|Year : 2012 | Volume
| Issue : 3 | Page : 308-313
Identification of serotypes and virulence markers of Escherichia coli isolated from human stool and urine samples in Egypt
KM Osman1, AM Mustafa2, M Elhariri1, GS AbdElhamed3
1 Department of Microbiology, Cairo University, Egypt
2 Center of Excellence in Biotechnology, King Saud University, Kingdom of Saudi Arabia
3 Sigma Pharmaceutical Company, Egypt
|Date of Submission||20-Jan-2012|
|Date of Acceptance||04-Apr-2012|
|Date of Web Publication||8-Aug-2012|
K M Osman
Department of Microbiology, Cairo University
Purpose: Haemorrhagic colitis and haemolytic-uremic syndrome are associated with Shiga-toxin producing Escherichia coli (STEC). There are others DEC (Diarrhoeagenic E. coli) pathotypes responsible for outbreaks and others toxins associated to these. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include E. coli haemolysin (hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. Materials and Methods: In this study, the PCR assay was used to detect 12 E. coli genes associated with virulence (stx1, stx2, hylA, Flic h7 , stb, F41, K99, sta, F17, LT-I, LT-II and eaeA). Results: A total of 108 E. coli strains were serotyped into 64 typable strains. The investigated strains from the stool, 8/80 (10%) strains were O 164:K, while the 56/110 strains isolated from the urine were O126:K71 (44/110, 40%) and O 86:K 61 (12/110, 11%). The distribution pattern of the detected virulence genes was observed to be in the following order: F17 (10% from the stool and 44% from the urine), Sta (10% from the stool), hylA (10% from the stool and 44% from the urine), Stb (44% from the urine) and stx1 (27% from the urine). The 8 faecal strains encoded a combination of the F17, Sta and hylA genes, while the 56 urine strains encoded a combination of the F17 0+ Stb + hylA (44/110, 40%) and Stx1 only (12/60, 20%). Conclusion: This is the first report on the molecular characterization of E. coli diarrhoeagenic strains in Egypt and the first report on the potential role of E. coli in diarrhoea and urinary tract infections in a localized geographic area where the people engage in various occupational activities.
Keywords: E. coli virulence markers, human diarrhoea, pathogenic E. coli, urine
|How to cite this article:|
Osman K M, Mustafa A M, Elhariri M, AbdElhamed G S. Identification of serotypes and virulence markers of Escherichia coli isolated from human stool and urine samples in Egypt. Indian J Med Microbiol 2012;30:308-13
|How to cite this URL:|
Osman K M, Mustafa A M, Elhariri M, AbdElhamed G S. Identification of serotypes and virulence markers of Escherichia coli isolated from human stool and urine samples in Egypt. Indian J Med Microbiol [serial online] 2012 [cited 2013 May 19];30:308-13. Available from: http://www.ijmm.org/text.asp?2012/30/3/308/99492
| ~ Introduction|| |
Escherichia More Details coli is a genetically heterogeneous group of bacteria whose members are typically nonpathogens and are a part of the normal microflora of the intestinal tract of humans and animals. Among the six recognized diarrhoeagenic categories of E. coli [enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), and enteroaggregative E. coli (EAEC); diffuse adhering E. coli (DAEC)] ETEC is the most common, particularly in the developing world. 
While both commensal and uropathogenic E. coli (UPEC) strains colonize the large intestines of humans, only UPEC strains are primarily selected for growth in the urinary tract.  E. coli strains have evolved to cause a variety of human diseases including sepsis, meningitis, diarrhoea and urinary tract infections (UTIs).  These organisms are serotypically diverse, spanning over 250 serotypes based on O, H and K antigens.  Strains of E. coli associated with infections of the urinary tract are referred to as UPEC strains and are a subset of strains called extraintestinal pathogenic E. coli strains, which cause UTI, sepsis and meningitis.  As with other organisms, UPEC strains possess an arsenal of virulence factors that specifically contribute to their ability to cause disease in the human urinary tract.  Genes encoding haemolysin, and fimbriae for example, have been identified . Information on the epidemiology of E. coli intestinal pathogens and their association with diarrhoeal disease and UTIs is limited because no diagnostic testing is available in countries with limited resources.
The present study was an endeavour aimed to characterize and compare pathogenic E. coli strains isolated in a restricted geographic area which has diversity in occupational activities in Cairo from the stool and urine in relation to serotypes and several genotypic virulence markers. The genes stx1, stx2, hylA, Flic h7 , stb, F41, K99, sta, F17, LT-I, LT-II and eaeA were tested by PCR. 
| ~ Materials and Methods|| |
Sampling was established at the University of Cairo, Faculty of Medicine, Abou ElReesh Hospital during the period June 2008 - March 2009 following ethical approval from the Faculty of Medicine, Cairo University, Research Ethics Committee. Due to the large number of patients who come to the hospitals for evaluation of diarrhoea, the study enrolment was on patients (n0 = 80) of various ages and sexes affected with diarrhoea. In this study, we made the assumption that children with severe gastroenteritis would be taken to hospital outpatient clinics more regularly than children with mild or moderate forms of the disease. After informed consent was obtained, a physical examination was performed and a detailed questionnaire that collected information on the present illness along with the patient's history of diarrhoeal diseases and selected demographic information was completed. The urine of patients (n = 110) of various ages and sexes with uncomplicated human urinary tract infections (UTIs) was also collected. Consecutive consenting patients who met the following criteria were included: (i) clinical and laboratory evidence of uncomplicated cystitis (ii) no antimicrobial treatment within 21 days of presentation; (iii) E. coli as the sole urine microorganism and (iv) E. coli isolated from the rectal swab.
Rectal swabs were directly plated on Chromogenic agar (BBL CHROMagar O157 (CHROM, Becton Dickinson, USA) and incubated at 37°C overnight (O157:H7 appear as mauve cells, while non-O157 VTEC typically present as either pink or blue colonies). Colonies typical of E. coli were streaked onto eosin methylene blue (EMB) agar (Becton Dickinson, Sparks, MD, USA) which were incubated at 37°C for 18 to 20 h. Colonies with a metallic sheen on EMB agar were picked and streaked onto MacConkey agar (Becton Dickinson). After 24 to 48 h of incubation (37°C), as many as 30 isolated typical pink colonies (as available) of putative E. coli were randomly picked from each MacConkey agar plate and the isolates were tested by the indole, methyl red, Voges-Proskauer and Simmons citrate (IMVic) tests for confirmation of E. coli. The E. coli isolates were stored in 5% glycerol-supplemented broth at −70°C until use.
Urine culture was done using chromogenic agar (BBL CHROMagar O157 (CHROM, Becton Dickinson, USA), followed by conventional identification. E. coli isolates were stored in 5% glycerol in Trypticase soy broth (TSB, Cat. No. 257107 Becton, Dickinson and Company, USA) at −70°C until use.
E. coli strains were screened by using a variety of screening methods as described below.
E. coli strains were biochemically confirmed and submitted to slide agglutination tests using polyvalent and monovalent sera against serogroups O26, O25, O86, O111, O78, O119, O126, O127, O164, O157 and O158. Commercially available antisera in the Central Laboratories of Ministry of Public Health, Egypt, were used.
Vero cell assay
Preparation of cell-free culture filtrates
E. coli isolates were grown in brain heart infusion broth for 8 h at 41°C. Then 5 ml of each isolate were subcultured into 50 ml Casamino acid-yeast extract-salts (CAYE) medium (2% Casamino acids/0.6% yeast extract/43 mM NaCl/38 mM K 2 HPO 4 /0.25% glucose/0.1% (vol/vol) trace salts solution consisting of 203 mM MgSO 4 /25 mM MnCl 2 /18 mM FeCl 3 ). The cells were allowed to grow aerobically at 37°C. After 18-20 h, the cells were removed by centrifugation at 12,000 × g for 15 min at 4°C. The cell pellet was resuspended in 0.5 ml sterile phosphate buffered saline (PBS; 0.1 M, pH 7.2). The supernatant was filter-sterilized by using 0.22-μm filters (Millipore, USA). The cells were ultrasonically disrupted continuously for 2 min in an ice bath using a sonicator (Tomy Seiko Co, Ltd, Tokyo, Japan), which was again centrifuged to remove debris. Supernatant was filter-sterilized. Both the culture supernatant and cell lysate were used for the assay.
Vero cell assay
The cytotoxic effect of the strains was assayed on Vero cells in 96-well flat-bottom tissue culture plates (NUNC, Intermade, Denmark), as previously described.  The cells were observed microscopically for 72 h and the cytotoxicity titres determined; the highest toxin dilution that caused lysis of 50% of the cell monolayer was taken as the titre.
Haemolytic activity of the strains was investigated by streaking the strains on tryptic soy agar (Difco, USA) plates containing 5% washed and unwashed O group human blood cells. The haemolytic activity was observed from 3 to 18 h of incubation at 37°C. 
Congo red dye uptake
The ability to take up Congo red dye was determined on agar plates supplemented with 50 mg/ml of Congo red dye. Five microlitres of each suspension was streaked onto the plates and incubated at 37°C for 24 h. Orange colonies were considered positive, different intensities in the dye uptake were expressed as +, ++ and +++.
Detection of E. coli virulence determinants
Twelve virulence determinants (stx1, stx2, hylA, Flic h7 , stb, F41, K99, sta, F17, LT-I, LT-II and eaeA) from E. coli isolates in this study were detected by PCR. These targets were part of a large set of virulence genes described by Osman et al.  Reference strains used were E. coli ATCC35150 (O157:H7, stx1, stx2, eae, hly) and S. aureus ATCC29737 (negative control).
Bacterial strains grown overnight in nutrient broth (Sigma Chemical Co, St Louis, MO, USA) at 37°C were pelleted by centrifugation at 1200 g for 10 min. The pellet was resuspended in 250 μl of sterile distilled water, and the bacteria lysed by boiling for 10 min. The lysate was centrifuged as before and 200 μl of the supernatant were used directly as the template for the PCR.  DNA samples were dissolved in Tris-EDTA buffer (10 mM Tris, 1 mM EDTA at pH 8.0), and the DNA concentration was determined in micrograms per millilitre at an optical density reading of A260 . The template DNA concentration used was 200 ng/ml.
PCR screening was only analysed for the typable isolates. A total of 64 E. coli isolates were subjected to PCR, performed in an Eppendorf Mastercycler (Ependorf AG, Hamburg, Germany). stx1, stx2, hylA, Flic h7 , stb, F41, K99, sta, F17, LT-I, LT-II and eaeA genes were detected using the primers and PCR conditions described in [Table 1]. An initial denaturation step at 95°C for 3 min (stx1, stx2, hylA, Flic h7 , stb, F41, K99, sta, LT-I, LT-II and eaeA genes) and 94°C for 5 min (F17) was carried out. The primer mixtures were prepared with slight modification according to the instructions supplied with the AmpliTaq kit (Applied Biosystems, Foster City, California, USA). The amplified DNA products were separated by electrophoresis on a 1·5% agarose gel, stained with ethidium bromide and detected under ultraviolet light.
|Table 1: Virulence factor targets and primers, including nucleotide sequences, reference, and PCR conditions|
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| ~ Results|| |
E. coli were isolated in 8 of 80 collected faecal samples (10%) and in 100 of 110 urine samples (91%). Among the 100 E. coli strains analysed, the somatic antigen (serogroup) belonged to 3 O serogroups: O 164 from the stool (8/80, 10%) and O86 (12/110, 11%) and O126 (44/110, 40%) from the urine. The 64 isolates contained genes encoding for serotypes O 164:K-, O126:K71 and O86:K61, which are frequently associated with human infections.
Congo red assay was used as a phenotypic marker for the invasive and non-invasive E. coli. In the present study, all of the 110 tested strains for the CR-binding affinities were 100% positive. The binding activity of the CR dye was found to be variable in their affinity according to their serotypes. The isolates were not able to produce any cytopathic effect on the Vero cells although they were 100% haemolytic. The 64 serogrouped isolates were submitted to PCR to detect stx1, stx2, hylA, Flic h7, eae, F41, K99, Sta, Stb, F17, LT-I and LT-II genes.
The genes that were not detected included eaeA, stx2, F41, K99, LT-I, LT-II and Flic h7. The distribution of E. coli serotypes, virulence genes, gene association and combinations and virulence genes encoding serotypes in pathogenic E. coli recovered from human stool and urine are recorded in [Table 2].
|Table 2: The Distribution of E. coli serovars, virulence genes, gene association and combinations and virulence genes encoding serotypes in pathogenic E. coli recovered from human stool and urine|
Click here to view
The distribution pattern of the detected virulence genes was observed to be in the following order: F17 (10% from the stool and 44% from the urine), Sta (10% from the stool), hylA (10% from the stool and 44% from the urine), Stb (44% from the urine) and stx1 (27% from the urine). The rest of the assayed genes were not detected eaeA, stx2, F41, K99, LT-I, LT-II and Flic h7.
The faecal isolates carried the F17 + Sta + hylA genes in conjunction (8/80, 10%). Twelve of the urine isolates were positive for the stx1 gene (12/44, 27%), while 44/110 isolates (40%) were detected to carry the F 17 , Stb and hylA genes in combination.
The genes, F17 + hylA + Sta encoded serogroup O164:K- (8/80 from the stool; 10%). The urine isolates revealed that the gene stx1 encoded O126:K71 (12/44, 27%); F 17 , Stb and hylA encoded O126:K71 (36/44, 82%); and F 17 , Stb and hylA encoded serogroup O86:K61 (12/56, 21.4%).
| ~ Discussion|| |
Diarrhoea is not a specific disease and is by far the most common medical problem among developed tropical and subtropical countries. Pathogenic E. coli is one of the most common causes of acute watery diarrhoea among children and adults in the developing world, causing ≈400 million diarrhoeal episodes and 380,000 deaths in children <5 years of age every year  Many strains of diarrhoeagenic E coli (DEC) primarily affect developing nations due to inadequate sanitary conditions. Statistics on the prevalence of the strains vary by location and surveillance activity. The incidences of diarrhoeal illnesses caused by the different categories of DEC were examined mainly in Latin America, Africa, southern and Southeast Asia, and the Middle East , . Globally, studies have shown that DEC in nontravellers are not fixed and may vary by geographic region of residence, and factors that make the host more vulnerable such as occupation and increased ingestion of contaminated food may change over time. ,, The observed differences between recovery rates from the stools of diarrhoeal patients in developing countries indicates that there are important regional differences in the prevalence of the DEC. ,
It is interesting to indicate that the prevalent serogroups O86, O126 and O164 found in our study were also isolated from a Maasai community in Kenya 
The diarrhoea caused by ETEC strains is mediated by one or two plasmid-encoded enterotoxins; the heat-stable toxin (ST) and/or the heat-labile toxin (LT).  Approximately one third of all pathogenic E. coli strains isolated globally reportedly produce ST only, one-third produce LT and ST, and one-third produce LT only  The significance of E. coli as an enteric pathogen has been underestimated, because the virulence factors of DEC have not been evaluated. This report could be important for future studies of the epidemiology of DEC in Egypt.
In the present investigation, the virulent nature of E. coli was determined by Congo red binding on agar plate, where highly virulent strains showed orange/pink colour colonies, suggesting that plasmid DNA and its associated virulence factors are intact, whereas white colour colonies indicate non-virulent bacteria. It has been reported that the ability of E. coli strains to bind Congo red is affected by the levels of curli fibres expressed at the bacterial cell surface  Kai-Larsen et al.  provided evidence that curli are present on E. coli in fresh urine of infected patients. Curli are highly aggressive surface fibres assembled by E. coli. Curli belong to a class of fibres known as amyloids  and are involved in adhesion to surfaces, cell aggregation and, finally, biofilm development formed by bacteria from the family Enterobacteriaceae. Uropathogenic E. coli forms biofilm-like structures on and inside host cells and the ability to form biofilms has been related to persistence of bacteria in the urinary tract.  As with other organisms, uropathogenic E. coli (UPEC) strains possess an arsenal of virulence factors that specifically contribute to their ability to cause disease in the human urinary tract. E. coli strains producing fimbriae of the F17 family and haemolysin (hly) cause intestinal and extraintestinal disease in animals and humans were found in all of the isolates tested  Analysis of the virulence markers in our study indicated that the non-O157 faecal and urine strains carried the F17 and hylA genes in common.
The toxin genes stx1, stx2, LT and ST are the main pathogenic elements of STEC and ETEC strains. These strains are intestinal E. coli and cause diarrhoea in infected individuals. This study revealed a high prevalence of non-O157 ST-harbouring E. coli in Egypt and is contrary to the reports from Brazil, Ethiopia, Argentina, Arizona, New Caledonia and Bangladesh  where a much lower incidence was reported.
Stx toxin-producing E. coli have rarely been isolated from the urine samples. However, the results obtained by Nazemi et al.  found that, 1% was carrying both stx1 and stx2 genes. The present study is the first report of detecting stx1 gene among E. coli isolates from UTI. In the present investigation, the stx-PCR-positive strain which was VCA - (serotype O126:K71, [Table 2]) was classified as a strain carrying non-expressed stx genes. This suggests that these strains did not produce Stx encoded by the stx gene that might have been undetectable by PCR stx screening as previously indicated  A paradoxical finding in our study was that the stx1-harbouring strain did not demonstrate the production of Stx in the VCA cytotoxicity. A similar observation was encountered in previous studies of stx1 strains isolated from faecal samples 
It is widely accepted that the source of E. coli causing most UTIs is the colonic flora of affected individuals  . A plausible route of transmission for any bacterium colonizing the gut is a faecal-oral one. If the faecal-oral route is a possible means by which urovirulent E. coli come to inhabit the human intestine, it would seem important to determine what food might serve as their vehicle. Speculation has long existed regarding a food-borne origin for ExPEC strains as a candidate vehicle based on the findings of others , that show transmission of avian E. coli from poultry to humans or similarities between avian E. coli and uropathogenic E. coli (UPEC). Interpretation of our results must be tempered by the fact that a limited sample of human faecal and UPEC, all isolated from the same geographic location, was examined in this study. Principally, pathogenic E. coli is transmitted through the consumption of contaminated foods such as raw or undercooked ground meat products and raw milk. , Yet, humans may acquire pathogenic E. coli infections from other sources, possibly vegetables, fruit juice and contaminated drinking water. 
Geographic factors play a role where contamination rates appeared to be higher in an area more than another. Outpatient visitors to Faculty of Medicine, Abou ElReesh Hospital, where the strains were obtained and subjected to analyses in the present investigation, are people who engage in various occupational activities. Therefore, the potential sources of contamination vary. Kasr AlAini area is about 4 k sq area and is a very busy commercial area where a wide diversity of activities, and which are abundant in the area, in the form of health-care facilities, microbiological research laboratories, diagnostic laboratories, blood banks, medical and dental clinics, commercial clinical laboratories, hospitals, restaurants, grocery stores, butcheries, pet animal shops, restaurants, dairies, abattoirs, cafeterias, fresh juice shops, fresh fruit and vegetable retail shops and shops selling live poultry, which always generated a wide variety of waste components.
Occupational exposure to food retail shops (vegetables and fruit) has been shown to increase risk of infection with E. coli, and are subject to major risk factor for illness. Personnel who prepare, transport, store and serve food handler reusable shopping bags grocery bags, plastic bags poses a serious threat to public health, especially from coliform bacteria including E. coli [Charles Gerba, http://www.consumeraffairs.com/news04/2010/06/grocery_bags.html#ixzz1BfYvnIty]
The occupational and public health risks associated with the components of the solid waste stream (these types of waste are referred to as health-care waste or HCW) have not been well assessed. Most exposures to biological hazards from health-care wastes occur at waste treatment facilities or other locations where workers manually handle untreated waste in the cases of sewage overflow from the sewers in the area.
In conclusion, our study is the first molecular study to demonstrate virulence markers carrying at least one virulence-related property affecting the virulence of DEC from faeces and urine in Egypt. Serogrouping-based diagnosis of DEC should be restricted to those serogroups that are most likely to be associated with virulent strains (e.g. O86, O126, O164, etc.). The microbiological laboratories in Egypt only test for serotypes to detect DEC, the development of widely available tests for DEC, including molecular diagnostic methods must be implemented. Furthermore, national basic research studies of the epidemiology of DEC in Egypt must include not just the city area but also the outer urban and rural areas. We recommend greater collaboration with veterinary specialists schooled in population medicine, zoonosis prevention and control, food hygiene, animal production and agriculturists.
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[Table 1], [Table 2]