Molecular Identification
Methods
Confirmation of identity for
commonly used laboratory strains
should ideally be done at the level
of genotypic analysis’…...
Pharmacopeial Forum, Volume 30 (5)
2004 Microbiological Best
Laboratory Practices, Page 1717)
Overview
Current phenotypic methods/
Problems
Molecular case studies in
industry
PCR/Sequencing techniques
•
•
Species level ID and
Commercial systems
Strain level ID and
Commercial system
Phenotypic Methods
Morphology
Growth Characteristics
Serotyping
Phage typing
Bacteriocin
Biochemical Characteristics
phenol red carbohydrate, catalase production, oxidase
production, tests, methyl red test, nitrate
reduction,starch hydrolysis, tryptophan hydrolysis,
hydrogen sulfide production, citrate utilization, and litmus
milk
Microbiological Analyses
Create microbe
on plate
Streak on
fresh plate
Gram Stain
Vitek
Biolog
Problems With
Microbiological Testing in
QA Laboratories
• Gram staining, messy, time
consuming
• Biolog/Vitek time consuming 2-3
days
• Large amount of isolates
unidentifiable
• Only identifiable to genus, species
level
• Some non-specific, moulds take 23 weeks, many repeats
Microbiological Testing
in Industry
How can Molecular Methods
Improve Testing?
•
•
•
•
•
Sterility testing –
Environmental testing –
Raw material testing –
Water testing –
Personnel monitoring –
When Genotyping
Necessary?
Sterility Test positive……
Requires
Investigation, corrective and
preventative action (best case)
Pharmaceutical Case
Study Investigation 1
Result - Biolog
Sterility test
positive
Paenibacillus
plyymxya 99%
Tester isolate Bacillus subtilis
93%
Environmental Paenibacillus
isolate
macerans 97%
16S Sequencing
Results
Bacillus pumilus 99%
(R)
Bacillus pumilus 100%
(F)
Bacillus pumilus 99%
(R)
Bacillus pumilus
98%(F)
Bacillus circulans (R)
99%
Bacillus circulans(F)
98%
Environmental Paenibacillus pabuli Paenibacillus pabuli
isolate
100%
(R) 98%
Paenibacillus pabuli
(F) 98%
Pharmaceutical Case
Study Investigation 1
PFGE
• The PFGE
results showed
identical
banding
patterns by
not1 digest and
are the same
strain of
Bacillus pumilus
Pharmaceutical Case
Study Investigation 2
Number
% Relatedness
(Forward and
Reverse)
99% F - 99% R
Area
Location
Test
BoxAIR
PCR
1.
16S
Sequencing
ID
M. luteus
Micro
R400P
Trays
2.
M. luteus
99% F - 100% R
Micro
R400P
Trays
3.
M. luteus
99% F - 99% R
Micro
R400P
Sterilins
4.
M. luteus
99% F - 100% R
Micro
R400P
Drawers
5.
M. luteus
99% F – Reverse
not tested
Micro
R400P
6.
M. luteus
100% F - 99% R
Micro
R400P
7.
M. luteus
Micro
R400P
8.
M. luteus
99% F –
sequencing
failure R
99% F - 100% R
Micro
R400P
9.
M. luteus
99% F - 99% R
Micro
R400P
10.
M. luteus
99% F - 100% R
Micro
R400P
11.
M. luteus
99% F - 99% R
Micro
R404
30-35
Degree
Inc
30-35
Degree
Inc
30-35
Degree
Inc
Test tube
racks
Test tube
racks
Wrists and
Cheeks
Grey Floor
Different
Strain
Different
Strain
Different
Strain
Different
Strain
Different
Strain
12.
M. luteus
99% F - 100% R
Micro
IPA
17.
M. luteus
99% F - 99% R
18.
M. luteus
99% F - 99% R
Sterility
Positive
Day1
Sterility
Positive
Day 14
Different
Strain
Different
Strain
N/A
Composite
sprayer
N/A
Different
Strain
Different
Strain
Different
Strain
Different
Strain
Different
Strain
Identical
N/A
N/A
Identical
Pharmaceutical Case
Study Investigation 2
BoxAIR
M
M
1
2
3
4
6
7
8
9
10
11
12
13
14
16
17
18
PCR
Sequencing Method
Automatic Sequencing
Species Level ID
Sequencing rRNA Genes
411
completed
bacterial
genomes
Structural
Conserved regions
Universal
Not laterally
transferred
How is Ribosomal DNA
Sequencing Carried Out?
• Ribosomal 18S/16S or 28S/23S
Sequencing
Pre-analysis by BlastN
Penicillium corylophilum.seq
Probe 1.seq
Penicillium hirsutum.seq
Penicillium chrysog enum.seq
3.6
2
0
Commercial Systems
– Species level ID
Species Level IDs
Microseq – Applied Biosystems
Sherlock (DNA) –
Midi Inc.
Riboprinter (strain level?) – DuPont
Qualicon
Bax (food pathogens, 0157 etc. )
– DuPont Qualicon
Microseq and SherlockDNA
3 Databases
Fungal – D2 region of large rRNA subunit
Bacterial – 16S full sequence (1500bp), 16S
partial sequence (500bp)
Microseq Software and Database adheres to
good manufacturing practice (CGMP)
regulations (21 CFR part 11 for audit trails)
Strain Level IDs
•
•
•
•
Sequencing ITS region
- Strain level identification
RFLP Restriction Fragment Length Polymorphism
– Identification and differentiation
PFGE Pulse Field Gel Electrophoresis
– Epidemiological studies
BoxAIR-PCR BoxA Inverse Repeat PCR
– Identification and differentiation
Commercial System
Ribotytping
-RFLP principle
Sequencing to Strain Level
Identification
• ITS (Internal transcribed
spacer regions)
•
between 18S and 28S
• Or 16S and 23S
RFLP
RFLP (Restriction Fragment Length
Polymorphism)
Restriction digest of
a) direct extracted DNA
b) amplified selected genes via PCR
Example of a PFGE Analysis on
Staphylococcus aureus.
PFGE
BOXAIR-PCR
BOX-PCR (Amplification of BOX-cassettes)
Rapid detection method with high reproducibility.
Commercial System Strain level ID
adheres to good
manufacturing
practice (CGMP)
regulations (21
CFR part 11 for
audit trails)
Riboprinter
Ribotyping
•
Riboprinter Batch
System Report
Summary
• Species Level
ID
• Sequencing
16S/5S/23S
genes
(bacteria)
• Sequencing
18S/5.8S/28S
(fungal)
• Strain Level ID
• Sequencing ITS region
• PFGE Pulse Field Gel
Electrophoresis
• RFLP Restriction Fragment
Length Polymorphism
• BoxAIR-PCR BoxA Inverse
Repeat PCR
• Ribotytping