Characterization of non-O157 enterohemorrhagic 
Most E. coli strains are harmless . By acquiring mobile genetic elements, through horizontal gene transfer, normally harmless E. coli can rapidly transform into a pathogen with a high adaptive capacity. Besides the prominent role of E. coli to be a contributor to intestinal diarrheal diseases, the majority of pathogenic isolates also result in extra-intestinal illnesses which are known as extraintestinal pathogenic E. coli (ExPEC) . They can also lead to more than two million annual fatalities . Feces and wastewater treatment of plants can both release infectious E. coli into the environment . Raw milk and cheese can be a significant source of hazardous pathogenic E. coli to people . There are nine different pathotypes of Diarrheagenic E. coli strains, including enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely-adhering E. coli (DAEC), Shiga toxin-producing E. coli (STEC), adherent-invasive E. coli (AIEC), and cell-detaching E.coli (CDEC) . The STEC are pathogenic bacteria that infect people through contaminated food and water. Shiga toxins (stx) are produced during STEC infections. Infection outcomes are determined by an array of strain and host variable and symptoms can range from mild to severe bloody diarrhoea, with HUS being a possible consequence. The EHEC is a prominent category of STEC that can produce either one or more stx which make up the main virulence characteristic of this pathogroup of E. coli . This pathotype causes a variety of infections, from barely detectable diarrhea to more serious manifestations such as the HC and the emergence of the potentially fatal HUS. EHEC infections are the leading factor of children’s acute renal failure in many countries, making kids and infants the most vulnerable patients . Whereas not all STEC induce HC or HUS, EHEC is generally reserved for those that do . The serotype O157:H7 is regarded as the prototype of this pathogenic group as it was the initial cause the HC and HUS cases in the 80 s and since then it has become a contributor to many outbreaks across the globe . While O157:H7 is the most prominent EHEC serotype, non-O157 EHEC strains are now recognized as hazardous causes of disease. O26, O103, O111, and O145 are the most prevalent non-O157 O-serogroups in Germany, accounting for one-third of EHEC outbreaks including severe diarrhea and life-threatening HUS . These serogroups' detection rate has increased worldwide . This increase may have been caused by increased awareness among labs or the creation of improved detection procedures, however, it may have been a result of the greater prevalence of infections with these pathogens that are linked to human and animal disease . Toxin production, the formation of biofilm, iron acquisition systems, serum resistance ability, capsules, and adhesins are just a few of the many virulence factors that E. coli possesses . These factors encourage tissue colonization, cause damage, and promote the spread of diseases. Due to these virulence traits, the microorganism can colonize anatomical environments, override host defense mechanisms, and start causing an inflammatory response in the host . Shiga toxin 1 and 2, intimin, and enterohemolysin play vital roles in the O157 EHEC pathogenic mechanism . The genus Escherichia is represented by a wide range of species including E. coli , E. albertii , E. fergusonii , and five cryptic Escherichia clades (I–V) . Because of the nucleotide identity identified between clade I and E. coli strains, Escherichia clade I should be regarded as a subspecies of E. coli . Based on the most recent phylogenetic assortment, E. coli strains have been assembled into eight phylogenetic classes that are A, B1, B2, C, D, E, F, and clade I . Previous studies have shown that nonpathogenic strains belonged to A and B1 phylogenetic groups, meanwhile pathogenic ones belonged to B2 and D phylogenetic groups  and . As a result, phylogenetic clustering of E. coli strains is useful for displaying the relationship between phylotypes and diseases induced by the organism. Determining the source of infection and the specific types of pathogens using various approaches such as molecular typing is a necessity to investigate the prevalence of hospitalized infections . Moreover, accurate and quick molecular detection techniques are required to recognize pathogenic E. coli in food and animals in order to improve food safety and human health, as well as to minimize geographical extent of outbreaks . Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique serves as DNA fingerprinting tool for assessing bacterial clonal variability . As an outcome, ERIC-PCR was applied to examine the genetic similarity of EHEC isolates obtained from diverse sources. This also allows for the evaluation of the variable possible contamination sources . Traditional molecular typing techniques such as phage typing, antibiotic resistance patterns analysis and plasmid profiling might be labor intensive, complex, difficult to distinguish between strains with high genetic similarity and are poorly transportable because they index variation that is hard to compare across laboratories. As a result, in epidemiological investigations, these approaches are not ideal for determining the source of infection . To solve these issues, sequence-based typing methods have become the gold standard for epidemiological monitoring to study microbial population genetics. Multi-locus Sequence Typing (MLST) is a typing method that is widely used for clinically relevant bacterial species, and online databases have been developed, allowing data to be analyzed simply . The MLST technique generates an allelic profile by analyzing the sequences of seven housekeeping genes. The produced allelic profile is summarized by the assignment of a sequence type in an electronic database. The closely related species are categorized into clonal complexes . Bearing in mind the possibility of transmission of pathogenic E. coli to humans through consumption of contaminated food, this study aims to detect the prevalence of non-O157 EHEC isolates among different clinical, food and sewage sources in Egypt. In addition, monitoring of various virulence determinants is important to detect the potential of pathogenesis of these emerging strains.   Specimens’ collection During the period from November 2018 to April 2020, a total of 118 clinical samples were collected, including 41 and 77 isolates from urine and stool, respectively and a total of 217 samples from different food sources including 93 animal product (meat, luncheon, smoked turkey, beef burger, ground beef and pastrami), 89 dairy products such as (cheese, yogurt and raw milk), 11 Fresh vegetables isolates (colored pepper, lemon and tomatoes), 15 chicken isolates (cooked and roasted), two fish isolates and seven sewage water isolates (supplementary Table 1). Urine and stool samples were obtained from Mansoura university hospitals and private medical analysis laboratories from separate human cases after a written informed consent from the participant. The experimental protocol used in this research adheres to the ethical guidelines and principles of care, use, and handling of human subjects in medical research established by "The Research Ethics Committee, Faculty of Pharmacy, Mansoura University, Egypt" (code: 2022–126), which is governed by the World Medical Association's Code of Ethics (Declaration of Helsinki). Food samples (meat and dairy products) were obtained from one hundred eighty-two butchers’ shops and supermarkets, chicken isolates were obtained from fifteen poultry shops and fish isolates from two separate fish shop in Mansoura and Damietta cities, Egypt. Sewage isolates were obtained from sewage water from different places in Mansoura and Damietta cities. The fecal, meat, food samples were collected in sterile plastic containers and the milk, yogurt, sewage water and urine samples were collected in sterile falcons. Each individual sample was kept cool in icebox and transferred directly to the Microbiology and Immunology laboratory at the Faculty of Pharmacy, Mansoura university and stored at 4 °C till use; all samples were processed as fast as possible to avoid deterioration and contamination of samples. Isolation and identification of E. coli The collected specimens were cultivated for isolation of E. coli in nutrient broth media that contain yeast extract (0.2%), peptone (0.5%), sodium chloride (0.5%) and incubated for 24 h at 37 °C. Then, appropriate inoculum subjected to subsequent isolation on MacConkey's agar selective media plates which were incubated at 37 °C for 24 h. All plates were then examined for colony morphology. The suspected bacterial colonies that were identified by observation of lactose fermentation on MacConkey`s agar media and negative Gram staining were picked up and inoculated for further confirmation on Eosin methylene blue (EMB) agar plates . Colonies with the characteristic green metallic sheen on EMB agar were subjected to additional confirmation using biochemical tests . Food and sewage water samples were isolated according to Dickson et al. 1995 , where food samples (approximately 5 g) and liquid samples about 5 mL were homogenized before isolation and suspended in ratio of 1:10 in peptone buffered saline and incubated with shaking at 37ºC for 24–48h, then plating 100 µL of each agitated sample onto MacConkey's agar plates. Produced colonies were sub-cultured into nutrient agar slant and were subjected to various biochemical tests intended for conventional identification of E. coli . Detection of O-serogroups The confirmed E. coli isolates were submitted to Center of food analysis, Faculty of Veterinary Medicine, Benha university, Egypt. The serotyping was performed using sets of E. coli antisera for rapid diagnosis (Denka Seiken Co., Japan) . Phenotypic identification of some virulence determinants of EHEC isolates Biofilm assay using microtiter plate method Formation of biofilm was determined quantitatively using a 96 well flat-bottomed polystyrene microtiter plate . A pure colony from an overnight culture streaked on tryptic soy agar (TSA) (Sigma) was sub-cultured into 5 mL tryptic soy broth (TSB) (Sigma) supplemented with 0.25% anhydrous glucose for 24 h at 37 °C. Aliquots of 200 µL of pre-adjusted culture (OD 600 = 0.25) were placed in four adjacent wells, and a negative control containing only TSB was included. After incubation of the plate at 37 °C without shaking for 18–24 h, the contents of the wells were aspirated and rinsed three times with 200µL PBS (pH = 7.4) to eliminate any non-adherent cells. The adherent cells were fixed using 150µL of absolute methanol for 15 min, then left to dry. The fixed biofilm was then stained with 150µL of 1% w/v crystal violet for 20 min. Following that, the plate was rinsed three times with distilled water and dried in the air. The stained biofilm was resolubilized by adding 150µL of (33% v/v) glacial acetic acid per well, and the OD of the tested isolates was determined at 490 nm using a microtiter plate reader. The mean of the four ODs for each isolate was calculated. The capacity of biofilm formation was assessed according to Schönborn et al., 2017 . Briefly, cut-off optical density (ODc) was set as three standard deviations above the mean OD of the negative control. Strains were categorized as: non-biofilm producer, NBP (OD ≤ ODc); weak biofilm producer, WBP (ODc < OD WBP ≤ 2 × ODc); moderate biofilm producer, MBP (2 × ODc < OD MBP ≤ 4 × ODc) and strong biofilm producer, SBP (OD SBP > 4 × ODc). Swimming motility assay Motility evaluation of EHEC isolates was performed according to Murinda et al., 2002, shields and Cathcart, 2012 . Shortly, the bacterial culture was tested for motility using triphenyltetrazolium chloride (TTC) media. After 24–48 h of incubation at 37 °C, growth was indicated by the appearance of red color. As motility occurred, red color was visible surrounding the inoculation area. Hemagglutination assay All isolates were tested for their ability to agglutinate human erythrocytes using slide agglutination technique  using human red blood cells (RBCs) (O-type) obtained from Blood Bank Gastroenterology Hospital, Mansoura university. Isolates were inoculated in nutrient broth media and incubated at 37°C for 48 h. Suspension of RBCs was prepared by washing of human RBCs (O-type) three times with physiological saline (0.9% NaCl) then, resuspended in saline to final conc of 3%. One drop of the bacterial culture was blended with one drop of the RBC suspension on a clean glass slide, and the mixture was vigorously stirred by a sterile tip to promote agglutination. The presence of erythrocyte clumping within 5 min of mixing was considered a positive agglutination result. Screening of hemolysin activity EHEC isolates' hemolytic activity was determined by streaking onto 5% blood agar plates. Plates were evaluated after incubation for 24 h at 37 °C . Hemolysin production was determined quantitatively according to Rossignol et al., 2008 . All EHEC isolates were cultured in TSB at 37 °C for 24 h with shaking and bacterial extract was obtained by centrifugation at 10,000 rpm for 10 min. A mixture of 500µL RBC suspension (2% O-type RBCs in 10 mM Tris HCl, pH 7.4) and 500µL bacterial extract previously prepared according to Rossignol was incubated for 2 h at 37 °C. A positive control (T) using 500µL 0.1% sodium dodecyl sulphate (SDS) and a negative control (B) using 500µL TSB with 500µL RBC suspension were included. After centrifugation at 10,000 rpm for 10 min, the amount of hemoglobin liberated in each sample (X) was measured at 540 nm. Three replicates were performed for each isolate. Hemolysis percentage was determined as follow: [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$\%= ((-)/(-))  100$$ Hemolysis % = ( ( X - B ) / ( T - B ) ) × 100 Serum resistance assay Serum resistance of the strains was analyzed, as described by Vandekerchove et al., 2005 using a turbidimetric assay . In a 96-well microtiter plate, 50 μL of bacterial culture (OD600 = 0.1) was combined with 150 μL of normal human serum obtained from Blood Bank Gastroenterology Hospital, Mansoura university. Using a microplate reader, the initial and final absorbance (after 3 h of incubation at 37 °C) were measured at 600 nm. The absorbance of each isolate was estimated by taking the average of three replicates. The percentage of remaining absorbance relative to the initial absorbance was calculated. The strain is classified as serum resistant (SR) if the remaining absorbance after 3 h was higher than 150%, intermediate resistant (IR) if it was between 125 and 150%, slow-intermediate resistant (S-I) if percent was between 100 and 125% and classified as serum sensitive (SS) if it was less than 100%. Molecular detection of some virulence genes Genomic DNA extraction The DNA templates were extracted from tested isolates by boiling method previously reported by Said et al., 2018 . Concisely, purified E. coli colonies were suspended in 100 μL of sterile DNase free water in 0.2 mL sterile PCR tube and subjected to heat block in thermocycler at 95 °C for 10 min, followed by centrifugation at 10,000 rpm for 3 min and stored in −20°C. Supernatant served as DNA template for all the following PCR sets. Screening of some toxin genes by PCR Shiga toxin I (stx1), Shiga toxin II (stx2), intimin (eae) and hemolysin (ehxA, ehlyA, hlyA, ehlyA and sheA) genes were screened among the tested isolates using PCR. The reaction mixture of a total volume 25 μL consisted of 12.5 μL master mix (DreamTaq Green master), 2 μL of template DNA, 0.5µL from each primer listed in supplementary Table 2 (Invitrogen TM, UK) and the final volume completed with DNase free water. stx gene subtyping Subtyping of stx1 and stx2 genes was carried out as previously reported  using primer pairs listed in supplementary Table 2. Amplification was carried out using a DNA thermocycler with a predetermined cycling condition: initial denaturation at 94 °C for 5 min followed by 35 cycles each consisted of denaturation at 94 °C for 40 s, annealing at the temperature as stated in supplementary Table 2 and extension at 72 °C for 1 min, then final extension at 72 °C for 10 min. The amplified products were electrophoresed on 1.25% agarose gel stained with ethidium bromide and photographed under ultraviolet light (UV) light and compared with a DNA marker (Gene Ruler 100 bp, Thermo Fisher Scientific Tm, UK) to detect their sizes. Clermont’s phylogenetic typing Phylotyping of EHEC isolates was performed as previously described by Clermont et al., 2013 . Amplicons were electrophoresed using 1.25% agarose gel stained with ethidium bromide and photographed under UV light. Phylotypes of EHEC isolates were assigned as: A, B1, B2, C, D, E, F, and Clade I. Molecular typing by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) Molecular genotyping of isolates was performed using (ERIC-PCR) using specific primers (supplementary Table 2) . A total reaction of 25 µL consisted of 12.5µL of Dream Taq™ Green PCR Master Mix (2x), 0.5 µL of ERIC-1 (10 µM), 0.5 µL of ERIC-2 (10 µM), 8.5 µL of nuclease-free water and 3µL of template DNA. Amplification was conducted with the following conditions: initial denaturing at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 40 s annealing at 48 °C for 1 min, extension at 72 °C for 1.5 min, then the reaction was terminated by a final extension of 72 °C for 10 min. The amplified DNA fragments were separated on 2% agarose gel and compared with 100 bp plus DNA marker (Thermo Fisher Scientific™, UK) Cat. No. (SM0323). Following this, the gel was examined and evaluated using gelJ software. A similarity matrix was generated using Dice's coefficient and subsequently, the corresponding dendrogram was created utilizing the unweighted-pair group method with arithmetic averages (UPGMA) . Multi locus sequence typing (MLST) Purification of PCR products Amplification of MLST genes was performed using the previously described protocol  using the primers listed in (supplementary Table 2). PCR reaction was performed in a 100 μL reaction mixture containing 12 μL of the template DNA, 50 μL of Dream Taq™ Green PCR master mix (2X), each of the forward and reverse primer 2 μL and 34 μL of nuclease-free water. The standard cycling procedure was performed following the conditions: initial denaturation for 2 min at 95 °C, 35 cycles of denaturation at 95 °C for 1 min, annealing at variable temperature stated in (supplementary Table 2) for 30 s, extension at 72 °C for 2 min, and final extension at 72 °C for 7 min. The PCR products were analyzed by gel electrophoresis on 1.25% agarose gel stained with ethidium bromide and photographed under UV light, then the products with the target size were precisely cut from the gel and purified using Gene Jet Gel Extraction Kit (Thermo Scientific™). DNA sequencing The purified PCR products of the MLST were sequenced according to the protocol of applied biosystems big dye terminator v3.1 DNA sequencing reaction at Colors Medical Lab, Cairo, Egypt. Nucleotide sequences of the housekeeping genes were submitted to the E. coli MLST Database ( https://pubmlst.org/bigsdb?db=pubmlst_escherichia_seqdef ), and the EnteroBase Database ( https://enterobase.warwick.ac.uk/species/ecoli/allele_st_search ) to determine the sequence types and clonal complex. Statistical analysis and data interpretation The statistical analysis of the data was performed using GraphPad Prism (version 5.01), which involved the application of the chi-square test and Fisher's exact test, and Monte Carlo test. The significance of the obtained results was judged at p -value < 0.05.   During the period from November 2018 to April 2020, a total of 118 clinical samples were collected, including 41 and 77 isolates from urine and stool, respectively and a total of 217 samples from different food sources including 93 animal product (meat, luncheon, smoked turkey, beef burger, ground beef and pastrami), 89 dairy products such as (cheese, yogurt and raw milk), 11 Fresh vegetables isolates (colored pepper, lemon and tomatoes), 15 chicken isolates (cooked and roasted), two fish isolates and seven sewage water isolates (supplementary Table 1). Urine and stool samples were obtained from Mansoura university hospitals and private medical analysis laboratories from separate human cases after a written informed consent from the participant. The experimental protocol used in this research adheres to the ethical guidelines and principles of care, use, and handling of human subjects in medical research established by "The Research Ethics Committee, Faculty of Pharmacy, Mansoura University, Egypt" (code: 2022–126), which is governed by the World Medical Association's Code of Ethics (Declaration of Helsinki). Food samples (meat and dairy products) were obtained from one hundred eighty-two butchers’ shops and supermarkets, chicken isolates were obtained from fifteen poultry shops and fish isolates from two separate fish shop in Mansoura and Damietta cities, Egypt. Sewage isolates were obtained from sewage water from different places in Mansoura and Damietta cities. The fecal, meat, food samples were collected in sterile plastic containers and the milk, yogurt, sewage water and urine samples were collected in sterile falcons. Each individual sample was kept cool in icebox and transferred directly to the Microbiology and Immunology laboratory at the Faculty of Pharmacy, Mansoura university and stored at 4 °C till use; all samples were processed as fast as possible to avoid deterioration and contamination of samples.   E. coli The collected specimens were cultivated for isolation of E. coli in nutrient broth media that contain yeast extract (0.2%), peptone (0.5%), sodium chloride (0.5%) and incubated for 24 h at 37 °C. Then, appropriate inoculum subjected to subsequent isolation on MacConkey's agar selective media plates which were incubated at 37 °C for 24 h. All plates were then examined for colony morphology. The suspected bacterial colonies that were identified by observation of lactose fermentation on MacConkey`s agar media and negative Gram staining were picked up and inoculated for further confirmation on Eosin methylene blue (EMB) agar plates . Colonies with the characteristic green metallic sheen on EMB agar were subjected to additional confirmation using biochemical tests . Food and sewage water samples were isolated according to Dickson et al. 1995 , where food samples (approximately 5 g) and liquid samples about 5 mL were homogenized before isolation and suspended in ratio of 1:10 in peptone buffered saline and incubated with shaking at 37ºC for 24–48h, then plating 100 µL of each agitated sample onto MacConkey's agar plates. Produced colonies were sub-cultured into nutrient agar slant and were subjected to various biochemical tests intended for conventional identification of E. coli .   The confirmed E. coli isolates were submitted to Center of food analysis, Faculty of Veterinary Medicine, Benha university, Egypt. The serotyping was performed using sets of E. coli antisera for rapid diagnosis (Denka Seiken Co., Japan) .   Biofilm assay using microtiter plate method Formation of biofilm was determined quantitatively using a 96 well flat-bottomed polystyrene microtiter plate . A pure colony from an overnight culture streaked on tryptic soy agar (TSA) (Sigma) was sub-cultured into 5 mL tryptic soy broth (TSB) (Sigma) supplemented with 0.25% anhydrous glucose for 24 h at 37 °C. Aliquots of 200 µL of pre-adjusted culture (OD 600 = 0.25) were placed in four adjacent wells, and a negative control containing only TSB was included. After incubation of the plate at 37 °C without shaking for 18–24 h, the contents of the wells were aspirated and rinsed three times with 200µL PBS (pH = 7.4) to eliminate any non-adherent cells. The adherent cells were fixed using 150µL of absolute methanol for 15 min, then left to dry. The fixed biofilm was then stained with 150µL of 1% w/v crystal violet for 20 min. Following that, the plate was rinsed three times with distilled water and dried in the air. The stained biofilm was resolubilized by adding 150µL of (33% v/v) glacial acetic acid per well, and the OD of the tested isolates was determined at 490 nm using a microtiter plate reader. The mean of the four ODs for each isolate was calculated. The capacity of biofilm formation was assessed according to Schönborn et al., 2017 . Briefly, cut-off optical density (ODc) was set as three standard deviations above the mean OD of the negative control. Strains were categorized as: non-biofilm producer, NBP (OD ≤ ODc); weak biofilm producer, WBP (ODc < OD WBP ≤ 2 × ODc); moderate biofilm producer, MBP (2 × ODc < OD MBP ≤ 4 × ODc) and strong biofilm producer, SBP (OD SBP > 4 × ODc). Swimming motility assay Motility evaluation of EHEC isolates was performed according to Murinda et al., 2002, shields and Cathcart, 2012 . Shortly, the bacterial culture was tested for motility using triphenyltetrazolium chloride (TTC) media. After 24–48 h of incubation at 37 °C, growth was indicated by the appearance of red color. As motility occurred, red color was visible surrounding the inoculation area. Hemagglutination assay All isolates were tested for their ability to agglutinate human erythrocytes using slide agglutination technique  using human red blood cells (RBCs) (O-type) obtained from Blood Bank Gastroenterology Hospital, Mansoura university. Isolates were inoculated in nutrient broth media and incubated at 37°C for 48 h. Suspension of RBCs was prepared by washing of human RBCs (O-type) three times with physiological saline (0.9% NaCl) then, resuspended in saline to final conc of 3%. One drop of the bacterial culture was blended with one drop of the RBC suspension on a clean glass slide, and the mixture was vigorously stirred by a sterile tip to promote agglutination. The presence of erythrocyte clumping within 5 min of mixing was considered a positive agglutination result. Screening of hemolysin activity EHEC isolates' hemolytic activity was determined by streaking onto 5% blood agar plates. Plates were evaluated after incubation for 24 h at 37 °C . Hemolysin production was determined quantitatively according to Rossignol et al., 2008 . All EHEC isolates were cultured in TSB at 37 °C for 24 h with shaking and bacterial extract was obtained by centrifugation at 10,000 rpm for 10 min. A mixture of 500µL RBC suspension (2% O-type RBCs in 10 mM Tris HCl, pH 7.4) and 500µL bacterial extract previously prepared according to Rossignol was incubated for 2 h at 37 °C. A positive control (T) using 500µL 0.1% sodium dodecyl sulphate (SDS) and a negative control (B) using 500µL TSB with 500µL RBC suspension were included. After centrifugation at 10,000 rpm for 10 min, the amount of hemoglobin liberated in each sample (X) was measured at 540 nm. Three replicates were performed for each isolate. Hemolysis percentage was determined as follow: [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$\%= ((-)/(-))  100$$ Hemolysis % = ( ( X - B ) / ( T - B ) ) × 100 Serum resistance assay Serum resistance of the strains was analyzed, as described by Vandekerchove et al., 2005 using a turbidimetric assay . In a 96-well microtiter plate, 50 μL of bacterial culture (OD600 = 0.1) was combined with 150 μL of normal human serum obtained from Blood Bank Gastroenterology Hospital, Mansoura university. Using a microplate reader, the initial and final absorbance (after 3 h of incubation at 37 °C) were measured at 600 nm. The absorbance of each isolate was estimated by taking the average of three replicates. The percentage of remaining absorbance relative to the initial absorbance was calculated. The strain is classified as serum resistant (SR) if the remaining absorbance after 3 h was higher than 150%, intermediate resistant (IR) if it was between 125 and 150%, slow-intermediate resistant (S-I) if percent was between 100 and 125% and classified as serum sensitive (SS) if it was less than 100%.   Formation of biofilm was determined quantitatively using a 96 well flat-bottomed polystyrene microtiter plate . A pure colony from an overnight culture streaked on tryptic soy agar (TSA) (Sigma) was sub-cultured into 5 mL tryptic soy broth (TSB) (Sigma) supplemented with 0.25% anhydrous glucose for 24 h at 37 °C. Aliquots of 200 µL of pre-adjusted culture (OD 600 = 0.25) were placed in four adjacent wells, and a negative control containing only TSB was included. After incubation of the plate at 37 °C without shaking for 18–24 h, the contents of the wells were aspirated and rinsed three times with 200µL PBS (pH = 7.4) to eliminate any non-adherent cells. The adherent cells were fixed using 150µL of absolute methanol for 15 min, then left to dry. The fixed biofilm was then stained with 150µL of 1% w/v crystal violet for 20 min. Following that, the plate was rinsed three times with distilled water and dried in the air. The stained biofilm was resolubilized by adding 150µL of (33% v/v) glacial acetic acid per well, and the OD of the tested isolates was determined at 490 nm using a microtiter plate reader. The mean of the four ODs for each isolate was calculated. The capacity of biofilm formation was assessed according to Schönborn et al., 2017 . Briefly, cut-off optical density (ODc) was set as three standard deviations above the mean OD of the negative control. Strains were categorized as: non-biofilm producer, NBP (OD ≤ ODc); weak biofilm producer, WBP (ODc < OD WBP ≤ 2 × ODc); moderate biofilm producer, MBP (2 × ODc < OD MBP ≤ 4 × ODc) and strong biofilm producer, SBP (OD SBP > 4 × ODc).   Motility evaluation of EHEC isolates was performed according to Murinda et al., 2002, shields and Cathcart, 2012 . Shortly, the bacterial culture was tested for motility using triphenyltetrazolium chloride (TTC) media. After 24–48 h of incubation at 37 °C, growth was indicated by the appearance of red color. As motility occurred, red color was visible surrounding the inoculation area.   All isolates were tested for their ability to agglutinate human erythrocytes using slide agglutination technique  using human red blood cells (RBCs) (O-type) obtained from Blood Bank Gastroenterology Hospital, Mansoura university. Isolates were inoculated in nutrient broth media and incubated at 37°C for 48 h. Suspension of RBCs was prepared by washing of human RBCs (O-type) three times with physiological saline (0.9% NaCl) then, resuspended in saline to final conc of 3%. One drop of the bacterial culture was blended with one drop of the RBC suspension on a clean glass slide, and the mixture was vigorously stirred by a sterile tip to promote agglutination. The presence of erythrocyte clumping within 5 min of mixing was considered a positive agglutination result.   EHEC isolates' hemolytic activity was determined by streaking onto 5% blood agar plates. Plates were evaluated after incubation for 24 h at 37 °C . Hemolysin production was determined quantitatively according to Rossignol et al., 2008 . All EHEC isolates were cultured in TSB at 37 °C for 24 h with shaking and bacterial extract was obtained by centrifugation at 10,000 rpm for 10 min. A mixture of 500µL RBC suspension (2% O-type RBCs in 10 mM Tris HCl, pH 7.4) and 500µL bacterial extract previously prepared according to Rossignol was incubated for 2 h at 37 °C. A positive control (T) using 500µL 0.1% sodium dodecyl sulphate (SDS) and a negative control (B) using 500µL TSB with 500µL RBC suspension were included. After centrifugation at 10,000 rpm for 10 min, the amount of hemoglobin liberated in each sample (X) was measured at 540 nm. Three replicates were performed for each isolate. Hemolysis percentage was determined as follow: [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$\%= ((-)/(-))  100$$ Hemolysis % = ( ( X - B ) / ( T - B ) ) × 100   Serum resistance of the strains was analyzed, as described by Vandekerchove et al., 2005 using a turbidimetric assay . In a 96-well microtiter plate, 50 μL of bacterial culture (OD600 = 0.1) was combined with 150 μL of normal human serum obtained from Blood Bank Gastroenterology Hospital, Mansoura university. Using a microplate reader, the initial and final absorbance (after 3 h of incubation at 37 °C) were measured at 600 nm. The absorbance of each isolate was estimated by taking the average of three replicates. The percentage of remaining absorbance relative to the initial absorbance was calculated. The strain is classified as serum resistant (SR) if the remaining absorbance after 3 h was higher than 150%, intermediate resistant (IR) if it was between 125 and 150%, slow-intermediate resistant (S-I) if percent was between 100 and 125% and classified as serum sensitive (SS) if it was less than 100%.   Genomic DNA extraction The DNA templates were extracted from tested isolates by boiling method previously reported by Said et al., 2018 . Concisely, purified E. coli colonies were suspended in 100 μL of sterile DNase free water in 0.2 mL sterile PCR tube and subjected to heat block in thermocycler at 95 °C for 10 min, followed by centrifugation at 10,000 rpm for 3 min and stored in −20°C. Supernatant served as DNA template for all the following PCR sets. Screening of some toxin genes by PCR Shiga toxin I (stx1), Shiga toxin II (stx2), intimin (eae) and hemolysin (ehxA, ehlyA, hlyA, ehlyA and sheA) genes were screened among the tested isolates using PCR. The reaction mixture of a total volume 25 μL consisted of 12.5 μL master mix (DreamTaq Green master), 2 μL of template DNA, 0.5µL from each primer listed in supplementary Table 2 (Invitrogen TM, UK) and the final volume completed with DNase free water. stx gene subtyping Subtyping of stx1 and stx2 genes was carried out as previously reported  using primer pairs listed in supplementary Table 2. Amplification was carried out using a DNA thermocycler with a predetermined cycling condition: initial denaturation at 94 °C for 5 min followed by 35 cycles each consisted of denaturation at 94 °C for 40 s, annealing at the temperature as stated in supplementary Table 2 and extension at 72 °C for 1 min, then final extension at 72 °C for 10 min. The amplified products were electrophoresed on 1.25% agarose gel stained with ethidium bromide and photographed under ultraviolet light (UV) light and compared with a DNA marker (Gene Ruler 100 bp, Thermo Fisher Scientific Tm, UK) to detect their sizes. Clermont’s phylogenetic typing Phylotyping of EHEC isolates was performed as previously described by Clermont et al., 2013 . Amplicons were electrophoresed using 1.25% agarose gel stained with ethidium bromide and photographed under UV light. Phylotypes of EHEC isolates were assigned as: A, B1, B2, C, D, E, F, and Clade I. Molecular typing by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) Molecular genotyping of isolates was performed using (ERIC-PCR) using specific primers (supplementary Table 2) . A total reaction of 25 µL consisted of 12.5µL of Dream Taq™ Green PCR Master Mix (2x), 0.5 µL of ERIC-1 (10 µM), 0.5 µL of ERIC-2 (10 µM), 8.5 µL of nuclease-free water and 3µL of template DNA. Amplification was conducted with the following conditions: initial denaturing at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 40 s annealing at 48 °C for 1 min, extension at 72 °C for 1.5 min, then the reaction was terminated by a final extension of 72 °C for 10 min. The amplified DNA fragments were separated on 2% agarose gel and compared with 100 bp plus DNA marker (Thermo Fisher Scientific™, UK) Cat. No. (SM0323). Following this, the gel was examined and evaluated using gelJ software. A similarity matrix was generated using Dice's coefficient and subsequently, the corresponding dendrogram was created utilizing the unweighted-pair group method with arithmetic averages (UPGMA) .   The DNA templates were extracted from tested isolates by boiling method previously reported by Said et al., 2018 . Concisely, purified E. coli colonies were suspended in 100 μL of sterile DNase free water in 0.2 mL sterile PCR tube and subjected to heat block in thermocycler at 95 °C for 10 min, followed by centrifugation at 10,000 rpm for 3 min and stored in −20°C. Supernatant served as DNA template for all the following PCR sets.   Shiga toxin I (stx1), Shiga toxin II (stx2), intimin (eae) and hemolysin (ehxA, ehlyA, hlyA, ehlyA and sheA) genes were screened among the tested isolates using PCR. The reaction mixture of a total volume 25 μL consisted of 12.5 μL master mix (DreamTaq Green master), 2 μL of template DNA, 0.5µL from each primer listed in supplementary Table 2 (Invitrogen TM, UK) and the final volume completed with DNase free water.   gene subtyping Subtyping of stx1 and stx2 genes was carried out as previously reported  using primer pairs listed in supplementary Table 2. Amplification was carried out using a DNA thermocycler with a predetermined cycling condition: initial denaturation at 94 °C for 5 min followed by 35 cycles each consisted of denaturation at 94 °C for 40 s, annealing at the temperature as stated in supplementary Table 2 and extension at 72 °C for 1 min, then final extension at 72 °C for 10 min. The amplified products were electrophoresed on 1.25% agarose gel stained with ethidium bromide and photographed under ultraviolet light (UV) light and compared with a DNA marker (Gene Ruler 100 bp, Thermo Fisher Scientific Tm, UK) to detect their sizes.   Phylotyping of EHEC isolates was performed as previously described by Clermont et al., 2013 . Amplicons were electrophoresed using 1.25% agarose gel stained with ethidium bromide and photographed under UV light. Phylotypes of EHEC isolates were assigned as: A, B1, B2, C, D, E, F, and Clade I.   Molecular genotyping of isolates was performed using (ERIC-PCR) using specific primers (supplementary Table 2) . A total reaction of 25 µL consisted of 12.5µL of Dream Taq™ Green PCR Master Mix (2x), 0.5 µL of ERIC-1 (10 µM), 0.5 µL of ERIC-2 (10 µM), 8.5 µL of nuclease-free water and 3µL of template DNA. Amplification was conducted with the following conditions: initial denaturing at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 40 s annealing at 48 °C for 1 min, extension at 72 °C for 1.5 min, then the reaction was terminated by a final extension of 72 °C for 10 min. The amplified DNA fragments were separated on 2% agarose gel and compared with 100 bp plus DNA marker (Thermo Fisher Scientific™, UK) Cat. No. (SM0323). Following this, the gel was examined and evaluated using gelJ software. A similarity matrix was generated using Dice's coefficient and subsequently, the corresponding dendrogram was created utilizing the unweighted-pair group method with arithmetic averages (UPGMA) .   Purification of PCR products Amplification of MLST genes was performed using the previously described protocol  using the primers listed in (supplementary Table 2). PCR reaction was performed in a 100 μL reaction mixture containing 12 μL of the template DNA, 50 μL of Dream Taq™ Green PCR master mix (2X), each of the forward and reverse primer 2 μL and 34 μL of nuclease-free water. The standard cycling procedure was performed following the conditions: initial denaturation for 2 min at 95 °C, 35 cycles of denaturation at 95 °C for 1 min, annealing at variable temperature stated in (supplementary Table 2) for 30 s, extension at 72 °C for 2 min, and final extension at 72 °C for 7 min. The PCR products were analyzed by gel electrophoresis on 1.25% agarose gel stained with ethidium bromide and photographed under UV light, then the products with the target size were precisely cut from the gel and purified using Gene Jet Gel Extraction Kit (Thermo Scientific™). DNA sequencing The purified PCR products of the MLST were sequenced according to the protocol of applied biosystems big dye terminator v3.1 DNA sequencing reaction at Colors Medical Lab, Cairo, Egypt. Nucleotide sequences of the housekeeping genes were submitted to the E. coli MLST Database ( https://pubmlst.org/bigsdb?db=pubmlst_escherichia_seqdef ), and the EnteroBase Database ( https://enterobase.warwick.ac.uk/species/ecoli/allele_st_search ) to determine the sequence types and clonal complex. Statistical analysis and data interpretation The statistical analysis of the data was performed using GraphPad Prism (version 5.01), which involved the application of the chi-square test and Fisher's exact test, and Monte Carlo test. The significance of the obtained results was judged at p -value < 0.05.   Amplification of MLST genes was performed using the previously described protocol  using the primers listed in (supplementary Table 2). PCR reaction was performed in a 100 μL reaction mixture containing 12 μL of the template DNA, 50 μL of Dream Taq™ Green PCR master mix (2X), each of the forward and reverse primer 2 μL and 34 μL of nuclease-free water. The standard cycling procedure was performed following the conditions: initial denaturation for 2 min at 95 °C, 35 cycles of denaturation at 95 °C for 1 min, annealing at variable temperature stated in (supplementary Table 2) for 30 s, extension at 72 °C for 2 min, and final extension at 72 °C for 7 min. The PCR products were analyzed by gel electrophoresis on 1.25% agarose gel stained with ethidium bromide and photographed under UV light, then the products with the target size were precisely cut from the gel and purified using Gene Jet Gel Extraction Kit (Thermo Scientific™).   The purified PCR products of the MLST were sequenced according to the protocol of applied biosystems big dye terminator v3.1 DNA sequencing reaction at Colors Medical Lab, Cairo, Egypt. Nucleotide sequences of the housekeeping genes were submitted to the E. coli MLST Database ( https://pubmlst.org/bigsdb?db=pubmlst_escherichia_seqdef ), and the EnteroBase Database ( https://enterobase.warwick.ac.uk/species/ecoli/allele_st_search ) to determine the sequence types and clonal complex.   The statistical analysis of the data was performed using GraphPad Prism (version 5.01), which involved the application of the chi-square test and Fisher's exact test, and Monte Carlo test. The significance of the obtained results was judged at p -value < 0.05.   Isolation, identification, and serotyping of EHEC Out of the 335 clinical, food and sewage water specimens collected, 105 (31%) isolates of E. coli were identified. The prevalence of the identified isolates among sources was as follows: 19/41 human urine, 46/77 human stool, 23/81 cheese, 2/6 yogurt, 2/2 raw milk, 7/27 cattle meat, 1/5 beef burger, 1/3 pastrami, 1/15chicken, 1/2 fish, 2/7 sewage water. No E. coli isolates were obtained from vegetables, sausage, or luncheon. Serological typing classified the isolated E. coli into four pathotypes. They were distributed as 31 EHEC, 19 EPEC, 11 ETEC, 4 EIEC among clinical isolates and 21 EHEC, 9 EPEC, 8 ETEC, 2 EIEC among food and sewage water isolates. Thus, 52 (49.5%) of the identified isolates were assigned as EHEC which were subjected to further investigation throughout the study. The prevalence of EHEC among E. coli isolated from urine and stool was 57.9% and 43.5%, respectively. Concerning food isolates, it was detected among 62.5% and 52.17% of E. coli isolated from meat and cheese, respectively. Also, all E. coli isolated from yogurt and sewage water were EHEC. Among the detected 52 EHEC isolates, nine serotypes were identified. O111: H2 (23%) was the highest detected serotype followed by O91: H21 (21.2%) and O26: H11(19.2%). Moreover, other serotypes; O55: H7 (11.5%), O117:H4 (9.6%), O126: H21 (9.6%), O113: H4 (1.92%), O121: H7 (1.92%) and O103: H4 (1.92%); were detected. No significant difference was noticed between clinical, food and sewage water isolates concerning the distribution of serotypes (Table ). Phenotypic identification of virulence determinants among EHEC isolates Biofilm formation capacity Biofilm formation capacity was observed in 51/52 (98%) of EHEC isolates. Concerning clinical isolates, 30/31 (96.77%) isolates formed biofilm. These isolates were classified as strong (1, 3.3% isolate), moderate (5, 16.66% isolates), weak (24, 80% isolates) – biofilm producers. All food and sewage water EHEC isolates were biofilm producers. They were classified equally into strong, moderate, and weak—biofilm producers (7, 33.3% isolates each) (Table ). Statistical analysis has revealed that strong biofilm formation capacity was significantly higher among EHEC from food and sewage water sources than from clinical isolates ( P = 0.003) while weak biofilm formation capacity was significantly higher among clinical isolates ( P = 0.001). Phenotypic screening of motility, blood agglutination, hemolysin, and serum resistance Regarding motility testing, 46/52 (88.5%) EHEC isolates showed growth diffusion with red color appeared around the stab line representing motility. While all clinical EHEC isolates were significantly motile, only 71% of food and sewage water EHEC isolates were motile ( P = 0.001). Noticeably, our results revealed that all non-motile isolates were obtained from food sources. For blood agglutination, we found that 18/52 (34.6%) isolates had the ability to agglutinate human RBCs within 5 min of vigorous stirring (Table ). Blood agglutination was more observed among clinical isolates [14 (45%)] as compared to food and sewage water isolates [4 (19%)]. Hemolysis activity (β-hemolysis) was observed in only one yogurt isolate; the remaining 51 isolates (98%) were non-hemolytic (Table ). Among the 52 EHEC isolates, 48 (92.3%) isolates were serum resistant. 29 (93.5%) clinical isolates showed serum resistance [6 (20.69%) serum resistant, 5 (17.24%) intermediate, 18 (62.07%) slow intermediates. Serum resistance was detected in 19 (90.5%) of food and sewage water isolates [7 (36.84%) serum resistant, 2 (10.53%) intermediate, and 10 (52.63%) slow intermediates]. No significant difference was noticed between clinical, food and sewage water EHEC isolates regarding blood agglutination, hemolysis, and serum resistance (Table ). Molecular detection of some virulence genes Molecular detection of stx1, stx2, eae and hemolysin genes All EHEC isolates were tested for toxin associated genes by PCR technique and were shown in (supplementary Fig. 1). Shiga toxin I ( stx1 ) gene was harbored by only two clinical isolates. Subtyping of the two stx1 positive isolates showed that both were of stx 1a subtype. No stx 1c or stx 1d subtypes were detected. Shiga toxin II ( stx2 ) gene was more prevalent than stx1 gene as it was detected among 39/52 (75%) isolates. Stx2 positive clinical isolates (29/31,93.5%) were significantly more abundant than stx2 positive food and sewage water isolates (10/21, 47.6%) ( P = 0.001). Regarding stx positive subtypes, three subtypes were obtained: stx2b , stx2d and stx2g. Among the 39 stx2 positive isolates, stx2g was the highest detected subtype (17/39, 43.59%) followed by stx2b (8/39, 20.5%). stx2d was the least detected subtype (2/39, 5.13%) . None of the isolates were subtyped as stx2a , stx2c , stx2e or stx2f. Worth mentioning, five isolates harbored a combination of stx2b and stx2g subtypes simultaneously. Seventeen of stx2 positive isolates could not be assigned to any of the tested subtypes. Significant difference in the prevalence of stx2g and un- subtyped isolates was found at ( P = 0.019) and ( P = 0.0008), respectively between isolates from different sources (Table ). Additionally, eae gene was detected in 19/52 (36%) isolates. They were distributed as follows: 12/31 (39%) of clinical isolates and 7/21(33%) of food isolates, respectively. Ten out of 20 (50%) EHEC urine isolates and 2/11 (18%) EHEC stool isolates were eae positive. Regarding food isolates, we found that dairy products harbored eae gene more frequently than meat product where 6/14 (42.8%), 1/5 (20%) of dairy and meat products isolates, respectively were eae positive. Hemolysin genes were detected among 47/52 (90%) EHEC isolates. Silent hemolysin ( sheA ) gene was detected in 44/52 (84.6%) isolates. The detection rate was higher among clinical isolates (28/31,90.3%) than food and sewage water isolates (16/21, 76.2%). While α hemolysin ( hlyA ) gene was harbored by only 4/52 (7.7%) isolates which were of clinical origin. Enterohemolysin-a ( ehlyA ) and enterohemolysin-x ( ehxA ) genes were not detected in any isolate (Table ). Virulence genes detected among EHEC isolates were represented in supplementary Fig. 1. Virulence genes profile Gene profiles and their distribution among EHEC isolates were investigated. Fifteen different gene combinations of the tested virulence genes were obtained among the 52 EHEC (Table ). The most prevalent profile was stx2, sheA (10/52,19.2%) followed by stx2, stx2g, sheA, eae (6/52, 11.5%) and stx2, sheA, eae and stx2, stx2g, sheA (5/52, 9.6% each). Nine profiles were unique as they were detected in only one isolate each. One meat product isolate and one sewage water isolate did not harbor any of the tested genes. Clinical isolates showed higher diversity as they revealed 14 different profiles, while only nine profiles were shown by food and sewage water isolates. Phylogenetic groups Phylogenetic typing, based on Clermont’s typing, showed diversity among EHEC isolates. The tested isolates were assigned into seven phylogroups. B1 and C were the predominant phylogroups (15/52,28.8% each) followed by A and D (5/52, 9.6% each), B2 and E (3/52,5.77% each). Only two (3.8%) isolates were assigned to F phylogroup. The distribution of the detected phylogroups among EHEC isolates revealed no correlation between the phylotypes of isolates and their source p > 0.05 (Table ). Genotyping by using ERIC-PCR The tested EHEC isolates were genotyped into 46 different patterns using ERIC-PCR. The largest pattern (P27) comprised four isolates that were obtained from food sources including cheese and meat samples. The clonal relationship between EHEC isolates was analyzed using GelJ software and UPGMA clustering analysis (Fig. ). Results revealed that the majority of isolates had shown similarity of > 70%. The obtained dendrogram clustered the isolates into sixteen clusters. In addition to a group of four (EM1, EM2, EC2 and EC5) and another pair (EC6 and EC9) of food isolates, two pairs of clinical isolates; (CS14 and CS15) and (CU5 and CS3); showed 100% similarity. Multiple sequence analysis of the seven house-keeping genes The analysis of each gene among the three selected isolates is shown in supplementary Table 3. Allele profiles, sequence typing and clonal complex of the selected EHEC isolates The allelic number, the corresponding sequence type and clonal complex were designated by using MLST website. Our results had shown that the three isolates were assigned to different three sequence types (ST) depending on a specific combination of the allele numbers determined automatically using the Pubmlst website, where EC9 was assigned to ST120, CS9 was assigned to ST394 of clonal complex (CC) ST394 Cplx and CU11 was assigned to ST70 (supplementary Table 3). Association between isolates, phylogroups, and phenotypic & genotypic characters Correlation matrix and hierarchical clustering with heat map (Fig. ) were utilized to detect associations between the phenotypic & genotypic features and origin of the isolates to find any potential correlation between the isolates. Heat map classified isolates into two clusters comprising 20 patterns. Of our 52 EHEC isolates, 21 isolates showed 70% similarity.9 groups of isolates showed identical patterns in several traits that are: (EM2, EW2), (CU2, CU3, CU4, CU5, CU6, CS17, CS18, CS20, EM1 and EM4), (EC1, EW1), (EC3, EC5, EM3), (CS2, CS4, CS6, CS8), (CU11, CS3), (CS1, CS10, CS19), (CS5, CS9, CS12) and (EC9, EC11).   Out of the 335 clinical, food and sewage water specimens collected, 105 (31%) isolates of E. coli were identified. The prevalence of the identified isolates among sources was as follows: 19/41 human urine, 46/77 human stool, 23/81 cheese, 2/6 yogurt, 2/2 raw milk, 7/27 cattle meat, 1/5 beef burger, 1/3 pastrami, 1/15chicken, 1/2 fish, 2/7 sewage water. No E. coli isolates were obtained from vegetables, sausage, or luncheon. Serological typing classified the isolated E. coli into four pathotypes. They were distributed as 31 EHEC, 19 EPEC, 11 ETEC, 4 EIEC among clinical isolates and 21 EHEC, 9 EPEC, 8 ETEC, 2 EIEC among food and sewage water isolates. Thus, 52 (49.5%) of the identified isolates were assigned as EHEC which were subjected to further investigation throughout the study. The prevalence of EHEC among E. coli isolated from urine and stool was 57.9% and 43.5%, respectively. Concerning food isolates, it was detected among 62.5% and 52.17% of E. coli isolated from meat and cheese, respectively. Also, all E. coli isolated from yogurt and sewage water were EHEC. Among the detected 52 EHEC isolates, nine serotypes were identified. O111: H2 (23%) was the highest detected serotype followed by O91: H21 (21.2%) and O26: H11(19.2%). Moreover, other serotypes; O55: H7 (11.5%), O117:H4 (9.6%), O126: H21 (9.6%), O113: H4 (1.92%), O121: H7 (1.92%) and O103: H4 (1.92%); were detected. No significant difference was noticed between clinical, food and sewage water isolates concerning the distribution of serotypes (Table ).   Biofilm formation capacity Biofilm formation capacity was observed in 51/52 (98%) of EHEC isolates. Concerning clinical isolates, 30/31 (96.77%) isolates formed biofilm. These isolates were classified as strong (1, 3.3% isolate), moderate (5, 16.66% isolates), weak (24, 80% isolates) – biofilm producers. All food and sewage water EHEC isolates were biofilm producers. They were classified equally into strong, moderate, and weak—biofilm producers (7, 33.3% isolates each) (Table ). Statistical analysis has revealed that strong biofilm formation capacity was significantly higher among EHEC from food and sewage water sources than from clinical isolates ( P = 0.003) while weak biofilm formation capacity was significantly higher among clinical isolates ( P = 0.001). Phenotypic screening of motility, blood agglutination, hemolysin, and serum resistance Regarding motility testing, 46/52 (88.5%) EHEC isolates showed growth diffusion with red color appeared around the stab line representing motility. While all clinical EHEC isolates were significantly motile, only 71% of food and sewage water EHEC isolates were motile ( P = 0.001). Noticeably, our results revealed that all non-motile isolates were obtained from food sources. For blood agglutination, we found that 18/52 (34.6%) isolates had the ability to agglutinate human RBCs within 5 min of vigorous stirring (Table ). Blood agglutination was more observed among clinical isolates [14 (45%)] as compared to food and sewage water isolates [4 (19%)]. Hemolysis activity (β-hemolysis) was observed in only one yogurt isolate; the remaining 51 isolates (98%) were non-hemolytic (Table ). Among the 52 EHEC isolates, 48 (92.3%) isolates were serum resistant. 29 (93.5%) clinical isolates showed serum resistance [6 (20.69%) serum resistant, 5 (17.24%) intermediate, 18 (62.07%) slow intermediates. Serum resistance was detected in 19 (90.5%) of food and sewage water isolates [7 (36.84%) serum resistant, 2 (10.53%) intermediate, and 10 (52.63%) slow intermediates]. No significant difference was noticed between clinical, food and sewage water EHEC isolates regarding blood agglutination, hemolysis, and serum resistance (Table ).   Biofilm formation capacity was observed in 51/52 (98%) of EHEC isolates. Concerning clinical isolates, 30/31 (96.77%) isolates formed biofilm. These isolates were classified as strong (1, 3.3% isolate), moderate (5, 16.66% isolates), weak (24, 80% isolates) – biofilm producers. All food and sewage water EHEC isolates were biofilm producers. They were classified equally into strong, moderate, and weak—biofilm producers (7, 33.3% isolates each) (Table ). Statistical analysis has revealed that strong biofilm formation capacity was significantly higher among EHEC from food and sewage water sources than from clinical isolates ( P = 0.003) while weak biofilm formation capacity was significantly higher among clinical isolates ( P = 0.001).   Regarding motility testing, 46/52 (88.5%) EHEC isolates showed growth diffusion with red color appeared around the stab line representing motility. While all clinical EHEC isolates were significantly motile, only 71% of food and sewage water EHEC isolates were motile ( P = 0.001). Noticeably, our results revealed that all non-motile isolates were obtained from food sources. For blood agglutination, we found that 18/52 (34.6%) isolates had the ability to agglutinate human RBCs within 5 min of vigorous stirring (Table ). Blood agglutination was more observed among clinical isolates [14 (45%)] as compared to food and sewage water isolates [4 (19%)]. Hemolysis activity (β-hemolysis) was observed in only one yogurt isolate; the remaining 51 isolates (98%) were non-hemolytic (Table ). Among the 52 EHEC isolates, 48 (92.3%) isolates were serum resistant. 29 (93.5%) clinical isolates showed serum resistance [6 (20.69%) serum resistant, 5 (17.24%) intermediate, 18 (62.07%) slow intermediates. Serum resistance was detected in 19 (90.5%) of food and sewage water isolates [7 (36.84%) serum resistant, 2 (10.53%) intermediate, and 10 (52.63%) slow intermediates]. No significant difference was noticed between clinical, food and sewage water EHEC isolates regarding blood agglutination, hemolysis, and serum resistance (Table ).   Molecular detection of stx1, stx2, eae and hemolysin genes All EHEC isolates were tested for toxin associated genes by PCR technique and were shown in (supplementary Fig. 1). Shiga toxin I ( stx1 ) gene was harbored by only two clinical isolates. Subtyping of the two stx1 positive isolates showed that both were of stx 1a subtype. No stx 1c or stx 1d subtypes were detected. Shiga toxin II ( stx2 ) gene was more prevalent than stx1 gene as it was detected among 39/52 (75%) isolates. Stx2 positive clinical isolates (29/31,93.5%) were significantly more abundant than stx2 positive food and sewage water isolates (10/21, 47.6%) ( P = 0.001). Regarding stx positive subtypes, three subtypes were obtained: stx2b , stx2d and stx2g. Among the 39 stx2 positive isolates, stx2g was the highest detected subtype (17/39, 43.59%) followed by stx2b (8/39, 20.5%). stx2d was the least detected subtype (2/39, 5.13%) . None of the isolates were subtyped as stx2a , stx2c , stx2e or stx2f. Worth mentioning, five isolates harbored a combination of stx2b and stx2g subtypes simultaneously. Seventeen of stx2 positive isolates could not be assigned to any of the tested subtypes. Significant difference in the prevalence of stx2g and un- subtyped isolates was found at ( P = 0.019) and ( P = 0.0008), respectively between isolates from different sources (Table ). Additionally, eae gene was detected in 19/52 (36%) isolates. They were distributed as follows: 12/31 (39%) of clinical isolates and 7/21(33%) of food isolates, respectively. Ten out of 20 (50%) EHEC urine isolates and 2/11 (18%) EHEC stool isolates were eae positive. Regarding food isolates, we found that dairy products harbored eae gene more frequently than meat product where 6/14 (42.8%), 1/5 (20%) of dairy and meat products isolates, respectively were eae positive. Hemolysin genes were detected among 47/52 (90%) EHEC isolates. Silent hemolysin ( sheA ) gene was detected in 44/52 (84.6%) isolates. The detection rate was higher among clinical isolates (28/31,90.3%) than food and sewage water isolates (16/21, 76.2%). While α hemolysin ( hlyA ) gene was harbored by only 4/52 (7.7%) isolates which were of clinical origin. Enterohemolysin-a ( ehlyA ) and enterohemolysin-x ( ehxA ) genes were not detected in any isolate (Table ). Virulence genes detected among EHEC isolates were represented in supplementary Fig. 1.   stx1, stx2, eae and hemolysin genes All EHEC isolates were tested for toxin associated genes by PCR technique and were shown in (supplementary Fig. 1). Shiga toxin I ( stx1 ) gene was harbored by only two clinical isolates. Subtyping of the two stx1 positive isolates showed that both were of stx 1a subtype. No stx 1c or stx 1d subtypes were detected. Shiga toxin II ( stx2 ) gene was more prevalent than stx1 gene as it was detected among 39/52 (75%) isolates. Stx2 positive clinical isolates (29/31,93.5%) were significantly more abundant than stx2 positive food and sewage water isolates (10/21, 47.6%) ( P = 0.001). Regarding stx positive subtypes, three subtypes were obtained: stx2b , stx2d and stx2g. Among the 39 stx2 positive isolates, stx2g was the highest detected subtype (17/39, 43.59%) followed by stx2b (8/39, 20.5%). stx2d was the least detected subtype (2/39, 5.13%) . None of the isolates were subtyped as stx2a , stx2c , stx2e or stx2f. Worth mentioning, five isolates harbored a combination of stx2b and stx2g subtypes simultaneously. Seventeen of stx2 positive isolates could not be assigned to any of the tested subtypes. Significant difference in the prevalence of stx2g and un- subtyped isolates was found at ( P = 0.019) and ( P = 0.0008), respectively between isolates from different sources (Table ). Additionally, eae gene was detected in 19/52 (36%) isolates. They were distributed as follows: 12/31 (39%) of clinical isolates and 7/21(33%) of food isolates, respectively. Ten out of 20 (50%) EHEC urine isolates and 2/11 (18%) EHEC stool isolates were eae positive. Regarding food isolates, we found that dairy products harbored eae gene more frequently than meat product where 6/14 (42.8%), 1/5 (20%) of dairy and meat products isolates, respectively were eae positive. Hemolysin genes were detected among 47/52 (90%) EHEC isolates. Silent hemolysin ( sheA ) gene was detected in 44/52 (84.6%) isolates. The detection rate was higher among clinical isolates (28/31,90.3%) than food and sewage water isolates (16/21, 76.2%). While α hemolysin ( hlyA ) gene was harbored by only 4/52 (7.7%) isolates which were of clinical origin. Enterohemolysin-a ( ehlyA ) and enterohemolysin-x ( ehxA ) genes were not detected in any isolate (Table ). Virulence genes detected among EHEC isolates were represented in supplementary Fig. 1.   Gene profiles and their distribution among EHEC isolates were investigated. Fifteen different gene combinations of the tested virulence genes were obtained among the 52 EHEC (Table ). The most prevalent profile was stx2, sheA (10/52,19.2%) followed by stx2, stx2g, sheA, eae (6/52, 11.5%) and stx2, sheA, eae and stx2, stx2g, sheA (5/52, 9.6% each). Nine profiles were unique as they were detected in only one isolate each. One meat product isolate and one sewage water isolate did not harbor any of the tested genes. Clinical isolates showed higher diversity as they revealed 14 different profiles, while only nine profiles were shown by food and sewage water isolates.   Phylogenetic typing, based on Clermont’s typing, showed diversity among EHEC isolates. The tested isolates were assigned into seven phylogroups. B1 and C were the predominant phylogroups (15/52,28.8% each) followed by A and D (5/52, 9.6% each), B2 and E (3/52,5.77% each). Only two (3.8%) isolates were assigned to F phylogroup. The distribution of the detected phylogroups among EHEC isolates revealed no correlation between the phylotypes of isolates and their source p > 0.05 (Table ).   The tested EHEC isolates were genotyped into 46 different patterns using ERIC-PCR. The largest pattern (P27) comprised four isolates that were obtained from food sources including cheese and meat samples. The clonal relationship between EHEC isolates was analyzed using GelJ software and UPGMA clustering analysis (Fig. ). Results revealed that the majority of isolates had shown similarity of > 70%. The obtained dendrogram clustered the isolates into sixteen clusters. In addition to a group of four (EM1, EM2, EC2 and EC5) and another pair (EC6 and EC9) of food isolates, two pairs of clinical isolates; (CS14 and CS15) and (CU5 and CS3); showed 100% similarity.   The analysis of each gene among the three selected isolates is shown in supplementary Table 3. Allele profiles, sequence typing and clonal complex of the selected EHEC isolates The allelic number, the corresponding sequence type and clonal complex were designated by using MLST website. Our results had shown that the three isolates were assigned to different three sequence types (ST) depending on a specific combination of the allele numbers determined automatically using the Pubmlst website, where EC9 was assigned to ST120, CS9 was assigned to ST394 of clonal complex (CC) ST394 Cplx and CU11 was assigned to ST70 (supplementary Table 3). Association between isolates, phylogroups, and phenotypic & genotypic characters Correlation matrix and hierarchical clustering with heat map (Fig. ) were utilized to detect associations between the phenotypic & genotypic features and origin of the isolates to find any potential correlation between the isolates. Heat map classified isolates into two clusters comprising 20 patterns. Of our 52 EHEC isolates, 21 isolates showed 70% similarity.9 groups of isolates showed identical patterns in several traits that are: (EM2, EW2), (CU2, CU3, CU4, CU5, CU6, CS17, CS18, CS20, EM1 and EM4), (EC1, EW1), (EC3, EC5, EM3), (CS2, CS4, CS6, CS8), (CU11, CS3), (CS1, CS10, CS19), (CS5, CS9, CS12) and (EC9, EC11).   The allelic number, the corresponding sequence type and clonal complex were designated by using MLST website. Our results had shown that the three isolates were assigned to different three sequence types (ST) depending on a specific combination of the allele numbers determined automatically using the Pubmlst website, where EC9 was assigned to ST120, CS9 was assigned to ST394 of clonal complex (CC) ST394 Cplx and CU11 was assigned to ST70 (supplementary Table 3).   Correlation matrix and hierarchical clustering with heat map (Fig. ) were utilized to detect associations between the phenotypic & genotypic features and origin of the isolates to find any potential correlation between the isolates. Heat map classified isolates into two clusters comprising 20 patterns. Of our 52 EHEC isolates, 21 isolates showed 70% similarity.9 groups of isolates showed identical patterns in several traits that are: (EM2, EW2), (CU2, CU3, CU4, CU5, CU6, CS17, CS18, CS20, EM1 and EM4), (EC1, EW1), (EC3, EC5, EM3), (CS2, CS4, CS6, CS8), (CU11, CS3), (CS1, CS10, CS19), (CS5, CS9, CS12) and (EC9, EC11).   The emergence of O157 and non-O157 EHEC foodborne pathogens has become a major concern worldwide . They cause serious disease outbreaks and severe illness in humans such as diarrhea, HC and potentially fatal HUS . The great attention to non-O157 EHEC could be related to the increased prevalence of these organisms in human and animal infections, as well as increased public awareness of the danger of infection caused by non-O157 serogroups . Children under the age of five are more vulnerable and at danger of dying from diarrhea caused by E. coli infection . The goal of this study was to explore the prevalence of EHEC among different sources in Egypt and investigate their molecular characteristics. In addition, the distribution of virulence factors among these isolates was also investigated. A total of 52 (49.5%) EHEC isolates had been identified (Table ). The prevalence of EHEC was similar in both clinical (31/65, 47.7%), food and sewage water (21/40, 52.5%) isolates, where none of these isolates was O157. On the other hand, Ahmed et al. (2017) reported that 33% of their EHEC isolates was O157 . In Germany, one-third of non-O157 HUS-causing EHEC was associated with O26, O103, O111, and O145 serogroups . Serogroups O26, O111, O103, and O145 accounted for one-third of human infections other than O157:H7 such as diarrhea and HUS, in Germany and caused global outbreaks . Of these serogroups, O26, O103 and O111 were detected in our study. Similarly, Ahmed et al. (2017) reported the presence of these three serogroups among their EHEC isolates. A high prevalence of EHEC was observed among our urine and stool isolates (Table ), where prevalence in urine was greater than that of stool. Some studies reported variable rates of isolation of EHEC from clinical sources in Iran (30.77%)  and South Africa (0.4%) . Meat products are a substantial cause of human EHEC infections . Rahimi, et al. 2012 reported the presence of EHEC in meat products in Netherland (10.4%), England (13.4%), and Iran (8.2%) . Similarly, EHEC was detected in 14.3% of all meat samples in our study. Interestingly, researchers detected similar rates of EHEC in animal products at 9.5%, . None of our vegetable samples harbored EHEC, despite the detection of EHEC in vegetables from different countries . Raw milk and raw-milk products have been implicated in many diseases and even fatalities . Several countries reported the detection of variable levels of stx gene in their milk products, which is the most crucial virulence for EHEC-associated infections . It was found that 7 out of 12 EHEC cheese isolates were stx gene positive. This finding is alarming and necessitates strict control over the cheese industry to prevent the spread of this organism. Thankfully, yogurt samples that were identified as EHEC among our isolates did not harbor stx gene. E. coli is known to persist in natural environments due to the formation of biofilm . The majority (98.1%) of our non-O157 EHEC isolates were biofilm producers, which is consistent with several previous studies . Strong biofilm production was observed among 3.2% of clinical isolates, where 9% of urine isolates were strong biofilm producers (Table ). A recent study in Egypt, reported that 22.7% of urine isolates were strong biofilm producers . Many bacterial pathogens rely on flagellar motility in the early stages of infection. While Sherfi, et al. 2013 concluded that E. coli motility and indole production are related, only 88.5% of our indole-positive isolates were motile . Hemagglutination (HA) of erythrocytes is believed to be a major virulence in E. coli strains that cause extraintestinal illnesses in human . A higher detection rate of HA was found among clinical isolates (45%) than in food isolates (19%). HA was observed at 60% from our fecal isolates while at 18% from our urine isolates (Table ). It was reported that HA was observed at 25% from urine isolates while at 4% from fecal isolates . Prolonged presence of hemolytic E. coli strains in the host could potentially lead to the onset of extra-intestinal infections , bloodstream infection and sepsis . Beta-hemolytic activity was detected in a yogurt isolate and not detected in clinical isolates (Table ). However, previous studies observed higher percent of hemolytic activity including 42.2%, 25%, 16.8% in E. coli isolates for  and , respectively. Serum resistance characteristic enables E. coli to evade the complement system and increases risk of developing septic shock and mortality . In this study, 92.3% of EHEC isolates showed varying levels of serum resistance (Table ). The ability to harbor stx gene is a trait shared by EHEC/STEC isolates. In our research, stx2 gene (75%) was more prevalent than stx1 (3.8%) and a percent of 3.8% of EHEC isolates harbored both genes (Table ). This result is supported by Sallam et al.,2013 in that stx1 and stx2 genes were found in the isolated EHEC strains in 46.7% and 86.7%, respectively  and by Jajarmi et al., 2017 who reported that stx1 (52%), stx2 (64%) and 16% of EHEC isolates that harbored both genes were detected among their isolates  . The stx1 was found among isolated STEC at low rates 0.16%, 5.2% as reported by Kargar M, Homayoon M. 2015 and Tarazi , et al. 0.2021, respectively  and . It has been reported that STEC strains that produce stx2 gene are more likely to cause HUS and could cause more severe neurological symptoms in piglets than strains producing just stx1 or both stx1 and stx2 , whereas stx1 -producing strains induce only diarrhea without systemic complications . Subtyping of the stx1 + isolates showed that they were of stx 1a subtype. The same finding was reported by Elsayed et al who found that no stx1c was detected in non-o157 E. coli isolates . The stx2 group is composed of stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g subtypes . Our results indicated that stx2g (43.6%) was the most prevalent subtype among stx2 positive isolates followed by stx2b (20.5%) then stx2d (5.1%), while stx2a, stx2c, stx2e and stx2f were not detected among our isolates (Table ). Elsayed et al. , 2021  demonstrated that no stx2a , stx2d , stx2f , or s tx2g subtypes were detected in their isolates. In contrast, Jajarmi et al., identified stx1a (52%), stx2a (44%), stx2c (44%), and stx2d (30%) in selected STEC isolates . Supporting our findings, stx2c have been detected often in isolates from HUS patients, whereas stx2d is often isolated from cases of uncomplicated diarrhea . The stx2e subtype is predominantly isolated from pigs and pork products . Schmidt et al., 2000 came to a conclusion that stx2f appears to be closely related to STEC of bird and pigeons origins . The eae gene is one of the most common pathogenic genes found in the environment  that makes STEC strains more virulent for humans. However, eae-lacking STEC strains caused a minority of sporadic cases of HUS . Several putative non intimin based adhesion has been described, such as long polar fimbriae and STEC auto-agglutinating adhesin in non-O157:H7 STEC . In our study eae gene was detected in 19/52 (36%) isolates. The hemolysin gene that is one of the key pathogenic components of E. coli was found in 92% of our EHEC isolates and its subtypes were detected. Of which, silent hemolysin gene (sheA) was the most prevalent one. Alpha- hemolysin ( hlyA ) gene was detected in 7.7% of clinical isolates only (Table ). Similarly, low prevalence of hlyA gene was reported in 2.25%, 0.9% of cheese and raw milk, respectively . Fifteen different combinations of virulence genes were detected among our 52 EHEC isolates . The most prevalent profile is stx2, sheA (19.2%) followed by stx2, stx2g, sheA, eae (11.5%), stx2, sheA, eae (9.6%) and stx2, stx2g, sheA (9.6%) (Table ). Eklund M, et al . 2002 reported 11 combinations in Finland . Furthermore, we detected stx gene subtypes combinations among clinical isolates only, where four isolates harbored ( stx2b with stx2g) while (stx1a with stx2g) and ( stx1a with stx2b and stx2g) were harbored each by only one isolate, which is similar to Elsayed M, et al.2021 . Various combinations were previously detected . Supporting our findings Dong, et al.2017 reported that stx1d , stx2e, and stx2f were not detected among E. coli isolates . A multiplex PCR system was carried out to classify E. coli strains into phylogenetic groups. Most of our EHEC isolates belonged to non-pathogenic B1 and C phylogroups, and few strains belonged to group E, F (Table ) which is supported by Rúgeles et al . , 2010 . In our EHEC isolates 8/52 belonged to pathogenic B2 and D phylogroups and they were of clinical origin and all harbored stx2 and hemolysin genes while only five of them harbored eae gene. The ERIC-PCR was applied to examine the genetic similarity of EHEC isolates obtained from diverse sources. The isolates were genotyped into forty-six different patterns using ERIC-PCR. Similarity of 100% was observed between many isolates which may be indicative of similar origin of dissemination of similar isolates (Fig. ). Opposing our finding that there was no correlation between ERIC patterns and serotypes as O55:H7 isolates were highly diverged and found in 6 different groups (P1, P6, P12, P18, P19, and P33), Dalla costa et al. reported that related clones like O55:H7 and O157:H7 displayed similar ribotypes and clustered together in a dendrogram . Furthermore, Nicholas Waters et al. demonstrated the relationship between whole-genome phylogeny and the phylotypic classification and concluded that phylotypes and classification at the whole-genome are correlated . On the other hand, our study advocated that there was no correlation between phylogeny and ERIC clusters. The MLST technique is used to study genetic relatedness of isolates, and closely related species can be categorized into clonal complexes . The selection of isolates for MLST was based on using two isolates from clinical source, both belonged to pathogenic phylogroup (D) with one isolate from food source that belonged to non-pathogenic phylogroup (A). The three selected EHEC isolates were classified into three different sequence types demonstrating a clonal complex diversity among the isolates. The EC9 isolated from cheese was assigned to endemic clone ST120 belonging to commensal phylogenetic group A. This endemic clone of ST120 has been previously reported in China . This necessitates immediate action to prevent the spread of this clone. The ST120 strains identified from cormorants were CTX-M-15 producers and belonged to commensal phylogenetic group B1 . Regarding CS9 isolate from stool, it belonged to pathogenic D phylogroup and ST394 (clonal complex: ST394 Cplx). Zahra et al., 2018 detected ST394 from sewage around Pakistan . Alarmingly, ST394 has been associated with sporadic diarrheal outbreaks in various countries . The ST394 that was previously isolated from raw milk belonging to phylogroup D was confirmed to be CTX-M15-producing . Urine isolate (CU11) in our study was assigned to epidemic clone ST70 and belonged to pathogenic D phylogroup. Alarmingly, ST70 was previously detected in the acute intensive care unit, Venezuela indicating that its ability to induce frequent outbreaks . The three detected STs were previously characterized as emerging ESBLs that are capable of degrading expanded-spectrum cephalosporins and monobactams  and resistant to aminoglycosides and fluoroquinolones  as well as resistance to gentamicin and ciprofloxacin  leaving just a few reliable alternative therapies . By comparing results of the three selected isolates by two different typing techniques; ERIC typing method that classified them into 3 variable patterns (P3, P4 and P19) and MLST that classified them into three different sequence types (ST70, ST120, ST394) demonstrating a genetic diversity among the isolates and ensures accuracy of both techniques. Associations between phenotypic, genotypic features and serotypes of isolates (Fig. ) revealed that CU4, CU5 isolates that showed similarity in several traits were obtained from urine and both belonging to pathogenic phylogroup B2 and CS9, CS12 isolates that were obtained from stool showed similarity in several traits are belonging to pathogenic phylogroup D. In conclusion, our study advocated that cheese and meat products represent dangerous threats as source of EHEC infections. Various serotypes were detected among clinical, food and sewage water isolates including: O111: H2, O91: H21, O26: H11, O55: H7, O117: H4, O126: H21, O113: H4, O121: H7 and O103: H4 and this represents a potential risk for public health. It is worrying that 58% of our cheese isolates harbored stx2 gene that is the most important virulence trait for EHEC infections along with 77% of total EHEC isolates having “ stx2 , hemolysin and or eae ” gene combination making them highly virulent. The MLST technique was applied, ST394 detected has been associated with acute and chronic sporadic diarrheal outbreaks in both developed and developing countries, detected epidemic clone ST70 was found to be capable of inducing frequent outbreaks. The endemic ST120 detected in this work was isolated from cheese necessitating critical action to prevent spread of this clone and it is our duty to emphasize the importance of strict control over cheese factories, since they pose the threat of infection with hazardous microorganisms. Our study advocated that pathogenic phylogroups B2 and D were found solely within clinical isolates. With high rate of stx2 gene detection in clinical isolates, representing a major threat for the dissemination of EHEC among hospitals and community-acquired E. coli isolates. Alarmingly, clinical EHEC isolates harbored high virulence genes score and combinations that is causing a public health problem. Our findings might have a substantial influence on the development of preventive strategies for E. coli infections through the identification of potential sources that could serve as a vehicle for the transmission of these pathogenic bacteria.   Supplementary Material 1. Supplementary Material 2. Supplementary Material 3. Supplementary Material 4. Supplementary Material 5.   