DOI: 10.5061/dryad.fj6q573xq
title: Off-target integron activity leads to rapid plasmid compensatory evolution in response to antibiotic selection pressure

description: Integrons are mobile genetic elements that have played an important role
 in the dissemination of antibiotic resistance. As shown previously (Souque
 et al, 2021), the integron can generate under stress combinatorial
 variation in resistance cassette expression by cassette re-shuffling,
 accelerating the evolution of resistance. However, the flexibility of the
 integron integrase site recognition motif hints at potential off-target
 effects of the integrase on the rest of the genome that may have important
 evolutionary consequences. Here we test this hypothesis by selecting for
 increased piperacillin resistance populations of P.aeruginosa with a
 mobile integron containing a hard-to-mobilise beta-lactamase cassette to
 minimize the potential for adaptive cassette re-shuffling. We found that
 integron activity can both decrease overall survival rate but also improve
 the fitness of the surviving populations. Off-target inversions mediated
 by the integron accelerated plasmid adaptation by disrupting costly
 conjugative genes otherwise mutated in control populations lacking a
 functional integrase. Plasmids containing integron-mediated inversions
 were associated with lower plasmid costs and higher stability than
 plasmids carrying mutations, albeit at a cost of reduced conjugative
 ability. These findings highlight the potential for integrons to create
 structural variation that can drive bacterial evolution, and they provide
 an interesting example showing how antibiotic pressure can drive the loss
 of conjugative genes.
description: Antibiotic minimum inhibitory concentration (MIC) - MICs of
 integrons arrays / MICs of evolved populations / MICs of evolved plasmids
 in ancestral background Minimum inhibitory concentrations (MIC) for each
 antibiotic were determined following the broth microdilution method from
 the Clinical and Laboratory Standards Institute (CLSI) guidelines.
 5x105 c.f.u bacteria inocula were prepared in cation-adjusted
 Mueller- Hinton Broth 2 (MH2) using individual colonies
 grown on selective agar and incubated in doubling concentrations of
 antibiotics for 20h in three technical replicates. Cultures optical
 density (OD600) was then read using a Biotek Synergy plate reader and
 wells were considered empty when the overall OD600 was under 0.1. The MIC
 for each assay was defined as the minimal concentration in which growth
 was inhibited in all three technical replicates. The final MICs values are
 the average of two to three replicate assays (from separately prepared
 inocula, on different days). Growth curve assays (Fitness of evolved populations
 / fitness of evolved plasmids in ancestral
 background) Growth rates in the absence of antibiotics were
 determined using a Biotek Synergy plate reader. Populations were
 inoculated in similar conditions to the MIC assay (5x105 c.f.u bacteria
 inocula prepared in cation-adjusted (MH2) from individual colonies) and
 followed for 24h at 37*C with periodic shaking, with OD600 measurements
 taken every 15 minutes. Intrinstic growth rate were determined using the
 Growthcurver package  (Sprouffske and Wagner, 2016). Each measurement was done in three technical
 replicates (separate wells from the same inocula) and replicated three
 times over different days (biological replicates).
 Evolutionary ramp experiment
 (survival data) 90 populations of each strain (PA01:WTA4 and
 PA01:ΔintA4) were initially inoculated from individual colonies grown on
 selective agar in MH2 at a piperacillin concentration of 1/8 MIC. The
 outer wells of every plate were kept as negative controls and strain
 distribution among the plates was kept balanced to limit plate effects.
 Plates were kept at 37°C with shaking at 225 rpm. Every day the
 populations were diluted 1/10 000 in a doubling concentration of MIC until
 reaching x8 MIC. As controls, 15 populations of each strain were
 transferred either without antibiotic or at a constant piperacillin
 concentration of 1/8 MIC. The OD of each population was measured each day
 and a population was considered extinct when its OD fell below 0.1 after
 20h of incubation.  Conjugation rate
 Determination of the conjugation
 rates of the ancestral and evolved plasmids was performed using filter
 mating with a rifampicin resistant PA01 acceptor (D512G rpoB mutant
 from (Qi et al.,
 2014)). Populations of donor
 and recipients were grown from overnight for 3h30 with (donors) and
 without (recipient) antibiotics, washed and resuspended in NaCl (0.9%).
 Donor and recipient populations were mixed at a 1:1 ratio, deposited on
 filter on agar plate without antibiotic alongside pure donor/recipients
 control and incubated for 1h. Populations were then resuspended in NaCl
 0.9%. Concentrations of donors, recipients and transconjugants were
 determined at both T0 and Tf using appropriate antibiotics (ceftazidime
 100mg/L, rifampicin 64mg/L and both combined). Conjugation rate was
 calculated using the Simonsen method (Simonsen et al., 1990). Plasmid persistence
 Populations with ancestral and
 evolved plasmids were inoculated from single colonies in the presence of
 antibiotic and passaged for 6 days in the absence of antibiotic with a
 similar dilution (1:10 000) as the experimental evolution experiment.
 After 6 days the ratio of plasmid containing cells was determined by
 serial dilution plating with (for the cfu of the plasmid containing cells)
 and without ceftazidime (for the cfu of the entire
 population).  Transcription data
 All RNA samples were treated with the TURBO DNA-free Kit
 (ThermoFisher) to eliminate genomic DNA. Concentration of the RNA samples
 was quantified using the Quantifluor RNA system (Promega). cDNA was
 synthesized from 100ng of RNA templates using random primers from the
 GoScript Reverse Transcription Mix (Promega). qPCR was carried out on the
 StepOnePlus Real-time PCR platform (Applied Biosystems) using the iTaq
 Universal SYBR Green Supermix. The cassette, as well as two reference
 genes (actpA and acp), were amplified using the primers described in
 Supplementary Table S2 in two technical replicates for each extraction.
 Standard curves for the pair of cassette primers was included in each PCR
 using restriction enzyme-digested gDNA as template, and used to quantify
 the amount of target cDNA in each sample to control for inter-run
 variations. For each biological replicate the cassette transcript levels
 were normalized based on the geometric means of the two internal reference
 genes, using the first array A1 as a reference, before division by its
 plasmid copy number. The biological replicates were then averaged for each
 array and normalised by the expression of the best array for each gene.
 Transcription data for the aadB gene is reproduced from the following
 dataset: https://doi.org/10.5061/dryad.rv15dv469