Recombinant Escherichia coli Probable phosphatidylethanolamine transferase Mcr-1 (mcr1)

What is the mechanism by which MCR-1 confers colistin resistance?

MCR-1 functions as a phosphoethanolamine (PEA) transferase that modifies bacterial lipid A, reducing its affinity for colistin and related polymyxins. The enzyme catalyzes the transfer of a PEA moiety from phosphatidylethanolamine to lipid A phosphate groups. This covalent modification alters the negative charge of the bacterial outer membrane, reducing the binding affinity of the positively charged colistin molecules.

The catalytic mechanism involves:

• Utilization of phosphatidylethanolamine (PE) as a substrate

• Generation of diacylglycerol and association of PEA to the MCR-1 active site

• Transfer of the PEA moiety to glucosamine phosphate groups of lipid A

MCR-1 contains a transmembrane domain that anchors it to the bacterial membrane, positioning the catalytic domain in the periplasm where it can access and modify lipid A on newly synthesized LPS molecules. The MCR-1 protein requires zinc ions in its catalytic site for enzymatic activity, and structural studies have confirmed its similarity to other phosphoethanolamine transferases like those found in Neisseria species (LptA/EptA) .

What bacterial species harbor mcr-1 and how prevalent is this gene globally?

The mcr-1 gene has been identified in multiple species of Enterobacteriaceae, including:

• Escherichia coli

• Salmonella enterica

• Klebsiella pneumoniae

• Enterobacter aerogenes

• Enterobacter cloacae

• Cronobacter sakazakii

• Shigella sonnei

• Kluyvera species

• Citrobacter species

• Raoultella ornithinolytica

Following its initial discovery in China in 2015, mcr-1 has rapidly spread globally. As of 2017, the gene had been detected in more than 30 countries across 5 continents in less than a year after its identification . The gene has been found in isolates from humans, animals (particularly livestock), and environmental samples, indicating its wide dissemination across ecological niches.

The prevalence varies by region and setting, with higher rates typically observed in agricultural settings where colistin has been used extensively as a growth promoter in animal husbandry. The gene has been detected in both clinical and community settings, highlighting its potential threat to public health .

What plasmid types commonly carry mcr-1 and how does this impact dissemination?

The mcr-1 gene has been identified on various plasmid types, with certain incompatibility groups being particularly important for its dissemination:

The plasmid backbone plays a crucial role in transfer efficiency and host range. When comparing different plasmid types carrying mcr-1, researchers found that "the content of plasmid backbone had an influence on efficiency" of transfer . Some plasmid types are more readily transferable via conjugation, facilitating rapid spread between bacterial strains and species.

Co-localization with other resistance genes on the same plasmid is particularly concerning. The mcr-1 gene has been found to coexist with genes encoding extended-spectrum β-lactamases (ESBLs) and carbapenemases, including the notorious NDM-1 and its variants (NDM-5 and NDM-9) . This co-localization facilitates co-selection and persistence of mcr-1 even in the absence of colistin selection pressure.

How can recombinant MCR-1 be expressed and purified for biochemical studies?

Successfully expressing and purifying MCR-1 presents challenges due to its transmembrane domain. Here is a validated methodological approach:

Expression System Selection:

• Low-copy number vectors (such as pACYCDuet-1) carrying mcr-1 with its native promoter provide physiological expression levels

• Arabinose-inducible systems like pBAD24 allow controlled expression

• Careful consideration of host strain is essential; BW25113 has been successfully used

Protein Engineering Strategies:

• The full-length MCR-1 containing the transmembrane domain can be expressed in membrane fractions

• Addition of a C-terminal 6x-His tag facilitates purification while preserving activity

• Soluble forms of MCR-1 (without the transmembrane domain) can be engineered for structural studies

Purification Protocol:

• Bacterial cell lysis under mild conditions to preserve membrane integrity

• Membrane fraction isolation via differential centrifugation

• Detergent solubilization of membrane proteins (selection of appropriate detergent is critical)

• Immobilized metal affinity chromatography using the His-tag

• Further purification via size exclusion chromatography if needed

Verification Methods:

• SDS-PAGE for purity assessment

• Western blot using anti-6x-His antibodies for identity confirmation

• Mass spectrometry for definitive identification

• Phosphoethanolamine transferase activity assays to confirm functionality

This approach has successfully yielded active MCR-1 protein for biochemical and structural studies, as reported in the literature: "We over-expressed the membrane protein MCR-1 and purified it to homogeneity... MS-based identification further confirmed the identity of the recombinant MCR-1 trans-membrane protein" .

What experimental approaches can assess the impact of MCR-1 on bacterial fitness?

Evaluating the fitness impact of MCR-1 requires multiple complementary approaches:

Growth Kinetics Analysis:

• Comparison of growth curves between isogenic strains with and without mcr-1

• Assessment of lag phase duration, exponential growth rate, and maximum cell density

• Testing in both rich and nutrient-limited media to capture condition-dependent effects

Competition Assays:

• Co-culture of mcr-1-positive and mcr-1-negative strains at defined ratios

• Monitoring population dynamics over multiple generations

• Calculation of selection coefficients and competitive indexes

Membrane Integrity Assessments:

• Sensitivity to detergents (SDS) at various concentrations

• Susceptibility to antibiotics that normally don't penetrate the outer membrane (vancomycin, rifampicin)

• Combination treatments to assess membrane permeabilization (e.g., "a lower concentration of SDS is capable of potentiating vancomycin into MCR-1-expressing cells")

Microscopy Approaches:

• Transmission electron microscopy to examine cellular morphology

• Fluorescence microscopy with stress reporters (e.g., IbpA-TC-FlAsH labeling for protein aggregates)

• Live/dead staining to assess viability under various conditions

Long-term Viability Studies:

• Flow cytometry with propidium iodide staining to quantify dead cells

• Extended stationary phase survival monitoring

• Assessment of physiological parameters under stress conditions

How can researchers effectively express mcr-1 at different levels to study dose-dependent effects?

Controlling mcr-1 expression levels is crucial for studying dose-dependent effects on bacterial physiology and colistin resistance. Several effective approaches have been validated:

Chromosomal Integration with Variable Promoters:

• λ-Red recombination can integrate mcr-1 into the bacterial chromosome with promoters of different strengths

• This approach has successfully generated "seven constructs with more than 200-fold mcr-1 transcriptional expression differences"

• Chromosomal integration provides stable, single-copy expression without plasmid maintenance concerns

Inducible Expression Systems:

• Arabinose-inducible pBAD vectors allow titratable expression by varying inducer concentration

• IPTG-inducible systems with lac or tac promoters provide controlled expression

• Tetracycline-responsive systems offer alternative regulation mechanisms

Native Promoter Variants:

• Natural regulatory mutations in the mcr-1 promoter region can be exploited

• Studies have identified variants with single polymorphisms in the -10 promoter or Shine-Dalgarno regions that alter expression levels

• These variants have been shown to "fine-tune the expression of mcr-1, allowing E. coli to reduce the fitness cost of mcr-1 while simultaneously increasing colistin resistance"

Plasmid Copy Number Manipulation:

• Different plasmid backbones with varying copy numbers affect mcr-1 expression

• Studies have used pSEVA121, a mini-RK2 derived expression vector with "similar copy number (~4–6 per cell) to natural plasmids that carry mcr-1 (typically 2-5 per cell)"

Important Considerations:

• "Over-high expression of mcr-1 cannot be tolerated" by bacteria

• Expression level directly correlates with colistin MICs, with higher expression leading to higher resistance

• Different expression levels have varying impacts on bacterial fitness and morphology

This methodological diversity allows researchers to precisely control mcr-1 expression and systematically investigate the relationship between expression level, resistance phenotype, and fitness effects.

How does mcr-1 expression modulate bacterial inflammatory responses?

MCR-1 expression significantly alters the interaction between bacteria and host immune cells, modulating inflammatory responses in several important ways:

Effects on Inflammatory Signaling Pathways:

• Recombinant mcr-1-expressing E. coli significantly modulates p38-MAPK and Jun N-terminal protein kinase (JNK) activation

• pNF-κB nuclear translocation is altered compared to mcr-1-negative strains

• These pathways are central to inflammatory responses, suggesting MCR-1 interferes with host immune signaling

Impact on Cytokine Expression:

• MCR-1-positive strains alter the expression of genes for key proinflammatory cytokines:

  • Tumor necrosis factor alpha (TNF-α)

  • Interleukin-12 (IL-12)

  • Interleukin-1β (IL-1β)

• These changes may help mcr-1-positive bacteria evade effective immune clearance

Inflammasome Activation:

• Caspase-1 activity is significantly less activated by mcr-1-positive E. coli strains than by mcr-1-negative counterparts

• IL-1β secretion is reduced during infection with mcr-1-positive strains

• This suggests MCR-1 enables bacteria to "escape recognition by cytosolic receptors and inflammasome activation"

Cellular Damage Mitigation:

• MCR-1-producing bacteria "limit the caspase-1 activation, the cellular membrane damage, and the massive release of inflammatory molecules"

• This may represent "a common mechanism used by MCR-1-producing bacteria to prolong their survival within human hosts"

These findings have significant implications for virulence and persistence of mcr-1-positive pathogens. By dampening inflammatory responses, these bacteria may evade immune clearance while establishing infections. This represents an additional advantage beyond colistin resistance that could contribute to the success and spread of mcr-1-positive strains.

How do environmental conditions influence MCR-1 activity and expression?

Environmental factors significantly impact MCR-1 activity and expression, with important implications for resistance phenotypes and bacterial fitness:

pH Effects:

• "Growth in media with low pH dramatically increases both MCR-1-dependent phosphoethanolamine modification of lipid A as well as colistin-resistance activity"

• In vitro transferase and lipid A reconstitution assays demonstrate that "MCR-1 is highly active at acidic pH"

• This pH-dependence may be relevant in environmental niches with acidic conditions or during infection of acidic host compartments

Stress Response Activation:

• MCR-1 induces a Cpx-dependent envelope stress response (ESR) in E. coli

• This response helps bacteria maintain membrane integrity under stress conditions

• A "conserved motif within MCR-1 induces components of the ESR to confer resilience to stimuli commonly encountered in the environment"

Nutritional Status:

• MCR-1 expression causes specific physiological changes during stationary phase

• Starvation-induced shrinkage is more pronounced in MCR-1-expressing cells

• Protein aggregation increases in MCR-1-expressing cells during stationary phase

Regulatory Adaptation:

• Environmental selection pressures drive evolutionary fine-tuning of mcr-1 expression

• "Regulatory mutations were associated with increased mcr-1 stability in pig farms following a ban on the use of colistin as a growth promoter that decreased colistin consumption by 90%"

• These adaptations allow bacteria to "reduce the fitness cost of mcr-1 while simultaneously increasing colistin resistance"

Antimicrobial Peptide Exposure:

• "Acquiring MCR-1 also renders strains more resistant to antimicrobial peptides"

• This suggests that environmental niches with high concentrations of host defense peptides might select for mcr-1 maintenance

These findings indicate that MCR-1 provides adaptive advantages beyond colistin resistance, potentially explaining the persistence of mcr-1 even after reduction in colistin usage. The environmental modulation of MCR-1 activity highlights the importance of considering ecological contexts when studying antimicrobial resistance mechanisms.

What is the relationship between MCR-1 expression levels and colistin MICs?

The relationship between MCR-1 expression levels and colistin resistance is complex and influenced by multiple factors:

Direct Correlation with Expression Level:

• Colistin MICs increase with increasing MCR-1 expression levels

• In E. coli strains carrying mcr-1 on the chromosome with promoters of different strengths, a direct relationship was observed between expression level and resistance

• The highest achievable MIC appears to be around 8 μg/mL, representing a practical upper limit for mcr-1-mediated resistance

Expression Level Limitations:

• "Over-high expression of mcr-1 cannot be tolerated" by bacteria

• This suggests an inherent toxicity of MCR-1 when expressed beyond certain thresholds

• The relationship follows a saturation curve rather than continuing indefinitely

Comparison Between Species:

• In K. pneumoniae, colistin MICs increased 256- to 4,096-fold for mcr-1-negative strains but only 16- to 256-fold for mcr-1-harboring transformants when selected for high-level resistance

• For E. coli, colistin MICs increased 4- to 64-fold, but only 2- to 16-fold for their mcr-1-harboring transformants

• This species-dependent variation suggests different interactions between MCR-1 and endogenous resistance mechanisms

Regulatory Optimization:

• Regulatory mutations in the mcr-1 promoter region can fine-tune expression

• These mutations allow "bacteria to reduce the fitness cost of mcr-1 while simultaneously increasing colistin resistance"

• Fitness was strongly correlated with mcr-1 expression (r² = 0.68, p = 0.006), highlighting the value of optimizing expression levels

Clinical Relevance:

• Bacteria demonstrate colistin MICs of 4–8 μg/mL at mcr-1 expression levels similar to those found in clinical isolates

• This indicates that natural selection has optimized expression levels to balance resistance and fitness costs

• The European Committee on Antimicrobial Susceptibility Testing (EUCAST) defines colistin resistance as MIC > 2 μg/mL, meaning mcr-1 expression typically confers clinical resistance

These findings demonstrate that the relationship between MCR-1 expression and colistin resistance is not simply linear but is constrained by bacterial physiology and fitness costs associated with high-level expression.

Why do studies show conflicting results regarding fitness costs of mcr-1?

The scientific literature contains contradictory findings regarding mcr-1-associated fitness costs, with some studies reporting significant impacts while others observe minimal effects. Several factors contribute to these discrepancies:

Plasmid Backbone Influence:

• Different plasmid types carrying mcr-1 impose variable fitness burdens

• "The content of plasmid backbone had an influence on efficiency" when comparing different plasmids

• Some studies report that IncI2-type plasmids can actually "increase the fitness of host E. coli DH5-α cells"

Expression Level Variation:

• Studies using high-copy plasmids or strong inducible promoters may observe greater fitness costs

• Studies with native promoters or chromosomal integration may show reduced impacts

• Regulatory mutations that fine-tune expression can "reduce the fitness cost of mcr-1 while simultaneously increasing colistin resistance"

Species-Specific Effects:

• "Plasmid-borne mcr-1 did not reduce fitness in E. coli but impaired fitness in K. pneumoniae"

• Different bacterial species may vary in their ability to tolerate the physiological disruptions caused by MCR-1

• In K. pneumoniae, "the coexistence of mcr-1 and chromosomal mutations imposed a fitness burden on HLCR mutants"

Experimental Conditions:

• Rich laboratory media may mask fitness costs that would be apparent under resource-limited conditions

• "Natural conditions are rarely ideal for microbial growth, especially compared with the rich media used in laboratory settings"

• Short-term experiments may not capture the long-term evolutionary dynamics

Methodology Differences:

• Various metrics for assessing fitness (growth rate, competition assays, long-term survival)

• Different control strains used for comparison

• Variable experimental durations (immediate effects versus long-term adaptation)

To resolve these contradictions, researchers should:

• Clearly specify plasmid types, copy numbers, and expression levels

• Test multiple fitness parameters under diverse environmental conditions

• Use isogenic strains that differ only in mcr-1 presence/absence

• Conduct both short-term and long-term experiments

• Consider species-specific effects rather than generalizing across bacterial taxa

What challenges exist in developing effective inhibitors against MCR-1?

Developing inhibitors against MCR-1 presents several significant challenges that researchers must navigate:

Structural Considerations:

• MCR-1 is a membrane-anchored protein with a transmembrane domain

• The active site contains zinc ions crucial for catalytic activity

• Crystal structures reveal similarity to other phosphoethanolamine transferases

• Designing inhibitors that can access the periplasmic catalytic domain presents challenges

Target Specificity:

• MCR-1 belongs to a family of phosphoethanolamine transferases

• Inhibitors must distinguish between bacterial targets and host enzymes with similar functions

• Cross-inhibition of other bacterial enzymes may lead to unintended consequences

Chemical Properties of Inhibitors:

• Compounds must penetrate the bacterial outer membrane to reach MCR-1

• For pyrazolones identified as potential inhibitors, molecular docking studies predict binding affinities for MCR-1

• Balancing potency, membrane permeability, and stability presents challenges

Resistance Development:

• Bacteria may develop resistance to MCR-1 inhibitors through mutations

• The search results mention "three different types of defects in CRISPR-Cas9 system lead to escape mutants"

• Inhibitor design must consider potential resistance mechanisms

Validation Approaches:

• In vitro enzymatic assays with purified MCR-1

• Cellular assays measuring colistin susceptibility restoration

• Molecular dynamics simulations to understand inhibitor-enzyme interactions

• Animal models to assess in vivo efficacy

Promising Approaches:

• Pyrazolones have been identified as "able to restore colistin susceptibility of mcr-1-producing bacteria"

• CRISPR-Cas9 systems can efficiently eliminate mcr-1-harboring plasmids

• Targeting regulatory mechanisms that control mcr-1 expression

• Exploiting MCR-1-induced membrane disruptions with combination therapies

Overcoming these challenges requires multidisciplinary approaches combining structural biology, medicinal chemistry, microbiology, and computational modeling to develop effective strategies against mcr-1-mediated colistin resistance.

How do chromosomal mutations interact with mcr-1 to develop high-level colistin resistance?

The interaction between chromosomal mutations and mcr-1 in developing high-level colistin resistance (HLCR) reveals complex evolutionary pathways with species-specific patterns:

Species-Specific Mutation Patterns:

• In K. pneumoniae HLCR mutants, amino acid alterations predominantly occur in:

  • crrB (most common)

  • phoQ

  • crrA

  • pmrB

  • mgrB

  • phoP

• In E. coli HLCR mutants, genetic alterations mostly occur in:

  • pmrB

  • phoQ

• These differences suggest distinct evolutionary pathways to high-level resistance

Impact of mcr-1 on HLCR Development:

• HLCR is "more likely to occur in K. pneumoniae strains than E. coli strains when exposed to colistin"

• For K. pneumoniae, colistin MICs increased 256- to 4,096-fold for mcr-1-negative strains but only 16- to 256-fold for mcr-1-harboring transformants

• For E. coli, colistin MICs increased 4- to 64-fold, but only 2- to 16-fold for their mcr-1-harboring transformants

• This suggests mcr-1 may actually limit the development of extremely high-level resistance

Survival Advantage:

• "mcr-1 improved the survival rates of both E. coli and K. pneumoniae strains when challenged with relatively high concentrations of colistin"

• This survival advantage may facilitate the initial steps toward developing HLCR

Fitness Implications:

• "The coexistence of mcr-1 and chromosomal mutations imposed a fitness burden on HLCR mutants of K. pneumoniae"

• This fitness cost may explain why extremely high-level resistance (MICs >64 μg/mL) is relatively rare in clinical settings

Evolutionary Perspective:

• MCR-1 provides moderate resistance (MICs 4-8 μg/mL) with manageable fitness costs

• Chromosomal mutations can provide higher resistance but often with greater fitness penalties

• The combination may not be advantageous in many environments, explaining why mcr-1 typically confers low to moderate resistance levels

Understanding these interactions helps explain the observed patterns of colistin resistance in clinical isolates and highlights the complex evolutionary tradeoffs between resistance level and bacterial fitness.

What promising approaches exist for counteracting mcr-1-mediated colistin resistance?

Several innovative approaches show promise for combating mcr-1-mediated colistin resistance:

CRISPR-Cas9 Systems:

• Plasmid-mediated CRISPR-Cas9 systems can "efficiently resensitize E. coli to colistin"

• These systems can eliminate mcr-1-harboring plasmids with high efficiency

• Can also "protect the recipient from plasmid-borne mcr-1 transfer via conjugation"

• Research has found "no significant correlation between sgRNA lengths and curing efficiency"

Chemical Inhibitors:

• Pyrazolones have been identified as compounds that can "restore colistin susceptibility of mcr-1-producing bacteria"

• Molecular docking studies predict binding affinities of these compounds for MCR-1

• Virtual screening approaches have identified compounds with "high binding affinities for MCR-1/MCR-3, as predicted by molecular docking"

Combination Therapies:

• MCR-1 expression increases bacterial membrane permeability

• This vulnerability can be exploited with combination treatments

• "A lower concentration of SDS is capable of potentiating vancomycin into MCR-1-expressing cells"

• This approach could allow the use of antibiotics not typically effective against Gram-negative bacteria

Exploiting Fitness Costs:

• "MCR-1-dependent lipid remodelling compromises the viability of Escherichia coli"

• "Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1"

• Strategies targeting these vulnerabilities could selectively eliminate mcr-1-positive strains

Protease Activation:

• MCR-1 production induces a Cpx-dependent envelope stress response

• "Transactive expression of DegP" can reverse some MCR-1 effects

• "Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1"

pH-Based Strategies:

• MCR-1 activity is influenced by environmental pH

• Understanding the pH-dependence of MCR-1 could lead to novel intervention strategies

• Manipulating local pH in infection sites might modulate MCR-1 activity

These diverse approaches provide multiple avenues for combating mcr-1-mediated colistin resistance, which may be particularly valuable given the rapid global spread of this resistance mechanism.

What novel detection methods could improve mcr-1 surveillance in clinical settings?

Effective surveillance of mcr-1 requires advanced detection methods that are sensitive, specific, and feasible for routine clinical implementation:

Improved Molecular Approaches:

• Real-time PCR (qPCR) with optimized primers for mcr-1 variants

• Multiplex PCR systems targeting multiple mcr variants simultaneously

• Digital PCR for absolute quantification of mcr-1 copy numbers

• LAMP (Loop-mediated isothermal amplification) for resource-limited settings

Next-Generation Sequencing Applications:

• Whole genome sequencing for comprehensive characterization

• Targeted sequencing of plasmids to track mcr-1 transfer

• Metagenomic approaches for direct detection in complex samples

• k-mer based SNP analysis to identify plasmid relationships

Plasmid Tracking Tools:

• "New tools need to be developed to allow diagnostic laboratories to detect mcr-1 and AMR plasmid transmission events in hospitals and other relevant settings"

• Plasmid MLST (Multi-Locus Sequence Typing) for tracking plasmid spread

• Integron detection tools like "Integron Finder v2.0"

• Conjugation monitoring systems to track horizontal gene transfer

Phenotypic-Genotypic Combined Approaches:

• Rapid phenotypic screening followed by molecular confirmation

• Automated systems combining antimicrobial susceptibility testing with genetic detection

• MALDI-TOF MS applications for rapid screening of colistin resistance

Data Integration Systems:

• Centralized databases linking phenotypic and genotypic data

• Alert systems for unusual resistance patterns

• Geographic information systems to track spread patterns

• One Health surveillance platforms integrating human, animal, and environmental data

Current surveillance may underestimate mcr-1 prevalence, as highlighted by evidence suggesting "multiple transmission events of mcr-1-carrying plasmids, despite the gene only being identified from a relatively small set of mcr-1 positive strains from a single medical center" . Improved detection methods are essential for accurate prevalence estimates and effective antimicrobial stewardship.

How might regulatory evolution of mcr-1 impact resistance management strategies?

Regulatory evolution of mcr-1 has significant implications for antimicrobial resistance management strategies:

Fine-Tuning of Expression:

• "Regulatory evolution has fine-tuned the expression of mcr-1, allowing E. coli to reduce the fitness cost of mcr-1 while simultaneously increasing colistin resistance"

• Single polymorphisms within the -10 promoter or Shine-Dalgarno region can significantly alter expression levels

• All regulatory variants studied showed higher fitness than wild-type strains

Resistance-Fitness Relationship:

• Fitness was strongly correlated with increased resistance (r² = 0.65, p = 0.009)

• This demonstrates the ability of regulatory mutations to optimize mcr-1 expression without fitness trade-offs

• Challenging the traditional assumption that resistance necessarily imposes fitness costs

Implications for Antibiotic Stewardship:

• Regulatory mutations were associated with increased mcr-1 stability in pig farms "following a ban on the use of colistin as a growth promoter that decreased colistin consumption by 90%"

• This suggests that reducing antibiotic use alone may be insufficient once regulatory adaptation has occurred

• More comprehensive approaches may be needed to eliminate resistant strains

Plasmid Transfer Dynamics:

• "Conjugative plasmids have transferred low-cost/high-resistance mcr-1 alleles across an incredible diversity of E. coli strains, further stabilising mcr-1 at the species level"

• This horizontal transfer of optimized regulatory variants accelerates adaptation

• Surveillance should monitor not just mcr-1 presence but also regulatory sequences

Management Strategy Implications:

• Monitor regulatory sequences, not just mcr-1 presence

• Consider fitness-neutral resistance as particularly challenging to eliminate

• Implement more aggressive interventions targeting mcr-1 carriers

• Develop combination approaches rather than relying solely on antibiotic restriction

• Focus on preventing initial establishment of mcr-1 in new environments

These findings suggest that "regulatory evolution and plasmid transfer can combine to stabilise resistance and limit the impact of reducing antibiotic consumption" . Effective management strategies must account for this evolutionary dimension of antimicrobial resistance.