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An oxidative stress-induced bypass mechanism confers antibiotic resistance in 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.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Mecillinam is a beta-lactam antibiotic that specifically inhibits the essential penicillin binding protein PBP2. Mutations in >100 genes can confer resistance to mecillinam but in clinical isolates of Escherichia coli from urinary tract infections, mutations in the cysB gene are the major cause of resistance. However, how cysB mutations confer resistance is unknown. We used a combination of proteomics and genetic analysis to examine the mechanism of resistance. Results show that cysB mutations cause an oxidative stress response and change expression of more than 450 genes, among them the PBP1B, LpoB and FtsZ proteins, which show increased levels. Addition of reducing agents to a cysB mutant converted it to full susceptibility, with an associated down-regulation of PBP1B, LpoB and FtsZ. Artificial over-expression of either PBP1B or LpoB in a wild type E. coli conferred mecillinam resistance, and conversely, inactivation of either the mrcB (encodes PBP1B) or lpoB gene, made cysB mutants susceptible. These results together show that expression of the proteins PBP1B and LpoB is both necessary and sufficient to confer mecillinam resistance.

We propose a model whereby cysB mutants confer mecillinam resistance by inducing an oxidative stress response that causes an up-regulation of the PBP1B and LpoB proteins. These two proteins can then together substitute for the function of the mecillinam-sensitive PBP2 protein. Our results provide new insights into how antibiotic resistance can be conferred by a bypass mechanism that is induced by changed redox state and gene expression.

Keyword [en]
Antibiotic resistance, mecillinam, Escherichia coli, penicillin binding protein, cell wall, cysB, lpoB, ftsZ, cysteine, redox state
National Category
Medical and Health Sciences
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-330815OAI: oai:DiVA.org:uu-330815DiVA: diva2:1146921
Funder
Swedish Research Council
Available from: 2017-10-04 Created: 2017-10-04 Last updated: 2017-10-05
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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