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

Abstract

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.

New Arctic Bacterial Isolates with Relevant Enzymatic Potential

“…On the contrary, the isolates WG29 and WG35 grouped within one cluster according to MALDI-TOF MS Biotyper did not reflect 100% similarity according to the 16S rDNA analysis. This is in accordance with the opinion of Strejcek et al [ 25 ], who established that, although whole-cell MALDI-TOF-based identification of bacterial isolated is in general agreement with the results of 16S rRNA gene sequence analysis, high accordance was confirmed merely up to the genus level. Nevertheless, at the species level, the agreement appeared to be limited.…”

16S rRNA gene sequencing and MALDI-TOF mass spectrometry based comparative assessment and bioprospection of psychrotolerant bacteria isolated from high altitudes under mountain ecosystem

“…Strejcek et al [44] also compared whole-cell MALDI-TOF MS with 16S rRNA gene analysis for identification of recurrent bacterial isolates. The authors confirmed the potential of MALDI-TOF MS as a rapid screening method in environmental microbiology for determining similarities between bacterial isolates at genus level with various limitations such as low number of peaks, availability of reference mass spectra of only well-studied and described strains in database, etc.…”

Introducing SPeDE: High-Throughput Dereplication and Accurate Determination of Microbial Diversity from Matrix-Assisted Laser Desorption–Ionization Time of Flight Mass Spectrometry Data

“…We compared the outcome of SPeDE dereplication for the benchmark data set to that of previously described methods based on (i) the global Pearson product moment correlation and the unweighted pair group method with arithmetic mean (UPGMA) clustering proposed by Ghyselinck and colleagues (21) and (ii) the cosine similarity/UPGMA clustering described by Strejcek and colleagues (22). The results are given in Table S3 and Table S4, respectively.…”

“…The method of Ghyselinck et al (21) returned the most OIUs, retrieved the least diversity from the benchmark data set, and had the overall longest processing time (Table 1). The method of Strejcek and colleagues (22), based on a proposed cosine similarity cutoff of 92%, was the best performing in terms of overall dereplication ratio, with a reduction of 97% of the spectra for the benchmark data set. However, the analysis also returned 15 OIUs which included multiple genetically distinct strains, with 1 OIU merging strains sharing less than 92% genome-wide ANI (Table 1).…”

“…Methods based on global similarity measures combined with hierarchical clustering of mass spectra have been developed to uncouple dereplication from identification (15, 20). A shortcoming of this strategy is that the identification of redundant profiles relies either on visual inspection of dendrograms or on the use of a predefined distance cutoff value to delineate clusters of similar spectra (21, 22). Predefined cutoff values do not consider the variability of profiles between taxa and thus need to be adjusted according to the taxonomic composition of the samples.…”