Rapid Test for the Detection of Pathogens in Dairy Products

Rapid Test for the Detection of Pathogens in Dairy Products June 13, 2013 Bioq. Mariano Veltri Product Manager for BioMonitoring

Content From classical culture to rapid testing Overview Merck Millipore rapid detection solutions General principle of real-time PCR Overview Merck Millipore real-time PCR solutions Pathogenic E.coli testing from ground beef using RapidCult TM Other Pathogens in Food: - Salmonella - Listeria - Bacillus Cereus and Cronobacter Sakazakii 2

Food Pathogens Microorganisms Enrichment Rapid Cultivation Identification Salmonella Cronobacter DCM Immunology Singlepath DCM DCM Bactident Listeria E. coli O157 Bac. cereus RTU Real Time PCR Foodproof MMB RTU Lateral Flow RT PCR Staph. aureus Campylobacter Enterococci RTU Enterobacteriaceae Vibrio Pseudo. aerug 24-48 h 20-90 min 24-48 h 24-48 h

Food & Beverage / Enrichment media Food & - Key products for QC Lab 1.07228. Buffered Peptone Water ( Salmonella) 1.10266. Lauryl Sulphate Broth ( Coliforms in Water) 1.10398. FRASER Broth ( Listeria) 1.10549. LEB (antibiotics incl.); ( Listeria) 1.14582. mec Broth /mtsb ( Enterohemorrhagic E.coli) 1.07661. Lactose Broth ( Salmonella) 4

Food & Beverage / Plating Agar Key products for QC Lab 1.05463. Plate Count Agar ( Total Count) 1.05463 Chromocult Coliform Agar ( Coliforms & E.coli) 1.01406. VRB Agar ( Coliforms & E.coli) 1.16000. YGC Agar ( Yeasts) 5

ISO 11133 ISO 11133-1 Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media - Part 1 ISO 11133-2 Microbiology of food and animal feeding stuffs Practical guidelines on performance testing of culture media - Part 2 This new standard describes for the first time in history of Microbiology the definition of quality control for commercial culture media. Quantitative testing and reporting of results (CoA) with recovery rates is the new preferred testing method 6

ISO 11133 Part 2 Practical Guideline on Performance testing of culture media - Certificate of Analysis Quality Control of Plating Agar Productivity Selectivity > 70% for non selective media > 50% for selective media Most important change! 7

Methods for use in performance testing of culture media Quality Control of Selective Liquid Media Target bacteria Unwanted 10*6 to 10*8 c.f.u per ml below 10*4 c.f.u per ml Extremely important to avoid false-negative results

Methods for use in performance testing of culture media Quality Control of Selective Liquid Media RVS / TTn / Selenite Cystine broth Quality control Minimum level must be 10*5 c.f.u Test strains Inoculum Growth after 24 hours Escherichia coli ATCC 25922 Approx. 99 % < 10 % Salmonella typhimurium ATCC 14028 Approx. 1 % > 90 %

Specification of Tryptic Soy Agar Merck COA in Compliance with ISO 11133 more than 70 % Recovery Rate for each test strain used 1 Page 10

From classical culture to rapid testing

RAPID TESTING 7 6 5 4 3 2 1 log 10 cfu REAL TIME TEST 1 2 Culture +PCR/ REAL TIME PCR BIOCHIP Culture + ELISA SHORTENED CULTURE 3 4 5 Classical Culture + ID 6 7 DAYS 12

Pre-Conditions for Using Rapid Tests All rapid testing methods are fully dependent on the quality of the corresponding non/selective enrichment media If an enrichment medium does not allow growth of a minimum level of bacteria necessary for detection, a false-negative results will occur! Minimum levels PCR: 10 3-10 4 CFU / ml Minimum levels Immunoassays: 10 5 CFU / ml 13

Content From classical culture to rapid testing Overview Merck Millipore rapid detection solutions General principle of real-time PCR Overview Merck Millipore real-time PCR solutions Pathogenic E.coli testing from ground beef using RapidCult TM Other Pathogens in Food: - Salmonella - Listeria - Bacillus Cereus and Cronobacter Sakazakii 14

Overview rapid detection solutions Innovative Immunoassays Lateral FlowTests - Singlepath and Duopath Superior Granulated Dehydrated Culture Media Broad range of preparation & detection kits for real-time PCR 15

Superior Granulated Culture Media Safe Minimizes air-borne toxins, allergens and workplace contaminations Accurate Prevents component separation and clumping Fast Dissolves rapidly in water Easy Ensure easier handling by superior flow properties & non-sticking media Reliable Homogenous distribution of ingredients assures high reproducibility No dust Powder vs Granulated media No clumping 16

General Application of Lateral Flow Tests 1 ADD MEDIUM 2 ADD SAMPLE 3 BLENDING 4 INCUBATE 5 PIPETTE SAMPLE 6 PLACE SAMPLE NEGATIVE POSITIVE Read after 20-25 min 17

Innovative Immunoassays Lateral Flow Tests Fast Definite results within 20 30 minutes Easy to use Clear yes/no results after simple sample application Safe Positive control and specially adapted enrichment media for precise, reliable results Economic Rapid results save operating costs Immunochromatographic principle: Antibody-linked colloidal gold particles react with its complimentary antigenic determinant to provide a visual reaction read-out 18

Principle of Lateral Flow Test antibody gold particle bacterium 19 membrane

Advantages of Lat. Flow tests vs. ELISA 1. Control reaction in-built in LFT. No need for separate reaction to demonstrate proper function, as necessary for ELISA. Can save costs esp. when low numbers of samples to be tested 2. LFT s are 1-step tests. After application of test sample to sample port, NO further pipetting steps necessary. No separate color reaction for LFT s necessary to make reactions visible. 3. LFT are provide results faster than ELISA 20 Only incubate test on lab bench for 20 30 min and read result.

Merck s Lateral Flow Test Portfolio Listeria: Salmonella: E. coli O157&VTEC: Campylobacter: Bacillus cereus: Legionella: LEB Tetrathionate mec + n Bolton Broth M.Y.P. Agar CYE Agar Base Fraser Rappaport.-Vas. mtsb + n CCD Agar CGY Broth BCYE Suppl. UVM Selenit Cystin CT-SMAC BHI GVPC Suppl. PALCAM Salmosyst SMAC CYE-Plates OXFORD M Broth BHI GVPC-Plates Chromocult XLT 4, XLD, CAYE Anaerocult C Listeria Agar Rambach 21

Content From classical culture to rapid testing Overview Merck Millipore rapid detection solutions General principle of real-time PCR Overview Merck Millipore real-time PCR solutions Pathogenic E.coli testing from ground beef using RapidCult TM - Other Pathogens in Food: - Salmonella - Listeria - Bacillus Cereus and Cronobacter Sakazakii 22

What is real-time PCR? PCR = Polymerase Chain Reaction Specific amplification of DNA fragments from bacteria, viruses, human, animal or plant cells & the simultaneous automated detection of this amplified DNA on the computer monitor by fluorescence measurement = real-time 23 Presentation title in footer 00 Month 0000

PCR: Polymerase Chain Reaction Invented by Karl Mullis, 1979 Nobel Price in Chemistry,1993 Specific amplification of DNA fragments from bacteria, viruses, human, animal or plant cells & simultaneous automated detection of these amplified DNA by fluorescence measurement Real-Time PCR www.forensic-science.org 24

Basic principle T T A C G A C C T T A G C C T G A C G A G A T G A C T G A G C A C A T T C A G C A C T G A C T G C A G A T G C T G G A A T C G G A C T G C T C T A C G A C C T T A G C C T G A C G A G A T T G A C T G A G C A C A T T C A G C A C T G A C T G C A G A T G T C A T C G A A C A C T C T G A G A C C T G C A T C C G A G A T T G A C T G A G C A C A T T C A G C A C T G A C T G C A G T A C G A C C T T A G C C T G A C G A G A T G C T G G A A T C G G A C T G C T C A T T G A C C T G A G C A C A T A T C A G A G C C A T C T A T G C A C T G C A C G A G T A C T G C A T C G G C A T T A T G A T T C T A C G A G C G G A C A C T T C A T G C G A C T T G A C T G C A G G A G A G T G G A A C G A C T G T C T A C T G A A C G C A C C T C T A T C A G A A C C T C T T A G C C A T C G C A C A G A G G A T T G A C C T A G C A C A T T A C T T G A C T G A G A C T T G A C G C T G A G C A A T C T A G A T T C T G C A G C G A C A C T A T C A C T G G C A T C A T G C C G T A C G C T A G C A T G G C A T G A C T T G T G C A T G C G A A G A T G A C T C T A C T C G T C G A T G A A T G C G A C C G A C T C G T G C A C T T C T C G C T A C G A C C T T A G C C T G A C G A G T A C A G T T G A C C T G C A G T C T G T A C A T G T A T G C A C G A G T T C A T C G C T A G C G T A G A A C T G C A A G G G T A C G A C C T T A C T G A C A T T G A C A C T T G G C A G T C A G A T C A G G T T T C A T A C G T A A G A C A C G T G C T G A A G C C A C T T T C T G C A T C A C G C T C A G G C G A T G C T G G A A T C G A T T G A C A T G C A G C A T C A T G T C A G C A C C T G A C T G C A G T A C G A C C T A G C C T G A C G A T G C T G G A A T G C T G G G A A A T C G G T A G C T C G C T C A T T G A C C T G A G C A T C A C T G T A C A C G C G T C T A A C G T C G C T A G C C A T C G G C A G A T G C T G A G T T G A A C T G T A G C A C G A T G T C A G C A T C T G G C A C T G C A G A T G C T G G A A T C G G A C T G C T C 72 C Taq PCR 95 C Taq 55 C 25

26 PCR Principle of Amplification

Detection formats Hybridisation Probes TaqMan Probes (5 Nuclease) 27 Probes remain intact Probes get destroyed

Real-time PCR: Online data measurement Real-Time PCR Classical PCR Ct-value Threshold Cycle (C t ) value Agarose gel electrophoresis Staining Photography Advantage High specifity Low risk of false positive results 28

PCR workflow Start 20 min 40 min 90-120 min 130 min Enrichment, Culture or Single Colony Sample preparation PCR Set-up PCR run Detection & Analysis 29

Real Time PCR Sample preparation From manual to full automated handling ShortPrep SamplePrep RoboPrep <30 min <60 min 30

Content From classical culture to rapid testing Overview Merck Millipore rapid detection solutions General principle of real-time PCR Overview Merck Millipore real-time PCR solutions Pathogenic E.coli testing from ground beef using RapidCult TM Other Pathogens in Food: - - - Salmonella - - Listeria - Bacillus Cereus and Cronobacter Sakazakii 31

Broad range of real-time thermal cycler Corbett/QIAGEN Rotor-Gene 3000 Agilent/Stratagene Mx3000p BioRad IQ5 Cepheid SmartCycler BioRad myiq ABI 7500 Roche LC480 Eppendorf realplex ABI 7900 Roche LightCycler 1.5 Roche LightCycler 2.0 ABI 7000 ABI StepOne System ABI 7700 32

Test for several bacteria in 1 PCR run Salmonella L. mono., L. Genus E. coli O157 Campylobacter Quant. GMO screening RR Soya Quant. Identical Thermoycler conditions 96 well plate PCR Setup at room temperature 33

Benefits of Real-Time PCR: Saving of labour / time Applying real-time PCR, all samples are read by just one mouse-click Clear results, no hidden colonies in the background Objective results; no personal influence of lab manager who reads the plates 34

Real-time PCR product portfolio Foodproof & MMB detection kits Presence/absence tests of pathogenic bacteria Quantitative investigation of pathogenic bacteria Large scale identification of spoilage organisms Detection and quantification of gene modified organisms 35

Foodproof Detection Kits: For Roche LightCycler 1.x, 2.0 Salmonella Listeria monocytogenes Listeria Genus E. coli 0157 Campylobacter Hybridization Probes Kits Enterobacteriaceae plus E. sakazakii E. sakazakii E. coli and Shigella Beer Screening bacteria Saccharomyces cerevisiae var. diastaticus GMO Maize Quantification GMO Soya Quantification GMO Screening 36

Foodproof 5 Nuclease Kits: For TaqMan Instruments & LC 480 Salmonella spp. Listeria monocytogenes Listeria Genus E. coli O157 Campylobacter quantitatively Enterobacteriaceae + E. sakazakii Brucella Dekkera (= Brettanomyces: Wine spoilage) Alicyclobacillus GMO Screening (4 events multiplexed!!!) GMO Soya Quantification GMO Maize Quantification 37

Foodproof Sample Preparation Modules I Rapid & Convenient Kits foodproof ShortPrep I Kit (for Salmonella kits) foodproof ShortPrep II Kit (for E. coli O157, Campylobacter & Listeria) foodproof ShortPrep III Kit (for beer screening kit) Kits for difficult matrices foodproof Sample Preparation Kit I (Gram-neg. bacteria in raw & processed foods) foodproof Sample Preparation Kit II (Gram-pos. bacteria in raw & processed foods) foodproof GMO Sample Preparation Kit Rapid Kits (with bulk reagents) = Value for Money foodproof StarPrep One Kit (for all Enterobacteriaceae) foodproof StarPrep Two Kit (for Gram-positive bacteria) Available Q1 / 2013 foodproof StarPrep Four Kit (for yeasts) foodproof Magnetic Separation Kit I (for Gram-negative bacteria) 38

Foodproof products from Merck for various instruments Brand label: foodproof Lightcycler (Roche): foodproof Salmonella Detection Kit - Hybridization Probes (LC 1.x, 2.0) - TaqMan-Instruments: foodproof Salmonella Detection Kit - 5 Nuclease - NO kit based on SYBR Green from Merck!!!! 39

Highest Quality Assurance with foodproof real-time PCR kits All foodproof real-time PCR kits contain: Internal Amplification Control (IAC) present in every capillary / well and every test PCR run positive control (PC) as a separate reaction. Target DNA from kit added. PCR Negative control (NC) as a separate reaction. Ultra-pure H 2 O from kit added. Taq / UNG enzyme Mix present in every capillary / well and every test 40 No false-negative results from food matrix No false-negative results from defective kit No false-positive results due to contaminated kit No false-positive results due to carry-over contamination

Prevention of Carry-over Contamination Mechanism of Uracil N Glycosylase Native DNA Amplified DNA DNA-Matrix DNA-Matrix a a t taa a t a aa t aaa t aa t a aa t t ata a a t taa a t a aa t aaa t aa t a aa t t ata t t a at t t a t t ta t t t a t t a t t t aa tat t t a a t t t a t t t a t t t a t t a t t t aa tat PCR mit Thymin PCR with thymine PCR with uracil a a t taa a t a aa t aaa t aa t a aa t t ata t t a at t t a t t ta t t t a t t a t t t aa tat u ua auu u a u uua uuua uu a u uu aa uau a au uaa a u a aau aaau aa u a aa uu aua Uracil-N-Glycosylase a a t taa a t a aa t aaa t aa t a aa t t ata t t a at t t a t t t a t t t a t t at t t aa tat u ua auu u a u uua uuua uu a u uu aa uau a au uaa a u a aau aaau aa u a aa uu aua a a t taa a t a aa t aaa t aa t a aa t t ata t t a at t t a t t t a t t t a t t a t t t aa tat 41

Prevention of Carry-over Contamination Necessary Kit component UNG Sample Enrichment DNA Extraction Purification Contamination by old amplified GAUC-DNA possible UNG - Glycosylase step PCR Detection UNG degrades ONLY old amplified GAUC-DNA UNG cannot degrade native GATC-DNA UNG is inactivated before new PCR starts (during initial denaturation of DNA) no false positive results possible due to lab contamination 42

Content From classical culture to rapid testing Overview Merck Millipore rapid detection solutions General principle of real-time PCR Overview Merck Millipore real-time PCR solutions Pathogenic E.coli testing from ground beef using RapidCult TM Other Pathogens in Food: - - - Salmonella - - Listeria - Bacillus Cereus and Cronobacter Sakazakii 43

44 EHEC s: Emerging Pathogens

EHEC s: Emerging Pathogens Highly pathogenic enterohaemorrhagic E. coli (EHEC) strains like verotoxin (= shiga-toxin) producing (VTEC/STEC) strains gain importance Serovars: O157:H7, O26, O103, O111, O145 and other strains are of interest Production of verotoxins is the most common criteria for detection: Verotoxin 1 (VT1, SLT1, Stx1) and 2 (VT2, SLT2, Stx2) EHEC strains produce either VT1 or VT2 only or even both VT2 is causative agent for severe diseases, like hemolytic uremic syndrome (HUS) Infection with E. coli O157 are known as Hamburger Disease 45

Features of Rapidcult E. coli Fast Reduced time to result from more than 18 down to 8-12 hours Flexible Incubation for 12 or less up to 22 hours to fit with optimal workflow Efficient Pooled samples: 25 g and 375 g are validated High yield Increased growth of all E. coli due to special composition Reliable Validation with Singlepath E.coli 0157 & foodproof E. coli O157 Detection Kit consistent with USDA-FSIS guidance High-quality Comprehensive inclusivity study: superior growth of all E.coli strains tested Granulated medium meet ISO 11133 guidelines 46

ISO Standard for testing of E.coli 0157 in Food Day 1 25 g/ml TEST SAMPLE 225 ml m-ec or m-tsb 37 C FOR 24 h Day 2 Sorbitol (+) are non pathogenic E.coli Caution: Some E.c. O157 are Sorbitol (+) -> false-neg. result CT-SMAC Agar 37 C / 24 h Fluorocult E.coli 0157 Agar Day 3 47 Confirmation by culture and biochemical tests required

One broth detects all EHEC strains Enrichment* 25g/375g in pre-warm Rapidcult 8-22h/42 C * USDA FIS validated Classical culture media Lateral Flow Test* Real-time PCR* Post-enrichment After immunoconcentration streak out on 160 µl in Sample Port DNA Extraction StarPrep One Kit CAYE Broth & supplement PCR Screening foodproof E.coli 0157 Detection Kit Duopath Verotoxin CT-SMAC Agar Incubation: 24h/35-37 C Conformation e.g. Fluorocult E.coli 0157 Result: 48 h Result: 8h 20 min Result: 10 h Result: 14 h 48

Rapidcult results with real-time PCR Sensitive screening for most important virulence genes: eae, stx 1 and stx 2 with new foodproof STEC Screening Kit prototype version After 8h enrichment using Rapidcult E.coli The virulence patterns of more than 50 STEC strains were identified correctly strains included: O26 (n=11), O103 (n=3), O111 (n=3), O121 (n=1), O145 (n=3), O157 (n=14) and others (n=15). O104 as the cause of the severe outbreak in Germany in 2011 included 8h enrichment for 25g of ground beef shown for E. coli O157, O26, O103, O111 and O104 compared to the Microbiology Laboratory Guidebook USDA-FSIS,(MLG 5.05B ) and ISO/PRF TS 13136 Tests for O45, O121 and O45 ongoing 49

Rapidcult results with real-time PCR Real-time PCR detection of important virulence genes from E. coli 0157:H - after 8 h RapidCult incubation 50

Content From classical culture to rapid testing Overview Merck Millipore rapid detection solutions General principle of real-time PCR Overview Merck Millipore real-time PCR solutions Pathogenic E.coli testing from ground beef using RapidCult TM Other Pathogens in Food: - Salmonella - Listeria - Bacillus Cereus and Cronobacter Sakazakii 51

From presence/absence to quantitative risk assessment Salmonella Listeria 52 Cronobacter Sakazakii Bacillus cereus CDC Public Health Image Library

53 Salmonella in Food

Day 1 ISO Standard 6579 for Detection of Salmonella 25 g/ml test sample in 225 ml BPW 16-20 h / 35-37 C 3 days Day 2 0.1 ml BPW to 10 ml RVS Broth 24 h at 41.5 C h 1 ml BPW to 10 ml MKTTn Broth 24 h at 37 C Day 3 XLD Agar 24 h / 35-37 C 1 plate 14 cm / 2 plates 9 cm Any other Salmonella Agar 1 plate 14 cm / 2 plates 9 cm XLD Agar 24 h / 35-37 C 1 plate 14 cm / 2 plates 9 cm Any other Salmonella Agar 1 plate 14 cm / 2 plates 9 cm Day 4 Interpretating of Growth on Plates 11 media Day 5 Day 6 54 For confirmation take 5 suspected colonies from each plate and streak onto Nutrient agar 18-24 h / 35-37 C Biochemical / Serological Confirmation Tests TSI / Urea / Lysin / -Gal / VP / Indol

Cultural reference method ISO 6579:2002 Annex D Non-selective pre-enrichment 25g/225ml BPW 18+2h/37 C Selective enrichment 3 drops/0.1ml on MRSV* 24h/41,5 C Negative plates: Further 24h incubation Confirmation Selective plating on XLD Biochemical Identification *Modified Semi-Solid Rappaport-Vassiliadis Medium Detection principle: Motility of Salmonellae to migrate into the semi-solid medium 55

Day 1 Day 2 Rapid Testing Singlepath Salmonella - Screening 25 g/ml TEST SAMPLE In 225 ml Buff. Peptone Water 35-37 C for 16-20 h 0.1 ml BPW to 9.9 ml RVS Broth 42 C for 24 h only 2 days Day 3 NO Salmonella not present YES Salmonella present only 2 media Day 4 If positive, confirmation by culture and biochemical tests required STREAK OUT ONTO RAMBACH 37 C FOR 24 h 56

Detection of Salmonella by RT-PCR: Within ONLY 24 h and only BPW Conventional Non-selective enrichment in BPW 16 20h 1. Solid media 24h Selective enrichment 24h Biochemical verification 4h Isolation/Incubation 24h 2. Solid Media 24h Serological verification 4h Non-selective enrichment in BPW 16 24h Sample preparation 0.5h Amplification/Detection Time savings: PCR 1h neg. result > 2 d pos. result > 4 d 57

Real-time PCR-Analysis Non-selective pre-enrichment 25g/225ml BPW 18+2h/37 C Dilute possible PCR inhibitiors Selective enrichment 3 drops/0.1ml on MRSV* 24h/41,5 C Negative plates: Further 24h incubation Confirmation Selective plating on XLD Biochemical Identification Subcultivation 1:10 0.5 ml BPW in 5 ml prewarmed BHI * 3h/37 C DNA extraction StarPrep One Kit PCR foodproof Salmonella Kit Time: 3-5 days Time: 1.5 days 58 * Brain Heart Infusion

Results on Milk Amplification Plots LC480II spiked with 12,9 cfu Positive control Samples 1c 18 h / 18+3 h Negative control 59

Listeria in Food Milk and milk products: Soft cheese, raw milk, pasteurized milk, ice cream, fresh cream 60

ISO Standard 11290-2004 for testing of Listeria in Food and Animal feeds Day 1 STREAK OUT ON ALOA + PALCAM 37 C FOR 24-48h 25 g/ml TEST SAMPLE IN 225 ml 1/2 STRENGTH FRASER 30 C FOR 24h Day 3-4 0.1 ml IN 10 ml FRASER 35 OR 37 C FOR 48h Day 3-6 Day 4-7 STREAK OUT ON ALOA + PALCAM 37 C FOR 24-48h All colonies showing a blue-green color with opaque halo / grey-green color with a black zone are presumptive L. monocytogenes colonies (typical colonies) and counted as such. Suspect colonies must be confirmed. BIOCHEMICAL CONFIRMATION: GRAM (+), CATALASE (+), MOTILITY (+), CAMP (+), HAEMOLYSIS (+), GLUCOSE (+), RHAMNOSE (+), XYLOSE (-)

Chromocult Listeria Selective Agar Appearance of L. monocytogenes on Chromocult : blue-green colonies = -D-glucosidase activity opaque halo = phosphatidylinosit-phospholipase C activity Listeria monocytogenes Listeria spp. 62

Chromocult Listeria Selective Agar Chromocult Listeria Selective Agar complies with Agar Listeria according to Ottaviani and Agosti (ALOA ) in line with the recommendations of ISO 11290 (2004) and FDA / BAM (2003) 1.00427 Chromocult Listeria Selective Agar Base 1.00432 Chromocult Listeria Selective Supplement Contains 4 different antibiotics 1.00439 Chromocult Listeria Enrichment Supplement Contains L-α- phosphatidylinositol 63

Rapid Testing Singlepath Listeria Lateral Flow Test for the SPECIFIC detection of all Listeria species For screening of environmental and food samples. Very useful for screening in food plants acc HACCP guidelines US-food companies screen for Listeria spp. For the confirmation of presumptive Listeria spp. positive colonies on Listeria selective agars like Chromocult Listeria Agar ALOA ) PALCAM, Oxford Enrichment media used: Fraser, LEB, UVM 64

Rapid Testing Singlepath L mono Worldwide first Lateral Flow Test for the SPECIFIC detection of Listeria monocytogenes For screening of environmental and food samples Very useful for screening in food plants acc HACCP guidelines European food companies screen for Listeria monocytogenes For the confirmation of presumptive positive colonies on Listeria selective agars like Chromocult Listeria Agar ALOA ) PALCAM, Oxford Enrichment media used: Fraser, LEB, UVM 65

Singlepath L mono / Singlepath Listeria - Screening Day 1 25 g/ml TEST SAMPLE IN 225 ml 1/2 STRENGTH FRASER 30 C FOR 24h Day 2 0,1 ml IN 10 ml LEB or Full Fraser OR UVM 30 / 37 C FOR 21-24 h 160µl Day 3 L. monocytogenes not present L. monocytogenes present Listeria spp. not present Confirmation ALOA 37 C for 24-48h

Singlepath L mono - Confirmation suspend 1-3 presumptive colonies in 250 µl BHI or CASO or Fraser or PALCAM Broth ALOA Agar 1 h at 37 C PALCAM Agar OXFORD Agar 150 µl L. monocytogenes not confirmed L. monocytogenes confirmed Confirmation within 1,5 h Fastest confirmation assay available on the market

Detection of Listeria spp. & L. monocytogenes by real-time PCR: Within ONLY 24 h Conventional Non-selective enrichment ½ Fraser 24 h Biochemical verification 4 h Selective enrichment Full Fraser 24 48 h 1. Solid media 24 h Isolation/Incubation 24 h Serological verification 4 h PCR Non-selective enrichment ½ Fraser 24 h Sample preparation 0.5 h Amplification/Detection 1 h by foodproof Listeria Genus or by foodproof Listeria monocytogenes

69 Cronobacter spp.

Rapid Detection of Cronobacter spp. Enterobacter sakazakii is renamed since 2008 New name is: Cronobacter spp. nov.

Cronobacter spp. (formerly E. sakazakii) Considered as a clear species since 1980. Renamed in 2008 due to pheno-and genotypic diversity. C. spp. is an opportunistic pathogen. High risk for newborns, especially low-birth weight infants. Causes severe neonatal sepsis, meningitis Powdered infant formula (PIF) is major vehicle and cause of Cronobacter spp. illness. Very resistant to osmotic & dry stress. Can survive >2 years in PIF 71

Cronobacter spp. EC Regulation 2073 Absence of Enterobacter sakazakii in 10 g sample Milk powder Mandatory for all infant formula producer 72

Cronobacter spp.: Current FDA method 1. pre-enrichment in BPW 2. enrichment in EE broth, 3. plating + isolation on violet red bile glucose agar (VRBGA) 4. biochemical identification of typical colonies is time consuming and requires up to 7 days FDA method only yielded 26% positive results in a comparative study with old ISO draft (40%) 73 Dr. A. Bubert, Merck KGaA

ISO TS 22964 for isolation of E. sakazakii Day 1 Day 2 Test sample in BPW (1:10) 37 C for 16-20 h 0,1 ml 10 ml Modified LST Broth Vancomycin Medium 45 C for 22-26 h Day 3 Bottleneck: Not all E. sak. can grow under these conditions Streak onto Enterobacter sakazakii Isolation Agar 44 C for 22-26 h Day 4 Biochemical Confirmation

foodproof Enterobact. plus E. sakazakii Kit Features Unique real-time PCR kit for detection of all Enterobacteria AND Cronobacter spp. simultaneously with ONLY 1 reaction Highest reliability for Cronobacter Screening in powdered Infant Formula and other babyfoods Very useful for Hygiene Monitoring of Enterobacteriaceae Kit is designed according to latest ISO standards for realtime PCR to prevent false-positive and false-negative results Kit does NOT require mlst enrichment step 75

Cronobacter spp. PCR vs. Conventional Detection Conventional (ISO 22964:2006) Pre-Enrichment 16 20 h Enrichment 22-26 h Isolation 22-26 h Biochemical Identification 44-48 h PCR Pre-Enrichment in BPW for 18 h Subcultivation in BPW 3 h DNA Isolation 0.5 h Amplification/Detection 1 h Time to result : 1 day only 76

Detection of Enterobacteriaceae / Cronobacter spp. by RT-PCR Enrichment 20-24 h DNA-Extraction PCR 1 Enterobacteriaceae/Cronobacter PCR 2 Salmonella Internal Pos. Control Enterobacteriaceae Cronobacter Positive Negative Result Positive Result 77 Enterobacteriaceae neg. Cronobacter neg. E. coli neg. Salmonella neg. Coliforms neg.

Reagent D for dead-live discrimination Reagent D efficiently eliminates amplification of DNA from dead bacteria. Mechanism: Reagent D invades ONLY into dead bacteria and binds to DNA. DNA is not amplifiable any more. 78 Reagent D is a light sensitive substance. Exposure to visible light leads to covalent binding of this substance to DNA and prevents the DNA from being amplified via PCR. Works until 10 6 dead bacteria / gram food Currently validated for Enterobacteriaeceae only Only Merck offers Reagent D for dead-live discrimination

No false-positive results even at high Entero- bacteriaceae concentrations using Reagent D Without Reagent D with Reagent D 79

Advantages of foodproof Enterobacteriaceae plus E. sakazakii Detection Kit Unique 5 in - 1 Test: - Negative result of Enterobacteriaceae reaction means: No Enterobacteriaceae No Cronobacter spp. (formerly E. sakazakii) No Salmonella No E. coli No Coliforms Unique kit which can discriminate between live and dead bacteria Unique kit which give no false-positive results for Enterobacteriaceae These unique features save time and costs 80

foodproof Enterobacteriaceae plus E. sakazakii Detection Kit vs. Sybr green system Sybr green real-time PCR System: - Only for the detection of E. sakazakii - Disadvantage: High rate of false-positive (due to SYBR Green) results foodproof Enterobacteriaceae plus E. sakazakii - Detects ALL Cronobacter spp. strains - Advantage: No false-positive or false-negative results Foodproof kit is successfully validated by Nestlé, Switzerland!! - Nestlé recommends their babyfood plants to use foodproof kit - ISO is working on a revision of C. sakazakii method because the existing method 81 works not very well. Up to now there is no improved method available.

MMB Food Pathogen System MMB Food Pathogen Prep Kits MMB Bacteria Prep Kit MMB DNA/RNA Virus Prep Kit MMB Food Pathogen Detection Kits MMB Staphylococcus aureus (V), (R), (LC) Kit MMB Vibrio cholerae (V), (R), (LC) Kit MMB Legionella pneumophilia (V), (R), (LC) Kit MMB Clostridium perfringens (V), (R), (LC) Kit MMB Bacillus cereus (V), (R), (LC) Kit MMB Legionella Screening (V), (R), (LC) Kit MMB Norovirus (V),(R),(LC) Kit LC = For Lightcycler Systems R = ROX for BioRad Systems V = VIC for Applied Biosystems, Agilent, Eppendorf, QIAGEN 82

MMB Food Pathogen Prep Kits 2 x 50 extractions Membrane filter technology Content: 1 x Lysis Buffer 1 x Binding Buffer 1 x Wash Buffer 1 x Elution Buffer 2.0 ml Receiver tube for washing 1.5 ml Receiver tube for elution Spin filter 83

MMB Food Pathogen Prep Kits procedure 1. Preparation of food matrix for example in 225 ml Buffered Peptone Water Incubation 16-20 hours 25 g chocolate 2. DNA Isolation with MMB Food Pathogen Prep Kit Lysis Wash Wash again Elute Sample DNA 84

Bacillus cereus detection General Environmentally widespread Gram-positive, spore-forming, motile rod Cause gastrointestinal (e.g. diarrhea) as well as non-gastrointestinal diseases (e.g. septicemia,endocarditis, infections of the central nervous system) Self-limiting and of short duration, relative low number of registered cases; < 1% Contamination risk by meat and milk products, vegetables, soups, spices and especially baby food Ability to produce one or more toxins: Cytotoxic enterotoxins are produced by almost 95% of isolates Pre-enrichment : Tryptone soya broth (TSB) PCR detection: B. cereus, B. mycoides, B. thuringiensis, B. anthracis 85

Duopath Cereus Enterotoxins Worldwide first Lateral Flow Test for the SPECIFIC detection of Bacillus cereus via their enterotoxins For screening of foods (tested with cream of wheat, spinach, coffee white, egg powder, chocolate powder) For confirmation of enterotoxin-producing presumptive positive B. cereus colonies on M.Y.P. Agar CGY Broth (+ 1% Glucose) for the enhancement of toxin production ~ 90% of B. cereus are NHE toxin positive; ~ 50% of B. cereus are HBL toxin positive Advantage: Broad coverage of nearly all B. cereus (except emetic strains) due to detection of 2 enterotoxins 86

Singlepath Emetic Toxin MRK Worldwide FIRST Rapid Test for detection of emetic Bacillus cereus Singlepath does not detect Emetic toxin but a protein which is co-produced with Emetic Toxin in B. cereus = Surrogate Marker. For screening of foods (tested with soup, rice) For confirmation of emetic B. cereus on agar plates Test successfully evaluated by Kagawa Food Univ. Japan NOW available!!!! Singlepath Emetic is already standard method for testing UHT milk in Malaysia) 87

Test Protocols Screening of B. cereus in foods Duopath Cereus Enterotoxins AND / OR Singlepath Emetic Toxin MRK Day 1 Rapid Screening (>100 CFU / g) 10 g/ml sample into 90 ml CGY Broth + 1% Glucose or BHI Sensitive Screening (>1 CFU / g) 200 µl 20 ml CGY + 1% Glucose 18 24 h at 30 C 200 µl 18 24 h at 30 C 6 h at 30 C in 20 ml CGY + 1% Glucose Day 2 NO B. cereus not present 150 µl 150 µl YES B. cereus present 88 Emetic B. cereus not present Emetic B. cereus present

Confirmation: B. cereus by Duopath Cereus Enterotoxins Emetic B. cereus by Singlepath Emetic TOX MRK suspend 1-3 presumptive colonies into 1 ml CGY Broth + 1% Glucose, 4 h at 30 C Day 1 NO 150 µl M.Y.P. Agar YES B. cereus not present B. cereus present Emetic B. cereus not present Emetic B. cereus present 89

Clostridium perfringens detection General Anaerobic, Gram-positive, spore-forming rod Widely distributed in the environment and frequently in the intestines of humans and domestic and feral animals Spores persist in soil, sediments, and areas subject to human or animal fecal pollution - heat resistant and not killed by ordinary cooking Poisoning associated with temperature abuse of prepared foods Growth in foods with high starch or protein content, such as cooked beans, meat products, thick soups, and gravy 8 22 h after consumption of foods containing large numbers of bacteria capable of producing food poisoning toxins Pre-enrichment: Thioglycolate broth PCR detection: Positive reference C. perfringens ATCC 13124 90

Norovirus detection General Norovirus particles with diameter of ~ 35 nm, highly stable in environment Capside & a single strand RNA, 7300 7700 nucleotides Cause of 90 % of non bacterial gastroenteritis Outbreaks in community facilities, e.g. hospitals, rest or nursery homes Symptoms: vomiting, diarrhoea and nausea Minimal infection dose is 10-100 virus particles Food incl. shellfish (oysters, clams, mussels), salads, peeled fruits, cold meats, sandwiches, dairy products, egg dishes, ice and fecally contaminated water or of surfaces of foods and food preparation Pre-enrichment: None, direct detection RT-PCR detection: Genotypes of genogroup I & genogroup II, dominant in outbreaks 91

Staphylococcus aureus detection General Facultative anaerobic Gram-positive coccus, which is non-motile, catalase and coagulase positive. Some strains produce staphylococcal enterotoxins (SEs), the causative agents of food poisoning. Common symptoms: nausea, vomiting, retching, abdominal cramping, and diarrhe.a Exist in air, dust, sewage, water, milk, and food, as well as on food equipment, environmental surfaces, humans, and animals. Able to grow in a wide range of temperatures (7 48.5 C, with an optimum of 30 37 C), ph (4.2 to 9.3, with an optimum of 7 7.5) and sodium chloride concentrations (up to 15% NaCl) Foods include meat and meat products, poultry and egg products, salads, bakery products (e.g. cream-filled pastries, cream pies, and chocolate eclairs), sandwich fillings and milk and dairy products Pre-enrichement : Tryptone soya broth (TSB) / PCR detection : MRSA Typ I, II, III, IV, IVa, IVb,IVc and other S. aureus species, not detection of other Staphylococci 92

Vibrio cholerae detection General Gram-negative, asporogenous, motile rods, straight or comma-shaped Strictly aqueous organism, brackish and marine waters are natural environments Transmission is fecal-oral, indirectly via polluted water supplies or irrigation water or contaminated shellfish that is raw or undercooked Infection of the small intestines caused by cholera enterotoxin (CT) producing Vibrio cholerae of serogroups O1 and O139 Mild symptoms, 10% of patients may experience classical cholera symptoms with profuse watery diarrhea ( rice-water stool ) and often vomiting Pre-enrichment: Alkaline peptone water with 2% sodium chloride (APW) PCR detection: Positive reference V. cholerae Mop 413 & 415 - Mop a putative protease modulating virulence, no detection of other Vibrio species 93

Questions? Veronica.gregoret@merck.com Mariano.veltri@merckgroup.com Muchas Gracias 94