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Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli
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 Biochemistry and Microbiology.
2015 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 59, no 3, 1723-1732 p.Article in journal (Refereed) Published
Abstract [en]

Amdinocillin (mecillinam) is a beta-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates of Escherichia coli and to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 x 10(-8) to 2 x 10(-5) per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. The cysB gene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of the cysB gene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates of E. coli.

Place, publisher, year, edition, pages
2015. Vol. 59, no 3, 1723-1732 p.
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-252716DOI: 10.1128/AAC.04819-14ISI: 000352550000043PubMedID: 25583718OAI: oai:DiVA.org:uu-252716DiVA: diva2:811298
Available from: 2015-05-11 Created: 2015-05-11 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Mechanisms and Dynamics of Mecillinam Resistance in Escherichia coli
Open this publication in new window or tab >>Mechanisms and Dynamics of Mecillinam Resistance in Escherichia coli
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The introduction of antibiotics in healthcare is one of the most important medical achievements with regard to reducing human morbidity and mortality. However, bacterial pathogens have acquired antibiotic resistance at an increasing rate, and due to a high prevalence of resistance to some antibiotics they can no longer be used therapeutically. The antibiotic mecillinam, which inhibits the penicillin-binding protein PBP2, however, is an exception since mecillinam resistance (MecR) prevalence has remained low. This is particularly interesting since laboratory experiments have shown that bacteria can rapidly acquire MecR mutations by a multitude of different types of mutations.

In this thesis, I examined mechanisms and dynamics of mecillinam resistance in clinical and laboratory isolates of Escherichia coli. Only one type of MecR mutations (cysB) was found in the clinical strains, even though laboratory experiments demonstrate that more than 100 genes can confer resistance Fitness assays showed that cysB mutants have higher fitness than most other MecR mutants, which is likely to contribute to their dominance in clinical settings.

To determine if the mecillinam resistant strains could compensate for their fitness cost, six different MecR mutants (cysB, mrdA, spoT, ppa, aspS and ubiE) were evolved for 200-400 generations. All evolved mutants showed increased fitness, but the compensation was associated with loss of resistance in the majority of cases. This will also contribute to the rarity of clinical MecR isolates with chromosomal resistance mutations.

How MecR is mediated by cysB mutations was previously unclear, but in this thesis I propose and test a model for the mechanism of resistance. Thus, inactivation of CysB results in cellular depletion of cysteine that triggers an oxidative stress response. The response alters the intracellular levels of 450 proteins, and MecR is achieved by the increase of two of these, the LpoB and PBP1B proteins, which rescue the cells with a mecillinam-inhibited PBP2.

Mecillinam is used for UTI treatments and to investigate mecillinam resistance in a more host-like milieu, MecR strains were grown in urine and resistance was examined. Interestingly, this study showed that neither laboratory, nor clinical cysB mutants are resistant in urine, most likely because the cysteine present in the urine phenotypically reverts the bacteria to susceptibility. These findings suggest that mecillinam can be used to treat also those clinical strains that are identified as MecR in standard laboratory tests, and that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant results.

In summary, the work described in this thesis has increased ourgeneral knowledge of mecillinam resistance and its evolution. Hopefully this knowledge can be put to good use in clinical settings to reduce the negative impact of antibiotic resistance.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 69 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1375
Keyword
Mecillinam, Antibiotic resistance, Escherichia coli, Urinary tract infections, Fitness, Penicillin binding proteins, cysteine biosynthesis
National Category
Medical and Health Sciences Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-330856 (URN)978-91-513-0090-0 (ISBN)
Public defence
2017-11-24, A1:111a, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2017-11-01 Created: 2017-10-05 Last updated: 2017-11-01

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Thulin, ElisabethAndersson, Dan I.

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