Bugs to Bar Codes in 8 hours

1
Bugs to Bar Codes in 8 hours

• Ann Krywiuk
• Molecular Microbiology Specialist

2
Agenda

• Introduction to RiboPrinter
• Process overview
• Results
• Applications
• Summary

3
RiboPrinter system from DuPont Qualicon,
distributed by ThermoFisher
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
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
Ribotyping Example Using EcoRI
STRAIN 1
EcoRI cuts the DNA into different sized fragments
(A E) according to the location of the
restriction sites that match the enzyme.
7
Ribotyping Example Using EcoRI
STRAIN 2
The same enzyme cuts the DNA of each individual
isolate into a different number or size of
fragments for differentiation.
8
Differences Produce Below-Species Differentiation
Strain 1
Strain 2

B
B
D
D
Conserved fragments
C
C

A
A
Variation
Increasing Fragment Size
E
E
same 16S sequence different RiboPrint patterns
9
Detection Raw Image
10
RiboPrint Pattern Automatically Generated for
Analysis
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
RiboPrint Patterns Clearly Demonstrate Genera
Differentiation
13
RiboPrint Patterns Provide Conserved Characters
that Show Species Variation
Listeria
14
RiboPrint Patterns Show Non-Conserved Characters
that Discriminate Below the Species Level
Listeria monocytogenes
15
RiboPrinter System Batch Report
16
Ribotyping
17
Ribotyping
18
How the RiboPrinter System Works

• Automated processing
• DNA Preparation
• Separation and Transfer
• Membrane Processing
• Detection
• Automated analysis
• Identification
• Characterisation

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
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
RiboPrinter System Batch Report
22
Tracking Contamination in Production

• S. epidermidis(Personnel Hands)
• S. epidermidis(Raw Material)
• S. epidermidis(Finished Product)

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
Solutions for Detection, Tracking, and Tracing

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
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
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
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
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.

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 11th 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
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
Solving Post-therapy Infection Questions
Pre-therapy isolate
Post-therapy isolate
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
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
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
Molecular epidemiology in an integrated HIC
program

• 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.

• Proactive surveillance for rapid, systematic
  determination of clonality

Immediate reporting of results to infection
control practitioners
Focused action, i.e., intervention
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
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
RiboPrinter System proves competitive results
incorrect and product recall is avoided

• 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

• 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
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 didnt 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
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