity for chloramphenicol . Isolates were resistance to Ampicillin and Amoxicillin, 5 isolates were resistance to Ampicillin, Chloramphenicol and Amoxicillin while 2 isolates were resistance to Ampicillin, Chloramphenicol, Tetracycline and Amoxicillin.
Acute diarrhoea remains as one of the most prevalent diseases affecting young children in developing countries. The findings of the study revealed that Shigella sp is one of the bacteria associated with acute diarrhoea in children. It is highly prevalent (32.4%) among children diagnosed with acute diarrhea. Sensitivity test of Shigella sp against some antibiotics showed sensitivity to ciprofloxacin, Levofloxacin, Erythromycin and Gentamicin and resistant to Ampicillin and Amoxicillin. Proper environmental cleanliness, good personal hygiene and use balance diet is recommended for children as well as antibiotics such as ciprofloxacin, Gentamicin and Levofloxacin should be used in treatment of acute diarrhoea. It is also recommended that; further studies on genetic makeup of the isolated should be conducted to determine the resistant genes.
The authors wish to thank Ministry of Health, Kano State for the ethical approval for the study. Thanks to staff pathology Department of Murtala Muhammad Specialist Hospital for sample provisions.
- Yeshwondm M, Gesit M, Asaye B, Kassu D, Surafel F. Isolation and Antibiotic Susceptibility Patterns of Shigella and Salmonella among Under 5 Children with Acute Diarrhoea: A Cross-Sectional Study at Selected Public Health Facilities in Addis Ababa, Ethiopia. Clin Microbiol. 2015; 4: 1-7.
- O'Ryan M, Prado V, Pickering LK. A Millennium update on pediatric diarrheal illness in the developing world. Semin Pediatr Infect Dis. 2005; 16: 125-136. PubMed: https://pubmed.ncbi.nlm.nih.gov/15825143/
- Altinier V, Martin G. Diarrhea Caused by Shiga-Toxigenic Escherichia coli, other Pathogens and HUS. Effects of Bismuth Hydroxide Gel. Gastroenterol Hepatol Open Access. 2017; 6: 1-4.
- Mandomando IM, Macet EV, Ruiz J, Sanz S, Abacassamo F, et al. Etiology of diarrhea in children younger than 5 years of age Admitted in a rural hospital of southern mozambique. Am J Trop Med Hyg. 2007; 76: 522-527. PubMed: https://pubmed.ncbi.nlm.nih.gov/17360878/
- Riddle MS, DuPont HL, Connor BA. ACG Clinical Guideline: Diagnosis, Treatment, and Prevention of Acute Diarrheal Infections in Adults. Am J Gastroenterol. 2016; 111: 602-22. PubMed: https://pubmed.ncbi.nlm.nih.gov/27068718/
- Vargas M, Casals C, Schellenberg D, Urassa H, Kahigwa E, Ruiz J, Vila J. Etiology of Diarrhea In Children Less than Five Years of Age In Ifakara, Tanzaia. Am J Trop Med Hyg. 2004; 70: 536-539. PubMed: https://pubmed.ncbi.nlm.nih.gov/15155987/
- Gebreegziabher G, Asrat D, Yimtubezinash W, Hagos T. Isolation and Antimicrobial Susceptibility Profile of Shigella and Salmonella species from Children with Acute Diarrhoea in Mekelle Hospital and Semen Health Center, Tigray, Ethiopia. Ethiop J Sci. 2018; 28:197-206. PubMed: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016337/
- Yan H, Li L, Alam MJ, Shinoda S, Miyoshi S. Prevalence and antimicrobial resistance of Salmonella in retail foods in northern China. Int J Food Microbiol. 2010; 143: 230–234. PubMed: https://pubmed.ncbi.nlm.nih.gov/20797804/
- Ameya G, Tsalla T, Getu F, Getu E. Antimicrobial susceptibility pattern, and associated factors of Salmonella and Shigella infections among under five children in Arba Minch, South Ethiopia. Ann Clin Microbiol Antimicrob. 2018; 17: 1. PubMed: https://pubmed.ncbi.nlm.nih.gov/29391040/
- Assefa A, Girma M. Prevalence and antimicrobial susceptibility patterns of Salmonella and Shigella isolates among children aged below five years with diarrhea attending Robe General Hospital and Goba Referral Hospital, South East Ethiopia. Tropical Diseases, Travel Medicine and Vaccines. 2019; 5: 19. PubMed: https://pubmed.ncbi.nlm.nih.gov/31832223/
- Gebrekidan A, Dejene TA, Kahsay G, Wasihun AG. Prevalence and antimicrobial susceptibility patterns of Shigella among acute diarrheal outpatients in Mekelle hospital, Northern Ethiopia. BMC Res Notes. 2015; 8: 611. PubMed: https://pubmed.ncbi.nlm.nih.gov/26508303/
- Taneja N, Mewara A, Kumar A, Verma G, Sharma M. Cephalosporin resistant Shigella flexneri over 9 years (2001-09) in India. J Antimicrob Chemother. 2012; 67: 1347–1353. PubMed: https://pubmed.ncbi.nlm.nih.gov/22410619/
- Sengupta S, Chattopadhyay MK. Antibiotic resistance of bacteria: A global challenge. Resonance. 2012; 17: 177-191.
- Goossens H, Ferech M, Vander Stichele R, Elseviers M, ESAC Project Group. Outpatient antibiotic use in Europe and association with resistance: a cross-national database study. The Lancet. 2005; 365: 579-587. PubMed: https://pubmed.ncbi.nlm.nih.gov/15708101/
- Center for Disease Control and Prevention (CDC). Antibiotic resistance threats in the United States. U.S Department of Health and Human Services. 2013; 1-112.
- Isenbarger DW, Hoge CW, Srijan A, Pitarangsi C, Vithayasai N, et al. Comparative antibiotic resistance of diarrheal pathogens from Vietnam and Thailand, 1996-1999. Emerg Infect Dis, 2002; 8: 175-180. PubMed: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3369583/
- Asrat D. Shigella and Salmonella serogroups and their antibiotic susceptibility patterns in Ethiopia, East Mediterr Health J. 2008; 14: 760-767.
- National Population Commission (NPC) National population census result, Abuja Nigeria. 2006
- Presscot LM, Harley, JP, Klein DA. Microbiology 6th (Edn). McGraw Hill International edition New York, 2005
- Cheesbrough M. District Laboratory Practice in Tropical Countries. Cambridge university press, 2nd ed. Part 2: 2006; 97-105.
- Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 1996; 45: 493-496. PubMed: https://pubmed.ncbi.nlm.nih.gov/5325707/
- European committee on antimicrobial susceptibility testing (EUCAST) breakpoint for interpretation of MICs and zone diameters. 2015.
- Morales LR, Cancio JA, Cairncross S, Huttly S. Impact of drainage and sewerage on diarrhoea in poor urban areas in Salvador, Brazil. Trans R Soc Trop Med Hyg. 2003; 97: 153-158. PubMed: https://pubmed.ncbi.nlm.nih.gov/14584367/
- Elmanama AA, Abdelateef N. Antimicrobial Resistance of Enteric Pathogens Isolated from Acute Gastroenteritis Patients in Gaza strip, Palestine. Int Arabic J Antimicrobial Agents. 2012; 2: 4.
- Sheikh AF, Moosavian M, Abdi M, Heidary M, Shahi F, et al. Prevalence and antimicrobial resistance of Shigella species isolated from diarrheal patients in Ahvaz, southwest Iran. Infect Drug Resist. 2019; 12: 249-253. PubMed: https://pubmed.ncbi.nlm.nih.gov/30774392/
- Obi CL, Coker AO, Epoke J, Ndip RN. Enteric bacterial pathogen in stools of resident of urban and rural region in Nigeria. J Diarrhoeal Dis Res. 1997; 15: 241-247. PubMed: https://pubmed.ncbi.nlm.nih.gov/9661320/
- Lopez MD, Gonzales ES, Valdes DM, Fragoso AT, Albizu CJ. Wales, 1992-2003. Food control. 1996; 18: 766-772.
- Madigan MT, Martinko JM, Stahl DA, Clark DP. Brock Biology of Microorganisms. 13th ed. San Francisco: Pearson Education, Inc., Publishing. 2012.