The giant panda gut harbors a high diversity of lactic acid bacteria revealed by a novel culturomics pipeline

ABSTRACT Some lactic acid bacteria (LAB) can provide significant health benefits, which are critically important for the conservation of endangered animals, such as giant pandas. However, little is known about the diversity and culturability of LAB in the giant panda gut microbiota. To understand the roles of LAB in giant panda conservation, it is critical to culture bacterial strains of interest. In this study, we established a pipeline to culture bacterial strains using enrichment of target bacteria with different liquid media and growth conditions. Then, the strains were isolated in solid media to study their functions. Using 210 samples from the culture enrichment method and 138 culture-independent samples, we obtained 1120 amplicon sequencing variants (ASVs) belonging to Lactobacillales. Out of the 1120 ASVs, 812 ASVs from the culture enrichment approach were twofold more diverse than 336 ASVs from the culture-independent approach. Many ASVs of interest were not detected in the culture-independent approach. Using this pipeline, we isolated many relevant bacterial strains and established a giant panda gut bacteria strain collection that included strains with low-abundance in culture-independent samples and included most of the giant panda LAB described by other researchers. The strain collection consisted of 60 strains representing 35 species of 12 genera. Thus, our pipeline is powerful and provides guidance in culturing gut microbiota of interest in hosts such as the giant panda. IMPORTANCE Cultivation is necessary to screen strains to experimentally investigate microbial traits, and to confirm the activities of novel genes through functional characterization studies. In the long-term, such work can aid in the identification of potential health benefits conferred by bacteria and this could aid in the identification of bacterial candidate strains that can be applied as probiotics. In this study, we developed a pipeline with low-cost and user-friendly culture enrichment to reveal the diversity of LAB in giant pandas. We compared the difference between culture-independent and culture enrichment methods, screened strains of interest that produced high concentrations of short-chain fatty acids (SCFAs), and we investigated the catalog of virulence factors, antibiotic resistance, butyrate and lactate synthesis genes of the strains at a genomic level. This study will provide guidance for microbiota cultivation and a foundation for future research aiming to understand the functions of specific strains.

Figure S4: The Venn chart of all observed ASVs: all ASVs for every culture media.The details of culture media are shown in Table S2.S3.The number indicated the correlations (r 2 ).Here, only the positive correlation between the important ASVs and SCFAs are shown (P < 0.05).

Supplemental Table Legends
Table S1: the information of samples of culture-independent method in this study.
Table S2: the information of culture media in this study.
Table S3: the information of samples of culture-enrichment method and the statistical analysis of SCFAs among culture media.(a separate file) Table S4: the relative abundance and taxonomy of ASVs of culture-independent and culture-enrichment methods.(a separate file) Table S5: the ANI values (> 0.95) and closest species of genomes from JSpecies.
Table S6: the ANI values (< 95%) and taxonomy of genomes from JSpecies.Here showed the identification of TYGS and results of CheckM.The details of culture media showed in table S2. Figure S7: the comparisons of SCFAs among culture media.The details of statistic analysis showed in Table S3.
Continued Figure S7:   The tree was based on sequences from ldhA using the Maximum Likelihood method based on the Poisson correction model in MEGA X.The percentage of trees in which the associated taxa clustered together is shown next to the branches.The number at the end of name for every strain showed the code of every copy of ldhA in corresponding genome.

Figure S1 :
Figure S1: the pipeline of this study.

Figure S2 :
Figure S2: the relative abundance on genus level for culture-independent method.Every column denoted one sample.

Figure S6 :
Figure S6: The PCoA of unweighted (A) and weighted (B) UniFrac analysis and the PCA of Bray-Curtis distance (C) of culture-enriched samples based on culturing days.

Figure S7 :
Figure S7: Comparisons of SCFAs among culture media.The details of statistical analysis are shown in TableS3.

Figure S8 :
Figure S8: Phylogenetic distribution of 12 Lactobacillus plantarum genomes following the results of TYGS in culture-enriched cohort.

Figure S9 :
Figure S9: Phylogeny of Butyrate kinase (Buk).The tree was based on sequences from Buk using the Maximum Likelihood method based on the Poisson correction model in MEGA X.The percentage of trees in which the associated taxa clustered together is shown next to the branches.The number at the end of name for every strain showed the code of every copy of Buk in corresponding genome.

Figure S10 :
Figure S10: Phylogeny of Lactate dehydrogenase A (encoded by ldhA).The tree was based on sequences from ldhA using the Maximum Likelihood method based on the Poisson correction model in MEGA X.The percentage of trees in which the associated taxa clustered together is shown next to the branches.The number at the end of name for every strain showed the code of every copy of ldhA in corresponding genome.

Figure S1 :
Figure S1: the pipeline of this study.

Figure
Figure S2: the relative abundance of genus level for culture-independent method.Every column denoted one sample.

Figure
Figure S8：Phylogenetic distribution of 12 Lactobacillus plantarum genomes following the results of TYGS in culture-enriched cohort.

Figure S9 :
Figure S9: Phylogeny of Butyrate kinase (Buk).The tree was based on sequences from Buk using the Maximum Likelihood method based on the Poisson correction model in MEGA X.The percentage of trees in which the associated taxa clustered together is shown next to the branches.The number at the end of name for every strain showed the code of every copy of Buk in corresponding genome.

Figure S10 :
Figure S10: Phylogeny of Lactate dehydrogenase A (encoded by ldhA).The tree was based on sequences from ldhA using the Maximum Likelihood method based on the Poisson correction model in MEGA X.The percentage of trees in which the associated taxa clustered together is shown next to the branches.The number at the end of name for every strain showed the code of every copy of ldhA in corresponding genome.

Figure S11 :
Figure S11: Pearson's correlations of different ASVs in culture-enrichment with SCFAs.The number indicated the correlations (r 2 ).Here only showed the positive correlation between the important ASVs and SCFAs (P < 0.05).

Table S1 :
The information of samples of culture-independent method in this study.

Table S2 :
The information of culture media in this study.

Table S5 :
the ANI values (> 0.95) and closest species of genomes from JSpecies.

Table S6 :
the ANI values (< 95%) and taxonomy of genomes from JSpecies.Here showed the identification of TYGS and results of CheckM.