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Römhild, R., Linkevicius, M. & Andersson, D. I. (2020). Molecular mechanisms of collateral sensitivity to the antibiotic nitrofurantoin. PLoS biology, 18(1), Article ID e3000612.
Open this publication in new window or tab >>Molecular mechanisms of collateral sensitivity to the antibiotic nitrofurantoin
2020 (English)In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 18, no 1, article id e3000612Article in journal (Refereed) Published
Abstract [en]

Antibiotic resistance increasingly limits the success of antibiotic treatments, and physicians require new ways to achieve efficient treatment despite resistance. Resistance mechanisms against a specific antibiotic class frequently confer increased susceptibility to other antibiotic classes, a phenomenon designated collateral sensitivity (CS). An informed switch of antibiotic may thus enable the efficient treatment of resistant strains. CS occurs in many pathogens, but the mechanisms that generate hypersusceptibility are largely unknown. We identified several molecular mechanisms of CS against the antibiotic nitrofurantoin (NIT). Mutants that are resistant against tigecycline (tetracycline), mecillinam (beta-lactam), and protamine (antimicrobial peptide) all show CS against NIT. Their hypersusceptibility is explained by the overexpression of nitroreductase enzymes combined with increased drug uptake rates, or increased drug toxicity. Increased toxicity occurs through interference of the native drug-response system for NIT, the SOS response, with growth. A mechanistic understanding of CS will help to develop drug switches that combat resistance.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2020
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-406492 (URN)10.1371/journal.pbio.3000612 (DOI)000510743800002 ()31986134 (PubMedID)
Funder
Swedish Research Council, 2017-01527
Available from: 2020-03-11 Created: 2020-03-11 Last updated: 2020-03-11Bibliographically approved
Puértolas-Balint, F., Warsi, O., Linkevicius, M., Tang, P.-C. & Andersson, D. I. (2020). Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli. Journal of Antimicrobial Chemotherapy, 75(2), 300-308
Open this publication in new window or tab >>Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli
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2020 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 75, no 2, p. 300-308Article in journal (Refereed) Published
Abstract [en]

Objectives

To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli.

Methods

Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined.

Results

Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)=16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX=32–64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline.

Conclusions

Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.

National Category
Microbiology Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-396064 (URN)10.1093/jac/dkz434 (DOI)000515116900006 ()31633764 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-10-29 Created: 2019-10-29 Last updated: 2020-04-01Bibliographically approved
Olivenza, D. R., Nicoloff, H., Antonia Sanchez-Romero, M., Cota, I., Andersson, D. I. & Casadesus, J. (2019). A portable epigenetic switch for bistable gene expression in bacteria. Scientific Reports, 9, Article ID 11261.
Open this publication in new window or tab >>A portable epigenetic switch for bistable gene expression in bacteria
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 11261Article in journal (Refereed) Published
Abstract [en]

We describe a portable epigenetic switch based on opvAB, a Salmonella enterica operon that undergoes bistable expression under DNA methylation control. A DNA fragment containing the opvAB promoter and the opvAB upstream regulatory region confers bistability to heterologous genes, yielding OFF and ON subpopulations. Bistable expression under opvAB control is reproducible in Escherichia coli, showing that the opvAB switch can be functional in a heterologous host. Subpopulations of different sizes can be produced at will using engineered opvAB variants. Controlled formation of antibiotic-resistant and antibiotic-susceptible subpopulations may allow use of the opvAB switch in the study of bacterial heteroresistance to antibiotics.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-392133 (URN)10.1038/s41598-019-47650-2 (DOI)000478575000048 ()31375711 (PubMedID)
Funder
Swedish Research Council, 2017-01527
Available from: 2019-09-02 Created: 2019-09-02 Last updated: 2019-09-02Bibliographically approved
Rosenkilde, C. E. H., Munck, C., Porse, A., Linkevicius, M., Andersson, D. I. & Sommer, M. O. A. (2019). Collateral sensitivity constrains resistance evolution of the CTX-M-15 beta-lactamase. Nature Communications, 10, Article ID 618.
Open this publication in new window or tab >>Collateral sensitivity constrains resistance evolution of the CTX-M-15 beta-lactamase
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2019 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 618Article in journal (Refereed) Published
Abstract [en]

Antibiotic resistance is a major challenge to global public health. Discovery of new antibiotics is slow and to ensure proper treatment of bacterial infections new strategies are needed. One way to curb the development of antibiotic resistance is to design drug combinations where the development of resistance against one drug leads to collateral sensitivity to the other drug. Here we study collateral sensitivity patterns of the globally distributed extended-spectrum beta-lactamase CTX-M-15, and find three non-synonymous mutations with increased resistance against mecillinam or piperacillin-tazobactam that simultaneously confer full susceptibility to several cephalosporin drugs. We show in vitro and in mice that a combination of mecillinam and cefotaxime eliminates both wild-type and resistant CTX-M-15. Our results indicate that mecillinam and cefotaxime in combination constrain resistance evolution of CTX-M-15, and illustrate how drug combinations can be rationally designed to limit the resistance evolution of horizontally transferred genes by exploiting collateral sensitivity patterns.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-378536 (URN)10.1038/s41467-019-08529-y (DOI)000457862200003 ()30728359 (PubMedID)
Funder
EU, Horizon 2020, 638902Swedish Research Council, 2017-01527Novo Nordisk, NNF10CC1016517
Available from: 2019-03-22 Created: 2019-03-22 Last updated: 2019-03-22Bibliographically approved
Knopp, M., Gudmundsdottir, J. S., Nilsson, T., Konig, F., Warsi, O., Rajer, F., . . . Andersson, D. I. (2019). De Novo Emergence of Peptides That Confer Antibiotic Resistance. mBio, 10(3), Article ID e00837-19.
Open this publication in new window or tab >>De Novo Emergence of Peptides That Confer Antibiotic Resistance
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2019 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 3, article id e00837-19Article in journal (Refereed) Published
Abstract [en]

The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from non-coding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes. IMPORTANCE De novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2019
Keywords
Escherichia coli, antibiotic resistance, aminoglycosides, de novo, gene evolution, membrane potential, peptides
National Category
Microbiology Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-390534 (URN)10.1128/mBio.00837-19 (DOI)000473596500078 ()31164464 (PubMedID)
Funder
Swedish Research Council, 2017-01527Knut and Alice Wallenberg Foundation, KAW 2013-0006Knut and Alice Wallenberg Foundation, KAW 2015.0069
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
Balaban, N. Q., Helaine, S., Lewis, K., Ackermann, M., Aldridge, B., Andersson, D. I., . . . Zinkernagel, A. (2019). Definitions and guidelines for research on antibiotic persistence. Nature Reviews Microbiology, 17(7), 441-448
Open this publication in new window or tab >>Definitions and guidelines for research on antibiotic persistence
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2019 (English)In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 17, no 7, p. 441-448Article, review/survey (Refereed) Published
Abstract [en]

Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-390000 (URN)10.1038/s41579-019-0196-3 (DOI)000471747200008 ()30980069 (PubMedID)
Funder
EU, European Research Council, 681619NIH (National Institute of Health), R01GM 091875
Available from: 2019-08-05 Created: 2019-08-05 Last updated: 2019-08-05Bibliographically approved
Ge, X., Oliveira, A., Hjort, K., Bergfors, T., Gutiérrez-de-Terán, H., Andersson, D. I., . . . Åqvist, J. (2019). Inhibition of translation termination by small molecules targeting ribosomal release factors. Scientific Reports, 9, Article ID 15424.
Open this publication in new window or tab >>Inhibition of translation termination by small molecules targeting ribosomal release factors
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 15424Article in journal (Refereed) Published
Abstract [en]

The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Medicinal Chemistry Structural Biology
Identifiers
urn:nbn:se:uu:diva-396310 (URN)10.1038/s41598-019-51977-1 (DOI)000492832300009 ()31659219 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish National Infrastructure for Computing (SNIC)
Available from: 2019-11-01 Created: 2019-11-01 Last updated: 2019-11-18Bibliographically approved
Andersson, D. I., Nicoloff, H. & Hjort, K. (2019). Mechanisms and clinical relevance of bacterial heteroresistance. Nature Reviews Microbiology, 17(8), 479-496
Open this publication in new window or tab >>Mechanisms and clinical relevance of bacterial heteroresistance
2019 (English)In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 17, no 8, p. 479-496Article, review/survey (Refereed) Published
Abstract [en]

Antibiotic heteroresistance is a phenotype in which a bacterial isolate contains subpopulations of cells that show a substantial reduction in antibiotic susceptibility compared with the main population. Recent work indicates that heteroresistance is very common for several different bacterial species and antibiotic classes. The resistance phenotype is often unstable, and in the absence of antibiotic pressure it rapidly reverts to susceptibility. A common mechanistic explanation for the instability is the occurrence of genetically unstable tandem amplifications of genes that cause resistance. Due to their instability, low frequency and transient character, it is challenging to detect and study these subpopulations, which often leads to difficulties in unambiguously classifying bacteria as susceptible or resistant. Finally, in vitro experiments, mathematical modelling, animal infection models and clinical studies show that the resistant subpopulations can be enriched during antibiotic exposure, and increasing evidence suggests that heteroresistance can lead to treatment failure.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390413 (URN)10.1038/s41579-019-0218-1 (DOI)000475479100007 ()31235888 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-08-12 Created: 2019-08-12 Last updated: 2019-08-12Bibliographically approved
Warsi, O. M., Lundin, E., Lustig, U., Näsvall, J. & Andersson, D. I. (2019). Selection for novel metabolic capabilities in Salmonella enterica. Evolution, 73(5), 990-1000
Open this publication in new window or tab >>Selection for novel metabolic capabilities in Salmonella enterica
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2019 (English)In: Evolution, ISSN 0014-3820, E-ISSN 1558-5646, Vol. 73, no 5, p. 990-1000Article in journal (Refereed) Published
Abstract [en]

Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.

National Category
Evolutionary Biology Microbiology
Research subject
Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-379227 (URN)10.1111/evo.13713 (DOI)000467303100009 ()30848832 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-03-14 Created: 2019-03-14 Last updated: 2019-06-10Bibliographically approved
Nicoloff, H., Hjort, K., Levin, B. R. & Andersson, D. I. (2019). The high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification. Nature Microbiology, 4(3), 504-514
Open this publication in new window or tab >>The high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification
2019 (English)In: Nature Microbiology, E-ISSN 2058-5276, Vol. 4, no 3, p. 504-514Article in journal (Refereed) Published
Abstract [en]

When choosing antibiotics to treat bacterial infections, it is assumed that the susceptibility of the target bacteria to an antibiotic is reflected by laboratory estimates of the minimum inhibitory concentration (MIC) needed to prevent bacterial growth. A caveat of using MIC data for this purpose is heteroresistance, the presence of a resistant subpopulation in a main population of susceptible cells. We investigated the prevalence and mechanisms of heteroresistance in 41 clinical isolates of the pathogens Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Acinetobacter baumannii against 28 different antibiotics. For the 766 bacteria-antibiotic combinations tested, as much as 27.4% of the total was heteroresistant. Genetic analysis demonstrated that a majority of heteroresistance cases were unstable, with an increased resistance of the subpopulations resulting from spontaneous tandem amplifications, typically including known resistance genes. Using mathematical modelling, we show how heteroresistance in the parameter range estimated in this study can result in the failure of antibiotic treatment of infections with bacteria that are classified as antibiotic susceptible. The high prevalence of heteroresistance with the potential for treatment failure highlights the limitations of MIC as the sole criterion for susceptibility determinations. These results call for the development of facile and rapid protocols to identify heteroresistance in pathogens.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-379034 (URN)10.1038/s41564-018-0342-0 (DOI)000459201400019 ()30742072 (PubMedID)
Funder
Swedish Research Council, 2017-01527
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2019-03-11 Created: 2019-03-11 Last updated: 2019-03-11Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0001-6640-2174

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