Exploring the Potential of Micrococcus luteus Culture Supernatant With Resuscitation-Promoting Factor for Enhancing the Culturability of Soil Bacteria.

Marco Antonio Lopez Marin,Jachym Suman,Jiri Santrucek,Petra Junkova,Michal Strejcek,Ondrej Uhlik

doi:10.3389/fmicb.2021.685263

Marco Antonio Lopez Marin, Jachym Suman + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2021.685263

Copy DOI

Abstract

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.

Highlights

The isolation of microorganisms in pure cultures has remained one of the main pillars of microbiology since its beginnings
The reaction of soil bacteria to supernatant containing resuscitation-promoting factor (SRpf) was assessed by community changes in soil suspensions through 16S rRNA amplicon sequencing, while the diversity on plates was assessed by 16S rRNA gene sequencing after dereplication with MALDI-TOF MS (Figure 1)
The number of colony-forming units (CFUs) on plates for each of the three bioaugmented strains was higher for the SRpf-treated extractions than for the controls, i.e., ISRpftreated extractions and phosphate-buffered saline (PBS) (Figure 2)

Summary

INTRODUCTION

The isolation of microorganisms in pure cultures has remained one of the main pillars of microbiology since its beginnings. Because microorganisms do not live in isolation in their natural environments, but are a part of a complex community, the addition of components produced by other organisms may induce the growth of fastidious bacteria Signaling components such as siderophores and quorum sensing compounds have been shown to increase the culturability of bacteria in vitro (Bruns et al, 2002, 2003; D’Onofrio et al, 2010). Growth-promoting factors can increase bacterial culturability by inducing their resuscitation or facilitating their replication (Mukamolova et al, 1999) In their environments, bacteria can subsist dormant as a defense mechanism for coping with deleterious environmental conditions (Mukamolova et al, 2003). The reaction of soil bacteria to SRpf was assessed by community changes in soil suspensions through 16S rRNA amplicon sequencing, while the diversity on plates was assessed by 16S rRNA gene sequencing after dereplication with MALDI-TOF MS (Figure 1)

MATERIALS AND METHODS

RESULTS

DATA AVAILABILITY STATEMENT