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Evolution and Mechanisms of Tigecycline Resistance in Escherichia coli
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibiotic resistance is an ongoing global medical crisis and we are in great need of new antibacterial agents to combat rapidly emerging resistant pathogens. Tigecycline is one of few drugs that have been introduced into medicine during the last two decades. It is a broad-spectrum third generation tetracycline that is active against multidrug-resistant bacteria that cause complicated infections.

In this thesis I examined the development of tigecycline resistance in Escherichia coli and associated in vitro and in vivo fitness effects. Selections of spontaneous E. coli mutants revealed relatively high accumulation rates of changes in the multidrug efflux system AcrAB-TolC regulation network and in heptose biosynthesis and transport pathways important for lipopolysaccharide (LPS) synthesis. Both groups of mutations led to reduced susceptibility to tigecycline and slower growth compared to the wild-type bacteria. Additional in vitro fitness assays and in vivo competitions showed that LPS mutants were less fit than efflux mutants, providing a possible explanation for why up-regulation of multidrug efflux pumps is the main tigecycline resistance mechanism reported in clinical isolates.

Tigecycline was designed to evade the two most common tetracycline resistance mechanisms conferred by Tet proteins, efflux and ribosomal protection. However, tigecycline is a substrate for the tetracycline modifying enzyme Tet(X). Screening of Tet protein mutant libraries showed that it is possible to select Tet mutants with minimal inhibitory concentrations of tigecycline that reach clinically relevant levels. Mutations in Tet proteins that permitted a better protection from tigecycline frequently exhibited reduced activity against earlier generations of tetracyclines, except for the Tet(X) enzyme mutants, which were better at inactivating all tested tetracyclines. This is particularly worrisome because different variants of Tet(X) have recently spread to multidrug-resistant pathogens through horizontal gene transfer. Therefore, Tet(X) mutants with improved activity threaten the medical future of tetracyclines.

Multidrug resistance is easily disseminated through horizontally spreading conjugative plasmids. pUUH239.2 is an example of a successful conjugative plasmid that caused the first clonal outbreak of extended spectrum β-lactamase-producing Klebsiella pneumoniae in Scandinavia. This plasmid was formed after rearrangements between two different plasmid backbones and it carries resistance genes to multiple antibiotic classes, heavy metals, and detergents.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1121
Keyword [en]
Tigecycline, Bacterial resistance, Fitness, Escherichia coli, AcrAB, LPS, Tet proteins, Tet(A), Tet(K), Tet(M), Tet(X), tet genes, pUUH239.2
National Category
Medical and Health Sciences Natural Sciences
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-259226ISBN: 978-91-554-9285-4 (print)OAI: oai:DiVA.org:uu-259226DiVA: diva2:843670
Public defence
2015-09-25, B42, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2015-08-26 Created: 2015-07-30 Last updated: 2015-10-01
List of papers
1. Mechanisms and fitness costs of tigecycline resistance in Escherichia coli
Open this publication in new window or tab >>Mechanisms and fitness costs of tigecycline resistance in Escherichia coli
2013 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 12, 2809-2819 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: To stepwise select tigecycline-resistant Escherichia coli mutants in vitro, determine the mutation rates, identify the resistance mechanisms, determine the resistance level and cross-resistance to other antibiotic classes, evaluate the fitness costs of tigecycline resistance mechanisms and investigate if the same in vitro-identified target genes were mutated in clinical isolates.

Methods: Spontaneous mutants with reduced susceptibility to tigecycline were selected on agar plates supplemented with tigecycline. Resistance levels and cross-resistance were evaluated by performing MIC assays and determining mutation rates using Luria-Delbruck fluctuation tests. Mutant fitness was estimated by measuring exponential growth rates, lag phase and total yield. Illumina whole-genome sequencing was used to identify mutations increasing MICs of tigecycline.

Results: Spontaneous mutants with reduced susceptibility to tigecycline were selected at a rate of similar to 10-8 to 10-6 per cell per generation; however, the clinical MIC breakpoint was not reached. The resistance level of tigecycline was low and some of the mutants had elevated MICs of hydrophobic drugs (chloramphenicol, erythromycin and novobiocin) or decreased MICs of SOS response inducers (ciprofloxacin and nitrofurantoin). Mutations were identified in efflux regulatory network genes (lon, acrR and marR) or lipopolysaccharide core biosynthesis pathway genes (lpcA, rfaE, rfaD, rfaC and rfaF). Mutations in the same target genes were found in clinical isolates.

Conclusions: Tigecycline selects for low-level resistance mutations with relatively high mutation rates and the majority of them come with a substantial fitness cost. Further in vivo experiments are needed to evaluate how these mutations affect bacterial virulence and ability to establish a successful infection.

Keyword
E. coli, bacterial resistance, LPS core biosynthesis, AcrAB efflux system
National Category
Medical and Health Sciences Natural Sciences
Identifiers
urn:nbn:se:uu:diva-212839 (URN)10.1093/jac/dkt263 (DOI)000326980000015 ()
Available from: 2013-12-18 Created: 2013-12-16 Last updated: 2017-12-06Bibliographically approved
2. Fitness of Escherichia coli mutants with reduced susceptibility to tigecycline
Open this publication in new window or tab >>Fitness of Escherichia coli mutants with reduced susceptibility to tigecycline
2016 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, 1307-1313 p.Article in journal (Refereed) Published
Abstract [en]

The objective of this study was to determine the fitness of Escherichia coli mutants with reduced susceptibility to tigecycline after exposure to adverse conditions in vitro and in vivo. Survival in response to low pH, bile salts, oxidative stress and human serum was examined for E. coli mutants with reduced susceptibility to tigecycline due to single mutations that caused increased efflux (marR, lon) or impaired LPS (rfaC, rfaE, lpcA). An in vitro competition assay was used to determine growth fitness defects. Competitive fitness was assessed using mouse infection models. MICs, exponential growth rates and expression levels of efflux-related genes were measured for genetically reconstructed double and triple mutants. The LPS mutants were 48-85-fold more susceptible to bile salts compared with the ERN mutants and the WT. As shown by in vitro competitions, the fitness reduction was 0.3%-13% for ERN mutants and similar to 24% for LPS mutants. During in vivo survival experiments, LPS mutants were outcompeted by the WT strain in the thigh infection model. Constructed double ERN and LPS mutants showed additive and synergistic increases in tigecycline MICs. Generally, reduced susceptibility to tigecycline caused a decrease in fitness under stressful in vitro and in vivo conditions with ERN mutants being fitter than LPS mutants. When combined, ERN mutations caused a synergistic increase in the MIC of tigecycline. These findings could explain why clinical resistance to tigecycline in E. coli is mainly associated with up-regulation of the AcrAB efflux system.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-257804 (URN)10.1093/jac/dkv486 (DOI)000376291300024 ()26851608 (PubMedID)
Funder
Swedish Research Council Formas, 2013-5476-25194-9EU, European Research Council, 282004 EvoTAR
Available from: 2015-07-30 Created: 2015-07-08 Last updated: 2017-12-04Bibliographically approved
3. Potential of tetracycline resistance proteins to evolve tigecycline resistance
Open this publication in new window or tab >>Potential of tetracycline resistance proteins to evolve tigecycline resistance
2016 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 60, no 2, 789-796 p.Article in journal (Refereed) Published
Abstract [en]

Tigecycline is a glycylcycline antibiotic active against multidrug-resistant bacterial pathogens. The objectives of our study were to examine the potential of the Tet(A), Tet(K), Tet(M), and Tet(X) tetracycline resistance proteins to acquire mutations causing tigecycline resistance and to determine how this affects resistance to earlier classes of tetracyclines. Mutations in all four tet genes caused a significant increase in the tigecycline MIC in Escherichia coli, and strains expressing mutant Tet(A) and Tet(X) variants reached clinically relevant MICs (2 mg/liter and 3 mg/liter, respectively). Mutations predominantly accumulated in transmembrane domains of the efflux pumps, most likely increasing the accommodation of tigecycline as a substrate. All selected Tet(M) mutants contained at least one mutation in the functionally most important loop III of domain IV. Deletion of leucine 505 of this loop led to the highest increase of the tigecycline MIC (0.5 mg/liter) among Tet(M) mutants. It also caused collateral sensitivity to earlier classes of tetracyclines. A majority of the Tet(X) mutants showed increased activity against all three classes of tetracylines. All tested Tet proteins have the potential to acquire mutations leading to increased MICs of tigecycline. As tet genes are widely found in pathogenic bacteria and spread easily by horizontal gene transfer, resistance development by alteration of existing Tet proteins might compromise the future medical use of tigecycline. We predict that Tet(X) might become the most problematic future Tet determinant, since its weak intrinsic tigecycline activity can be mutationally improved to reach clinically relevant levels without collateral loss in activity to other tetracyclines.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-257805 (URN)10.1128/AAC.02465-15 (DOI)000369159800009 ()
Funder
EU, European Research Council, 282004Swedish Research Council, K2013-56X-13025-15- 5Swedish Research Council, 2013-5476-25194-9Swedish Research Council, K2013-99X-22208-01-5
Available from: 2015-07-30 Created: 2015-07-08 Last updated: 2017-12-04Bibliographically approved
4. Transfer of an Escherichia coli ST131 multiresistance cassette has created a Klebsiella pneumoniae-specific plasmid associated with a major nosocomial outbreak
Open this publication in new window or tab >>Transfer of an Escherichia coli ST131 multiresistance cassette has created a Klebsiella pneumoniae-specific plasmid associated with a major nosocomial outbreak
Show others...
2012 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 67, no 1, 74-83 p.Article in journal (Refereed) Published
Abstract [en]

Objectives:

To characterize the complete sequence, horizontal spread and stability of the CTX-M-15-encoding multiresistance plasmid of a Klebsiella pneumoniae strain involved in a large nosocomial outbreak.

Methods:

The 220 kbp plasmid pUUH239.2 was completely sequenced using 454 technology. The conjugational host range, conjugation frequencies, plasmid stability and fitness cost of plasmid carriage were studied in vitro. Conjugational spread during the outbreak was assessed retrospectively by multiplex PCR screening, restriction fragment length polymorphism and PFGE.

Results:

Plasmid pUUH239.2 encodes resistance to β-lactams (blaCTX-M-15, blaTEM-1 and blaOXA-1), aminoglycosides [aac-(6′)-1b-cr and aadA2], tetracyclines [tet(A) and tetR], trimethoprim (dhfrXII), sulphonamides (sul1) quaternary ammonium compounds (qacEΔ1), macrolides [mph(A)-mxr-mphR(A)] and heavy metal ions (silver, copper and arsenic). The plasmid consists of a backbone, highly similar to the K. pneumoniae plasmid pKPN3, and a 41 kbp resistance region, highly similar to the resistance regions of plasmids pEK499 and pC15-1a previously isolated from Escherichia coli strains belonging to the outbreak lineage ST131 (where ST stands for sequence type). The pUUH239.2 plasmid is stable in K. pneumoniae but unstable in E. coli and confers a fitness cost when introduced into a naive host cell. Transfer of pUUH239.2 from the outbreak K. pneumoniae clone to the E. coli of the patients’ intestinal floras has occurred on multiple occasions during the outbreak.

Conclusions:

The plasmid pUUH239.2 is a composite of the pKPN3 K. pneumoniae plasmid backbone and the blaCTX-M-15-encoding multiresistance cassette associated with the internationally recognized outbreak strain E. coli ST131. The resulting plasmid differs in stability between K. pneumoniae and E. coli, and this has probably limited the spread of this plasmid during the outbreak.

Keyword
O25:H4-ST131, ESBL, outbreak, horizontal transfer, fitness-cost, evolution, CTX-M-15
National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-169437 (URN)10.1093/jac/dkr405 (DOI)000300833700012 ()
Available from: 2012-02-29 Created: 2012-02-29 Last updated: 2017-12-07

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