| 1. |
- Hughes, Diarmaid, 1956-, et al.
(författare)
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Measurements of heme levels and respiration rate in Salmonella enterica serovar typhimurium
- 2010. - 2
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Ingår i: Antibiotic Resistance Protocols. - Totowa, NJ : Humana Press. - 9781603272780 - 9781603272797 ; , s. 105-112
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Bokkapitel (refereegranskat)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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| 2. |
- Macvanin, Mirjana, et al.
(författare)
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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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Ingår i: Antibiotic Resistance Protocols. - Totowa, NJ : Humana Press. - 9781603272780 - 9781603272797 ; , s. 95-103
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Bokkapitel (refereegranskat)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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| 3. |
- Macvanin, Mirjana, et al.
(författare)
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Fusidic acid-resistant EF-G perturbs the accumulation of ppGpp
- 2000
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 37:1, s. 98-107
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Tidskriftsartikel (refereegranskat)abstract
- Reductions in growth rate caused by fusidic acid-resistant EF-G mutants in Salmonella typhimurium correlate strongly with increased mean cell size. This is unusual because growth rate and cell size normally correlate positively. The global transcription regulator molecule ppGpp has a role in co-ordinating growth rate and division, and its basal level normally correlates inversely with cell size at division. We show that fusidic acid-resistant EF-G mutants have perturbed ppGpp basal levels during steady-state growth and perturbed induced levels during starvation. One mutation, fusA1, associated with the slowest growth rate and largest cell size, causes a reduction in the basal level of ppGpp to one-third of that found in the wild-type strain. Other fusA mutants with intermediate or wild-type growth rates and cell sizes have either normal or increased basal levels of ppGpp. There is an inverse relationship between the basal level of ppGpp in vivo and the degree to which translation dependent on mutant EF-G is inhibited by ppGpp in vitro. This enhanced interaction between mutant EF-G and ppGpp correlates with an increased KM for GTP. Our results suggest that mutant EF-G modulates the production of ppGpp by the RelA (PSI) pathway. In conclusion, fusidic acid-resistant EF-G mutations alter the level of ppGpp and break the normal relationship between growth rate and cell size at division. It would not be surprising if other phenotypes associated with these mutants, such as loss of virulence, were also related to perturbations in ppGpp levels effected through altered transcription patterns.
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| 4. |
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| 5. |
- Macvanin, Mirjana, et al.
(författare)
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Fusidic acid-resistant mutants of Salmonella enterica serovar typhimurium have low levels of heme and a reduced rate of respiration and are sensitive to oxidative stress
- 2004
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 48:10, s. 3877-3883
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in the translation elongation factor G (EF-G) make Salmonella enterica serovar Typhimurium resistant to the antibiotic fusidic acid. Fusr mutants are hypersensitive to oxidative stress and rapidly lose viability in the presence of hydrogen peroxide. We show that this phenotype is associated with reduced activity of two catalase enzymes, HPI (a bifunctional catalase-hydroperoxidase) and HPII (a monofunctional catalase). These catalases require the iron-binding cofactor heme for their activity. Fusr mutants have a reduced rate of transcription of hemA, a gene whose product catalyzes the first committed step in heme biosynthesis. Hypersensitivity of Fusr mutants to hydrogen peroxide is abolished by the presence of -aminolevulinic acid, the precursor of heme synthesis, in the growth media and by the addition of glutamate or glutamine, amino acids required for the first step in heme biosynthesis. Fluorescence measurements show that the level of heme in a Fusr mutant is significantly lower than it is in the wild type. Heme is also an essential cofactor of cytochromes in the electron transport chain of respiration. We found that the rate of respiration is reduced significantly in Fusr mutants. Sequestration of divalent iron in the growth media decreases the sensitivity of Fusr mutants to oxidative stress. Taken together, these results suggest that Fusr mutants are hypersensitive to oxidative stress because their low levels of heme reduce both catalase activity and respiration capacity. The sensitivity of Fusr mutants to oxidative stress could be associated with loss of viability due to iron-mediated DNA damage in the presence of hydrogen peroxide. We argue that understanding the specific nature of antibiotic resistance fitness costs in different environments may be a generally useful approach in identifying physiological processes that could serve as novel targets for antimicrobial agents.
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| 6. |
- Macvanin, Mirjana, et al.
(författare)
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Fusidic acid-resistant mutants of Salmonella enterica serovar Typhimurium with low fitness in vivo are defective in RpoS induction
- 2003
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 47:12, s. 3743-3749
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Tidskriftsartikel (refereegranskat)abstract
- Mutants of Salmonella enterica serovar Typhimurium resistant to fusidic acid (Fusr) have mutations in fusA, the gene encoding translation elongation factor G (EF-G). Most Fusr mutants have reduced fitness in vitro and in vivo, in part explained by mutant EF-G slowing the rate of protein synthesis and growth. However, some Fusr mutants with normal rates of protein synthesis still suffer from reduced fitness in vivo. As shown here, Fusr mutants could be similarly ranked in their relative fitness in mouse infection models, in a macrophage infection model, in their relative hypersensitivity to hydrogen peroxide in vivo and in vitro, and in the amount of RpoS production induced upon entry into the stationary phase. We identify a reduced ability to induce production of RpoS (sigmas) as a defect associated with Fusr strains. Because RpoS is a regulator of the general stress response, and an important virulence factor in Salmonella, an inability to produce RpoS in appropriate amounts can explain the low fitness of Fusr strains in vivo. The unfit Fusr mutants also produce reduced levels of the regulatory molecule ppGpp in response to starvation. Because ppGpp is a positive regulator of RpoS production, we suggest that a possible cause of the reduced levels of RpoS is the reduction in ppGpp production associated with mutant EF-G. The low fitness of Fusr mutants in vivo suggests that drugs that can alter the levels of global regulators of gene expression deserve attention as potential antimicrobial agents.
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| 7. |
- Macvanin, Mirjana, et al.
(författare)
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Hyper-susceptibility of a fusidic acid-resistant mutant of Salmonella to different classes of antibiotics
- 2005
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 247:2, s. 215-220
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Fusidic acid resistance (FusR) in Salmonella enterica serovar Typhimurium is caused by mutations in fusA, encoding elongation factor G (EF-G). Pleiotropic phenotypes are observed in FusR mutants. Thus, the fusA1 allele (EF-G P413L) is associated with slow growth rate, reduced ppGpp and RpoS levels, reduced heme levels, and increased sensitivity to oxidative stress. The fusA1–15 allele, (EF-G P413L and T423I) derived from fusA1 in a selection for growth rate compensation, is partially compensated in each of these phenotypic defects but maintains its resistance to fusidic acid. We show here that the fusA1 allele is associated with sensitivity to ultraviolet light and increased susceptibility to the inhibitory action of several unrelated antibiotic classes (β-lactam, fluoroquinolone, aminoglycoside, rifampicin, and chloramphenicol). The fusA1–15 allele, in contrast, is less susceptible to UV and to other antibiotics than fusA1. The hyper-susceptibility to multiple antibiotics associated with fusA1 and fusA1–15 is revealed in a novel growth competition assay at sub-MIC concentrations, but not in a standard MIC assay.
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| 8. |
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| 9. |
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| 10. |
- Macvanin, Mirjana, et al.
(författare)
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The physiological cost of antibiotic resistance
- 2003
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Ingår i: Recent research developments in microbiology. - Trivandrum, India : Research Signpost. ; 7, s. 607-629
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Forskningsöversikt (populärvet., debatt m.m.)
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