DOI: 10.5061/dryad.b5mkkwhpg
title: Shifts and rebound in microbial community function following repeated introduction of a novel species

description: Natural microbial communities continually encounter novel species that may
 successfully establish or simply be transient, yet both outcomes can alter
 the resident community composition and function. Preserving natural
 microbial communities and innovating synthetic ones requires insight on
 the immediate and long-term impact of species introductions on both
 composition and function. For instance, it remains unclear whether there
 are gradual and long-term impacts from repeated introductions where the
 introduced species fails to establish – so-called failed invaders. To
 investigate the persistent impacts by failed invaders, we present an
 experimental test of community stability over multiple generations against
 repeated novel species introduction. We propagated a natural microbial
 community from a traditional fermented milk beverage for approximately 100
 generations, with or without, repeated introduction of Escherichia coli at
 each transfer. Community function was determined by metabolic profiling,
 and we observed alterations therein immediately after E. coli
 introduction, followed by recovery, or rebound once ceased. In contrast to
 this proxy of community function, changes in the bacterial community
 composition were never detected. Our results evidence that community
 composition and function do not necessarily respond in parallel to an
 introduced species, potentially due to genotypic changes below species
 level detection or metabolic plasticity. Our work shows an ability for
 functional recovery in microbial communities and contributes insight on
 long-term community stability to sustained disturbances.
description: # Shifts and rebound in microbial community function following repeated
 introduction of a novel species
 [https://doi.org/10.5061/dryad.b5mkkwhpg](https://doi.org/10.5061/dryad.b5mkkwhpg) ## Description of the data and file structure: ### Shifts and rebound in microbial community function following repeated introduction of a novel species DOI: 10.1111/oik.10880 **General notes:** Two experiments were run in parallel, so datasets here include: * Experiment 1 = repeated E. coli invasion (i.e., the data relevant for this manuscript).  * Experiment 2 = propagation of top/bottom Mabisi [this was not used for any manuscript nor thesis chapter, concerns variable "SPACE"] **Bacterial community profiles:** * fall_2022_communities_v1.R (includes description of experimental variables) * output_barcoded_mabisi_2plates_0.95_clustering.csv (bionformatic pipeline output: "abundance","cluster","sample","total_count") * fall_2022_META.csv (match experimental variables to each sequencing barcode: TRANSFER, COMMUNITY, REP, SPACE, SAMPLE, PLATE) * new_names.csv (for renaming cluster names) * unique.csv (to define number of unique clusters) * colours.csv (to assign specific colours for each cluster) **Metabolic profiles:** * fall_2022_gcms_v3.R * gcms_raw_dec2022.xlsx (includes read.me tab explaining variables) * aroma_names.csv (full name of aromas) **Acidity:** * fall_2022_ph_v2.R * fall_2022_ph.xlsx (raw pH values, includes read.me tab explaining variables) ***E. coli*** (growing alone in BHI and milk): * feb2024_ecoli_cfus.R * feb2024_ecoli.xlsx (pH and raw plate counts after 72 hours, includes read.me tab explaining variables) **Description of variables:** TREATMENT: transfering from top 20mL (top), bottom 20mL (bottom), normally (con), tube sealed and kept on spinner (mix), ecoli added at every transfer (inv), ecoli line recovering with no ecoli added (rec) TRANSFER: 0 = day of inoculation. Fermentation regime: 72hrs @ 28 C, backslopped (t1), 24 hrs @ 4 C, 72 hours @28 C, backslopped (t2). Repeat. t02 refers to end-product of t01 REP: replicate 1 to 6 CODE: combination of treatment + replicate SPACE: area of tube sampled from (i.e., upper 20mL = up, lower 20mL = low, entire tube mixed = all) ### FILES & VARIABLES: #### File: fall2022\_META.csv **Description:** META data for: Shifts and rebound in microbial community function following repeated introduction of a novel species (DOI: 10.1111/oik.10880) ##### Variables: * TRANSFER:  0 = day of inoculation. Fermentation regime: 72hrs @ 28 C, backslopped (t1), 24 hrs @ 4 C, 72 hours @28 C, backslopped (t2). Repeat. t02 refers to end-product of t01 * COMMUNITY: transferring from top 20mL (top), bottom 20mL (bottom), normally (con), tube sealed and kept on spinner (mix), ecoli added at every transfer (inv), ecoli line recovering with no ecoli added (rec) * REP: biological replicate 1 to 6 * SPACE: area of tube sampled from (i.e., upper 20mL = up, lower 20mL = low, entire tube mixed = all). will be removed, because this data is from a separate experiment.  * CODE: combination of COMMUNITY + REP * PLATE: from MinIon sequencing. Plate 1 removed, as described in methods.  * SAMPLE: number 001 to 192 for barcoding * barcode: bc01 to bc86, from MinIon sequencing. #### File: fall\_2022\_communities\_v1.R **Description:** R script for community composition analysis.  It includes description of experimental variables. #### File: colours.csv **Description:** used to assign specific colours for each cluster in community composition analysis with "fall_2022_communities_v1.R".  #### File: new\_names.csv **Description:** used for renaming cluster names in community composition analysis with "fall_2022_communities_v1.R. " #### File: unique.csv **Description:** used to define number of unique clusters in community composition analysis with "fall_2022_communities_v1.R. " #### File: output\_barcoded\_mabisi\_2plates\_0.95\_clustering.csv **Description:** bionformatic pipeline output from full 16S rRNA Oxford Nanopore sequencing.  ##### Variables: * abundance: relative abundance  * cluster: a custom database was produced using vsearch to cluster 16S rRNA sequences at a 95% similarity threshold.Details of sequence compositions for main clusters are found in Table S3 from Leale et al. 2023 (Leale et al. 2023). * sample: combination of plate (1 or 2) and barcode (bc01 to bc96).  * total_count: number of sequence reads  ##### Note on NAs: First column is simply blank and contains no variables, thus may show as NAs. #### File: feb2024\_ecoli\_cfus.R **Description:** R script to analyse pH and raw plate counts after 72 hours of E. coli in BHI broth and milk. #### File: feb2024\_ecoli.xlsx **Description:** pH and raw plate counts after 72 hours. File includes read.me tab explaining variables. ##### Variables: | bhi\_6 | raw colony count of glycerol stock into BHI broth after 72hrs, 37 C, shaken. "bhi\_6" refers to 10^-6 dilution VRB media | | :-------- | :------------------------------------------------------------------------------------------------------------------------------------------------- | | treatment | E. coli added from glycerol to BHI broth, then broth culutre to mik. Negative is nothing added to broth or milk. | | milk\_4 | raw colony count of E. coli culture taken from BHI into milk, fermented for 72hrs, 28 C, unshaken. "milk\_4" refers to 10^-4 dilution on VRB media | | milk\_pH | pH of milk after 72 hrs of fermentation at 28 C, unshaken.  | ##### Note on NAs: Empty cells (i.e., NAs) were agar medias x dilution levels which were not plated and therefore no data was collected for these cells.  #### File: fall\_2022\_gcms\_v3.R **Description:** R script to analyse metabolic profiles of raw data from GC-MS machine. #### File: aroma\_names.csv **Description:** full name of metabolic compounds (i.e., aromas)  #### File: gcms\_raw\_dec2022.xlsx **Description:** metabolic profiles of raw data from GC-MS machine. File includes read.me tab explaining variables.  ##### Sheets / Variables: | Sheet | | | :-------- | :-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | raw\_100 | samples 1-100 (includes incorrect 94). Raw data output from Chromeleon - untouched  | | raw\_180 | samples 101-180. Raw data output from Chromeleon - untouched  | | raw\_2 | retention time and value removed, n.a.s replaced for 0.001 | | forR.001 | data transposed, sample names added, aromas replaced for A01-A29.  zeros replaced for 0.001  | | | | | variable | | | treatment | transferring from top 20mL (top), bottom 20mL (bottom), normally (con), tube sealed and kept on spinner (mix), ecoli added at every transfer (inv), ecoli line recovering with no ecoli added (rec) | | transfer | 0 = day of inoculation. Fermentation regime: 72hrs @ 28 C, backslopped (t1), 24 hrs @ 4 C, 72 hours @28 C, backslopped (t2). Repeat.  | | code | combination of treatment + replicate  | | space | area of tube sampled from (i.e., upper 20mL = up, lower 20mL = low, entire tube mixed = all) | | rep | replicate 1-6 | ##### Note on NAs: NAs in sheet "raw100" and "raw180" were compounds below detection level (i.e., flat line/ no peak in GC-MS plot).  ### File: fall\_2022\_ph\_v2.R **Description:**  R script for analysing acidity over time.  ### File: fall\_2022\_ph.xlsx **Description:** raw pH values, includes read.me tab explaining variables.  ##### Variables: | variable | description | | :-------- | :------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | date | date of pH measuring | | treatment | transfering from top 20mL (top), bottom 20mL (bottom), normally (con), tube sealed and kept on spinner (mix), ecoli added at every transfer (inv), ecoli line recovering with no ecoli added (rec) | | transfer | 0 = day of inoculation. Fermentation regime: 72hrs @ 28 C, backslopped (t01), 24 hrs @ 4 C, 72 hours @28 C, backslopped (t02). Repeat. So, t02 refers to end product of t01. | | code | combination of treatment + replicate  | | space | area of tube sampled from (i.e., upper 20mL = up, lower 20mL = low, entire tube mixed = all) | | rep | replicate 1-6 | | ph | acidity measured at room temperature | ##### Note on NAs: NAs are missing pH values of one replicate lost during the experiment ("ecoli1"). One other pH value that was mistakenly not measured and the sample thrown away.  ## Code/software R version 4.3.2 (R Core Team 2023) was used with R Studio.  ## Access information Other publicly accessible locations of the data: * Raw amplicon sequence data for the communities are available under NCBI BioProject PRJNA1096163 with each barcode available separately under SAMN40628846 - SAMN40629034. * Source data files and codes also available on GitHub: [https://github.com/amleale/ecoli_mabisi_2022](https://github.com/amleale/ecoli_mabisi_2022)