Sudan Journal of Medical Sciences Volume 17, Issue no. 1, DOI 10.18502/sjms.v17i1.10689 Production and Hosting by Knowledge E Original Article Isolation, Extraction, and Characterization of Verotoxin-producing Escherichia coli O157:H7 from Diarrheal Stool Samples Muhammad Musthafa Poyil1, Ponmurugan Karuppiah2,3*, Suresh S. S. Raja4, P. Sasikumar5 1Department of Basic Medical Sciences, College of Medicine, Prince Sattam bin Abdulaziz University, Al-Kharj, Saudi Arabia 2Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia 3Department of Microbiology, K.S. Rangasamy College of Arts and Science, Tiruchengode, Namakkal, Tamil Nadu, India 4Department of Microbiology, Government Arts and Science College, Perambalur, Tamil Nadu, India 5Department of Microbiology, The Madura College, Madurai, Tamil Nadu, India Abstract Background: Escherichia coli O157:H7 infection causes hemorrhagic colitis and is diagnosed based on symptoms such as cramps, stomach pain, and watery diarrhea. Shiga-like toxins (Verotoxin) produced by Escherichia coli O157:H7 damages endothelial cells of both kidney and brain, causing renal dysfunction and neurological problems. Methods: The present study focuses on identifying the prevalence of Verotoxin- producing Escherichia coli O157:H7 among diarrheal inpatients at Erode Government Hospital, India, and its antibiogram. Further, the Verotoxins were characterized by using SDS-PAGE analysis. A total of 123 diarrheal stool samples were collected, and 37 of them (30.08% of the total samples) were found to have the presence of E. coli. The organisms were identified based on their colony morphology on various media, cell morphology, and biochemical tests. The Shiga-like toxin production was identified by non-fermentation of sorbitol on SMAC agar plates. Confirmation of Shiga-like toxin was performed using agglutination assay. Results: In total, 12 isolates showed agglutination and these isolates were confirmed to be E. coli O157:H7. The molecular weight of the Verotoxin was found to be between 20 and 29 kD. The antibiogram profile of the four isolated strains against 10 standard antibiotics was determined. Conclusion: The results of this study show the occurrence of drug resistance on hemorrhagic colitis causing E. coli O157:H7. Keywords: E. coli, Verotoxin, diarrhea, hemorrhagic colitis How to cite this article: Muhammad Musthafa Poyil, Ponmurugan Karuppiah*, Suresh S. S. Raja, P. Sasikumar (2022) “Isolation, Extraction, and Characterization of Verotoxin-producing Escherichia coli O157:H7 from Diarrheal Stool Samples,” Sudan Journal of Medical Sciences, vol. 17, Issue no. 1, pages 116–127. DOI 10.18502/sjms.v17i1.10689 Page 116 Corresponding Author: Muhammad Poyil; email: pmusthu@gmail.com Received 15 September 2021 Accepted 19 February 2022 Published 31 March 2022 Production and Hosting by Knowledge E Muhammad Musthafa Poyil et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Editor-in-Chief: Prof. Mohammad A. M. Ibnouf http://www.knowledgee.com mailto:pmusthu@gmail.com https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ Sudan Journal of Medical Sciences Muhammad Musthafa Poyil et al 1. Introduction Escherichia coli is a predominant pathogenic microorganism that causes various condi- tions such as urinary tract infections, diarrhea, pneumonia, meningitis, and bacteremia. Escherichia coli-synthesizing toxins with a cytolytic effect on Vero cells were primar- ily termed as Verotoxin-producing E. coli (VTEC) [1]. Verotoxin and Shiga toxin were observed to be slightly similar in structures and the Shiga toxin-producing E. coli were termed as Shiga toxin-producing E. coli (SETC). Among the strains of SETC, the clinically isolated hemorrhagic colitis (HC)-causing serotypes were designated as enterohemorrhagic E. coli (EHEC) [2]. The virulent nature of EHEC is due to the bacteriophage-induced Shiga toxin. This toxin regulates diarrhea and HC to hemolytic uremic syndrome (HUS) as well. Stx 1 and Stx 2 are the two types of immunologically varied Shiga toxins synthesized in EHEC. Stx 1 was similar to the Shiga toxin synthesized by Shigella dysenteriae. Another virulent factor includes an external membrane protein known as intimin, which was found to be vital for colonizing the intestine. Enterohemolysin is encoded by a 60Mda plasmid called p O157. Enterohemolysin functions by lysis of RBC and providing an iron source to the pathogen [3]. The toxin produced by S. dysenteriae type 1 that is active on Vero cells is named Shiga toxin; two related toxins produced by E. coli O157:H7 are named Shiga-like toxins 1 and 2 (SLT-1 and SLT-2) or Verotoxins 1 and 2 (VT-1 and VT-2). Shiga toxin and Shiga- like toxin (SLT–1) are immunologically, biologically, and structurally closely related or identical. SLT–2 is also biologically (activity on Vero cells) and structurally (A and B subunit structures) related to but differs immunologically from Shiga toxin and SLT-1. The assay on Vero cells seems to be a useful method for their detection [4, 5]. The fecal–oral route was the common method of transmission. Oftentimes, humans are infected through carriers such as animals and fomites. Birds are identified to be evident vectors for transmission. Pathogen outbreaks are mostly related to the con- sumption of poorly cooked or packaged animal products; in particular ground beef, however, unpasteurized milk and dried meats, alfalfa sprouts, and other infected veg- etable products have often triggered epidemics [6]. Escherichia coli O157:H7 infection causes HC; this infection is generally diagnosed based on symptoms such as cramp, stomachache, and watery diarrhea accompanied by bloody diarrhea which may be absent in the primary stages and dehydration may be probable. The infection is observed to be self-limiting and lasts about a week in healthy DOI 10.18502/sjms.v17i1.10689 Page 117 Sudan Journal of Medical Sciences Muhammad Musthafa Poyil et al adults [7]. Severe complications may occur in a small percentage of cases. Shiga-like toxins (Verotoxin) produced by E. coli O157:H7 damages endothelial cells of both kidney and brain, causing renal dysfunction and neurological problems. HUS can occur in 5– 10% of infected patients – generally, the week after the onset of diarrhea and is evaluated by renal dysfunction, which can lead to enduring injury and hemolytic anemia. Severe complications like seizures colonic perforation, hypertension, strokes, pancreatitis, and coma may also be observed. Few patients exhibit lifelong Insulin-dependent diabetes mellitus. Although HUS can impact all age groups, children <10 years old are highly prone to this disease [8]. Therefore, the main objectives of the present study were to find out the occurrence of verotoxin-producing strains of E. coli O157:H7 among diarrheal patients, to perform the antibiogram analysis of the isolated strains, and to characterize them using SDS-PAGE analysis. 2. Materials and Methods 2.1. Sample collection and processing A total of 123 human diarrheal stool samples were collected in a sterile wide-mouth container and processed for microbial isolation. Stool samples were collected from inpatients at the Erode Government Hospital, India. The fecal samples were homoge- nized (10 gm of feces in 100 ml of Trypticase soy broth) immediately after collection from both the infected person and animal [9]. It was incubated for 14 hr in Shaker at 37ºC and then a loop full of culture was plated on MacConkey agar incubated at 37ºC for 24 hr. 2.2. Identification of E. coli Presumptive E. coli colonies were biochemically confirmed. The biochemical tests performed were oxidase test, indole production test, methyl red test, Voges–Proskauer test, citrate utilization test, Triple Sugar Iron Agar test, Gram’s staining and motility analysis. The lactose-fermenting colonies were examined for hemolysis in blood agar, fermentation of lactose in MacConkey agar, and in Eosin methylene blue agar. DOI 10.18502/sjms.v17i1.10689 Page 118 Sudan Journal of Medical Sciences Muhammad Musthafa Poyil et al 2.3. Antibiotic susceptibility test The culture was tested against 13 antibiotics. The antibiotics were Ampicillin, Amoxi- cillin, Ciprofloxacin, Chloramphenicol, Gentamicin, Kanamycin, Neomycin, Nalidixic acid, Penicillin-G, Polymyxin-B, Rifampicin, Streptomycin, and Tetracycline 2.4. Preliminary confirmation of Shiga-like toxin-producing E. coli The confirmed E. coli colonies were plated on sorbitol MacConkey agar (SMAC) by using the streak plate technique to confirm the Shiga-like toxin-producing E. coli. After plating, the plates were incubated at 37ºC for 24 hr [10]. 2.5. Serotyping for conformation of Shiga-like toxin-producing E. coli Escherichia coli O and H antigens were investigated based on standard protocols. Agglutination assay was performed on glass slides by adding a drop (20 𝜇l) of antiserum in PBS with a loop full of bacteria. Agglutination reactions were examined within a few seconds. Bacterial culture in 1 drop of PBS was used as control. Agglutination reac- tions were evaluated by live bacterial cultures, and confirmation tests were repeatedly performed by heating the bacterial cultures for 1 hr at 100ºC. 2.6. Production of Verotoxin/Shiga-like toxin by E. coli Cultures were oxygenated to the maximum by aeration and vigorous agitation at 300 rpm on a rotatory shaker for higher toxin production. After this, 1 ml of culture was transported to modified Syncase Broth (MSB) [11] for the detection and purification of the toxin. To 50 ml of the stock solution, 5 ml of 2% (W/V) glucose and 0.04% of tryptophan were added. 2.7. Extraction of Shiga-like toxins The supernatant from overnight cultures was taken. Seventy percent ammonium sulfate solution was added and incubated overnight for precipitation. The pellet was suspended in 250 ml of 10 mm Tris HCl (pH – 7.4) and centrifuged at 11,950 rpm at 4ºC. The final cell pellet was resuspended in 10 ml of 10 mM Tris HCl (pH – 7.4) and dialyzed DOI 10.18502/sjms.v17i1.10689 Page 119 Sudan Journal of Medical Sciences Muhammad Musthafa Poyil et al Figure 1: Identification of Verotoxin-producing E. coli O157:H7 in Sorbiltol MacConkey agar (SMAC) plates. extensively against 10 mM Tris (pH – 7.4) for at least 24 hr. The dialyzed ammonium sulfate precipitate was characterized using SDS – PAGE to determine the molecular weight of the extracted toxin. 3. Results 3.1. Isolation and identification of E. coli A total of 123 samples were collected both from the diarrheal stool. Bacteria that produce Verotoxin were isolated by using enrichment culture method followed by streak plate method by using trypticase soy agar medium. In the present study, over 37 serotypes (30.08%) of E. coli were isolated and the colonies on agar medium were analyzed based on their colony morphology, cell morphology, and biochemical tests. In biochemical tests, the isolates were observed to be gram-negative rods, motile. Tryptophan was breaking down by the production of indole. Methyl red was produced. Citrate was not utilized and the result of Voges–Proskauer test was negative. Oxidase was produced. And almost all the sugars were fermented by this organism such as glucose, lactose, mannitol, and mannose, and it produced negative results in the urease test. The results are presented in Table 1. On comparing the results with Bergey’s Manual of Determinative Bacteriology, the isolate was identified as E. coli. Most strains ferment lactose and thus grow as smooth, glossy, pink colonies on MacConkey agar and produce a clear zone of hemolysis on the blood agar medium. They produce greenish metallic sheen on Eosin Methylene Blue agar. DOI 10.18502/sjms.v17i1.10689 Page 120 Sudan Journal of Medical Sciences Muhammad Musthafa Poyil et al Table 1: Biochemical characterization. Biochemical test Results Gram staining G (–)ve Motility test + Indole Production test + Methyl red test + Voges–Proskauer test – Citrate utilization test – Oxidase test – TSI test A/A H2S production test – Glucose fermentation test – Lactose fermentation test + Mannitol fermentation test + Maltose fermentation test + Urease Test – Sorbitol fermentation test S+ = V–; S– = V+ +: positive; –: negative; V–: Verotoxin negative; V+: Verotoxin positive. Table 2: Antibiotic susceptibility. Bacterial isolates Antibiotics$/Zone of inhibition A Ac Cf C G K N Na P Pb R S T A1 17.00 (S) 18.00 (S) 21.00 (S) 11.00 (R) 18.00 (S) 18.00 (S) 13.00 (I) 0.00 (R) 0.00 (R) 0.00 (R) 10.00 (S) 13.00 (I) 17.00 (I) A2 15.00 (I) 10.00 (R) 16.00 (I) 15.00 (I) 17.00 (S) 13.00 (R) 17.00 (S) 9.00 (R) 0.00 (R) 0.00 (R) 9.00 (S) 13.00 (I) 10.00 (R) A3 17.00 (S) 19.00 (R) 19.00 (I) 21.00 (S) 17.00 (S) 14.00 (I) 15.00 (I) 0.00 (R) 0.00 (R) 0.00 (R) 11.00 (S) 17.00 (S) 10.00 (R) A4 17.00 (S) 19.00 (S) 20.00 (S) 12.00 (R) 19.00 (S) 17.00(S) 14.00 (I) 0.00 (R) 0.00 (R) 0.00 (R) 10.00 (S) 18.00 (S) 12.00 (R) $A: Ampicillin; Ac: Amoxicillin; Cf: Ciprofloxacin; C: Chloramphenicol; G: Gentamicin; K: Kanamycin; N: Neomycin; Na: Nalidixic acid; P: Penicillin-G; Pb: Polymyxin-B; R: Rifampicin; S: Streptomycin; T: Tetracycline. Total=37 E. coli E.coli O157: H7 32.43% 67.57% Figure 2: Occurrence of E. coli O157:H7 in human diarrheal samples. DOI 10.18502/sjms.v17i1.10689 Page 121 Sudan Journal of Medical Sciences Muhammad Musthafa Poyil et al Lane 1 : Protein molecular size marker Lane 2: Verotoxin in LB broth Lane 3: Verotoxin in MSB Figure 3: SDS-PAGE analysis of Verotoxin. 3.2. Preliminary confirmation of Shiga-like toxin-producing E. coli After incubation for 24 hr, the small, pale yellow colored (Negative), round colonies appeared on the sorbitol MacConkey agar medium. While the non-Verotoxin-producing E. coli ferment sorbitol and produce pink-colored colonies, Verotoxin-producing E. coli fail to ferment sorbitol and produce white-colored colonies. In this study, all 37 isolates were Verotoxin-positive and failed to ferment sorbitol. The colonies of this isolated strain on SMAC medium were colorless which permits presumptive identification of this organism (Figure 1). All E. coli O157:H7 strains identified to date have been observed to produce Verotoxin and do not ferment sorbitol [10]. 3.3. Confirmation of Shiga-like toxin-producing E. coli Thirty-seven isolates were further tested according to the standard procedures of serotyping; of them, twelve isolates were confirmed as O157 and H7 (Figure 2). Agglu- tination results show that antibody typing reagent supports the required sensitivity and specificity for identifying the E. coli serotype O 157:H7. DOI 10.18502/sjms.v17i1.10689 Page 122 Sudan Journal of Medical Sciences Muhammad Musthafa Poyil et al 3.4. Characterization of Shiga-like toxin The Shiga-like toxin was produced with the use of MSB and it was extracted by ammonium sulfate precipitation method. The protein was extracted from E. coli which are isolated from animal and human fecal samples. The molecular weight of the protein was between 20,000 and 29,000 Da (Figure 3). On comparing to LB broth, the band which was observed in MSB gave a better result [12]. 3.5. Antibiotic susceptibility test The four isolated strains were subjected to antibiogram analysis against 10 antibiotics. After incubation, the results were noted. The size of the zone was compared with the Zone size-interpretative chart (Table 2). 4. Discussion A cross-sectional study observed that the major pathogenic organism causing the disease was E. coli O157:H7, a species that was not previously reported as a human pathogen. Since then, E. coli O157:H7 has caused large food-borne outbreak infections worldwide. The center for disease control and prevention (CDC) has recently identified that E. coli O157:H7 is the fourth most predominant diarrhea-causing bacteria next to Campylobacter sp., Salmonella sp., and Shigella sp. This pathogen is a gram-negative motile rods belonging to the family of Enterobacteriaceae and was the primary reason for the HC in humans. Escherichia coli O157:H7 infection occurs globally and E. coli O157:H7 remains to be viable for more than three months in feces and soil, and infections can occur from weeks to months in widely used acidic foods such as mayonnaise, sausage, apple cider, and cheddar even at refrigeration temperature [13]. Thus, the present study concentrates on the identification of E. coli O157:H7 from diarrheal stool samples. Of the 123 samples, strains from 37 samples were found to have E. coli. Escherichia coli was confirmed by colony morphology on various media and produced green metallic sheen on EMB agar plates. Further, it was confirmed by several biochemical tests. The Shiga-like toxin production was identified by non-fermentation of sorbitol on SMAC agar plates. Confirmation of Shiga-like toxin was performed using agglutination assay. Twelve isolates showed agglutination and these isolates were confirmed to be E. coli O157:H7. DOI 10.18502/sjms.v17i1.10689 Page 123 Sudan Journal of Medical Sciences Muhammad Musthafa Poyil et al Escherichia coli O157:H7 has been reported in domestic animals like cattle, sheep, goats, pigs, dogs, and poultry. Cattle are the major reservoir for the pathogen and young animals shed E. coli in the fecal. Pathogens in feces can remain for weeks to months and can also be intermittent. Many types of STEC other than O157:H7 have been observed more commonly in cattle and most predominantly in fecal samples [14]. Similar results were observed for sheep and goats which carry other strains of STEC [15]. These observations denote that few STEC strains are mutated for colonizing the intestinal layer of their animal hosts and remain permanently for several years [16]. The effect of Shiga toxin and Shiga-like toxin 2 has been observed to differ from cell type due to the Gb3 receptors, which is present in certain tissues like sensory neuron and renal cell. In such cells, the concentration of receptor and interaction was regulated by factors like tumor necrosis factor (TNF), interleukins, and fatty acid content of the membranes. Shiga-like toxin and Shiga-like toxin 2 differ in their effects on different tissues. Moreover, both two types have been shown to induce cell death. Shiga-like toxins were characterized by SDS-PAGE analysis [17]. The strains were cultivated in MSB and supernatants were subjected to ammonium sulfate precipitation and dialysis. The purified proteins were characterized using SDS-PAGE analysis. The molecular weight of the toxins was found to be between 20 and 29 kDa. Such toxins have several interesting effects at the cellular level. Once the Shiga-like toxins have been enclosed, it is invariably transported through the Golgi apparatus to the rough endoplasmic reticulum where ribosomes are their major targets. The Shiga-like toxin also inhibits protein synthesis and regulates cytokines such as Il-1, Il-6, and Il-8. The Shiga-like toxin is also observed to express TNF-7, inducing F-acting de-polymerization and activating α kinase. Recent studies have shown that Shiga-like toxin is cytotoxic for primary cultures of human colonic cells. Shiga toxin can induce vascular cellular damage, as capillary lesions observed in hemolytic-uremic syndrome resemble those found in cerebral vessels of animals administered with these toxins [17, 18]. Further from the antibiogram analysis, the strains were found to be resistant to most of the commonly used antibiotics. This indicates the occurrence of drug resistance in Verotoxin-producing E. coli O157:H7. 5. Conclusion A total of 123 samples were collected both from diarrheal stools and strains from 37 samples (43.02%) were found to be E. coli. The organisms were identified based on DOI 10.18502/sjms.v17i1.10689 Page 124 Sudan Journal of Medical Sciences Muhammad Musthafa Poyil et al their colony morphology on various media, cell morphology, and biochemical tests. The Shiga-like toxin production was identified by non-fermentation of sorbitol on SMAC agar plates. Confirmation of Shiga-like toxin was performed using agglutination assay. Twelve isolates showed agglutination and these isolates were confirmed to be E. coli O157:H7. The molecular weight of the Verotoxin was found to be between 20 and 29 kDa. The antibiogram profile of the four isolated strains against 10 standard antibiotics was determined. Acknowledgements The authors are grateful to the Deanship of Scientific Research, Prince Sattam bin Abdu- laziz University, Al-Kharj, Saudi Arabia for its support and encouragement in conducting the research and publishing this report. Ethical Considerations Competing Interests The authors declare no conflict of interest. Availability of Data and Material All relevant data of this study are available to any interested researchers upon reason- able request to the corresponding author. Funding None. 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Identification and molecular characteristics of verotoxin-producing Escherichia coli (VTEC) from bovine and pig carcasses isolated in Poland during 2014–2018. Food Microbiology, vol. 92, p. 103587. DOI 10.18502/sjms.v17i1.10689 Page 127 Introduction Materials and Methods Sample collection and processing Identification of E. coli Antibiotic susceptibility test Preliminary confirmation of Shiga-like toxin-producing E. coli Serotyping for conformation of Shiga-like toxin-producing E. coli Production of Verotoxin/Shiga-like toxin by E. coli Extraction of Shiga-like toxins Results Isolation and identification of E. coli Preliminary confirmation of Shiga-like toxin-producing E. coli Confirmation of Shiga-like toxin-producing E. coli Characterization of Shiga-like toxin Antibiotic susceptibility test Discussion Conclusion Acknowledgements Ethical Considerations Competing Interests Availability of Data and Material Funding References