Prevalence and Molecular Characterization of Diarrheagenic Escherichia coli in Southern Khyber Pakhtunkhawa, Pakistan

Abstract

Diarrheagenic Escherichia coli (DEC) pathotypes are ranked third among the list of infectious agents, due to high morbidity/mortality rate in developed and developing countries. DEC transmission is linked to the ingestion of contaminated food and water. Fecal wastes from domestic animals, wildlife and humans are added to the water bodies via leakage or surface runoff. Humans also contaminate water bodies by adding poorly treated or untreated sewage effluents, spilling of septic tanks etc. Due to poorly developed sewage system monsoons rains in Pakistan resulted in heavy floods across the country affecting 21 million people in 2010. During the said calamity resulting in 5.3 million medical consultations, where 708, 891 13% were made only for acute diarrhea. Irrigation of agricultural farms with untreated water results in contamination of food products during harvesting, processing and handling. Foodborne diarrheal outbreaks were reported due to consumption of green leafy vegetables, drinking water and meat sources. Complications of Escherichia coli (E. coli) pathotypes infections are ranging from mild to severe life threatening diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. This study was conducted to assess the prevalence and molecular characterization of Diarrheagenic E. coli in Southern Khyber Pakhtunkhawa, Pakistan. Diarrheagenic E. coli pathotypes were isolated from diarrheal stool specimens, vegetables, meat and water sources collected for present study (2010-2012). DEC strains were isolated using (1) MacConkey agar, (2) Eosin Methylene blue agar (EMB), (3) Sorbitol MacConkey agar and (4) Cefixime Tellurite Sorbitol MacConkey Agar. Furthermore, E. coil strains were identified through biochemical tests. Colorless, non-sorbitol fermenting E. coli strains on Sorbitol MacConkey agar were confirmed as E. coli O157:H7 using DrySpot E. coli O157:H7 agglutination Kit (Oxoid, UK). Diarrheagenic E. coli pathotypes were also tested for antibiotic resistance using Kirby-Bauer disc ix diffusion method. Characteristics virulence factors DEC pathotypes i.e. (1) stx1, (2) stx2, (3) stx2c, (4) eae, (5) tir, (6) hlyA, (7) est (8) elt and (9) bfpA were detected using Multiplex PCR. Amplification conditions comprised of 94°C for 6 min, followed by 35 cycles of 94°C for 50 s, 57°C for 40 s and 72°C for 50 s, and finally 72°C for 3 min. Amplicons were analyzed by electrophoresis on agarose 1.5% w⁄v gels using standard conditions, followed by staining with ethidium bromide. (1) Water, (2) Meat and (3) Vegetable samples collected during the present study were processed for the isolation and identification of diarrheagenic E. coli pathotypes using differential media, as described above. Multiplex PCR was used the detection of virulence genes i.e. stx1, stx2, stx2c, eae, tir, hlyA, bfpA, heat stable toxin (ST) and heat labile toxin (LT). Amplification conditions comprised of 94°C for 6 min, followed by 35 cycles of 94°C for 50 s, 57°C for 40 s and 72°C for 50 s, and finally 72°C for 3 min. Amplicons were analyzed by electrophoresis on agarose 1.5% w⁄ v gels using standard conditions, followed by staining with ethidium bromide. Prevalence of diarrheagenic E. coli was found as high as 57% (515/900) during the present study. Based on stool physiology, 54.4% E. coli strains were isolated from watery diarrheal samples, compared to 37.6% from mucoid stool and 8% from bloody diarrheal stool specimens. Of group I, 155 (30%) isolates were confirmed as E. coli strains. Similarly, number and percentage isolation of DEC strains from each respective age group is as follows: Group II 137 (26.6%), Group III 59 (11.4%), Group IV 47 (9.2%), Group V 44 (8.6%), Group VI 42 (8.2%), and Group VII 31 (6%). However, none of the age groups achieved statistical significance. E. coli strains were obtained from 40% male patients compared to 60% strains from female patients. The frequency of E. coli strains recovered different units of hospitals were as; 31.8%, 52%, 3.15% and 12.94% from medical ward, Pediatrics ward, OPD and ICU, respectively. From outdoor x patients 44.5% samples were collected from refugee camps survey. E. coli strains from age Group I, was 14.1% where identified as E. coli O157:H7, followed by Group II: 12.4%, Group III: 13.5%, Group IV: 10.6%, Group V: 9%, Group VI: 14.2% and Group VII: 6.4%. During the present study, 11.8% (61/515) strains were isolated during winter season, 21.3% (110/515) during spring season, 47% (242/515) during summer season and 19.8% during autumn season. Samples from pond water were contaminated with 28% (14/50) E. coli strains, where 14% (2/14) were identified as serotype O157:H7. Tap water was found to be free of serotype O157:H7 contamination. Sewage water samples were contaminated with 62% E. coli strain, where 16.13% (5/31) were identified as serotype O157:H7. Similarly, 38% (19/50) irrigation water samples were contaminated with E. coli pathotypes, where 13.8% (3/19) were identified as serotype O157:H7. DEC strains 64 isolated from water sources were randomly selected for pathogroup specific molecular characterization. Enterotoxigenic E. coli were identified as 56.25% (36/64) of pathogroups including a combination of heat stable toxin (ST) and heat labile toxin (LT) gene. Enteropathogenic E. coli were identified as 28% (18/64) comprising of 72% (13/18) typical enteropathogenic E. coli and 28% (6/18) atypical E. coli. Only 15.6% (10/64) were identified as E. coli O157:H7 serotype carrying stx1 and stx2 genes. Beef samples were contaminated with 58% of (29/50) E. coli strains and 20.9% (6/29) of these were identified as serotype E. coli O157:H7. Chicken meat samples were contaminated with 16% (8/50) E. coli strains and none of them was identified as serotype E. coli O157:H7. Sheep meat samples were contaminated with 21.5% (3/14) serotype E. coli O157:H7. Goat meat samples were contaminated with 34 % (17/50) E. coli pathotypes, where only one among 17 (5.9%) was confirmed as serotype E. coli O157:H7. xi (1) Mixed salad samples were contaminated with 54% of (27/50) E. coli strains, and 14.8% (4/27) of them were identified as E. coli O157:H7. (2) Cucumber samples were contaminated with 46% (23/50) E. coli strains and 30.4% (7/23) were confirmed as E. coli O157:H7. (3) Spinach samples were contaminated with 28% (14/50) E. coli strains and 28.57% (4/14) were identified as E. coli O157:H7. (4) Lettuce samples were contaminated with 40% (20/50) E. coli strains and 15% (3/20) were identified as E. coli O157:H7. STEC isolates (100%) were positive for the presence of stx1, stx2. tir, hly and eae genes. Among Shiga toxin, Bacteriophage Insertion (SBI) 100% stx1 bacteriophage was found to be inserted in yehV gene (both right and left side insertion). stx2 was completely invaded by the bacteriophages at wrbA site, and similar invasions were observed at sbsB occupied by stx2C bacteriophage. Out of 300 DEC isolates, randomly selected for antibiogram development, included 150 (50%) E. coli from outpatients, 75 (25%) from medical ward and 75 (25%) from pediatrics ward. 94% isolates were found sensitive to imipenem, cefuroxime was the second most effective antibiotic (55%). The maximum resistance (92%) was observed against tetracycline, followed by ampicillin 83% and ciprofloxacin 81%. In case of β-lactam antibiotics, high resistance (78%) was observed against amoxicillin/clavulanic acid. Sewage water and industrial effluents needs prior treatment before entering into to the environment. Drinking water needs to be boiled before intake. Firm adherence to the prescribed drugs can decrease trends in antibiotics resistance. Oral rehydration therapy is strongly recommended to minimize extensive dehydration leading to kidney failure. It is further, recommended that the use of antibiotics in food, animals should follow prudent guidelines, to minimize spread of resistant bacteria. xii Keywords: Escherichia coli (E. coli), Diarrheagenic E. coli (DEC), Enterohaemorrheagic E. coli (EHEC), Diarrhaegenic E. coli (DEC), Shiga Toxin producing gene (stx)