# Difficult-to-culture micro-organisms specifically isolated using the liquid-liquid co-culture method – towards the identification of bacterial species and metabolites supporting their growth

## TL;DR

A new method called liquid-liquid co-culture helps grow hard-to-culture bacteria like Waltera spp. by using supportive bacteria such as E. coli and Bacteroides.

## Contribution

The study introduces a novel co-culture method to isolate and grow difficult-to-culture bacteria and identifies their supporting bacterial partners.

## Key findings

- Waltera spp., Roseburia spp., and Phascolarctobacterium faecium were successfully isolated using liquid-liquid co-culture.
- Bacteroides thetaiotaomicron and Escherichia coli were identified as key supporting bacteria for Waltera spp. growth.
- Growth of Waltera spp. required continuous co-culture and not just metabolite addition, indicating a symbiotic relationship.

## Abstract

Micro-organisms that are difficult to culture were isolated using liquid-liquid co-culture. Waltera spp. only formed colonies through continuous co-culture in liquid media in the presence of Escherichia coli and Bacteroides spp.

In this study, the liquid-liquid co-culture method was applied using faecal samples and specific bacterial species as growth-supporting bacteria. We aimed to isolate new, difficult-to-culture bacterial species using metabolites produced by supportive bacteria to promote the growth of small bacteria selected using filter treatment. This study aimed to identify the supporting bacteria and their metabolites that promote the growth of these isolates. Analysis of the 16S rRNA gene sequences of the isolates obtained by co-culture revealed that they were Waltera spp., Roseburia spp. and Phascolarctobacterium faecium. Roseburia spp. and Waltera spp. were isolated from several faecal samples, suggesting that they were specifically isolated using this culture method. We focused on Waltera spp. isolated from several faecal samples with unique shapes, from long to short or thin cells. The growth of Waltera spp. was not promoted by co-culture on the agar medium, suggesting that growth was only promoted by liquid-liquid co-culture. The growth of the selected small-sized Waltera spp. was promoted by co-culture, whereas the growth of the unfiltered long-cell Waltera sp. strain was suppressed by co-culture. The selected small Waltera spp. did not grow when the supporting bacterial supernatant was added, suggesting that the supporting bacteria and Waltera spp. had a symbiotic relationship through the continuous exchange of metabolites. Co-cultured supporting bacteria (diluted faecal samples) with selected small-sized Waltera spp. were predominantly Bacteroides thetaiotaomicron and Escherichia coli, compared with monoculture diluted faecal samples. We further confirmed the growth of filtered Waltera spp. by co-culturing them with B. thetaiotaomicron and E. coli. Additionally, when B. thetaiotaomicron and E. coli were co-cultured with the selected small Waltera spp., some nutrients and metabolites were reduced. Decreased metabolites were added to the medium, and selected small-sized Waltera spp. were cultured, but Waltera spp. did not grow. Therefore, it was again strongly suggested that continuous co-culturing with the supporting bacteria was important for the growth of Waltera spp. The liquid-liquid co-culture method used in this study can be used to isolate new and unique bacterial species from any environment, not just the gut microbiome. Furthermore, this co-culture method helped identify supporting bacteria and understand metabolite variations.