https://imcjms.com Ibrahim Medical College Journal of Medical Science https://imcjms.com/registration/journal_full_text/66 Tue, 02 Aug 2016 11:57:46 +0000 Enteropathogenic Escherichia coli (EPEC) mediated infantile diarrhoea among children is an important cause of morbidity and mortality in developing countries. The antimicrobial susceptibility pattern of EPEC strains isolated from children under 5 years of age was studied. Stool samples from 272 patients with diarrhoea were collected from two tertiary care hospitals. Out of 272 stool samples, 20 (7.35%) isolates were identified as EPEC on the basis of presence of bfpA gene detected by polymerase chain reaction and antibiotic susceptibility testing was performed on these EPEC strains by Kirby-Bauer disc diffusion method. The antimicrobial susceptibility test revealed that the EPEC isolates were highly resistant to ampicillin (100%), nalidixic acid (95%) and tetracycline (95%) and were sensitive to ceftazidime (95%), cefotaxime (90%), ceftriaxone (95%), imipenem (100%) and levofloxacin (85%). Isolation of EPEC is of great importance since they are responsible for acute diarrhoeal diseases in large number of children under the age of five years. The high antimicrobial resistance observed in our study indicates indiscriminate or improper use of antimicrobials, besides the risks of self-medication. Introduction   Stool or rectal swab (R/S) samples were collected from Sir Salimullah Medical college and Mitford Hospital (SSMC & MH) and Dhaka Shishu Hospital (DSH) from 272 patients under 5 years of age, presenting with acute diarrhoea and who did not take any antibiotic during the last 30 days.6 All the samples were collected within the period from January to December 2011. Standard microbiological techniques were followed for culture and isolation of Escherichia coli (Esch. coli). Detection of bfpA genes of EPEC by PCR assay   Susceptibility of isolated EPEC strains to different antibiotics was determined by Kirby-Bauer disc-diffusion techniqueas specified by the National Committee for Clinical Laboratory Standards (NCCLS).8 The antibiotic discs used in this study were Ampicillin (10 µg), ceftazidime (30 µg), cefoxitin (30 µg), cefotaxime (30 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), gentamycin (10 µg), imipenem (10 µg), levofloxacin (5 µg), nalidixic acid (30 µg), piperacillin tazobactum (110 µg), tetracycline (30 µg ), cotrimoxazole (trimethoprim sulphamethoxazole) (25 µg).5 All the antibiotic discs used for the study were obtained from Oxoid Ltd. Bashingstore Hampaire, UK. Inoculum standardization   Within 15 minutes after standardization of inoculums, a sterile cotton swab was immersed into bacterial suspension. The swab was then streaked evenly on the surface of the plate in three different planes by rotating the plate to get a uniform distribution of inoculum. The inoculum was allowed to dry for 15 minutes at room temperature with lid closed. The antimicrobial discs were then placed on the inoculum surface by a sterile fine pointed forceps 10-15 mm away from the edge of the Petri dishes and 24 mm gap between the discs. The plates were incubated at 37º C for 24 hours. Measurement of inhibition zone   The zone of inhibition in growth produced by each antimicrobial agent on the test organisms were categorized into sensitive (S) and resistant (R) according to NCCLS.8 Results           Discussion A total of 272 samples either stool or rectal swab collected from the patients with diarrhoea were investigated. The EPEC strains identified on the basis of presence of bfpA gene detection by PCR. In this study Esch. coli were isolated from all specimens. All Esch. coli isolates were investigated by PCR to detect the presence of bfpA gene. The bfpA genes were identified in 20 (7.35%) Esch. coli isolates. Similar result was reported by Iman et al., (2011).11 Around 3.2% EPEC diarrhoea was reported from Thailand in 2004,12 6.6% from Vietnam in 2005,13 15.8% from India in 2008.14 Lower isolation rate of EPEC in our study could be due the fact that only bfp A gene was detected by PCR and other genes like eaeA gene was not considered. Also, lower isolation rates might be due to inclusion of breast fed children. Breast milk (colostrum) from mothers living in endemic areas have been reported to contain high levels of immunoglobulin A (IgA) antibodies against the EPEC virulence factors. Other reasons could be increased awareness about food and hand hygiene, resulting from intensive education programs carried out by the media after H5N1 (Avian flu) and H1N1 (Swine flu) outbreaks in 2006 and 2008 respectively.11 Another cause of lower isolation rate of EPEC in the present study was probablly for not detecting EPEC by serotyping as polyvalent and specific monovalent antisera for EPEC sero-groups were not available. The susceptibility test results showed that, most of the EPEC strains were multidrug resistance. EPEC strains were highly susceptible to ceftazidime (95%), cefotaxime (90%), ceftriaxone (95%), imipenem (100%), levofloxacin (85%). The most effective beta-lactam antibiotics were ceftazidime, ceftriaxone, imipenem and piperacillin-tazobactam. Such results may indicate that the isolated strains of EPEC were not extended-spectrum beta-lactamase producers, since resistance to third generation cephalosporin was not observed.17 The results imply that the strains were likely to have originated from the community, which supports the observation of low levels of resistance to such drugs.18,19 Sensitivity was moderately high for gentamicin (80%) and piperacillin tazobactum (60%). Moreover, such antimicrobials are generally used in hospitals, and bacteria resistant to theses agents originating from the community are not expected.20 Several studies in different parts of the world showed similar sensitivity pattern of EPEC.5,21 Low levels of resistance against levofloxacin were observed in this study. The literature has reported varying rates of resistance against both levofloxacin and ciprofloxacin, which can be explained by the use of these drugs in some countries as a treatment for enteric infections.22,23 However, studies to assess the role of these antimicrobial agents in the treatment of EPEC infections in children are needed. It has been shown that the treatment of diarrhoea caused by EPEC with antibiotics, specifically co-trimoxazole, decreases the duration and intensity of the diarrhoea.24   1.    Nataro J, and. Kaper J. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 1998; 11: 142–201. 3.    Jordi V, Martha V, Climent C, Honorato U, Mshinda H, David S, Joaquim G. Antimicrobial Resistance of diarrheagenic Escherichia coli Isolated from Children under the Age of 5 Years from Ifakara, Tanzania. American Society for Microbiology 1999; 43(12):  3022–3024. 5.    Khairun N, Dilruba A, Johirul I, Lutful F, Anowar M. Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting. Bang J Med Microbiol 2007; 01(02): 38-42. 7.    Svenungsson B, Lagergren A, Erik E, Birgitta E, Hedlund K, Karnell A, Lofdahl S. Enteropathogens in adult patients with diarrhoea and healthy control subjects: A 1-Year prospective study in a Swidish Clinic for infectous disease. Clin Inf Dis 2000; 30: 770-78. 9.    Voetsch A, Angulo F, Rabatsky T. Laboratory practices for stool-specimen culture for bacterial pathogens, including Escherichia coli 0157:H7, in the Food Net sites. Clin infect Dis 2004; 38(3): 190-197. 11.  Iman K, Emad A, Entsar A and Rania S. Enteropathogenic Escherichia coli Associated with Diarrhoea in Children in Cairo. Egypt The Scientific World J 2011; 11: 2613–2619. 13.  Nguyen R, Taylor L, Tauschek M. Atypical enteropathogenic Escherichia coli infection and prolonged diarrhoea in children. Emerge infect Dis 2006; 12(4): 597-603. 15.  Murray C and A. Lo´pez D. Mortality by cause for eight regions of the world: global burden of disease study. Lancet 1997; 349: 1269–1276. 17. Goyal, A, Prasad K, Prasad A, Gupta S, Ghoshal U, and Ayyagari A. Extended spectrum β-lactamase in Escherichia coli, Klebsiella pneumonia and associated risk factors. Indian MedJ 2009; 129: 695-700. 19.  Erb A, Stürmer T, Marre R and Brenner H. Prevalence of antibiotic resistance in Escherichia coli: overview of geographical, temporal and methodological variations. Eur. J. Clin. Microbiol.Infect. Dis. 2007; 26: 83-90. 21. Patrícia G, Vânia L, and Cláudio G. Occurrence and Antimicrobial Drug Susceptibility Patterns of Commensal and Diarrheagenic Escherichia coli in Fecal Microbiota from Children With and Without Acute Diarrhoea. The J of Microbiol. 2011; 10: 2102-3213. 23. Yang, C, Lin M, Lin C, Huang Y, Hsu C and Liou L. Characterization of antimicrobial resistance patterns and integrons in human fecal Escherichia coli in Taiwan. J. Infect.Dis. 2009; 62: 177-181. 2026 Ibrahim Medical College. All rights reserved.