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

MorphologyGrowth CharacteristicsSerotypingPhage typingBacteriocin Biochemical Characteristicsphenol 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 StainBiologVitek

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 2-3 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 16S Sequencing

Results Sterility test positive

Paenibacillus plyymxya 99%

Bacillus pumilus 99% (R) Bacillus pumilus 100% (F)

Tester isolate Bacillus subtilis 93%

Bacillus pumilus 99% (R) Bacillus pumilus 98%(F)

Environmental isolate

Paenibacillus macerans 97%

Bacillus circulans (R) 99% Bacillus circulans(F) 98%

Environmental isolate

Paenibacillus pabuli 100%

Paenibacillus pabuli (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 2Number 16S Sequencing ID

% Relatedness (Forward and Reverse)

Area Location Test BoxAIR PCR

1. M. luteus 99% F - 99% R Micro R400P Trays Different Strain

2. M. luteus 99% F - 100% R Micro R400P Trays Different Strain

3. M. luteus 99% F - 99% R Micro R400P Sterilins Different Strain

4. M. luteus 99% F - 100% R Micro R400P Drawers Different Strain

5. M. luteus 99% F – Reverse not tested

Micro R400P 30-35 Degree Inc

Different Strain

6. M. luteus 100% F - 99% R Micro R400P 30-35 Degree Inc

Different Strain

7. M. luteus 99% F – sequencing failure R

Micro R400P 30-35 Degree Inc

Different Strain

8. M. luteus 99% F - 100% R Micro R400P Test tube racks

Different Strain

9. M. luteus 99% F - 99% R Micro R400P Test tube racks

Different Strain

10. M. luteus 99% F - 100% R Micro R400P Wrists and Cheeks

Different Strain

11. M. luteus 99% F - 99% R Micro R404 Grey Floor Different Strain

12. M. luteus 99% F - 100% R Micro IPA Composite sprayer

Different Strain

17. M. luteus 99% F - 99% R Sterility Positive Day1

N/A N/A Identical

18. M. luteus 99% F - 99% R Sterility Positive Day 14

N/A N/A Identical

Pharmaceutical Case Study Investigation 2

BoxAIRM 1 2 3 4 6 7 8 9 10 11 12 13 14 16 17 18 M

PCR

Sequencing Method

Automatic Sequencing

Species Level ID

411 completed bacterial genomes

Sequencing rRNA Genes

Structural

Conserved regions

Universal

Not laterally transferred

How is Ribosomal DNA Sequencing Carried Out?

• Ribosomal 18S/16S or 28S/23SSequencing

0

3.6

2

Penicillium corylophilum.seqProbe 1.seqPenicillium hirsutum.seqPenicillium chrysogenum.seq

Pre-analysis by BlastN

Commercial Systems – Species

level IDSpecies Level IDsMicroseq – Applied Biosystems

Sherlock (DNA) – Midi Inc.

Riboprinter (strain level?) – DuPont Qualicon

Bax (food pathogens, 0157 etc. ) – DuPont Qualicon

Microseq and Sherlock-DNA

3 Databases Fungal – D2 region of large rRNA subunitBacterial – 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 differentiationCommercial SystemRibotytping-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 DNAb) amplified selected genes via

PCR

Example of a PFGE Analysis on Staphylococcus aureus.

PFGE

                                                                                                        

                                                     

 

                                                              

                            Figure 2. Example of a real PFGE; drug

resistant Staphylococcus aureus. The molecular weight markers are digested

lambda phage (λ) and are given in kb. See the CDC web page with the original data .

 

BOXAIR-PCR

BOX-PCR (Amplification of BOX-cassettes)Rapid detection method with high reproducibility.

Commercial System - Strain level ID

Riboprinter

adheres to good manufacturing practice (CGMP) regulations (21 CFR part 11 for audit trails)

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