Rapid identification of bacterial isolates from wheat roots by high resolution whole cell MALDI-TOF MS analysis

Stets, Maria Isabel; Pinto, Artur Soares; Huergo, Luciano F.; Souza, Emanuel Maltempi de; Guimarães, Vandeir Francisco; Alves, Alexessander Couto; Steffens, Maria; Monteiro, Rose A.; Pedrosa, Fábio de Oliveira; Cruz, Leonardo M.

doi:10.1016/j.jbiotec.2013.04.001

Cited by 37 publications

(16 citation statements)

References 39 publications

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“…Moreover, the values of some of these parameters have not been specified in the literature. For each of these reasons, we evaluated five parameters which are frequently reported in the literature [37]–[41]. The five factors which are adjusted through the Bruker FlexControl software (version 3.0; Bruker Daltonics) during automated data acquisition included: A) peak selection mass range; B) base peak S:N; C) base peak intensity; D) base peak minimum resolution; and E) number of shots summed (Table 1).…”

Section: Methodsmentioning

confidence: 99%

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A Designed Experiments Approach to Optimization of Automated Data Acquisition during Characterization of Bacteria with MALDI-TOF Mass Spectrometry

2014

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MALDI-TOF MS has been shown capable of rapidly and accurately characterizing bacteria. Highly reproducible spectra are required to ensure reliable characterization. Prior work has shown that spectra acquired manually can have higher reproducibility than those acquired automatically. For this reason, the objective of this study was to optimize automated data acquisition to yield spectra with reproducibility comparable to those acquired manually. Fractional factorial design was used to design experiments for robust optimization of settings, in which values of five parameters (peak selection mass range, signal to noise ratio (S:N), base peak intensity, minimum resolution and number of shots summed) commonly used to facilitate automated data acquisition were varied. Pseudomonas aeruginosa was used as a model bacterium in the designed experiments, and spectra were acquired using an intact cell sample preparation method. Optimum automated data acquisition settings (i.e., those settings yielding the highest reproducibility of replicate mass spectra) were obtained based on statistical analysis of spectra of P. aeruginosa. Finally, spectrum quality and reproducibility obtained from non-optimized and optimized automated data acquisition settings were compared for P. aeruginosa, as well as for two other bacteria, Klebsiella pneumoniae and Serratia marcescens. Results indicated that reproducibility increased from 90% to 97% (p-value0.002) for P. aeruginosa when more shots were summed and, interestingly, decreased from 95% to 92% (p-value 0.013) with increased threshold minimum resolution. With regard to spectrum quality, highly reproducible spectra were more likely to have high spectrum quality as measured by several quality metrics, except for base peak resolution. Interaction plots suggest that, in cases of low threshold minimum resolution, high reproducibility can be achieved with fewer shots. Optimization yielded more reproducible spectra than non-optimized settings for all three bacteria.

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Section: Methodsmentioning

confidence: 99%

“…Specifically, in the literature, many mass ranges have been used for peak selection, for example, 2 to 20 kDa [11], [18], [40], [41], [45], 2 to 6 kDa [37], 3 to 20 kDa [38], and 7 to 10 kDa [2]. Base peak S:N is usually set as 2 or 3 in literature [2], [11], [38].…”

Section: Methodsmentioning

confidence: 99%

A Designed Experiments Approach to Optimization of Automated Data Acquisition during Characterization of Bacteria with MALDI-TOF Mass Spectrometry

2014

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“…Similarly, 43% of bacterial isolates from two waste disposal sites from non-ferrous metal industry were not reliably identified using MALDI TOF MS and fewer than 20% were identified at species-level (Kopcakova et al, 2014). Despite these limitations, MALDI TOF MS based characterizations have been done for microorganisms from non-clinical sources including, air samples, sewage sludge, biogas plants, ballast water, coastal caves, marine sponges, spinach-processing plant, plant rhizosphere, plant root nodules, wine, and waste disposal sites from non-ferrous metal industry (Dieckmann et al, 2005; Uhlik et al, 2011; Emami et al, 2012; Hausdorf et al, 2013; Stets et al, 2013; Angelakis et al, 2014; Busquets et al, 2014; Kopcakova et al, 2014; Stantscheff et al, 2014; Usbeck et al, 2014). In a comprehensive analysis 5085 isolates of aerobic, heterotrophic and extremely halophilic bacteria and archea from eight solar salterns distributed among different locations in the Spanish Mediterranean, Canary Islands' Atlantic and Chilean Pacific coasts were retrieved combining large-scale cultivation, and identified using MALDI-TOF MS and 16S rRNA gene analysis (Viver et al, 2015).…”

Section: Application Of Maldi Tof Ms In Microbial Ecology Studiesmentioning

confidence: 99%

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry (MALDI-TOF MS) Based Microbial Identifications: Challenges and Scopes for Microbial Ecologists

2016

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Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based biotyping is an emerging technique for high-throughput and rapid microbial identification. Due to its relatively higher accuracy, comprehensive database of clinically important microorganisms and low-cost compared to other microbial identification methods, MALDI-TOF MS has started replacing existing practices prevalent in clinical diagnosis. However, applicability of MALDI-TOF MS in the area of microbial ecology research is still limited mainly due to the lack of data on non-clinical microorganisms. Intense research activities on cultivation of microbial diversity by conventional as well as by innovative and high-throughput methods has substantially increased the number of microbial species known today. This important area of research is in urgent need of rapid and reliable method(s) for characterization and de-replication of microorganisms from various ecosystems. MALDI-TOF MS based characterization, in our opinion, appears to be the most suitable technique for such studies. Reliability of MALDI-TOF MS based identification method depends mainly on accuracy and width of reference databases, which need continuous expansion and improvement. In this review, we propose a common strategy to generate MALDI-TOF MS spectral database and advocated its sharing, and also discuss the role of MALDI-TOF MS based high-throughput microbial identification in microbial ecology studies.

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“…Furthermore, the crop response to inoculation under field conditions heavily depends on the combination of the plant genotype and the bacterial strain (18)(19)(20), stressing the need for methodologies to evaluate the success of plant colonization accurately at a high resolution. Previously, we used whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis to differentiate species of Azospirillum, including several closely related A. brasilense strains (21). However, this method is not quantitative, requires growth on a culture medium, and is time and labor-intensive.…”

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confidence: 99%

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR

Stets

Alquéres

Souza

et al. 2015

Appl Environ Microbiol

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bAzospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N 2 fixation. However, one of the most important factors for achieving an increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for the strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on a comparison of the genomic sequences within the same species. The draft genome sequences of A. brasilense FP2 and Sp245 were aligned, and FP2-specific regions were filtered and checked for other possible matches in public databases. Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were specific for the A. brasilense FP2 strain. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and nonsterile growth conditions. In addition, coinoculations with other plant growth-promoting bacteria in wheat were performed under nonsterile conditions. The results showed that A. brasilense FP2 inoculated into wheat roots is highly competitive and achieves high cell numbers (ϳ10 7 CFU/g [fresh weight] of root) in the rhizosphere even under nonsterile conditions and when coinoculated with other rhizobacteria, maintaining the population at rather stable levels for at least up to 13 days after inoculation. The strategy used here can be applied to other organisms whose genome sequences are available.A zospirillum is one of the most important genera of plant growth-promoting rhizobacteria found worldwide under a variety of environmental and soil conditions (1). The diazotroph Azospirillum brasilense is the best-studied species of the genus, is found in close association with many agriculturally important crops, and exerts beneficial effects on plant growth and productivity (2-4). Nitrogen fixation (5, 6) and the production of the auxin 3-indoleacetic acid (IAA) by many representatives of the genus Azospirillum are related to the growth promotion effects observed in inoculated plants, such as increases in root length and the numbers of root hairs and lateral roots (3).The biotechnological use of A. brasilense inoculants in Latin American and in Brazil, in particular, has increased in recent years (7). Strain FP2 is a spontaneous mutant of A. brasilense Sp7 (8). Strain Sp7 has been shown to be capable of stimulating the growth of several members of the family Poaceae and increasing the productivities of wheat and maize crops (2). Strain FP2 can also promote the growth of wheat (9) and enhance maize and wheat productivity under field conditions (unpublished data). Most of the A. brasilense inoculants in B...

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Rapid identification of bacterial isolates from wheat roots by high resolution whole cell MALDI-TOF MS analysis

Cited by 37 publications

References 39 publications

A Designed Experiments Approach to Optimization of Automated Data Acquisition during Characterization of Bacteria with MALDI-TOF Mass Spectrometry

A Designed Experiments Approach to Optimization of Automated Data Acquisition during Characterization of Bacteria with MALDI-TOF Mass Spectrometry

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry (MALDI-TOF MS) Based Microbial Identifications: Challenges and Scopes for Microbial Ecologists

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR

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