Image_1_Whole-Cell MALDI-TOF MS Versus 16S rRNA Gene Analysis for Identification and Dereplication of Recurrent Bacterial Isolates.PDF

<p>Many ecological experiments are based on the extraction and downstream analyses of microorganisms from different environmental samples. Due to its high throughput, cost-effectiveness and rapid performance, Matrix Assisted Laser Desorption/Ionization Mass Spectrometry with Time-of-Flight detector (MALDI-TOF MS), which has been proposed as a promising tool for bacterial identification and classification, could be advantageously used for dereplication of recurrent bacterial isolates. In this study, we compared whole-cell MALDI-TOF MS-based analyses of 49 bacterial cultures to two well-established bacterial identification and classification methods based on nearly complete 16S rRNA gene sequence analyses: a phylotype-based approach, using a closest type strain assignment, and a sequence similarity-based approach involving a 98.65% sequence similarity threshold, which has been found to best delineate bacterial species. Culture classification using reference-based MALDI-TOF MS was comparable to that yielded by phylotype assignment up to the genus level. At the species level, agreement between 16S rRNA gene analysis and MALDI-TOF MS was found to be limited, potentially indicating that spectral reference databases need to be improved. We also evaluated the mass spectral similarity technique for species-level delineation which can be used independently of reference databases. We established optimal mass spectral similarity thresholds which group MALDI-TOF mass spectra of common environmental isolates analogically to phylotype- and sequence similarity-based approaches. When using a mass spectrum similarity approach, we recommend a mass range of 4–10 kDa for analysis, which is populated with stable mass signals and contains the majority of phylotype-determining peaks. We show that a cosine similarity (CS) threshold of 0.79 differentiate mass spectra analogously to 98.65% species-level delineation sequence similarity threshold, with corresponding precision and recall values of 0.70 and 0.73, respectively. When matched to species-level phylotype assignment, an optimal CS threshold of 0.92 was calculated, with associated precision and recall values of 0.83 and 0.64, respectively. Overall, our research indicates that a similarity-based MALDI-TOF MS approach can be routinely used for efficient dereplication of isolates for downstream analyses, with minimal loss of unique organisms. In addition, MALDI-TOF MS analysis has further improvement potential unlike 16S rRNA gene analysis, whose methodological limits have reached a plateau.</p

Top OTUs detected in ¹³C-DNA after incubation of soil with ¹³C-biphenyl, ¹³C-benzoate, and ¹³C-naphthalene.

<p>Identification was performed by mothur-implemented RDP reference files <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040653#pone.0040653-Wang1" target="_blank">[78]</a> and the closest type strain was determined by RDP Seqmatch with the representative sequence of each OTU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040653#pone.0040653-Cole1" target="_blank">[76]</a>. The entire dataset is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040653#pone.0040653.s002" target="_blank">Table S1</a>.</p>a<p>Relative abundance of sequences.</p>b<p>Identification of OTU based on identification of the representative at the level of genus as determined by RDP classifier (using 50% threshold).</p>c<p>Determined by RDP Seqmatch.</p>d<p>Score represents S_ab score – the number of (unique) 7-base oligomers shared between the sequence data and a given RDP sequence divided by the lowest number of unique oligos in either of the two sequences.</p>e<p>Refers to samples where the same OTU was detected.</p

Additional file 1: Table S1. of Complete genome sequence of Pseudomonas alcaliphila JAB1 (=DSM 26533), a versatile degrader of organic pollutants

Description of five regions harboring genes for aromatic compound degradation pathways were identified in the JAB1 genome. (DOCX 34 kb

Number of sequences obtained after sequence processing, after subtracting sequences detected also in control DNA (valid sequences), and after normalizing.

<p>Number of sequences obtained after sequence processing, after subtracting sequences detected also in control DNA (valid sequences), and after normalizing.</p

Analysis of inorganic nutrients and contaminants available in the soil.

<p>Results shown are averages from 5 independently measured samples (performed commercially).</p>a<p>Based on method CZ_SOP_D06_07_121.</p>b<p>Based on method US EPA 8082.</p>c<p>Based on methods EPA 8270, EPA 8131, EPA 8091.</p>d<p>Based on method US EPA 200.7.</p

Bacterial strains used for the preparation of the mock community.

<p>Bacterial strains used for the preparation of the mock community.</p