Transcript Pathogen workshop - lamsam

Bugs to Bar Codes in 8 hours
Ann Krywiuk
Molecular Microbiology Specialist
Agenda
• Introduction to RiboPrinter®
• Process overview
• Results
• Applications
• Summary
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RiboPrinter system from DuPont
Qualicon, distributed by ThermoFisher
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
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What Is Ribotyping?
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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
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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.
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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.
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Differences Produce Below-Species Differentiation
Strain #1
Strain #2
B
B
D
D
C
C
A
Increasing
Fragment
Size
A
E
E
}
}
Conserved
fragments
Variation
same 16S sequence; different RiboPrint™ patterns
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Detection – Raw Image
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RiboPrint™ Pattern Automatically Generated for Analysis
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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
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RiboPrint™ Patterns Clearly Demonstrate Genera Differentiation
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RiboPrint™ Patterns Provide Conserved Characters that Show
Species Variation
Listeria
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RiboPrint™ Patterns Show Non-Conserved Characters that Discriminate
Below the Species Level
Listeria monocytogenes
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RiboPrinter® System Batch Report
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Ribotyping
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Ribotyping
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How the RiboPrinter® System Works
Automated processing
•DNA Preparation
•Separation and Transfer
•Membrane Processing
•Detection
Automated analysis
•Identification
•Characterisation
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Automated Pattern Analysis - Identification
• Incoming pattern is
compared to a database
of known reference
patterns
• Match provides accurate
sample identification
0.85 similarity
threshold
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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
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RiboPrinter® System Batch Report
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Tracking Contamination in Production
• S. epidermidis
(Personnel Hands)
• S. epidermidis
(Raw Material)
• S. epidermidis
(Finished Product)
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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
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Solutions for Detection, Tracking, and Tracing
Environmental Sample
Final Product
Pre- Op Sample
Air Sample
Foot Matt
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A
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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
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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
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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.
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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
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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.
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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.
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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.
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Solving Post-therapy Infection Questions
Pre-therapy
isolate
Post-therapy
isolate
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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
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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
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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
• Enterobacteriaceae
• Staphylococcus aureus
• Coagulase-negative staphylococci
• Pseudomonas aeruginosa
• Streptococcus pneumoniae
• Acinetobacter baumanii
• Emerging trend - antibiotic resistance
• methicillin-resistant staphylococci
• vancomycin-resistant enterococci
• aminoglycoside-resistant
Pseudomonas species
• Common Hospital Infection Control Practices (HIC) include clinical
observation and microbiological analysis for ID & susceptibility
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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
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Immediate reporting of
results to infection control
practitioners
Focused action,
i.e., intervention
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
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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.
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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.
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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.
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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
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Thank you
Ann Krywiuk – Molecular Microbiology Specialist