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Reversion of High-level Mecillinam Resistance to Susceptibility in Escherichia coli During Growth in Urine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Social Sciences, Department of Statistics.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2017 (English)In: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 23, 111-118 p.Article in journal (Refereed) Published
Abstract [en]

Mecillinam (amdinocillin) is a β-lactam antibiotic used to treat uncomplicated urinary tract infections (UTIs). We have previously shown that inactivation of the Escherichia coli cysB gene is the major cause of mecillinam resistance (Mec(R)) in clinical isolates. In this study, we used different E. coli strains (laboratory and clinical isolates) that were Mec(R) due to cysB mutations to determine how mecillinam susceptibility was affected during growth in urine compared to growth in the commonly used growth medium Mueller Hinton (MHB). We also examined mecillinam susceptibility when bacteria were grown in urine obtained from 48 different healthy volunteers. Metabolome analysis was done on the urine samples and the association between the mecillinam susceptibility patterns of the bacteria and urine metabolite levels was studied. Two major findings with clinical significance are reported. First, Mec(R)E. coli cysB mutant strains (both laboratory and clinical isolates) were always more susceptible to mecillinam when grown in urine as compared to laboratory medium, with many strains showing complete phenotypic susceptibility in urine. Second, the degree of reversion to susceptibility varied between urine samples obtained from different individuals. This difference was correlated with osmolality such that in urine with low osmolality the Mec(R) mutants were more susceptible to mecillinam than in urine with high osmolality. This is the first example describing conditional resistance where a genetically stable antibiotic resistance can be phenotypically reverted to susceptibility by metabolites present in urine. These findings have several important clinical implications regarding the use of mecillinam to treat UTIs. First, they suggest that mecillinam can be used to treat also those clinical strains that are identified as Mec(R) in standard laboratory tests. Second, the results suggest that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant susceptibility testing results. Third, these findings imply that changes in patient behavior, such as increased water intake or use of diuretics to reduce urine osmolality and increased intake of cysteine, might induce antibiotic susceptibility in an infecting Mec(R)E. coli strain and thereby increase treatment efficiency.

Place, publisher, year, edition, pages
2017. Vol. 23, 111-118 p.
Keyword [en]
Conditional resistance, Escherichia coli, Mecillinam, Metabolomics, Urinary tract infection, Urine
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-330802DOI: 10.1016/j.ebiom.2017.08.021ISI: 000410740900018PubMedID: 28855073OAI: oai:DiVA.org:uu-330802DiVA: diva2:1146835
Funder
Swedish Research Council, 2012-03482
Available from: 2017-10-04 Created: 2017-10-04 Last updated: 2017-12-07Bibliographically 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, ElisabethEriksson, MånsAndersson, Dan I

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