Biology subjects

MacLean, C.

Publications and source records attributed to MacLean, C..

17 recordsLinked to original sources

bioRxiv2026

DNA-damaging combination treatments impose genotype-specific constraints on hypermutator evolvability

Bacterial hypermutator strains drive rapid evolution of antibiotic resistance in chronic infections. Inspired by cancer therapy approaches that exploit synthetic lethality by targeting DNA repair deficiencies in hypermutator tumours, we tested whether pairing a conventional antibiotic with a secondary DNA-damaging agent could constrain hypermutator evolution in bacteria. Using high-throughput experimental evolution of Escherichia coli repair-deficient strains, we evolved populations under carbapenem selection in combination with ciprofloxacin or mitomycin C. Strains lacking oxidative damage repair, double-strand break repair, or transcription-coupled repair showed significantly reduced evolvability, particularly under constant antibiotic pressure and increasing genotoxic stress. However, mismatch repair (MMR) hypermutators, the predominant clinical genotype, did not show reduced evolvability under these combination treatments. This is consistent with pathway orthogonality: MMR does not repair the structural DNA lesions induced by ciprofloxacin or mitomycin C, and the elevated mutation supply of MMR-deficient strains may allow rapid adaptation despite background DNA damage. Our findings demonstrate that combination strategies can constrain the evolvability of specific repair-deficient genotypes in vitro, but success requires matching DNA damage type to specific repair vulnerabilities. This work establishes proof of principle for genotype-directed antimicrobial strategies that exploit DNA repair vulnerabilities to constrain hypermutator evolution. SignificanceThis work demonstrates that combining DNA-damaging agents with antibiotics can constrain resistance evolution in specific hypermutator genotypes, but not others. These findings establish that evolution-informed antimicrobial strategies must be genotype-specific, opening new paths for precision approaches to delay or prevent the evolution of antibiotic resistance by exploiting DNA repair vulnerabilities.

microbiology↗

bioRxiv2025

Plasmid dependent phage eliminate pathogenic bacteria and antibiotic resistance plasmids from the chicken gut microbiome

Conjugative plasmids are a key reservoir of antimicrobial resistance (AMR) in commensal and pathogenic bacteria within the gut microbiome. Plasmid-dependent phage (PDPs) are a promising therapeutic option directly targeting AMR plasmids, but the efficacy of PDP therapy in vivo is poorly understood. Here, we show that treatment with the PRD1 phage eliminated Salmonella enterica carrying a broad-host range IncP AMR plasmid (RP4) from the chicken gut microbiome. Moreover, PRD1 treatment prevented the transfer of the RP4 plasmid to other members of the gut microbiome, whilst causing only minimal disruption to microbiome composition. Our results demonstrate that PDP therapy rapidly eliminates AMR from a natural gut microbiome by killing pathogenic bacteria and blocking AMR plasmid transmission to commensals, highlighting the conjugative pilus as a promising antimicrobial target.

microbiology↗

bioRxiv2025

Trade-offs between phage resistance and conjugative ability shape the ecological and evolutionary response of a multidrug resistance plasmid to plasmid-dependent phage

Phage therapy is a promising alternative to antibiotics to treat multidrug resistant infections. Plasmid dependent phages (PDPs) are particularly attractive as therapeutics because they can both kill targeted pathogen cells, whilst also potentially preventing the further spread of antibiotic resistance genes encoded by plasmids. However, we lack experimental studies of the ecological and evolutionary response of multidrug resistance plasmids against plasmid dependent phage treatment under ecologically relevant scenarios allowing plasmid conjugation. We experimentally evolved populations of E. coli carrying the multidrug resistance RP4 plasmid with the PRD1 PDP under conditions where conjugation was associated with either strong or weak benefits. When opportunities for conjugation were rare, PRD1 only transiently suppressed the conjugative plasmid population due to the rapid evolution of PRD1 resistant plasmids that lacked conjugative ability. Increasing the ecological opportunity for conjugation enhanced plasmid suppression and delayed the evolution of PRD1 resistant plasmids. PRD1 resistance was associated with reduced conjugative ability, but this trade-off was complex due to the heterogeneous impacts of resistance mutations on pilus production and conjugative ability. Mutations and IS element insertions in conjugation genes caused a wide range of PRD1 resistance phenotypes, ranging from complete resistance (virB4) to partial resistance (trbB, trbL). Bioinformatic analysis of publicly available IncP plasmid sequences showed that truncated variants of VirB4 protein are common in natural populations, suggesting that plasmid-dependent phages are an important selective pressure in microbial communities. Our results demonstrate an evolutionary trade-off between conjugative ability and phage resistance that cannot be easily circumvented by plasmids. Targeting multidrug resistance plasmids with PDPs is likely to drive loss of conjugation limiting the transfer of antibiotic resistance genes in bacterial communities.

evolutionary biology↗

bioRxiv2025

Plasmids link antibiotic resistance genes and phage defense systems in E. coli

Phage therapy has been proposed as an alternative to antibiotics to treat resistant infections. However, we have a limited understanding of how antibiotic resistance genes (ARGs) associate with bacterial phage defense systems (PDSs). Here, we explore the relationship between ARGs and PDSs in a sample of 2,559 plasmids originating from 1,044 E. coli isolates, representing a snapshot of clinical and non-clinical diversity in Oxfordshire, UK (2008-2020). In total, we identify 3,193 ARGs and 14,013 PDSs (180 unique types). We demonstrate that E. coli plasmids are enriched for ARGs and PDSs (both p<0.001), with a bias towards toxin-antitoxin/abortive-infection, TIR-domain and CBASS systems (all q<0.025). We proceed to show that ARGs and PDSs are physically linked by plasmids (p<0.001). Together, our results suggest that phage therapy may inadvertently select for antibiotic resistant bacteria, and that antibiotic use may similarly drive resistance to phage.

evolutionary biology↗

bioRxiv2025

Chromosomal capture of beneficial genes drives plasmids towards ecological redundancy

Plasmids are a ubiquitous feature of bacterial genomes, but the evolutionary forces driving genes to become associated with plasmids are poorly understood. To address this problem, we compared the fitness effects of chromosomal and plasmid genes in the plant symbiont Rhizobium leguminosarum. Here we show that plasmids are depleted in beneficial genes compared to the chromosome, and this effect is stronger for ancient plasmids compared to recently acquired plasmids. These findings support the hypothesis that evolution drives beneficial genes to become localized to the bacterial chromosome, resulting in a gradual decay in the ecological value of plasmids. These findings question the ecological importance of plasmids and highlight the challenge of understanding how plasmids persist over the long term.

evolutionary biology↗

bioRxiv2022

Regulatory fine-tuning and horizontal gene transfer stabilize mobile colistin resistance

Antibiotic resistance tends to carry fitness costs, making it difficult to understand how resistance can be stably maintained in pathogen populations over the long-term. Here, we investigate this problem in the context of mcr-1, a fitness-costly gene that confers resistance to the last-resort antibiotic, colistin. Here we show that regulatory evolution has fine-tuned the expression of mcr-1, allowing E. coli to reduce the cost of mcr-1 while simultaneously increasing colistin resistance. Conjugative plasmids have transferred low cost/high resistance mcr-1 alleles across an incredible diversity of E. coli strains, further stabilizing mcr-1 at the species level. Crucially, 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%. Our study shows how the rapid evolution and horizontal transmission of resistance genes can combine to stabilize resistance and reduce the efficiency of interventions aimed at reducing AMR by limiting antibiotic consumption.

evolutionary biology↗

bioRxiv2022

Antibiotic heteroresistance generated by multi-copy plasmids

Heteroresistance - in which a clonal bacterial population contains a cell subpopulation with higher resistance to antibiotics than the main population - is a growing clinical problem that complicates susceptibility determination and threatens therapeutic success. Despite the high prevalence of heteroresistance in clinical settings, the underlying genetic mechanisms that stably maintain heterogeneous bacterial populations are poorly understood. Using fluorescence microscopy, single-cell microfluidics, and quantitative image analysis, we show that random replication and segregation of multicopy plasmids produce populations of bacterium Escherichia coli MG1655 in which cells with low-and high-plasmid copy numbers stably co-exist. By combining stochastic simulations of a computational model with high-throughput single-cell measurements of blaTEM-1 expression, we show that copy number variability confers the bacterial population with transient resistance to a lethal concentration of a {beta} -lactam antibiotic. Moreover, this surviving, high plasmid copy minority is capable of regenerating a heterogeneous bacterial population with low and high plasmid copy numbers through segregational instability, rapidly alleviating the fitness burden of carrying large numbers of plasmids. Our results provide further support for the tenet that plasmids are more than simple vehicles for horizontal transmission of genetic information between cells, as they can also drive bacterial adaptation in dynamic environments by providing a platform for rapid amplification and attenuation of gene copy number that can accelerate the rate of resistance adaptation and can lead to treatment failure.

microbiology↗

bioRxiv2022

Off-target integron activity leads to rapid plasmid compensatory evolution in response to antibiotic selection pressure

Integrons are mobile genetic elements that have played an important role in the dissemination of antibiotic resistance. Under stress, the integron can generate 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.

evolutionary biology↗

bioRxiv2022

Pre-existing chromosomal polymorphisms in pathogenic E. coli potentiate the evolution of antibiotic resistance by MCR-1 plasmid acquisition

Bacterial pathogens show high levels of standing genetic diversity, but the influence of this diversity on the evolution of antibiotic resistance remains unclear. Here we address this problem in the context of colistin, a last line of defense antibiotic. Using experimental evolution, we show that a plasmid carrying the MCR-1 colistin resistance gene dramatically increases the ability of E. coli populations to evolve high-level colistin resistance by acquiring mutations in lpxC, an essential chromosomal gene involved in lipopolysaccharide biosynthesis. Crucially, lpxC mutations increase colistin resistance in the presence of the MCR-1 gene, but decrease the resistance of wild-type cells, revealing positive sign epistasis for antibiotic resistance. Analysis of public genomic datasets shows that lpxC polymorphisms are common in pathogenic E. coli carrying MCR-1, highlighting the clinical relevance of this interaction. Importantly, lpxC diversity is high in pathogenic E. coli from regions with no history of MCR-1 acquisition, suggesting that pre-existing lpxC polymorphisms have potentiated the evolution of high-level colistin resistance by MCR-1 acquisition. More broadly, these findings highlight the importance of standing genetic variation and plasmid/chromosomal interactions in the evolutionary dynamics of antibiotic resistance.

evolutionary biology↗

bioRxiv2022

The evolution of colistin resistance increases bacterial resistance to host antimicrobial peptides and virulence

Antimicrobial peptides (AMPs) offer a promising solution to the antibiotic resistance crisis. However, an unresolved serious concern is that the evolution of resistance to therapeutic AMPs may generate cross-resistance to host AMPs, compromising a cornerstone of the innate immune response. We systematically tested this hypothesis using globally disseminated mobile colistin resistance (MCR) that has been selected by the use of colistin in agriculture and medicine. Here we show that MCR provides a selective advantage to E. coli in the presence of key AMPs from humans and agricultural animals by increasing AMP resistance. Moreover, MCR promotes bacterial growth in human serum and increases virulence in a Galleria mellonella infection model. Our study shows how the anthropogenic use of AMPs can drive the accidental evolution of resistance to the innate immune system of humans and animals. These findings have major implications for the design and use of therapeutic AMPs and they suggest that MCR will be difficult to eradicate, even if colistin use is withdrawn.

microbiology↗

bioRxiv2021

Localized hypermutation drives the evolution of unstable colistin resistance in small populations of Pseudomonas aeruginosa

Colistin has emerged as an important last line of defence for the treatment of infections caused by antibiotic resistant Gram-negative pathogens. Here we investigate the responses of {approx}1,000 populations of an MDR strain of P. aeruginosa to a high dose of colistin. Colistin exposure resulted in rapid cell death, but a sub-set of populations eventually recovered due to the outgrowth of heteroresistant cells. Genome sequencing revealed that heteroresistance was primarily driven by mutations in the PmrAB two-component system that occurred at a rate ({approx}2x10-5 per cell division) that was 103-104 fold higher than typical resistance mutation rates. Crucially, this elevated mutation rate was only found in pmrB, demonstrating that hypermutability is localized to this gene. PmrAB provides resistance to antimicrobial peptides that are involved in host immunity, suggesting that this pathogen may have evolved a high mutation rate as an adaption to generate mutants that are resistant to host antimicrobial peptides that are secreted during infection. Interestingly, we found no mutations in 1/3 of populations that recovered from colistin treatment, suggesting that phenotypic plasticity and/or persister cells contribute to the ability of Pseudomonas to adapt to colistin.

microbiology↗

bioRxiv2021

Evolutionary processes driving the rise and fall of Staphylococcus aureus ST239, a dominant hybrid pathogen

Staphylococcus aureus ST239 has been one of the most successful epidemic MRSA strains, and one of the leading causes of healthcare-associated MRSA infections. Here we investigate the evolution of ST239 using a combination of computational and experimental approaches. ST239 is thought to have emerged by a large scale chromosomal replacement event in which an ST8 clone acquired approximately 600 kb of DNA from an ST30 clone. Analysis of large-scale genomic data sets allowed us to confirm and refine the model of the origin of ST239. Importantly, we found that ST239 originated between the 1920s and 1945, implying that this MRSA lineage evolved at least 14 years before the clinical introduction of methicillin. Molecular evolution within ST239 has been dominated by purifying selection, although we found some evidence that the acquired region of the genome has evolved rapidly as a result of relaxed selective constraints. Crucially, we found that ST239 isolates have low competitive ability relative to both ST30 and ST8, demonstrating that this hybrid lineage is characterized by low fitness. We also found evidence of positive selection in a small number of genes involved in antibiotic resistance and virulence, suggesting that ST239 has evolved towards an increasingly pathogenic lifestyle. Collectively, these results support the view that low fitness has driven the recent decline of ST239, and highlight the challenge of using evolutionary approaches to understand the dynamics of pathogenic bacteria.

evolutionary biology↗

bioRxiv2020

Graded calcium spikes differentially signal neurotransmitter input in cerebrospinal fluid contacting neurons of mouse spinal cord

The action potential and its all-or-none nature is fundamental to neural communication. Canonically the action potential is initiated once voltage-gated Na+ channels are activated, and their rapid kinetics of activation and inactivation give rise to the action potentials all-or-none nature. Here we show that cerebrospinal fluid contacting neurons (CSFcNs) surrounding the central canal of the mouse spinal cord employ a different strategy. Rather than using voltage-gated Na+ channels to generate binary spikes, CSFcNs use two different types of voltage-gated Ca2+ channel, enabling spikes of different amplitude. T-type Ca2+ channels generate small amplitude spikes, whereas large amplitude spikes require high voltage-activated Cd2+ sensitive Ca2+ channels. We show that these different amplitude spikes signal input from different transmitter systems; purinergic inputs evoke smaller T-type dependent spikes while cholinergic inputs evoke large T-type independent spikes. Different synaptic inputs to CSFcNs can therefore be signalled by the spike amplitude.

neuroscience↗

bioRxiv2020

CRISPR-Cas systems restrict horizontal gene transfer in Pseudomonas aeruginosa

CRISPR-Cas systems provide bacteria and archaea with an adaptive immune system that targets foreign DNA. However, the xenogenic nature of immunity provided by CRISPR-Cas raises the possibility that these systems may constrain horizontal gene transfer. Here we test this hypothesis in the opportunistic pathogen Pseudomonas aeruginosa, which has emerged an important model system for understanding CRISPR-Cas function. Across the diversity of P. aeruginosa, active CRISPR-Cas systems are associated with smaller genomes and a reduced GC content, suggesting that CRISPR-Cas inhibits the acquisition of foreign DNA. Although phage are the major target of CRISPR-Cas spacers, more than 80% of isolates with an active CRISPR-Cas system have spacers that target integrative conjugative elements (ICE) or the conserved conjugative transfer machinery used by plasmids and ICE. Consistent with these results, genomes containing active CRISPR-Cas systems harbor a lower abundance of both prophage and ICE. Crucially, spacers in genomes with active CRISPR-Cas systems map to ICE and phage that are integrated into the chromosomes of closely related genomes lacking CRISPR-Cas immunity, providing direct evidence that CRISPR-Cas constrains horizontal gene transfer in these lineages. In conclusion, we find that CRISPR-Cas acts as an important constraint to horizontal gene transfer, suggesting that CRISPR-Cas may constrain the ability of this pathogen to adapt to new niches and stressors.

genomics↗

bioRxiv2020

Rapid evolution drives the rise and fall of carbapenem resistance during an acute Pseudomonas aeruginosa infection

It is well established that antibiotic treatment selects for resistance in pathogenic bacteria. However, the evolutionary responses of pathogen populations to antibiotic treatment during infections remain poorly resolved, especially in acute infections. Here we map the evolutionary responses to treatment in high definition through genomic and phenotypic characterization of >100 isolates from a patient with P. aeruginosa pneumonia. Antibiotic therapy (meropenem, colistin) caused a rapid crash of the P. aeruginosa population in the lung, but this decline was followed by the spread of meropenem resistance mutations that restrict antibiotic uptake (oprD) or modify LPS biosynthesis (wbpM). Low fitness strains with high-level meropenem resistance (oprD) were then replaced by high fitness strains with anti-resistance mutations in the MexAB-OprM efflux pump, causing a rapid decline in resistance to both meropenem and a collateral loss of resistance to a broad spectrum of antibiotics. In contrast, we did not observe any evolutionary responses to antibiotic treatment in the intestinal population of P. aeruginosa. Carbapenem antibiotics are key to the treatment of infections caused by Gram negative pathogens, and our work highlights the ability of natural selection to drive both the rapid rise and fall of carbapenem resistance during acute infections.

evolutionary biology↗

bioRxiv2020

Integron activity accelerates the evolution of antibiotic resistance

Mobile integrons are widespread genetic platforms that allow bacteria to modulate the expression of antibiotic resistance cassettes by shuffling their position from a common promoter. Antibiotic stress induces the expression of an integrase that excises and integrates cassettes, and this unique recombination and expression system is thought to allow bacteria to evolve on demand in response to antibiotic pressure. To test this hypothesis, we inserted a custom three cassette integron into P. aeruginosa, and used experimental evolution to measure the impact of integrase activity on adaptation to gentamicin. Crucially, integrase activity accelerated evolution by increasing the expression of a gentamicin resistance cassette through duplications and by eliminating redundant cassettes. Importantly, we found no evidence of deleterious off-target effects of integrase activity. In summary, integrons accelerate resistance evolution by rapidly generating combinatorial variation in cassette composition while maintaining genomic integrity.

microbiology↗

bioRxiv2018

Stochastic bacterial population dynamics prevent the emergence of antibiotic resistance within the mutant selection window

A better understanding of how antibiotic exposure impacts the evolution of resistance is crucial for designing more sustainable treatment strategies. The conventional approach to relating antibiotic dose to resistance evolution within a bacterial population is to measure the range of concentrations over which resistant strain(s) are selectively favoured over a sensitive strain - the "mutant selection window". Here, we instead investigate how antibiotic concentration impacts the initial establishment of resistance from single cells, mimicking the clonal expansion of a resistant lineage following mutation or horizontal gene transfer. Using two Pseudomonas aeruginosa strains carrying distinct resistance plasmids, we show that single resistant cells have <5% probability of outgrowth at antibiotic concentrations as low as 1/8th of the resistant strains minimum inhibitory concentration. This low probability of establishment is due to detrimental effects of antibiotics on resistant cells, coupled with the inherently stochastic nature of cell division and death on the single-cell level, which leads to loss of many nascent resistant lineages. Our findings suggest that moderate doses of antibiotics, within the traditional mutant selection window, may be more effective at preventing de novo emergence of resistance than predicted by deterministic approaches. Significance statementThe emergence of antibiotic resistance poses a critical threat to the efficacy of antibiotic treatments. A resistant bacterial population must originally arise from a single cell that mutates or acquires a resistance gene. This single cell may, by chance, fail to successfully reproduce before it dies, leading to loss of the nascent resistant lineage. Here we show that antibiotic concentrations that selectively favour resistance are nonetheless sufficient to reduce the chance of outgrowth from a single cell to a very low probability. Our findings suggest that lower antibiotic concentrations than previously thought may be sufficient to prevent, with high probability, emergence of resistance from single cells.

microbiology↗