Peter Schierack

Brandenburgische Technische Universität Cottbus, Kottbus, Brandenburg, Germany

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Publications (56)144.59 Total impact

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    ABSTRACT: Due to increased release or production of Shiga toxin by Enterohemorrhagic Escherichia coli (EHEC) after exposure to antimicrobial agents, the role of antimicrobial agents in EHEC mediated infections remains controversial. Probiotics are therefore rapidly gaining interest as an alternate therapeutic option. The well-known probiotic strain Escherichia coli Nissle 1917 (EcN) was tested in vitro to determine its probiotic effects on growth, Shiga toxin (Stx) gene expression, Stx amount and associated cytotoxicity on the most important EHEC strains of serotype O104:H4 and O157:H7. Following co-culture of EcN:EHEC in broth for 4 and 24 h, the probiotic effects on EHEC growth, toxin gene expression, Stx amount and cytotoxicity were determined using quantitative real time-PCR, Stx-ELISA and Vero cytotoxicity assays.Probiotic EcN strongly reduced EHEC numbers (cfu) of O104:H4 up to (68%) and O157:H7 to (72.2%) (P < 0.05) in LB broth medium whereas the non-probiotic E. coli strain MG1655 had no effect on EHEC growth. The level of stx expression was significantly down-regulated, particularly for the stx2a gene. The stx down-regulation in EcN co-culture was not due to reduced numbers of EHEC. A significant inhibition in Stx amounts and cytotoxicity were also observed in sterile supernatants of EcN:EHEC co-cultures.These findings indicate that probiotic EcN displays strong inhibitory effects on growth, Shiga toxin gene expression, amount and cytotoxicity of EHEC strains. Thus, EcN may be considered as a putative therapeutic candidate, in particular against EHEC O104:H4 and O157:H7.
    International Journal of Medical Microbiology. 10/2014;
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    ABSTRACT: The international consensus for the classification of antiphospholipid syndrome (APS) requires clinical and laboratory criteria to be considered at an equal level for diagnosing APS. Thus, detection of antiphospholipid antibodies (aPL) being a hallmark of APS has been the object of intensive investigation over the past 40 years. However, appropriate detection of aPL still remains a laboratory challenge due to their heterogeneity comprising autoantibodies reactive to different phospholipid-binding plasma proteins, such as beta-2 glycoprotein I (β2GPI) and prothrombin. The relevance of aPL interacting with phospholipids other than cardiolipin (CL, diphosphatidylglycerol), such as phosphatidylserine (PS), remains elusive with regard to the diagnosis of APS. Recently, the concept of aPL profiling has been introduced to assess the risk of thrombotic complications in patients with APS. New assay techniques, apart from enzyme-linked immunosorbent assays (ELISAs) recommended by the international consensus for the classification of APS, have been proposed for multiplexing of aPL testing. Line immunoassays (LIAs) employing a novel hydrophobic solid phase for the simultaneous detection of different aPL seem to be an intriguing alternative. We evaluated a novel multiplex LIA employing a hydrophobic membrane coated with different phospholipid (PL)-binding proteins or PLs. The performance characteristics of this new multiplexing assay technique demonstrated its usefulness for aPL profiling.
    Lupus 10/2014; 23(12):1262-4. · 2.78 Impact Factor
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    ABSTRACT: Dear Editor, With interest we read the paper by Juste et al1 proposing the amount of zymogen-granule membrane glycoprotein 2 (GP2) on the surface of intestinal bacteria as a Crohn's disease (CD) marker. Indeed, a decreased GP2 level was found on microbes in patients with CD as compared to those of healthy controls. GP2 is a homologue to the urinary Tamm–Horsefall protein demonstrating an antimicrobial function by binding type 1-fimbriated uropathogenic Escherichia coli (UPEC). Likewise, GP2 seems to interact with intestinal bacteria as a specific receptor of bacterial type-1 fimbriae (FimH) on intestinal microfold cells that are partaking in immune responses against such microbes.2 GP2 is overexpressed in the inflamed intestine of patients with CD and has an immunomodulating role in innate and acquired immune responses.3 ,4 Interestingly, GP2 was identified as autoantigen of pancreatic antibodies in CD.4 Altogether, these findings indicate two major GP2 sources (pancreatic/intestinal) and support a role for GP2 in the interaction between the immune system and intestinal microbiota.3 Thus, loss of tolerance to GP2 could play a role in CD's pathophysiology supposed to be exacerbated by preceding intestinal infections. In general, the findings by Juste et al1 may be explained by a lower pancreatic GP2 secretion, an impaired GP2 binding to bacteria, or by a higher prevalence of bacteria with poor or no GP2 binding in patients with CD.
    Gut 07/2014; · 10.73 Impact Factor
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    ABSTRACT: The edition of this Special Issue was commenced in 2013 at the occasion of the 60th anniversary of the elucidation of DNA structure. This milestone has completely changed biological and medical sciences and, more recently, has triggered the development of sophisticated instrumentation. The discovery of the polymerase chain reaction (PCR) has further promoted this trend and now allows the analysis of a DNA sequences even at a level of a single molecule. Similarly, various miniaturized chip-based approaches have been introduced in the past 10 years. The transition from a laboratory scale to a microscale implies many advantages. These include, in particular, reduced sample volumes, reduced costs, shorter assay times, faster heating/cooling rates, higher throughput, and the integration of processing module cascades. The chip approach enabled, in particular, the development of sophisticated PCR assays. These include single-well and multi-well continuous flow PCR devices, stationary chamber-based PCR chips, and planar microbead-based chips. Most of these systems have common components such as miniaturized reaction chambers, heating/cooling units, and an analyzer/detector unit. Some of the devices evolved from the end-point quantification to the multiplex real-time monitoring of amplification reactions and high-resolution analysis of melting curves. These technologies contributed to breakthroughs that were envisioned years before. In particular, PCR in microfluidics and isothermal amplification have radically changed medical diagnostics and life sciences in general. Any development in field is based on the efforts of experts from highly different fields, examples being molecular biology, surface chemistry, microfluidics, and of course engineering. Systems are becoming smaller and more widely applicable, for example to diagnostics, environmental monitoring, and life sciences. This Special Issue gives a representative selection of the progress made in this field and in technologies related to affinity biosensors in a wider sense. The Issue includes reviews and original papers. Many of them are based on presentations given at the 7th Senftenberg Innovationsforum on Multiparameter Diagnostics in April 2013 which was organized by Brandenburg University of Technology Cottbus - Senftenberg (Germany). We have mainly included manuscripts on new analytical techniques, on end-user applications, new instrumentation, sensors and materials, with a main focus on the analysis of nucleic acids. The first part (4 reviews) gives an overview on current technologies such as extraction, amplification and detection of DNA in microfluidic chip-based and microbead-based assays. This includes recent developments in solid-phase enzymatic assays and the application in multiparametric diagnostics. The second part (Chip-based technologies for amplification, detection and analyses of nucleic acids) mainly focuses on end-point and real-time PCR techniques. Articles cover topics ranging from fundamental research to commercial applications. Part 3 (Application of chip-based technologies for amplification, detection and analyses) covers aspects of reference gene selection, quantification of gene expression, PCR in microfluidics, microarray technology in PCR, conventional and isothermal amplification methods as well as fluorescent detection. The final part (Chip-based technologies for affinity sensing) focuses on novel techniques for use in affinity sensing which have a wide application potential including analysis of nucleic acids. We are confident that this Special Issue represents a timely overview on this field and will fuel novel ideas for microanalytical systems. Last, but not least, we would like to thank the authors of this Special Issue for their excellent articles. We also are grateful to the reviewers for their numerous constructive comments on these manuscripts. We truly hope that readers will enjoy reading the result of this collective effort. V. M. Mirsky, S. Rödiger, P. Schierack, D. Roggenbuck (Editors) Brandenburg University of Technology, Cottbus – Senftenberg (Germany)
    Microchimica Acta 04/2014; · 3.43 Impact Factor
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    ABSTRACT: Autoantibodies are well-established biomarkers of autoimmune rheumatic diseases. Their detection in the routine clinical laboratory provides key information for early and differential diagnosis, prognosis, and monitoring of disease-activity. However, the currently existing variety of laboratory tests and methods as well as different diagnostic and clinical algorithms of autoimmune testing is the reason for lack of reproducibility and harmonization across immunological laboratories. Novel technical developments in the area of digital analysis of immunofluorescent images paved the way for the improvement of intra- and interlaboratory standardizationof test results. Additionally, digital immunofluorescence analysis enables the simultaneous multiparametric combination of cell- or tissue-based indirect immunofluorescence screening tests with confirmatory testing employing microparticles covered with purified autoantigens in one reaction environment.
    Medical Immunology 03/2014; 16(3):221-226.
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    ABSTRACT: Enteropathogenic Escherichia coli (EPEC) are recognized as important intestinal pathogens that frequently cause acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastro-intestinal disorders. While both in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria including pathogenic E. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infection steps including adhesion, microcolony formation and attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagellae, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific for EcN but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of these inhibitory factor. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence associated proteins of EPEC, but not their expression.
    Infection and immunity 02/2014; · 4.21 Impact Factor
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    ABSTRACT: Anti-neutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of small vessel vasculitis, so called ANCA-associated vasculitis. The international consensus requires testing by indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils (ethN) as screening followed by confirmation with enzyme-linked immunosorbent assays (ELISAs). This study evaluates the combination of cell- and microbead-based digital IIF analysis of ANCA in one reaction environment by the novel multiplexing CytoBead technology for simultaneous screening and confirmatory ANCA testing. Sera of 592 individuals including 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 77 with inflammatory bowel syndrome, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis and 125 blood donors were tested for cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA) by classical IIF and ANCA to proteinase 3 (PR3) and myeloperoxidase (MPO) by ELISA. These findings were compared to respective ANCA results determined by automated multiplex CytoBead technology using ethN and antigen-coated microbeads for microbead immunoassays. There was a good agreement for PR3- and MPO-ANCA and a very good one for P-ANCA and C-ANCA by classical and multiplex analysis (Cohen's kappa [κ] = 0.775, 0.720, 0.876, 0.820, respectively). The differences between classical testing and CytoBead analysis were not significant for PR3-ANCA, P-ANCA, and C-ANCA (p<0.05, respectively). The prevalence of confirmed positive ANCA findings by classical testing (IIF and ELISA) compared with multiplex CytoBead analysis (IIF and microbead immunoassay positive) resulted in a very good agreement (κ = 0.831) with no significant difference of both methods (p = 0.735). Automated endpoint-ANCA titer detection in one dilution demonstrated a very good agreement with classical analysis requiring dilution of samples (κ = 0.985). Multiplexing by CytoBead technology can be employed for simultaneous screening and quantitative confirmation of ANCA. This novel technique provides fast and cost-effective ANCA analysis by automated digital IIF for the first time.
    PLoS ONE 01/2014; 9(9):e107743. · 3.53 Impact Factor
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    ABSTRACT: Heterogeneous nuclear ribonucleoproteins (hnRNPs) are potent autoantigenic targets in systemic autoimmune rheumatic diseases (SARD). Loss of tolerance to the RA33 complex consisting of hnRNP A2 and its alternatively spliced variants B1 and B2 has been the interest of rheumatologists. A novel ELISA for the detection of anti-hnRNP B1 autoantibodies has been developed to investigate the prevalence thereof in 397 patients with SARD, including patients with rheumatoid arthritis (RA), spondyloarthropathy (SPA), juvenile chronic arthritis, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjögren's syndrome (SS), in comparison to 174 controls. Anti-hnRNP B1 autoantibodies were significantly more prevalent in patients with SARD than controls (47/397, 11.8% versus 2/174, 1.1%; P < 0.001). In particular, anti-hnRNP B1 were found more frequently in the disease cohorts than in the controls and were present in 24/165 (14.5%) patients with RA, 6/58 (10.3%) SPA, 11/65 (16.9%) SSc, and 4/50 (8.0%) SLE. In RA patients, anti-hnRNP B1 autoantibodies correlated significantly with C-reactive protein levels and erythrocyte sedimentation rate, while in patients with SSc it was associated with features of arterial wall stiffness and presence of hypertension. Anti-hnRNP B1 autoantibodies occur in SARD and seem to be correlated with distinct clinical characteristics in patients with RA and SSc.
    Research Journal of Immunology 01/2014; 2014:516593.
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    ABSTRACT: Microbead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. Contains 211 references.
    Microchimica Acta 01/2014; · 3.43 Impact Factor
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    ABSTRACT: Abstract Crohn's disease (CrD) and ulcerative colitis (UC) are the main inflammatory bowel diseases (IBD). IBD-specific humoral markers of autoimmunity in the form of autoantibodies have been reported first in the late 1950s by demonstrating the occurrence of autoimmunity in UC, while humoral autoimmunity in CrD can be traced back to the 1970s. Ever since, the pathophysiological role of autoimmune responses in IBDs has remained poorly understood. Notwithstanding, autoreactive responses play a major role in inflammation leading to overt IBD. In CrD, approximately 40% of patients and <20% of patients with UC demonstrate loss of tolerance to antigens of the exocrine pancreas. Glycoprotein 2 (GP2) has been identified as a major autoantigenic target of the so-called pancreatic antibodies. The previously unsolved contradiction of pancreatic autoreactivity and intestinal inflammation in IBD was elucidated by demonstrating the expression of GP2 at the site thereof. Intriguingly, GP2 has been reported to be a receptor on microfold cells of intestinal Peyer's patches, which are believed to represent the origin of CrD inflammation. The development of immunoassays for the detection of antibodies to GP2 has paved the way to investigate the association of such antibodies with the clinical phenotype in CrD. Given the recently discovered immunomodulating role of GP2 in innate and adaptive intestinal immunity, this association can shed further light on the pathophysiology of IBD. In this context, the association of anti-GP2 autoantibodies as novel CrD-specific markers with the clinical phenotype in CrD will be discussed in this review.
    Clinical Chemistry and Laboratory Medicine 11/2013; · 3.01 Impact Factor
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    ABSTRACT: Different strategies of colonization or infection by E. coli result in formation of certain adhesion patterns which help also in classifying intestinal E. coli into pathotypes. Little is known about adhesion patterns and host- and tissue adaption of commensal E. coli and about E. coli originating in clinically healthy hosts carrying pathotype-specific virulence-associated genes. Adhesion pattern of E. coli (n = 282) from humans and from 18 animal species were verified on intestinal human Caco-2 and porcine IPEC-J2 cells and, furthermore, for comparison on human urinary bladder 5637, porcine kidney PK-15 epithelial and HEp-2 cells. The analysis was carried out on 150,000 images of adhesion assays.Adhesion patterns were very diverse; 88 isolates were completely non-adherent, whereas 194 adhered to at least one cell line with the dominant adhesion patterns "diffusely distributed" and "microcolony formation". Adhesion patterns "chains" and "clumps" were also visible. Chain formation was mediated by the presence of epithelial cells. Clump formation was very specific on only the 5637 cell line. All enteropathogenic (eae+) E. coli (EPEC; n = 14) were able to form microcolonies which was cell line specific for each isolate. Most EPEC formed microcolonies on intestinal IPEC-J2 and Caco-2 but several also on urinary tract cells. Shigatoxin-producing (stx+) E. coli (n = 10) showed no specific adhesion patterns. E. coli isolates were highly diverse. Commensal and pathogenic isolates can adhere in various forms, including diffuse distribution, microcolonies, chains and clumps. Microcolony formation seems to be a global adhesion strategy also for commensal E. coli.
    Gut Pathogens 11/2013; 5(1):31. · 2.74 Impact Factor
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    ABSTRACT: Feed supplementation with the probiotic Enterococcus (E.) faecium to piglets has been found to reduce pathogenic gut microorganisms. As Escherichia (E.) coli is among the most important pathogens in pig production, we performed a comprehensive analyses to gain further insight on the influence of E. faecium NCIMB 10415 on porcine intestinal E. coli.A total of 1,436 E. coli were isolated from three intestinal habitats (mucosa, digesta, feces) of probiotic-supplemented and non-supplemented (control) piglets. E. coli were characterized via Pulsed-field gel electrophoresis (PFGE) for clonal analysis. The high diversity of E. coli was reflected by 168 clones. Multi-locus sequence typing (MLST) was used to determine the phylogenetic backgrounds which revealed 79 sequence types (STs). Pathotypes of E. coli were further defined using multiplex-PCR for virulence-associated genes.While these analyses discerned only few significant differences in the E. coli population between the feeding groups, analyses distinguishing clones that were uniquely isolated either in the probiotic, the control or in both groups revealed clear effects at the habitat level. Interestingly, ExPEC-typical clones adhering to the mucosa were significantly reduced in the probiotic group.Our data show a minor influence of E. faecium on the overall population of E. coli in healthy piglets. In contrast, this probiotic has a profound effect on mucosa-adherent E. coli. This finding further substantiates a specific effect of E. faecium strain NCIMB 10415 in piglets against pathogenic E. coli in the intestine. In addition, these data question the relevance of data based on sampling fecal E. coli only.
    Applied and Environmental Microbiology 10/2013; · 3.95 Impact Factor
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    ABSTRACT: Analysis of phosphorylated histone protein H2AX (γH2AX) foci is currently the most sensitive method to detect DNA double-strand breaks (DSB). This protein modification has the potential to become an individual biomarker of cellular stress, especially in the diagnosis and monitoring of neoplastic diseases. To make γH2AX foci analysis available as a routine screening method, different software approaches for automated immunofluorescence pattern evaluation have recently been developed. In this study, we used novel pattern recognition algorithms on the AKLIDES(®) platform to automatically analyze immunofluorescence images of γH2AX foci and compared the results with visual assessments. Dose- and time-dependent γH2AX foci formation was investigated in human peripheral blood mononuclear cells (PBMCs) treated with the chemotherapeutic drug etoposide (ETP). Moreover, the AKLIDES system was used to analyze the impact of different immunomodulatory reagents on γH2AX foci formation in PBMCs. Apart from γH2AX foci counting the use of novel pattern recognition algorithms allowed the measurement of their fluorescence intensity and size, as well as the analysis of overlapping γH2AX foci. The comparison of automated and manual foci quantification showed overall a good correlation. After ETP exposure, a clear dose-dependent increase of γH2AX foci formation was evident using the AKLIDES as well as Western blot analysis. Kinetic experiments on PBMCs incubated with 5 μM ETP demonstrated a peak in γH2AX foci formation after 4 to 8 h, while a removal of ETP resulted in a strong reduction of γH2AX foci after 1 to 4 h. In summary, this study demonstrated that the AKLIDES system can be used as an efficient automatic screening tool for γH2AX foci analysis by providing new evaluation features and facilitating the identification of drugs which induce or modulate DNA damage. © 2013 International Society for Advancement of Cytometry.
    Cytometry Part A 09/2013; · 3.71 Impact Factor
  • Clinical Chemistry and Laboratory Medicine 08/2013; · 3.01 Impact Factor
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    ABSTRACT: Intestinal colonization is influenced by the ability of the bacterium to inhabit a niche which is based on the expression of colonization factors. E. coli carries a broad range of virulence-associated genes (VAGs) which contribute to intestinal (inVAGs) and extraintestinal infection (exVAGs). Moreover, initial evidence indicates that inVAGs and exVAGs support intestinal colonization.We developed new screening tools to genotypically and phenotypically characterize E. coli originating in humans, domestic pigs and 17 wild mammalian and avian species. We analyzed 317 isolates for the occurrence of 44 VAGs using a novel multiplex PCR microbead assay (MPMA), and for adhesion to four epithelial cell lines using a new adhesion assay. We correlated data for the definition of new adhesion genes.InVAGs were identified only sporadically, particularly in roe deer (Capreolus capreolus) and European hedgehog (Erinaceus europaeus). The prevalence of exVAGs depended on isolation from a specific host. Human UPEC carried exVAGs with the highest prevalence followed by badger (Meles meles) and roe deer isolates. Adhesion was found to be very diverse. Adhesion was specific to cells, host and tissue, though also unspecific. Occurrence of the following VAGs was associated with a higher adhesion rate to one or more cell lines: afa/dra/daaD, tsh, vat, ibeA, fyuA, mat, sfa/foc, malX, pic, irp2 and papC.Summarized, we established new screening methods which enabled us to characterize large amounts of E. coli. We defined reservoirs for potential pathogenic E. coli. We also identified a very broad range of colonization strategies and defined potential new adhesion genes.
    Applied and Environmental Microbiology 07/2013; · 3.95 Impact Factor
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    ABSTRACT: Following the Europe-wide ban of antimicrobial growth promoters, feed supplementation with zinc has increased in livestock breeding. In addition to possible beneficial effects on animal health, feed supplementation with heavy metals is known to influence the gut microbiota and might promote the spread of antimicrobial resistance via co-selection or other mechanisms. As Escherichia coli is among the most important pathogens in pig production and often displays multi-resistant phenotypes, we set out to investigate the influence of zinc feed additives on the composition of the E. coli populations in vivo focusing on phylogenetic diversity and antimicrobial resistance. In a piglet feeding trial, E. coli were isolated from ileum and colon digesta of high dose zinc-supplemented (2500ppm) and background dose (50ppm) piglets (control group). The E. coli population was characterized via pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) for the determination of the phylogenetic background. Phenotypic resistance screening via agar disk diffusion and minimum inhibitory concentration testing was followed by detection of resistance genes for selected clones. We observed a higher diversity of E. coli clones in animals supplemented with zinc compared to the background control group. The proportion of multi-resistant E. coli was significantly increased in the zinc group compared to the control group (18.6% vs. 0%). For several subclones present both in the feeding and the control group we detected up to three additional phenotypic and genotypic resistances in the subclones from the zinc feeding group. Characterization of these subclones suggests an increase in antimicrobial resistance due to influences on plasmid uptake by zinc supplementation, questioning the reasonability of zinc feed additives as a result of the ban of antimicrobial growth promoters.
    International journal of medical microbiology: IJMM 06/2013; · 4.54 Impact Factor
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    ABSTRACT: Background: Antibodies executing catalytic activity are referred to as antibody enzymes or short “abzymes” and may have diagnostic relevance. Despite several reports on the occurrence of DNase abzymes in systemic autoimmune rheumatic diseases, conclusive data about DNase activity of antibodies in patients with spondyloarthritides (SpAs) are lacking. Objectives: DNase IgG activity was determined in 315 patients with SpAs (99 patients with PsA, 160 patients with ReA associated with Chlamydia trachomatis urogenital infections, 56 patients with AS) and 69 healthy persons as control group. Methods: Electrophoretically and immunochemically homogeneous IgGs were obtained from patients and control sera by a multi-step purification method including complete coagulation of blood samples and treatment with 0.75% ethacridine lactate solution in a ratio of 1:2 for the removal of precipitated serum components. Affinity chromatography employing protein A-Sepharose was applied. The basic method for DNase activity measurement relies upon the capacity of rivanol to form a clot with DNA, reversely proportional to nucleic acid depolymerisation on DNase action [1]. Results: Levels of DNase IgG activity in patients with SpAs were significantly higher (p<0.001) compared to donors while DNase IgG activity in patients with PsA exceeded the levels of patients with ReA and AS. Assay performance characteristics of abzyme activity assessment were calculated for PsA differentiation from ReA and AS by receiver operating curve (ROC) analysis. Patients suffering from PsA could readily be differentiated from AS by DNase abzyme activity detection demonstrating positive likelihood ratios greater than 5.0 for DNase activity per 1mg of purified IgG or per 1 ml of serum. All patients with SpA demonstrated a positive correlation of DNase IgG activity with the number of swollen joints (r=0.65) and pain grade (r=0.59). In patients with PsA, a similar correlation between the degree of DNase IgG activity and synovitis was detected (r=0.64). In ReA patients, we found positive correlations between DNase IgG levels and disease activity index (r=0.53), C-reactive protein (CRP) levels (r=0.58), total CD2 positive T cell count (r=0.71), CD4 positive T helper count (r=0.70). Likewise, in patients with AS, multiple moderate positive correlations of DNase IgG activity were established with the stage of spondylitis (r=0.44), BASRI (r=0.67), and occipital wall distance (r=0.55; for all p < 0.001). Conclusions: DNase IgG activity appears to be elevated in patients suffering from spondyloarthritides in particular psoriatic arthritis, reactive arthritis, and ankylosing spondylitis in comparison with healthy persons, whereas the highest DNase IgG activity has been determined in patients with psoriatic arthritis. Assessment of patient sera for DNase abzyme activity might be an intriguing new serological approach for the differential diagnosis of SpA. References: Gabibov AG, Gololobov GV, Makarevich OI, Schourov DV, Chernova EA, Yadav RP. DNA-hydrolyzing autoantibodies. Appl Biochem Biotechnol. 1994;47:293-302. Acknowledgements: AK and MV were supported by the Belorussian Republican Foundation and Fundamental Research (BRFFR (04-04-81017): the Belorussian State Scientific Technical Program “Treatment and Diagnostic Technologies” (01.12.2006-2008) and the Russian Foundation of Basic Research (RFBR). Disclosure of Interest: None Declared
    EULAR 2013; 06/2013
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    ABSTRACT: Escherichia coli have become the enterobacteriaceae species most affected by extended-spectrum β-lactamases (ESBLs) in view of the emergence of CTX-M-type ESBLs. These CTX-M-positive E. coli have been reported in numerous regions worldwide. Virulence determinants of already reported CTX-M-positive E. coli were investigated. To gain insights into the mechanism underlying this phenomenon, we assessed serogroup, susceptibility pattern and diversity of virulence profiles within a collection of nine bla CTX-M-positive E. coli strains and their virulent determinant using miniaturized DNA microarray techniques. The nine ESBL-positive E. coli isolates were from eight male and one female patient(s) selected for study based on previous work. Virulence potential was inferred by detection of 63 virulence factor (VF) genes. Four (44.4%) of the 9 E. coli isolates exhibited the same set of core characteristics: serotype O8:Hnt, while all were positive for OXA-1, ciprofloxacin resistance. Five of the isolates exhibited highly similar (91% to 100%) VF profiles. The findings describe a broadly disseminated, bla CTX-M-positive and virulent E. coli serogroup with highly homogeneous virulence genotypes, suggesting recent emergence in this zone. Understanding how this clone has emerged and successfully disseminated within the hospital and community, including across national boundaries, should be a public health priority.
    European journal of microbiology & immunology. 06/2013; 3(2):120-125.

Publication Stats

565 Citations
144.59 Total Impact Points

Institutions

  • 2013–2014
    • Brandenburgische Technische Universität Cottbus
      Kottbus, Brandenburg, Germany
    • Belarusian Medical Academy of Post-Graduate Education
      Myenyesk, Minsk, Belarus
  • 2011–2013
    • Fachhochschule der Wirtschaft
      Paderborn, North Rhine-Westphalia, Germany
  • 2012
    • Otto-von-Guericke-Universität Magdeburg
      • Institute for Molecular and Clinical Immunology
      Magdeburg, Saxony-Anhalt, Germany
  • 2003–2012
    • Freie Universität Berlin
      • • Institute of Microbiology and Epizootics
      • • Institute of Chemistry and Biochemistry
      Berlin, Land Berlin, Germany
    • Humboldt-Universität zu Berlin
      Berlín, Berlin, Germany
  • 2009
    • Bundesamt für Verbraucherschutz
      Brunswyck, Lower Saxony, Germany
  • 2006
    • Bundesinstitut für Risikobewertung
      Berlín, Berlin, Germany