19th European Congress of Clinical Microbiology and Infectious Diseases, Helsinki/Finland, 16 - 19 May 2009

Application of MALDI TOF mass spectrometry for Helicobacter pylori study.

Elena N. Ilina1, Vladimir A. Vereshchagin1, Marina V. Serebryakova1, Vera V. Chelysheva1, Alexandra D. Borovskaya1,
P510
5 0 y
Kuvat T. Momynaliev 1, Thomas Maier2, Markus Kostrzewa2, and Vadim M. Govorun1

¹Research Institute for Physical-Chemical Medicine, Moscow, Russia

² Bruker Daltonics GmbH, Bremen, Germany

Table 1. List of strains which were analysed in presented Table 2. Comparative analysis of H. pylori strains J99 and 26695

Abstract study.

Isolation region
g /
peak lists. The most reproducible peaks with registration frequency
are equal or more than 0.7 for 10 different spectra are included
only. Identical peaks are selected by grey. Ribosomal proteins are
Results
no. Description indicated by bold type.
type
Objectives. The applicability of MALDI TOF mass spectrometry techniques for investigation of a highly variable bacterium additional information
such as Helicobacter pylori was studied. Escherichia coli Average m/z for J99 Average m/z for 26695 Description Initial MALDI-TOF analysis for H. pylori was performed using reference strains J99 and 26695 (fig. 1). Accuracy and
Methods. H. pylori were grown on Columbia agar plates (BioMerieux, France) at 37oC and 5% CO2 for 48 hours. Fresh
1 Ec DH5α Laboratory strain 4320 4320 RL36 reproducibility criterion were similar to those for E. coli (data not shown), but significant differences between mass profiles of
bacterial colonies were transferred into 300 µl of water. After precipitation with ethanol (900 µl) and centrifugation the pellet was
5246 5246 RL34 the two reference strains were found (tabl. 2). Some of spectra differences may be explained by amino acid changes
suspended in 20 μL of 50% acetonitrile, 35% formic acid, and analyzed in a microflexTM (Bruker Daltonics, Germany) using a Helicobacter pylori
described for corresponding proteins (RL32, RL29, RL24, and RS16). Most of the identical peaks were matched to ribosomal
saturated solution of α-CHCA as matrix. Spectra analysis and species identification was done using flexAnalysis 2.4 and Clonal group subgroup 5515 5529 RL32
proteins of H. pylori except of four. For the peak with m/z 6948 a MS/MS spectrum was obtained. This peak was assigned to
MALDI Biotyper 2.0 software (Bruker Daltonics, Germany). Mass spectra of protein fragmentation were obtained by an Ultraflex 5541 5541
1 Hp J99 hpAfrica1 Laboratory strain histidine-rich, metal binding polypeptide (Hpn) (Gilbert et al., 1995).
II MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Germany) equipped with Smart BeamTM laser. 6066 6066 RL33 Mass spectra for the 17 clinical strains of H. pylori generally showed less peaks (7 - 13 peaks per strain) with high
Results. 17 clinical starins as well as two laboratory strains of H .pylori were analyzed by MALDI TOF MS. Mass spectra 2 Hp 26695 hpEurope Laboratory strain
6798* 6798* RL28 variability between strains. Only six peaks (m/z 4319, 5245, 5527, 5539, 6064, and 6948) were common for more than half of
collected were found containing 7 - 13 significant peaks per sample, and only five protein signals were identical for more than
3 Hp_6y hpEurope Yakutia/ clinical strain
6912 6912 the strains and therefore can be noted as H. pylori - specific peaks.
70% of strains. Four of them were matched to ribosomal proteins. The fifth reproducible peak with m/z 6948 was assigned to
hpEurope In spite of this, we found that for a certain H. pylori strain under the same cultivation and measuring conditions its mass
histidine-rich metal binding polypeptide by MS/MS. In spite of evident intra species protein heterogeneity of H .pylori the mass 4 Hp_7y Yakutia/ clinical strain 6946 6946 Hpn
spectrum was not dramatically changed. For H. pylori reference strains 26695 the correlation values between mass spectra
spectra collected for a particular strain under the several cultivations were reproducible.
reproducible Moreover
Moreover, all clinical strains were 5 Hp_17y h E Ai
hpEastAsia h Sib i
hspSiberia Yakutia/ clinical strain 7129* 7129* RL35 collected during ten passages were in the range from 0.85 to 0.95 (average 0.92). Similar data were obtained for two clinical
perfectly identified as H. pylori by MALDI Biotyper 2.0 software using a database containing mass spectra from different
6 Hp_18y hpEastAsia hspSiberia Yakutia/ clinical strain 7652 7652 RL31 strains.
bacterial strains (n=3290) including H. pylori 26695 and J99.
7752 7683 RL29 To examine if DBP using MALDI-TOF mass spectrometry for species identification can be applied for H. pylori the
Conclusion. MALDI TOF MS fingerprinting is a suitable tool for H. pylori species identification and typing and could help in 7 Hp_57y hpEastAsia hspSiberia Yakutia/ clinical strain
BioTyper 2.0 software (Bruker Daltonics, Germany) has been used. The characteristic main-spectra, constructed for each
better understanding of transmission pathways of this bacterium. 7915 7905 RL24
8 Hp_44y hpEastAsia hspSiberia Yakutia/ clinical strain clinical strain based on sets of raw DBP data (twenty per sample), were compared with a separated BioTyper library
8482 8482 RS21
9 Hp_92y hpEastAsia hspSiberia Yakutia/ clinical strain containing 3290 main spectra of bacterial strains from 1239 different species including H. pylori 26695 and J99. In spite of
8657 evident differences between MALDI profiles, all clinical strains were correctly identified as H. pylori (tabl. 3), although the

Introduction 10

11
Hp_1m

Hp_4m
hpAsia2

hpEastAsia hspSiberia
Mongolia/ clinical strain

Mongolia/ clinical strain

8971 8985
9114
RS16 identification scores partially were below threshold generally set for secure species identification (log(score)=2.0).
At the same time we have not found a relationship between H. pylori strains features (genotyping data, geographical
region of isolation) and distribution of their profiles according to the dendrogram constructed based on similarity scores by
12 Hp_37m hpEastAsia hspSiberia Mongolia/ clinical strain 9129
H. pylori possesses a highly natural variability and the availability of accurate tools for species identification and BioTyper software (fig. 3). This fact could be easily explained by contemporary mutability (unsteadiness) of H. pylori strains
epidemiological characterization could help scientific community to better understand the transmission pathways and virulence 13 Hp_3t hpEastAsia hspAmerind Tuva/ clinical strain 9278 under the host exposure. But it should be mentioned that the group we analyzed was not large enough for a reliable
mechanisms of this bacteria. Direct bacterial profiling (DBP) using matrix-assisted laser desorption/ionisation time of flight 14 Hp 13t
Hp_13t hpEastAsia hspAmerind Tuva/ clinical strain 10065 10065 RS20 epidemiological study. Maybe a broadcast investigation of clinically H. pylori strains and extensive analysis for group-specific
(MALDI-TOF) mass-spectrometry is a novel fast and accurate approach for identification and subtyping of bacterial species. It is 10260 10260 signals in larger datasets would reveal different results.
based on the comparison of specific mass-spectra of the cellular components mixture, mainly proteins and peptides, obtained 15 Hp_18t hpEastAsia hspAmerind Tuva/ clinical strain
10384
directly from “whole” cells without preliminary separation of cellular components. Possibilities of this approach were 16 Hp_a51 hpEastAsia hspSiberia Altai/ clinical strain
demonstrated for many of gram positive as well as gram negative bacteria species. One of the questions is whether only one 10414
17 Hp_a58 hpEastAsia hspAmerind Altai/ clinical strain
protocol for all different species can be used. In particular, the method is interesting for highly variable bacterial species such as 10448 10448 RS18
Helicobacter pylori. Low information content of according mass spectra was shown in some previous studies (Nilsson, 1999; 18 Hp_a62 hpEastAsia hspAmerind Altai/ clinical strain 10543 RS19
Winkler, 1999). 19 Hp_a63 hpEastAsia hspSiberia Altai/ clinical strain 10557
The goal of this study was to improve the DBP protocol for H. pylori measuring and to demonstrate the applicability of
MALDI Biotyper technique for H. pylori species identification.

Methods
H. pylori references (n = 2) as well as clinical strains (n = 17) collected from different parts of the Russian Federation
were analyzed (tabl. 1). A laboratory strain of Escherichia coli DH5α (Life Technologies, UK) was used for calibration and
instrument parameter optimization. Before analysis bacteria were grown in dedicated conditions according to CDC protocols.
For MALDI-TOF mass spectrometry analysis single colonies of fresh bacterial cultures have been used.
Bacterial cells were transferred from the plate into an extraction 0.5 ml tube (Eppendorf, Germany) with a 1.0 l plastic
loop (FL medical, Italy). In this case the volume of bacterial cells corresponded to 5 - 10 mg, approximately. Then 50 l of
extraction solution consisting of 50 % acetonitrile (Sigma-Aldrich, Germany), 2.5 % trifluoroacetic acid (Sigma-Aldrich,
Germany), 47.5 % water were added (Fluka, Switzerland). The mixture was resuspended well and centrifuged (12000 rpm for 1 Figure 1. MALDI-TOF mass spectra of whole-cell extract of H. pylori strain J99 (A) and 26695 (B). The peak of histidine-rich
min). Supernatant was used for further MALDI-TOF mass spectrometric analysis. A saturated solution of -cyano-4-hydroxy metal binding polypeptide (Hpn) is indicated by an arrow. The relative intensities of the ions are shown on the Υ axis and the
cinnamic acid (CHCA) (Bruker Daltonics, Germany) was prepared in 1.0 ml of 50 % acetonitrile, 2.5 % trifluoroacetic acid, 47.5 mass to charge ratios are shown on the X axis.
% water. 1 l of the bacterial cell lysate was deposited onto a sample spot of a steel MALDI target plate (MSP 96 target ground
steel; Bruker Daltonics,
Daltonics Germany) and was allowed to air dry at room temperature. Finally 2 l of CHCA matrix solution were
temperature Finally,
deposited onto the dried matrix and allowed to dry at room temperature. Mass spectra were recorded on a Microflex MALDI-
TOF mass spectrometer (Bruker Daltonics, Germany) equipped with a N2 337 nm laser. For visual spectra inspection the
flexAnalysis 2.4 software (Bruker Daltonics, Germany) was used. To construct and compare characteristic main spectra of
certain strains the sets of DBP mass spectra were processed using the BioTyper 2.0 software (Bruker Daltonics, Germany).
Conclusions
Strain Detected Species log(Score) Table 3. Examples of identification results for DBP approach is well applicable for the identification and typing of such variable bacteria as H.
Helicobacter pylori 26695_ce PGM 2.298 three different H. pylori clinical strains using
Hp_13t the BioTyper 2.0 software (Bruker Daltonics, pylori, especially with further extension of database content by additional H. pylori reference
Helicobacter pylori_J99_PGM 1.849
Lactobacillus ingluviei 15946T_DSM 1.069 Germany) and dedicated library containing the spectra to reflect the broad variability of H. pylori profile fingerprint spectra. If the intra-specific
Helicobacter pylori 26695_ce PGM 2.04 3290 main spectra generated from different diversity of the profile spectra can be used for epidemiological purposes has to be examined
Hp_a63 clinically relevant microorganisms. Thresholds
Helicobacter
e cob c e pylori
py o _J99_
J99 PGM
G 1.432
.
for spectra adjusting and score calculation
more inin-depth
depth.
Clostridium tertium 1048_NCTC 541_BOG 1.142
have been set at 50 and 5, respectively, and Figure 2. A score oriented dendrogram of MALDI-TOF mass spectra profiles was generated by the MALDI BioTyper 2.0
Hp_57y Helicobacter pylori 26695_ce PGM 1.88
intensity correction function was 0.25. software with the following settings for peak picking: lower bound 3000, upper bound 11000, max. peaks - 100 and threshold 0.001.
Helicobacter pylori_J99_PGM 1.519
H. pylori strains are listed in accordance with table 1. Mass spectra from E. coli strains were taken of a separate database containing
Campylobacter coli 10090_03 NVU 1.217
3290 main spectra generated from different clinically relevant microorganisms. The dendrogram was generated with the following
settings: distance measure was set at Euclidian and linkage at complete. Acknowledgments
This work was supported by a grant of the Sächsische Aufbaubank (SAB10634).

Research Institute for Physical-Chemical Medicine, Malaya Pirogovskaya st., 1a, 119992, Moscow, Russia. ilinaen@gmail.com