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A bacterial species is best characterized after its isolation in a pure culture. This is an arduous endeavor for many soil microorganisms, but it can be simplified by several techniques for improving culturability: for example, by using growth-promoting factors. We investigated the potential of a Micrococcus luteus culture supernatant containing resuscitation-promoting factor (SRpf) to increase the number and diversity of cultured bacterial taxa from a nutrient-rich compost soil. Phosphate-buffered saline and inactivated SRpf were included as controls. After agitation with SRpf at 28°C for 1 day, the soil suspension was diluted and plated on two different solid, oligotrophic media: tenfold diluted Reasoner’s 2A agar (R2A) and soil extract-based agar (SA). Colonies were collected from the plates to assess the differences in diversity between different treatments and cultivation media. The diversity on both R2A and SA was higher in the SRpf-amended extracts than the controls, but the differences on R2A were higher. Importantly, 51 potentially novel bacterial species were isolated on R2A and SA after SRpf treatment. Diversity in the soil extracts was also determined by high-throughput 16S rRNA amplicon sequencing, which showed an increase in the abundance of specific taxa before their successful cultivation. Conclusively, SRpf can effectively enhance the growth of soil bacterial species, including those hitherto uncultured.

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.