Publications by authors named "Anders Folkesson"

29 Publications

  • Page 1 of 1

Effect of tetracycline treatment regimens on antibiotic resistance gene selection over time in nursery pigs.

BMC Microbiol 2019 12 2;19(1):269. Epub 2019 Dec 2.

DTU BioEngineering, Technical University of Denmark, Kongens Lyngby, Denmark.

Background: The majority of antimicrobials given during the production of pigs are given to nursery pigs. The influence of antimicrobial use on the levels of antimicrobial resistant (AMR) genes is important to quantify to be able to assess the impact of resistance on the food chain and risk to human and animal health.

Results: This study investigated the response on the levels of nine AMR genes to five different treatment strategies with oxytetracycline, and the dynamics of gene abundance over time by following 1167 pigs from five different farms in Denmark. The results showed no significant difference between treatments and an increase in abundance for the efflux pump encoding tet(A) gene and the genes encoding the ribosomal protection proteins tet(O) and tet(W) tetracycline resistant genes following treatment, while tet(M) showed no response to treatment. However, it was also observed that the levels of tet(O), tet(W), and ermB in some farms would drift more over time compared to a single treatment-course with antibiotic.

Conclusion: This study underlines the large variation in AMR levels under natural conditions and the need for increased investigation of the complex interactions of antimicrobial treatment and other environmental and managerial practices in swine production on AMR gene abundance.
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http://dx.doi.org/10.1186/s12866-019-1619-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889206PMC
December 2019

Evolution of TEM-type extended-spectrum β-lactamases in Escherichia coli by cephalosporins.

J Glob Antimicrob Resist 2019 12 30;19:32-39. Epub 2019 Apr 30.

Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.

Objectives: This study was conducted to examine the molecular mechanisms responsible for the evolution of TEM-type extended-spectrum β-lactamases (ESBLs) following selective pressure from four third-generation cephalosporins, namely ceftazidime, cefotaxime, ceftriaxone and ceftibuten. In addition, selective enrichment for ESBL detection in environmental samples was investigated.

Methods: Using experimental evolution, resistant variants were isolated and mutations in TEM-1 were examined by DNA sequencing. Resistance levels and the development of cross-resistance were determined for ESBL-producing isolates by Etest and disk diffusion assay. Selective plating with or without prior growth in selective broth was used to examine the approach of selective enrichment for ESBL detection.

Results: The third-generation cephalosporins ceftazidime, cefotaxime and ceftriaxone selected for ESBLs, whereas ceftibuten did not. All ESBL variants additionally remained susceptible to ceftibuten. DNA sequencing of the TEM-1 coding sequence of mutants revealed mutations not previously isolated through selection. This indicates that the potential for ESBL evolution is much broader than can be inferred from sequence analysis of clinical samples alone. The results also indicate that selective enrichment for enhanced detection of ESBL-producers may give unreliable results owing to the selection of spontaneous mutations in narrow-spectrum β-lactamases resulting in TEM-type ESBL-producers.

Conclusion: These results help explain the molecular changes responsible for evolution of TEM-type ESBLs and meanwhile question the appropriate use of selective enrichment for detection of ESBLs in environmental samples.
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http://dx.doi.org/10.1016/j.jgar.2019.03.010DOI Listing
December 2019

Continuing occurrence of vancomycin resistance determinants in Danish pig farms 20 years after removing exposure to avoparcin.

Vet Microbiol 2019 May 6;232:84-88. Epub 2019 Apr 6.

Technical University of Denmark, Department of Biotechnology and Biomedicine, Kemitorvet Building 204, 2800, Kgs. Lyngby, Denmark. Electronic address:

Vancomycin-resistant Enterococcus spp. is a major health problem worldwide and livestock have been implicated in constituting a reservoir for the transmission of vancomycin resistance to zoonotic pathogens. Vancomycin resistance determinants can be situated on mobile genetic elements and transferred between bacterial species The livestock reservoir must therefore be included in a risk assessment of the vancomycin resistance burden. Avoparcin, a vancomycin analogue, has not been used in Danish pig production for over 20 years and vancomycin has never been used. The objective of this study was to screen faecal samples from Danish pig farms for nine selected vancomycin resistance determinants. We found at least four different vancomycin resistance determinants in all screened Danish pig farms (665 finisher farms and 78 sow farms). The vancomycin resistance determinants present in vanB or vanG clusters were found at significantly different levels in sow and finisher farms. However, vanA was not detected in any of the farms. In conclusion, vancomycin resistance determinants are still present in Danish pig production 25 years after the ban on avoparcin use.
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http://dx.doi.org/10.1016/j.vetmic.2019.04.007DOI Listing
May 2019

Send more data: a systematic review of mathematical models of antimicrobial resistance.

Antimicrob Resist Infect Control 2018 29;7:117. Epub 2018 Sep 29.

1Department of Applied Mathematics and Computer Science, Technical University of Denmark, Asmussens Allé Building 303B, 2800 Kgs. Lyngby, Denmark.

Background: Antimicrobial resistance is a global health problem that demands all possible means to control it. Mathematical modelling is a valuable tool for understanding the mechanisms of AMR development and spread, and can help us to investigate and propose novel control strategies. However, it is of vital importance that mathematical models have a broad utility, which can be assured if good modelling practice is followed.

Objective: The objective of this study was to provide a comprehensive systematic review of published models of AMR development and spread. Furthermore, the study aimed to identify gaps in the knowledge required to develop useful models.

Methods: The review comprised a comprehensive literature search with 38 selected studies. Information was extracted from the selected papers using an adaptation of previously published frameworks, and was evaluated using the TRACE good modelling practice guidelines.

Results: None of the selected papers fulfilled the TRACE guidelines. We recommend that future mathematical models should: a) model the biological processes mechanistically, b) incorporate uncertainty and variability in the system using stochastic modelling, c) include a sensitivity analysis and model external and internal validation.

Conclusion: Many mathematical models of AMR development and spread exist. There is still a lack of knowledge about antimicrobial resistance, which restricts the development of useful mathematical models.
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http://dx.doi.org/10.1186/s13756-018-0406-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162961PMC
September 2019

Persistence of antimicrobial resistance genes from sows to finisher pigs.

Prev Vet Med 2018 Jan 6;149:10-14. Epub 2017 Nov 6.

National Veterinary Institute, Technical University of Denmark, Kemitorvet, Building 204, 2800 Kgs. Lyngby, Denmark.

Antimicrobial resistance in pigs has been under scrutiny for many years. However, many questions remain unanswered, including whether the initial antimicrobial resistance level of a pig will influence the antimicrobial resistance found at slaughter. Faecal samples from finishers pigs from 681 farms and from sows from 82 farms were collected, and levels of seven antimicrobial resistance genes, ermB, ermF, sulI, sulII, tet(M), tet(O), and tet(W), were quantified by high-capacity qPCR. There were 40 pairs of observations where the finishers were born in the farms of the sows. The objective of this study was to evaluate whether the levels of AMR genes found in finisher pigs at slaughter were associated with the levels in the farm where the finishers were born, and whether the levels of the AMR genes were equal in the sow and finisher pig populations. We found a significant positive correlation between the levels of AMR genes in finishers and the sows in the farms where the pigs were born for some of the genes (ermB (ρ=0.47, p-value=0.002), ermF (ρ=0.41, p-value=0.03), and tet(O) (ρ=0.33, p-value=0.04)). Furthermore, there were significant differences between AMR gene levels for the sow and finisher populations for ermB, ermF, sulI, sulII, tet(O), and tet(W), though not for tet(M).
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http://dx.doi.org/10.1016/j.prevetmed.2017.11.007DOI Listing
January 2018

Association between selected antimicrobial resistance genes and antimicrobial exposure in Danish pig farms.

Sci Rep 2017 08 29;7(1):9683. Epub 2017 Aug 29.

Division for Diagnostics & Scientific Advice, National Veterinary Institute, Technical University of Denmark, Kemitorvet Building 204, 2800 Kgs. Lyngby, Denmark.

Bacterial antimicrobial resistance (AMR) in pigs is an important public health concern due to its possible transfer to humans. We aimed at quantifying the relationship between the lifetime exposure of antimicrobials and seven antimicrobial resistance genes in Danish slaughter pig farms. AMR gene levels were quantified by qPCR of total-community DNA in faecal samples obtained from 681 batches of slaughter pigs. The lifetime exposure to antimicrobials was estimated at batch level for the piglet, weaner, and finisher periods individually for the sampled batches. We showed that the effect of antimicrobial exposure on the levels of AMR genes was complex and unique for each individual gene. Several antimicrobial classes had both negative and positive correlations with the AMR genes. From 10-42% of the variation in AMR gene levels could be explained in the final regression models, indicating that antimicrobial exposure is not the only important determinant of the AMR gene levels.
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http://dx.doi.org/10.1038/s41598-017-10092-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575052PMC
August 2017

Effect of Tetracycline Dose and Treatment Mode on Selection of Resistant Coliform Bacteria in Nursery Pigs.

Appl Environ Microbiol 2017 06 31;83(12). Epub 2017 May 31.

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

This study describes the results of a randomized clinical trial investigating the effect of oxytetracycline treatment dose and mode of administration on the selection of antibiotic-resistant coliform bacteria in fecal samples from nursery pigs. Nursery pigs (pigs of 4 to 7 weeks of age) in five pig herds were treated with oxytetracycline for -induced diarrhea. Each group was randomly allocated to one of five treatment groups: oral flock treatment with a (i) high (20 mg/kg of body weight), (ii) medium (10 mg/kg), or (iii) low (5 mg/kg) dose, (iv) oral pen-wise (small-group) treatment (10 mg/kg), and (v) individual intramuscular injection treatment (10 mg/kg). All groups were treated once a day for 5 days. In all groups, treatment caused a rise in the numbers and proportions of tetracycline-resistant coliform bacteria right after treatment, followed by a significant drop by the time that the pigs left the nursery unit. The counts and proportions of tetracycline-resistant coliforms did not vary significantly between treatment groups, except immediately after treatment, when the highest treatment dose resulted in the highest number of resistant coliforms. A control group treated with tiamulin did not show significant changes in the numbers or proportions of tetracycline-resistant coliforms. Selection for tetracycline-resistant coliforms was significantly correlated to selection for ampicillin- and sulfonamide-resistant strains but not to selection for cefotaxime-resistant strains. In conclusion, the difference in the dose of oxytetracycline and the way in which the drug was applied did not cause significantly different levels of selection of tetracycline-resistant coliform bacteria under the conditions tested. Antimicrobial resistance is a global threat to human health. Treatment of livestock with antimicrobials has a direct impact on this problem, and there is a need to improve the ways that we use antimicrobials in livestock production. We hypothesized that antibiotic resistance development following treatment of diarrhea in nursery pigs could be reduced either by lowering the dose of oxytetracycline or by replacing the commonly used practice of flock treatment with individual or small-group treatments, since this would reduce the number of pigs treated. However, the study showed no significant difference between treatment groups with respect to the number or proportion of tetracycline-resistant coliforms selected. The most important conclusion is that under practical field conditions, there will be no added value, in terms of lowering resistance development, by exchanging flock treatment for individual or small-group treatment of nursery pigs. The reason for the lack of an effect of single-animal treatment is probably that such animals share the environment with treated animals and take up resistant bacteria from the environment.
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http://dx.doi.org/10.1128/AEM.00538-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5452818PMC
June 2017

A sampling and metagenomic sequencing-based methodology for monitoring antimicrobial resistance in swine herds.

J Antimicrob Chemother 2017 02 8;72(2):385-392. Epub 2016 Nov 8.

Research Group for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Søltofts Plads, Building 221, 2800 Kgs Lyngby, Denmark

Objectives: Reliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. Quantification of AMR is mostly done using culture-based techniques, but metagenomic read mapping shows promise for quantitative resistance monitoring.

Methods: We evaluated the ability of: (i) MIC determination for Escherichia coli; (ii) cfu counting of E. coli; (iii) cfu counting of aerobic bacteria; and (iv) metagenomic shotgun sequencing to predict expected tetracycline resistance based on known antimicrobial consumption in 10 Danish integrated slaughter pig herds. In addition, we evaluated whether fresh or manure floor samples constitute suitable proxies for intestinal sampling, using cfu counting, qPCR and metagenomic shotgun sequencing.

Results: Metagenomic read-mapping outperformed cultivation-based techniques in terms of predicting expected tetracycline resistance based on antimicrobial consumption. Our metagenomic approach had sufficient resolution to detect antimicrobial-induced changes to individual resistance gene abundances. Pen floor manure samples were found to represent rectal samples well when analysed using metagenomics, as they contain the same DNA with the exception of a few contaminating taxa that proliferate in the extraintestinal environment.

Conclusions: We present a workflow, from sampling to interpretation, showing how resistance monitoring can be carried out in swine herds using a metagenomic approach. We propose metagenomic sequencing should be part of routine livestock resistance monitoring programmes and potentially of integrated One Health monitoring in all reservoirs.
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http://dx.doi.org/10.1093/jac/dkw415DOI Listing
February 2017

Biofilm as a production platform for heterologous production of rhamnolipids by the non-pathogenic strain Pseudomonas putida KT2440.

Microb Cell Fact 2016 Oct 24;15(1):181. Epub 2016 Oct 24.

Department of Systems Biology, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark.

Background: Although a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used.

Results: In the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C fatty acids.

Conclusion: This study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without hampering biofilm capabilities. These findings expands the possibilities of cultivation setups and paves the way for employing biofilm flow systems as production platforms for biochemicals, which as a consequence of physiochemical properties are troublesome to produce in conventional fermenter setups, or for production of compounds which are inhibitory or toxic to the production organisms.
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http://dx.doi.org/10.1186/s12934-016-0581-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5075983PMC
October 2016

The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is shaped by strong epistatic interactions.

Nat Commun 2016 10 3;7:13002. Epub 2016 Oct 3.

National Veterinary Institute, Technical University of Denmark, Frederiksberg, Denmark.

Colistin is an antimicrobial peptide that has become the only remaining alternative for the treatment of multidrug-resistant Gram-negative bacterial infections, but little is known of how clinical levels of colistin resistance evolve. We use in vitro experimental evolution and whole-genome sequencing of colistin-resistant Pseudomonas aeruginosa isolates from cystic fibrosis patients to reconstruct the molecular evolutionary pathways open for high-level colistin resistance. We show that the evolution of resistance is a complex, multistep process that requires mutation in at least five independent loci that synergistically create the phenotype. Strong intergenic epistasis limits the number of possible evolutionary pathways to resistance. Mutations in transcriptional regulators are essential for resistance evolution and function as nodes that potentiate further evolution towards higher resistance by functionalizing and increasing the effect of the other mutations. These results add to our understanding of clinical antimicrobial peptide resistance and the prediction of resistance evolution.
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http://dx.doi.org/10.1038/ncomms13002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494192PMC
October 2016

Determining the optimal number of individual samples to pool for quantification of average herd levels of antimicrobial resistance genes in Danish pig herds using high-throughput qPCR.

Vet Microbiol 2016 Jun 22;189:46-51. Epub 2016 Apr 22.

National Veterinary Institute, Technical University of Denmark, Frederiksberg, Denmark. Electronic address:

The primary objective of this study was to determine the minimum number of individual fecal samples to pool together in order to obtain a representative sample for herd level quantification of antimicrobial resistance (AMR) genes in a Danish pig herd, using a novel high-throughput qPCR assay. The secondary objective was to assess the agreement between different methods of sample pooling. Quantification of AMR was achieved using a high-throughput qPCR method to quantify the levels of seven AMR genes (ermB, ermF, sulI, sulII, tet(M), tet(O) and tet(W)). A large variation in the levels of AMR genes was found between individual samples. As the number of samples in a pool increased, a decrease in sample variation was observed. It was concluded that the optimal pooling size is five samples, as an almost steady state in the variation was observed when pooling this number of samples. Good agreement between different pooling methods was found and the least time-consuming method of pooling, by transferring feces from each individual sample to a tube using a 10μl inoculation loop and adding 3.5ml of PBS, approximating a 10% solution, can therefore be used in future studies.
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http://dx.doi.org/10.1016/j.vetmic.2016.04.017DOI Listing
June 2016

Persistence and drug tolerance in pathogenic yeast.

Curr Genet 2017 Feb 19;63(1):19-22. Epub 2016 May 19.

National Veterinary Institute, Technical University of Denmark, Kongens Lyngby, Denmark.

In this review, we briefly summarize the current understanding of how fungal pathogens can persist antifungal treatment without heritable resistance mutations by forming tolerant persister cells. Fungal infections tolerant to antifungal treatment have become a major medical problem. One mechanism leading to drug recalcitrance is the formation of antifungal persister cells. These cells have wild-type genotype with the ability to survive exposure to antifungal agents due to changed membrane composition, upregulated stress response, and enhanced cell wall integrity. Knowledge of the mechanisms regulating entry and exit of the persister phenotype is limited, but it has recently been shown that the inhibition of the growth regulating TORC1 pathway induces fungal persistence. The phenotypic properties of persister cells and the involvement of the TORC1 pathway indicate that persister cells are quiescent in G0 of the cell cycle. This knowledge leads us to suggest that the identified shared drug-tolerance mechanisms of persister and quiescent cells may serve as a foundation for developing novel treatment strategies that are independent of growth mode against systemic fungal infections.
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http://dx.doi.org/10.1007/s00294-016-0613-3DOI Listing
February 2017

A common mechanism involving the TORC1 pathway can lead to amphotericin B-persistence in biofilm and planktonic Saccharomyces cerevisiae populations.

Sci Rep 2016 Feb 23;6:21874. Epub 2016 Feb 23.

National Veterinary Institute, Technical University of Denmark, Frederiksberg, Denmark.

Fungal infections are an increasing clinical problem. Decreased treatment effectiveness is associated with biofilm formation and drug recalcitrance is thought to be biofilm specific. However, no systematic investigations have tested whether resistance mechanisms are shared between biofilm and planktonic populations. We performed multiplexed barcode sequencing (Bar-seq) screening of a pooled collection of gene-deletion mutants cultivated as biofilm and planktonic cells. Screening for resistance to the ergosterol-targeting fungicide amphotericin B (AmB) revealed that the two growth modes had significant overlap in AmB-persistent mutants. Mutants defective in sterol metabolism, ribosome biosynthesis, and the TORC1 and Ras pathways showed increased persistence when treated with AmB. The ras1, ras2 and tor1 mutants had a high-persister phenotype similar to wild-type biofilm and planktonic cells exposed to the TORC1 pathway inhibitor rapamycin. Inhibition of TORC1 with rapamycin also increased the proportion of persisters in Candida albicans and Candida glabrata. We propose that decreased TORC1-mediated induction of ribosome biosynthesis via Ras can lead to formation of AmB-persister cells regardless of whether the cells are in planktonic or biofilm growth mode. Identification of common pathways leading to growth mode-independent persister formation is important for developing novel strategies for treating fungal infections.
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http://dx.doi.org/10.1038/srep21874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763212PMC
February 2016

Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa.

Int J Antimicrob Agents 2016 Jan 25;47(1):48-55. Epub 2015 Oct 25.

National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark. Electronic address:

Combination therapy with several antibiotics is one strategy that has been applied in order to limit the spread of antimicrobial resistance. We compared the de novo evolution of resistance during combination therapy with the β-lactam ceftazidime and the fluoroquinolone ciprofloxacin with the resistance evolved after single-drug exposure. Combination therapy selected for mutants that displayed broad-spectrum resistance, and a major resistance mechanism was mutational inactivation of the repressor gene mexR that regulates the multidrug efflux operon mexAB-oprM. Deregulation of this operon led to a broad-spectrum resistance phenotype that decreased susceptibility to the combination of drugs applied during selection as well as to unrelated antibiotic classes. Mutants isolated after single-drug exposure displayed narrow-spectrum resistance and carried mutations in the MexCD-OprJ efflux pump regulator gene nfxB conferring ciprofloxacin resistance, or in the gene encoding the non-essential penicillin-binding protein DacB conferring ceftazidime resistance. Reconstruction of resistance mutations by allelic replacement and in vitro fitness assays revealed that in contrast to single antibiotic use, combination therapy consistently selected for mutants with enhanced fitness expressing broad-spectrum resistance mechanisms.
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http://dx.doi.org/10.1016/j.ijantimicag.2015.09.014DOI Listing
January 2016

Saccharomyces cerevisiae biofilm tolerance towards systemic antifungals depends on growth phase.

BMC Microbiol 2014 Dec 4;14:305. Epub 2014 Dec 4.

Department of Systems Biology, Technical University of Denmark, Kgs, Lyngby, Denmark.

Background: Biofilm-forming Candida species cause infections that can be difficult to eradicate, possibly because of antifungal drug tolerance mechanisms specific to biofilms. In spite of decades of research, the connection between biofilm and drug tolerance is not fully understood.

Results: We used Saccharomyces cerevisiae as a model for drug susceptibility of yeast biofilms. Confocal laser scanning microscopy showed that S. cerevisiae and C. glabrata form similarly structured biofilms and that the viable cell numbers were significantly reduced by treatment of mature biofilms with amphotericin B but not voriconazole, flucytosine, or caspofungin. We showed that metabolic activity in yeast biofilm cells decreased with time, as visualized by FUN-1 staining, and mature, 48-hour biofilms contained cells with slow metabolism and limited growth. Time-kill studies showed that in exponentially growing planktonic cells, voriconazole had limited antifungal activity, flucytosine was fungistatic, caspofungin and amphotericin B were fungicidal. In growth-arrested cells, only amphotericin B had antifungal activity. Confocal microscopy and colony count viability assays revealed that the response of growing biofilms to antifungal drugs was similar to the response of exponentially growing planktonic cells. The response in mature biofilm was similar to that of non-growing planktonic cells. These results confirmed the importance of growth phase on drug efficacy.

Conclusions: We showed that in vitro susceptibility to antifungal drugs was independent of biofilm or planktonic growth mode. Instead, drug tolerance was a consequence of growth arrest achievable by both planktonic and biofilm populations. Our results suggest that efficient strategies for treatment of yeast biofilm might be developed by targeting of non-dividing cells.
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http://dx.doi.org/10.1186/s12866-014-0305-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258017PMC
December 2014

Genetic basis for Saccharomyces cerevisiae biofilm in liquid medium.

G3 (Bethesda) 2014 Jul 9;4(9):1671-80. Epub 2014 Jul 9.

Department of Biology, University of Copenhagen, Copenhagen, Denmark

Biofilm-forming microorganisms switch between two forms: free-living planktonic and sessile multicellular. Sessile communities of yeast biofilms in liquid medium provide a primitive example of multicellularity and are clinically important because biofilms tend to have other growth characteristics than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the Σ1278b background and found 71 genes that were essential for biofilm development. Quantitative northern blots further revealed that AIM1, ASG1, AVT1, DRN1, ELP4, FLO8, FMP10, HMT1, KAR5, MIT1, MRPL32, MSS11, NCP1, NPR1, PEP5, PEX25, RIM8, RIM101, RGT1, SNF8, SPC2, STB6, STP22, TEC1, VID24, VPS20, VTC3, YBL029W, YBL029C-A, YFL054C, YGR161W-C, YIL014C-A, YIR024C, YKL151C, YNL200C, YOR034C-A, and YOR223W controlled biofilm through FLO11 induction. Almost all deletion mutants that were unable to form biofilms in liquid medium also lost the ability to form surface-spreading biofilm colonies (mats) on agar and 69% also lost the ability to grow invasively. The protein kinase A isoform Tpk3p functioned specifically in biofilm and mat formation. In a tpk3 mutant, transcription of FLO11 was induced three-fold compared with wild-type, but biofilm development and cell-cell adhesion was absent, suggesting that Tpk3p regulates FLO11 positive posttranscriptionally and negative transcriptionally.The study provides a resource of biofilm-influencing genes for additional research on biofilm development and suggests that the regulation of FLO11 is more complex than previously anticipated.
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http://dx.doi.org/10.1534/g3.114.010892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169159PMC
July 2014

Bactericidal antibiotics increase hydroxyphenyl fluorescein signal by altering cell morphology.

PLoS One 2014 19;9(3):e92231. Epub 2014 Mar 19.

Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg C, Denmark.

It was recently proposed that for bactericidal antibiotics a common killing mechanism contributes to lethality involving indirect stimulation of hydroxyl radical (OH•) formation. Flow cytometric detection of OH• by hydroxyphenyl fluorescein (HPF) probe oxidation was used to support this hypothesis. Here we show that increased HPF signals in antibiotics-exposed bacterial cells are explained by fluorescence associated with increased cell size, and do not reflect reactive oxygen species (ROS) concentration. Independently of antibiotics, increased fluorescence was seen for elongated cells expressing the oxidative insensitive green fluorescent protein (GFP). Although our data question the role of ROS in lethality of antibiotics other research approaches point to important interplays between basic bacterial metabolism and antibiotic susceptibility. To underpin such relationships, methods for detecting bacterial metabolites at a cellular level are needed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092231PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960231PMC
December 2015

Draft Genome Sequences of Pseudomonas aeruginosa B3 Strains Isolated from a Cystic Fibrosis Patient Undergoing Antibiotic Chemotherapy.

Genome Announc 2013 Oct 17;1(5). Epub 2013 Oct 17.

Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark.

Pseudomonas aeruginosa frequently establishes chronic infections in the airways of patients suffering from cystic fibrosis (CF). Here, we report the draft genome sequences of four P. aeruginosa B3 strains isolated from a chronically infected CF patient undergoing antibiotic chemotherapy.
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http://dx.doi.org/10.1128/genomeA.00804-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798451PMC
October 2013

The synthetic amphipathic peptidomimetic LTX109 is a potent fungicide that disturbs plasma membrane integrity in a sphingolipid dependent manner.

PLoS One 2013 12;8(7):e69483. Epub 2013 Jul 12.

Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.

The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069483PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709891PMC
February 2014

High in vitro antimicrobial activity of β-peptoid-peptide hybrid oligomers against planktonic and biofilm cultures of Staphylococcus epidermidis.

Int J Antimicrob Agents 2013 Jan 13;41(1):20-7. Epub 2012 Nov 13.

Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.

An array of β-peptoid-peptide hybrid oligomers displaying different amino acid/peptoid compositions and chain lengths was studied with respect to antimicrobial activity against Staphylococcus epidermidis both in planktonic and biofilm cultures, comparing the effects with those of the common antibiotic vancomycin. Susceptibility and time-kill assays were performed to investigate activity against planktonic cells, whilst confocal laser scanning microscopy was used to investigate the dynamics of the activity against cells within biofilms. All tested peptidomimetics were bactericidal against both exponentially growing and stationary-phase S. epidermidis cells with similar killing kinetics. At the minimum inhibitory concentration (MIC), all peptidomimetics inhibited biofilm formation, whilst peptidomimetics at concentrations above the MIC (80-160μg/mL) eradicated young (6-h-old) biofilms, whilst even higher concentrations were needed to eradicate mature (24-h-old) biofilms completely. Chiral and guanidinylated hybrids exhibited the fastest killing effects against slow-growing cells and had more favourable antibiofilm properties than analogues only containing lysine or lacking chirality in the β-peptoid residues. However, the results of the mature biofilm killing assay indicated more complex structure-activity relationships. Cytotoxicity assays showed a clear correlation between oligomer length and cell toxicity within each subclass of peptides, but all possessed a high differential toxicity favouring killing of bacterial cells. This class of peptidomimetics may constitute promising antimicrobial alternatives for the prevention and treatment of multidrug-resistant S. epidermidis infections.
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http://dx.doi.org/10.1016/j.ijantimicag.2012.09.014DOI Listing
January 2013

Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective.

Nat Rev Microbiol 2012 Dec 13;10(12):841-51. Epub 2012 Nov 13.

Department of Systems Biology, Technical University of Denmark, 2800 Lyngby, Denmark.

The airways of patients with cystic fibrosis (CF) are nearly always infected with many different microorganisms. This environment offers warm, humid and nutrient-rich conditions, but is also stressful owing to frequent antibiotic therapy and the host immune response. Pseudomonas aeruginosa is commonly isolated from the airways of patients with CF, where it most often establishes chronic infections that usually persist for the rest of the lives of the patients. This bacterium is a major cause of mortality and morbidity and has therefore been studied intensely. Here, we discuss how P. aeruginosa evolves from a state of early, recurrent intermittent colonization of the airways of patients with CF to a chronic infection state, and how this process offers opportunities to study bacterial evolution in natural environments. We believe that such studies are valuable not only for our understanding of bacterial evolution but also for the future development of new therapeutic strategies to treat severe chronic infections.
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http://dx.doi.org/10.1038/nrmicro2907DOI Listing
December 2012

Evolution and diversification of Pseudomonas aeruginosa in the paranasal sinuses of cystic fibrosis children have implications for chronic lung infection.

ISME J 2012 Jan 30;6(1):31-45. Epub 2011 Jun 30.

Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark.

The opportunistic pathogen Pseudomonas aeruginosa is a frequent colonizer of the airways of patients suffering from cystic fibrosis (CF). Depending on early treatment regimens, the colonization will, with high probability, develop into chronic infections sooner or later, and it is important to establish under which conditions the switch to chronic infection takes place. In association with a recently established sinus surgery treatment program for CF patients at the Copenhagen CF Center, colonization of the paranasal sinuses with P. aeruginosa has been investigated, paralleled by sampling of sputum from the same patients. On the basis of genotyping and phenotypic characterization including transcription profiling, the diversity of the P. aeruginosa populations in the sinuses and the lower airways was investigated and compared. The observations made from several children show that the paranasal sinuses constitute an important niche for the colonizing bacteria in many patients. The paranasal sinuses often harbor distinct bacterial subpopulations, and in the early colonization phases there seems to be a migration from the sinuses to the lower airways, suggesting that independent adaptation and evolution take place in the sinuses. Importantly, before the onset of chronic lung infection, lineages with mutations conferring a large fitness benefit in CF airways such as mucA and lasR as well as small colony variants and antibiotic-resistant clones are part of the sinus populations. Thus, the paranasal sinuses potentially constitute a protected niche of adapted clones of P. aeruginosa, which can intermittently seed the lungs and pave the way for subsequent chronic lung infections.
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http://dx.doi.org/10.1038/ismej.2011.83DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246239PMC
January 2012

A Mig-14-like protein (PA5003) affects antimicrobial peptide recognition in Pseudomonas aeruginosa.

Microbiology (Reading) 2011 Sep 23;157(Pt 9):2647-2657. Epub 2011 Jun 23.

Center for Systems Microbiology, DTU-Systems Biology, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark.

The evolution of antibiotic resistance in pathogenic bacteria is a growing global health problem which is gradually making the treatment of infectious diseases less efficient. Antimicrobial peptides are small charged molecules found in organisms from the complete phylogenetic spectrum. The peptides are attractive candidates for novel drug development due to their activity against bacteria that are resistant to conventional antibiotics, and reports of peptide resistance are rare in the clinical setting. Paradoxically, many clinically relevant bacteria have mechanisms that can recognize and respond to the presence of cationic antimicrobial peptides (CAMPs) in the environment by changing the properties of the microbial surface thereby increasing the tolerance of the microbes towards the peptides. In Pseudomonas aeruginosa an essential component of this inducible tolerance mechanism is the lipopolysaccharide modification operon arnBCADTEF-PA3559 which encodes enzymes required for LPS alterations leading to increased antimicrobial peptide tolerance. The expression of the operon is induced by the presence of CAMPs in the environment but the molecular mechanisms underlying the cellular recognition of the peptides are poorly elucidated. In this work, we investigate the factors influencing arnB expression by transposon mutagenesis and arnB promoter green fluorescent protein reporters. We have identified a novel gene encoding a Mig-14-like protein that is required for recognition of the CAMPs colistin and Novispirin G10 by P. aeruginosa. Moreover, we show that this gene is also required for the formation of CAMP-tolerant subpopulations in P. aeruginosa hydrodynamic flow chamber biofilms.
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http://dx.doi.org/10.1099/mic.0.049445-0DOI Listing
September 2011

Evolutionary dynamics of bacteria in a human host environment.

Proc Natl Acad Sci U S A 2011 May 25;108(18):7481-6. Epub 2011 Apr 25.

Department of Systems Biology, Technical University of Denmark, 2800 Lyngby, Denmark.

Laboratory evolution experiments have led to important findings relating organism adaptation and genomic evolution. However, continuous monitoring of long-term evolution has been lacking for natural systems, limiting our understanding of these processes in situ. Here we characterize the evolutionary dynamics of a lineage of a clinically important opportunistic bacterial pathogen, Pseudomonas aeruginosa, as it adapts to the airways of several individual cystic fibrosis patients over 200,000 bacterial generations, and provide estimates of mutation rates of bacteria in a natural environment. In contrast to predictions based on in vitro evolution experiments, we document limited diversification of the evolving lineage despite a highly structured and complex host environment. Notably, the lineage went through an initial period of rapid adaptation caused by a small number of mutations with pleiotropic effects, followed by a period of genetic drift with limited phenotypic change and a genomic signature of negative selection, suggesting that the evolving lineage has reached a major adaptive peak in the fitness landscape. This contrasts with previous findings of continued positive selection from long-term in vitro evolution experiments. The evolved phenotype of the infecting bacteria further suggests that the opportunistic pathogen has transitioned to become a primary pathogen for cystic fibrosis patients.
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http://dx.doi.org/10.1073/pnas.1018249108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088582PMC
May 2011

Biofilm induced tolerance towards antimicrobial peptides.

PLoS One 2008 Apr 2;3(4):e1891. Epub 2008 Apr 2.

Infection Microbiology Group, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark.

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001891PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2270907PMC
April 2008

Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms.

J Bacteriol 2007 Jan 13;189(1):28-37. Epub 2006 Oct 13.

Center for Biomedical Microbiology, BioCentrum-DTU, Building 301, The Technical University of Denmark, DK-2800 Lyngby, Denmark.

During Pseudomonas aeruginosa flow cell biofilm development, the cell population differentiates into a nonmotile subpopulation which forms microcolonies and a migrating subpopulation which eventually colonizes the top of the microcolonies, resulting in the development of mushroom-shaped multicellular structures. The cap-forming subpopulation was found to develop tolerance to membrane-targeting antimicrobial agents, such as the cyclic cationic peptide colistin and the detergent sodium dodecyl sulfate. The stalk-forming subpopulation, on the other hand, was sensitive to the membrane-targeting antibacterial agents. All biofilm-associated cells were sensitive to the antibacterial agents when tested in standard plate assays. A mutation eliminating the production of type IV pili, and hence surface-associated motility, prevented the formation of regular mushroom-shaped structures in the flow cell biofilms, and the development of tolerance to the antimicrobial agents was found to be affected as well. Mutations in genes interfering with lipopolysaccharide modification (pmr) eliminated the biofilm-associated colistin tolerance phenotype. Experiments with a PAO1 strain harboring a pmr-gfp fusion showed that only the cap-forming subpopulation in biofilms treated with colistin expresses the pmr operon. These results suggest that increased antibiotic tolerance in biofilms may be a consequence of differentiation into distinct subpopulations with different phenotypic properties.
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http://dx.doi.org/10.1128/JB.00720-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797205PMC
January 2007

Components of the peptidoglycan-recycling pathway modulate invasion and intracellular survival of Salmonella enterica serovar Typhimurium.

Cell Microbiol 2005 Jan;7(1):147-55

Mikrobiologiskt och Tumörbiologiskt Centrum, Karolinska Institutet, S-17177 Stockholm, Sverige, Sweden.

beta-Lactam resistance in enteric bacteria is frequently caused by mutations in ampD encoding a cytosolic N-acetylmuramyl- l-alanine amidase. Such mutants are blocked in murein (peptidoglycan) recycling and accumulate cytoplasmic muropeptides that interact with the transcriptional activator ampR, which de-represses beta-lactamase expression. Salmonella enterica serovar Typhimurium, an extensively studied enteric pathogen, was used to show that mutations in ampD decreased the ability of S. typhimurium to enter a macrophage derived cell line and made the bacteria more potent as inducers of inducible nitric oxide synthase (iNOS), as compared with the wild-type. ampG mutants, defective in the transport of recycled muropeptides across the cytoplasmic membrane, behaved essentially as the wild-type in invasion assays and in activation of iNOS. As ampD mutants also have reduced in vivo fitness in a murine model, we suggest that the cytoplasmic accumulation of muropeptides affects the virulence of the ampD mutants.
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http://dx.doi.org/10.1111/j.1462-5822.2004.00443.xDOI Listing
January 2005

Immunogenic properties of the Salmonella atypical fimbriae in BALB/c mice.

Vaccine 2004 Mar;22(11-12):1448-56

Department of Pharmacy, Biomedical Centre, Uppsala University, Box 580, Uppsala SE-751 23, Sweden.

Components of the Salmonella atypical fimbriae (Saf) were investigated for potential inclusion in a Salmonella vaccine. Recombinant histidine-tagged SafB chaperone complexed with SafD adhesin was expressed in Escherichia coli and purified. Starch microparticles were used, as an adjuvant and recombinant cholera toxin B subunit (rCTB) was included as a mucosal antigen-uptake enhancer. BALB/c mice were immunized orally or subcutaneously with SafB/D- and rCTB-conjugated microparticles and nasally or subcutaneously with SafB/D mixed with rCTB. The systemic and mucosal immune responses were studied, and an oral challenge with Salmonella enteritidis was performed. All immunized groups except that receiving oral immunization responded with high IgM-IgG titers to SafB/D. Analysis of the subclass ratio (IgG1/IgG2a+IgG2b) indicated a mixed Th1 and Th2 response, with Th1 predominating. The mucosal response, measured as specific IgA/total IgA (from fecal samples), was significantly greater than that in the untreated control group only in the group receiving intranasal immunization (P<0.05). Spleens were removed 6 days after oral challenge and Salmonella colony-forming units (CFU) were counted. The group immunized subcutaneously with SafB/D- and rCTB-conjugated microparticles had significantly lower CFU counts than the untreated control group (P<0.05).
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http://dx.doi.org/10.1016/j.vaccine.2003.10.012DOI Listing
March 2004

The Salmonella enterica subspecies I specific centisome 7 genomic island encodes novel protein families present in bacteria living in close contact with eukaryotic cells.

Res Microbiol 2002 Oct;153(8):537-45

Mikrobiologiskt och Tumörbiologiskt Centrum, Karolinska Institutet, Stockholm, Sweden.

We have determined the genetic structure of the Salmonella enterica centisome 7 genomic island (SCI) located at the aspV loci in S. enterica subspecies I strains. The 47-kb long genomic island encodes 37 putative proteins, including the previously described saf fimbrial operon and the sinR transcriptional regulator. Other open reading frames (designated sci A to Z) in the island encode putative proteins with homologies to virulence-associated proteins in a number of gram-negative bacteria such as Pseudomonas aeruginosa, Yersinia pestis and enterohemorrhagic Escherichia coli, bacteria that have the ability to interact with and manipulate eukaryotic cells. The Sci proteins have putative cytoplasmic, periplasmic and outer membrane localizations pointing to a role in extracellular processes such as secretion or organelle biosynthesis. The genes encoding Sci-like proteins are clustered in all sequenced bacterial genomes available in the databases and a core set can be defined by the presence of genes encoding proteins with similarity to the SciB, C, G, H, I, O proteins. The SCI genomic island DNA sequences are restricted to Salmonella strains belonging to S. enterica subspecies I and deletion of the entire island affects the ability of the organisms to enter eukaryotic cells.
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http://dx.doi.org/10.1016/s0923-2508(02)01348-7DOI Listing
October 2002