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Evolution of Escherichia coli rifampicin resistance in an antibiotic-free environment during thermal stress.

ABSTRACT: Beneficial mutations play an essential role in bacterial adaptation, yet little is known about their fitness effects across genetic backgrounds and environments. One prominent example of bacterial adaptation is antibiotic resistance. Until recently, the paradigm has been that antibiotic resistance is selected by the presence of antibiotics because resistant mutations confer fitness costs in antibiotic free environments. In this study we show that it is not always the case, documenting the selection and fixation of resistant mutations in populations of Escherichia coli B that had never been exposed to antibiotics but instead evolved for 2000 generations at high temperature (42.2°C).We found parallel mutations within the rpoB gene encoding the beta subunit of RNA polymerase. These amino acid substitutions conferred different levels of rifampicin resistance. The resistant mutations typically appeared, and were fixed, early in the evolution experiment. We confirmed the high advantage of these mutations at 42.2°C in glucose-limited medium. However, the rpoB mutations had different fitness effects across three genetic backgrounds and six environments.We describe resistance mutations that are not necessarily costly in the absence of antibiotics or compensatory mutations but are highly beneficial at high temperature and low glucose. Their fitness effects depend on the environment and the genetic background, providing glimpses into the prevalence of epistasis and pleiotropy.

SUBMITTER: Rodriguez-Verdugo A

PROVIDER: S-EPMC3598500 | biostudies-other | 2013 Feb

REPOSITORIES: biostudies-other

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Evolution of Escherichia coli rifampicin resistance in an antibiotic-free environment during thermal stress.

Rodríguez-Verdugo Alejandra A Gaut Brandon S BS Tenaillon Olivier O

BMC evolutionary biology 20130222

<h4>Background</h4>Beneficial mutations play an essential role in bacterial adaptation, yet little is known about their fitness effects across genetic backgrounds and environments. One prominent example of bacterial adaptation is antibiotic resistance. Until recently, the paradigm has been that antibiotic resistance is selected by the presence of antibiotics because resistant mutations confer fitness costs in antibiotic free environments. In this study we show that it is not always the case, doc ...[more]

PMID: 23433244

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Project description:Escherichia coli K-12 W3110 grows in the presence of membrane-permeant organic acids that can depress cytoplasmic pH and accumulate in the cytoplasm. We conducted experimental evolution by daily diluting cultures in increasing concentrations of benzoic acid (up to 20 mM) buffered at external pH 6.5, a pH at which permeant acids concentrate in the cytoplasm. By 2,000 generations, clones isolated from evolving populations showed increasing tolerance to benzoate but were sensitive to chloramphenicol and tetracycline. Sixteen clones grew to stationary phase in 20 mM benzoate, whereas the ancestral strain W3110 peaked and declined. Similar growth occurred in 10 mM salicylate. Benzoate-evolved strains grew like W3110 in the absence of benzoate, in media buffered at pH 4.8, pH 7.0, or pH 9.0, or in 20 mM acetate or sorbate at pH 6.5. Genomes of 16 strains revealed over 100 mutations, including single-nucleotide polymorphisms (SNPs), large deletions, and insertion knockouts. Most strains acquired deletions in the benzoate-induced multiple antibiotic resistance (Mar) regulon or in associated regulators such as rob and cpxA, as well as the multidrug resistance (MDR) efflux pumps emrA, emrY, and mdtA Strains also lost or downregulated the Gad acid fitness regulon. In 5 mM benzoate or in 2 mM salicylate (2-hydroxybenzoate), most strains showed increased sensitivity to the antibiotics chloramphenicol and tetracycline; some strains were more sensitive than a marA knockout strain. Thus, our benzoate-evolved strains may reveal additional unknown drug resistance components. Benzoate or salicylate selection pressure may cause general loss of MDR genes and regulators. IMPORTANCE:Benzoate is a common food preservative, and salicylate is the primary active metabolite of aspirin. In the gut microbiome, genetic adaptation to salicylate may involve loss or downregulation of inducible multidrug resistance systems. This discovery implies that aspirin therapy may modulate the human gut microbiome to favor salicylate tolerance at the expense of drug resistance. Similar aspirin-associated loss of drug resistance might occur in bacterial pathogens found in arterial plaques.

Project description:Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 microg/ml), kanamycin (25 microg/ml), and ofloxacin (10 microg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.

Project description:Background and Objectives:Metallic antimicrobial materials are of growing interest due to their potential to control pathogenic and multidrug-resistant bacteria. Yet we do not know if utilizing these materials can lead to genetic adaptations that produce even more dangerous bacterial varieties. Methodology:Here we utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance. Results:By day 10 of evolution, increased gallium resistance was evident in populations cultured in medium containing a sublethal concentration of gallium. Furthermore, these populations showed increased resistance to ionic silver and iron (III), but not iron (II) and no increase in traditional antibiotic resistance compared with controls and the ancestral strain. In contrast, the control populations showed increased resistance to rifampicin relative to the gallium-resistant and ancestral population. Genomic analysis identified hard selective sweeps of mutations in several genes in the gallium (III)-resistant lines including: fecA (iron citrate outer membrane transporter), insl1 (IS30 tranposase) one intergenic mutations arsC ?/? yhiS; (arsenate reductase/pseudogene) and in one pseudogene yedN ?; (iapH/yopM family). Two additional significant intergenic polymorphisms were found at frequencies > 0.500 in fepD ?/? entS (iron-enterobactin transporter subunit/enterobactin exporter, iron-regulated) and yfgF ?/? yfgG (cyclic-di-GMP phosphodiesterase, anaerobic/uncharacterized protein). The control populations displayed mutations in the rpoB gene, a gene associated with rifampicin resistance. Conclusions:This study corroborates recent results observed in experiments utilizing pathogenic Pseudomonas strains that also showed that Gram-negative bacteria can rapidly evolve resistance to an atom that mimics an essential micronutrient and shows the pleiotropic consequences associated with this adaptation. Lay summary:We utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance.

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Evolution of Escherichia coli rifampicin resistance in an antibiotic-free environment during thermal stress.

Publications

Evolution of Escherichia coli rifampicin resistance in an antibiotic-free environment during thermal stress.

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