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  • 1.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella2018In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 108, no 6, p. 697-710Article in journal (Refereed)
    Abstract [en]

    Bacteria can have multiple copies of a gene at separate locations on the same chromosome. Some of these gene families, including tuf (translation elongation factor EF-Tu) and rrl (ribosomal RNA), encode functions critically important for bacterial fitness. Genes within these families are known to evolve in concert using homologous recombination to transfer genetic information from one gene to another. This mechanism can counteract the detrimental effects of nucleotide sequence divergence over time. Whether such mechanisms can also protect against the potentially lethal effects of mobile genetic element insertion is not well understood. To address this we constructed two different length insertion cassettes to mimic mobile genetic elements and inserted these into various positions of the tuf and rrl genes. Wemeasured rates of recombinational repair that removed the inserted cassette and studied the underlying mechanism. Our results indicate that homologous recombination can protect the tuf and rrl genes from inactivation by mobile genetic elements, but forinsertions within shorter gene sequences the efficiency of repair is very low. Intriguingly, we found that physical distance separating genes on the chromosome directly affects the rate of recombinational repair suggesting that relative location will influence the ability of homologous recombination to maintain homogeneity.

  • 2.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Measuring Homologous Recombination Rates between Chromosomal Locations in Salmonella2019In: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, no 3, article id e3159Article in journal (Refereed)
    Abstract [en]

    Homologous recombination between two similar DNA molecules, plays an important role in the repair of double-stranded DNA breaks. Recombination can occur between two sister chromosomes, or between two locations of similar sequence identity within the same chromosome. The assay described here is designed to measure the rate of homologous recombination between two locations with sequence similarity within the same bacterial chromosome. For this purpose, a selectable/counter-selectable genetic cassette is inserted into one of the locations and homologous recombination repair rates are measured as a function of recombinational removal of the inserted cassette. This recombinational repair process is called gene conversion, non-reciprocal recombination. We used this method to measure the recombination rates between genes within gene families and to study the stability of mobile genetic elements inserted into members of gene families.

  • 3.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, Biomed Ctr, Dept Med Biochem & Microbiol, Uppsala, Sweden.
    Operon Concatenation Is an Ancient Feature That Restricts the Potential to Rearrange Bacterial Chromosomes2019In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 36, no 9, p. 1990-2000Article in journal (Refereed)
    Abstract [en]

    The last common ancestor of the Gammaproteobacteria carried an important 40-kb chromosome section encoding 51 proteins of the transcriptional and translational machinery. These genes were organized into eight contiguous operons (rrnB-tufB-secE-rpoBC-str-S10-spc-alpha). Over 2 Gy of evolution, in different lineages, some of the operons became separated by multigene insertions. Surprisingly, in many Enterobacteriaceae, much of the ancient organization is conserved, indicating a strong selective force on the operons to remain colinear. Here, we show for one operon pair, tufB-secE in Salmonella, that an interruption of contiguity significantly reduces growth rate. Our data show that the tufB-secE operons are concatenated by an interoperon terminator-promoter overlap that plays a significant role regulating gene expression. Interrupting operon contiguity interferes with this regulation, reducing cellular fitness. Six operons of the ancestral chromosome section remain contiguous in Salmonella (tufB-secE-rpoBC and S10-spc-alpha) and, strikingly, each of these operon pairs is also connected by an interoperon terminator-promoter overlap. Accordingly, we propose that operon concatenation is an ancient feature that restricts the potential to rearrange bacterial chromosomes and can select for the maintenance of a colinear operon organization over billions of years.

  • 4.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Having your cake and eating it - Staphylococcus aureus small colony variants can evolve faster growth rate without losing their antibiotic resistance2017In: MICROBIAL CELL, ISSN 2311-2638, Vol. 4, no 8, p. 275-277Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus can produce small colony variants (SCVs) during infections. These cause significant clinical problems because they are difficult to detect in standard microbiological screening and are associated with persistent infections. The major causes of the SCV phenotype are mutations that inhibit respiration by inactivation of genes of the menadione or hemin biosynthesis pathways. This reduces the production of ATP required to support fast growth. Importantly, it also decreases cross-membrane potential in SCVs, resulting in decreased uptake of cationic compounds, with reduced susceptibility to aminoglycoside antibiotics as a consequence. Because SCVs are slow-growing (mutations in men genes are associated with growth rates in rich medium similar to 30% of the wild-type growth rate) bacterial cultures are very susceptible to rapid takeover by faster-growing mutants (revertants or suppressors). In the case of reversion, the resulting fast growth is obviously associated with the loss of antibiotic resistance. However, direct reversion is relatively rare due to the very small genetic target size for such mutations. We explored the phenotypic consequences of SCVs evolving faster growth by routes other than direct reversion, and in particular whether any of those routes allowed for the maintenance of antibiotic resistance. In a recent paper (mBio 8: e00358-17) we demonstrated the existence of several different routes of SCV evolution to faster growth, one of which maintained the antibiotic resistance phenotype. This discovery suggests that SCVs might be more adaptable and problematic that previously thought. They are capable of surviving as a slow-growing persistent form, before evolving into a significantly faster-growing form without sacrificing their antibiotic resistance phenotype.

  • 5.
    Cao, Sha
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 3, article id e00358-17Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus is known to generate small colony variants (SCVs) that are resistant to aminoglycoside antibiotics and can cause persistent and recurrent infections. The SCV phenotype is unstable, and compensatory mutations lead to restored growth, usually with loss of resistance. However, the evolution of improved growth, by mechanisms that avoid loss of antibiotic resistance, is very poorly understood. By selection with serial passaging, we isolated and characterized different classes of extragenic suppressor mutations that compensate for the slow growth of small colony variants. Compensation occurs by two distinct bypass mechanisms: (i) translational suppression of the initial SCV mutation by mutant tRNAs, ribosomal protein S5, or release factor 2 and (ii) mutations that cause the constitutive activation of the SrrAB global transcriptional regulation system. Although compensation by translational suppression increases growth rate, it also reduces antibiotic susceptibility, thus restoring a pseudo-wild-type phenotype. In contrast, an evolutionary pathway that compensates for the SCV phenotype by activation of SrrAB increases growth rate without loss of antibiotic resistance. RNA sequence analysis revealed that mutations activating the SrrAB pathway cause upregulation of genes involved in peptide transport and in the fermentation pathways of pyruvate to generate ATP and NAD(+), thus explaining the increased growth. By increasing the growth rate of SCVs without the loss of aminoglycoside resistance, compensatory evolution via the SrrAB activation pathway represents a threat to effective antibiotic therapy of staphylococcal infections. IMPORTANCE Small colony variants (SCVs) of Staphylococcus aureus are a significant clinical problem, causing persistent and antibiotic-resistant infections. However, SCVs are unstable and can rapidly evolve growth-compensated mutants. Previous data suggested that growth compensation only occurred with the loss of antibiotic resistance. We have used selection with serial passaging to uncover four distinct pathways of growth compensation accessible to SCVs. Three of these paths (reversion, intragenic suppression, and translational suppression) increase growth at the expense of losing antibiotic resistance. The fourth path activates an alternative transcriptional program and allows the bacteria to produce the extra ATP required to support faster growth, without losing antibiotic resistance. The importance of this work is that it shows that drug-resistant SCVs can evolve faster growth without losing antibiotic resistance.

  • 6.
    De Rosa, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lu, Lu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Zamaratski, Edouard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Szałaj, Natalia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wadensten, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lenhammar, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Roos, Annette K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Karlen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)2017In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 3, p. 897-911Article in journal (Refereed)
    Abstract [en]

    Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.

  • 7.
    Jeannot, Frédéric
    et al.
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Taillier, Thomas
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Despeyroux, Pierre
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Renard, Stéphane
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Rey, Astrid
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Mourez, Michaël
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Poeverlein, Christoph
    Sanofi Aventis Deutschland GmbH, Integrated Drug Discovery, R&D, Ind Pk Hoechst, D-65926 Frankfurt, Germany.
    Khichane, Imène
    Sanofi R&D, Analyt Sci, LGCR, 13 Quai Jules Guesde, F-94400 Vitry Sur Seine, France.
    Perrin, Marc-Antoine
    Sanofi R&D, Analyt Sci, LGCR, 13 Quai Jules Guesde, F-94400 Vitry Sur Seine, France.
    Versluys, Stéphanie
    Evotec France, 195 Route Espagne,BP 13669, F-31036 Toulouse 1, France.
    Stavenger, Robert A.
    GlaxoSmithKline, Antibacterial DPU, 1250 Collegeville Rd, Collegeville, PA 19426 USA.
    Huang, Jianzhong
    GlaxoSmithKline, Antibacterial DPU, 1250 Collegeville Rd, Collegeville, PA 19426 USA.
    Germe, Thomas
    John Innes Ctr, Dept Biol Chem, Norwich Res Pk, Norwich NR4 7UH, Norfolk, England.
    Maxwell, Anthony
    John Innes Ctr, Dept Biol Chem, Norwich Res Pk, Norwich NR4 7UH, Norfolk, England.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bacqué, Eric
    Sanofi R&D, Therapeut Area Infect Dis, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France.
    Imidazopyrazinones (IPYs): Non-Quinolone Bacterial Topoisomerase Inhibitors Showing Partial Cross-Resistance with Quinolones2018In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, no 8, p. 3565-3581Article in journal (Refereed)
    Abstract [en]

    In our quest for new antibiotics able to address the growing threat of multidrug resistant infections caused by Gram-negative bacteria, we have investigated an unprecedented series of non-quinolone bacterial topoisomerase inhibitors from the Sanofi patrimony, named IPYs for imidazopyrazinones, as part of the Innovative Medicines Initiative (IMI) European Gram Negative Antibacterial Engine (ENABLE) organization. Hybridization of these historical compounds with the quinazolinediones, a known series of topoisomerase inhibitors, led us to a novel series of tricyclic IPYs that demonstrated potential for broad spectrum activity, in vivo efficacy, and a good developability profile, although later profiling revealed a genotoxicity risk. Resistance studies revealed partial cross-resistance with fluoroquinolones (FQs) suggesting that IPYs bind to the same region of bacterial topoisomerases as FQs and interact with at least some of the keys residues involved in FQ binding.

  • 8.
    Juhas, Mario
    et al.
    Univ Zurich, Inst Med Microbiol, Gloriastr 30, CH-8006 Zurich, Switzerland.
    Widlake, Emma
    Cardiff Univ, Sch Med, Div Infect & Immun, Cardiff CF14 4XN, S Glam, Wales.
    Teo, Jeanette
    Natl Univ Singapore Hosp, Dept Lab Med, 5 Lower Kent Ridge Rd, Singapore 119074, Singapore.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tyrrell, Jonathan M.
    Cardiff Univ, Sch Med, Div Infect & Immun, Cardiff CF14 4XN, S Glam, Wales.
    Polikanov, Yury S.
    Univ Illinois, Coll Liberal Arts & Sci, Dept Biol Sci, 900 South Ashland Ave,MBRB 4170, Chicago, IL 60607 USA.
    Ercan, Onur
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Petersson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Aboklaish, Ali F.
    Cardiff Univ, Sch Med, Div Infect & Immun, Cardiff CF14 4XN, S Glam, Wales.
    Rominski, Anna
    Univ Zurich, Inst Med Microbiol, Gloriastr 30, CH-8006 Zurich, Switzerland.
    Crich, David
    Wayne State Univ, Dept Chem, 5101 Cass Ave, Detroit, MI 48202 USA.
    Bottger, Erik C.
    Univ Zurich, Inst Med Microbiol, Gloriastr 30, CH-8006 Zurich, Switzerland.
    Walsh, Timothy R.
    Cardiff Univ, Sch Med, Div Infect & Immun, Cardiff CF14 4XN, S Glam, Wales.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hobbie, Sven N.
    Univ Zurich, Inst Med Microbiol, Gloriastr 30, CH-8006 Zurich, Switzerland.
    In vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii2019In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 74, no 4, p. 944-952Article in journal (Refereed)
    Abstract [en]

    Objectives: Widespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants.

    Methods: A thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity.

    Results: MIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin.

    Conclusions: Its superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics.

  • 9.
    Khan, David D.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lagerbäck, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lustig, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nielsen, Elisabet I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Friberg, Lena E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    A mechanism-based pharmacokinetic/pharmacodynamic model allows prediction of antibiotic killing from MIC values for WT and mutants2015In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 70, no 11, p. 3051-3060Article in journal (Refereed)
    Abstract [en]

    Objectives: In silico pharmacokinetic/pharmacodynamic (PK/PD) models can be developed based on data from in vitro time-kill experiments and can provide valuable information to guide dosing of antibiotics. The aim was to develop a mechanism-based in silico model that can describe in vitro time-kill experiments of Escherichia coli MG1655 WT and six isogenic mutants exposed to ciprofloxacin and to identify relationships that may be used to simplify future characterizations in a similar setting. Methods: In this study, we developed a mechanism-based PK/PD model describing killing kinetics for E. coli following exposure to ciprofloxacin. WT and six well-characterized mutants, with one to four clinically relevant resistance mutations each, were exposed to a wide range of static ciprofloxacin concentrations. Results: The developed model includes susceptible growing bacteria, less susceptible (pre-existing resistant) growing bacteria, non-susceptible non-growing bacteria and non-colony-forming non-growing bacteria. The non-colony-forming state was likely due to formation of filaments and was needed to describe data close to the MIC. A common model structure with different potency for bacterial killing (EC50) for each strain successfully characterized the time-kill curves for both WT and the six E. coli mutants. Conclusions: The model-derived mutant-specific EC50 estimates were highly correlated (r(2) = 0.99) with the experimentally determined MICs, implying that the in vitro time-kill profile of a mutant strain is reasonably well predictable by the MIC alone based on the model.

  • 10.
    Khan, David
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lagerbäck, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Malmberg, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Kristoffersson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gullberg, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nielsen, Elisabet I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Friberg, Lena E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Predicting mutant selection in competition experiments with ciprofloxacin-exposed Escherichia coli2018In: International Journal of Antimicrobial Agents, ISSN 0924-8579, E-ISSN 1872-7913, Vol. 51, no 3, p. 399-406, article id S0924-8579(17)30392-8Article in journal (Refereed)
    Abstract [en]

    Predicting competition between antibiotic-susceptible wild-type (WT) and less susceptible mutant (MT) bacteria is valuable for understanding how drug concentrations influence the emergence of resistance. Pharmacokinetic/pharmacodynamic (PK/PD) models predicting the rate and extent of takeover of resistant bacteria during different antibiotic pressures can thus be a valuable tool in improving treatment regimens. The aim of this study was to evaluate a previously developed mechanism-based PK/PD model for its ability to predict in vitro mixed-population experiments with competition between Escherichia coli (E. coli) WT and three well-defined E. coli resistant MTs when exposed to ciprofloxacin. Model predictions for each bacterial strain and ciprofloxacin concentration were made for in vitro static and dynamic time–kill experiments measuring CFU (colony forming units)/mL up to 24 h with concentrations close to or below the minimum inhibitory concentration (MIC), as well as for serial passage experiments with concentrations well below the MIC measuring ratios between the two strains with flow cytometry. The model was found to reasonably well predict the initial bacterial growth and killing of most static and dynamic time–kill competition experiments without need for parameter re-estimation. With parameter re-estimation of growth rates, an adequate fit was also obtained for the 6-day serial passage competition experiments. No bacterial interaction in growth was observed. This study demonstrates the predictive capacity of a PK/PD model and further supports the application of PK/PD modelling for prediction of bacterial kill in different settings, including resistance selection.

  • 11.
    Lannergård, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Norström, Tobias
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Delgado, Alejandro
    University of New Mexico.
    Gustafson, John E
    University of New Mexico.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Genetic Complexity of Fusidic Acid-Resistant Small Colony Variants (SCV) in Staphylococcus aureus2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 11, p. e28366-Article in journal (Refereed)
    Abstract [en]

    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.

  • 12.
    Nielsen, Elisabet I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Khan, David D.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lustig, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Friberg, Lena E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Can a pharmacokinetic/pharmacodynamic (PKPD) model be predictive across bacterial densities and strains?: External evaluation of a PKPD model describing longitudinal in vitro data2017In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 72, no 11, p. 3108-3116Article in journal (Refereed)
    Abstract [en]

    Background: Pharmacokinetic/pharmacodynamic (PKPD) models developed based on data from in vitro time-kill experiments have been suggested to contribute to more efficient drug development programmes and better dosing strategies for antibiotics. However, for satisfactory predictions such models would have to show good extrapolation properties. Objectives: To evaluate if a previously described mechanism-based PKPD model was able also to predict drug efficacy for higher bacterial densities and across bacterial strains. Methods: A PKPD model describing the efficacy of ciprofloxacin on Escherichia coli was evaluated. The predictive performance of the model was evaluated across several experimental conditions with respect to: (i) bacterial start inoculum ranging from the standard of similar to 10(6) cfu/mL up to late stationary-phase cultures; and (ii) efficacy for seven additional strains (three laboratory and four clinical strains), not included during the model development process, based only on information regarding their MIC. Model predictions were performed according to the intended experimental protocol and later compared with observed bacterial counts. Results: The mechanism-based PKPD model structure developed based on data from standard start inoculum experiments was able to accurately describe the inoculum effect. The model successfully predicted the time course of drug efficacy for additional laboratory and clinical strains based on only the MIC values. The model structure was further developed to better describe the stationary phase data. Conclusions: This study supports the use of mechanism-based PKPD models based on preclinical data for predictions of untested scenarios.

  • 13.
    Szałaj, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Lu, Lu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Benediktsdottir, Andrea
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Zamaratski, Edouard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Olanders, Gustav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Hedgecock, Charles
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Karlen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Erdélyi, Máté
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mowbray, Sherry L
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Boronic ester-linked macrocyclic lipopeptides as serine protease inhibitors targeting Escherichia coli type I signal peptidase.2018In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 157, p. 1346-1360Article in journal (Refereed)
    Abstract [en]

    Type I signal peptidase, with its vital role in bacterial viability, is a promising but underexploited antibacterial drug target. In the light of steadily increasing rates of antimicrobial resistance, we have developed novel macrocyclic lipopeptides, linking P2 and P1' by a boronic ester warhead, capable of inhibiting Escherichia coli type I signal peptidase (EcLepB) and exhibiting good antibacterial activity. Structural modifications of the macrocyclic ring, the peptide sequence and the lipophilic tail led us to 14 novel macrocyclic boronic esters. It could be shown that macrocyclization is well tolerated in terms of EcLepB inhibition and antibacterial activity. Among the synthesized macrocycles, potent enzyme inhibitors in the low nanomolar range (e.g. compound 42f, EcLepB IC50 = 29 nM) were identified also showing good antimicrobial activity (e.g. compound 42b, E. coli WT MIC = 16 μg/mL). The unique macrocyclic boronic esters described here were based on previously published linear lipopeptidic EcLepB inhibitors in an attempt to address cytotoxicity and hemolysis. We show herein that structural changes to the macrocyclic ring influence both the cytotoxicity and hemolytic activity suggesting that the P2 to P1' linker provide means for optimizing off-target effects. However, for the present set of compounds we were not able to separate the antibacterial activity and cytotoxic effect.

1 - 13 of 13
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