DOI: 10.5061/dryad.wm37pvn2b
title: Data from: Cross-feeding constrains the evolution of antibiotic resistance in <em>Escherichia coli</em>

description: The rise of antibiotic-resistant bacteria poses a critical global health
 threat. Previous studies on the evolution of bacterial antibiotic
 resistance have often overlooked the fact that bacteria frequently exist
 within complex microbial communities, where they engage in mutually
 beneficial interactions through nutrient exchange. Here, we studied the
 evolution of antibiotic resistance in an obligate cross-feeding
 population. After 15 days of selection under kanamycin, both co-cultures
 and mono-cultures of auxotrophic strains evolved significantly higher
 resistance. The increase in resistance was significantly lower in the
 co-culture consortium than in each mono-culture consortium. Strains in the
 co-cultures showed higher levels of amino acid secretion and a reduced
 growth rate than those in the mono-cultures. This suggests a higher cost
 of amino acid exchange in the co-cultures. Exogenous amino acids
 supplementation reduced the cost of metabolic exchange and intensified
 competition between the two strains due to niche overlap. This further
 resulted in decreased kanamycin resistance in both the mono-evolved and
 the co-evolved populations under the kanamycin-free environment. The
 interaction among the two auxotrophic strains shifted from positive to
 negative in co-evolved population. This supports the stress gradient
 hypothesis, which posits that facilitation is more common in stressful
 environments, whereas competition dominates when conditions are more
 benign. Our findings demonstrate that environmental stress and nutrient
 availability can modulate bacterial antibiotic resistance by altering
 microbial interactions and fitness dynamics within communities.
description: # Data from: Cross-feeding constrains the evolution of antibiotic
 resistance in *Escherichia coli* Dataset DOI:
 [10.5061/dryad.wm37pvn2b](https://doi.org/10.5061/dryad.wm37pvn2b) ##
 Description of the data and file structure Reference Information
 \===================== Provenance for this README
 \-------------------------- \* File name: README_file.txt File/Folder
 Details \=================== Details for: Data.pdf
 \--------------------------------------- \* Description: a series of
 tables containing population densities, population minimum antibiotic
 inhibitory concentration of kanamycin, amino acid sercetion concentration,
 relative gene expression levels and area under the growth curve of
 bacterial population in the study. \* Format(s): .pdf \* Size(s): 1.14 MB
 Details for: Data.pdf__Figure1 \---------------------------------------
 *Explanation: This is the design of the evolutionary experiment, and it
 does not include the experimental data. Details for: Data.pdf__Figure2
 \--------------------------------------- *Methods: Monoculture and
 coculture populations were transferred into fresh M9 medium every 72 h
 with kanamycin or without knanmycin, all the populations were subjected to
 five serial passages over 15 days. Minimum antibiotic inhibitory
 concentration (MIC) of kanamycin were measured at both the initial and the
 final transfer stages. \* Variables: \* Group: Mono-culture of ΔL,
 mono-culture of ΔA, and a co-culture of ΔL and ΔA \* Kanamycin
 concentration used in each transfer: transfer 1 were 0.25μg/mL and
 0.5μg/mL, transfer 2 were 0.5μg/mL and 1μg/mL, transfer 3 were 1μg/mL and
 2μg/mL, transfer 4 were 2μg/mL and 4μg/mL, transfer 5 were 4μg/mL and
 8μg/mL. \* ΔMIC was calculated by subtracting the MIC values of the
 initial transfer populations from those of their respective descendants
 (the final transfer population). Details for: Data.pdf__Figure3
 \--------------------------------------- *methods: ΔL and ΔA strains were
 separate out from the monocultures and cocultures which evolved under
 kanamycin. ΔL and ΔA strains were cocultured with or without supplementing
 with amino acids. MIC values were determined in different cocultures. \*
 Variables: *ΔL+ΔA-CO: the ΔL and ΔA strain was all separate out from the
 cocultures. *ΔL+ΔA-MO: the ΔL and ΔA strain was all separate out from the
 monocultures. +: the coculture pupolation was supplemented with amino
 acids. Details for: Data.pdf__Figure4
 \--------------------------------------- *methods: ΔL and ΔA strains were
 separate out from the monocultures and cocultures which evolved under
 kanamycin, then monocultured in M9 medium with supplementing amino acids
 for 24 h and 800 rpm at 37 °C in a multifunctional microplate reader to
 generate growth curves. The growth rate and doubling time were calculated
 from the growth curves using r software. Real-time quantitative PCR was
 performed using a QuantiNovaTM SYBR® Green PCR kit to quantify the
 expression of key genes in the arginine and lysine metabolism pathway. The
 arginine production levels of the ΔL strain evolved under antibiotic
 stress were measured using high performance liquid chromatography. \*
 Variables: *ΔL-MO: ΔL strain was separate out from the monocultures.
 *ΔA-MO: ΔA strain was separate out from the monocultures. *ΔL-CO: ΔL
 strain was separate out from the cocultures. *ΔA-CO: ΔA strain was
 separate out from the cocultures. Details for: Data.pdf__Figure5
 \--------------------------------------- *methods: ΔL and ΔA strains were
 separate out from the monocultures and cocultures which evolved in the
 absence of kanamycin. ΔL and ΔA strains were cocultured with or without
 supplementing with amino acids. MIC values were determined in different
 cocultures. \* Variables: *ΔL+ΔA-CO: the ΔL and ΔA strain was all separate
 out from the cocultures. *ΔL+ΔA-MO: the ΔL and ΔA strain was all separate
 out from the monocultures. +: the coculture pupolation was supplemented
 with amino acids. Details for: Data.pdf__Figure6
 \--------------------------------------- *methods: Area under the growth
 curve of ΔL and ΔA strains in both mono-cultures and co-cultures with and
 without supplemental amino acids were calculated. The ΔL and ΔA strains
 that coevolved in the absence of the antibiotic were pre-cultured
 overnight, washed three times with 0.85% NaCl to thoroughly remove any
 residual amino acids from the pre-culture medium, and resuspended in 0.85%
 NaCl to the same optical density (OD600). Then the cultures were
 monocultured and cocultured for 36 h in M9 medium supplemented with and
 without lysine or arginine. The corresponding abundance of each strain
 (CFU) was estimated by plating these cultures onto selective agar culture
 medium over the course of 36 h. \* Variables: *ΔL-MO: ΔL strain was
 separate out from the monocultures. *ΔA-MO: ΔA strain was separate out
 from the monocultures. *ΔL-CO: ΔL strain was separate out from the
 cocultures. *ΔA-CO: ΔA strain was separate out from the cocultures. +: the
 pupolation was supplemented with amino acids. *AUC: area under the growth
 curve, which was caculated by GraphPad Prism 8. Details for:
 Data.pdf__FigureS1 \--------------------------------------- *methods: The
 ancestral ΔL and ΔA strain were monocultured in M9 medium which contained
 0.08 g/L, 0.09 g/L, 0.1 g/L, 0.11 g/L, 0.12 g/L, 0.13 g/L, 0.14 g/L, 0.15
 g/L, 0.20 g/L, 0.25 g/L and 0.30 g/L lysine or arginine. All populations
 were cultured for 36 h and 800 rpm at 37 °C in a multifunctional
 microplate reader, the OD600 value was measured at hourly intervals. The
 concentration that reached the maximum optical density (Max OD600) was
 considered as the most appropriate supplement concentration of exogenous
 amino acid. \* Variables: *Max OD600: the maximum optical density. Details
 for: Data.pdf__FigureS2 \--------------------------------------- *methods:
 The ancestral ΔL strain、ancestral ΔA strain and wild type strain were
 monocultured in M9 medium without supplementing amino acids. All
 populations were cultured for 24 h and 800 rpm at 37 °C in a
 multifunctional microplate reader, the OD600 value was measured at hourly
 intervals. \* Variables: *ΔL-ANC: ancestral ΔL strain. *ΔA-ANC: ancestral
 ΔA strain. *Wild-type: Escherichia coli K-12 MG1655 Details for:
 Data.pdf__FigureS3 \--------------------------------------- *methods:
 Molecular validation of ΔL and ΔA strain. Gel was run at 150 V for 15min,
 the wild type gene band of lysA and argH were at 1762 bp and 1873 bp, ΔL
 and ΔA strains gene band of were at 500bp. \* Variables: *ΔL-ANC:
 ancestral ΔL strain. *ΔA-ANC: ancestral ΔA strain. *Wild-type: Escherichia
 coli K-12 MG1655 *ΔL-K5-C: ΔL strain was separate out from the cocultures
 under kanamycin at the final transfer. *ΔL-K5-M: ΔL strain was separate
 out from the monocultures under kanamycin at the final transfer. *ΔA-K5-C:
 ΔA strain was separate out from the cocultures under kanamycin at the
 final transfer. *ΔA-K5-M: ΔA strain was separate out from the monocultures
 under kanamycin at the final transfer. *ΔL-T5-C: ΔL strain was separate
 out from the cocultures without kanamycin at the final transfer. *ΔL-T5-M:
 ΔL strain was separate out from the monocultures without kanamycin at the
 final transfer. *ΔA-T5-C: ΔA strain was separate out from the cocultures
 without kanamycin at the final transfer. *ΔA-T5-M: ΔA strain was separate
 out from the monocultures without kanamycin at the final transfer. Details
 for: Data.pdf__FigureS4 \--------------------------------------- *methods:
 The ancestral ΔL and ΔA strain were monocultured or cocultuerd in M9
 medium. All populations were cultured for 64 h and 800 rpm at 37 °C in a
 multifunctional microplate reader, the OD600 value was measured at hourly
 intervals. Doubling time were derived from specific growth rates used the
 package Growthcurver in R-version 4.3.1, relative fitness was expressed as
 the ratio of the Malthusian parameter (m) in which the initial culture
 OD600 was annotated as N0 and the culture OD600 at time i was annotated as
 Ni. mi=[ln(Ni/N0)]/i \* Variables: *ΔL-ANC: monocultures of ancestral ΔL
 strain. *ΔA-ANC: monocultures of ancestral ΔA strain. *ΔL+ΔA-ANC:
 cocultures of ancestral ΔA and ΔLstrain. +: the pupolation was
 supplemented with amino acids. Details for: Data.pdf__FigureS5
 \--------------------------------------- *methods: Ancestral ΔL and ΔA
 strain were monocultured and cocultured under 12 different kanamycin
 concentrations. After 36 h incubation, the OD600 were measured using a
 microplate reader, and the MIC was determined by GraphPad Prism 8
 software. \* Variables: *ΔL-ANC: monocultures of ancestral ΔL strain.
 *ΔA-ANC: monocultures of ancestral ΔA strain. *ΔL+ΔA-ANC: cocultures of
 ancestral ΔA and ΔLstrain. +: the pupolation was supplemented with amino
 acids. Details for: Data.pdf__FigureS6
 \--------------------------------------- *methods: Ancestral ΔL and ΔA
 strain, ΔL and ΔA strain which evolved under kanamycin at final transfer,
 were cocultured for 36h, the OD600 value was measured at hourly intervals.
 Growth rate were caculated using the package Growthcurver in R-version
 4.3.1. \* Variables: *ΔL+ΔA-ANC: cocultures of ancestral ΔA and ΔLstrain.
 *ΔL+ΔA-CO: cocultures of ΔA and ΔLstrain, which coevolved under kanamycin
 at the final transfer. *ΔL+ΔA-MO: cocultures of ΔA and ΔLstrain, which
 monoevolved under kanamycin at the final transfer. *Max OD600: maximum
 optical density Details for: Data.pdf__FigureS7
 \--------------------------------------- *methods: ΔL and ΔA strain which
 derived from kanamycin at the initial and final transfer were cocultured
 for 24h, the OD600 value was measured at hourly intervals. \* Variables:
 *ΔL+ΔA-CO: cocultures of ΔA and ΔLstrain, which coevolved under kanamycin.
 *ΔL+ΔA-MO: cocultures of ΔA and ΔLstrain, which monoevolved under
 kanamycin. +: the pupolation was supplemented with amino acids.
 *Kanamycin: the strains were evolved under kanamycin. Details for:
 Data.pdf__FigureS8 \--------------------------------------- *methods: ΔL
 and ΔA strain which derived from kanamycin at the final transfer were
 monocultured or cocultured for 24h, the OD600 value was measured at hourly
 intervals. \* Variables: *ΔL+ΔA-CO: cocultures of ΔA and ΔLstrain, which
 coevolved under kanamycin. *ΔL+ΔA-MO: cocultures of ΔA and ΔLstrain, which
 monoevolved under kanamycin. *Kanamycin: the strains were evolved under
 kanamycin. Details for: Data.pdf__FigureS9
 \--------------------------------------- *methods: RNA extraction of ΔL
 that evolved under antibiotic was performed using the RNA iso plus
 extraction solution. The relative expression levels of the target genes
 were normalized using a reference gene (16S rRNA). Gene expression was
 measured using 2-ΔΔCT method. \* Variables: *ΔL-CO: ΔL strain was separate
 out from the cocultures which evolved under kanamycin. *ΔA-CO: ΔA strain
 was separate out from the cocultures which evolved under kanamycin.
 Details for: Data.pdf__FigureS10 \---------------------------------------
 *methods: RNA extraction of ΔL and ΔLA that evolved without antibiotic was
 performed using the RNA iso plus extraction solution. The relative
 expression levels of the target genes were normalized using a reference
 gene (16S rRNA). Gene expression was measured using 2-ΔΔCT method. \*
 Variables: *ΔL-CO: ΔL strain was separate out from the cocultures which
 evolved without kanamycin. *ΔA-CO: ΔA strain was separate out from the
 cocultures which evolved without kanamycin. ### Files and variables ####
 File: Data.pdf **Description:** a series of tables containing population
 densities, population minimum antibiotic inhibitory concentration of
 kanamycin, amino acid serection concentration, relative gene expression
 levels and area under the growth curve of bacterial population in the
 study.