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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
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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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.

Place, publisher, year, edition, pages
2013. Vol. 68, no 6, 1319-1326 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-197878DOI: 10.1093/jac/dkt044ISI: 000319468900016PubMedID: 23478794OAI: oai:DiVA.org:uu-197878DiVA: diva2:614631
Available from: 2013-04-05 Created: 2013-04-05 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Mechanisms and Dynamics of Carbapenem Resistance in Escherichia coli
Open this publication in new window or tab >>Mechanisms and Dynamics of Carbapenem Resistance in Escherichia coli
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
carbapenem, antibiotic resistance, fitness cost, ESBLs, penicillin-binding proteins, gene amplification
National Category
Biochemistry and Molecular Biology Genetics Microbiology
Identifiers
urn:nbn:se:uu:diva-221432 (URN)978-91-554-8950-2 (ISBN)
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
2. Multidrug-Resistant Escherichia coli and Klebsiella pneumoniae: Treatment, Selection and International Spread
Open this publication in new window or tab >>Multidrug-Resistant Escherichia coli and Klebsiella pneumoniae: Treatment, Selection and International Spread
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The prevalence of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases (ESBLs) and carbapenemases is increasing worldwide. Therapeutic options for infections with these bacteria are limited not only by the production of ESBLs and carbapenemases, which confer resistance to cephalosporins and carbapenems, but also by frequent co-resistance to other antibiotics. The overall aim of this thesis was to obtain a better understanding of multidrug-resistant E. coli and K. pneumoniae in relation to epidemiology, selection and susceptibility to antibiotic therapy.

In a prospective study ESBL-producing E. coli was found to spread easily through international travel. Twenty-four of 100 Swedes travelling outside Northern Europe acquired ESBL-producing E. coli in the intestinal flora. The risk was highest for travelers visiting India and those suffering from gastroenteritis during travel.

To minimize selection of ESBL-producing K. pneumoniae during a hospital outbreak with these bacteria, an educational antibiotic intervention was performed at Uppsala University Hospital in 2006. The primary aim of the intervention was to reduce the consumption of parenteral cephalosporins. An immediate and radical reduction of cephalosporins was demonstrated with interrupted time series analysis. The outbreak declined during 2007 and no increased resistance to replacement antibiotics was detected.

The impact of ESBL production on the antibacterial activity of ertapenem was studied in time-kill experiments. It was shown that porin-deficient subpopulations with reduced susceptibility to ertapenem frequently emerged in ESBL-producing E. coli during exposure to ertapenem at concentrations simulating human pharmacokinetics.

Further, the antibacterial effects of antibiotic combinations against four strains of K. pneumoniae producing carbapenemases of the metallo-beta-lactamase type were studied in time-kill experiments. Double and triple combinations of aztreonam, fosfomycin, meropenem, rifampin and colistin at clinically relevant static concentrations were effective despite that the bacteria were frequently resistant to the individual drugs. These results indicate that there is a largely unexplored potential of antibiotic combination therapy for multidrug-resistant K. pneumoniae.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 840
Keyword
Escherichia coli, Klebsiella pneumoniae, extended-spectrum beta-lactamases, carbapenemases, metallo-beta-lactamases, synergy, antibiotic interventions
National Category
Infectious Medicine Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-182897 (URN)978-91-554-8537-5 (ISBN)
Public defence
2012-12-15, Gustavianum, Auditorium minus, Akademigatan 3, Uppsala, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2012-11-23 Created: 2012-10-18 Last updated: 2015-09-30Bibliographically approved

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Tängdén, ThomasAdler, MarlenCars, OttoSandegren, LinusLöwdin, Elisabeth

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