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

Forensic microbiology: whodunnit? Corné HW Klaassen Dept. Medical Microbiology & Infectious Diseases

Core activities MMIZ molecular diagnostics  Detection  Identification,  Typing of potentially pathogenic micro-organisms  Bacteria, fungi/yeasts, parasites using molecular (DNA/RNA) based techniques

Typing: a working definition  The ability to discriminate between different ‘individuals’ of the same species Damn! Now your footprint will discrminate you Shoot! A from the rest of us! hole in my shoe!  Not to confuse with ‘identification’: to determine to which species a micro-organism belongs.

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.

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’

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)

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

Amplified Fragment Length P olymorphism )

AFLP example: Aspergillus fumigatus No prior sequence information neccessary!

Multilocus sequence typing: MLST

Multilocus sequence typing: MLST Usually 7 markers are used

MLST for S. aureus Ruimy et al. JB, 2008

Microsatellites: principle

Microsatellites: multiplex option

MLVA / VNTR / microsatellites Schouls et al., PLoS One, 2009

Whole genome / Next generation sequencing: De Been et al. JCM, 2015

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 …. 

Hospital acquired infection? Hey neighbour: Don’t what appears know: to be your same as problem? yours? Acinetobacter Acinetobacter baumannii baumannii

Hospital acquired infection? AFLP: indistinguishable genomes  Possible transmission or exposure to same source Acinetobacter Acinetobacter baumannii baumannii

Hospital acquired infection? Hey neighbour: Don’t what appears know: to be your same as problem? yours? Enterococcus faecium Enterococcus faecium Vancomycin R Vancomycin R

Hospital acquired infection? MLST: indistinguishable genomes  but … Enterococcus faecium Enterococcus faecium Vancomycin R Vancomycin R

Hospital acquired infection? Strain carries Strain carries ‘vanB’ gene ‘vanA’ gene No transmission!  Enterococcus faecium Enterococcus faecium Vancomycin R Vancomycin R

Hospital acquired infection? Hey neighbour: Don’t what appears know: to be your same as problem? yours? Klebsiella pneumoniae ESBL Klebsiella pneumoniae ESBL blaCTX-M pos blaCTX-M pos

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

Hospital acquired infection? Hey neighbour: Don’t what appears know: to be your same as problem? yours? Pseudomonas aeruginosa Pseudomonas aeruginosa carbapenemase R (blaVIM-2) carbapenemase R (blaVIM-2)

Hospital acquired infection? blaVIM-2 blaVIM-2 on chromosome on plasmid! No transmission!  Pseudomonas aeruginosa Pseudomonas aeruginosa carbapenemase R (blaVIM-2) carbapenemase R (blaVIM-2)

Hospital acquired infection? blaVIM-2 blaVIM-2 on plasmid on plasmid Possible transmission of plasmid!  More likely to occur in the environment than between patients Pseudomonas putida Pseudomonas aeruginosa carbapenemase R (blaVIM-2) carbapenemase R (blaVIM-2)

 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