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| 2. |
- Hammarlöf, Disa L, et al.
(författare)
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Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu
- 2008
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 70:5, s. 1194-1209
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Tidskriftsartikel (refereegranskat)abstract
- Salmonella enterica with mutant EF-Tu (Gln125Arg) has a low level of EF-Tu, a reduced rate of protein synthesis and an extremely slow growth rate. Eighty independent suppressor mutations were selected that restored normal growth. In some cases (n = 7) suppression was due to mutations in tufA but, surprisingly, in most cases (n = 73) to mutations in rne, the gene coding for RNase E. These rne mutations alone had only modest effects on growth rate. Fifty different suppressor mutations were isolated in rne, all located in or close to the N-terminal endonucleolytic half of RNase E. Steady state levels of several mRNAs were lower in the mutant tuf strain but restored to wild-type levels in the tuf-rne double mutant. In contrast, the half-lives of mRNAs were unaffected by the tuf mutation. We propose a model where the tuf mutation causes the ribosome following RNA polymerase to pause, possibly in a codon-specific manner, exposing unshielded nascent message to RNase E cleavage. Normal growth rate can be restored by increasing EF-Tu activity or by reducing RNase E activity. Accordingly, RNase E is suggested to act at two distinct stages in the life of mRNA: early, on the nascent transcript; late, on the complete mRNA.
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| 5. |
- Komp Lindgren, Patricia, et al.
(författare)
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Biological cost of single and multiple norfloxacin resistance mutations in Escherichia coli implicated in urinary tract infections
- 2005
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 49:6, s. 2343-2351
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to fluoroquinolones in urinary tract infection (UTIs)caused by Escherichia coli is associated with multiple mutations,typically those that alter DNA gyrase and DNA topoisomeraseIV and those that regulate AcrAB-TolC-mediated efflux. We askedwhether a fitness cost is associated with the accumulation ofthese multiple mutations. Mutants of the susceptible E. coliUTI isolate Nu14 were selected through three to five successivesteps with norfloxacin. Each selection was performed with theMIC of the selected strain. After each selection the MIC wasmeasured; and the regions of gyrA, gyrB, parC, and parE, previouslyassociated with resistance mutations, and all of marOR and acrRwere sequenced. The first selection step yielded mutations ingyrA, gyrB, and marOR. Subsequent selection steps yielded mutationsin gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associatedmutations were identified in almost all isolates after selectionsteps 1 and 2 but in less than 50% of isolates after subsequentselection steps. Selected strains were competed in vitro, inurine, and in a mouse UTI infection model against the startingstrain, Nu14. First-step mutations were not associated withsignificant fitness costs. However, the accumulation of threeor more resistance-associated mutations was usually associatedwith a large reduction in biological fitness, both in vitroand in vivo. Interestingly, in some lineages a partial restorationof fitness was associated with the accumulation of additionalmutations in late selection steps. We suggest that the relativebiological costs of multiple mutations may influence the evolutionof E. coli strains that develop resistance to fluoroquinolones.
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| 6. |
- Kugelberg, Elisabeth, et al.
(författare)
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Establishment of a superficial skin infection model in mice by using Staphylococcus aureus and Streptococcus pyogenes
- 2005
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 49:8, s. 3435-3441
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- A new animal model for the purpose of studying superficial infections is presented. In this model, an infection is established by disruption of the skin barrier by partial removal of the epidermal layer by tape stripping and subsequent application of the pathogens Staphylococcus aureus and Streptococcus pyogenes. The infection and the infection route are purely topical, in contrast to those used in previously described animal models in mice, such as the skin suture-wound model, where the infection is introduced into the deeper layers of the skin. Thus, the present model is considered more biologically relevant for the study of superficial skin infections in mice and humans. Established topical antibiotic treatments are shown to be effective. The procedures involved in the model are simple, a feature that increases throughput and reproducibility. This new model should be applicable to the evaluation of novel antimicrobial treatments of superficial infections caused by S. aureus and S. pyogenes.
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| 7. |
- Lannergård, Jonas, et al.
(författare)
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Genetic Determinants of Resistance to Fusidic Acid among Clinical Bacteremia Isolates of Staphylococcus aureus
- 2009
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 53:5, s. 2059-2065
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to fusidic acid in Staphylococcus aureus is caused by mutation of the elongation factor G (EF-G) drug target (FusA class) or by expression of a protein that protects the drug target (FusB and FusC classes). Recently, two novel genetic classes of small-colony variants (SCVs) were identified among fusidic acid-resistant mutants selected in vitro (FusA-SCV and FusE classes). We analyzed a phylogenetically diverse collection of fusidic acid-resistant bacteremia isolates to determine which resistance classes were prevalent and whether these were associated with particular phylogenetic lineages. Each isolate was shown by DNA sequencing and plasmid curing to carry only one determinant of fusidic acid resistance, with approximately equal frequencies of the FusA, FusB, and FusC genetic classes. The FusA class (mutations in fusA) were distributed among different phylogenetic types. Two distinct variants of the FusC class (chromosomal fusC gene) were identified, and FusC was also distributed among different phylogenetic types. In contrast, the FusB class (carrying fusB on a plasmid) was found in closely related types. No FusE-class mutants (carrying mutations in rplF) were found. However, one FusA-class isolate had multiple mutations in the fusA gene, including one altering a codon associated with the FusA-SCV class. SCVs are frequently unstable and may undergo compensatory evolution to a normal growth phenotype after their initial occurrence. Accordingly, this normal-growth isolate might have evolved from a fusidic acid-resistant SCV. We conclude that at least three different resistance classes are prevalent among fusidic acid-resistant bacteremia isolates of S. aureus.
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| 8. |
- Lannergård, Jonas, et al.
(författare)
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Identification of the Genetic Basis for Clinical Menadione-Auxotrophic Small-Colony Variant Isolates of Staphylococcus aureus
- 2008
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 52:11, s. 4017-4022
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Tidskriftsartikel (refereegranskat)abstract
- Small-colony variants (SCVs) of Staphylococcus aureus are associated with persistent infections and may be selectively enriched during antibiotic therapy. Three pairs of clonally related S. aureus isolates were recovered from patients receiving systemic antibiotic therapy. Each pair consisted of an isolate with a normal phenotype and an isolate with an SCV phenotype. These SCVs were characterized by reduced susceptibility to gentamicin, reduced hemolytic activity, slow growth, and menadione auxotrophy. Sequencing of the genes involved in menadione biosynthesis revealed mutations in menB, the gene encoding naphthoate synthase, in all three strains with the SCV phenotype. The menB mutations were (i) a 9-bp deletion from nucleotides 55 to 63, (ii) a frameshift mutation that resulted in a premature stop codon at position 230, and (iii) a point mutation that caused the amino acid substitution Gly to Val at codon 233. Fluctuation tests showed that growth-compensated mutants arose in the SCV population of one strain, strain OM1b, at a rate of 1.8 x 10(-8) per cell per generation. Sequence analyses of 23 independently isolated growth-compensated mutants of this strain revealed alterations in the menB sequence in every case. These alterations included reversions to the wild-type sequence and intragenic second-site mutations. Each of the growth-compensated mutants showed a restoration of normal growth and a loss of menadione auxotrophy, increased susceptibility to gentamicin, and restored hemolytic activity. These data show that mutations in menB cause the SCV phenotype in these clinical isolates. This is the first report on the genetic basis of menadione-auxotrophic SCVs determined in clinical S. aureus isolates.
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| 9. |
- 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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| 10. |
- Marcusson, Linda L, et al.
(författare)
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Interplay in the Selection of Fluoroquinolone Resistance and Bacterial Fitness
- 2009
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 5:8, s. e1000541-
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Tidskriftsartikel (refereegranskat)abstract
- Fluoroquinolones are antibacterial drugs that inhibit DNA Gyrase and Topoisomerase IV. These essential enzymes facilitate chromosome replication and RNA transcription by regulating chromosome supercoiling. High-level resistance to fluoroquinolones in E. coli requires the accumulation of multiple mutations, including those that alter target genes and genes regulating drug efflux. Previous studies have shown some drug-resistance mutations reduce bacterial fitness, leading to the selection of fitness-compensatory mutations. The impact of fluoroquinolone-resistance on bacterial fitness was analyzed in constructed isogenic strains carrying up to 5 resistance mutations. Some mutations significantly decreased bacterial fitness both in vitro and in vivo. We identified low-fitness triple-mutants where the acquisition of a fourth resistance mutation significantly increased fitness in vitro and in vivo while at the same time dramatically decreasing drug susceptibility. The largest effect occurred with the addition of a parC mutation (Topoisomerase IV) to a low-fitness strain carrying resistance mutations in gyrA (DNA Gyrase) and marR (drug efflux regulation). Increased fitness was accompanied by a significant change in the level of gyrA promoter activity as measured in an assay of DNA supercoiling. In selection and competition experiments made in the absence of drug, parC mutants that improved fitness and reduced susceptibility were selected. These data suggest that natural selection for improved growth in bacteria with low-level resistance to fluoroquinolones could in some cases select for further reductions in drug susceptibility. Thus, increased resistance to fluoroquinolones could be selected even in the absence of further exposure to the drug.
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