Dataset Information

In situ identification of plant-invasive bacteria with MALDI-TOF mass spectrometry.

ABSTRACT: Rhizobia form a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes. The study of rhizobial populations in nature involves the collection of large numbers of nodules found on roots or stems of legumes, and the subsequent typing of nodule bacteria. To avoid the time-consuming steps of isolating and cultivating nodule bacteria prior to genotyping, a protocol of strain identification based on the comparison of MALDI-TOF MS spectra was established. In this procedure, plant nodules were considered as natural bioreactors that amplify clonal populations of nitrogen-fixing bacteroids. Following a simple isolation procedure, bacteroids were fingerprinted by analysing biomarker cellular proteins of 3 to 13 kDa using Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry. In total, bacteroids of more than 1,200 nodules collected from roots of three legumes of the Phaseoleae tribe (cowpea, soybean or siratro) were examined. Plants were inoculated with pure cultures of a slow-growing Bradyrhizobium japonicum strain G49, or either of two closely related and fast-growing Sinorhizobium fredii strains NGR234 and USDA257, or with mixed inoculants. In the fully automatic mode, correct identification of bacteroids was obtained for >97% of the nodules, and reached 100% with a minimal manual input in processing of spectra. These results showed that MALDI-TOF MS is a powerful tool for the identification of intracellular bacteria taken directly from plant tissues.

SUBMITTER: Ziegler D

PROVIDER: S-EPMC3355115 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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In situ identification of plant-invasive bacteria with MALDI-TOF mass spectrometry.

Ziegler Dominik D Mariotti Anna A Pflüger Valentin V Saad Maged M Vogel Guido G Tonolla Mauro M Perret Xavier X

PloS one 20120517 5

Rhizobia form a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes. The study of rhizobial populations in nature involves the collection of large numbers of nodules found on roots or stems of legumes, and the subsequent typing of nodule bacteria. To avoid the time-consuming steps of isolating and cultivating nodule bacteria prior to genotyping, a protocol of strain identification based on the comparison of MALDI-TOF MS spectra was established ...[more]

PMID: 22615938

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Project description:Dermatophytoses represent a major health burden in animals and man. Zoophilic dermatophytes usually show a high specificity to their original animal host but a zoonotic transmission is increasingly recorded. In humans, these infections elicit highly inflammatory skin lesions requiring prolonged therapy even in the immunocompetent patient. The correct identification of the causative agent is often crucial to initiate a targeted and effective therapy. To that end, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents a promising tool. The objective of this study was to evaluate the reliability of species identification of zoophilic dermatophytes using MALDI-TOF MS. The investigation of isolates from veterinary clinical samples suspicious of dermatophytoses suggests a good MALDI-TOF MS based identification of the most common zoophilic dermatophyte Microsporum canis. Trichophyton (T.) spp. usually achieved scores only around the cutoff value for secure species identification because of a small number of reference spectra. Moreover, these results need to be interpreted with caution due to the close taxonomic relationship of dermatophytes being reflected in very similar spectra. In our study, the analysis of 50 clinical samples of hedgehogs revealed no correct identification using the provided databases, nor for zoophilic neither for geophilic causative agents. After DNA sequencing, adaptation of sample processing and an individual extension of the in-house database, acceptable identification scores were achieved (T. erinacei and Arthroderma spp., respectively). A score-oriented distance dendrogram revealed clustering of geophilic isolates of four different species of the genus Arthroderma and underlined the close relationship of the important zoophilic agents T. erinacei, T. verrucosum and T. benhamiae by forming a subclade within a larger cluster including different dermatophytes. Taken together, MALDI-TOF MS proofed suitable for the identification of zoophilic dermatophytes provided fresh cultures are used and the reference library was previously extended with spectra of laboratory-relevant species. Performing independent molecular methods, such as sequencing, is strongly recommended to substantiate the findings from morphologic and MALDI-TOF MS analyses, especially for uncommon causative agents.

Project description:The insufficient quality of products containing beneficial live bacteria in terms of content and viability of labelled microorganisms is an often-reported problem. The aim of this work was to evaluate the quality of dietary supplements containing viable bacteria available in Slovenian pharmacies using plate counting, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and species- or subspecies-specific PCR with DNA isolated from consortia of viable bacteria, from individual isolates, or directly from the products. Twelve percent of the products (3 of 26) contained insufficient numbers of viable bacteria. Eighty-three of the labelled species (111 in total) were confirmed by PCR with DNA from the product; 74% of these were confirmed by PCR with DNA from viable consortium, and 65% of these were confirmed by MALDI-TOF MS analysis of colonies. Certain species in multi-strain products were confirmed by PCR with DNA from viable consortia but not by MALDI-TOF MS, suggesting that the number of isolates examined (three per labelled strain) was too low. With the exception of Lacticaseibacillus casei and closely related species (Lacticaseibacillus rhamnosus and Lacticaseibacillus zeae), PCR and MALDI-TOF identification results agreed for 99% of the isolates examined, although several MALDI-TOF results had lower score values (1.700-1.999), indicating that the species identification was not reliable. The species L. zeae, which appeared in 20 matches of the Biotyper analysis, was identified as L. rhamnosus by PCR. The MALDI-TOF MS analysis was also unsuccessful in detecting Lactobacillus acidophilus La-5 and Bacillus coagulans due to missing peaks and unreliable identification, respectively. Mislabelling was detected by both methods for two putative L. casei strains that turned out to belong to the species Lacticaseibacillus paracasei. PCR remains more successful in subspecies-level identification as long as the database of MALDI-TOF MS spectra is not expanded by building in-house databases. The lack of positive PCR results with viable consortia or colonies, but positive PCR results with DNA isolated directly from the products observed in 10% (11/112) of the labelled strains, suggests the presence of non-culturable bacteria in the products. MALDI-TOF MS is a faster and simpler alternative to PCR identification, provided that a sufficient number of colonies are examined. Generation of in-house library may further improve the identification accuracy at the species and sub-species level.

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Dataset Information

In situ identification of plant-invasive bacteria with MALDI-TOF mass spectrometry.

Publications

In situ identification of plant-invasive bacteria with MALDI-TOF mass spectrometry.

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