DOI: 10.5061/dryad.2jm63xszx
title: Differential selection for survival and for growth in adaptive laboratory evolution experiments with benzalkonium chloride

description: Biocides are used to control microorganisms across different applications,
 but emerging resistance may pose risks for those applications. Resistance
 to biocides has commonly been studied using adaptive laboratory evolution
 (ALE) experiments with growth at subinhibitory concentrations linked to
 serial subculturing. It has been shown recently that E. coli adapts to
 repeated lethal stress imposed by the biocide benzalkonium chloride (BAC)
 by increased survival (i.e. tolerance) and not by evolving the ability to
 grow at increased concentrations (i.e. resistance). Here, we investigate
 the contributions of evolution for tolerance as opposed to resistance for
 the outcome of ALE experiments with E. coli exposed to BAC. We find that
 BAC concentrations close to the half maximal effective concentration
 (EC50, 4.36 µg mL-1) show initial killing (~40%) before the population
 resumes growth. This indicates that cells face a two-fold selection
 pressure: for increased survival and for increased growth. To disentangle
 the effects of both selection pressures, we conducted two ALE experiments:
 (i) one with initial killing and continued stress close to the EC50 during
 growth and (ii) another with initial killing and no stress during growth.
 Phenotypic characterization of adapted populations showed that growth at
 higher BAC concentrations was only selected for when BAC was present
 during growth. Whole genome sequencing revealed distinct differences in
 mutated genes across treatments. Treatments selecting solely for survival
 led to mutations in genes for metabolic regulation (cyaA) and cellular
 structure (flagella fliJ), while treatments selecting for growth and
 survival led to mutations in genes related to stress response (hslO and
 tufA). Our results demonstrate that serial subculture ALE experiments with
 an antimicrobial at sub-inhibitory concentrations can select for increased
 growth and survival. This finding has implications for the design of ALE
 experiments to assess resistance risks of antimicrobials in different
 scenarios such as disinfection, preservation, and environmental pollution.
description: The dataset contains microbial growth data in the presence and
 absence of the biocide benzalkonium chloride. The data was measured as
 increase of OD over time in a microtiter plate reader (Fig. 2A, Fig. 3B
 & C) or as counts of colony forming units (cfu) (Fig. 2B &
 C and Fig. 3A). In addition, antimicrobial susceptibility data regarding
 different antibiotics and benzalkonium chloride for <em>E.
 coli</em> populations that evolved under benzalkonium chloride
 exposure are provided. The details of the underlying experiments and data
 processing are described in the Methods section of the linked manuscript.
 The provided data constitutes results of processed growth data, which was
 used to create the graphs shown in Figure 2 and Figure 3 in the
 manuscript.
description: # Differential selection for survival and for growth in adaptive
 laboratory evolution experiments with benzalkonium chloride
 [https://doi.org/10.5061/dryad.2jm63xszx](https://doi.org/10.5061/dryad.2jm63xszx) ## Description of the data and file structure ### Files and variables #### File: Figure2A.xlsx **Description:** Dose-response curve for growth rate as a function of benzalkonium chloride (BAC) exposure for E. coli K12 MG1655 ##### Variables BAC [µg/mL] µ maximal specific growth rate [h] #### File: Figure2B.xlsx **Description:** viable cell density [cfu/mL] (cfu=colony forming units) at different times [h] at 0 µg/mL and 4 µg/mL BAC exposure ##### Variables time [h] treatment 1: 0 g/mL BAC treatment 2: 4 g/mL BAC #### File: Figure2C.xlsx **Description:** %cfu (colony forming units) relative to cfu at t0 with and without BAC exposure after 2 h ##### Variables Treatment1: at 0 g/mL BAC Treatment2: at 4 g/mL BAC #### File: Figure3A.xlsx **Description:** viable cell density [cfu/mL] (cfu=colony forming units) at 0 h and after 2 h of exposure with 4 g/mL BAC ##### Variables Treatment1: Ctrl S Treatment2: S Treatment3: Ctrl SG Treatment4: SG S-survival treatment SG-survival and growth treatment CTRL-control evolutionary lineages that evolved without BAC exposure under S or SG protocol 1 to 6 are replicate evolutionary lineages Data for ancestor is shown in Figure 2B and 2C #### File: Figure3B.xlsx **Description:** specific growth rate [h-1] at 4 g mL-1 BAC exposure for the ancestor and different evolutionary lineages ##### Variables Treatment1: ancestor Treatment2: Ctrl S Treatment3: S Treatment4: Ctrl SG Treatment5: SG S-survival treatment SG-survival and growth treatment CTRL-control evolutionary lineages that evolved without BAC exposure under S or SG protocol 1 to 6 are replicate evolutionary lineages #### File: Figure3C.xlsx **Description:** time [h] to first increase in OD ##### Variables Treatment1: ancestor Treatment2: Ctrl S Treatment3: S Treatment4: Ctrl SG Treatment5: SG S-survival treatment SG-survival and growth treatment CTRL-control evolutionary lineages that evolved without BAC exposure under S or SG protocol 1 to 6 are replicate evolutionary lineages #### File: Figure3D.xlsx **Description:** antimicrobial susceptibility of the replicate evolved lineages relative to the ancestor for different antibiotics and benzalkonium chloride (BAC) ##### Variables Treatment1: S Treatment2: SG Measured for 4 antibiotics and benzalkonium chloride (BAC) absolute data is provided in Table S1