Whats in a Name The Advantages of Genotypic Microbial ID

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Whats in a Name?The Advantages of Genotypic
Microbial ID
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Overview

• Microbial ID Programs
• Technologies
• Phenotypic vs. Genotypic
• Regulatory Considerations
• Introduction to RiboPrinter
• Microbial Mapping
• Microbial Source Tracking
• Applications

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Micro ID Program Objective

• To provide information that will help exclude
  microbes from products
• It is very useful in
• Investigating microbial contamination
• Finding the root cause
• Determining corrective actions

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Characterization to Genus and Species

• EM isolates in ISO 5 areas
• Alert and action level isolates
• Water
• Pharmaceutical ingredients
• Finished products
• EM

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Critical Micro ID Program

• Media fill failures
• Sterility test failures
• Microbial Limit Test failures
• Product stability failures
• Product complaints

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PhenotypicMicrobial ID Methods

• Utilize expressed gene products to distinguish
  among different organisms
• Morphological
• Biochemical
• Chemo-Taxonomic

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Nucleic Acid BasedMicrobial ID Methods

• Microbial genotype characteristics of highly
  conserved regions employed
• Genotypic
• Restriction fragment patterns
• Phylogenic
• 16S RNA sequences

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Phenotypic ID Methods

• Growth-Based
• Measure biochemical or physiological parameters
  that reflect growth of microorganisms
• Artifact-Based
• Detect cellular composition of microbes grown on
  specified media

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Growth-Based IDBiochemical

• API
• Packaged strips containing substrates
• 20E
• 20NE
• Staph
• Strep
• Coryne
• NH
• Anaerobes
• Yeast

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Growth-Based IDBiochemical (cont.)

• Vitek
• Miniaturized API concept transferred onto cards
  coupled with an automated incubation and reading
  system
• ANI Anaerobes Micrococcus
• BAC Bacillus
• GPI Gram()
• GNI/GNI Gram( -) Oxidase( -)
• NFC Gram( -) Oxidase()
• YBC Yeast

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Biochemical Tests Process

• Day 0 -Subculture
• Day 1 -Gram stain
• -Preliminary tests
• -Suspension preparation
• -Set up API strip or Vitek card
• -Incubate
• Day 2 -Read API strip or Vitek report
• Day 3 -Read API strip and interpret results

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Artifact Based ID

• MIDI (Sherlock Microbial Identification System)
• Harvest 24 hr culture
• Extract Preparation
• Saponification
• Methylation
• Extraction
• Wash
• GC Analysis
• Pattern Recognition

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Nucleic Acid Based IDRibosomal RNA Sequencing

• MicroSeq
• Microbial DNA is amplified
• First 500 base pairs of the 16S rRNA gene are
  sequenced
• Sequence is compared to a 16S rDNA database

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MicroSeq Process

• Microbial isolate plated
• DNA extraction (1 hr)
• PCR (3 hrs)
• Preparation of Sequencing Reactions (1 hr)
• Cycle Sequencing (10-15 hrs)
• Analysis (1 hr)
• Journal of Clinical Microbiology Vol.36,No.12
  P.3674, 1998

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Nucleic Acid Based ID (Ribotyping)

• RiboPrinter (Automated - 8hrs)
• Bacterial DNA cut into fragments
• Separation by gel electrophoresis
• Hybridization with DNA probe
• Chemiluminescent agent added
• Fragments pattern digitized
• RiboPrint compared to patterns database

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Phenotypic vs. Genotypic

• Phenotypic
• Dependent on growth conditions
• Subjective
• Time consuming
• API - 2-3 days
• Vitek - 2 days
• MIDI - 3-4 days

• Genotypic
• Independent of growth conditions
• More reliable
• Fast
• Ribotyping - 8.5 hrs
• 16s Sequencing - 21 hrs

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Microbial ID Methods Database

• API 600
• Vitek 800
• MIDI 2000
• MicroSeq 2,400
• Accugenix 2,151
• RiboPrinter 6,000
• USP Pharmacopeial Forum Vol.30(5)2004
• Microbial Identification in the Pharaceutical
  Industry

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Limitations of Phenotypic Methods

• Labor intensive protocols
• Need for multiple systems (Gram -/)
• Limited ability to differentiate microbes below
  species
• Results often unreliable and irreproducible
• Subjective interpretation
• Limited databases

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Ongoing Round-Robin Study

• 23 isolates sent to 40 labs for phenotypic ID
• Only 3 were consistently identified
• Up to 9 different genera reported for a single
  isolate
• Duplicate samples sent to 36 labs
• 21 labs could not tell the strains were the same
• 10 labs reported different genera
• 10 labs reported the same genus but different
  species
• Organized by Dr. Clive Thompson of Yorkshire
  Environmental, U.K.

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Regulatory Considerations
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Sterile Drug Products Produced by Aseptic
Processing - FDA Guidance for Industry 2004

• Genotypic methods have been shown to be more
  accurate and precise than traditional biochemical
  and phenotypic techniques. These methods are
  especially valuable for investigation into
  failures (e.g. sterility test, media fill
  contamination)

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Sterile Drug Products Produced by Aseptic
Processing - FDA Guidance for Industry 2004

• Microbiological monitoring data should be
  reviewed to determine if the organism is also
  found in the laboratory and production
  environments, personnel, or product bioburden.
  Advanced identification methods (e.g. nucleic
  acid based) are valuable for investigational
  purposes. When comparing results from
  environmental monitoring and sterility positives,
  both identifications should be performed using
  the same methodology.

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USP lt71gt Sterility Test

• To invalidate a positive sterility test
• After determination of the identity of the
  microorganisms isolated from the test, the growth
  of this species may be unequivocally ascribed to
  faults with respect to material and/or technique
  used in conducting a sterility procedure

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How a Microbiologist Views Bacteria
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How Manufacturing and QA View Bacteria
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RiboPrinter System
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RiboPrinter System

• DuPont Qualicon
• Characterization capability
• Fully automated process
• Results in 8 hours
• Fully automated data analysis
• no subjective data interpretation
• flexible search, sort features reports

t
t
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Ribotyping
Based on ribosomal RNA genes

• Highly conserved regions
• Very stable
• Variable number and position

Listeria monocytogenes
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Loading the Disposables
DNA prep pack
Sample carrier
Membrane
Gel cassette
Membrane processing base with inserts
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How Does it Work?

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

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Automated Membrane Processing
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Digitized Image
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Image Processing
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Output Strain Level Genetic Fingerprinting
RiboPrint Pattern
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Ribotyping Probe

• Derived from E. coli rRNA ribosomal operon
• Approximately 6.5 kB
• Contains sequences encoding for
• 16S rRNA
• Spacer region including Glu-tRNA
• 23S rRNA
• 5S rRNA

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Ribotyping Using EcoR1
PROBE
5S
23S rRNA
16S rRNA
Gene Z
Gene X
EcoRI Sites
A
B
C
D
E
B
RiboPrint pattern generated by EcoRI restriction
of one ribosomal RNA operon
D
Increasing Fragment Size
C
A
E
3.1
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Principles of Ribotyping
STRAIN 1
PROBE
5S
23S rRNA
16S rRNA
Gene Z
Gene X
Restriction Sites
A
B
STRAIN 1
RiboPrint pattern generated using an infrequent
cutter (one ribosomal RNA operon shown)
B
Increasing Fragment Size
A
5
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Principles of Ribotyping (cont.)
STRAIN 2
PROBE
5S
23S rRNA
16S rRNA
Gene Z
Gene X
Restriction Sites
A
B
STRAIN 2
STRAIN 1
Two strains, using same enzyme, generate
different RiboPrint patterns
B
B
Increasing Fragment Size
A
A
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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
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Using Multiple Enzymes

• Standard enzymes are EcoRI PvuII
• Other enzymes can be used
• to cut organism DNA not amenable to EcoRI or
  PvuII digestion
• when even greater discrimination is needed

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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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DuPont ID Database

• Contains over 6448 RiboPrint patterns
  representing 194 genera, and over 1500
  species/serotypes
• Updates taxonomy consistent with the
  International Journal of Systemic and
  Evolutionary Microbiology
• Captures all taxonomic changes in audit trail

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Automated Pattern Analysis Characterization

• Provides information below the species level
• Allows comparison of isolates without knowing the
  names
• Tells you if isolates are the same or different

0.90 sim
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Identification Characterization
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RiboPrinter System Batch Report
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Data Management
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Automated Nearest Neighbor Searches
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Sample Pattern Comparisons
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RiboGroup Clustering of Identical Strains
(Strain History)
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RiboPrint Patterns Clearly Demonstrate Genera
Differentiation
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RiboPrint Patterns - Conserved Regions Show
Species Variation
Listeria
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RiboPrint Patterns - Non-Conserved Regions
Discriminate Below the Species Level
Listeria monocytogenes
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Ribotyping Dual Benefit

• Speciation Based on information from conserved
  regions
• Strain tracking - Full operon/intergenic
  information to differentiate below the species
  level

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Microbial Mapping
Products
Environment
Microbial mapping using your own custom database
Personnel
Equipment
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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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Quality Control

• Routine IDs, Microbial Mapping, OOS
  Investigations
• Final Products
• Raw Materials
• Environment/Water

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Custom Database Building

• Methylobacterium radiotolerans
• Biofilm Bug
• Product Contaminant 2003
• EM Isolate 4
• Control Strain

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Increasing Lab Efficiency

• Reduces/eliminates reliance on data log books
• no searching through notebooks to reference
  source information
• Viewing data from different angles brings trends
  to surface, especially when you are not sure what
  you are looking for

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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 appropriate corrective
  actions

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Microbial Mapping
Microbial Mapping involves creating a sampling
plan by sampling hot spots, sites of probable
cross-contamination, evaluating interventions and
pathways of movement through a plant, a process,
or a value chain.
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Focused Process Mapping
Mapping onto critical parts of the process
enables people to see how contamination can
occur.
Staphylococcus warneri
Staphylococcus epidermidis
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More Efficient Corrective Action Strategies
Microbial Mapping looks at the locations of
matched genetic fingerprints and pathways of
movement through a plant, a process, or a value
chain
Bacillus subtilis
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Analyzing Process Flow

By analyzing the processes and flow you can trace
how bacteria move for example wheels of
equipment, raw and finished product, people
traffic patterns
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Tracking Sources of Contamination
S. epidermidis (Personnel Gloves) S.
epidermidis (Raw Material) S. epidermidis
(Finished Product)
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Putting Your Data to Work

• Accurate, Reproducible, Strain level Data
• Linked Sample Source Information
• Powerful, Easy To Use, Data-base Analysis
  Software
• Improved Understanding
• Improved Efficiency
• Reduced Incidents
• Reduced Testing

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Other Applications

• Assess efficacy of sanitization programs
• Research and development
• Ensure culture integrity
• Clinical trial support
• Expedite accurate decision making

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Whats in a Name?

• Unless it has been assigned by reproducible,
  definitive DNA typing, the organism might have
  the wrong name
• Gene-based IDs reliable regardless of name

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• Thank You
• QA
• Pacific BioLabs
• [phone]
• info_at_PacificBioLabs.com