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 Biolog Vitek

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

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

Pharmaceutical Case Study Investigation 2 BoxAIR M 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/23S Sequencing Pre-analysis by BlastN

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 Sherlock- DNA 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 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