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Mechanisms and Dynamics of Carbapenem Resistance in Escherichia coli
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Linus Sandegren)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The emergence of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae worldwide has led to an increased use of carbapenems and may drive the development of carbapenem resistance. Existing mechanisms are mainly due to acquired carbapenemases or the combination of ESBL-production and reduced outer membrane permeability. The focus of this thesis was to study the development of carbapenem resistance in Escherichia coli in the presence and absence of acquired β-lactamases. To this end we used the resistance plasmid pUUH239.2 that caused the first major outbreak of ESBL-producing Enterobacteriaceae in Scandinavia.

Spontaneous carbapenem resistance was strongly favoured by the presence of the ESBL-encoding plasmid and different mutational spectra and resistance levels arose for different carbapenems. Mainly, loss of function mutations in the regulators of porin expression caused reduced influx of antibiotic into the cell and in combination with amplification of β-lactamase genes on the plasmid this led to high resistance levels. We further used a pharmacokinetic model, mimicking antibiotic concentrations found in patients during treatment, to test whether ertapenem resistant populations could be selected even at these concentrations. We found that resistant mutants only arose for the ESBL-producing strain and that an increased dosage of ertapenem could not prevent selection of these resistant subpopulations. In another study we saw that carbapenem resistance can even develop in the absence of ESBL-production. We found mutants in export pumps and the antibiotic targets to give high level resistance albeit with high fitness costs in the absence of antibiotics. In the last study, we used selective amplification of β-lactamases on the pUUH239.2 plasmid by carbapenems to determine the cost and stability of gene amplifications. Using mathematical modelling we determined the likelihood of evolution of new gene functions in this region. The high cost and instability of the amplified state makes de novo evolution very improbable, but constant selection of the amplified state may balance these factors until rare mutations can establish a new function.

In my studies I observed the influence of β-lactamases on carbapenem resistance and saw that amplification of these genes would further contribute to resistance. The rapid disappearance of amplified arrays of resistance genes in the absence of antibiotic selection may lead to the underestimation of gene amplification as clinical resistance mechanism. Amplification of β-lactamase genes is an important stepping-stone and might lead to the evolution of new resistance genes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 998
Keyword [en]
carbapenem, antibiotic resistance, fitness cost, ESBLs, penicillin-binding proteins, gene amplification
National Category
Biochemistry and Molecular Biology Genetics Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-221432ISBN: 978-91-554-8950-2 (print)OAI: oai:DiVA.org:uu-221432DiVA: diva2:712041
Public defence
2014-06-05, B42, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2014-05-13 Created: 2014-03-31 Last updated: 2014-06-30Bibliographically approved
List of papers
1. Influence of acquired β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli
Open this publication in new window or tab >>Influence of acquired β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli
2013 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 1, 51-59 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: To investigate the influence of plasmid-borne β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli and the fitness costs associated with resistance. Methods: Stepwise selection of carbapenem-resistant mutants with or without the extended-spectrum β-lactamase (ESBL)-encoding plasmid pUUH239.2 was performed. Mutation rates and mutational pathways to resistance were determined. In vitro-selected and constructed mutants were characterized regarding the MICs of the carbapenems, porin expression profiles, growth rates and the presence of mutations in the porins ompC/ompF and their regulatory genes. The influence of the plasmid-encoded β-lactamases TEM-1, OXA-1 and CTX-M-15 on resistance development was determined. Results: Results show that E. coli readily developed reduced carbapenem susceptibility and clinical resistance levels by a combination of porin loss and increased β-lactamase expression, especially towards ertapenem. All tested β-lactamases (CTX-M-15, TEM-1 and OXA-1) contributed to reduced carbapenem susceptibility in the absence of porin expression. However, complete loss of porin expression conferred a 20% fitness cost on the bacterial growth rate. Increased β-lactamase expression through spontaneous gene amplification on the plasmid was a major resistance factor. Conclusions: Plasmid-encoded β-lactamases, including non-ESBL enzymes, have a strong influence on the frequency and resistance level of spontaneous carbapenem-resistant mutants. The fitness cost associated with the loss of OmpC/OmpF in E. coli most likely reduces the survivability of porin mutants and could explain why they have not emerged as a clinical problem in this species.

Keyword
Fitness cost, Gene amplification, Mutations, Plasmids
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-192026 (URN)10.1093/jac/dks368 (DOI)000312646300010 ()
Available from: 2013-01-24 Created: 2013-01-15 Last updated: 2014-05-13Bibliographically approved
2. Frequent emergence of porin-deficient subpopulations with reduced carbapenem susceptibility in ESBL-producing Escherichia coli during exposure to ertapenem in an in vitro pharmacokinetic model
Open this publication in new window or tab >>Frequent emergence of porin-deficient subpopulations with reduced carbapenem susceptibility in ESBL-producing Escherichia coli during exposure to ertapenem in an in vitro pharmacokinetic model
Show others...
2013 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 6, 1319-1326 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES:

Ertapenem resistance is increasing in Enterobacteriaceae. The production of extended-spectrum β-lactamases (ESBLs) and reduced expression of outer membrane porins are major mechanisms of resistance in ertapenem-resistant Klebsiella pneumoniae. Less is known of ertapenem resistance in Escherichia coli. The aim of this study was to explore the impact of ESBL production in E. coli on the antibacterial activity of ertapenem.

METHODS:

Two E. coli strains, with and without ESBL production, were exposed to ertapenem in vitro for 48 h at concentrations simulating human pharmacokinetics with conventional and higher dosages.

RESULTS:

Isolates with non-susceptibility to ertapenem (MICs 0.75-1.5 mg/L) were detected after five of nine time-kill experiments with the ESBL-producing strain. All of these isolates had ompR mutations, which reduce the expression of outer membrane porins OmpF and OmpC. Higher dosage did not prevent selection of porin-deficient subpopulations. No mutants were detected after experiments with the non-ESBL-producing strain. Compared with other experiments, experiments with ompR mutants detected in endpoint samples showed significantly less bacterial killing after the second dose of ertapenem. Impaired antibacterial activity against E. coli with ESBL production and ompR mutation was also demonstrated in time-kill experiments with static antibiotic concentrations.

CONCLUSIONS:

The combination of ESBL production and porin loss in E. coli can result in reduced susceptibility to ertapenem. Porin-deficient subpopulations frequently emerged in ESBL-producing E. coli during exposure to ertapenem at concentrations simulating human pharmacokinetics. Inappropriate use of ertapenem should be avoided to minimize the risk of selection of ESBL-producing bacteria with reduced susceptibility to carbapenems.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-197878 (URN)10.1093/jac/dkt044 (DOI)000319468900016 ()23478794 (PubMedID)
Available from: 2013-04-05 Created: 2013-04-05 Last updated: 2015-09-30Bibliographically approved
3. Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli
Open this publication in new window or tab >>Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli
2016 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, 1188-1198 p.Article in journal (Refereed) Published
Abstract [en]

The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of beta-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of beta-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. beta-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.

National Category
Biochemistry and Molecular Biology Microbiology
Identifiers
urn:nbn:se:uu:diva-221428 (URN)10.1093/jac/dkv475 (DOI)000376291300008 ()26869688 (PubMedID)
Funder
Swedish Research Council Formas, 2013-5476-25194-9EU, European Research Council, 282004
Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-04-13Bibliographically approved
4. High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms
Open this publication in new window or tab >>High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms
Show others...
2014 (English)In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, no 6, 1526-1535 p.Article in journal (Refereed) Published
Abstract [sv]

An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different sub-models, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kbp of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modelling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be off-set by positive selection for novel beneficial functions.

National Category
Microbiology Biochemistry and Molecular Biology Genetics
Identifiers
urn:nbn:se:uu:diva-221431 (URN)10.1093/molbev/msu111 (DOI)000337067400019 ()
Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2014-07-15Bibliographically approved

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