Microbiology Meets Mass Spectrometry

1. The newest technique for identification
of bacteria, yeast and mycobacteria in
the clinical microbiology laboratory!

2.  US Microbiology laboratories mostly in the era
of agar plates, Gram stains and biochemical reactions
 Automated biochemical identification instrumentation
(Vitek2, Phoenix and Microscan) are not able to identify
many of the newly reclassified and/or rarely isolated
organisms in clinical specimens
 This results in alternative testing methods that
can be labor intensive and costly
There must be something better!!

3.  16S rRNA Gene Sequencing
 Accurate and precise but it is not ready for prime time
 Currently labor intensive and far too expensive for routine use
in the clinical microbiology laboratory ($$/isolate)
 MALDI-TOF Mass Spectrometry
 Provides accurate and extensive identification information
 Not labor saving but very inexpensive to do daily testing
($0.50/isolate)
 Instruments in the $250,000 – $350,000 range!

4.  Mass Spectrometry creates a unique molecular fingerprint
of an organism
 Measures highly abundant proteins that are found in all
microorganisms and create measurable peaks
 The characteristic patterns of these highly abundant
protein peaks are used to reliably and accurately identify
a particular microorganism
 The pattern created is compared to a computer database
to determine the identity of the microorganism – high %
to the species level.
 Can identify aerobic & anaerobic bacteria,
fungi, and mycobacteria

5. (1) Requires only 1 colony of bacteria or yeast
Age of colony can be 18 hr – 3 days old
No need to Gram stain – MALDI does not
determine results by gram stain rxn
(2) Smear organism on one circle of target plate
(3) Add one drop of matrix solution (alpha
cyano4 hydroxycinnamnic acid) onto each circle
(4) Dry at room temp
(5) Place target plate in the MALDI instrument

6. Two instruments currently:
Bruker MALDI Biotyper
Vitek MS
Target plate is positioned in
instrument
Bruker
Plate insertion
Vitek MS

7.  Matrix Assisted Laser Desorption Ionization
 MALDI
Desorption: Matrix
solution absorbs the UV
laser light with
ablation of the top layer of
the matrix, this leads to a
hot plume containing many
molecules
Ionization: or Protonation
occurs in the hot plume
creating single and multiply
charged ions
Target plate

8. Time of Flight (TOF)
Based on Mass and Charge of Proteins
Plots the Time of flight (TOF) based on measuring the size and
charge of the proteins hitting the detector plate, creating peaks
The plot peaks detected on the detector plate are matched to the
computer data base and develops and identification score

9. Biochemical Method MALDI-TOF
 Lactose fermenting GNR
on MacConkey agar
 Vitek AST test card
 TAT @ 6-8 hours
 Materials cost @ $4.00
 Labor neutral
 Lactose fermenting GNR on
MacConkey agar
 MALDI-TOF
 TAT @ 10 min
 Cost @ $ 0.50
 Labor comparable

10. Biochemical
identification workup
Small grey colony
 Gram stain = small GNR
 Vitek 2 = No ID (8hr)
 Next day new subcultures
required (36 hr)
 NF Crystal ID test (48 hr)
 Cardiobacterium hominis
 Cost @ $12.00 materials
 TAT @ 48 hours
MALDI TOF Identification
Small grey colony
MALDI-TOF testing
Cardiobacterium hominis
Cost @ $ 0.25-0.50 materials
TAT 10 minutes

11.  Based on a price of $4.00 for a VITEK2 GNS card, save
@ $3.50 per Gram negative identification replacing
Vitek identification with MALDI-TOF
 Tests/month Savings $$/year
 1000 $3,500 $42,000
 2000 $7,000 $84,000
 4000 $14,000 $168,000
 There is also the potential for elimination of
numerous other tests and kits for organism
identification used in the laboratory

12.  That would truly effect TAT
 Direct testing from positive blood culture
bottle
 Techniques and kits available for processing
positive blood cultures to eliminate the blood and
concentrate organisms
@ 80% of pathogens can be identified in direct
testing
 Considerably higher % of identification for Gram
negative rods than Gram positive cocci
 Costs @ $10-12 per specimen identification
 Less expensive than comparable PCR identification
methods used on blood cultures (>=$65)

13.  No! But it is very good!
 The purchase price of the instrument is $$$
but very little expense moving forward in
specimen testing
 Few inexpensive consumables (pipette tips,
chemicals)
 Disposable target plates (Vitek MS only)
 Maintenance agreement
 Still need growth on plated media for
organism identification (18 hr delay) – large
enough colonies to spot onto target plate
 The data base spans most disciplines of
microbiology – so you do not need other
instruments or kits to identify unusual
organisms. Simplifies inventory and ordering
of testing supplies.

14.  Cost effective – low consumable costs
 Green
 Rapid turnaround time and high throughput
 Easy to perform/ instrument requires
virtually no maintenance
 Automated, inter-laboratory reproducibility
 Single colony requirement
 Low exposure risk – due to sample
inactivation
 Identifies aerobic and anaerobic bacteria,
fungi, and Mycobacteria

15.  No susceptibility information
 No current protocols for direct testing of clinical
specimens (for example urine)
 Sometimes need repeat analyses
 Databases need to be updated/maintained
 Financial loss on purchased equipment
Equipment cost can limit laboratory purchase
 Potential instrument downtime (single instrument
owned by laboratory due to cost)