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Biological cost of single and multiple norfloxacin resistance mutations in Escherichia coli implicated in urinary tract infections
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2005 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 49, no 6, 2343-2351 p.Article in journal (Refereed) Published
Abstract [en]

Resistance to fluoroquinolones in urinary tract infection (UTIs)caused by Escherichia coli is associated with multiple mutations,typically those that alter DNA gyrase and DNA topoisomeraseIV and those that regulate AcrAB-TolC-mediated efflux. We askedwhether a fitness cost is associated with the accumulation ofthese multiple mutations. Mutants of the susceptible E. coliUTI isolate Nu14 were selected through three to five successivesteps with norfloxacin. Each selection was performed with theMIC of the selected strain. After each selection the MIC wasmeasured; and the regions of gyrA, gyrB, parC, and parE, previouslyassociated with resistance mutations, and all of marOR and acrRwere sequenced. The first selection step yielded mutations ingyrA, gyrB, and marOR. Subsequent selection steps yielded mutationsin gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associatedmutations were identified in almost all isolates after selectionsteps 1 and 2 but in less than 50% of isolates after subsequentselection steps. Selected strains were competed in vitro, inurine, and in a mouse UTI infection model against the startingstrain, Nu14. First-step mutations were not associated withsignificant fitness costs. However, the accumulation of threeor more resistance-associated mutations was usually associatedwith a large reduction in biological fitness, both in vitroand in vivo. Interestingly, in some lineages a partial restorationof fitness was associated with the accumulation of additionalmutations in late selection steps. We suggest that the relativebiological costs of multiple mutations may influence the evolutionof E. coli strains that develop resistance to fluoroquinolones.

Place, publisher, year, edition, pages
2005. Vol. 49, no 6, 2343-2351 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96328DOI: 10.1128/AAC.49.6.2343-2351.2005OAI: oai:DiVA.org:uu-96328DiVA: diva2:170868
Available from: 2007-11-01 Created: 2007-11-01 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Mutations and Mutation Rate in the Development of Fluoroquinolone Resistance
Open this publication in new window or tab >>Mutations and Mutation Rate in the Development of Fluoroquinolone Resistance
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The emergence of multidrug resistant bacteria world wide is a serious problem, and very few new drugs are under development. The selection of resistant bacteria is affected by factors such as mutation rate, biological fitness cost and the rate of fitness compensation. This thesis is focused on how mutation rate affects resistance to fluoroquinolones and on exploring a dosing strategy that might slow resistance development.

In a set of urinary tract Escherichia coli isolates MIC values above the breakpoint for the fluoroquinolones norfloxacin and ciprofloxacin carried at least three resistance-associated mutations. In these isolates the number of resistance mutations correlated with the mutation rate. During step-wise selection for decreased susceptibility to fluoroquinolones, the accumulation of mutations in E. coli was associated with an increasing biological cost both in vitro and in vivo. However, in some lineages an additional selection step for resistance was associated with a partial restoration of fitness. During step-wise selections we found, as expected, that reduced ciprofloxacin susceptibility frequently hitchhiked with a strong mutator phenotype. More surprisingly, we also found that reduced susceptibility was frequently associated with the emergence of rifampicin-resistant populations. We hypothesise that this correlation reflects selection for fitness-compensating mutations in RNA polymerase.

Mutant prevention concentration (MPC) dosing has been proposed as a strategy to reduce the selection of resistant bacterial populations. Based on limited data it had been thought that MPC might be a simple multiple of MIC, which can easily be determined. However, we showed for a collection of susceptible urinary tract E. coli that MPC could not be predicted from MIC and must be measured directly for relevant populations. Using an in vitro kinetic model we also showed that the pharmacodynamic index that best predicted prevention of resistance development in wild type E. coli was an AUC/MPC of > 22 for ciprofloxacin.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 102 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 357
Keyword
Microbiology, Mutation rate, Fluoroquinolone, Resistance, Biological fitness, Urinary tract infection, Escherichia coli, MPC, Mikrobiologi
Identifiers
urn:nbn:se:uu:diva-8275 (URN)978-91-554-6997-9 (ISBN)
Public defence
2007-12-01, B21, BMC, Husargatan 3, 75124 Uppsala, 10:15
Opponent
Supervisors
Available from: 2007-11-01 Created: 2007-11-01Bibliographically approved
2. Resistance to Fluoroquinolones in Escherichia coli: Prevention, Genetics and Fitness Costs
Open this publication in new window or tab >>Resistance to Fluoroquinolones in Escherichia coli: Prevention, Genetics and Fitness Costs
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibiotic-resistant bacteria are increasingly a major healthcare problem but very few new classes of antibiotics have been discovered or launched in recent decades. Approaches to dealing with the problem include learning how bacteria evolve to resistance and improving dosing regimens with current antibiotics so as to reduce the selection of resistant bacteria.

This thesis presents studies examining whether antibiotic dosing at high levels can prevent the selection of fluoroquinolone-resistant mutants in Escherichia coli. It also addresses the genetics of fluoroquinolone resistance in E. coli in relation to fitness costs for the resistant bacteria, and the evolution of E. coli to reduce the costs of resistance.

The mutant prevention concentration (MPC) of ciprofloxacin was measured for a set of clinical urinary tract infection E. coli strains showing that MPC could not be predicted from the minimum inhibitory concentration (MIC). Results from an in vitro kinetic model showed that an AUC/MPC >22 for ciprofloxacin was the single best pharmacodynamic index that predicted prevention of resistance emergence in the wild-type. Simulating currently approved dosing regimens for three different fluoroquinolones it was found that only a few were effective in preventing the selection of a small sub-population of pre-existing mutants.

Step-wise selection of fluoroquinolone resistance showed that the accumulation of mutations usually reduced bacterial fitness in vitro and in vivo. Systematic construction of isogenic resistant strains confirmed this result and revealed that some combinations of resistance mutations mutually compensate and increase both resistance and fitness. It was discovered that mutations altering RNA polymerase could ameliorate the fitness costs of fluoroquinolone resistance. Thus, the major fitness cost of fluoroquinolone resistance is due to defective transcription.

The finding that fluoroquinolone resistance mutations can increase resistance while mutually compensating their fitness costs, shows that resistance to fluoroquinolones can continue to evolve in the absence of antibiotic selection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 76 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 302
Keyword
Biology, Fluoroquinolone, Escherichia coli, Resistance, Gyrase, Topoisomerase IV, MPC, Fitness compensation, Urinary Tract Infection, Biologi
Identifiers
urn:nbn:se:uu:diva-7851 (URN)978-91-554-6884-2 (ISBN)
Public defence
2007-05-18, B21, BMC, Husargatan 3, 751 24 Uppsala, 10:15
Opponent
Supervisors
Available from: 2007-04-27 Created: 2007-04-27 Last updated: 2011-07-18Bibliographically approved

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