Abstract Book of EAVLD2012 - eavld congress 2012

S4 - O - 04
MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY
(MALDI-TOF MS) IN A VETERINARY DIAGNOSTIC LABORATORY
Annet E. Heuvelink, Refke Peerboom, Erik van Engelen, Abdul A. Hassan
GD Animal Health Service, Deventer, The Netherlands
MALDI-TOF, bacterial identification, veterinary laboratory
Introduction
Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass
Spectrometry (MALDI-TOF MS) has emerged as a new tool for
fast and reliable identification of microorganisms (). We evaluated
the performance of MALDI-TOF MS for the routine identification
of bacteria in our veterinary diagnostic laboratory.
Materials & methods
Mass spectra were acquired using a Microflex LT mass
spectrometer and analysed by MALDI Biotyper 3.0 software
(Bruker Daltonik GmbH, Bremen, Germany).
First, we determined the accuracy of MALDI-TOF MS
identification by testing 66 bacterial strains of culture collections,
in duplicate spots on the MALDI target plate. Second, we
performed a prospective validation study, including 262 isolates
collected in the routine laboratory: 156 isolates from post-mortem
examinations of mammals and 106 mastitis pathogens from
cow’s milk. The isolates were identified by conventional
biochemical methods in parallel with MALDI-TOF MS, measured
in duplicate spots. A selection of Gram-positive cocci, including
coagulase-negative staphylococci (CNS) and Streptococcus
uberis isolates, were tested by both direct colony testing and the
extended direct transfer method described by the manufacturer,
comprising a rapid partial extraction with 70% aqueous formic
acid. Finally, the repeatability and the robustness of identification
of bacteria by MALDI-TOF MS were evaluated. The repeatability
was determined with five reference strains, by testing 10 cfu from
one plate, in one run, by one technician. The robustness was
evaluated by testing the effect of (a) refrigerated storage of fresh
cultures grown on agar plates (for 24 or 48 h), (b) the use of
selective solid media, and (c) prolonged incubation (for 48 or 72
h) either or not followed by a refrigeration step. For each of the
species included in the robustness test, one reference strain and
five field isolates were tested.
some isolates, especially those of S. uberis, more not reliable
identifications were obtained when performed with cultures stored
for 48 h in the refrigerator or incubated for 48 h, and especially
for 72 h prior to identification.
Discussion & conclusions
The present results show that MALDI-TOF MS is a reliable and
rapid method for identification of most bacterial species
encountered in our routine veterinary laboratory. No
misidentifications occurred, only “no reliable identifications”. For
the majority of the isolates direct colony testing can be performed
without extraction. For identification of presumptive CNS isolates,
it is recommended to use the simple partial extraction procedure
with 70% aqueous formic acid following the instructions of the
manufacturer.
One of the additional benefits of MALDI-TOF MS analysis is the
identification of CNS species. Species level identification of CNS
will help to elucidate sources, transmission mechanisms and the
impact of different CNS species on cow health, productivity and
milk quality, and this knowledge may lead to species-specific
infection control measures.
References
1. Bizzini A, Greub G. Matrix-assisted laser desorption ionization
time-of-flight mass spectrometry, a revolution in clinical microbial
identification. Clin. Microbiol. Infect. 2010, 16(11), 1614-1619.
2. Wieser A, Schneider L, Jung J, Schubert S. MALDI-TOF MS
in microbiological diagnostics-identification of microorganisms
and beyond (mini review). Appl. Microbiol. Biotechnol. 2012,
93(3), 965-974.
Results
Of the 66 reference strains tested, 79% were correctly identified
to species level. The remaining strains were identified to genus
level (14%) or could not be identified (7%). The latter strains
belonged to species not included in the BioTyper 3.0 database.
For 90% of the 156 isolates from post-mortem examinations of
mammals matching identifications at species level were obtained
between MALDI-TOF MS and biochemical methods, 8% were
correctly identified to genus level, and for 2% no reliable
identifications were achieved. Eighty-nine% of the MALDI-TOF
MS identifications of 106 mastitis pathogens were concordant
with standard biochemical identifications. Ten% of the mastitis
pathogens were identified to species level by MALDI-TOF MS
(mainly CNS), while standard biochemical identifications of these
isolates were at genus level only. For one isolate the MALDI-TOF
MS result diverged from the biochemical identification; MALDI-
TOF MS identified the isolate as Klebsiella pneumoniae, a
qualitatively good result at species level, while the API 20E
(bioMérieux, Marcy l’Etoile, France) result was Klebsiella
oxytoca, with a doubtful profile. Most bacteria tested in this study
could be successfully identified at species level using the direct
colony testing method. However, for 70% of the CNS isolates the
partial extraction procedure showed to be superior. For S. uberis
the superiority of the extended direct transfer method appeared
to be less pronounced, being observed for only 32% of the
isolates.
The repeatability of MALDI-TOF MS identification of the
reference strains of Escherichia coli, Clostridium perfringens,
Staphylococcus aureus, and Salmonella Typhimurium was 100%.
For the Streptococcus suis reference strain it was 90%, with 10%
being not reliably identified.
In general, MALDI-TOF MS identification results were not altered
when performed with cultures stored for 24 or 48 h at
refrigeration temperatures or with cultures tested after 48 or 72 h
of incubation, nor when selective agars were used. However, for