Choice of β-Lactam Resistance Pathway Depends Critically on Initial Antibiotic Concentration

Ruelens, Philip; Visser, Jaap

doi:10.1128/aac.00471-21

Cited by 7 publications

(10 citation statements)

References 20 publications

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“…We also see that, when two different amino acid substitutions for a given position are tested (A237S vs A237T, R164H vs R164S), only the most frequent one in CCED has a substantial effect on cefotaxime resistance on its own in our analysis. This implies that less frequent substitutions may be more dependent on a network of interactions to produce a large increase in resistance, consistent with their lower frequency , or the fact that their selection may have been driven by a different lactam antibiotic (R164S primarily confers resistance to ceftazidime) or by a lower antibiotic concentration …”

Section: Results and Discussionmentioning

confidence: 99%

“…This implies that less frequent substitutions may be more dependent on a network of interactions to produce a large increase in resistance, consistent with their lower frequency 18,27 or the fact that their selection may have been driven by a different lactam antibiotic (R164S primarily confers resistance to ceftazidime 30 ) or by a lower antibiotic concentration. 31 Next, we determined the IC 50 for a total of 206 TEM mutants�101 simple ones (i.e., involving 2 mutations) and 105 complex ones (i.e., involving 3 mutations). The results of the test are shown in Table S2.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

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Experimental and In Silico Analysis of TEM β-Lactamase Adaptive Evolution

et al. 2022

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Multiple mutations often have non-additive (epistatic) phenotypic effects. Epistasis is of fundamental biological relevance but is not well understood mechanistically. Adaptive evolution, i.e., the evolution of new biochemical activities, is rich in epistatic interactions. To better understand the principles underlying epistasis during genetic adaptation, we studied the evolution of TEM-1 β-lactamase variants exhibiting cefotaxime resistance. We report the collection of a library of 487 observed evolutionary trajectories for TEM-1 and determine the epistasis status based on cefotaxime resistance phenotype for 206 combinations of 2–3 TEM-1 mutations involving 17 positions under adaptive selective pressure. Gain-of-function (GOF) mutations are gatekeepers for adaptation. To see if GOF phenotypes can be inferred based solely on sequence data, we calculated the enrichment of GOF mutations in the different categories of epistatic pairs. Our results suggest that this is possible because GOF mutations are particularly enriched in sign and reciprocal sign epistasis, which leave a major imprint on the sequence space accessible to evolution. We also used FoldX to explore the relationship between thermodynamic stability and epistasis. We found that mutations in observed evolutionary trajectories tend to destabilize the folded structure of the protein, albeit their cumulative effects are consistently below the protein’s free energy of folding. The destabilizing effect is stronger for epistatic pairs, suggesting that modest or local alterations in folding stability can modulate catalysis. Finally, we report a significant relationship between epistasis and the degree to which two protein positions are structurally and dynamically coupled, even in the absence of ligand.

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Section: Results and Discussionmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

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Experimental and In Silico Analysis of TEM β-Lactamase Adaptive Evolution

et al. 2022

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Section: Discussionmentioning

confidence: 99%

“…If many genotypes have equivalently high fitness above a threshold value of resistance, there are many factors that could determine which variants eventually predominate. For example, mutation bias (44), trade-offs between resistance and growth (45), and collateral sensitivity to other antibiotics (46, 47) could be important. Selection for resistant types in natural environments may be further complicated by the striking inverse relationship between resistance and fitness in spatially structured sub-MIC environments – a phenomenon which is associated with filamentation of susceptible types as previously reported (25).…”

Section: Discussionmentioning

confidence: 99%

The fitness landscape of TEM-1 β-lactamase is stratified and inverted by sublethal concentrations of cefotaxime

Farr

Pesce

Zwart

et al. 2022

Preprint

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Adaptive evolutionary processes are constrained by the availability of mutations which cause a fitness benefit — a concept that may be illustrated by ′fitness landscapes ′ which map the relationship of genotype space with fitness. Experimentally derived landscapes have demonstrated a predictability to evolution by identifying limited ′mutational routes ′ that evolution by natural selection may take between low and high–fitness genotypes. However, such studies often utilise indirect measures to determine fitness. We estimated the competitive fitness of each mutant relative to all of its single–mutation neighbours to describe the fitness landscape of three mutations in a β–lactamase enzyme at sub–lethal concentrations of the antibiotic cefotaxime in a structured and unstructured environment. We found that in the unstructured environment the antibiotic selected for higher–resistance types — but with an equivalent fitness for subsets of mutants, despite substantial variation in resistance — resulting in a stratified fitness landscape. In contrast, in a structured environment with low antibiotic concentration, antibiotic–susceptible genotypes had a relative fitness advantage, which was associated with antibiotic–induced filamentation. These results cast doubt that highly resistant genotypes have a unique selective advantage in environments with sub–inhibitory concentrations of antibiotics, and demonstrate that direct fitness measures are required for meaningful predictions of the accessibility of evolutionary routes.

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Ecological feedback limits the spread of an adaptive β-lactamase mutant

Ruelens,

de Ridder,

de Visser

et al. 2023

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Antibiotic resistance in bacterial infections is a growing global health concern, with β-lactamases playing a pivotal role. Understanding the origin and spread of β-lactamases can provide insights for developing novel drugs and therapies. However, complexity arises by the fact that they can affect their own selective advantage through antibiotic degradation, which also shields nearby susceptible bacteria. To address this eco-evolutionary feedback, we investigated the interplay between feedbacks and the spread of bacterial strains expressing different β-lactamase enzymes conferring varying levels of resistance to the cephalosporin cefotaxime (CTX). Specifically, we conducted head-to-head competitions between two related clinically relevant β-lactamase mutants, TEM-19 and TEM-52, and varied CTX concentration and cell density to explore the impact of these factors on competitive fitness and coexistence. Results revealed that at certain antibiotic treatment regimes, culture density, which modulated the eco-evolutionary feedback strength, determined whether resistance alleles could invade the population and replace or coexist with susceptible alleles. Furthermore, using a population dynamic model based solely on measured parameters, we investigated the underlying mechanisms of the observed dynamics. By validating our model with experimental data, we show that it can largely predict the equilibrium frequencies based on dose-dependent growth rates and antibiotic degradation activities of the individual strains. Our study emphasizes the importance of considering ecological feedbacks in the evolution of antibiotic resistance, even at the level of individual proteins, and highlights the potential of a predictive model applicable to clinically relevant conditions.

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Choice of β-Lactam Resistance Pathway Depends Critically on Initial Antibiotic Concentration

Cited by 7 publications

References 20 publications

Experimental and In Silico Analysis of TEM β-Lactamase Adaptive Evolution

Experimental and In Silico Analysis of TEM β-Lactamase Adaptive Evolution

The fitness landscape of TEM-1 β-lactamase is stratified and inverted by sublethal concentrations of cefotaxime

Ecological feedback limits the spread of an adaptive β-lactamase mutant

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