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| 2. |
- Allander, Lisa, et al.
(författare)
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Evaluation of In Vitro Activity of Double-Carbapenem Combinations against KPC-2-, OXA-48-and NDM-Producing Escherichia coli and Klebsiella pneumoniae
- 2022
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Ingår i: Antibiotics. - : MDPI. - 2079-6382. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- Double-carbapenem combinations have shown synergistic potential against carbapenemase-producing Enterobacterales, but data remain inconclusive. This study evaluated the activity of double-carbapenem combinations against 51 clinical KPC-2-, OXA-48-, NDM-1, and NDM-5-producing Escherichia coli and Klebsiella pneumoniae and against constructed E. coli strains harboring genes encoding KPC-2, OXA-48, or NDM-1 in an otherwise isogenic background. Two-drug combinations of ertapenem, meropenem, and doripenem were evaluated in 24 h time-lapse microscopy experiments with a subsequent spot assay and in static time-kill experiments. An enhanced effect in time-lapse microscopy experiments at 24 h and synergy in the spot assay was detected with one or more combinations against 4/14 KPC-2-, 17/17 OXA-48-, 2/17 NDM-, and 1/3 NDM-1+OXA-48-producing clinical isolates. Synergy rates were higher against meropenem- and doripenem-susceptible isolates and against OXA-48 producers. NDM production was associated with significantly lower synergy rates in E. coli. In time-kill experiments with constructed KPC-2-, OXA-48- and NDM-1-producing E. coli, 24 h synergy was not observed; however, synergy at earlier time points was found against the KPC-2- and OXA-48-producing constructs. Our findings indicate that the benefit of double-carbapenem combinations against carbapenemase-producing E. coli and K. pneumoniae is limited, especially against isolates that are resistant to the constituent antibiotics and produce NDM.
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| 4. |
- Allander, Lisa
(författare)
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β-lactam combinations against multidrug-resistant Enterobacterales : Exploring combination effects and resistance development
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The β-lactam antibiotics are a cornerstone in treating bacterial infections, but the increasing prevalence of antibiotic resistance worldwide threatens their effectiveness. The main driver of β-lactam resistance is the production of β-lactamases, which are bacterial enzymes that inactivate the antibiotic. Moreover, resistance to multiple antibiotic classes is common in β-lactamase producing bacteria, further limiting treatment options. At the same time, few novel antibacterial agents are reaching the market. To address this challenge, antibiotic combination therapy is employed to enhance the effects of existing drugs against multidrug-resistant bacteria. Yet, there is a lack of knowledge regarding which antibiotics to combine to achieve the best effect. The investigations in this thesis evaluate the potential and limitations of combinations involving β-lactams, β-lactamase inhibitors and colistin against multidrug-resistant Enterobacterales in vitro. In the first paper, we investigated resistance mechanisms to three commonly used β-lactam/β-lactamase inhibitor combinations (BLBLIs) in an Escherichia coli strain encoding multiple β-lactamases. We found that β-lactamase gene amplifications were a key driver of resistance, with variations in the amplification pattern depending on the BLBLI combination. Clinical resistance could be reached by gene amplifications for ampicillin-sulbactam and piperacillin-tazobactam, whereas ceftazidime-avibactam resistance required multiple genetic changes. In the second paper, we evaluated the efficacy of double-carbapenem combinations against E. coli and Klebsiella pneumoniae producing carbapenemases (KPC-2, OXA-48, NDM-1, and NDM-5). Synergistic effects were most commonly observed against OXA-48-producing strains, whereas the efficacy was low against KPC-2 and negligible against NDM producers. In the third and fourth papers, we evaluated the antibacterial activity of colistin in combination with BLBLIs. Considering that reduced membrane permeability is associated with decreased susceptibility towards BLBLIs, adding colistin may be beneficial since its membrane-disrupting effect may increase the entry of other drugs. In paper three, we showed synergistic effects with colistin and ceftazidime-avibactam against a KPC-2-producing K. pneumoniae strain with porin deficiencies. However, when systematically assessing the impact of porin loss on the synergistic potential of colistin in combination with BLBLIs in paper four, we did not find any clear association between porin loss and synergy. These studies provide insight into the therapeutic potential and limitations of combinations, including β-lactam antibiotics against strains with different setups of resistance genes. More research is required to understand how to best use the newly introduced BLBLI combinations to preserve their activity and enhance the value of the available antibiotics for future generations.
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| 5. |
- Andersson, Evalena, et al.
(författare)
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Structure of bacteriophage T4 endonuclease II mutant E118A, a tetrameric GIY-YIG enzyme
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 397:4, s. 1003-1016
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Tidskriftsartikel (refereegranskat)abstract
- Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16Da, 136aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P21 with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context.
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| 7. |
- Carlson, Karin, et al.
(författare)
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Bacteriophage T4 endonuclease II: concerted single-strand nicks yield double-strand cleavage.
- 2004
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Ingår i: Mol Microbiol. - : Wiley. ; 52, s. 1403-1411
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Tidskriftsartikel (refereegranskat)abstract
- In vivo, endonuclease II (EndoII) of coliphage T4 cleaves sites with conserved sequence elements (CSEs) to both the left and the right of the cleaved bonds, 16 bp altogether with some variability tolerated. In vitro, however, single-strand nicks in the lower strand predominate at sites containing only the left-side CSE that determines the precise position of lower strand nicks. Upper strand nick positions vary both in vivo and in vitro. A 24 bp substrate was nicked with the same precision as in longer substrates, showing that the conserved sequence suffices for precise nicking by EndoII. Using DNA ligase in vitro, we found that EndoII nicked both strands simultaneously at an in vivo-favoured site but not at an in vitro-favoured site. This indicates that the right-side CSE at in vivo-favoured sites is important for simultaneous nicking of both strands, generating double-strand cleavage. Separate analysis of the two strands following in vitro digestion at two in vitro-favoured sites showed that EndoII nicked the lower strand about 1.5-fold faster than the upper strand. In addition, the upper and lower strands were nicked independently of each other, seldom resulting in double-strand cleavage. Thus, cleavage by EndoII is the fortuitous outcome of two separate nicking events.
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| 9. |
- Karvanen, Matti, 1975-, et al.
(författare)
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Colistin is Extensively Lost during Standard in Vitro Experimental Conditions
- 2017
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 61:11
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Tidskriftsartikel (refereegranskat)abstract
- Colistin adheres to a range of materials, including plastics in labware. The loss caused by adhesion influences an array of methods detrimentally, including MIC assays and in vitro time-kill experiments. The aim of this study was to characterize the extent and time course of colistin loss in different types of laboratory materials during a simulated time-kill experiment without bacteria or plasma proteins present. Three types of commonly used large test tubes, i.e., soda-lime glass, polypropylene, and polystyrene, were studied, as well as two different polystyrene microplates and low-protein-binding microtubes. The tested concentration range was 0.125 to 8 mg/liter colistin base. Exponential one-phase and two-phase functions were fitted to the data, and the adsorption of colistin to the materials was modeled with the Langmuir adsorption model. In the large test tubes, the measured start concentrations ranged between 44 and 102% of the expected values, and after 24 h, the concentrations ranged between 8 and 90%. The half-lives of colistin loss were 0.9 to 12 h. The maximum binding capacities of the three materials ranged between 0.4 and 1.1 μg/cm2, and the equilibrium constants ranged between 0.10 and 0.54 ml/μg. The low-protein-binding microtubes showed start concentrations between 63 and 99% and concentrations at 24 h of between 59 and 90%. In one of the microplates, the start concentrations were below the lower limit of quantification at worst. In conclusion, to minimize the effect of colistin loss due to adsorption, our study indicates that low-protein-binding polypropylene should be used when possible for measuring colistin concentrations in experimental settings, and the results discourage the use of polystyrene. Furthermore, when diluting colistin in protein-free media, the number of dilution steps should be minimized.
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