Fig. 1
MALDI-TOF spectrum of Actinobacillus pleuropneumoniae type strain S4074T
The MALDI-TOF mass spectrometers designed for microbial identification purpose generally are equipped with databases for the identification of bacteria of primary clinical importance in human medicine and hence lack data for many animal pathogens or opportunists such as species of the family Pasteurellaceae. A comprehensive database of MALDI-TOF spectra has recently been developed using a well-characterized strain collection representing type strains and field isolates of a large range of species and also subspecies of Pasteurellaceae that are of current relevance in veterinary infectious disease diagnostics [10]. The data obtained revealed that MALDI-TOF is able to discriminate most of Pasteurellaceae species tested, while only a few closely related species or subspecies needed additional tests for accurate identification. The current chapter describes the establishment of a MALDI-TOF spectrum database of Pasteurellaceae and the method for rapid bacterial identification to be used in diagnostic laboratories. The method is basically adaptable to any other bacterial family, which might be lacking in the commercial databases provided with the MALDI-TOF instruments. Note that MALDI-TOF databases are adapted to the specific mass spectrometers and are not normalized and are applicable to the specific instrument only. Therefore there are currently no online databases for MALDI-TOF-based identification of bacteria available. Spectra are electronically transferable from one instrument to another of the same brand, but adjustments are necessary as there are minor differences from one instrument to another.
2 Materials
Prepare all solutions using ultrapure water and analytical grade reagents. Preparation and storage of reagents is at room temperature unless otherwise indicated.
2.1 Chemicals
1.
0.9 % (w/v) NaCl (=154 mM).
2.
Ethanol, absolute.
3.
70 % (v/v) formic acid.
4.
Acetonitrile.
5.
Organic solvent (OS): 50 % acetonitrile, 2.5 % trifluoroacetic acid (TFA) in H2O.
6.
Matrix solution (10 mg/ml): dissolve 10 mg of α-cyano-4-hydroxycinnamic acid in 1 ml of OS and vortex thoroughly. The matrix solution should be saturated, i.e., some crystals might still be visible even after a few minutes. It can be kept in the dark at room temperature up to 1 week. Aliquots of pre-weighted powder can be kept at −20 °C and be supplemented with OS just prior to use.
7.
Appropriate growth medium for species to be included in database or to be analyzed, e.g., trypticase soy agar supplemented with 5 % sheep erythrocytes or chocolate agar (see Note 1 ).
2.2 Equipment
1.
MALDI-TOF mass spectrometer for biotyping, such as Bruker Daltonik Microflex LT or Shimadzu AXIMA Microorganism Identification System.
2.
General laboratory equipment such as micro-centrifuge.
3 Methods
Procedures can be carried out at room temperature unless otherwise specified. They are given for the Bruker Daltonik Microflex LT (Bruker Daltonik GmbH, Bremen, Germany), using the “Flex control” software, and might vary slightly with other instruments.
3.1 Generation of Reference Spectra for the Establishment of a New or for Upgrading a Database
1.
For each species of Pasteurellaceae family, grow the type strain and three to four confirmed field strains on appropriate medium, at the appropriate temperature (generally 37 °C) under suitable conditions, such as aerobic, microaerophilic, or capnophilic conditions. Ideally, use field strains for which the species has been confirmed by sequence analysis of the 16S rRNA and housekeeping genes [5, 6].
2.
Suspend a few colonies in 300 μl deionized H2O in an Eppendorf 1.5 ml tube.
3.
Add 900 μl of absolute ethanol and mix well.
4.
Centrifuge for 2 min in a micro-centrifuge at full speed (15,000 × g).
5.
Discard liquid and centrifuge again for 2 min at full speed.
6.
Remove all liquid with a pipette and air-dry the bacterial pellet for 20–30 min. Suspend the pellet in 50 μl of formic acid 70 % and verify the full suspension of the bacteria.
7.
Add 50 μl of acetonitrile and mix well.
8.
Centrifuge for 2 min in a micro-centrifuge at full speed.
9.
Keep the supernatant for the production of MALDI-TOF spectra.
10.
Spot 1 μl aliquots of the supernatant onto the steel target support of the MALDI-TOF mass spectrometer in eight replicas (see Note 2 ).
11.
Let the samples on steel target support air-dry (2–5 min).
12.
13.
Let the samples on steel target support air-dry (2–5 min).
14.
Include a calibration with a bacterial test standard (BTS), which is, in general, a specific strain of Escherichia coli provided by the supplier of the MALDI-TOF instrument.
15.
Produce reference spectra with the MALDI-TOF mass spectrometer by measuring each spot three times with standard settings resulting in a total of 24 spectra for each strain (Fig. 1). Run one to three BTS calibration spots with each plate read.
16.
Import the spectra into the “Flex analysis” software and process it according to the manufacturer’s recommendations.
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