1. Bugs to Bar Codes in 8 hours Ann Krywiuk Molecular Microbiology Specialist

2. 2 Agenda Introduction to RiboPrinter® Process overview Results Applications Summary

3. RiboPrinter system from DuPont Qualicon, distributed by ThermoFisher

4. 4 Simultaneously Identifies and Characterises Virtually Any Bacterium Results identify samples below the species level Identifies bacteria regardless of environmental stresses, gram stain results or media and growth conditions Ability to differentiate virtually any bacterium beyond the species level Learning database increases continuously Definitive, DNA-based results Based on scientific data Little subjective data interpretation

5. 5 What Is Ribotyping? Method of “fingerprinting” genomic DNA Based on differences in ribosomal RNA genes Stable, highly conserved regions Variable number and position Focuses on restriction fragments that contain all or part of the genes coding for the 16S and 23S rRNA Variations in position and intensity of rRNA bands are used to identify and classify the bacteria Listeria monocytogenes

6. 6 Ribotyping: Example Using EcoRI EcoRI cuts the DNA into different sized fragments (A – E) according to the location of the restriction sites that match the enzyme. STRAIN #1

7. 7 Ribotyping: Example Using EcoRI The same enzyme cuts the DNA of each individual isolate into a different number or size of fragments for differentiation. STRAIN #2

8. 8 Differences Produce Below-Species Differentiation B D C E Strain #1 B A E C Strain #2 same 16S sequence; different RiboPrint ™ patterns A D } } Conserved fragments Variation Increasing Fragment Size

9. 9 Detection – Raw Image

10. 10 RiboPrint™ Pattern Automatically Generated for Analysis

11. 11 Data Analysis: Identification Incoming patterns compared to a continually improving database of known reference patterns Pattern upgrades are supplied periodically (uploaded from CD) Collaborations with leading pharma companies and culture collections including ATCC, DSMZ (Germany) and Riken (Japan) Match provides accurate sample identification - Similarity threshold is 0.85

12. 12 RiboPrint™ Patterns Clearly Demonstrate Genera Differentiation

13. 13 RiboPrint™ Patterns Provide Conserved Characters that Show Species Variation Listeria

14. 14 RiboPrint™ Patterns Show Non-Conserved Characters that Discriminate Below the Species Level Listeria monocytogenes

15. 15 RiboPrinter® System Batch Report

16. 16 Ribotyping

17. 17 Ribotyping

18. 18 How the RiboPrinter ® System Works Automated processing DNA Preparation Separation and Transfer Membrane Processing Detection Automated analysis Identification Characterisation

19. 19 Automated Pattern Analysis - Identification Incoming pattern is compared to a database of known reference patterns Match provides accurate sample identification 0.85 similarity threshold

20. 20 Automated Pattern Analysis - Characterisation Allows you to compare samples without knowing the identity of the organisms Provides information below the species level Tells you if samples are the SAME or DIFFERENT

21. 21 RiboPrinter® System Batch Report

22. 22 Tracking Contamination in Production S. epidermidis (Personnel Hands) S. epidermidis (Raw Material) S. epidermidis (Finished Product)

23. 23 APPLICATION - QA/QC Map microbial ecology of production facilities Routine monitoring of microbial flora in production facility for subtle changes Address sterility failures by tracking sources of contamination Develop strategies for corrective actions

24. 24 Solutions for Detection, Tracking, and Tracing A 20 19 46 12 13 45 48 47 5 4 56 6 49 3 18 38 17 22 51 7 23 50 30 31 40 21 37 2 28 26 43 34 35 10 11 15 55 33 27 1 3932 Final Product Pre-Op Sample Air Sample FootMatt 54 5352 24 Environmental Sample

25. 25 Importance of Strain Level Mapping Identifies the normal ecology within a facility as well as the incoming raw materials by creating a library of patterns Surveillance from environmental/product samples allows historical threats to be spotted quickly reducing risk Allows for root cause identification of source to be pinpointed through matching fingerprints Provides information for effective corrective actions

26. 26 Enterobacter sakazakii in infant formula Problem A European producer of infant formula found that its finished product was consistently contaminated with low levels of Enterobacter sakazakii The company realised that the bacteria could grow rapidly in reconstituted milk, and they wanted to understand how the product became contaminated A survey of raw materials and the environment at each site yielded several hundred samples of Enterobacter sakazakii The QC team performed some preliminary screening using biochemical methods, but those data were not helpful in tracking the contamination

27. 27 Enterobacter sakazakii in infant formula Action The team decided to use the RiboPrinter® Microbial Characterisation System for more information The RiboPrinter® system was able to discriminate among many of the Enterobacter sakazakii strains below the species level, which allowed the company to learn how the contamination was being spread throughout the factories. For example, 30 strains of the organism found in one factory clustered into 8 RiboGroup pattern sets By examining this level of characterization and identification, the investigators could see which strains were confined to a single site and which were common to all three factories.

28. 28 Enterobacter sakazakii in infant formula Important trends became evident The use of portable vacuum cleaners presented a cross-contamination hazard Personnel movement was a factor in the transfer of some strains of Enterobacter sakazakii “Resident" florae of a particular type of Enterobacter sakazakii were able to evade standard cleaning and sanitation regimes. This source of contamination was considered significant Result The company changed its cleaning practices to address these issues The QC team monitors and recognizes that changes in the subspecies florae may indicate a problem in process or hygiene practices The team can then quickly direct cleaning crews and engineers to the area of concern

29. 29 Enterobacter sakazakii in infant formula The RiboPrinter® system was invaluable in helping this business quickly, definitively address the source and route of contamination throughout its complex manufacturing operation. The plant environment yielded a number of strains of Enterobacter sakazakii. The distinctiveness of their RiboPrint® patterns allowed the manufacturer to track the sources of contamination.

30. 30 Information to Support Clinical Trials with FDA Alcon manufacturers high-quality eye care products and therapies. By using RiboPrinter® instead of traditional biochemical processes, they are able to pinpoint contamination and determine treatment efficacy more accurately and with more confidence. In a comparison study, researchers characterized 11 ocular isolates of Corynebacterium identified as sp. pseudodiphteriticum by biochemical methods (API and Biolog). RiboPrint patterns characterized 10 of the isolates into 4 ribogroups, while the 11 th sample was determined to not be pseudodiphteriticum at all. During a sterility test failure, two isolates of staph were identified as S. haemolyticus using a fatty-acid profile (MIDI) and Vitek. One sample was from the sterility test and the other from a line workers lab coat. The RiboPrinter verified the samples as S. haemolyticus, but also as two different strains which shows the failed test was not the result of contamination by the worker.

31. 31 Information to Support Clinical Trials with FDA To measure treatment efficacy, three ocular isolates from 2 different hospitals were identified as 3 different strains of Streptococcus pneumoniae by the RiboPrinter. This indicates the two isolates from the same facility were from different sources. Post-therapy patterns matched the pre-therapy patterns in all three cases, indicating the treatment was not effective rather than a re-infection. Conversely, pre- and post-treatment nosocomial isolates from a conjunctivitis patient were identified as Staphylococcus epidermidis by biochemical testing. RiboPrint patterns confirmed the test and identified the two samples as different strains, ruling out the possibility of a drug-resistant strain of bacteria.

32. 32 Solving Post-therapy Infection Questions Post-therapy isolate Pre-therapy isolate

33. 33 Understanding and Controlling Multi-site Contamination A global manufacturer of biotech therapies observed a sudden and dramatic spike in contamination across 3 different manufacturing sites Traditional biochemical testing indicated the contamination was the same genus and species across all three sites Assumed 3 different strains and each site initiated separate investigation to determine the source Newly installed RiboPrinter showed QC department that a single strain was responsible for the contamination across the three sites Investigation quickly focused on those activities that all three sites had in common Environmental monitoring quickly identified the inter-site laundry service as the source of contamination

34. 34 Microbial Mapping to Proactively Manage Contamination Global pharmaceutical company uses RiboPrinter® systems on two continents to routinely monitor and manage the natural flora within their manufacturing and testing facilities Information is used to map the strains and locations of frequently occurring bacterial contaminants for use as a reference During an in-process sterility failure, the RiboPrinter was able to quickly ribotype the contaminant and determine the most likely source The water purification system was quickly identified as the source over within ~1 week. Lab management indicated the investigation could have easily spread over 4-5 months without the RiboPrinter and their ability to source track

35. 35 Nosocomial infections are a serious problem for hospitals Nosocomial infections contribute enormously to morbidity, mortality and excess costs among hospitalized patients, about $14B/year globally Major nosocomial pathogens Emerging trend - antibiotic resistance Common Hospital Infection Control Practices (HIC) include clinical observation and microbiological analysis for ID & susceptibility Enterobacteriaceae Staphylococcus aureus Coagulase-negative staphylococci Pseudomonas aeruginosa Streptococcus pneumoniae Acinetobacter baumanii methicillin-resistant staphylococci vancomycin-resistant enterococci aminoglycoside-resistant Pseudomonas species

36. 36 Molecular epidemiology in an integrated HIC program Proactive surveillance for rapid, systematic determination of clonality Immediate reporting of results to infection control practitioners Focused action, i.e., intervention Similar to other applications where environmental monitoring is crucial, the RiboPrinter can be a very valuable tool in helping hospitals monitor the level of natural flora and take effective corrective actions following an infection.

37. 37 Daniele, Inc. Relies on RiboPrinter® for Quality Assurance RiboPrinter System The RiboPrinter system allows Daniele to identify potential contamination sites and link them to bacteria found in other areas DeCesare used RiboPrinter to analyze microbial trends he was seeing in one plant. He traced the source to traffic issues and employee habits. By implementing new procedures and training, he resolved the trend in two weeks

38. 38 RiboPrinter® System proves competitive results incorrect and product recall is avoided Problem The makers of high-value, refrigerated and ready-to-use foods learned at 2 p.m. on a Friday that they could have a problem so serious it would be necessary to recall one of their products from four European countries. Over a three-day period, samples from the product had been tested using ELISA, latex agglutination, biochemical screening and a serology assay for examining antigens. All these tests told the company they had a Salmonella contamination. Product recall, company executives believed, was their only option.

39. 39 Action Before withdrawing product from the market, managers chose to have an independent laboratory confirm the contamination Confirming with BAX® System - By early Saturday, the BAX® assay had produced startling results: There were no Salmonella present in the tested samples. RiboPrinter® System Differentiates - Isolates were then run on the RiboPrinter® Microbial Characterization System to determine what the bacteria were. Within eight hours, RiboPrint® patterns of the isolate had been generated and matched to the system database pattern of Citrobacter freundii RiboPrinter® System proves competitive results incorrect and product recall is avoided RiboPrint® patterns for a common Salmonella species and Citrobacter freundii show how genetically similar the two organisms are. Biochemical tests could not distinguish between the pathogenic Salmonella and the innocuous Citrobacter.

40. 40 BAX® And RiboPrinter® Systems Prove Competitive Results Incorrect And Product Recall Is Avoided Result The company realised a savings of nearly $350,000 in lost product alone. This figure didn’t take into account untold savings in market share, customer/retailer confidence, press coverage and the overall impact on the industry that would have resulted from a product recall.

41. 41 Summary RiboPrinter® Identifies genus and species Characterises at strain level Allows tracking of same strain throughout plant to identify source of contamination Facilitates speedy implementation of corrective actions

42. Thank you Ann Krywiuk – Molecular Microbiology Specialist