Operational taxon pairs in microbial co-occurrence networks imply complicated bacterial associations
Min-Zhi Jiang, Haizhen Zhu, Nan Zhou, Chang Liu, Cheng‐Ying Jiang, Yulin Wang, Shuang‐Jiang Liu · Research Square · 2022
Abstract Background Microbial co-occurrence networks inferred from the abundance data of environmental and human gut samples are widely applied to describe microbial associations and to predict potential microbial interactions. However, the microbial associations and predicted interactions were hardly demonstrated with cultivated microbial isolates. Experimental demonstration of the microbial associations and the predicted interactions needs extensive isolation and cultivation of microbes and matches the isolates to operational taxon units (OTUs) from co-occurrence networks. The high workloads of bacterial isolation and cultivation, the shortage of methods to match cultivated bacterial isolates to co-occurrence network OTUs, and the complexity of the networks themselves were factors that constrained experimental demonstrations. Results Here, we integrated droplet microfluidics and bar-coding logistics for high-throughput bacterial isolation and cultivation from samples. The co-occurrence network complexity of samples was reduced by dilution of samples. Phylogenetic tree topology was applied to match bacterial isolates to zero-level OTUs (Zotus). We collected wetland samples from Beijing Olympic Park (BOP) including Potamogeton perfoliatus plants, their roots and root peripheral sediments. Droplets of series diluted homogenates of wetland samples were inoculated into 126 of 96-well plates containing R2A and TSB media. After 10 days of cultivation, 65 plates (each with > 30% wells of the 96 wells showed microbial growth) were elected to reconstruct co-occurrence networks. The prevalent co-occurred zero-level OTUs (Zotus) pairs were selected from the networks and were matched to bacterial isolates. We cultivated 129 bacterial isolates belonged to 15 taxa at species level. Thirty-six bacterial isolate combinations corresponding to 19 Zotus pairs were evaluated for interactions on agar plates. Results suggested that positively associated Zotu pairs in the co-occurrence network implied complicated relations including neutralism, competition, and mutualism. We further tested in broth the bacterial isolate combinations between BOP-1, BOP-5, BOP-11 or BOP-16 and BOP-108 that represented the Zotu1-Zotu10 pair. Results revealed that this Zotu1-Zotu10 pair had also competitive, neutral, or mutual relation, depending on bacterial isolate combination and on cultivation time. Conclusion This study established a workflow for experimental demonstration of relations between Zotu pairs from microbial co-occurrence networks. Using a wetland sample, we reconstructed microbial co-occurrence networks and cultivated extensively bacterial isolates, and further demonstrated that positively associated Zotu pairs had neutral, competitive, and mutual relations during bacterial cultivations on solid agar plates and in liquid broth.