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Asian Journal of Dairy and Food Research, volume 40 issue 1 (march 2021) : 1-7

Isolation and Molecular Characterization of Shigatoxigenic O157 and Non-O157 Escherichia coli in Raw Milk Marketed in Chittagong, Bangladesh

Md. Kauser-Ul Alam, Nazmul Sarwar, Shireen Akther, Monsur Ahmad, Paritosh Kumar Biswas
1Department of Food Processing and Engineering, Chittagong Veterinary and Animal Sciences University (CVASU), Chittagong-4225, Bangladesh.
Cite article:- Alam Kauser-Ul Md., Sarwar Nazmul, Akther Shireen, Ahmad Monsur, Biswas Kumar Paritosh (2021). Isolation and Molecular Characterization of Shigatoxigenic O157 and Non-O157 Escherichia coli in Raw Milk Marketed in Chittagong, Bangladesh . Asian Journal of Dairy and Food Research. 40(1): 1-7. doi: 10.18805/ajdfr.DR-178.

ABSTRACT

Background: Quality and microbial safety of milk is demanding day by day as it is considered as a host for pathogenic and spoilage microorganisms. In this study, isolation and molecular characterization of shigatoxigenic O157 and non-O157 Escherichia coli in raw milk marketed in Chittagong, Bangladesh were done on 186 raw milk samples in Bangladesh. 

Methods: MacConkey agar was initially used to screen for the presence of E. coli and the suspected growth as evidenced by large pink colonies on MacConkey agar. Finally the organism was verified by plating through Eosin Methylene Blue (EMB) agar (a selective medium for E. coli where it produces metallic sheen) and applying standard biochemical tests for E. coli. The presence of virulent genes, Shiga-like toxin (stx1 and stx2), Intimin (eaeA), O157 antigen rfbE and Enterohemorrhagic Escherichia coli (EHEC) Hemolysin (EHEC) hlyA in the contaminating E. coli population was determined by polymerase chain reaction (PCR) run on a thermocycler (Applied Biosystem, 2720 thermal cycler, Singapore). 

Result: Among the raw milk samples, 33 samples were identified as E.coli positive and among the isolates, 6 (18.18%) were identified as possible EHEC O157 and rest of the isolates (81.82%) were considered as probable non EHEC O157. About, 3.23% (186 samples) EHEC O157 was isolated from raw milk samples. Then all the 33 isolates were taken under PCR assay for the identification of five virulent genes Stx1, Stx2, eaeA, rfbE and hlyA. No virulent genes were found in non- EHEC O157 isolates, but 4 stx2 (66.67%) and 1 hlyA (16.67%) gene were observed in another 4 EHEC O157 isolates out of 6, but one isolates contained the both genes and hence the prevalence of STEC was 2.15% in raw milk. Result indicated poor hygienic standard of raw milk from uncontrolled environments and the increased public health risk of those consuming raw milk from such uncontrolled sources.

INTRODUCTION

E. coli is a normal inhabitant of the intestines of animals and humans. The most reported human infections are with entero-hemorrhagic E. coli (EHEC O157), asymptomatic reservoirs and excretors of which are ruminants, particularly cattle. Recovery of EHEC O157 from food should be of public health concern because it can lead to severe intestinal and extra intestinal diseases when consumed. Cattle, especially the young ones, have been conceived as a principal reservoir of E. coli O157:H7 (Zhao et al., 1995). Therefore, insufficient heat-treatment of raw milk possess a potential risk for infection (Betts, 2000) while processing conditions are very crucial for the survival of the bacterium in milk.
 
E. coli has acquired specific virulence attributes associated with diarrhoeal disease and extra-intestinal infections (Kaper et al., 2004; Nataro and Kaper, 1998; Russo and Johnson, 2000). Among the intestinal E. coli there are six well-described categories: enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC) (Nataro and Kaper, 1998). The group of extra-intestinal pathogenic E. coli comprises uropathogenic E. coli (UPEC), neonatal meningitis-associated E. coli (NMEC) and sepsis-causing E. coli (SEPEC) (Johnson and Russo, 2002, 2005; Russo and Johnson, 2000; Smith et al., 2007).  Enterohaemorrhagic E. coli (EHEC) groups are verotoxin-producing E. coli (VTEC) that able to induce haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) (Levine, 1987; Griffin and Tauxe, 1991). Report indicate that consumption of raw milk and various milk products related with occurrence of 1 to 5% of food infections and among that 53% of cases produced by enteropathogenic E. coli (EPEC) (Schrade and Yager, 2001).
 
The Stx (verocytotoxin-producing) family contains two subgroups: Stx1 and Stx2 (Paton and Paton, 1998). The production of Stx alone may not be sufficient for VTEC to cause disease (Beutin et al., 1995). Other virulence factors may play a role in VTEC pathogenicity, including intimin (encodedby the eaeA gene), which is required for intimate adherence of these pathogens to tissue culture cells and formation of the attaching and effacing (A/E) lesion (Mckee and O’Brien, 1996). The formation of A/E lesions is mediated by multiple genes called the Locus of Enterocyte Effacement (LEE) (McDaniel et al., 1995). Another virulence factor that contributes to VTEC pathogenicity is the 60-MDa plasmid borne enterohaemolysin A gene (encoded by the E-hlyA gene) (Paton and Paton, 1998). A gene, known as rfbE more specific for the O157 serotype has been identified (Desmarchelier et al., 1998). The various pathotypes of E. coli tend to be clonal groups that are characterized by shared O (lipopolysaccharide, LPS) and H (flagellar) antigens that define Serogroups (O antigen only) or Serotypes (O and H antigens) (Nataro and Kaper, 1998 and Whittam,1996). Serotypes include in EHEC groups are E. coli O157:H7, the non-motile organism E. coli O157:H- and members of other serogroups, particularly O26, O103, O111 and O145 but also O91, O104, O113, O117, O118, O121, O128 and others. The predominant serotype associated with human infection and death is O157:H7 (Levine, 1987; Griffin and Tauxe, 1991). MacConkey agar containing 1% sorbitol (SMAC), often with cefixime and potassium tellurite (CT-SMAC) (Zadik et al., 1993), is frequently used to identify EHEC based on lack of β-glucuronidase activity and the inability of rapid sorbitol fermentation. Plasmid profiling may aid in the genetic characterization and molecular screening of different serotypes of EHEC, e.g; E Coli O157 (Radu et al., 2001) and Non-O157 (Nazmul et al., 2012).
 
There are several milk-based commercial companies in Bangladesh and the demand for safe-fresh milk is increasing day by day. Taking into account this consideration, there are groups of milk vendors seen to collect raw liquid milk directly from the farmers and to sell it at the markets - may be local or city-based, or directly to the households. The producers themselves are also seen to do so. If the proper hygienic approaches are not followed through and/or the collected milk is adulterated with different means, or unconventional materials are used to enhance its shelf life, there is every possibility that milk can be contaminated with zoonotic pathogens, such as E. coli with its serious pathotypes: O157 serotype or non-O157 and shiga toxin producing ones and with many more organisms like as Campylobacter jejuni, Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus, Yersinia enterocolitica etc. In view of the these particulars, the present study was carried out to isolate and unravel the cultural/biochemical characteristics of E. coli from collected raw milk samples and to determine the molecular characterization of E. coli O157 and E. coli non-O157 using universal, specific, Stx1, Stx2, eaeA, rfbE and hlyAprimers.

MATERIALS AND METHODS

Selection of sample collection points
 
186 fluid raw milk samples (250 ml) were collected where 169 Samples were from fluid-milk marketing points of Chittagong city: Shikalbaha, Sholoshahar Railway Station, Jalalabad Market, Chittagong City Gate, Halishahar, Chittagong Port and Chittagong Batali Road and other 17 samples were from a dairy farm located in Shikalbaha September 2013 to March 2014.
 
Procedure of sampling
 
The samples were directly collected from the bulk sources of incoming fluid raw milk through proper mixing with the help of a plunger and dipper aseptically in a clean sterile bottle. Soon after collection the sample was kept into a cool box for ceasing the growth and activity of acid producing organisms and for shipment to the Department of Microbiology and Veterinary Public Health laboratory of Chittagong Veterinary and Animal Sciences University (CVASU), where the samples were kept at 0°C until investigation.
 
Bacteriological investigation
 
Serial dilution was carried out for the initial screening of E. coli of the collected samples. 100µl of each milk sample was transferred to 900 µl sterile peptone water (0.1%) and thoroughly mixed to give 1:10 dilution, the ‘first dilution’; serial dilutions were prepared by transferring one ml from first dilution (10-1) to 9 ml peptone water, (10-2) and so on (10-3, 10-4, …) as described by Harrigan and McCance (1976). Then each diluted milk sample (100 µl) was inoculated onto MacConkey agar medium (Oxoid, Basingstoke, Hampshire, UK), where E. coli produces large pink colour colony after incubation of 24 hrs at 37°C.
 
MacConkey agar medium was prepared according to the manufacturer instructions. Five large pink coloured cross-sectional colonies from MacConkey agar medium were homogenized and inoculated onto an Eosin Methylene Blue (EMB) (Oxoid, Basingstoke, Hampshire, UK) agar plate, incubated at 37°C for 24 h to verify whether such population produced colonies with metallic sheen, a diagnostic criterion for E. coli. (Dyes Eosin Y and Methylene Blue react with products released by E. coli from lactose or sucrose as carbon and energy source, forming metallic green sheen.) The isolates from MacConkey produced metallic sheen on EMB were considered as probable E.coli. A portion of a colony displaying characteristic metallic sheen on EMB was inoculated into TSB (Trypticase Soya Broth) broth, incubated at 37°C for 20 h and finally tested for standard biochemical tests for E. coli, e.g Catalase test, Indole, Methyl red, Voges-Proskauer test, Nitrate reduction, Urease production, Simmon’s citrate agar and various sugar fermentation tests. When a broth culture in TSB gave typical reaction was finally deduced as E. coli and then preserved at -80°C with 15% glycerin.
 
CT-SMAC (Oxoid, Basingstoke, Hampshire, UK) agar added with cefixime tellurite, which is a selected medium for EHEC O157, was initially used in this study to screen any probable E. coli O157 which is incapable of fermenting sorbitol and thus produces colorless colonies on medium. CT-SMAC was prepared according to the manufacturer’s instructions. Briefly, 25.75gm sorbitol MacConkey agar was weighed, mixed with 500 ml of distilled water and autoclaved. The medium in the flask was then placed in a hot water bath until become cooled to 50°C when 2 ml of cefixime tellurite (potassium tellurite 1.25mg and cefixime 0.05mg) was added to it. Cefixime-tellurite (CT) added medium was poured into petridishes at the amount of about 20 ml medium per petridish and the medium in petridishes are preserved at +4°C before the use, as recommended.
 
Growth of a probable EHEC O157 colony on a CT-SMAC agar plate was presumptively diagnosed if it was slightly transparent, colorless with a weak pale brownish appearance and with a diameter of 1mm. Five such cross-sectional colonies were picked up and transferred to a 10 ml test tube containing 5 ml of tryptic soy broth (TSB), incubated at 37°C for 6 h and preserved at -80°C with 15% glycerin until investigation, for observing more diversity at molecular level.
 
Screening the E. coli isolates of milk origin for the virulence genes - hlyA, stx1, stx2, eaeA and rfbE
 
The diversity of all the probable EHEC isolates as found producing colorless colonies on CT-SMAC were investigated based on the presence of five virulent genes - hlyA, stx1, stx2, eaeA and rfb by Polymerase Chain Reactions (PCR). The sequences of five oligonucleotide sets of primers, respectively, used for  hlyA, stx1, stx2, eaeA and rfbE genes are shown in Table 1. The reagents used for their PCR amplifications are listed in Table 2.
 

Table 1: Oligonucleotide primers used to detect hlyA, stx1, stx2,eaeA and rfbEgenes.


 

Table 2: Reagents used for PCR amplifications of the five genes - hlyA, stx1, stx2, eaeA and rfbE in the probable EHEC isolates as produced colorless colonies on CT-SMAC.


 
After thawing in room temperature, the preserved isolates were inoculated onto 5% citrated bovine blood agar and then incubated at 37°C for 24 hours. After that, 200 µl deionized water was taken in 1.5 ml Eppendorf tube for each isolate. With the help of an inoculating loop, a loop-full of fresh colonies were picked up and transferred to the Eppendorf tube. A homogeneous cell suspension was made. It was vortexed, boiled at 99°C for 15 minutes and then immediately placed upon ice. Bacterial cell wall breaks down during boiling and DNA is released. A ventilation hole was made in the lid of the Eppendorf tube using a needle. The boiled suspension was then centrifuged at 15000 rpm for 2 minutes and 100 µl of supernatant was taken in another Eppendorf tube. This collected supernatant was used as DNA template. According to the manufacturer’s instructions, a stock solution containing 100 Pmol of each primer was prepared by adding molecular grade water. Working solution having 20 Pmol concentrations of the primers were used in PCR.
 
The master mix for PCR of a gene was prepared according to the number of samples to be tested at a time. The proportions of different reagents used to prepare master mix for five different genes are shown in Table 3. Each PCR reaction was run with a final volume of 50 μl where 49 μl of master mix was added to 1 μl of DNA template. The readymade master mix containing Dream Taq DNA polymerase, dNTPs set, Dream Taq buffer (containing 20 mM Mgcl2) was also used to prepare PCR reaction mixture for PCR assay.
 

Table 3: Preparation of PCR master mixused for the identification of virulent genes.


 
PCR assays
 
PCR was run on a thermocycler (Applied Biosystem, 2720 thermal cycler, Singapore). The reactions conditions for the hlyA, stx1 and stx2 genes are listed in Table 4. The same reaction conditions were also used for the eaeAand rfbE genes.
 

Table 4: Cycling conditions used for the detection of three genes hly, stx1 and stx2 with the recommended primer sets (Nadine et al., 2003).


 
After the PCR amplification gel electrophoresis was performed. For gel electrophoresis a gel tray was prepared and proper set up was made by placing comb in position. Then 1% agarose solution (Seakem® Le agarose-Lonza) was prepared in 1x TE buffer by boiling on a microwave oven for 2 minutes. To make 1% agarose solution 50 μg of agarose powder was added to 50 ml of 1x TE buffer. The agarose was cooled to 40-50°C in a water bath and 1 drop of ethidium bromide with a concentration of 5 μg/ml ethidium bromide was added. Finally, the agarose was poured into the gel tray and allowed 20 minutes to solidify the gel.
 
An electrophoresis tank which was filled up with 1x TE buffer and then the gel was placed into it with the gel tray. Then the comb was removed carefully so that it could produce smooth holes in gel. After that 5 μl of each PCR product for a gene was mixed with approximately 1 μl of a loading dye (Thermo Scientific, fermentas international Inc) and loaded into a gel-hole. First hole was loaded with 1 kb DNA marker (O’Gene Rular 1 kb plus) to compare the amplicon size of a gene product. PCR product was loaded from the second hole.
 
Electrophoresis was done at 110 volts, 80 Amp for 20 minutes; gel was placed in a water bath for rinsing and examined on a UV trans illuminator (BDA digital, biometra GmbH, Germany). DNA product sizes of 165, 614, 779, 881 and 259 bp were considered for the presence of  hlyA, stx1, stx2, eaeAand rfbEgene, respectively.

RESULTS AND DISCUSSION

Proportion of milk samples positive with E. coli
 
Of the 169 raw market milk samples, 33 (18.3%) were found positive with E. coli and out of the 17 samples from a dairy farm 2 (11.8%) yielded E. coli characteristic growth of bacterial colonies produced on MacConkey and EMB agar plates based on which E. coli was delineated are shown in Fig 1 and 2, respectively. The results of the biochemical tests done with each of the isolate are shown in Table 5. These result are in conformity with other South Asian studies such as Surve et al., (2011), Dewangan et al., (2017) and Gupta et al., (2020).
 

Fig 1: Typical large pink colonies indicating the growth of E. coli onto Maconkey agar plates.


 

Fig 2: Colonies with typical metallic sheen onto EMB agar plates showing a homogenous growth of E. coli.


 
@table5
 
E. coli is an indicator organism to show if any sample/objects is contaminated with materials of fecal origins. E. coli, particularly those belonging to the shiga toxin producing O157 serotype or non-O157 but Shiga toxin producing ones themselves are pathogenic. Life threatening disease may be resulted because of consuming milk contaminated with Shiga toxin producing E. coli belonging to O157:H7 or O157: H- (Tarr et al., 2000 and Grant et al., 2011). This is not the only danger; the presence of E. coli in milk does indicate that it might also be contaminated with any other enteric pathogens, such as any members of shigella, vibrio and others, indicating if such contaminated milk is taken, it can lead to the development of any enteric disease.

While examining the fluid milk destined to be marketed at the chittagong metropolitan area it can be concluded that a substantial proportion, i.e one in every five milk samples is seemingly contaminated with E. coli here. Milk is usually taken, having boiled in Bangladesh. However, if it is taken raw without any heat-treatment it might pose a serious health-risk to the public health because of its contamination with any enteric pathogens of animal or human origins.
 
Presence of probable EHEC O157
 
E. coli from only six (18.2%) of the 33 positive samples yielded colorless colonies across the CT-SMAC, suggesting the probable presence of populations belonging to the serotype O157. Growth of probable E. coli O157, as evidenced by the colorless colonies on CT-SMAC compared to coloured colonies from other bacteria is shown in Fig 3. The yielding of colorless colonies on CT-SMAC from a milk sample in this study was considered as the probable presence of E. coli O157. This cannot be its confirmation until agglutination test is done using specific antiserum. Because of resource limitation this was not done in the study. Very virulent combination in Shiga toxin producing E. coli is the presence of both stx1 and stx2 genes plus the eaeA gene (Eelco et al., 2007). The findings of this study indicate that the presence of Shiga toxin producing E. coli in the fluid milk being marketed at Chittagong is very low and only four isolates from the study carried the stx2 gene. None of the 33 isolates resulted from the study neither had the stx1 nor the eaeA gene.
 

Fig 3: Growth of probable E. coli O157 (colourless colonies on CT-SMAC agar at the left plate


 
Shiga toxin producing E. coli
 
Of the probable E. coli O157 isolates only 4 (66.7%) had Stx2. The 779 bp sized amplicons indicating the presence of Stx2 are shown in Fig 4. None of the other virulent genes, such as Stx1, eaeA, hlyA and rfbE was found in any of the isolates investigated.

Therefore, although a substantial portion of milk samples carried E. coli the danger of having infections with Shiga toxin producing E.coli, by consuming fluid milk at Chittagong is low. While saying this low risk, however, the risk for infection with any other enteric pathogen(s) cannot be overlooked or underestimated because presence of E.coli in milk indicates the presence of any enteric pathogens.
 

Fig 4: Gel picture showing the results on screening stx2 (amplicon size: 779bp) in the E coli isolates.

CONCLUSION

About 18% of fluid milk marketed in Chittagong contains E. coli. Most virulent combination of genes: stx1, stx2 and eaeAin the contaminating E. coli seems to be absent; however, Shiga toxin 2 (stx2) producing E. coli could be found at a very low proportion, thus indicating some public health risk directly from the presence of E. coli in fluid milk. Its presence also indicates the presence of any other enteric pathogens. PCR based molecular epidemiological studies are required for detection of all types of pathogenic as well as zoonotic potential strains of E. coli isolates for future research.

ACKNOWLEDGEMENT

The authors gratefully acknowledge to the Poultry Research and Training Institute (PRTC), Chittagong Veterinary and Animal Sciences University, Bangladesh for providing the facilities to carry out this research and the Office of the University Grant Commission (UGC) of Bangladesh for funding this research.

REFERENCES


  1. Betts, G.D. (2000). Controlling E. coli O157. Nutrition and Food Science. 30(4):183-186.

  2. Desmarchelier, P.M., Bilge, S.S., Fegan, N., Mills, L., Vary, J.C., Tarr, P.I. (1998). A PCR specific for Escherichia coli O157 based on the rfb locus encoding O157 lipopolysaccharide. Journal of Clinical Microbiology. 36(6): 1801-1804.

  3. DesROSIERS, A.N.N.I.E., Fairbrother, J.M., Johnson, R.P., Desautels, C., Letellier, A. and Quessy, S., (2001). Phenotypic and genotypic characterization of Escherichia coli verotoxin-producing isolates from humans and pigs. Journal of food protection. 64(12): pp.1904-1911.

  4. Dewangan, P., Shakya*, S., Patyal, A., Gade, N. and Bhoomika, E. (2017). Prevalence and molecular characterization of extended-spectrum b-Lactamases (blaTEM) producing Escherichia coli isolated from humans and foods of animal origin in Chhattisgarh, India. Indian Journal of Animal Research. (51): 310-315.

  5. Eelco, F., Klerks, M.M., De Vos, O.J., Termorshuizen, A.J. and van Bruggen, A.H. (2007). Prevalence of Shiga toxin-producing Escherichia coli stx1, stx2, eaeA and rfbE genes and survival of E. coli O157: H7 in manure from organic and low-input conventional dairy farms. Applied and Environmental Microbiology. 73(7): pp.2180-2190.

  6. Franz, E., Klerks, M.M., De Vos, O.J., Termorshuizen, A.J., Van Bruggen, A.H. (2007). Prevalence of Shiga toxin-producing Escherichia coli stx1, stx2, eaeA and rfbE genes and survival of E. coli O157: H7 in manure from organic and low-input conventional dairy farms. Applied and Environmental Microbiology. 73(7): 2180-2190.

  7. Grant, M.A., Hedberg, C., Johnson, R., Harris, J., Logue, C.M., Meng, J., Sofos, J.N., Dickson, J.S. (2011). The Significance of Non-O157 Shiga Toxin-producing Escherichia coli in Food. Food Protection Trends. 33-45.

  8. Griffin, P.M., Tauxe, R.V. (1991). The epidemiology of infections caused by Escherichia coli O157: H7, other enterohemorrhagic E. coli and the associated hemolytic uremic syndrome. Epidemiologic Reviews. 13(1): 60-98.

  9. Gupta, P.K., Rai, D.C., Paswan, V.K., Panta, R. and Yadav, A.K. (2020). Study on Physico-chemical and microbial quality of raw milk collected from different places of assi region Food Research. 39(1): 1-9.

  10. Harrigan, W.F., MacCance, M.E. (1976): Laboratory Methods in Food and Dairy Microbiology. 1st edn. Academic Press, London. 221-238.

  11. Johnson, J.R. and Russo, T.A. (2002). Extraintestinal pathogenic Escherichia coli the other bad E coli. Journal of Laboratory and Clinical Medicine. 139(3): 155-162.

  12. Johnson, J.R. and Russo, T.A. (2005). Molecular epidemiology of extraintestinal pathogenic (uropathogenic) Escherichia coli. International Journal of Medical Microbiology. 295(6-7): pp.383-404.

  13. Kaper, J.B., Nataro, J.P. and Mobley, H.L., (2004). Pathogenic Escherichia coli. Nature reviews microbiology. 2(2): pp.123-140.

  14. Levine, M.M. (1987). Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic and enteroadherent. Journal of Infectious Diseases. 155:377-389.

  15. Manna, S.K., Brahmane, M.P., Das, R., Chandana, M. and Batabyal, S. (2006). Detection of Escherichia coli O157 in foods of animal origin by culture and multiplex polymerase chain reaction. Journal of Food Science and Technology-Mysore. 43(1): 77-79.

  16. McDaniel, T.K., Jarvis, K.G., Donnenberg, M.S. and Kaper, J.B., (1995). A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proceedings of the National Academy of Sciences. 92(5): pp.1664-1668.

  17. McKee, M.L. and O’Brien, A.D. (1996). Truncated enterohemorrhagic Escherichia coli (EHEC) O157: H7 intimin (EaeA) fusion proteins promote adherence of EHEC strains to HEp-2 cells. Infection and Immunity. 64(6): 2225-2233.

  18. Nataro, J.P. and Kaper, J.B. (1998). Diarrheagenic Escherichia coli. Clinical Microbiology Reviews. 11(1):142-201.

  19. Nazmul, M., Fazlul, M. and Rashid, M. (2012). Plasmid profile analysis of non-O157 diarrheagenic Escherichia coli in Malaysia. Indian Journal of Science. 1(2): 130-132.

  20. Oswald, E., Schmidt, H., Morabito, S., Karch, H., Marches, O. and Caprioli, A., (2000). Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant. Infection and immunity. 68(1). pp.64-71.

  21. Paton, A.W. and Paton, J.C., (1998). Detection and Characterization of Shiga Toxigenic Escherichia coli by using Multiplex PCR Assays forstx 1, stx 2, eaeA, Enterohemorrhagic E. coli hlyA, rfb O111 andrfb O157. Journal of clinical microbiology. 36(2): pp.598-602.

  22. Radu, S., Ling, O.W., Rusul, G., Karim, M.I.A. and Nishibuchi, M. (2001). Detection of Escherichia coli O157: H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses. Journal of Microbiological Methods. 46(2): 131-139.

  23. Russo, T.A. and Johnson, J.R. (2000). Proposal for a new inclusive designation for extraintestinal pathogenic isolates of Escherichia coli: ExPEC. The Journal of Infectious Diseases. 181(5): 1753-1754.

  24. Schrade, J.P., Yager, J. (2001). Implication of milk and milk products in food disease in France and in different industrialized countries. International Journal of Food Microbiology. 67: 1-17.

  25. Smith, J.L., Fratamico, P.M. and Gunther, N.W., (2007). Extraintestinal pathogenic Escherichia coli. Foodborne pathogens and disease. 4(2): pp.134-163.

  26. Smith, J.L., Fratamico, P.M., Gunther, N.W. (2001). Extraintestinal pathogenic Escherichia coli. Foodborne Pathogens and Disease. 4: 34-163.

  27. Surve, V.V., Patil, M.U. and Gaikwad, S. (2011). Microbiological quality of milk marketed in Goa state. Asian Journal of Dairy and Food Research. 30(1): 75-76.

  28. Tarr, P.I., Bilge, S.S., Vary, J.C., Jelacic, S., Habeeb, R.L., Ward, T.R., Baylor, M.R. and Besser, T.E., (2000). Iha: a novel Escherichia coli O157: H7 adherence-conferring molecule encoded on a recently acquired chromosomal island of conserved structure. Infection and Immunity. 68(3): 1400-1407.

  29. Whittam, T.S. (1996). Escherichia coli and Salmonella. ASM Press, Washington DC, USA. 2708-2720.

  30. Zadik, P.M., Chapman, P.A. and Siddons, C.A. (1993). Use of tellurite for the selection of verocytotoxigenic Escherichia coli O157. Journal of Medical Microbiology. 39(2): 155-158.

  31. Zhao, T., Doyle, M.P., Shere, J. and Garber, L. (1995). Prevalence of enterohemorrhagic Escherichia coli O157: H7 in a survey of dairy herds. Applied and Environmental Microbiology. 61(4): 1290-1293.
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