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Mechanisms and Dynamics of Mecillinam Resistance in Escherichia coli
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
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 [en]
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: urn:nbn:se:uu:diva-330856ISBN: 978-91-513-0090-0 (print)OAI: oai:DiVA.org:uu-330856DiVA: diva2:1147254
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
List of papers
1. Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli
Open this publication in new window or tab >>Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli
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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-252716 (URN)10.1128/AAC.04819-14 (DOI)000352550000043 ()25583718 (PubMedID)
Available from: 2015-05-11 Created: 2015-05-11 Last updated: 2017-10-05Bibliographically approved
2. Compensatory evolution in mecillinam resistant Escherichia coli
Open this publication in new window or tab >>Compensatory evolution in mecillinam resistant Escherichia coli
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Antibiotic resistance mutations typically have pleiotropic effects that reduce bacterial fitness, but secondary compensatory mutations may ameliorate these fitness costs. Mecillinam is a b-lactam antibiotic that is used for treatment of Urinary Tract Infections (UTIs) and in a previous screen of mecillinam resistant (MecR) clinical isolates of Escherichia coli mutations in the cysB gene were shown to be the main cause of MecR.  This is unexpected since over 100 different genes are known to confer MecR in laboratory settings. Mutations in cysB are less costly than most other types of MecR, which is likely to be a contributing factor to only cysB mutations being found in clinical MecR isolates.

To examine if the fitness costs associated with MecR can be compensated by second-site mutations, we performed a serial passage evolution experiment where six different MecR mutants (mrdA, ppa, ubiE, aspS, spoT and cysB) were grown without mecillinam for 200-400 generations. All evolved lineages showed increased fitness as compared to the parental MecR strain. Increase of fitness was associated with loss of resistance in all compensated mutants except in the mrdA mutant lineages. Our results show that compensation of the fitness cost of MecR mutations involved either a small mutational target or acquisition of several mutations. These factors are likely to contribute to the low frequency of MecR clinical isolates.

National Category
Medical and Health Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-330816 (URN)
Funder
Swedish Research Council
Available from: 2017-10-04 Created: 2017-10-04 Last updated: 2017-10-05
3. An oxidative stress-induced bypass mechanism confers antibiotic resistance in Escherichia coli
Open this publication in new window or tab >>An oxidative stress-induced bypass mechanism confers antibiotic resistance in Escherichia coli
(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
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:nbn:se:uu:diva-330815 (URN)
Funder
Swedish Research Council
Available from: 2017-10-04 Created: 2017-10-04 Last updated: 2017-10-05
4. Reversion of High-level Mecillinam Resistance to Susceptibility in Escherichia coli During Growth in Urine.
Open this publication in new window or tab >>Reversion of High-level Mecillinam Resistance to Susceptibility in Escherichia coli During Growth in Urine.
2017 (English)In: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 23, 111-118 p., S2352-3964(17)30338-9Article 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.

Keyword
Conditional resistance, Escherichia coli, Mecillinam, Metabolomics, Urinary tract infection, Urine
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-330802 (URN)10.1016/j.ebiom.2017.08.021 (DOI)28855073 (PubMedID)
Available from: 2017-10-04 Created: 2017-10-04 Last updated: 2017-10-05

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