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University of Gaza Faculty of Health Science Medical laboratory Sciences Department

University of Gaza Faculty of Health Science Medical laboratory Sciences Department. بسم الله الرحمن الرحيم . Lab 7 :. Isolation of E . col i 015 7 i n foods. Isolation of E . col i 015 7 i n foods. Objectives:.

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University of Gaza Faculty of Health Science Medical laboratory Sciences Department

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  1. University of Gaza Faculty of Health Science Medical laboratory Sciences Department بسم الله الرحمن الرحيم Lab 7 : IsolationofE.coli0157infoods

  2. IsolationofE.coli0157infoods Objectives: 1. Detect E. coli O157:H7 in food.2. Identify the pathogenic strain based on its biochemical and serological characteristics.3. practice a rapid test using an immunochromatographic technique.

  3. E.coli0157 • Escherichia coli 0157:H7 is an important foodborne pathogen causing symptoms ranging from mild diarrhea to hemorrhagic colitis and haemolyticuraemic syndrome. • Mostoutbreaks and sporadic cases have been associated with consumption of undercooked beef or dairy products such as untreated milk. • infections due to E. coli 0157 are increasing and wide ranges of foods have been associated with outbreaks. • These include cooked meat products, vegetables, salad vegetables, coleslaw, and acid based foods such as apple cider, yoghurt and fermented sausages. • Microbiological guidelines for ready to eat foods recommend the absence of E. coli 0157 in 25 grams.

  4. Most strains of E. coli 0157 do not ferment sorbitol within 24hrs and are B-glucuronidasenegative whereas most other serogroups of E. coli give positive reactions. • The method described has successfully isolated the organism from naturally contaminated and artificially inoculated foods. • E. coli 0157 is pathogenic to man, has a low infective dose and laboratory acquired infections have been reported. • Therefore, the isolation and identification of this organism must be carried out by trained personnel in a properly equipped laboratory, under the control of a qualified microbiologist.

  5. Application The method described is applicable to the detection of Escherichia coli 0157 in all foodtypes. • Description • The method has been shown to produce satisfactory results with artificially-contaminated meats (including beef, veal and pork), vegetables, dairy products, spices and environmental samples. • This method can be used successfully for the detection of E. coli O157in other foods, food ingredients and environmental samples.

  6. PRINCIPLE: • The detection of E. coli 0157 involves the selective enrichment with isolation from enrichment culture by immunomagnetic separation (IMS) followed by subculture to aselective agar. • The selective agar cultures are examined for characteristic colonies. • Confirmation of presumptive E. coli 0157 is by serological and biochemical tests.

  7. Materials and special equipment • Broths and agars (base media and supplements are commercially available) • Modified Tryptic Soy Broth with Novobiocin (mTSB-n) • Enterohemorrhagic E. coli (EHEC) Enrichment Broth (EEB) • Modified Hemorrhagic Coli Agar (mHC) with Tellurite and Cefsulodin • Modified Sorbitol MacConkey agar (SMAC) with Tellurite, Cefixime, and Cefsulodin 5. Purple broth base with cellobiose.

  8. 0.5% K2SO4 (needed for some spices and foods containing large amounts of spices). • 1N HCl and 1N NaOH. • pH meter or paper capable of distinguishing 0.3 to 0.5 pH units within a range of 6.0 to 7.5. • Stomacher, blender, or equivalent. • Control cultures (use ATTC cultures or equivalent) positive control: E. coli O157 (H7 or other serovars) negative control: E. coli (NOT an O157) • Incubators capable of maintaining 35 and 42°C Confirmation media and reagents (commercially available). • Trypticase Soy Agar with Yeast Extract (TSA-YE) • Rapid Identifcation kits • Latex Agglutination kits • IMVIC Reagents (see MFHPB-19) • urea agar slants • MUG (4-methylumbelliferyl-$-D-glucuronide) • BCIG (5-bromo-4-chloro-3-indolyl-$-D-glucuronide) either –Na or-CHX (cyclohexylammonium) salt. • O157 and H7 antisera

  9. Procedure • Handling of Sample Units • In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5oC) or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death. • Analyze sample units as soon as possible after their receipt in the laboratory. • Preparation for Analysis • Have ready sterile mTSB-n (and/or EHEC enrichment broth (EEB)). • Clean the surface of the working area with a suitable disinfectant.

  10. Preparation of Sample • To ensure a truly representative analytical unit agitate liquids or free flowing materials until the contents are homogeneous. • If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. • To reduce the workload, the analytical units may be combined for analysis. • It is recommended that a composite contain not more than five analytical units. • Note: When analyzing larger volumes, the enrichment broth should be prewarmedto 35oC.

  11. Prepare a 1:10 dilution of the food by aseptically adding 25 g or mL (the analytical unit) into 225 mL of the enrichment broth mTSB-n (and EEB if applicable). Stomach or blend. • A second primary enrichment broth started directly in EEB should be done when there is a high bacterial load of competing organisms in the sample. • Note : Some spices, such as onion and garlic powder are antimicrobial in nature. When analyzing spices or products containing large amounts of spices, add 0.5% K2SO4 to mTSB-n before autoclaving. Garlic especially affects E. coli O157, therefore garlic or garlic containing products need to be diluted 1:100. Other spices also may need to be analyzed using larger dilutions.

  12. After the addition of the sample to the broth adjust the pH of the mixture, if necessary, to 6.0 to 7.0 with 1N NaOH or 1N HCl. • A positive and a negative control should be set up at the same time. • Incubate the enrichment mixture and controls for 22-24 h at 42oC. • Either screen the enrichment broth for E. coli O157 by using rapid kits.

  13. Secondary Enrichment in EEB • Note : This step must be used when a rapid kit has identified the presence of E. coli O157 but it was not isolated when the primary enrichment broths, mTSB-n and/or EEB, were plated onto the selective agars. • E. coli O157 may be difficult to isolate from some samples with high ACC levels. After agitation of the enrichment broth, transfer 1 mL of the mTSB-n and/or EEB to 9 mL of EEB. Incubate 18-24 h at 35oC. Plate 0.1 mL of 10-4 to 10-6 dilutions (made in 0.1% peptone water) • from the enrichment broths onto the selective • agars, as below. • Follow confirmation steps for typical colonies.

  14. Selective Isolation • Plate dilutions of 10-4 to 10-6 from each enrichment broth onto mHC and SMAC agar plates. Incubate for 18-24 h at 42oC. Due to the increased selectivity of SMAC, the counts may be one log less than on mHC. • 2. On the mHC agar, typical E. coli O157:H7 colonies appear blue. On SMAC, typical E. coli O157:H7 colonies appear colorless, bear the tint of the medium or are gray to pink with Smokey centers. Other serovars of E. coli, including O157 (not H7) will be yellow on mHC and red on SMAC. Some of these sorbitol positive colonies may be pathogenic also.

  15. Use the purified colonies (step 1) and continue confirmation steps. Unless stipulated, incubate all tests at 35oC for 22-24 h. • Do a Gram stain. • Use rapid identification kits following manufacturers’ instructions. • Confirm isolates as an O157 using latex agglutination kits. • Streak suspect colonies onto Phenol red sorbitol agar with MUG (PSRA), and/or • Sorbitol MacConkey agar with BCIG. Incubate plates at 35oC for 22-24 h. • Inoculate IMViC tests and Urea slants). Incubate at 35oC. • Complete serological testing using O157 antisera and H7 antisera. • Follow manufacturer’s instructions. The isolate must be "resuscitated“ • in M broth or on motility agar several times (at least three times).

  16. Sorbitol-MacConkey agar (SMAC) • majority of E. coli O157 do not ferment sorbitol in contrast to most faecalE. coli • modification of SMAC : • supplemented with cefixime and tellurite (CT-SMAC) • suppress other sorbitol non-fermenting organisms • most non-O157 STEC ferment sorbitol

  17. ISO Standard for testing of E. coli O157 in Food

  18. CHROM agar for E.coli O157 Rapid and reliable detection of the enterohaemorrhagicE.coli O157. Easily distinguishable colonies due to the purple colouring they have acquired. Most other bacterial species are inhibited, giving blue or colorless colonies. Despite specifity is improved in comparison to Sorbitol MacConkey Agar, when direct isolation method is used, the false positive must be screened out by additional tests and candidates must be further studied for confirmation. E. coli O157 - purple other bacterial colonies - inhibited, blue or colorless

  19. Colonies appear as pink on CHROM agar • After incubation for 16–24 hours at 37°C, the plate should be examined for possible O157 colonies, which are colorless on SMAC or CT-SMAC and are mauve or pink on CHROM agar O157

  20. latex agglutination test To identify E. coli O157, a portion of a well-isolated colony (i.e., a distinct, single colony) should be selected from the culture plate and tested inO157-specific antiserum or O157 latex reagent as recommended by the manufacturer . Colonies that agglutinate with one of the O157-specific reagents and do not agglutinate with normal serum or control latex reagent are presumed to be E. coli O157.

  21. latex agglutination test anti-O157 anti-H7

  22. Identification with biochemical methods The colony in which O157 STEC are detected should be streaked onto SMAC or a nonselective agar medium such as tryptic soy agar (TSA), heart infusion agar (HIA), or blood agarand biochemically confirmed to be E. coli

  23. Singlepath® E. coli 0157 Test • For the rapid detection of E. coli O157 in food. • Sensitivity 97%, Specificity 98.4% ,Results In Just 20 Minutes! • Our new Singlepath® E. coli 0157 immunochromatographicaltest offers all the benefits of traditional test methods with the added benefits of speed, reliability and convenience. When used in conjunction with our EC Broth Modified (mEC Broth) or Tryptic Soy Broth Modified (mTSB) for enrichment, you can be sure that after a 24 hour incubation, the count of the target E. coli 0157 will be at levels that assure either a true positive or negative result from the rapid test. • The media already contain Novobiocin for convenience which suppresses the growth of Gram-positive microbial flora.

  24. Singlepath® E. coli 0157 Test Procedure Selective Enrichment • Add 25 g or 25 ml of the sample to 225 ml EC Broth Modified (mEC) or TrypticSoy Broth Modified (mTSB) • Incubate at 35-37 °C for 18-24h Detection • Transfer 160 μl from the enrichment to the circular sample port on the test • Read after: 20 min Confirmation If result is positive: Streak onto CT-SMAC Agar for confirmation. E.coli 0157 not present Negative E.coli 0157 present Positive

  25. Dynabeads® anti-E. coli O157 • Dynabeads® anti-E. coli O157 are supplied in a suspension of phosphate buffered saline (PBS) pH 7.4 with 0.1% bovine serum albumin (BSA) and 0.02% sodium azide. • Caution: Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. • Dynabeads® anti-E. coli O157 is designed for rapid selective separation of E. coli O157:H7 from food, water, or environmentalsamples. • Dynabeads® anti-E. coli O157 are designed for rapid, selective concentration of E. coli O157 directly from a pre-enriched sample aliquot using immunomagnetic separation (IMS). • Dynabeads® anti-E. coli O157 reacts with all E. coli O157 strains including pathogenic and non-pathogenic, sorbitol fermenting and non-sorbitol fermenting isolates.

  26. Dynabeads® anti-E. coli O157 • Dynabeads® anti-E. coli O157 are simply incubated with an aliquot of the pre-enriched sample, and the antibodies coated onto the beads will specifically bind the target bacteria. The bead-bacteria complexes are subsequently separated by applying a magnetic field. • Thewhole IMS process can be automated using a BeadRetriever™ instrument or performed manually. • Any food, water, feed, or environmental sample that has been pre-enriched for 6–18 hours in Buffered Peptone Water (BPW), TryptoneSoya Broth (TSB), or Brilliant-Green Bile Broth (BGBB) is suitable for IMS with Dynabeads® anti-E. coli O157.

  27. An electron micrograph showing E. coli O 157 bound to Dynabeads

  28. END OF LECTURE

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