Biology subjects

Gifford, D. R.

Publications and source records attributed to Gifford, D. R..

10 recordsLinked to original sources

bioRxiv2026

Fitness costs of antibiotic resistance mutations in rich media do not reliably predict those in infection-like environments

The acquisition of antibiotic resistance is typically associated with a fitness cost, reducing the competitive ability of cells in the absence of antibiotics. Our ability to predict these costs informs our ability to predict how likely it is that resistance will subside in the absence of treatment. Fitness costs are typically measured in laboratory cultures using a nutritionally rich medium for growth. Here, we tested the impact of infection-like environments on the prevalence and magnitude of fitness costs associated with antibiotic resistance mutations in the opportunistic pathogen Pseudomonas aeruginosa. We assessed two different clinically relevant infection niches, the lung and urinary tract, through the use of infection-niche mimicking media that models the chemical compositions of these niches. The fitness costs of resistance mutations in rich media did not reliably predict those in infection-like environments, on average costs were increased in the lung-mimicking environment and costly mutations were less prevalent in the urinary tract-mimicking environment. Synthetic human urine significantly enhanced the fitness of P. aeruginosa with resistance mutations in nfxB, encoding a multidrug efflux pump transcriptional repressor, and we identified oxalate as the likely component driving this effect. Our findings highlight the importance of environmental chemical composition for shaping the fitness costs of resistance in P. aeruginosa and suggest that nfxB mutants may have a selective advantage in the urinary tract, mediated by the presence of oxalate.

microbiology↗

bioRxiv2025

Evolutionary potential of the Escherichia coli antimutator ΔnudJ is reduced via altered mutational spectrum

The rate of spontaneous mutation is a key factor in determining the capacity of a population to adapt to a novel environment, for example a bacterial population exposed to antibiotics. Genetic and environmental factors controlling the mutation rate commonly also cause shifts in the relative rates of different mutational classes, i.e. the spectrum of spontaneous mutations. When the mutational spectrum is altered, the relatively enriched and depleted mutations may differ in their fitness effects. Here we aim to explore how a reduced mutation rate and altered mutational spectrum can contribute to adaptation in Escherichia coli. We measure mutation rates across a set of Nudix hydrolase deletants, finding multiple strains with an antimutator phenotype. We focus on the antimutator {Delta}nudJ which can cause a 6-fold mutation rate reduction relative to the wildtype with an altered mutational spectrum. Not only does nudJ deletion reduce the probability of antibiotic resistance arising but, in the case of rifampicin, the accessible resistance mutations under this altered mutation spectrum have greater fitness costs. While this effect is, so far, specific to this drug, it opens up the possibility of reducing the likelihood of resistance mutations establishing through simultaneous modification of mutation rate and spectrum. The identification and characterisation of antimutator alleles such as {Delta}nudJ provides potential targets for antievolution drugs which could be applied during antibiotic treatment to inhibit NudJ activity. Such an approach could suppress spontaneous resistance evolution both through fewer resistance mutations and by limiting access to the fittest mutations.

evolutionary biology↗

bioRxiv2024

Inflammation-like environments limit the loss of quorum sensing in Pseudomonas aeruginosa

Within-host environments are complex and multidimensional, making it challenging to link evolutionary responses of colonizing pathogens to causal selective drivers. Loss of quorum sensing (QS) via mutation of the master regulator, lasR, is a common adaptation of Pseudomonas aeruginosa during chronic infections. Here, we use experimental evolution in host-mimicking media to show that loss of QS is constrained by environmental factors associated with host inflammation. Specifically, environments combining oxidative stress and abundant free amino acids limited loss of QS, whereas QS loss was rapid in the absence of oxidative stress regardless of amino acids. Under oxidative stress, lasR mutations were contingent upon first decoupling regulation of oxidative stress responses from QS via mutations in the promoter region of the primary catalase, katA, or in the oxidative stress regulator, oxyR, enabling maintenance of oxidative stress tolerance. Together, our findings suggest that host inflammatory responses likely delay the loss of QS whilst colonizers undergo stepwise evolution, first adapting to survive lethal stressors before responding to other nutritional selective drivers that favour loss of QS.

evolutionary biology↗

bioRxiv2024

Evolutionary risk analysis of mutators for the development of nitrofurantoin resistance

Antimicrobial resistance is a significant global health crisis, with many antibiotics losing effectiveness within a decade of their introduction. The antibiotic nitrofurantoin, however, counters this trend, as it has sustained low resistance rates despite prolonged and widespread use. A key factor behind nitrofurantoins success is that resistance requires two independent inactivating mutations in separate genes, nfsA and nfsB. However, this inherent safeguard may be undermined by elevated mutation rates, a risk that remains unquantified for this antibiotic. Here, we investigated how mutation rates influence nitrofurantoin resistance in Escherichia coli using experimental evolution with both low-level and high-level mutator strains, alongside genomic analyses of uropathogenic nitrofurantoin-resistant clinical isolates. Under experimental conditions, increasing mutation rates increased the development of nitrofurantoin resistnace. Moreover, changes in the mutations that occurred also shifted the spectrum of resistance mutations from broad-impact frameshifts and indels to specific amino acid substitutions in the proteins active sites. Of the nitrofurantoin-resistant clinical isolates we analysed, nearly 40% harboured disruptive variants in DNA replication fidelity and repair genes--a prevalence at the higher end of what is typically observed in uropathogenic E. coli. These findings indicate that elevated mutation rates pose a risk to the continued efficacy of nitrofurantoin, highlighting the need for increased monitoring of mutator-driven resistance.

microbiology↗

bioRxiv2024

High-throughput method rapidly characterizes hundreds of novel antibiotic resistance mutations

A fundamental obstacle to tackling the antimicrobial resistance crsisis is identifying mutations that lead to resistance in a given genomic background and environment. We present a high-throughput technique - Quantitative Mutational Scan Sequencing (QMS-Seq) - that enables quantitative comparison of which genes are under antibiotic selection and captures how genetic background influences resistance evolution. We compared four E. coli strains exposed to ciprofloxacin, cycloserine, or nitrofurantoin and identified 975 resistance mutations, many in genes and regulatory regions not previously associated with resistance. QMS-Seq revealed that multi-drug and antibiotic-specific resistance are acquired through categorically different types of mutations, and that minor genotypic differences significantly influence evolutionary routes to resistance. By quantifying mutation frequency with single base pair resolution, QMS-Seq informs about the underlying mechanisms of resistance and identifies mutational hotspots within genes. Our method provides a way to rapidly screen for resistance mutations while assessing the impact of multiple confounding factors.

microbiology↗

bioRxiv2023

Antibiotic resistance alters the ability of Pseudomonas aeruginosa to invade the respiratory microbiome

The emergence and spread of antibiotic resistance in bacterial pathogens is a global health threat. One important unanswered question is how antibiotic resistance influences the ability of a pathogen to invade the host-associated microbiome. Here we investigate how antibiotic resistance impacts the ability of the opportunistic bacterial pathogen Pseudomonas aeruginosa to invade the respiratory microbiome, by measuring the ability of P. aeruginosa spontaneous antibiotic resistant mutants to invade pre-established cultures of commensal respiratory microbes. We find that commensal respiratory microbes tend to inhibit the growth of P. aeruginosa, and antibiotic resistance is a double-edged sword that can either help or hinder the ability of P. aeruginosa to overcome this inhibition. The directionality of this help or hinderance depends on both P. aeruginosa genotype and respiratory microbe identity. Antibiotic resistance facilitates the invasion of P. aeruginosa into Staphylococcus lugdunensis, yet impairs invasion into Rothia mucilaginosa and Staphylococcus epidermidis. Streptococcus species provide the strongest inhibition to P. aeruginosa invasion, and this is maintained regardless of antibiotic resistance genotype. Our study demonstrates how antibiotic resistance can alter the ability of a bacterial pathogen to invade the respiratory microbiome and suggests that attempts to manipulate the microbiome should focus on promoting the growth of commensals that can provide robust inhibition of both wildtype and antibiotic resistant pathogen strains.

microbiology↗

bioRxiv2023

Environmental and genetic influence on rate and spectrum of spontaneous mutations in Escherichia coli

Spontaneous mutations are the ultimate source of novel genetic variation on which evolution operates. Although mutation rate is often discussed as a single parameter in evolution, it comprises multiple distinct types of changes at the level of DNA. Moreover, the rates of these distinct changes can be independently influenced by genomic background and environmental conditions. Using fluctuation tests, we characterised the spectrum of spontaneous mutations in Escherichia coli grown in low and high glucose environments. These conditions are known to affect the rate of spontaneous mutation in wild-type MG1655, but not in a {Delta}luxS deletant strain --a gene with roles in both quorum sensing and the recycling of methylation products used in Escherichia colis DNA repair process. We find an increase in AT>GC transitions in the low glucose environment, suggesting that processes relating to the production or repair of this mutation could drive the response of overall mutation rate to glucose concentration. Interestingly, this increase in AT>GC transitions is maintained by the glucose non-responsive {Delta}luxS deletant. Instead, an elevated rate of GC>TA transversions, more common in a high glucose environment, leads to a net non-responsiveness of overall mutation rate for this strain. Our results show how relatively subtle changes, such as the concentration of a carbon substrate or loss of a regulatory gene, can substantially influence the amount and nature of genetic variation available to selection.

evolutionary biology↗

bioRxiv2020

Competition delays multi-drug resistance evolution during combination therapy

Combination therapies have shown remarkable success in preventing the evolution of resistance to multiple drugs, including HIV, tuberculosis, and cancer. Nevertheless, the rise in drug resistance still remains an important challenge. The capability to accurately predict the emergence of resistance, either to one or multiple drugs, may help to improve treatment options. Existing theoretical approaches often focus on exponential growth laws, which may not be realistic when scarce resources and competition limit growth. In this work, we study the emergence of single and double drug resistance in a model of combination therapy of two drugs. The model describes a sensitive strain, two types of single-resistant strains, and a double-resistant strain. We compare the probability that resistance emerges for three growth laws: exponential growth, logistic growth without competition between strains, and logistic growth with competition between strains. Using mathematical estimates and numerical simulations, we show that between-strain competition only affects the emergence of single resistance when resources are scarce. In contrast, the probability of double resistance is affected by between-strain competition over a wider space of resource availability. This indicates that competition between different resistant strains may be pertinent to identifying strategies for suppressing drug resistance, and that exponential models may overestimate the emergence of resistance to multiple drugs. A by-product of our work is an efficient strategy to evaluate probabilities of single and double resistance in models with multiple sequential mutations. This may be useful for a range of other problems in which the probability of resistance is of interest.

evolutionary biology↗

bioRxiv2019

Mutators drive evolution of multi-resistance to antibiotics

Antibiotic combination therapies are an approach used to counter the evolution of resistance; their purported benefit is they can stop the successive emergence of independent resistance mutations in the same genome. Here, we show that bacterial populations with mutators, organisms with defects in DNA repair, readily evolve resistance to combination antibiotic treatment when there is a delay in reaching inhibitory concentrations of antibiotic--under conditions where purely wild-type populations cannot. In populations subjected to combination treatment, we detected a remarkable amount of genomic diversity in resistance-determining mutations, multi-drug efflux pumps, and mutation-rate altering genes. However, using eco-evolutionary simulations, we demonstrate that only the initial mutator allele is required to explain multi-resistance evolution. Unexpectedly, mutators not only allowed multi-resistance to evolve under combination treatment where it was favoured, but also under single-drug treatments. Under both conditions, the mutator allele swept to fixation through hitch-hiking with single-drug resistance, enabling subsequent resistance mutations to emerge. Ultimately, our results suggest that mutators may hinder the utility of combination therapy when mutators are present. Additionally, by raising the rates of population mutation, selection for multi-resistance may have the unwanted side-effect of increasing the potential to evolve resistance to future antibiotic treatments. Significance statementThe global rise in antimicrobial resistance means that we urgently need new approaches to halt its spread. Antibiotic combination therapy, treatment involving more than one antibiotic, is a strategy proposed to do just that. Evolving resistance to combinations is thought to be exceedingly rare, as it would require two independent mutations to occur in the same genetic background before microbial growth is inhibited. We find that wild-type populations cannot achieve this, even when antibiotic concentrations increase gradually. However, populations with mutators, organisms with elevated mutation rates through DNA repair defects, can readily evolve multi-drug resistance under both single-drug and combination treatments. Further, hitch-hiking of mutator alleles alongside resistance increases the evolutionary potential for acquiring further resistance mutations. As mutators are commonly found in natural populations, including infection, our results suggest that combination therapy may not be as resilient a strategy against resistance evolution as was once thought.

evolutionary biology↗

bioRxiv2018

Opposing effects of population density and stress on Escherichia coli mutation rate

Evolution depends on mutations. For an individual genotype, the rate at which mutations arise is known to increase with various stressors (stress-induced mutagenesis - SIM) and decrease at high population density (density-associated mutation-rate plasticity - DAMP). We hypothesised that these two forms of mutation rate plasticity would have opposing effects across a nutrient gradient. Here we test this hypothesis, culturing Escherichia coli bacteria in increasingly rich media. We distinguish an increase in mutation rate with added nutrients through SIM (dependent on error-prone polymerases Pol IV and Pol V) and an opposing effect of DAMP (dependent on MutT, which removes oxidised G nucleotides). The combination of DAMP and SIM result in a mutation rate minimum at intermediate nutrient levels (which can support 7x108 cells ml-1). These findings demonstrate a strikingly close and nuanced relationship of ecological factors - stress and population density - with mutation, the fuel of all evolution.

evolutionary biology↗