MALDI TOF MASS SPECTROMETRY AND ITS FUTURE CLINICAL IMPACT Alex van Belkum Global Director, Research and Development Microbiology bioMérieux SA, La Balme Les Grottes, France Honorary Professor in molecular Microbiology Erasmus Medical Center Rotterdam, The Netherlands

“MODERN” CLINICAL MICROBIOLOGY PRACTICE

3

THE NAME GAME

CULTURE IS NOT DEAD!!

MASS SPECTROMETRY

SPECTRUM ACQUISITION

Let the spot dry

With 1µL-loop

take a small piece

of one colony

1µL HCCA matrix YEAST 0.5 µL formic acid

Sample scan on Preparation Station

BACTERIA MALDI TARGET

Let the spot dry

MS DATABASE CONCEPTS

Population based Strain based

Species represented by populations

Species specific characteristics can be

revealed

Many reference strains required

Species represented by individual

strains

Rapid database building

Strains not necessarily represent

typical isolates

MS DATABASE CONCEPTS

Population based Strain based

Bruker BioTyper VITEK MS (IVD) SARAMIS (RUO)

PATTERN MATCHING

Sample spectrum

Reference spectrum

Perfect match:

0 distance between

peaks

Less perfect match:

Sum of distances

between peaks

SARAMIS (RUO) v4.10

Analysis using

ReferenceSpectra

Study by Jean Philippe Charrier and co-workers

PATHOGEN TYPING??

PATHOGEN TYPING!!

Diversity of spectra depending on strain origin:

Trichophyton interdigitale

Zoophilic

Anthropophilic

14

Determination and characterization of β-lactamase activity in bacterial lysates using MALDI mass spectrometry Gero P. Hooff1*, Jeroen J.A. van Kampen2, Roland J.W. Meesters1, Alex van Belkum2,3, Wil H.F. Goessens2, Theo M. Luider1

ANTI-MICROBIAL SUSCEPTIBILITY TESTING

CIRCULATING PROTEASES IN ANTHRAX INFECTION

Late in infection large amounts of Lethal Factor (LF) toxin are produced

by Bacillus anthracis. LF is a protease attacking members of the

mitogen activated protein kinase kinases (MAPKK) and thereby blocking

cell signalling pathways. This suggests that artificial substrates for LF

might be used as diagnostic targets. Recognition site mimicking

peptides were developed.

Website P. Rizkallah, endorsed by MRC & NIH

VIRULENCE ASSESSMENT?

PROTEOLYSIS OF REPORTER PEPTIDES

Courtesy of Floris Bikker and Wendy Kaman,

TNO Defense, Rijswijk, The Netherlands

18

DIRECT BC TESTING

DIRECT TESTING OF URINE SAMPLES

Negative urine sample

Positive urine sample

E.coli 105 CFU/mL

E.coli calibrant ATCC 8739

MAIN ACTIVITIES

New protocols for molds, Mycobacteria and Nocardia (2 patents filed)

Adapted to the lab workflow (solid media for molds)

Allows Mycobacteria inactivation

VITEK MS database expansion

Clinical molds (Bellinzona, CBS, San Antonio)

Mycobacteria (HUG Genève, Durham)

Legionella (CNR, Bellinzona)

BSL3 species (CNR Brucella and Arizona university)

Strains from cystic fibrosis patients (HCL Lyon)

Environmental strains (Baseclear, Baxter)

Veterinary strains (Copenhague, LDV Perigueux)

New update planned in Q4 2013 : 1000 species in total

Inactivation/Extraction Method for Mycobacteria (Ethanol - Formic Acid 70% - Acetonitrile) - Solid Culture

Transfer suspension to

empty Eppendorf tube

(a) Please note that depending on the instrument, the centrifugation time may need to be adapted (for example 2’30 min in order to have 2 min at 10,000 rpm). Moreover, if

you need to put the tubes outside of the safety cabinet for the centrifugation steps, take care to clean the tubes with sporicidal product in order to avoid contamination.

Use a safety cabinet, wear laboratory coat and gloves

Use the appropriate procedure/product for disinfection

In 40 µ L FA [70%] +

2&3

3 3

Bead beat for 5 min and

then incubate at room

temperature for 10 min

Suspend one 1 µl

loopful of growth in

500 µl EtOH (in

Eppendorf tube

containing 0.5 mm

glass beads

Discard the EtOH

supernatant using a

Pasteur pipette

Add 10 µl 70% formic acid, (vortex, and

incubation at room temperature for 2-5 min

Add 10 µl acetonitrile and vortex

Centrifugation

2 min @

10,000 rpm (a)

Deposit 1 µl supernatant

Dry completely

Add 1 µl CHCA matrix

Dry completely

Analyze in VITEK MS

This step could be done on the bench outside the safety

cabinet

Centrifugation

2 min @

10,000 rpm (a)

ASSOCIATED RESEARCH

Antibiotic resistance

Detection of enzymatic activities (b-lactamase)

Analysis of cell wall components (porins)

Biomarker detection

Glycans as marker of fungal infections (Pr Poulain, Lille)

LPS modification

Detection of LPS modification linked to biofilm formation

(Institut Pasteur)

Detection of virulence factors (shiga-toxin)

Typing (Legionella)

(SOME) ACKNOWLEDGEMENTS

Microbiology R&D bMx Durham

Jay Hyman, John Walsh, Michael Dunne, Marc Van Nuenen

Microbiology R&D bMx La Balme Les Grottes

Sylvain Orenga, Geraldine Durand, Martin Welker, Sonia Chatellier

ESPCL Paris

Andrew Griffith

Geneva

Jacques Schrenzel

Rotterdam

Wil Goessens, Theo Luider

Boston

Howard Shapiro, Scot Manalis

Grenoble

Frederic Mallard

24

CURRENT bMx AND PAST ROTTERDAM FRIENDS AND COLLEAGUES