| 1. |
- Andersson, Dan I., et al.
(author)
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Antibiotic resistance and its cost : is it possible to reverse resistance?
- 2010
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In: Nature Reviews Microbiology. - : Springer Science and Business Media LLC. - 1740-1526 .- 1740-1534. ; 8:4, s. 260-271
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Research review (peer-reviewed)abstract
- Most antibiotic resistance mechanisms are associated with a fitness cost that is typically observed as a reduced bacterial growth rate. The magnitude of this cost is the main biological parameter that influences the rate of development of resistance, the stability of the resistance and the rate at which the resistance might decrease if antibiotic use were reduced. These findings suggest that the fitness costs of resistance will allow susceptible bacteria to outcompete resistant bacteria if the selective pressure from antibiotics is reduced. Unfortunately, the available data suggest that the rate of reversibility will be slow at the community level. Here, we review the factors that influence the fitness costs of antibiotic resistance, the ways by which bacteria can reduce these costs and the possibility of exploiting them.
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| 2. |
- Andersson, Dan I., et al.
(author)
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Biological roles of translesion synthesis DNA polymerases in eubacteria
- 2010
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In: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:3, s. 540-548
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Research review (peer-reviewed)abstract
- Biological systems are strongly selected to maintain the integrity of their genomes by prevention and repair of external and internal DNA damages. However, some types of DNA lesions persist and might block the replication apparatus. The universal existence of specialized translesion synthesis DNA polymerases (TLS polymerases) that can bypass such lesions in DNA implies that replication blockage is a general biological problem. We suggest that the primary function for which translesion synthesis polymerases are selected is to rescue cells from replication arrest at lesions in DNA, a situation that, if not amended, is likely to cause an immediate and severe reduction in cell fitness and survival. We will argue that the mutagenesis observed during translesion synthesis is an unavoidable secondary consequence of this primary function and not, as has been suggested, an evolved mechanism to increase mutation rates in response to various stresses. Finally, we will discuss recent data on additional roles for translesion synthesis polymerases in the formation of spontaneous deletions and in transcription-coupled TLS, where the coupling of transcription to TLS is proposed to allow the rescue of the transcription machinery arrested at DNA lesions.
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| 3. |
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| 4. |
- Brandis, Gerrit, 1985-, et al.
(author)
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Fitness-compensatory mutations in rifampicin-resistant RNA polymerase
- 2012
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In: Molecular Microbiology. - : Blackwell Publishing. - 0950-382X .- 1365-2958. ; 85:1, s. 142-151
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Journal article (peer-reviewed)abstract
- Mutations in rpoB (RNA polymerase β-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (RifR) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized RifR mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a RifR mutant.
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| 5. |
- Brandis, Gerrit, 1985-, et al.
(author)
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Genetic characterization of compensatory evolution in strains carrying rpoB Ser531Leu, the rifampicin resistance mutation most frequently found in clinical isolates
- 2013
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In: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 68:11, s. 2493-2497
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Journal article (peer-reviewed)abstract
- ObjectivesThe evolution of rifampicin resistance in Mycobacterium tuberculosis is a major threat to effective tuberculosis therapy. Much is known about the initial emergence of rifampicin resistance, but the further evolution of these resistant strains has only lately been subject to investigation. Although resistance can be caused by many different mutations in rpoB, among clinical M. tuberculosis isolates the mutation rpoB S531L is overwhelmingly the most frequently found. Clinical isolates with rpoB S531L frequently carry additional mutations in genes for RNA polymerase subunits, and it has been speculated that these are fitness-compensatory mutations, ameliorating the fitness cost of the primary resistance mutation. We tested this hypothesis using Salmonella as a model organism.MethodsWe created the rpoB S531L mutation in Salmonella and then evolved independent lineages with selection for mutants with increased relative fitness. Relative fitness associated with putative compensatory mutations was measured after genetic reconstruction in isogenic strains.ResultsCompensatory mutations were identified in genes coding for different subunits of RNA polymerase: rpoA, rpoB and rpoC. Genetic reconstructions demonstrated that each of these secondary mutations reduced the fitness cost of the rpoB S531L resistance mutation.ConclusionsThe compensatory mutations identified in Salmonella cluster in similar locations to the additional mutations found in M. tuberculosis isolates. These new data strongly support the idea that many of the previously identified rpoA, rpoB and rpoC mutations in rifampicin-resistant M. tuberculosis (rpoB S531L) are indeed fitness-compensatory mutations.
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| 6. |
- Hughes, Diarmaid, 1956-, et al.
(author)
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Measurements of heme levels and respiration rate in Salmonella enterica serovar typhimurium
- 2010. - 2
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In: Antibiotic Resistance Protocols. - Totowa, NJ : Humana Press. - 9781603272780 - 9781603272797 ; , s. 105-112
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Book chapter (peer-reviewed)abstract
- This chapter describes assays for the measurement of heme levels and the rate of respiration in bacteria. An assay of ALA supplementation is described, in which the effect of exogenous ALA in reversing sensitivity to hydrogen peroxide is an indication of a reduced flow through the heme biosynthesis pathway. A protocol for measurement of the relative amount of heme by a fluorescence assay, based on stripping the iron from the heme moiety, leaving a protoporphyrin molecule which fluoresces following excitation at 400 nm, is also provided. Finally, a method for the measurement of respiration (oxygen consumption) rate is provided. In this method, the respiration of the cell population is expressed as the specific respiration rate during one doubling time of the population.
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| 7. |
- Hughes, Diarmaid, 1956-, et al.
(author)
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Rifampicin Resistance : Fitness Costs and the Significance of Compensatory Evolution
- 2013
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In: Antibiotics. - : MDPI. - 2079-6382. ; 2:2, s. 206-216
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Research review (peer-reviewed)abstract
- Seventy years after the introduction of antibiotic chemotherapy to treat tuberculosis, problems caused by drug-resistance in Mycobacterium tuberculosis have become greater than ever. The discovery and development of novel drugs and drug combination therapies will be critical to managing these problematic infections. However, to maintain effective therapy in the long-term and to avoid repeating the mistakes of the past, it is essential that we understand how resistance to antibiotics evolves in M. tuberculosis. Recent studies in genomics and genetics, employing both clinical isolates and model organisms, have revealed that resistance to the frontline anti-tuberculosis drug, rifampicin, is very strongly associated with the selection of fitness compensatory mutations in the different subunits of RNA polymerase. This mode of resistance evolution may also apply to other drugs, and knowledge of the rates and mechanisms could be used to design improved diagnostics and by tracking the evolution of infectious strains, to inform the optimization of therapies.
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| 8. |
- Koskiniemi, Sanna, 1980-, et al.
(author)
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Effect of the translesion DNA polymerases, endonucleases and RpoS on mutation rates in Salmonella typhimurium
- 2010
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In: Genetics. - : Oxford University Press (OUP). - 0016-6731 .- 1943-2631. ; 185:3, s. 783-795
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Journal article (peer-reviewed)abstract
- It has been suggested that bacteria have evolved mechanisms to increase their mutation rate in response to various stresses and that the translesion DNA polymerase Pol IV under control of the LexA regulon and the alternative sigma factor RpoS are involved in regulating this mutagenesis. Here we examined in Salmonella enterica serovar Typhimurium LT2 the rates for four different types of mutations (rifampicin-, nalidixic acid- and chlorate-resistance and Lac+ reversion) during various growth conditions and with different levels of four translesion DNA polymerases (Pol II, Pol IV, Pol V and SamAB) and RpoS. Constitutive de-repression of the LexA regulon by a lexA(def) mutation increased mutation rates 1.5- to 12-fold and the contribution of the translesion DNA polymerases to this mutagenesis varied with the type of mutation examined. In contrast, for all four types of mutations examined the increase in mutation rate in the lexA(def) mutant required the presence of the LexA-controlled endonucleases UvrB, UvrC and Cho. With regard to the potential involvement of RpoS in mutagenesis, neither an increase in RpoS levels conferred by artificial over-expression from a plasmid nor long-term stationary phase incubation or slow growth caused an increase in any of the four mutation rates measured, alone or in combination with over-expression of the translesion DNA polymerases. In conclusion, mutation rates are remarkably robust and no combination of growth conditions, induction of translesion polymerases by inactivation of LexA or increased RpoS expression could confer an increase in mutation rates higher than the moderate increase caused by de-repression of the LexA regulon alone.
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| 9. |
- Lannergård, Jonas, et al.
(author)
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Genetic Complexity of Fusidic Acid-Resistant Small Colony Variants (SCV) in Staphylococcus aureus
- 2011
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:11, s. e28366-
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Journal article (peer-reviewed)abstract
- FusE mutants are fusidic acid-resistant small colony variants (SCVs) of Staphylococcus aureus that can be selected with aminoglycosides. All FusE SCVs have mutations in rplF, encoding ribosomal protein L6. However, individual FusE mutants including some with the same mutation in rplF display auxotrophy for either hemin or menadione, suggesting that additional mutations are involved. Here we show that FusE SCVs can be divided into three genetic sub-groups and that some carry an additional mutation, in one of the genes required for hemin biosynthesis, or in one of the genes required for menadione biosynthesis. Reversion analysis and genome sequencing support the hypothesis that these combinations of mutations in the rplF, hem, and/or men genes can account for the SCV and auxotrophic phenotypes of FusE mutants.
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| 10. |
- Macvanin, Mirjana, et al.
(author)
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Assays of sensitivity of antibiotic-resistant bacteria to hydrogen peroxide and measurement of catalase activity
- 2010. - 2
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In: Antibiotic Resistance Protocols. - Totowa, NJ : Humana Press. - 9781603272780 - 9781603272797 ; , s. 95-103
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Book chapter (peer-reviewed)abstract
- Bacteria, in common with other organisms that take advantage of aerobic respiration, generate and accumulate reactive oxygen species (ROS) that damage DNA, fatty acids, and proteins. In addition, intracellular pathogens like Salmonella enterica are exposed to an oxidate burst produced by host macrophages. The relative ability of aerobically growing bacteria to withstand oxidative stress and eliminate ROS has a large impact of their fitness in vitro and in vivo. Methods are described here to measure the viability and relative fitness of bacteria in the presence of hydrogen peroxide. A protocol for the determination of catalase activity, an important part of the ROS detoxification process, is also described.
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| 11. |
- Sandberg, Anne, et al.
(author)
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Intra- and Extracellular Activities of Dicloxacillin and Linezolid against a Clinical Staphylococcus aureus Strain with a Small-Colony-Variant Phenotype in an In Vitro Model of THP-1 Macrophages and an In Vivo Mouse Peritonitis Model
- 2011
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In: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 55:4, s. 1443-1452
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Journal article (peer-reviewed)abstract
- The small colony variant (SCV) phenotype of Staphylococcus aureus has been associated with difficult-to-treat infections, reduced antimicrobial susceptibility and intracellular persistence. This study represents a detailed intra- and extracellular investigation of a clinical wild type (WT) S. aureus strain and its counterpart SCV phenotype both in vitro and in vivo, using the THP-1 cell line model and mouse peritonitis model respectively. Both phenotypes infected the mouse peritoneum intra- and extracellularly. The SCV phenotype was less virulent, showed distinct bacterial clearance, reduced multiplication capacity and reduced internalization ability. Some of the SCV infected mice were, however, still culture positive up to 96 h post infection and the phenotype could spread to the mouse kidney and furthermore revert to the more virulent WT phenotype in both the mouse- peritoneum and kidney. The SCV phenotype is therefore, despite reduced virulence, an important player in the S. aureus pathogenesis. In the THP-1 cell line model, both dicloxacillin (DCX) and linezolid (LZD) reduced the intracellular inoculum of both phenotypes by approximately 1-1.5 log10 in vitro, while DCX was considerably more effective against extracellular bacteria. In the mouse peritonitis model, DCX and LZD were also able to control both the intra- and extracellular infection caused by either phenotype. Treatment with a single dose of DCX and LZD was, however, insufficient to clear the SCVs in the kidneys and the risk of recurrent infection remained. This stresses the importance of optimal dosing of the antibiotic when SCVs are present.
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