Publications by authors named "Andreas Burkovski"

112 Publications

Proteome Adaptation to Cell Culture Medium and Serum.

Proteomes 2021 Mar 13;9(1). Epub 2021 Mar 13.

Microbiology Division, Department of Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.

Host-pathogen interactions are often studied in vitro using primary or immortal cell lines. This set-up avoids ethical problems of animal testing and has the additional advantage of lower costs. However, the influence of cell culture media on bacterial growth and metabolism is not considered or investigated in most cases. To address this question growth and proteome adaptation of strain ISS3319 were investigated in this study. Bacteria were cultured in standard growth medium, cell culture medium, and fetal calf serum. Mass spectrometric analyses and label-free protein quantification hint at an increased bacterial pathogenicity when grown in cell culture medium as well as an influence of the growth medium on the cell envelope.
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http://dx.doi.org/10.3390/proteomes9010014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005964PMC
March 2021

Newly Isolated Animal Pathogen Is Cytotoxic to Human Epithelial Cells.

Int J Mol Sci 2021 Mar 29;22(7). Epub 2021 Mar 29.

Microbiology Division, Department of Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen and the widely distributed animal pathogen . In this study, strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of to different human epithelial cell lines and to an invertebrate animal model, larvae, comparable to diphtheria toxin-secreting Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.
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http://dx.doi.org/10.3390/ijms22073549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037504PMC
March 2021

Crystal structures of adenylylated and unadenylylated P protein GlnK from Corynebacterium glutamicum.

Acta Crystallogr D Struct Biol 2021 Mar 19;77(Pt 3):325-335. Epub 2021 Feb 19.

Division of Biotechnology, Department of Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestrasse 91, 91052 Erlangen, Germany.

P proteins are ubiquitous signaling proteins that are involved in the regulation of the nitrogen/carbon balance in bacteria, archaea, and some plants and algae. Signal transduction via P proteins is modulated by effector molecules and post-translational modifications in the P T-loop. Whereas the binding of ADP, ATP and the concomitant binding of ATP and 2-oxoglutarate (2OG) engender two distinct conformations of the T-loop that either favor or disfavor the interaction with partner proteins, the structural consequences of post-translational modifications such as phosphorylation, uridylylation and adenylylation are far less well understood. In the present study, crystal structures of the P protein GlnK from Corynebacterium glutamicum have been determined, namely of adenylylated GlnK (adGlnK) and unmodified unadenylylated GlnK (unGlnK). AdGlnK has been proposed to act as an inducer of the transcription repressor AmtR, and the adenylylation of Tyr51 in GlnK has been proposed to be a prerequisite for this function. The structures of unGlnK and adGlnK allow the first atomic insights into the structural implications of the covalent attachment of an AMP moiety to the T-loop. The overall GlnK fold remains unaltered upon adenylylation, and T-loop adenylylation does not appear to interfere with the formation of the two major functionally important T-loop conformations, namely the extended T-loop in the canonical ADP-bound state and the compacted T-loop that is adopted upon the simultaneous binding of Mg-ATP and 2OG. Thus, the P-typical conformational switching mechanism appears to be preserved in GlnK from C. glutamicum, while at the same time the functional repertoire becomes expanded through the accommodation of a peculiar post-translational modification.
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http://dx.doi.org/10.1107/S2059798321000735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919409PMC
March 2021

Pilot Study on the Use of a Laser-Structured Double Diamond Electrode (DDE) for Biofilm Removal from Dental Implant Surfaces.

J Clin Med 2020 Sep 21;9(9). Epub 2020 Sep 21.

Department of Prosthodontics, Saarland University, 66421 Homburg/Saar, Germany.

No proper treatment option for peri-implantitis exists yet. Based on previous studies showing the in vitro effectiveness of electrochemical disinfection using boron-doped diamond electrodes, novel double diamond electrodes (DDE) were tested here. Using a ceramic carrier and a laser structuring process, a clinically applicable electrode array was manufactured. Roughened metal discs ( = 24) made from Ti-Zr alloy were exposed to the oral cavities of six volunteers for 24 h in order to generate biofilm. Then, biofilm removal was carried out either using plastic curettes and chlorhexidine digluconate or electrochemical disinfection. In addition, dental implants were contaminated with ex vivo multispecies biofilm and disinfected using DDE treatment. Bacterial growth and the formation of biofilm polymer were determined as outcome measures. Chemo-mechanical treatment could not eliminate bacteria from roughened surfaces, while in most cases, a massive reduction of bacteria and biofilm polymer was observed following DDE treatment. Electrochemical disinfection was charge- and time-dependent and could also not reach complete disinfection in all instances. Implant threads had no negative effect on DDE treatment. Bacteria exhibit varying resistance to electrochemical disinfection with , sp., , , and surviving 5 min of DDE application at 6 V. Electrochemical disinfection is promising but requires further optimization with respect to charge quantity and application time in order to achieve disinfection without harming host tissue.
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http://dx.doi.org/10.3390/jcm9093036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565428PMC
September 2020

Cellular and Extracellular Proteome of the Animal Pathogen , a Close Relative of Zoonotic and .

Proteomes 2020 Aug 12;8(3). Epub 2020 Aug 12.

Microbiology Division, Department of Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany.

is a newly described animal pathogen, closely related to the emerging human pathogen and , a major pathogen of small ruminants. In this study, proteins of a whole cell and a shaving fraction and the exoproteome of strain W25 were analyzed as a first proteome study of this species. In total, 1305 proteins were identified out of 2013 proteins encoded by the W25 genome sequence and number of putative virulence factors were detected already under standard growth conditions including phospholipase D and sialidase. An up to now uncharacterized trypsin-like protease is by far the most secreted protein in this species, indicating a putative role in pathogenicity. Furthermore, the proteome analyses carried out in this study support the recently published taxonomical delineation of from the closely related zoonotic species.
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http://dx.doi.org/10.3390/proteomes8030019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564913PMC
August 2020

Phylogenomic characterisation of a novel corynebacterial species pathogenic to animals.

Antonie Van Leeuwenhoek 2020 Aug 4;113(8):1225-1239. Epub 2020 Jun 4.

Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne, UK.

The genus Corynebacterium includes species of biotechnological, medical and veterinary importance. An atypical C. ulcerans strain, W25, was recently isolated from a case of necrotizing lymphadenitis in a wild boar. In this study, we have analysed the genome sequence of this strain and compared the phenotypic and virulence properties with other corynebacterial pathogens. Phylogenomic analyses revealed that strain W25 belongs to a novel species along with PO100/5 and KL1196. The latter strains were isolated from a pig and a roe deer, respectively; hence, this species appears to be associated to animals. The isolate W25 is likely a non-toxigenic tox gene bearing strain and may have compromised abilities to adhere to pharyngeal and laryngeal epithelial cells due to potential loss of the gene functions in spaBC and spaDEF pilus gene clusters. A number of corynebacterial virulence genes are present including pld encoding phospholipase D. Therefore, this strain may be able to cause severe invasive infections in animals and zoonotic infections in humans.
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http://dx.doi.org/10.1007/s10482-020-01430-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334274PMC
August 2020

Electrochemical Disinfection of Dental Implants Experimentally Contaminated with Microorganisms as a Model for Periimplantitis.

J Clin Med 2020 Feb 9;9(2). Epub 2020 Feb 9.

Microbiology Division, Department of Biology, University of Erlangen-Nuremberg, 91058 Erlangen, Germany.

Despite several methods having been described for disinfecting implants affected by periimplantitis, none of these are universally effective and may even alter surfaces and mechanical properties of implants. Boron-doped diamond (BDD) electrodes were fabricated from niobium wires and assembled as a single instrument for implant cleaning. Chemo-mechanical debridement and air abrasion were used as control methods. Different mono-species biofilms, formed by bacteria and yeasts, were allowed to develop in rich medium at 37 °C for three days. In addition, natural multi-species biofilms were treated. Implants were placed in silicone, polyurethane foam and bovine ribs for simulating different clinical conditions. Following treatment, the implants were rolled on blood agar plates, which were subsequently incubated at 37 °C and microbial growth was analyzed. Complete electrochemical disinfection of implant surfaces was achieved with a maximum treatment time of 20 min for , , , , and , while in case of spore-forming and , a number of colonies appeared after BDD electrode treatment indicating an incomplete disinfection. Independent of the species tested, complete disinfection was never achieved when conventional techniques were used. During treatment with BDD electrodes, only minor changes in temperature and pH value were observed. The instrument used here requires optimization so that higher charge quantities can be applied in shorter treatment times.
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http://dx.doi.org/10.3390/jcm9020475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074531PMC
February 2020

Influence of In-Situ Electrochemical Oxidation on Implant Surface and Colonizing Microorganisms Evaluated by Scanning Electron Microscopy.

Materials (Basel) 2019 Nov 30;12(23). Epub 2019 Nov 30.

Division of Ultra-Hard Coatings, Department of Material Sciences, University of Erlangen-Nuremberg, 91058 Erlangen, Germany.

Peri-implantitis is a worldwide increasing health problem, caused by infection of tissue and bone around an implant by biofilm-forming microorganisms. Effects of peri-implantitis treatment using mechanical debridement, air particle abrasion and electrochemical disinfection on implant surface integrity were compared. Dental implants covered with bacterial biofilm were cleaned using mechanical debridement and air particle abrasion. In addition, implants were disinfected using a novel electrochemical technique based on an array of boron-doped diamond (BDD) coated electrodes. Following treatment and preparation, the implants were inspected by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). Mechanical debridement led to changes in surface topography destroying the manufacturer's medium-rough surface by scratch formation. Air particle abrasion led to accumulation of the abrasive used on the implant surface. With both treatment options, appearance of bacteria and yeasts was not affected. In contrast, electrochemical disinfection did not cause alterations of the implant surface but resulted in distorted microbial cells. Electrochemical disinfection of implant surfaces using BDD electrodes may constitute a promising treatment option for cleaning dental implant surfaces without negatively affecting materials and surface properties.
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http://dx.doi.org/10.3390/ma12233977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926823PMC
November 2019

Electrochemical Disinfection of Experimentally Infected Teeth by Boron-Doped Diamond Electrode Treatment.

J Clin Med 2019 Nov 21;8(12). Epub 2019 Nov 21.

Department of Prosthodontics, Saarland University, 66421 Homburg/Saar, Germany.

Disinfection and prevention of re-infection are the decisive treatment steps in endodontic therapy. In this study, boron-doped diamond (BDD) electrodes have been fabricated and used for disinfecting the root canals of extracted human teeth, which had been covered with bacterial biofilms formed by and . The growth of could be successfully impaired, achieving a complete disinfection after 8.5 min treatment time with the success of disinfection depending on the insertion depth of the electrode in the root canal. could completely be removed after 3.5 min treatment time. A clinically applicable electrode array led to complete disinfection after treatment times of 10 min for and 25 min for . BDD electrode application allowed for the improved disinfection of root canals and dentin tubules based on a continuous production of reactive oxygen species and their enhanced penetration of dentin tubules most likely due the formation of a continuous stream of small gas bubbles. The treatment times that are required here will be shortened in clinical application, as mechanical shaping of the canal system would precede the disinfection process.
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http://dx.doi.org/10.3390/jcm8122037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947473PMC
November 2019

Live cell imaging of macrophage/bacterium interaction demonstrates cell lysis induced by Corynebacterium diphtheriae and Corynebacterium ulcerans.

BMC Res Notes 2019 Oct 25;12(1):695. Epub 2019 Oct 25.

Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058, Erlangen, Germany.

Objectives: In frame of a study to characterize the interaction of human macrophage-like cells with pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, live cell imaging experiments were carried out and time lapse fluorescence microscopy videos were generated, which are presented here.

Data Description: The time lapse fluorescence microscopy data revealed new insights in the interaction of corynebacteria with human macrophage-like THP-1 cells. In contrast to uninfected cells and infections with non-pathogenic C. glutamicum used as a control, pathogenic C. diphtheriae and C. ulcerans showed highly detrimental effects towards human cells and induction of cell death of macrophages.
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http://dx.doi.org/10.1186/s13104-019-4733-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815039PMC
October 2019

Genome sequence of a pathogenic Corynebacterium ulcerans strain isolated from a wild boar with necrotizing lymphadenitis.

BMC Res Notes 2019 Oct 25;12(1):692. Epub 2019 Oct 25.

Institut für Bakterielle Infektionen und Zoonosen, Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Jena, Germany.

Objectives: Corynebacterium ulcerans can colonize a wide variety of animals and also humans are infected, typically by zoonotic transmission. Symptoms range from skin ulcers or systemic infections to diphtheria-like illness. In contrast, Corynebacterium pseudotuberculosis is widely distributed among herds of sheep, goats and other farm animals, where it causes high economic losses due to caseous lymphadenitis. Here we describe the genome sequence of an atypical C. ulcerans strain isolated from a wild boar with necrotizing lymphadenitis. This strain has similarities to C. pseudotuberculosis.

Data Description: Genome sequence data of C. ulcerans isolate W25 were generated, analyzed and taxonomical relationship to other Corynebacterium species as well as growth properties of the isolate were characterized. The genome of C. ulcerans W25 comprises 2,550,924 bp with a G+C content of 54.41% and a total of 2376 genes.
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http://dx.doi.org/10.1186/s13104-019-4704-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815020PMC
October 2019

Using Colistin as a Trojan Horse: Inactivation of Gram-Negative Bacteria with Chlorophyllin.

Antibiotics (Basel) 2019 Sep 20;8(4). Epub 2019 Sep 20.

Postgraduate Program in Health and Environment, University of Joinville Region, Rua Paulo Malschitzki, 10, Joinville 89219-710, Brazil.

Colistin (polymyxin E) is a membrane-destabilizing antibiotic used against Gram-negative bacteria. We have recently reported that the outer membrane prevents the uptake of antibacterial chlorophyllin into Gram-negative cells. In this study, we used sub-toxic concentrations of colistin to weaken this barrier for a combination treatment of and serovar Typhimurium with chlorophyllin. In the presence of 0.25 µg/mL colistin, chlorophyllin was able to inactivate both bacteria strains at concentrations of 5-10 mg/L for and 0.5-1 mg/L for Typhimurium which showed a higher overall susceptibility to chlorophyllin treatment. In accordance with a previous study, chlorophyllin has proven antibacterial activity both as a photosensitizer, illuminated with 12 mW/cm, and in darkness. Our data clearly confirmed the relevance of the outer membrane in protection against xenobiotics. Combination treatment with colistin broadens chlorophyllin's application spectrum against Gram-negatives and gives rise to the assumption that chlorophyllin together with cell membrane-destabilizing substances may become a promising approach in bacteria control. Furthermore, we demonstrated that colistin acts as a door opener even for the photodynamic inactivation of colistin-resistant (-positive) cells by chlorophyllin, which could help us to overcome this antimicrobial resistance.
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http://dx.doi.org/10.3390/antibiotics8040158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963628PMC
September 2019

Induction of Necrosis in Human Macrophage Cell Lines by and Strains Isolated from Fatal Cases of Systemic Infections.

Int J Mol Sci 2019 Aug 22;20(17). Epub 2019 Aug 22.

Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.

When infecting a human host, and are able to impair macrophage maturation and induce cell death. However, the underlying molecular mechanisms are not well understood. As a framework for this project, a combination of fluorescence microscopy, cytotoxicity assays, live cell imaging, and fluorescence-activated cell sorting was applied to understand the pathogenicity of two strains isolated from fatal cases of systemic infections. The results showed a clear cytotoxic effect of the bacteria. The observed survival of the pathogens in macrophages and, subsequent, necrotic lysis of cells may be mechanisms explaining dissemination of and to distant organs in the body.
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http://dx.doi.org/10.3390/ijms20174109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747468PMC
August 2019

Proteomics of Bordetella pertussis whole-cell and acellular vaccines.

BMC Res Notes 2019 Jun 10;12(1):329. Epub 2019 Jun 10.

Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058, Erlangen, Germany.

Objectives: Bordetella pertussis is the etiological agent of whooping cough, a bacterial infection of especially children, which may be fatal without treatment. In frame of studies to investigate putative effects of vaccination on host-pathogen interaction and clonal distribution of strains, in addition to Corynebacterium diphtheriae and Clostridium tetani toxoid vaccines, also whole-cell and acellular pertussis vaccines were analyzed by mass spectrometry.

Data Description: LC-MS/MS spectra were generated and analyzed using B. pertussis genome data and proteins present in whole-cell and acellular pertussis vaccines were identified. Subcellular localization of proteins and presence of signal peptides was determined bioinformatically.
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http://dx.doi.org/10.1186/s13104-019-4373-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558669PMC
June 2019

Beyond diphtheria toxin: cytotoxic proteins of Corynebacterium ulcerans and Corynebacterium diphtheriae.

Microbiology (Reading) 2019 08 4;165(8):876-890. Epub 2019 Jun 4.

> Microbiology Division, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.

Diphtheria toxin is one of the best investigated bacterial toxins and the major virulence factor of toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains. However, also diphtheria toxin-free strains of these two species can cause severe infections in animals and humans, indicating the presence of additional virulence factors. In this study, we present a first characterization of two proteins with cytotoxic effect in corynebacteria. A putative ribosome-binding protein (AEG80717, CULC809_00177), first annotated in a genome sequencing project of C. ulcerans strain 809, was investigated in detail together with a homologous protein identified in C. diphtheriae strain HC04 (AEX80148, CDHC04_0155) in this study. The corresponding proteins show striking structural similarity to Shiga-like toxins. Interaction of wild-type, mutant and complementation as well as overexpression strains with invertebrate model systems and cell lines were investigated. Depending on the presence of the corresponding genes, detrimental effects were observed in vivo in two invertebrate model systems, Caenorhabditis elegans and Galleria mellonella, and on various animal and human epithelial and macrophage cell lines in vitro. Taken together, our results support the idea that pathogenicity of corynebacteria is a multifactorial process and that new virulence factors may influence the outcome of potentially fatal corynebacterial infections.
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http://dx.doi.org/10.1099/mic.0.000820DOI Listing
August 2019

Of mice and men: Interaction of Corynebacterium diphtheriae strains with murine and human phagocytes.

Virulence 2019 12;10(1):414-428

a Department Biologie , Friedrich-Alexander-Universität Erlangen-Nürnberg , Erlangen , Germany.

Seven non-toxigenic C. diphtheriae strains and one toxigenic strain were analyzed with regard to their interaction with murine macrophages (BMM) and human THP-1 macrophage-like cells. Proliferation assays with BMM and THP-1 revealed similar intracellular CFUs for C. diphtheriae strains independent of the host cell. Strain ISS4060 showed highest intracellular CFUs, while the toxigenic DSM43989 was almost not detectable. This result was confirmed by TLR 9 reporter assays, showing a low signal for DSM43989, indicating that the bacteria are not endocytosed. In contrast, the non-pathogenic C. glutamicum showed almost no intracellular CFUs independent of the host cell, but was recognized by TLR9, indicating that the bacteria were degraded immediately after endocytosis. In terms of G-CSF and IL-6 production, no significant differences between BMM and THP-1 were observed. G-CSF production was considerably higher than IL-6 for all C. diphtheriae strains and the C. glutamicum did not induce high cytokine secretion in general. Furthermore, all corynebacteria investigated in this study were able to induce NFκB signaling but only viable C. diphtheriae strains were able to cause host cell damage, whereas C. glutamicum did not. The absence of Mincle resulted in reduced G-CSF production, while no influence on the uptake of the bacteria was observed. In contrast, when MyD88 was absent, both the uptake of the bacteria and cytokine production were blocked. Consequently, phagocytosis only occurs when the TLR/MyD88 pathway is functional, which was also supported by showing that all corynebacteria used in this study interact with human TLR2.
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http://dx.doi.org/10.1080/21505594.2019.1614384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527023PMC
December 2019

Proteomics of diphtheria toxoid vaccines reveals multiple proteins that are immunogenic and may contribute to protection of humans against Corynebacterium diphtheriae.

Vaccine 2019 05 26;37(23):3061-3070. Epub 2019 Apr 26.

Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. Electronic address:

Introduced for mass immunization in the 1920s, vaccines against diphtheria are among the oldest and safest vaccines known. The basic principle of their production is the inactivation of purified diphtheria toxin by formaldehyde cross-linking, which converts the potentially fatal toxin in a completely harmless protein aggregate, which is still immunogenic. Since in addition to diphtheria toxin also other proteins may be secreted by Corynebacterium diphtheriae during cultivation, we assumed that diphtheria toxoid might not be the only component present in the vaccine. To address this question, we established a protocol to reverse formaldehyde cross-linking and carried out mass spectrometric analyses. Different secreted, membrane-associated and cytoplasmic proteins of C. diphtheriae were detected in several vaccine preparations from across the world. Based on these results, bioinformatics and Western blot analyses were applied to characterize if these proteins are immunogenic and may therefore support protection against C. diphtheriae. In frame of this study, we could show that the C. diphtheriae toxoid vaccines induce antibodies against different C. diphtheriae proteins and against diphtheria toxin secreted by Corynebacterium ulcerans, an emerging pathogen which is outnumbering C. diphtheriae as cause of diphtheria-like illness in Western Europe.
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http://dx.doi.org/10.1016/j.vaccine.2019.04.059DOI Listing
May 2019

More than a Toxin: Protein Inventory of Toxoid Vaccines.

Proteomes 2019 Apr 16;7(2). Epub 2019 Apr 16.

Microbiology Division, Department of Biology, Friedrich-Alexander University Erlangen-Nuremberg, Staudtstr. 5, 91058 Erlangen, Germany.

is the etiological agent of tetanus, a life-threatening bacterial infection. The most efficient protection strategy against tetanus is a vaccination with the neurotoxin, which is inactivated by formaldehyde-crosslinking. Since we assumed that besides the tetanus toxin, other proteins of may also be present in toxoid preparations, we analyzed commercially available vaccines from different countries in respect to their protein content using mass spectrometry. In total 991 proteins could be identified in all five analyzed vaccines, 206 proteins were common in all analyzed vaccines and 54 proteins from the 206 proteins were potential antigens. The additionally present proteins may contribute at least partially to protection against infection by supporting the function of the vaccine against the devastating effects of the tetanus toxin indirectly. Two different label-free protein quantification methods were applied for an estimation of protein contents. Similar results were obtained with a Total Protein Approach (TPA)-based method and Protein Discoverer 2.2 software package based on the minora algorithm. Depending on the tetanus toxoid vaccine and the quantification method used, tetanus neurotoxin contributes between 14 and 76 % to the total protein content and varying numbers of other proteins were detected.
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http://dx.doi.org/10.3390/proteomes7020015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631180PMC
April 2019

Elimination of bacterial contaminations by treatment of water with boron-doped diamond electrodes.

World J Microbiol Biotechnol 2019 Mar 6;35(3):48. Epub 2019 Mar 6.

Professur für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058, Erlangen, Germany.

Boron-doped diamond electrodes can be used to generate reactive oxygen species directly at the electrode's surface. This property was used in this study for in-situ electrochemical oxidation to eliminate different bacteria, i.e. Escherichia coli, Pseudomonas fluorescens and Pseudomonas aeruginosa, as well as Bacillus subtilis spores from water samples. Application of low voltages in the rage from 4 to 10 V and short incubation times in the range of minutes allowed a complete disinfection of water contaminated with enterobacteria and freshwater microbes including nosocomial pathogens as well as a significant reduction of spores. A pilot reactor was constructed, which allowed to decrease microbial contamination of sewage plant effluent drastically. Boron-doped diamond electrodes allow efficient reduction of bacterial contaminations in water samples.
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http://dx.doi.org/10.1007/s11274-019-2624-yDOI Listing
March 2019

What an Mutant Can Teach Us About the Antibacterial Effect of Chlorophyllin.

Microorganisms 2019 Feb 22;7(2). Epub 2019 Feb 22.

Cell Biology Division: Gravitational Biology Group, Department of Biology, Friedrich-Alexander University Erlangen-Nuremberg, Staudtstraße 5, 91058 Erlangen, Germany.

Due to the increasing development of antibiotic resistances in recent years, scientists search intensely for new methods to control bacteria. Photodynamic treatment with porphyrins such as chlorophyll derivatives is one of the most promising methods to handle bacterial infestation, but their use is dependent on illumination and they seem to be more effective against Gram-positive bacteria than against Gram-negatives. In this study, we tested chlorophyllin against three bacterial model strains, the Gram-positive 168, the Gram-negative DH5α and strain NR698 which has a deficient outer membrane, simulating a Gram-negative "without" its outer membrane. Illuminated with a standardized light intensity of 12 mW/cm², showed high sensitivity already at low chlorophyllin concentrations (≤10⁵ cfu/mL: ≤0.1 mg/L, 10⁶⁻10⁸ cfu/mL: 0.5 mg/L), whereas DH5α was less sensitive (≤10⁵ cfu/mL: 2.5 mg/L, 10⁶ cfu/mL: 5 mg/L, 10⁷⁻10⁸ cfu/mL: ineffective at ≤25 mg/L chlorophyllin). NR698 was almost as sensitive as against chlorophyllin, pointing out that the outer membrane plays a significant role in protection against photodynamic chlorophyllin impacts. Interestingly, NR698 and can also be inactivated by chlorophyllin in darkness, indicating a second, light-independent mode of action. Thus, chlorophyllin seems to be more than a photosensitizer, and a promising substance for the control of bacteria, which deserves further investigation.
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http://dx.doi.org/10.3390/microorganisms7020059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406390PMC
February 2019

Detection and virulence potential of a phospholipase D-negative Corynebacterium ulcerans from a concurrent diphtheria and infectious mononucleosis case.

Antonie Van Leeuwenhoek 2019 Jul 15;112(7):1055-1065. Epub 2019 Feb 15.

Laboratory of Diphtheria and Corynebacteria of Clinical Relevance - Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro - UERJ, Av. 28 de Setembro, 87 - Fundos, 3° andar. Vila Isabel, Rio de Janeiro, RJ, 20.551, Brazil.

Diphtheria by Corynebacterium ulcerans is increasingly occurring in children, adolescents and adults. In addition to diphtheria toxin (DT), phospholipase D (PLD) is considered a virulence factor of C. ulcerans. In the present study, a first case of concurrent diphtheria by a PLD-negative C. ulcerans and infectious mononucleosis (IM) was verified. Clinical and microbiological profiles and binding properties to human Fibrinogen (Fbg), Fibronectin (Fn) and type I collagen (col I) biotinylated proteins and virulence to Caenorhabditis elegans were investigated for C. ulcerans strain 2590 (clinical isolate) and two control strains, including PLD-positive BR-AD22 wild type and PLD-negative ELHA-1 PLD mutant strains. MALDI-TOF assays and a multiplex PCR of genes coding for potentially toxigenic corynebacteria identified strain 2590 as non-DT producing. Interestingly, strain 2590 did not express PLD activity in the CAMP test although the presence of the pld gene was verified. PLD-negative 2590 and a PLD-positive 210932 strains showed similar affinity to Fbg, Fn and type I collagen. C. elegans were able to escape from C. ulcerans strains, independent of PLD and DT production. Higher mortality of nematodes was verified for PLD-negative strains. Additional studies concerning multifactorial virulence potential of C. ulcerans, including environmental conditions remain necessary.
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http://dx.doi.org/10.1007/s10482-019-01240-4DOI Listing
July 2019

Lack of evidence for the necessity of root canal obturation.

Quintessence Int 2019 ;50(1):22-28

Objective: Root canal obturation still is a relevant research topic and patients spend substantial amounts of financial resources for this step of endodontic treatment. Three experiments were conducted challenging the necessity of root canal obturation.

Method And Materials: Applying micro computed tomography, the volume of dentin tubules that cannot be instrumented during root canal therapy was determined. Using a simple biofilm model of human tooth segments, the effect of root canal obturation on the persistency of bacteria was evaluated and freshly extracted root canal treated teeth were examined for bacteria remaining in dentin.

Results: The volume of dentinal canals was found to be at least three times greater than the volume of the root canal itself. Bacterial growth was observed both in specimens with and without root canal obturation implying that the treatment rendered was ineffective in removing bacterial biofilm and the obturation material was incapable of hindering bacterial regrowth.

Conclusion: Despite showing adequate root canal obturation radiographically, persistent bacteria could be identified in all teeth extracted. While perfect disinfection of root canals is mandatory, root canal obturation seems questionable as current materials have no antibacterial activity, do not stabilize the tooth, and cannot seal the canal system if a coronal restoration is missing.
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http://dx.doi.org/10.3290/j.qi.a41335DOI Listing
May 2019

Biological Optical-to-Chemical Signal Conversion Interface: A Small-Scale Modulator for Molecular Communications.

IEEE Trans Nanobioscience 2019 01 18;18(1):31-42. Epub 2018 Sep 18.

Although many exciting applications of molecular communication (MC) systems are envisioned to be at microscale, the MC testbeds reported in the literature so far are mostly at macroscale. This may partially be due to the fact that controlling an MC system at microscale is challenging. To link the macroworld to the microworld, we propose and demonstrate a biological signal conversion interface that can also be seen as a microscale modulator. In particular, the proposed interface transduces an optical signal, which is controlled using a light-emitting diode, into a chemical signal by changing the pH of the environment. The modulator is realized using Escherichia coli bacteria as microscale entity expressing the light-driven proton pump gloeorhodopsin from Gloeobacter violaceus. Upon inducing external light stimuli, these bacteria locally change their surrounding pH level by exporting protons into the environment. To verify the effectiveness of the proposed optical-to-chemical signal converter, we analyze the pH signal measured by a pH sensor, which serves as a receiver. We develop an analytical parametric model for the induced chemical signal as a function of the applied optical signal. Using this model, we derive a training-based channel estimator that estimates the parameters of the proposed model to fit the measurement data based on a least square error approach. We further derive the optimal maximum likelihood detector and a suboptimal low-complexity detector to recover the transmitted data from the measured received signal. It is shown that the proposed parametric model is in good agreement with the measurement data. Moreover, for an example scenario, we show that the proposed setup is able to successfully convert an optical signal representing a sequence of binary symbols into a chemical signal with a bit rate of 1 bit/min and recover the transmitted data from the chemical signal using the proposed estimation and detection schemes. The proposed modulator may form the basis for future MC testbeds and applications at microscale.
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http://dx.doi.org/10.1109/TNB.2018.2870910DOI Listing
January 2019

The C-terminal coiled-coil domain of Corynebacterium diphtheriae DIP0733 is crucial for interaction with epithelial cells and pathogenicity in invertebrate animal model systems.

BMC Microbiol 2018 09 4;18(1):106. Epub 2018 Sep 4.

Microbiology Division, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.

Background: Corynebacterium diphtheriae is the etiologic agent of diphtheria and different systemic infections. The bacterium has been classically described as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of C. diphtheriae still remains unclear. Recently, the DIP0733 transmembrane protein was found to play an important role in the interaction with matrix proteins and cell surfaces, nematode colonization, cellular internalization and induction of cell death.

Results: In this study, we identified a number of short linear motifs and structural elements of DIP0733 with putative importance in virulence, using bioinformatic approaches. A C-terminal coiled-coil region of the protein was considered particularly important, since it was found only in DIP0733 homologs in pathogenic Corynebacterium species but not in non-pathogenic corynebacteria. Infections of epithelial cells and transepithelial resistance assays revealed that bacteria expressing the truncated form of C. diphtheriae DIP0733 and C. glutamicum DIP0733 homolog are less virulent, while the fusion of the coiled-coil sequence to the DIP0733 homolog from C. glutamicum resulted in increased pathogenicity. These results were supported by nematode killing assays and experiments using wax moth larvae as invertebrate model systems.

Conclusions: Our data indicate that the coil-coiled domain of DIP0733 is crucial for interaction with epithelial cells and pathogenicity in invertebrate animal model systems.
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http://dx.doi.org/10.1186/s12866-018-1247-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123952PMC
September 2018

Synthesis and characterization of manganese containing mesoporous bioactive glass nanoparticles for biomedical applications.

J Mater Sci Mater Med 2018 May 8;29(5):64. Epub 2018 May 8.

Department of Materials Science and Engineering, Institute of Biomaterials, University of Erlangen-Nuremberg, Cauerstr. 6, Erlangen, 91058, Germany.

Mesoporous bioactive glass (BG) nanoparticles based in the system: SiO-PO-CaO-MnO were synthesized via a modified Stöber process at various concentrations of Mn (0-7 mol %). The synthesized manganese-doped BG nanoparticles were characterized in terms of morphology, composition, in vitro bioactivity and antibacterial activity. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Brunauer-Emmett-Teller (BET) analysis confirmed that the particles had spherical morphology (mean particle size: 110 nm) with disordered mesoporous structure. Energy dispersive X-ray spectroscopy (EDX) confirmed the presence of Mn, Ca, Si and P in the synthesized Mn-doped BG particles. Moreover, X-ray diffraction (XRD) analysis showed that Mn has been incorporated in the amorphous silica network (bioactive glass). Moreover, it was found that manganese-doped BG particles form apatite crystals upon immersion in simulated body fluid (SBF). Inductively coupled plasma atomic emission spectroscopy (ICP-OES) measurements confirmed that Mn is released in a sustained manner, which provided antibacterial effect against Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. The results indicate that the incorporation of Mn in the bioactive glass network is an effective strategy to develop novel multifunctional BG nanoparticles for bone tissue engineering.
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http://dx.doi.org/10.1007/s10856-018-6070-4DOI Listing
May 2018

Surface and Extracellular Proteome of the Emerging Pathogen .

Proteomes 2018 Apr 17;6(2). Epub 2018 Apr 17.

Professur für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany.

is an emerging pathogen, which is increasingly recognized as an etiological agent of diphtheria, but can also evoke ulcers of the skin and systemic infections in humans. Besides man, the bacteria can colonize a wide variety of different animals, including cattle and pet animals, which might serve as a reservoir for human infections. In this study, surface-located proteins and the exoproteome of two strains were analyzed, since these may have key roles in the interaction of the pathogen with host cells. Strain 809 was isolated from a fatal case of human respiratory tract infection, while strain BR-AD22 was isolated from a nasal swap of an asymptomatic dog. While a very similar pattern of virulence factors was observed in the culture supernatant and surface protein fractions of the two strains, proteome analyses revealed a higher stability of 809 cells compared to strain BR-AD22. During exponential growth, 17% of encoded proteins of strain 809 were detectable in the medium, while 38% of the predicted proteins encoded by the BR-AD22 chromosome were found. Furthermore, the data indicate differential expression of phospholipase D and a cell wall-associated hydrolase, since these were only detected in strain BR-AD22.
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http://dx.doi.org/10.3390/proteomes6020018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027474PMC
April 2018

Argon Cold Plasma-A Novel Tool to Treat Therapy-resistant Corneal Infections.

Am J Ophthalmol 2018 06 24;190:150-163. Epub 2018 Mar 24.

Department of Ophthalmology, Friedrich-Alexander Universität Erlangen-Nürnberg, University Hospital Erlangen, Erlangen, Germany. Electronic address:

Purpose: To test whether therapy-resistant corneal infections can be successfully treated with argon cold plasma to reduce or eliminate pathogen microorganisms without affecting corneal cell viability.

Design: First-in-human case series and experimental study.

Methods: Cold plasma effects on viability of primary human corneal limbal epithelial cells were studied using exposure times from 0.5 to 10 minutes (metabolic activity, oxidative stress, apoptosis). Disinfective potential of cold plasma was tested against common pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans) on culture medium and evaluated by counting colony-forming units and optical density measurements, as well as against S aureus in a human cornea infection model. Additionally, in a first-in-human trial 4 patients with therapy-resistant corneal ulcers were treated to evaluate the clinical potential of cold plasma.

Results: Cells treated for 0.5-5 minutes completely recovered within 24 hours without changes in morphology; only 10-minute treatment impaired the cells permanently. No evident oxidative stress, apoptosis, or damage to the corneal structure could be found. All pathogens were susceptible to cold plasma treatments, with different levels of sensitivity. The condition of all 4 patients significantly improved after cold plasma treatment combined with antibiotic therapy.

Conclusions: Our results indicate that argon cold plasma treatment reduces or eliminates common pathogens without impairing corneal epithelial cells in vitro, ex vivo, and in direct application on patients' eyes. We conclude that argon cold plasma therapy offers a potential supplement or alternative therapy for therapy-resistant corneal infections. A larger, comparative study is necessary to further confirm these findings.
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http://dx.doi.org/10.1016/j.ajo.2018.03.025DOI Listing
June 2018

Complete Closed Genome Sequence of Nontoxigenic Invasive bv. mitis Strain ISS 3319.

Genome Announc 2018 Feb 1;6(5). Epub 2018 Feb 1.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom

The genome sequence of the human pathogen bv. mitis strain ISS 3319 was determined and closed in this study. The genome is estimated to have 2,404,936 bp encoding 2,257 proteins. This strain also possesses a plasmid of 1,960 bp.
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http://dx.doi.org/10.1128/genomeA.01566-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794954PMC
February 2018

The role of corynomycolic acids in Corynebacterium-host interaction.

Antonie Van Leeuwenhoek 2018 May 12;111(5):717-725. Epub 2018 Feb 12.

Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058, Erlangen, Germany.

Within the Actinobacteria, the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus form the so-called CMNR group, also designated as mycolic acid-containing actinomycetes. Almost all members of this group are characterized by a mycolic acid layer, the mycomembrane, which covers the cell wall and is responsible for a high resistance of these bacteria against chemical and antibiotic stress. Furthermore, components of the mycomembrane are crucial for the interaction of bacteria with host cells. This review summarizes the current knowledge of mycolic acid synthesis and interaction with components of the immune system for the genus Corynebacterium with an emphasis on the pathogenic species Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis and Corynebacterium ulcerans as well as the biotechnology workhorse Corynebacterium glutamicum.
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http://dx.doi.org/10.1007/s10482-018-1036-6DOI Listing
May 2018

Differential NtcA Responsiveness to 2-Oxoglutarate Underlies the Diversity of C/N Balance Regulation in .

Front Microbiol 2017 9;8:2641. Epub 2018 Jan 9.

Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Córdoba, Córdoba, Spain.

Previous studies showed differences in the regulatory response to C/N balance in with respect to other cyanobacteria, but no information was available about its causes, or the ecological advantages conferred to thrive in oligotrophic environments. We addressed the changes in key enzymes (glutamine synthetase, isocitrate dehydrogenase) and the gene (the global nitrogen regulator) involved in C/N metabolism and its regulation, in three model strains: MED4, SS120, and MIT9313. We observed a remarkable level of diversity in their response to azaserine, a glutamate synthase inhibitor which increases the concentration of the key metabolite 2-oxoglutarate, used to sense the C/N balance by cyanobacteria. Besides, we studied the binding between the global nitrogen regulator (NtcA) and the promoter of the gene in the same strains, and its dependence on the 2-oxoglutarate concentration, by using isothermal titration calorimetry, surface plasmon resonance, and electrophoretic mobility shift. Our results show a reduction in the responsiveness of NtcA to 2-oxoglutarate in , especially in the MED4 and SS120 strains. This suggests a trend to streamline the regulation of C/N metabolism in late-branching strains (MED4 and SS120), in adaptation to the rather stable conditions found in the oligotrophic ocean gyres where this microorganism is most abundant.
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http://dx.doi.org/10.3389/fmicb.2017.02641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767323PMC
January 2018