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Identification of Lactobacillus spp. isolates from children’s intestinal tissues
by MALDI-TOF MS profiling and automated ribotyping
Teshim Andrea1,2, Šedo Ondrej3, Švec Pavel2, Dráb Vladimír4, Sedláček Ivo2, Zdráhal Zbyněk3
(1) Division of Microbiology, Department of Experimental Biology, Masaryk University, Tvrdého 14, 602 00 Brno, CZ
(2) Czech Collection of Microorganisms, Department of Experimental Biology, Masaryk University, Tvrdého 14, 602 00 Brno, CZ
(3) Division of Functional Genomics and Proteomics, Department of Experimental Biology, Masaryk Univesity and CEITEC, Masaryk University, Kamenice 5, 625 00 Brno, CZ
(4) Dairy Research Institute, MILCOM a.s., Soběslavská 841, 390 01 Tábor, CZ
Introduction:
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers easily determinable peptide/protein fingerprints
for identification, typing and characterization of lactic acid bacteria strains, which can be useful in microbial technology as well as in clinical practice (to
compare clinical isolates and biotechnological strains, to distinguish probiotic strains from natural isolates, to monitor the use of patented strains, to
monitor the genetic stability of the strains).
Because of remarkable ecological and phenotypic diversity, high level of DNA homology and variability in G+C content
lactobacilli are continually reclassified (L. acidophilus group, L. casei-related taxa). Identification of some closely related
species often leads to ambiguous results even after their investigation by polyphasic taxonomy approaches.
L. plantarum
The aim of our study was to evaluate MALDI-TOF MS as a technique for identification of particular problematically distinguishable
Lactobacillus isolates and to compare obtained outcomes with results of automated ribotyping. With the use of MALDI-TOF MS
technique it was proved that bacteria are discriminated at the genus, species and even strain level. The particular sum of signals
that enables identification is constantly observed despite the culture age and culture conditions.
MALDI-TOF MS plate
Materials and Methods:
Bacterial strains: 57 Lactobacillus strains from biopsy samples of intestinal tissues of children patients suffering from familial adenomatous polyposis,
ulcerative colitis, Crohn's disease, bowel inflammation or celiac disease. Working cultures were cultured in 10 ml of deMan-Rogosa-Sharpe broth (Oxoid)
incubated anaerobically for 24 – 48 h at either 30 or 37 °C. Fresh bacteria cells from sediment were washed with water, sterilized with 75% ethanol and
extracted by 70 % formic acid and acetonitrile mixture (1:1 v/v).
MALDI-TOF MS analysis: supernatant was spotted onto a steel MALDI target plate, dried on air and overlayed with matrix solution
of HCCA acetonitrile/trifluoroacetic acid/water mixture (50:2.5/47.5 v/v). Mass spectra of Lactobacillus strains were obtained with
an Ultraflex III instrument and processed with BioTyper software (Bruker Daltonik) based on comparison with the database of
type and reference lactic acid bacteria MALDI-TOF MS profiles.
Automated ribotyping with EcoRI restriction enzyme was performed using the RiboPrinter identification system (DuPont Qualicon) according to the protocol
provided by the manufacturer. Obtained ribotype patterns were automatically categorized into ribogroups and identified by a DuPont Qualicon database DUP
2008 by using the RiboExplorer operating software.
Results:
In accordance with MALDI-TOF MS,
the RiboPrinter system identified only
32 strains to the species level (Fig. 2):
Lactobacillus salivarius (19 strains),
Lactobacillus paracasei (12) and
Lactobacillus gasseri (1).
One L. gasseri strain was assigned as
Aerococcus viridans and remaining
25 strains were not identified using
the ribotyping method as they did not
exceed the threshold of 85 % similarity
with the RiboPrinter reference
database entries.
Fig. 1:
Results of 57 Lactobacillus strains
identification based on MALDI-TOF
MS. Samples are classified to the type
culture of particular species.
Results:
Based on MALDI-TOF MS analysis,
Lactobacillus salivarius (28 strains),
Lactobacillus paracasei (11),
Lactobacillus rhamnosus (7), Lactobacillus mucosae (5),
Lactobacillus gasseri (2),
Lactobacillus plantarum (2), Lactobacillus paracasei (1)
and Lactobacillus oris (1) were reliably identified with
BioTyper log(score) > 2.3) (Fig. 1).
Conclusions: MALDI-TOF MS proved to be a suitable and highly reproducible tool for
identification of Lactobacillus spp. inhabiting human intestinal tract. All the 57 cultures were
identified to the species level. As compared with the RiboPrinter system identification, in this group
of 57 Lactobacillus samples MALDI-TOF MS technique offered more reliable outcomes.
Fig. 2
Acknowledgements: This work was supported by projects 2B08068, MSM0021622415, MSM0021622416 and LC06034.