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  • 1.
    Adler, Marlen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anjum, Mehreen
    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.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, p. 1188-1198Article in journal (Refereed)
    Abstract [en]

    The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of beta-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of beta-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. beta-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.

  • 2.
    Adler, Marlen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anjum, Mehreen
    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.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Influence of acquired β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 1, p. 51-59Article in journal (Refereed)
    Abstract [en]

    Objectives: To investigate the influence of plasmid-borne β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli and the fitness costs associated with resistance. Methods: Stepwise selection of carbapenem-resistant mutants with or without the extended-spectrum β-lactamase (ESBL)-encoding plasmid pUUH239.2 was performed. Mutation rates and mutational pathways to resistance were determined. In vitro-selected and constructed mutants were characterized regarding the MICs of the carbapenems, porin expression profiles, growth rates and the presence of mutations in the porins ompC/ompF and their regulatory genes. The influence of the plasmid-encoded β-lactamases TEM-1, OXA-1 and CTX-M-15 on resistance development was determined. Results: Results show that E. coli readily developed reduced carbapenem susceptibility and clinical resistance levels by a combination of porin loss and increased β-lactamase expression, especially towards ertapenem. All tested β-lactamases (CTX-M-15, TEM-1 and OXA-1) contributed to reduced carbapenem susceptibility in the absence of porin expression. However, complete loss of porin expression conferred a 20% fitness cost on the bacterial growth rate. Increased β-lactamase expression through spontaneous gene amplification on the plasmid was a major resistance factor. Conclusions: Plasmid-encoded β-lactamases, including non-ESBL enzymes, have a strong influence on the frequency and resistance level of spontaneous carbapenem-resistant mutants. The fitness cost associated with the loss of OmpC/OmpF in E. coli most likely reduces the survivability of porin mutants and could explain why they have not emerged as a clinical problem in this species.

  • 3.
    André, Malin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Center for Clinical Research Dalarna.
    Vernby, Åsa
    Berg, Johanna
    Lundborg, Cecilia Stalsby
    A survey of public knowledge and awareness related to antibiotic use and resistance in Sweden2010In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 65, no 6, p. 1292-1296Article in journal (Refereed)
    Abstract [en]

    To examine the level of knowledge about antibiotic treatment and awareness of antibiotic resistance among the general public in Sweden. A quantitative, cross-sectional interview study based on a structured questionnaire used during telephone interviews. The sample comprised 1000 randomly selected individuals aged 21-80 years throughout Sweden. Demographic data as well as level of agreement with various statements concerning antibiotics and antibiotic use were provided by the respondents. The response rate was 74.7%. Of the respondents, 19.1% agreed that antibiotics cure common colds more quickly; this belief was higher in those who had not previously received antibiotics. A high proportion, 80.7%, agreed that bacteria could become resistant to antibiotics. Trust in doctors was high, and significantly more respondents reported trusting the doctor not prescribing an antibiotic, 87.0%, than the doctor prescribing an antibiotic, 81.0%. The respondents showed some confusion surrounding the terms 'bacteria' and 'viruses', and the meaning of these in relation to the prescribing decision. The high level of trust in restrictive prescribing as well as the awareness of antibiotic resistance expressed by the Swedish public should be recognized by health professionals and utilized in future campaigns.

  • 4. Bengtsson, Stina
    et al.
    Naseer, Umaer
    Sundsfjord, Arnfinn
    Kahlmeter, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Sundqvist, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Sequence types and plasmid carriage of uropathogenic Escherichia coli devoid of phenotypically detectable resistance2012In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 67, no 1, p. 69-73Article in journal (Refereed)
    Abstract [en]

    Objectives

    Plasmids play a major role in the dissemination of antibiotic resistance, and several studies have shown the association between specific resistance mechanisms and certain plasmid types and/or Escherichia coli lineages. This study describes the distribution of plasmids, replicon types, sequence types (STs) and ST complexes (STCs) of E. coli devoid of phenotypic resistance to 24 antibiotics.

    Methods

    Eighty E. coli isolates from urinary tract infections from four European countries were selected because of their lack of phenotypically detectable antibiotic resistance. The isolates were characterized to the phylogenetic group level using PCR and to ST by multilocus sequence typing. Plasmid carriage was assessed using S1 nuclease PFGE profiling and PCR-based replicon typing.

    Results

    Plasmids were detected in only 38/80 (47%) of the isolates; one (n = 18), two (n = 14), three (n = 5) and four (n = 1) plasmids. Six different replicon types were identified, the most common being a combination of IncFII and IncFIB. Most isolates belonged to phylogenetic group B2 and STC73 (n = 20), STC95 (n = 7) and ST420 (n = 6). A high proportion of STC73 isolates (75%) was devoid of plasmids. No association could be found between specific STs and replicon type.

    Conclusions

    A large proportion of E. coli strains phenotypically devoid of antibiotic resistance were plasmid naive. Those isolates that harboured plasmids displayed replicon types similar to those of resistant isolates, but the distributions of STs and STCs were different. This may indicate chromosomally encoded mechanisms important for the stabilization of plasmids harbouring antibiotic resistance.

  • 5.
    Bonnedahl, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Drobni, P.
    Johansson, A.
    Hernandez, Jorge
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Melhus, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Stedt, J.
    Olsen, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Drobni, Mirva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Characterization, and comparison, of human clinical and black-headed gull (Larus ridibundus) extended-spectrum β-lactamase-producing bacterial isolates from Kalmar, on the southeast coast of Sweden2010In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 65, no 9, p. 1939-1944Article in journal (Refereed)
    Abstract [en]

    Antibiotic resistance is one of the great challenges for modern healthcare. In Gram-negative bacteria, CTX-M-type extended-spectrum beta-lactamases (ESBLs) have been rapidly spreading through Europe since the early 2000s. In Sweden, ESBL-producing Escherichia coli are still rare, but a 3-fold increase has been seen from 2004 to 2007. Enterobacteria and normal flora of wild animals, with or without antibiotic resistance traits, constitute a potential source of human infection and colonization. We studied wild birds with the aim to understand the environmental dissemination of antibiotic resistance and, focusing on clinically relevant resistance types, we made comparisons with human clinical samples. In this study, ESBL-producing human clinical isolates and isolates from juvenile black-headed gulls from Kalmar County hospital and the city of Kalmar, respectively, on the southeast coast of Sweden, were characterized and compared. Despite a low frequency of antibiotic resistance among the isolates from gulls, ESBL-producing E. coli isolates were found, two with bla(CTX-M-14) and one with bla(CTX-M-15). The same CTX-M types were dominant among human ESBL isolates. In addition, gull isolates were dispersed among the human samples in the PhenePlate (TM) clustering system, indicating that they neither differ from the human isolates nor form any separate clonal clustering. The finding of CTX-M-type ESBLs in E. coli isolated from black-headed gulls in Sweden, where 'background resistance' is low, is consistent with an ongoing environmental spread of these plasmid-borne resistance genes. The results indicate that a potential for transfer between the human population and environment exists even in countries with a low level of antibiotic resistance.

  • 6.
    Brandis, Gerrit
    et al.
    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.
    Genetic characterization of compensatory evolution in strains carrying rpoB Ser531Leu, the rifampicin resistance mutation most frequently found in clinical isolates2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 11, p. 2493-2497Article in journal (Refereed)
    Abstract [en]

    Objectives: The 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. Methods: We 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. Results: Compensatory 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. Conclusions: The 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.

  • 7.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pietsch, Franziska
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Alemayehu, Rahel
    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.
    Comprehensive phenotypic characterization of rifampicin resistance mutations in Salmonella provides insight into the evolution of resistance in Mycobacterium tuberculosis2015In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 70, no 3, p. 680-685Article in journal (Refereed)
    Abstract [en]

    Objectives: Mutations in the beta-subunit of RNA polymerase (RNAP), encoded by rpoB, are responsible for rifampicin resistance (Rif(R)). Although many mutations in rpoB can reduce susceptibility, only a few are frequent amongst Rif(R) clinical Mycobacterium tuberculosis (MTB) isolates. It has been suggested that there is a negative correlation between the fitness costs of Rif(R) mutations and their respective clinical frequency, but so far comparable fitness cost measurements have only been conducted for a very limited number of Rif(R) mutations. We tested this hypothesis using Salmonella and Mycobacterium smegmatis as model organisms. Methods: We constructed 122 different Rif(R) mutations in Salmonella. MICs and relative fitness costs in the presence and absence of rifampicin were determined for each mutant, including for a smaller number of Rif(R) M. smegmatis strains. Results were compared with available mutation frequency data from clinical MTB isolates. Results: (i) Rif(R) mutations frequently found in MTB isolates have a fitness cost in Salmonella Typhimurium and M. smegmatis. (ii) Clinically frequent Rif(R) mutations have a high rifampicin MIC. (iii) There is a strong correlation between the magnitude of the fitness cost of a Rif(R) mutation in Salmonella Typhimurium or M. smegmatis and the frequency with which that mutation is associated with secondary (putative compensatory) mutations in RNAP of clinical MTB isolates. Conclusions: This suggests that the success of Rif(R) mutations in clinical MTB isolates may be dependent not only on a low initial fitness cost, but rather the results of three factors: (i) a high rifampicin MIC; (ii) a relatively low initial fitness cost; and (iii) the ability to additionally acquire compensatory mutations selected to further reduce fitness cost.

  • 8. Chryssanthou, Erja
    et al.
    Loebig, Alissha
    Sjölin, Jan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Post-antifungal effect of amphotericin B and voriconazole against germinated Aspergillus fumigatus conidia2008In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 61, no 6, p. 1309-11Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: The post-antifungal effect (PAFE) of amphotericin B and voriconazole on germinated Aspergillus fumigatus conidia was studied using the BacT/Alert detection system based on fungal CO(2) production. METHODS: Germinated conidia of A. fumigatus were exposed to 1-10x MIC of amphotericin B for 1 and 4 h and to 2.5-40x MIC of voriconazole for 4 and 24 h. After removal of the drug by washing, similar numbers of exposed and control germlings were inoculated into Pedi-BacT culture bottles. CO(2) production was automatically monitored until the bottles signalled positive. The difference in time for positive signals in drug-exposed and control bottles was used to calculate the PAFE. RESULTS: The killing rate of amphotericin B against germlings was both concentration- and time-dependent, as has been previously found for actively growing hyphae. Similarly, voriconazole showed fungicidal effect after 24 h of exposure, but not after 4 h. Amphotericin B induced a long concentration- and time-dependent PAFE, whereas voriconazole resulted in a short and dose-independent PAFE that was significantly longer after 24 h than after 4 h of exposure. CONCLUSIONS: An automated method is presented for the determination of PAFE on filamentous fungi using quantifiable numbers of germinated conidia. In contrast to previous results obtained from conidia, this method could demonstrate a PAFE of amphotericin B on Aspergillus that shared characteristics similar to that on Candida spp.

  • 9.
    Clewe, Oskar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Aulin, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hu, Yanmin
    Coates, Anthony R M
    Simonsson, Ulrika S H
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    A multistate tuberculosis pharmacometric model: a framework for studying anti-tubercular drug effects in vitro2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 4, p. 964-974Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: Mycobacterium tuberculosis can exist in different states in vitro, which can be denoted as fast multiplying, slow multiplying and non-multiplying. Characterizing the natural growth of M. tuberculosis could provide a framework for accurate characterization of drug effects on the different bacterial states.

    METHODS: The natural growth data of M. tuberculosis H37Rv used in this study consisted of viability defined as cfu versus time based on data from an in vitro hypoxia system. External validation of the natural growth model was conducted using data representing the rate of incorporation of radiolabelled methionine into proteins by the bacteria. Rifampicin time-kill curves from log-phase (0.25-16 mg/L) and stationary-phase (0.5-64 mg/L) cultures were used to assess the model's ability to describe drug effects by evaluating different linear and non-linear exposure-response relationships.

    RESULTS: The final pharmacometric model consisted of a three-compartment differential equation system representing fast-, slow- and non-multiplying bacteria. Model predictions correlated well with the external data (R(2) = 0.98). The rifampicin effects on log-phase and stationary-phase cultures were separately and simultaneously described by including the drug effect on the different bacterial states. The predicted reduction in log10 cfu after 14 days and at 0.5 mg/L was 2.2 and 0.8 in the log-phase and stationary-phase systems, respectively.

    CONCLUSIONS: The model provides predictions of the change in bacterial numbers for the different bacterial states with and without drug effect and could thus be used as a framework for studying anti-tubercular drug effects in vitro.

  • 10.
    Dorlo, Thomas P. C.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Netherlands Canc Inst, Antoni Van Leeuwenhoek Hosp, Dept Pharm & Pharmacol, Louwesweg 6, NL-1066 EC Amsterdam, Netherlands..
    Kip, Anke E.
    Netherlands Canc Inst, Antoni Van Leeuwenhoek Hosp, Dept Pharm & Pharmacol, Louwesweg 6, NL-1066 EC Amsterdam, Netherlands..
    Younis, Brima M.
    Univ Khartoum, Inst Endem Dis, Khartoum, Sudan..
    Ellis, Sally J.
    Drugs Neglected Dis initiat, Geneva, Switzerland..
    Alves, Fabiana
    Drugs Neglected Dis initiat, Geneva, Switzerland..
    Beijnen, Jos. H.
    Netherlands Canc Inst, Antoni Van Leeuwenhoek Hosp, Dept Pharm & Pharmacol, Louwesweg 6, NL-1066 EC Amsterdam, Netherlands..
    Njenga, Simon
    Kenya Govt Med Res Ctr, Nairobi, Kenya..
    Kirigi, George
    Kenya Govt Med Res Ctr, Nairobi, Kenya..
    Hailu, Asrat
    Addis Ababa Univ, Addis Ababa, Ethiopia..
    Olobo, Joseph
    Makerere Univ, Kampala, Uganda..
    Musa, Ahmed M.
    Univ Khartoum, Inst Endem Dis, Khartoum, Sudan..
    Balasegaram, Manica
    Drugs Neglected Dis initiat, Geneva, Switzerland..
    Wasunna, Monique
    Drugs Neglected Dis initiat, Nairobi, Kenya..
    Karlsson, Mats O
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Khalil, Eltahir A. G.
    Univ Khartoum, Inst Endem Dis, Khartoum, Sudan..
    Visceral leishmaniasis relapse hazard is linked to reduced miltefosine exposure in patients from Eastern Africa: a population pharmacokinetic/pharmacodynamic study2017In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 72, no 11, p. 3131-3140Article in journal (Refereed)
    Abstract [en]

    Background: Low efficacy of miltefosine in the treatment of visceral leishmaniasis was recently observed in Eastern Africa. Objectives: To describe the pharmacokinetics and establish a pharmacokinetic/pharmacodynamic relationship for miltefosine in Eastern African patients with visceral leishmaniasis, using a time-to-event approach to model relapse of disease. Methods: Miltefosine plasma concentrations from 95 patients (48 monotherapy versus 47 combination therapy) were included in the population pharmacokinetic model using non-linear mixed effects modelling. Subsequently a time-to-event model was developed to model the time of clinical relapse. Various summary pharmacokinetic parameters (various AUCs, Time > EC50, Time > EC90), normalized within each treatment arm to allow simultaneous analysis, were evaluated as relapse hazard-changing covariates. Results: A two-compartment population model with first-order absorption fitted the miltefosine pharmacokinetic data adequately. Relative bioavailability was reduced (- 74%, relative standard error 4.7%) during the first week of treatment of the monotherapy arm but only the first day of the shorter combination regimen. Time to the relapse of infection could be described using a constant baseline hazard (baseline 1.8 relapses/year, relative standard error 72.7%). Miltefosine Time > EC90 improved the model significantly when added in a maximum effect function on the baseline hazard (half maximal effect with Time. > EC90 6.97 days for monotherapy). Conclusions: Miltefosine drug exposure was found to be decreased in Eastern African patients with visceral leishmaniasis, due to a (transient) initial lower bioavailability. Relapse hazard was inversely linked to miltefosine exposure. Significantly lower miltefosine exposure was observed in children compared with adults, further urging the need for implementation of dose adaptations for children.

  • 11.
    Fortin, Anny
    et al.
    Dafra Pharma Res & Dev, Slachthuisstr 30-7, Turnhout, Belgium.;McGill Univ, Dept Biochem, 1650 Cedar Ave, Montreal, PQ, Canada..
    Dorlo, Thomas P. C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Univ Utrecht, Utrecht Inst Pharmaceut Sci, Div Pharmacoepidemiol & Clin Pharmacol, Utrecht, Netherlands..
    Hendrickx, Sarah
    Univ Antwerp, Lab Microbiol Parasitol & Hyg LMPH, Univ Pl 1, Antwerp, Belgium..
    Maes, Louis
    Univ Antwerp, Lab Microbiol Parasitol & Hyg LMPH, Univ Pl 1, Antwerp, Belgium..
    Pharmacokinetics and pharmacodynamics of oleylphosphocholine in a hamster model of visceral leishmaniasis2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 7, p. 1892-1898Article in journal (Refereed)
    Abstract [en]

    Objectives: This study evaluated the pharmacokinetic properties of oleylphosphocholine (OlPC) in hamsters following a single oral dose. Its prophylactic activity was tested to establish exposure-activity relationships, while a 5+5 day oral regimen at 20 mg/kg with long post-treatment follow-up was performed to assess its curative potential. Methods: Single oral doses of 20, 50 and 100 mg/kg were administered for pharmacokinetic analysis while a 100 mg/kg single oral dose was given on day 7, 4 or 1, or 4 h prior to infection in the prophylactic efficacy study. The animals were infected on day 0 with Leishmania infantum and the resulting parasite burdens were measured in target organs on day 21. In the curative model, treatment started on day 21 post-infection at 20 mg/kg for 5+5 days and amastigote burdens were determined in target organs either on day 42 [10 days after the end of treatment (dpt)] or day 72 (40 dpt). Results: OlPC showed elimination t(1/2) of similar to 50 h and dose-proportional exposure. The prophylactic action of OlPC was in agreement with model-simulated drug exposures, showing dose-dependent residual activity. Interestingly, the 100 mg/kg single dose administered 4 days before infection (day -4) still reduced the overall parasite burden by similar to 50%. In the curative model, >99% clearance of infection was observed at 10 dpt in all OlPC-treated animals and remained so at 40 dpt. Conclusions: This study reveals that total plasma exposure (AUC(t-infinity)) correlates well with the prophylactic and curative efficacy of OlPC in the L. infantum hamster model.

  • 12.
    Goscinski, Gunilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Tano, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Löwdin, Elisabeth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Sjölin, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Propensity to release endotoxin after two repeated doses of cefuroxime in an in vitro kinetic model: Higher release after the second dose2007In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 60, no 2, p. 328-333Article in journal (Refereed)
    Abstract [en]

    Objectives: To study endotoxin release from two strains of Escherichia coli after exposure to two repeated doses of cefuroxime in an in vitro kinetic model.

    Methods: Cefuroxime in concentrations simulating human pharmacokinetics was added to the bacterial solution with a repeated dose after 12 h. In another experiment, tobramycin was given concomitantly with the second dose of cefuroxime. Samples for viable counts and endotoxin analyses were drawn before the addition of antibiotics and at 2 and 4 h after each dose.

    Results: The propensity to release endotoxin, expressed as log10 endotoxin release (EU)/log10 killed bacteria, was higher after the second than after the first dose, 0.80 ± 0.04 and 0.65 ± 0.01, respectively, in the ATCC strain and 0.80 ± 0.04 and 0.65 ± 0.02, respectively, in the clinical strain (P < 0.001). Endotoxin was released earlier after the second dose (P < 0.001). Addition of tobramycin at the second dose reduced the endotoxin release in comparison with that of cefuroxime alone (P < 0.001).

    Conclusions: The propensity to liberate endotoxin is higher after the second dose of cefuroxime than after the first, resulting in a higher release of endotoxin than expected from bacterial count. The release after the second dose can be reduced by the addition of tobramycin.

  • 13.
    Gustafsson, Ingegerd
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Sjölund, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Torell, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Johannesson, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Engstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Andersson, Dan I.
    Bacteria with increased mutation frequency and antibiotic resistance are enriched in the commensal flora of patients with high antibiotic usage2003In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 52, no 4, p. 645-650Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: We examined how prolonged antibiotic treatment affected the resistance and mutation frequency of human microflora isolated from intestine (Escherichia coli, enterococci spp.), pharynx (alpha-streptococci) and nostril (coagulase-negative staphylococci, CoNS).

    METHODS: Samples were collected from patients at the Center of Cystic Fibrosis (n=18) and the haematology ward (n=18) of the University Hospital, Uppsala, Sweden. The individually used amount of antibiotics for 1 year was recorded as the defined daily dose (DDD). Primary health care patients (n=30), with no antibiotic treatment for 1 year before sampling, were used as controls. Three isolates of each bacterium from each patient were examined. Antibiotic susceptibilities were determined by disc diffusion. Mutation frequencies to rifampicin resistance were measured on 30 independent cultures of each bacterial species from each individual by plating on rifampicin agar plates. For alpha-streptococci the mutation frequency to streptomycin resistance was also determined.

    RESULTS: Isolates from patients with high antibiotic use showed a pronounced shift towards increased resistance and a small but significant increase in the mutation frequency compared with isolates from the controls. For E. coli, enterococci and CoNS the increase in geometric mean mutation frequency in the patient group was 3-, 1.8- and 1.5-fold, respectively (P values 0.0001, 0.016 and 0.012). For alpha-streptococci there was a significant difference in geometric mean mutation frequency between patient and control groups for streptomycin resistance (P=0.024) but not for rifampicin resistance (P=0.74).

    CONCLUSIONS: High antibiotic use selected for commensals with highly increased resistance and a slight increase in mutation frequency.

  • 14.
    Hedenmalm, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Spigset, Olav
    Peripheral sensory disturbances related to treatment with fluoroquinolones1996In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 37, no 4, p. 831-837Article in journal (Refereed)
    Abstract [en]

    The symptoms and possible risk factors of peripheral sensory disturbances related to fluoroquinolones are reviewed on the basis of 37 reports submitted to the Swedish Adverse Drug Reactions Advisory Committee. In 25 patients (68%), symptoms occurred within 1 week after start of treatment. Paraesthesia was the most common complaint and occurred in 81% of the cases. Fifty-one per cent of the reports concerned numbness/hypoaesthesia, 27% pain/hyperaesthesia and 11% muscle weakness. Seventy-one per cent of the patients recovered within 2 weeks after drug discontinuation. Possible predisposing factors were impaired renal function, diabetes, lymphatic malignancy and treatment with another drug known to cause neuropathy.

  • 15.
    Hennig, Stefanie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Svensson, Elin M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Niebecker, Ronald
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fourie, P Bernard
    Weiner, Marc H
    Bonora, Stefano
    Peloquin, Charles A
    Gallicano, Keith
    Flexner, Charles
    Pym, Alex
    Vis, Peter
    Olliaro, Piero L
    McIlleron, Helen
    Karlsson, Mats O
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Population pharmacokinetic drug-drug interaction pooled analysis of existing data for rifabutin and HIV PIs2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, p. 1330-1340Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: Extensive but fragmented data from existing studies were used to describe the drug-drug interaction between rifabutin and HIV PIs and predict doses achieving recommended therapeutic exposure for rifabutin in patients with HIV-associated TB, with concurrently administered PIs.

    METHODS: Individual-level data from 13 published studies were pooled and a population analysis approach was used to develop a pharmacokinetic model for rifabutin, its main active metabolite 25-O-desacetyl rifabutin (des-rifabutin) and drug-drug interaction with PIs in healthy volunteers and patients who had HIV and TB (TB/HIV).

    RESULTS: Key parameters of rifabutin affected by drug-drug interaction in TB/HIV were clearance to routes other than des-rifabutin (reduced by 76%-100%), formation of the metabolite (increased by 224% in patients), volume of distribution (increased by 606%) and distribution to the peripheral compartment (reduced by 47%). For des-rifabutin, clearance was reduced by 35%-76% and volume of distribution increased by 67%-240% in TB/HIV. These changes resulted in overall increased exposure to rifabutin in TB/HIV patients by 210% because of the effects of PIs and 280% with ritonavir-boosted PIs.

    CONCLUSIONS: Given together with non-boosted or ritonavir-boosted PIs, rifabutin at 150 mg once daily results in similar or higher exposure compared with rifabutin at 300 mg once daily without concomitant PIs and may achieve peak concentrations within an acceptable therapeutic range. Although 300 mg of rifabutin every 3 days with boosted PI achieves an average equivalent exposure, intermittent doses of rifamycins are not supported by current guidelines.

  • 16.
    Khan, David D.
    et al.
    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.
    Nielsen, Elisabet I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    A pharmacokinetic-pharmacodynamic (PKPD) model based on in vitro time-kill data predicts the in vivo PK/PD index of colistin2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 7, p. 1881-1884Article in journal (Refereed)
    Abstract [en]

    Objectives: For antibiotics, extensive animal PKPD studies are often performed to evaluate the PK/PD driver for subsequent use when recommending dosing regimens. The aim of this work was to evaluate a PKPD model, developed based on in vitro time-kill data for colistin, in predicting the relationships between PK/PD indices and the bacterial killing previously observed in mice. Methods: An in silico PKPD model for Pseudomonas aeruginosa exposed to colistin was previously developed based on static in vitro time-kill data. The model was here applied to perform an in silico replication of an in vivo study where the effect of colistin on P. aeruginosa was studied in the thigh infection model. Concentration-time profiles of unbound colistin were predicted and used as input to drive the bacterial killing in the PKPD model. The predicted bacterial count at 24 h was related to each of the PK/PD indices and the results were compared with reported observations in vivo. Results: The model was found to adequately predict in vivo results from mice; both in terms of which PK/PD index best correlates to effect (fAUC/MIC) as well as the magnitude needed for a 2 log kill. The fAUC/MIC needed to achieve a 2 log reduction in bacterial counts after 24 h was here predicted to be 9 compared with 31 previously reported in vivo. Conclusions: This study provides further support that PKPD models based on longitudinal data can be a useful tool to make drug development more efficient within the infectious diseases area.

  • 17.
    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.

  • 18.
    Komp Lindgren, Patricia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Higgins, Paul G
    Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, 50935 Cologne, Germany .
    Seifert, Harald
    Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, 50935 Cologne, Germany .
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Prevalence of hypermutators among clinical Acinetobacter baumannii isolates2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 3, p. 661-665Article in journal (Refereed)
    Abstract [en]

    Objectives: The objectives of this study were to study the presence of mutators in a set of Acinetobacter baumannii isolates and to explore whether there is a correlation between mutation rates and antibiotic resistance.

    Methods: The variation in mutation rate was evaluated for 237 clinical A. baumannii isolates by determining the frequency of their mutation to rifampicin resistance. For each isolate, the antibiotic resistance profile was determined by disc diffusion and/or Etest. Isolates were divided into susceptible, resistant and MDR groups according to their resistance to five groups of different antibiotics. A comparison between differences in mutation frequency (f) and strain-specific factors was performed.

    Results: Of the 237 isolates 32%, 18% and 50% were classified as susceptible, resistant and MDR, respectively. The f of rifampicin resistance varied between 2.2×10210 and 1.2×1026 . Of the strains under investigation, 16% had an ≥2.5- to 166-fold higher f. The presence of mutators (definition ≥2.5-fold increase in f compared with ATCC 19606) in the MDR group (22%) was significantly higher (P,0.05) than that in the susceptible and resistant groups (11% and 7%, respectively). Furthermore, f was significantly higher in the MDR group compared with that in the susceptible and resistant groups.

    Conclusions: The facts that 26 of 37 mutator isolates (70%) in the population were MDR and that there was a significantly higher general f in isolates exhibiting an MDR profile suggest that hypermutability can be of advantage for the organism in a selective environment with extensive exposure to antimicrobials.

  • 19.
    Komp Lindgren, Patricia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Klockars, Oscar
    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.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Pharmacodynamic studies of nitrofurantoin against common uropathogens2015In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 70, no 4, p. 1076-1082Article in journal (Refereed)
    Abstract [en]

    Objectives

     To determine the pharmacokinetic/pharmacodynamic index that best correlates to nitrofurantoin's antibacterial effect, we studied nitrofurantoin activity against common causative pathogens in uncomplicated urinary tract infection (UTI).

    Methods 

    Five isolates [two Escherichia coli (one isolate producing the ESBL CTX-M-15), two Enterococcus faecium (including one that was vancomycin resistant) and one Staphylococcus saprophyticus] were used. The MICs of nitrofurantoin were determined by Etest. Time–kill curves with different concentrations of nitrofurantoin (based on multiples of isolate-specific MICs) were followed over 24 h. An in vitro kinetic model was used to simulate different time–concentration profiles, exposing E. coli to nitrofurantoin for varying proportions of the dosing interval. The outcome parameters reduction in cfu 0–24 h (Δcfu0–24) and the area under the bactericidal curve (AUBC), were correlated with time over MIC (T>MIC) and area under the antibiotic concentration curve divided by the MIC (AUC/MIC).

    Results 

    A bactericidal effect at varying static drug concentrations was achieved for all isolates. All isolates showed similar kill curve profiles. In the kinetic model, the effect of nitrofurantoin on E. coli displayed a 4 log reduction in cfu/mL within 6 h at 8 × MIC. The outcome parameters Δcfu0–24 and AUBC had a good correlation with T>MIC (R ≈ 0.83 and R ≈ 0.67, respectively), whereas log(AUC/MIC) was significantly poorer (R ≈ 0.39 andR ≈ 0.53, respectively).

    Conclusions 

    Nitrofurantoin was highly effective against E. coli and S. saprophyticus isolates; the killing effect against E. faecium was not as rapid, but still significant. Against E. coli, nitrofurantoin was mainly associated with a concentration-dependent action; this was confirmed in the kinetic model, in which T>MIC displayed the best correlation.

  • 20.
    Kostyanev, T.
    et al.
    Univ Antwerp, Dept Med Microbiol, Vaccine & Infect Dis Inst, B-2020 Antwerp, Belgium..
    Bonten, M. J. M.
    Univ Med Ctr Utrecht, Julius Ctr Hlth Sci & Primary Care, Utrecht, Netherlands..
    O'Brien, S.
    AstraZeneca, Infect Global Med Dev, Macclesfield, Cheshire, England..
    Steel, H.
    GlaxoSmithKline, Infect Dis Therapy Area Unit, London, England..
    Ross, S.
    GlaxoSmithKline, Infect Dis Therapy Area Unit, London, England..
    Francois, B.
    Ctr Hosp Univ Dupuytren, Limoges, France..
    Tacconelli, E.
    Univ Tubingen Hosp, DZIF TTU HAARBI, Infect Dis, Internal Med 1, Tubingen, Germany..
    Winterhalter, M.
    Jacobs Univ Bremen, Sch Sci & Engn, D-28759 Bremen, Germany..
    Stavenger, R. A.
    GlaxoSmithKline, Antibacterial Discovery Performance Unit, Collegeville, PA USA..
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Harbarth, S.
    Univ Geneva, Geneva, Switzerland.;Fac Med, Geneva, Switzerland..
    Hackett, J.
    AstraZeneca, Infect Global Med Dev, Gaithersburg, MD USA..
    Jafri, H. S.
    MedImmune, Gaithersburg, MD USA..
    Vuong, C.
    AiCuris GmbH & Co KG, Wuppertal, Germany..
    MacGowan, A.
    North Bristol NHS Trust & Publ Hlth England, Dept Infect Sci, Bristol Ctr Antimicrobial Res & Evaluat, Bristol, Avon, England..
    Witschi, A.
    Basilea Pharmaceut Int Ltd, Basel, Switzerland..
    Angyalosi, G.
    Novartis Pharma AG, Basel, Switzerland..
    Elborn, J. S.
    Queens Univ Belfast, Sch Med Dent & Biomed Sci, Belfast, Antrim, North Ireland..
    dewinter, R.
    Univ Med Ctr Utrecht, Julius Ctr Hlth Sci & Primary Care, Utrecht, Netherlands..
    Goossens, H.
    Univ Antwerp, Dept Med Microbiol, Vaccine & Infect Dis Inst, B-2020 Antwerp, Belgium.;Univ Antwerp Hosp, Lab Med Microbiol, Antwerp, Belgium..
    The Innovative Medicines Initiative's New Drugs for Bad Bugs programme: European public-private partnerships for the development of new strategies to tackle antibiotic resistance2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 2, p. 290-295Article, review/survey (Refereed)
    Abstract [en]

    Antibiotic resistance (ABR) is a global public health threat. Despite the emergence of highly resistant organisms and the huge medical need for new drugs, the development of antibacterials has slowed to an unacceptable level worldwide. Numerous government and non-government agencies have called for public-private partnerships and innovative funding mechanisms to address this problem. To respond to this public health crisis, the Innovative Medicines Initiative Joint Undertaking programme has invested more than a,not sign660 million, with a goal of matched contributions from the European Commission and the European Federation of Pharmaceutical Industries and Associations, in the development of new antibacterial strategies. The New Drugs for Bad Bugs (ND4BB) programme, an Innovative Medicines Initiative, has the ultimate goal to boost the fight against ABR at every level from basic science and drug discovery, through clinical development to new business models and responsible use of antibiotics. Seven projects have been launched within the ND4BB programme to achieve this goal. Four of them will include clinical trials of new anti-infective compounds, as well as epidemiological studies on an unprecedented scale, which will increase our knowledge of ABR and specific pathogens, and improve the designs of the clinical trials with new investigational drugs. The need for rapid concerted action has driven the funding of seven topics, each of which should add significantly to progress in the fight against ABR. ND4BB unites expertise and provides a platform where the commitment and resources required by all parties are streamlined into a joint public-private partnership initiative of unprecedented scale.

  • 21.
    Kubicek-Sutherland, Jessica Z.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lofton, Hava
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Vestergaard, Martin
    Univ Copenhagen, Dept Vet Dis Biol, Stigbojlen 4, DK-1870 Copenhagen C, Denmark..
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ingmer, Hanne
    Univ Copenhagen, Dept Vet Dis Biol, Stigbojlen 4, DK-1870 Copenhagen C, Denmark..
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Antimicrobial peptide exposure selects for Staphylococcus aureus resistance to human defence peptides2017In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 72, no 1, p. 115-127Article in journal (Refereed)
    Abstract [en]

    Background: The clinical development of antimicrobial peptides (AMPs) is currently under evaluation to combat the rapid increase in MDR bacterial pathogens. However, many AMPs closely resemble components of the human innate immune system and the ramifications of prolonged bacterial exposure to AMPs are not fully understood. Objectives: We show that in vitro serial passage of a clinical USA300 MRSA strain in a host-mimicking environment containing host-derived AMPs results in the selection of stable AMP resistance. Methods: Serial passage experimentswere conducted using steadily increasing concentrations of LL-37, PR-39 or wheat germ histones. WGS and proteomic analysis by MS were used to identify the molecular mechanism associated with increased tolerance of AMPs. AMP-resistant mutants were characterized by measuring in vitro fitness, AMP and antibiotic susceptibility, and virulence in a mouse model of sepsis. Results: AMP-resistant Staphylococcus aureus mutants often displayed little to no fitness cost and caused invasive disease in mice. Further, this phenotype coincided with diminished susceptibility to both clinically prescribed antibiotics and human defence peptides. Conclusions: These findings suggest that therapeutic use of AMPs could select for virulent mutants with crossresistance to human innate immunity as well as antibiotic therapy. Thus, therapeutic use of AMPs and the implications of cross-resistance need to be carefully monitored and evaluated.

  • 22. Kugelberg, Elisabeth
    et al.
    Löfmark, Sonja
    Wretlind, Bengt
    Andersson, Dan I
    Swedish Institute for Infectious Disease Control, Department of Bacteriology & Karolinska Institute, Stockholm .
    Reduction of the fitness burden of quinolone resistance in Pseudomonas aeruginosa2005In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 55, no 1, p. 22-30Article in journal (Refereed)
    Abstract [en]

    Objectives: Quinolone resistance in the opportunistic pathogen Pseudomonas aeruginosa is commonly caused by mutations that alter the target molecules DNAgyrase/topoisomerase IV, or cause activation of various efflux systems.We have analysed the effect of quinolone resistance caused by DNA gyrase/topoisomerase IV mutations on bacterial fitness.

    Methods: Norfloxacin-resistant mutants were isolated and by DNA sequencing the mutations conferring resistance were identified. Mutant fitnesswas determined by measuring growth rates in vitro. Mutants with reducedgrowth rates were serially passaged to obtain growth-compensatedmutants. The level of DNA supercoiling was determined by isolatingplasmid DNA from the susceptible, resistant and compensated mutants andcomparing the topoisomer distribution patterns by gel electrophoresis inthe presence of chloroquine.

    Results: Low-level resistance (4-48 mg/L) was caused by single mutations in gyrA or gyrB. Among these strains, three out of eight mutants showed lower fitness,whereas high-level resistant (>256 mg/L) mutants with doublemutations in gyrA and parC, parE, nfxB or unknown genes all showed areduced fitness. Slow-growing resistantmutants with a gyrA mutation had decreased DNA supercoiling. Afterserial passage in laboratory medium, mutant fitnesswas increased by compensatory mutation(s) that restored supercoiling tonormal levels. The compensatory mutation(s) was not located in any ofthe genes (gyrAB, topA, parCE, hupB, fis, hupN, himAD or PA5348) thatwere expected to affect supercoiling.

    Conclusions: Our results show that 'no cost' and compensatory mutations are common in quinolone-resistant P. aeruginosa.

  • 23.
    Lagerbäck, Pernilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Khine, Wei W. T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Giske, C. G.
    Karolinska Inst, Karolinska Univ Hosp, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Tängdén, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Evaluation of antibacterial activities of colistin, rifampicin and meropenem combinations against NDM-1-producing Klebsiella pneumoniae in 24 h in vitro time-kill experiments2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 8, p. 2321-2325Article in journal (Refereed)
    Abstract [en]

    To investigate the activity of colistin alone or in double and triple combination with rifampicin and meropenem against NDM-1-producing Klebsiella pneumoniae. Eight isolates of NDM-1-producing K. pneumoniae were exposed to clinically relevant antibiotic concentrations in 24 h time-kill experiments. Three colistin concentrations were used for two of the strains. Resistance development was assessed with population analysis and sequencing of the mgrB and pmrB genes. Initial killing was achieved with colistin alone, but with considerable regrowth at 24 h. Combinations including colistin and rifampicin were bacteriostatic or bactericidal against all strains. Colistin plus meropenem was bactericidal against one strain, but, overall, meropenem showed little additive effects. Higher concentrations of colistin did not enhance antibacterial activity. Resistant populations and deletion or mutations in the mgrB and pmrB genes were frequently detected in endpoint samples after exposure to colistin alone. Based on the results of this and previous studies, the combination of colistin and rifampicin seems promising and should be further explored in vivo and considered for clinical evaluation. Meropenem seems less useful in the treatment of infections caused by high-level carbapenem-resistant NDM-1-producing K. pneumoniae. Higher colistin concentrations did not result in significantly better activity, suggesting that combination therapy might be superior to monotherapy also when colistin is prescribed using high-dose regimens in accordance with current recommendations.

  • 24.
    Liakopoulos, Apostolos
    et al.
    CVI Wageningen Univ, Dept Bacteriol & Epidemiol, Lelystad, Netherlands..
    Mevius, Dik J.
    CVI Wageningen Univ, Dept Bacteriol & Epidemiol, Lelystad, Netherlands.;Univ Utrecht, Dept Infect Dis & Immunol, Fac Vet Med, Utrecht, Netherlands..
    Olsen, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bonnedahl, Jonas
    Kalmar Cty Hosp, Dept Infect Dis, Kalmar, Sweden..
    The colistin resistance mcr-1 gene is going wild2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 8, p. 2335-2336Article in journal (Refereed)
  • 25.
    Lignell, Anders
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Löwdin, Elisabeth
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Cars, Otto
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Sjölin, Jan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Characterization of the inhibitory effect of voriconazole on the fungicidal activity of amphotericin B against Candida albicans in an in vitro kinetic model2008In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 62, no 1, p. 142-8Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: The aim of the present investigation was to study and characterize the effect of voriconazole on the fungicidal activity of amphotericin B. METHODS: Four strains of Candida albicans susceptible to voriconazole were exposed to voriconazole and amphotericin B, either alone, simultaneously or sequentially in an in vitro kinetic model. Bolus doses resulting in voriconazole and amphotericin B concentrations of 0.005-5 and 2.5 mg/L, respectively, were administered. Antifungal-containing RPMI 1640 was eliminated and replaced by a fresh medium using a peristaltic pump, with a flow rate adjusted to obtain the desired half-lives. With two drugs tested, a computer-controlled dosing pump compensated for differences in the elimination rates. Using static time-kill methodology, one C. albicans strain was exposed to 5 mg/L voriconazole for varying durations followed by 2.5 mg/L amphotericin B after three repeated washes of voriconazole. RESULTS: Voriconazole and amphotericin B treatment alone resulted in fungistatic and fungicidal activities, respectively. Simultaneous administration of voriconazole and amphotericin B resulted in fungicidal activity, whereas only fungistatic activity was observed when repeated doses of amphotericin B were administered sequentially after voriconazole at 24-96 h. The inhibition of the fungicidal activity of amphotericin B was voriconazole dose-dependent, but seemed to be recovered once the voriconazole concentration fell below the MIC. The fungicidal activity was quickly regained after the removal of voriconazole, irrespective of the duration of voriconazole pre-exposure. CONCLUSIONS: Voriconazole inhibited the fungicidal effect of sequentially administered amphotericin B in a concentration- and time-dependent manner; the clinical significance of this needs further investigation.

  • 26.
    Linkevicius, Marius
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anderssen, Jytte Mark
    Statens Serum Institut.
    Sandegren, Linus
    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.
    Fitness of Escherichia coli mutants with reduced susceptibility to tigecycline2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, p. 1307-1313Article in journal (Refereed)
    Abstract [en]

    The objective of this study was to determine the fitness of Escherichia coli mutants with reduced susceptibility to tigecycline after exposure to adverse conditions in vitro and in vivo. Survival in response to low pH, bile salts, oxidative stress and human serum was examined for E. coli mutants with reduced susceptibility to tigecycline due to single mutations that caused increased efflux (marR, lon) or impaired LPS (rfaC, rfaE, lpcA). An in vitro competition assay was used to determine growth fitness defects. Competitive fitness was assessed using mouse infection models. MICs, exponential growth rates and expression levels of efflux-related genes were measured for genetically reconstructed double and triple mutants. The LPS mutants were 48-85-fold more susceptible to bile salts compared with the ERN mutants and the WT. As shown by in vitro competitions, the fitness reduction was 0.3%-13% for ERN mutants and similar to 24% for LPS mutants. During in vivo survival experiments, LPS mutants were outcompeted by the WT strain in the thigh infection model. Constructed double ERN and LPS mutants showed additive and synergistic increases in tigecycline MICs. Generally, reduced susceptibility to tigecycline caused a decrease in fitness under stressful in vitro and in vivo conditions with ERN mutants being fitter than LPS mutants. When combined, ERN mutations caused a synergistic increase in the MIC of tigecycline. These findings could explain why clinical resistance to tigecycline in E. coli is mainly associated with up-regulation of the AcrAB efflux system.

  • 27.
    Linkevicius, Marius
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    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.
    Mechanisms and fitness costs of tigecycline resistance in Escherichia coli2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 12, p. 2809-2819Article in journal (Refereed)
    Abstract [en]

    Objectives: To stepwise select tigecycline-resistant Escherichia coli mutants in vitro, determine the mutation rates, identify the resistance mechanisms, determine the resistance level and cross-resistance to other antibiotic classes, evaluate the fitness costs of tigecycline resistance mechanisms and investigate if the same in vitro-identified target genes were mutated in clinical isolates.

    Methods: Spontaneous mutants with reduced susceptibility to tigecycline were selected on agar plates supplemented with tigecycline. Resistance levels and cross-resistance were evaluated by performing MIC assays and determining mutation rates using Luria-Delbruck fluctuation tests. Mutant fitness was estimated by measuring exponential growth rates, lag phase and total yield. Illumina whole-genome sequencing was used to identify mutations increasing MICs of tigecycline.

    Results: Spontaneous mutants with reduced susceptibility to tigecycline were selected at a rate of similar to 10-8 to 10-6 per cell per generation; however, the clinical MIC breakpoint was not reached. The resistance level of tigecycline was low and some of the mutants had elevated MICs of hydrophobic drugs (chloramphenicol, erythromycin and novobiocin) or decreased MICs of SOS response inducers (ciprofloxacin and nitrofurantoin). Mutations were identified in efflux regulatory network genes (lon, acrR and marR) or lipopolysaccharide core biosynthesis pathway genes (lpcA, rfaE, rfaD, rfaC and rfaF). Mutations in the same target genes were found in clinical isolates.

    Conclusions: Tigecycline selects for low-level resistance mutations with relatively high mutation rates and the majority of them come with a substantial fitness cost. Further in vivo experiments are needed to evaluate how these mutations affect bacterial virulence and ability to establish a successful infection.

  • 28.
    Lofton, Hava
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anwar, Naeem
    Rhen, Mikael
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fitness of Salmonella mutants resistant to antimicrobial peptides2015In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 70, no 2, p. 432-440Article in journal (Refereed)
    Abstract [en]

    Objectives: To examine the effects of mutations in the waaY, phoP and pmrB genes, which confer resistance to antimicrobial peptides (AMPs), on fitness of Salmonella Typhimurium. Methods: Survival during low pH, oxidative stress, stationary-phase incubation, exposure to serum and bile and growth in mice and laboratory media were determined by time-kills, disc inhibition assays, competition experiments and optical density measurements. Results: Individual mutations in the waaY gene (involved in LPS core biosynthesis) and in the phoP and pmrB genes (part of two different two-component regulatory systems, phoPQ and pmrAB) conferred no or minor effects on bacterial survival during stressful in vitro conditions or in mice. In contrast, a waaY-phoP-pmrB triple mutant was compromised under most assay conditions. Conclusions: Results from this study show that AMP resistance can be cost-free, as assessed by several assays that attempt to mimic the conditions a bacterium might encounter within a host. Our findings imply that future therapeutic use of AMPs could select for fit mutants with cross-resistance to human defence peptides and that potential resistance development in response to therapeutic use of AMPs needs to be carefully monitored.

  • 29.
    Marcusson, Linda L
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Olofsson, Sara K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Komp Lindgren, Patricia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mutant prevention concentrations of ciprofloxacin for urinary tract infection isolates of Escherichia coli2005In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 55, no 6, p. 938-943Article in journal (Refereed)
    Abstract [en]

    Objectives: To measure the mutant prevention concentration (MPC) of ciprofloxacin for a set of urinary tract infection (UTI) Escherichia coli isolates with different levels of susceptibility and determine whether MPC can be predicted from MIC.

    Methods: MPC was defined as the lowest ciprofloxacin concentration that prevented the growth of resistant colonies when 1010 bacteria were spread on solid medium and incubated for 96 h at 37°C. MIC was measured by Etest. Bacteria surviving (persisting) at MPC were isolated and quantified from agar plugs taken after 96 h. The genes hipA and hipB were amplified by PCR from persisters and sequenced.

    Results: Isolates with MICs above the NCCLS breakpoint for ciprofloxacin resistance (4 mg/L) typically have MPCs greater than 32 mg/L. Isolates with MICs below the breakpoint for ciprofloxacin susceptibility (1 mg/L) have MPCs up to 5 mg/L. MPC/MIC is ∼16 for most susceptible isolates but there are several notable exceptions (MPC/MIC > 100). Resistant colonies arising one dilution step below MPC often had MIC > MPC. In every case tested, a proportion of cells survived (persisted), but did not grow into colonies, at MPC, without any increase in MIC.

    Conclusions: MPCs were determined for all ciprofloxacin-susceptible isolates. MPC is not accurately predicted from MIC. Colonies selected below MPC frequently have MIC > MPC, suggesting multiple mutations. A small fraction of cells from all strains tested survived for 96 h at MPC, without any associated increase in MIC. These survivors/persisters are not hipAB mutants.

  • 30.
    Meftahi, Nedra
    et al.
    Univ Tunis El Manar, Inst Pasteur Tunis, Lab Mol Microbiol Vaccinol & Biotechnol Dev, Unit Typing & Genet Mycobacteria, Tunis, Tunisia.
    Namouchi, Amine
    Univ Tunis El Manar, Inst Pasteur Tunis, Lab Mol Microbiol Vaccinol & Biotechnol Dev, Unit Typing & Genet Mycobacteria, Tunis, Tunisia.
    Mhenni, Besma
    Univ Tunis El Manar, Inst Pasteur Tunis, Lab Mol Microbiol Vaccinol & Biotechnol Dev, Unit Typing & Genet Mycobacteria, Tunis, Tunisia.
    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.
    Mardassi, Helmi
    Univ Tunis El Manar, Inst Pasteur Tunis, Lab Mol Microbiol Vaccinol & Biotechnol Dev, Unit Typing & Genet Mycobacteria, Tunis, Tunisia.
    Evidence for the critical role of a secondary site rpoB mutation in the compensatory evolution and successful transmission of an MDR tuberculosis outbreak strain2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 2, p. 324-332Article in journal (Refereed)
    Abstract [en]

    Background MDR Mycobacterium tuberculosis clinical strains that cause large outbreaks, particularly among HIV-negative patients, are likely to have undergone the most successful compensatory evolution. Hence, mutations secondary to the acquisition of drug resistance are worthy of consideration in these highly transmissible strains. Here, we assessed the role of a mutation within rpoBrpoB V615M, secondary to the rifampicin resistance-conferring mutation rpoB S531L, which is associated with a major MDR tuberculosis outbreak strain that evolved in an HIV-negative context in northern Tunisia.

    Methods Using BCG as a model organism, we engineered strains harbouring either the rpoB S531L mutation alone or the double mutation rpoB S531L, V615M. Individual and competitive in vitro growth assays were performed in order to assess the relative fitness of each BCG mutant.

    Results The rpoB V615M mutation was found to be invariably associated with rpoB S531L. Structural analysis mapped rpoB V615M to the same bridge helix region as rpoB compensatory mutations previously described in Salmonella. Compared with the rpoB single-mutant BCG, the double mutant displayed improved growth characteristics and fitness rates equivalent to WT BCG. Strikingly, the rpoB double mutation conferred high-level resistance to rifampicin.

    Conclusions Here, we demonstrated the fitness compensatory role of a mutation within rpoB, secondary to the rifampicin resistance mutation rpoB S531L, which is characteristic of an MDR M. tuberculosis major outbreak strain. The finding that this secondary mutation concomitantly increased the resistance level to rifampicin argues for its significant contribution to the successful transmission of the MDR-TB strain.

  • 31.
    Melhus, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Effects of amoxicillin on the expression of cytokines during experimental acute otitis media caused by non-typeable Haemophilus influenzae2001In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 48, no 3, p. 397-402Article in journal (Refereed)
    Abstract [en]

    Antibiotics are frequently prescribed when a diagnosis of acute otitis media (AOM) is made in childhood, but the effects of antibiotics on host-parasite interactions in the middle ear are not well defined. A rat model and PCR techniques were used to explore host responses during amoxicillin treatment of AOM caused by non-typeable Haemophilus influenzae (NTHi). The 5 day course of amoxicillin initiated at the otomicroscopic peak of the infection eradicated the bacteria and induced significant changes in the expression of cytokines. Interleukin (IL)-6, tumour necrosis factor-alpha and IL-10 were upregulated by the treatment, and the downregulation was slower than during the natural course. Amoxicillin inhibited the upregulation of transforming growth factor-beta, whereas IL-1alpha expression remained unaffected by the treatment. By comparing inflammatory host responses during treated and untreated NTHi AOM, new targets for modification of the course, or more specified and individualized treatments, may evolve.

  • 32.
    Mohamed, Ami F
    et al.
    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.
    Friberg, Lena E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    A pharmacokinetic/pharmacodynamic model developed for the effect of colistin on Pseudomonas aeruginosa in vitro with evaluation of population pharmacokinetic variability on simulated bacterial killing2014In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 69, no 5, p. 1350-1361Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: An optimized dosing regimen of the prodrug of colistin, colistin methanesulphonate (CMS), against resistant Pseudomonas aeruginosa is needed to ensure effective bacterial killing. The objectives of this study were to develop a pharmacokinetic (PK)/pharmacodynamic (PD) model that characterizes the time course of the antibacterial activity of colistin against P. aeruginosa in a static in vitro system and to perform simulations of different dosing regimens and dosing algorithms to evaluate the effect of interindividual variability and interoccasion variability in PK on bacterial killing.

    METHODS: Static in vitro time-kill curve experiments were conducted on two different strains of P. aeruginosa (MIC 1 and 1.5 mg/L). Mechanism-based PK/PD models were fitted in NONMEM7 and the final model was combined with a previously developed population PK model of CMS and colistin to perform simulations of variability based on different dosing algorithms.

    RESULTS: A model with compartments for growing and resting bacteria, with a function allowing the maximal bacterial killing of colistin to reduce upon increasing colistin exposure, characterized both the fast bactericidal effect and the adaptive resistance. The variability in PK was shown to translate into pronounced interoccasion variability in bacterial killing. A flat fixed loading dose was demonstrated to result in less variability than an algorithm based on weight.

    CONCLUSIONS: The developed PK/PD model described the growth, death and resistance development of P. aeruginosa in response to colistin for two different strains. Based on simulations, a flat fixed loading dose followed by an 8 or 12 hourly maintenance dose with an infusion duration of up to 2 h appeared adequate.

  • 33.
    Mohamed, Ami F
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Kristoffersson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Karvanen, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Nielsen, Elisabet
    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.
    Friberg, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Dynamic interaction of colistin and meropenem on a WT and a resistant strain of Pseudomonas aeruginosa as quantified in a PK/PD model2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, p. 1279-1290Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: Combination therapy can be a strategy to ensure effective bacterial killing when treating Pseudomonas aeruginosa, a Gram-negative bacterium with high potential for developing resistance. The aim of this study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model that describes the in vitro bacterial time-kill curves of colistin and meropenem alone and in combination for one WT and one meropenem-resistant strain of P. aeruginosa.

    METHODS: In vitro time-kill curve experiments were conducted with a P. aeruginosa WT (ATCC 27853) (MICs: meropenem 1 mg/L; colistin 1 mg/L) and a meropenem-resistant type (ARU552) (MICs: meropenem 16 mg/L; colistin 1.5 mg/L). PK/PD models characterizing resistance were fitted to the observed bacterial counts in NONMEM. The final model was applied to predict the bacterial killing of ARU552 for different combination dosages of colistin and meropenem.

    RESULTS: A model with compartments for growing and resting bacteria, where the bacterial killing by colistin reduced with continued exposure and a small fraction (0.15%) of the start inoculum was resistant to meropenem, characterized the bactericidal effect and resistance development of the two antibiotics. For a typical patient, a loading dose of colistin combined with a high dose of meropenem (2000 mg q8h) was predicted to result in a pronounced kill of the meropenem-resistant strain over 24 h.

    CONCLUSIONS: The developed PK/PD model successfully described the time course of bacterial counts following exposures to colistin and meropenem, alone and in combination, for both strains, and identified a dynamic drug interaction. The study illustrates the application of a PK/PD model and supports high-dose combination therapy of colistin and meropenem to overcome meropenem resistance.

  • 34. Mouton, Johan W.
    et al.
    Theuretzbacher, Ursula
    Craig, William A.
    Tulkens, Paul M.
    Derendorf, Hartmut
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Tissue concentrations: do we ever learn?2008In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 61, no 2, p. 235-237Article in journal (Refereed)
    Abstract [en]

    Over the last decades, numerous papers have appeared--and still are appearing--that describe concentrations in tissues in an effort to predict the efficacy of an antimicrobial agent based on these concentrations and MICs for microorganisms. A common method is to use measurements of concentrations in tissue homogenates, comparing these with values derived from the corresponding blood samples and on that basis draw conclusions with respect to the potential clinical use of the drug. This approach is not justifiable for a number of reasons that includes both pharmacokinetic as well as pharmacodynamic causes. This way of presenting data with the derived conclusions is often misleading and may ultimately be harmful in patient care.

  • 35.
    Nazir, Humera
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hasan, Fariha
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Can phylogenetic type predict resistance development?2011In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 66, no 4, p. 778-787Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES:

    To determine whether phylogenetic type is associated with the development of multidrug resistance to antibiotics.

    METHODS:

    Urinary tract infection (UTI) isolates from three hospitals in Pakistan were collected over a period of 10 months, and analysed in terms of causative bacterial species and drug susceptibility.

    RESULTS:

    Multidrug resistance was widespread and resistance frequencies were >50% for several of the most commonly used antibiotics, including ciprofloxacin and third-generation cephalosporins for Escherichia coli isolates. The great majority of E. coli isolates remained susceptible to meropenem and fosfomycin. Sixty E. coli isolates were analysed in detail to determine correlations between resistance phenotypes and genotypes, mutation rates and phylogenetic group. Most isolates had elevated mutation rates, suggesting this was being selected. The majority of ciprofloxacin-resistant isolates carried a specific set of mutations in the quinolone resistance-determining region of gyrA and parC (S83L, D87N, S80I and E84V). In addition, 67% of the ciprofloxacin-resistant E. coli isolates carried one or more horizontally transmissible determinants of resistance to ciprofloxacin, including aac(6')-Ib-cr, qepA, qnrA and qnrB. There was a significant correlation between resistance to third-generation cephalosporins, being an extended-spectrum β-lactamase producer, being resistant to ciprofloxacin and belonging to phylogenetic group B2.

    CONCLUSIONS:

    The data suggest that features of the bacterial genotype might facilitate the development of multidrug resistance in particular lineages. Better understanding of the mechanistic basis for correlations between drug resistance and genotype could potentially be exploited to develop molecular tools for the prediction of resistance development.

  • 36.
    Nazir, Humera
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hasan, Fariha
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Can phylogenetic type predict resistance development?2011In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 66, no 4, p. 778-787Article in journal (Refereed)
    Abstract [en]

    Objectives: To determine whether phylogenetic type is associated with the development of multidrug resistance to antibiotics. Methods: Urinary tract infection (UTI) isolates from three hospitals in Pakistan were collected over a period of 10 months, and analysed in terms of causative bacterial species and drug susceptibility. Results: Multidrug resistance was widespread and resistance frequencies were > 50% for several of the most commonly used antibiotics, including ciprofloxacin and third-generation cephalosporins for Escherichia coli isolates. The great majority of E. coli isolates remained susceptible to meropenem and fosfomycin. Sixty E. coli isolates were analysed in detail to determine correlations between resistance phenotypes and genotypes, mutation rates and phylogenetic group. Most isolates had elevated mutation rates, suggesting this was being selected. The majority of ciprofloxacin-resistant isolates carried a specific set of mutations in the quinolone resistance-determining region of gyrA and parC (S83L, D87N, S80I and E84V). In addition, 67% of the ciprofloxacin-resistant E. coli isolates carried one or more horizontally transmissible determinants of resistance to ciprofloxacin, including aac(6')-Ib-cr, qepA, qnrA and qnrB. There was a significant correlation between resistance to third-generation cephalosporins, being an extended-spectrum beta-lactamase producer, being resistant to ciprofloxacin and belonging to phylogenetic group B2. Conclusions: The data suggest that features of the bacterial genotype might facilitate the development of multidrug resistance in particular lineages. Better understanding of the mechanistic basis for correlations between drug resistance and genotype could potentially be exploited to develop molecular tools for the prediction of resistance development.

  • 37.
    Nicoloff, Herve
    et al.
    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.
    Indirect resistance to several classes of antibiotics in cocultures with resistant bacteria expressing antibiotic-modifying or -degrading enzymes2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 1, p. 100-110Article in journal (Refereed)
    Abstract [en]

    Objectives: The detection of indirect resistance in vitro suggests that coinfecting bacteria from the normal microbial flora may have a more important negative impact during antimicrobial therapy than is generally appreciated.Indirect resistance (IR), the ability of an antibiotic-resistant population of bacteria to protect a susceptible population, has been previously observed for beta-lactamase-producing bacteria and associated with antimicrobial treatment failures. Here, we determined whether other resistance determinants could cause IR in the presence of five other classes of antibiotics. Methods: A test was designed to detect IR and 14 antibiotic resistance genes were tested in the presence of 13 antibiotics from six classes. A bioassay was used to measure the ability of resistance-causing enzymes to decrease the concentration of active antibiotics in the medium. Results: We confirmed IR in the presence of beta-lactam antibiotics (ampicillin and mecillinam) when TEM-1A was expressed. We found that bacteria expressing antibiotic-modifying or -degrading enzymes Ere(A), Tet(X2) or CatA1 caused IR in the presence of macrolides (erythromycin and clarithromycin), tetracyclines (tetracycline and tigecycline) and chloramphenicol, respectively. IR was not observed with resistance determinants that did not modify or destroy antibiotics or with enzymes modifying aminoglycosides or degrading fosfomycin. IR was dependent on the resistance enzymes decreasing the concentration of active antibiotics in the medium, hence allowing nearby susceptible bacteria to resume growth once the antibiotic concentration fell below their MIC. Conclusions: IR was not limited to beta-lactamase-producing bacteria, but was also caused by resistant bacteria carrying cytoplasmic antibiotic-modifying or -degrading enzymes that catalyse energy-consuming reactions requiring complex cellular cofactors. Our results suggest that IR is common and further emphasizes that coinfecting agents and the human microflora can have a negative impact during antimicrobial therapy.

  • 38.
    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.

  • 39. Nielsen, Karen L.
    et al.
    Pedersen, Thomas M.
    Udekwu, Klas I.
    Petersen, Andreas
    Skov, Robert L.
    Hansen, Lars H.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Frimodt-Moller, Niels
    Fitness cost: a bacteriological explanation for the demise of the first international methicillin-resistant Staphylococcus aureus epidemic2012In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 67, no 6, p. 1325-1332Article in journal (Refereed)
    Abstract [en]

    Denmark and several other countries experienced the first epidemic of methicillin-resistant Staphylococcus aureus (MRSA) during the period 196575, which was caused by multiresistant isolates of phage complex 83A. In Denmark these MRSA isolates disappeared almost completely, being replaced by other phage types, predominantly only penicillin resistant. We investigated whether isolates of this epidemic were associated with a fitness cost, and we employed a mathematical model to ask whether these fitness costs could have led to the observed reduction in frequency. Bacteraemia isolates of S. aureus from Denmark have been stored since 1957. We chose 40 S. aureus isolates belonging to phage complex 83A, clonal complex 8 based on spa type, ranging in time of isolation from 1957 to 1980 and with varyous antibiograms, including both methicillin-resistant and -susceptible isolates. The relative fitness of each isolate was determined in a growth competition assay with a reference isolate. Significant fitness costs of 215 were determined for the MRSA isolates studied. There was a significant negative correlation between number of antibiotic resistances and relative fitness. Multiple regression analysis found significantly independent negative correlations between fitness and the presence of mecA or streptomycin resistance. Mathematical modelling confirmed that fitness costs of the magnitude carried by these isolates could result in the disappearance of MRSA prevalence during a time span similar to that seen in Denmark. We propose a significant fitness cost of resistance as the main bacteriological explanation for the disappearance of the multiresistant complex 83A MRSA in Denmark following a reduction in antibiotic usage.

  • 40.
    Niward, Katarina
    et al.
    Linkoping Univ Hosp, Dept Infect Dis, S-58185 Linkoping, Sweden.;Linkoping Univ, Dept Clin & Expt Med, Linkoping, Sweden..
    Aengeby, Kristian
    Karolinska Univ Hosp, Dept Clin Microbiol, Stockholm, Sweden.;Univ W Indies, Dept Microbiol, Kingston 7, Jamaica..
    Chryssanthou, Erja
    Karolinska Univ Hosp, Dept Clin Microbiol, Stockholm, Sweden.;Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Paues, Jakob
    Linkoping Univ Hosp, Dept Infect Dis, S-58185 Linkoping, Sweden.;Linkoping Univ, Dept Clin & Expt Med, Linkoping, Sweden..
    Bruchfeld, Judith
    Karolinska Univ Hosp, Infect Dis Unit, Stockholm, Sweden.;Karolinska Inst, Dept Med, Solna, Sweden..
    Jureen, Pontus
    Publ Hlth Agcy Sweden, Stockholm, Sweden..
    Giske, Christian G.
    Karolinska Univ Hosp, Dept Clin Microbiol, Stockholm, Sweden.;Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Kahlmeter, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Vaxjo Hosp, Dept Clin Microbiol, Vaxjo, Sweden..
    Schon, Thomas
    Kalmar Cty Hosp, Dept Clin Microbiol & Infect Dis, S-39185 Kalmar, Sweden.;Linkoping Univ, Dept Med Microbiol, S-58185 Linkoping, Sweden..
    Susceptibility testing breakpoints for Mycobacterium tuberculosis categorize isolates with resistance mutations in gyrA as susceptible to fluoroquinolones: implications for MDR-TB treatment and the definition of XDR-TB2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 2, p. 333-338Article in journal (Refereed)
    Abstract [en]

    Fluoroquinolones (FQs) are important in the treatment of MDR-TB and in the definition of XDR-TB. Our objective was to investigate how discrepancies in the phenotypic and genotypic methods for antimicrobial susceptibility testing could affect the interpretation of antimicrobial susceptibility test results. We analysed MICs of ofloxacin and levofloxacin in Middlebrook 7H10 broth (7H10) as well as sequencing of the quinolone resistance-determining region of the gyrA gene and the MTBDRsl assay in 75 resistant isolates, including MDR and XDR strains of Mycobacterium tuberculosis. Among 75 resistant isolates, 27 had mutations associated with FQ resistance. Among isolates with resistance mutations in gyrA, 26% (seven of 27) were susceptible to levofloxacin and ofloxacin by phenotypic testing at 1 mg/L and 2 mg/L. The most common mutation was in codon 94 and these isolates had significantly increased MICs of levofloxacin (2-8 mg/L) compared with isolates with mutations in codon 90 (0.25-2 mg/L, PaEuroS < aEuroS0.05). The sensitivity and specificity for the MTBDRsl assay compared with gyrA sequencing were 96% and 98%, respectively. Current critical concentrations may classify up to 26% of isolates with gyrA mutations as susceptible to FQs due to a close relationship between susceptible and resistant populations. These results should be considered while improving clinical breakpoints for M. tuberculosis and may have an impact on the definition of XDR-TB.

  • 41. Nordin, Sara L
    et al.
    Sonesson, Andreas
    Malmsten, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Mörgelin, Matthias
    Egesten, Arne
    The epithelium-produced growth factor midkine has fungicidal properties2012In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 67, no 8, p. 1927-1936Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES

    The skin encounters many potential pathogens present in the environment, where Candida spp. are among the most common causes of fungal infestation. Midkine (MK) is a heparin-binding growth factor that is constitutively produced in the epidermis and this study looks at the antifungal activity of MK, potential co-localization and mode of action of MK.

    METHODS AND RESULTS

    We show that MK is expressed in association with fungal infections of the skin. In vitro, MK showed strong fungicidal activity against Candida albicans and Candida parapsilosis. Scanning electron microscopy of fungi revealed blebbing and leakage of intracellular contents, indicating membrane interactions. Immunoelectron microscopy showed accumulation of MK in association with the membrane, but also a high degree of internalization, suggesting intracellular targets as well. Using liposome models mimicking fungal and human cell membranes (i.e. ergosterol- and cholesterol-containing membranes, respectively), MK was found to disrupt ergosterol-containing membranes to a higher degree than cholesterol-containing vesicles. Addition of increasing concentrations of salt caused a partial and dose-dependent decrease in the fungicidal activity exerted by MK in parallel with a decreased affinity for the yeast. However, at salt concentrations similar to those of an epithelial context (i.e. 50-100 mM), MK retained most of its fungicidal activity, in contrast to that of plasma (150 mM).

    CONCLUSIONS

    The findings suggest that MK plays a role in host defence against fungal infections and could serve as a template for development of novel antifungal treatments.

  • 42.
    Odenholt, Inga
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Pharmacodynamics of moxifloxacin and levofloxacin against Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli: simulation of human plasma concentrations after intravenous dosage in an in vitro kinetic model2006In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 58, no 5, p. 960-965Article in journal (Refereed)
    Abstract [en]

    Objectives: To compare in an in vitro kinetic model the pharmacodynamics of moxifloxacin and levofloxacin with a concentration-time profile simulating the human free non-protein bound concentrations of 400 mg moxifloxacin intravenous (iv) once daily, 500 mg levofloxacin iv once daily and 750 mg levofloxacin iv once daily against strains of Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli with variable susceptibility to fluoroquinolones.

    Methods: The strains used in the study included S. pneumoniae ATCC 6306 (native strain), S. pneumoniae 19397 (double mutation; gyrA and parC), S. pneumoniae 4241 (single mutation; parC), S. aureus ATCC 13709 (native strain), S. aureus MB5 (single mutation; gyrA), E. coli M12 (single mutation; gyrA), E. coli ATCC 25922 (native strain) and K. pneumoniae ATCC 29655 (native strain). The strains were exposed to moxifloxacin and levofloxacin in an in vitro kinetic model simulating the free human serum concentration-time profile of moxifloxacin 400 mg once daily, levofloxacin 500 mg once daily and 750 mg once daily. Repeated samples were taken regularly during 24 h and viable counts were carried out.

    Results and conclusions: A correlation was seen between both the area under the serum concentration curve and MIC (AUC/MIC) and the peak concentration/MIC (C-max/MIC) versus area under the bactericidal killing curve (AUBKC) or Delta log(0-24) cfu/mL. Compiling all data, an AUC/MIC of similar to 100 and a Cmax/MIC of 10 gave a maximal bactericidal effect for both levofloxacin and moxifloxacin. In accordance with the results from others, our study indicated that a lower AUC/MIC was needed for S. pneumoniae in comparison with the Gram-negative bacteria studied. Moxifloxacin yielded higher AUC/MIC and Cmax/MIC against the investigated Gram-positive bacteria in comparison with levofloxacin 500 mg once daily and 750 mg once daily.

  • 43.
    Olofsson, Sara K
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Marcusson, Linda L
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Komp Lindgren, Patricia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Selection of ciprofloxacin resistance in Escherichia coli in an in vitro kinetic model: relation between drug exposure and mutant prevention concentration2006In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 57, no 6, p. 1116-1121Article in journal (Refereed)
  • 44.
    Olofsson, Sara K
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Marcusson, Linda L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Strömbäck, Ann
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Dose-related selection of fluoroquinolone-resistant Escherichia coli2007In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 60, no 4, p. 795-801Article in journal (Refereed)
    Abstract [en]

    Objectives: To investigate the effects of clinically used doses of norfloxacin,ciprofloxacin and moxifloxacin on survival and selection inEscherichia coli populations containing fluoroquinolone-resistantsubpopulations and to measure the value of the pharmacodynamicindex AUC/mutant prevention concentration (MPC) that preventsthe growth of pre-existing resistant mutants.

    Methods: Mixed cultures of susceptible wild-type and isogenic single(gyrA S83L) or double (gyrA S83L, marR) fluoroquinolone-resistantmutants were exposed to fluoroquinolones for 24 h in an in vitrokinetic model. Antibiotic concentrations modelled pharmacokineticsattained with clinical doses.

    Results: All tested doses eradicated the susceptible wild-type strain.Norfloxacin 200 mg administered twice daily selected for bothsingle and double mutants. Ciprofloxacin 250 mg administeredtwice daily eradicated the single mutant, but not the doublemutant. For that, 750 mg administered twice daily was required.Moxifloxacin 400 mg once daily eliminated the single mutant,but did not completely remove the double mutant. The MPC ofciprofloxacin was determined and based on those dose simulationsthat eradicated mutant subpopulations, an AUC/MPCwild-type of35 prevented selection of the single mutant, whereas an AUC/MPCsinglemutant of 14 (equivalent to an AUC/MPCwild-type of 105) preventedselection of the double mutant.

    Conclusions: All tested clinical dosing regimens were effective in eradicatingsusceptible bacteria, but ciprofloxacin 750 mg twice daily wasthe only dose that prevented the selection of single- and double-resistantE. coli mutants. Thus, among approved fluoroquinolone dosingregimens, some are significantly more effective than othersin exceeding the mutant selection window and preventing theenrichment of resistant mutants.

  • 45.
    Papareddy, Praveen
    et al.
    Lund Univ, Dept Clin Sci, Div Dermatol & Venereol, Lund, Sweden.;Lund Univ, Dept Clin Sci, Div Infect Med, Lund, Sweden..
    Kasetty, Gopinath
    Lund Univ, Dept Clin Sci, Div Dermatol & Venereol, Lund, Sweden.;Lund Univ, Dept Clin Sci, Respiratory Med & Allergol, Lund, Sweden..
    Kalle, Martina
    Lund Univ, Dept Clin Sci, Div Dermatol & Venereol, Lund, Sweden..
    Bhongir, Ravi K. V.
    Lund Univ, Dept Clin Sci, Div Dermatol & Venereol, Lund, Sweden.;Lund Univ, Dept Clin Sci, Respiratory Med & Allergol, Lund, Sweden..
    Morgelin, Matthias
    Lund Univ, Dept Clin Sci, Div Infect Med, Lund, Sweden..
    Schmidtchen, Artur
    Lund Univ, Dept Clin Sci, Div Dermatol & Venereol, Lund, Sweden.;Nanyang Technol Univ, LKCMed, Dermatol, Singapore 639798, Singapore..
    Malmsten, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    NLF20: an antimicrobial peptide with therapeutic potential against invasive Pseudomonas aeruginosa infection2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 1, p. 170-180Article in journal (Refereed)
    Abstract [en]

    Objectives:Increasing resistance to antibiotics makes antimicrobial peptides interesting as novel therapeutics. Here, we report on studies of the peptide NLF20 (NLFRKLTHRLFRRNFGYTLR), corresponding to an epitope of the D helix of heparin cofactor II (HCII), a plasma protein mediating bacterial clearance. Methods: Peptide effects were evaluated by a combination of in vitro and in vivo methods, including antibacterial, anti-inflammatory and cytotoxicity assays, fluorescence and electron microscopy, and experimental models of endotoxin shock and Pseudomonas aeruginosa sepsis. Results: The results showed that NLF20 displayed potent antimicrobial effects against the Gram-negative bacteria Escherichia coli and P. aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus and the fungi Candida albicans and Candida parapsilosis. Importantly, this antimicrobial effect was retained in human blood, particularly for P. aeruginosa. Fluorescence and electron microscopy studies showed that the peptide exerted membrane-breaking effects. In an animal model of P. aeruginosa sepsis, NLF20 reduced bacterial levels, resulting in improved survival. Reduced mortality was also observed in experimental animal models of endotoxin shock, which was paralleled with modulated IFN-gamma, IL-10 and coagulation responses. Conclusions: Together, these results indicate that functional epitopes of HCII may have therapeutic potential against bacterial infection.

  • 46.
    Pietsch, Franziska
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergman, Jessica M.
    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.
    Marcusson, Linda L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zorzet, Anna
    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.
    Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects2017In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 72, no 1, p. 75-84Article in journal (Refereed)
    Abstract [en]

    Objectives: Resistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the beta-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism. Methods: Independent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action. Results: Mutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT-PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele. Conclusions: These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.

  • 47. Poulsen, Hanna O.
    et al.
    Johansson, Anders
    Granholm, Susanne
    Kahlmeter, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Sundqvist, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    High genetic diversity of nitrofurantoin- or mecillinam-resistant Escherichia coli indicates low propensity for clonal spread2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 9, p. 1974-1977Article in journal (Refereed)
    Abstract [en]

    The empirical treatment with trimethoprim or ciprofloxacin of urinary tract infections (UTIs) is now questioned, partly due to the global expansion of a few resistant clonal groups of Escherichia coli. In this study we investigated the clonal structure of 34 strains of E. coli (collected from non-pregnant women aged 1865 years with uncomplicated UTIs in Europe and Canada) resistant to either of two other common treatment alternatives for uncomplicated UTIs, mecillinam or nitrofurantoin, using multilocus sequence typing (MLST). The 34 isolates were, despite high levels of multiresistance, distributed all over the E. coli genetic diversity spectrum with little association of antibiotic resistance to specific clonal groups. The results of this study indicate a low probability of a future clonal spread of resistance to mecillinam and nitrofurantoin.

  • 48.
    Praski Alzrigat, Lisa
    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.
    Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli: Multiple Antibiotic-Resistance, Gram-Negative Bacteria, Multidrug Efflux Pump, Urinary-Tract-Infections, Fluoroquinolone Resistance, Quinolone Resistance, Mechanisms, Expression, Sequence, Soxs2017In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 2, no 11, p. 3016-3024Article in journal (Refereed)
    Abstract [en]

    Objectives: To determine whether the spectrum of mutations in marR in ciprofloxacin-resistant clinical isolates of Escherichia coli shows evidence of selection bias, either to reduce fitness costs, or to increase drug resistance. MarR is a repressor protein that regulates, via MarA, expression of the Mar regulon, including the multidrug efflux pump AcrAB-TolC. Methods: Isogenic strains carrying 36 different marR alleles identified in resistant clinical isolates, or selected for resistance in vitro, were constructed. Drug susceptibility and relative fitness in growth competition assays were measured for all strains. The expression level of marA, and of various efflux pump components, as a function of specific mutations in marR, was measured by qPCR. Results: The spectrum of genetic alterations in marR in clinical isolates is strongly biased against inactivating mutations. In general, the alleles found in clinical isolates conferred a lower level of resistance and imposed a lower growth fitness cost than mutations selected in vitro. The level of expression of MarA correlated well with the MIC of ciprofloxacin. This supports the functional connection between mutations in marR and reduced susceptibility to ciprofloxacin. Conclusions: Mutations in marR selected in ciprofloxacin-resistant clinical isolates are strongly biased against inactivating mutations. Selection favours mutant alleles that have the lowest fitness costs, even though these cause only modest reductions in drug susceptibility. This suggests that selection for high relative fitness is more important than selection for increased resistance in determining which alleles of marR will be selected in resistant clinical isolates.

  • 49.
    Pränting, Maria
    et al.
    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.
    Mechanisms and physiological effects of protamine resistance in Salmonella enterica serovar Typhimurium LT22010In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 65, no 5, p. 876-887Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: Protamines are cationic peptides that exert antimicrobial activity. We have examined the evolution of bacterial resistance to protamine sulphate and the resulting effects on fitness and physiology, with the objective of increasing knowledge about mechanisms of bacterial resistance to antimicrobial peptides. METHODS: Spontaneous, protamine-resistant Salmonella enterica serovar Typhimurium (i.e. Salmonella Typhimurium) LT2 mutants were isolated on agarose plates containing protamine sulphate. Resistance mutations were identified using transposon insertions and DNA sequencing. Peptide susceptibility was determined by broth dilution tests and antibiotic susceptibility using Etests. Fitness was determined as log-phase growth rates. Growth-compensated strains were isolated by serial passage through population bottlenecks followed by visual screening for large colonies. RESULTS: Protamine-resistant mutants appeared at a rate of 2.3 x 10(-7)/cell/generation. These mutants were 2-20 times more resistant to protamine than the parental strain and less susceptible to several other antimicrobials, including colistin, gentamicin, lactoferricin and human defensin HNP-1. The resistance mutations were mapped to genes involved in haem biosynthesis and respiration, and were associated with a reduction in bacterial fitness. Some mutants could, in the absence of protamine, be evolved to higher fitness by acquiring second-site compensatory mutations. CONCLUSIONS: Spontaneous mutants resistant to protamine sulphate were readily selected in Salmonella Typhimurium LT2. These mutants were less susceptible to several other peptides and antibiotics, and had the characteristics of small colony variants, a phenotype often associated with persistent and recurrent infections that are difficult to treat and which could be a strategy for bacteria to escape the killing effects of antimicrobial peptides.

  • 50.
    Pränting, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lööv, Camilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Burman, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Göransson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The cyclotide cycloviolacin O2 from Viola odorata has potent bactericidal activity against Gram-negative bacteria2010In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 65, no 9, p. 1964-1971Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES:

    To determine the antibacterial activity of small cyclic plant proteins, i.e. cyclotides, and the importance of the surface exposed charged residues for activity.

    METHODS:

    Prototypic cyclotides, including the Möbius kalata B1 and the bracelet cycloviolacin O2 (cyO2), were isolated using reversed-phase HPLC. Initial activity screenings were conducted using radial diffusion assays (RDAs) and MIC assays with Salmonella enterica serovar Typhimurium LT2, Escherichia coli and Staphylococcus aureus as test strains. For the most active peptide, cyO2, time-kill kinetics was determined in sodium phosphate buffer (containing 0.03% trypticase soy broth) against several Gram-negative and Gram-positive bacterial species. Charged residues in cyO2 were chemically modified and activity was determined in time-kill assays.

    RESULTS:

    CyO2 was the most active cyclotide and efficiently inhibited the growth of S. enterica serovar Typhimurium LT2 and E. coli in RDAs and MIC assays, while the other peptides were less active. In time-kill assays, cyO2 also had bactericidal activity against the Gram-negative species Klebsiella pneumoniae and Pseudomonas aeruginosa. In contrast, none of the cyclotides had high activity against S. aureus. Chemical masking of the charged Glu and Lys residues in cyO2 caused a near total loss of activity against Salmonella, while masking Arg caused a less pronounced activity reduction.

    CONCLUSIONS:

    CyO2 is a cyclotide with potent activity against Gram-negative bacteria. The charged residues in cyO2 are all required for optimum antibacterial activity. In combination with its previously demonstrated cytotoxic activity against cancer cells and the general stability of cyclotides, cyO2 provides a promising scaffold for future drug design.

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