Forensic microbiology: whodunnit? Corn HW Klaassen Dept. Medical - - PowerPoint PPT Presentation

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Forensic microbiology: whodunnit? Corn HW Klaassen Dept. Medical - - PowerPoint PPT Presentation

Dec 15, 2022 232 likes •528 views

Forensic microbiology: whodunnit? Corn HW Klaassen Dept. Medical Microbiology & Infectious Diseases Core activities MMIZ molecular diagnostics Detection Identification, Typing of potentially pathogenic micro-organisms

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Forensic microbiology: whodunnit?

Corné HW Klaassen

  • Dept. Medical Microbiology & Infectious Diseases
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Core activities MMIZ molecular diagnostics

  • Detection
  • Identification,
  • Typing of potentially pathogenic micro-organisms
  • Bacteria, fungi/yeasts, parasites

using molecular (DNA/RNA) based techniques

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Typing: a working definition

  • The ability to discriminate between different ‘individuals’ of the same

species

  • Not to confuse with ‘identification’: to determine to which species a

micro-organism belongs.

Shoot! A hole in my shoe! Damn! Now your footprint will discrminate you from the rest of us!

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Micro-organisms are easily spread

  • person to person contact!
  • surfaces, air, water
  • animals
  • (medical) devices (e.g. humidifiers, sterilization equipment)
  • food, drinks
  • contact lens fluids
  • etc.
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Rationale for typing

Clonal expansion or different ‘individuals’ of the same species?

  • Essential in potential outbreak scenario’s!
  • HAI impose a significant economical burden!
  • Efficacy of treatment / recurring infections?
  • Monitoring local, national or global spread of specific ‘clones’
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Molecular fingerprinting methods

  • ‘Old’ school (not in use anymore):
  • MLEE (multilocus enzyme electrophoresis)
  • PFGE (pulsed field gelelectrophoresis)
  • RAPD (random amplified polymorphic DNA)
  • AP-PCR (arbitrarily primed PCR)
  • ‘New’ school:
  • AFLP (amplified fragment length electrophoresis)
  • VNTR (variable number of tandem repeats)
  • Microsatellites / Short tandem repeats
  • MLVA (multilocus VNTR analysis)
  • MLST (multilocus sequence typing)
  • WGS (whole genome sequencing)
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Basic characteristics of typing methods

  • discriminatory power
  • typeability
  • reproducibility
  • speed
  • costs
  • ease of use
  • data interpretation, complexity
  • need for a (pure) culture, application directly on clinical specimens
  • genome coverage
  • species specific vs. generic
  • storage and exchange of data between labs
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Amplified Fragment Length Polymorphism)

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AFLP example: Aspergillus fumigatus

No prior sequence information neccessary!

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Multilocus sequence typing: MLST

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Multilocus sequence typing: MLST

Usually 7 markers are used

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MLST for S. aureus

Ruimy et al. JB, 2008

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Microsatellites: principle

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Microsatellites: multiplex option

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MLVA / VNTR / microsatellites

Schouls et al., PLoS One, 2009

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Whole genome / Next generation sequencing:

De Been et al. JCM, 2015

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Whole genome / Next generation sequencing:

  • Provides superior resolution compared to any other available

genotyping method

  • The ultimate genotyping / identification / characterization tool (???)
  • Currently still too expensive and slow for routine use 
  • Different challenges with respect to data analysis and interpretation
  • With a whole genome, you already have all answers, the questions will

come later …. 

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Hospital acquired infection?

Acinetobacter baumannii Acinetobacter baumannii

Hey neighbour: what appears to be your problem? Don’t know: same as yours?

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Hospital acquired infection?

Acinetobacter baumannii Acinetobacter baumannii AFLP: indistinguishable genomes  Possible transmission

  • r exposure

to same source

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Hospital acquired infection?

Enterococcus faecium Vancomycin R Enterococcus faecium Vancomycin R

Hey neighbour: what appears to be your problem? Don’t know: same as yours?

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Hospital acquired infection?

Enterococcus faecium Vancomycin R Enterococcus faecium Vancomycin R MLST: indistinguishable genomes  but …

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Hospital acquired infection?

Enterococcus faecium Vancomycin R Enterococcus faecium Vancomycin R Strain carries ‘vanA’ gene Strain carries ‘vanB’ gene No transmission! 

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Hospital acquired infection?

Klebsiella pneumoniae ESBL blaCTX-M pos Klebsiella pneumoniae ESBL blaCTX-M pos

Hey neighbour: what appears to be your problem? Don’t know: same as yours?

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Hospital acquired infection?

Klebsiella pneumoniae ESBL blaCTX-M pos Klebsiella pneumoniae ESBL blaCTX-M pos Strain carries ‘CTX-M-2 gene Strain carries ‘CTX-M-15’ gene No transmission!  Sanger sequencing:

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Hospital acquired infection?

Pseudomonas aeruginosa carbapenemase R (blaVIM-2) Pseudomonas aeruginosa carbapenemase R (blaVIM-2)

Hey neighbour: what appears to be your problem? Don’t know: same as yours?

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Hospital acquired infection?

Pseudomonas aeruginosa carbapenemase R (blaVIM-2) Pseudomonas aeruginosa carbapenemase R (blaVIM-2) blaVIM-2

  • n chromosome

blaVIM-2

  • n plasmid!

No transmission! 

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Hospital acquired infection?

Pseudomonas putida carbapenemase R (blaVIM-2) Pseudomonas aeruginosa carbapenemase R (blaVIM-2) blaVIM-2

  • n plasmid

blaVIM-2

  • n plasmid

Possible transmission

  • f plasmid!

More likely to occur in the environment than between patients

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  • Parameters used to reveal possible transmission of potentially

dangerous micro-organisms may include analysis of the

  • Genomic background (chromosome)
  • Mobile elements (plasmids, integrons etc.)
  • Individual genes (usually resistance genes)

…. using a wide variety of molecular methods