Alexander W Friedrich

Publications of Alexander W Friedrich

• Does nasal colonization with livestock-associated Methicillin-resistant Staphylococcus aureus (MRSA) in pig farmers persist after holidays from pig exposure?

  Authors: Robin Köck, Bea Loth, Mahir Köksal, Josef Schulte-Wülwer, Jürgen Harlizius, Alexander W Friedrich

  Applied and environmental microbiology. 03/2012;

  Livestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA) is frequently transmitted from pigs to farmers. This study analyzed whether an absence from direct contact with pigs duringLivestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA) is frequently transmitted from pigs to farmers. This study analyzed whether an absence from direct contact with pigs during holidays had an impact on nasal MRSA colonization rates of pig farmers. Overall, 59% of the farmers did not clear from MRSA colonization during their leave.

• Promiscuous Shiga toxin 2e and its intimate relationship to Forssman.

  Authors: Johannes Müthing, Iris Meisen, Wenlan Zhang, Martina Bielaszewska, Michael Mormann, Rolf Bauerfeind, M Alexander Schmidt, Alexander W Friedrich, Helge Karch

  Glycobiology. 01/2012;

  Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurologicalShiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurological disorders, and often fatal outcome. Stx2e-producing strains from the intestine of slaughtered pigs (n=3), feces of piglets with postweaning diarrhea or edema disease (n=12) and feces of humans with asymptomatic infections or mild diarrhea (n=13) were comparatively analyzed for the binding specificities of Stx2e to glycosphingolipids (GSLs) of the globo-series. Besides equivalent binding towards globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) we could demonstrate specific interaction of Stx2e preparations from human and porcine STEC isolates with Forssman GSL. Notably, Forssman GSL was recognized neither by structurally closely related Stx2 nor by Stx1 derived from human STEC isolates conferring Stx2e a unique recognition feature. Noteworthy, 7 (54%) of the 13 human and 8 (53%) of the 15 pig Stx2e samples exhibited cytotoxic action towards human brain microvascular endothelial cells. Our findings provide a basis for further exploring the functional role of the promiscuous receptor repertoire of Stx2e and the exact nature of the mechanisms that underlie different pathological outcomes of Stx2e-producing STEC in humans and pigs.

• Absence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex CC398 as a Nasal Colonizer of Pigs Raised in an Alternative System.

  Authors: Christiane Cuny, Alexander W Friedrich, Wolfgang Witte

  Applied and environmental microbiology. 12/2011; 78(4):1296-7.

  Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 isolated from pigs raised in conventional farms was previously reported. Here we report a study on 25 farms adheringLivestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 isolated from pigs raised in conventional farms was previously reported. Here we report a study on 25 farms adhering to an alternative system. LA-MRSA ST398 was not detected in nasal swabs from 178 pigs or from 89 humans working and living on these farms.

• The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Germany.

  Authors: Robin Köck, Alexander Mellmann, Frieder Schaumburg, Alexander W Friedrich, Frank Kipp, Karsten Becker

  Deutsches Ärzteblatt international. 11/2011; 108(45):761-7.

  For decades, methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of infection in hospitals and nursing homes (health care-associated MRSA, HA-MRSA). Beginning in the late 1990s,For decades, methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of infection in hospitals and nursing homes (health care-associated MRSA, HA-MRSA). Beginning in the late 1990s, many countries have also experienced a rising incidence of MRSA infection outside of the health care setting (community-associated MRSA, CA-MRSA). Moreover, animal reservoirs are increasingly considered to represent an important source of human MRSA acquisition. In this review article the authors describe the current epidemiological situation of MRSA in Germany. This review is based on pertinent articles published up to 2010 that were retrieved by a selective PubMed search, as well as on publications issued by national reference institutions up to 2010. There are about 132 000 cases of MRSA in German hospitals each year. MRSA is found in about 18% to 20% of all inpatient-derived culture specimens that are positive for S. aureus. CA-MRSA is not yet endemic in Germany; important risk factors for its acquisition include travel to high-prevalence areas and household contact with persons that harbor a CA-MRSA infection. Agricultural livestock is the main animal reservoir for MRSA, which is often zoonotically transmitted from animals to human beings by direct contact. However, both CA-MRSA and MRSA from animal reservoirs can be imported into hospitals and cause nosocomial infections. Hospitals and nursing homes were once the main reservoirs of MRSA, but new ones have now emerged outside of the healthcare setting. Efforts to prevent MRSA and limit its spread must rise to this new challenge.

• In vitro activity against Staphylococcus aureus of a novel antimicrobial agent, PRF-119, a recombinant chimeric bacteriophage endolysin.

  Authors: Evgeny A Idelevich, Christof von Eiff, Alexander W Friedrich, Domenico Iannelli, Guoqing Xia, Georg Peters, Andreas Peschel, Ingrid Wanninger, Karsten Becker

  Antimicrobial agents and chemotherapy. 09/2011; 55(9):4416-9.

  Antistaphylococcal activity of the novel chimeric endolysin PRF-119 was evaluated with the microdilution method. The MIC(50) and MIC(90) of 398 methicillin-susceptible Staphylococcus aureus isolatesAntistaphylococcal activity of the novel chimeric endolysin PRF-119 was evaluated with the microdilution method. The MIC(50) and MIC(90) of 398 methicillin-susceptible Staphylococcus aureus isolates were 0.098 μg/ml and 0.391 μg/ml, respectively (range, 0.024 to 0.780 μg/ml). Both the MIC(50) and MIC(90) values of 776 methicillin-resistant S. aureus isolates were 0.391 μg/ml (range, 0.024 to 1.563 μg/ml). All 192 clinical isolates of coagulase-negative staphylococci exhibited MIC values of >50 μg/ml. In conclusion, PRF-119 exhibited very good activity specifically against S. aureus.

• Livestock-associated methicillin-resistant Staphylococcus aureus in humans, Europe.

  Authors: Brigitte A G L van Cleef, Dominique L Monnet, Andreas Voss, Karina Krziwanek, Franz Allerberger, Marc Struelens, Helena Zemlickova, Robert L Skov, Jaana Vuopio-Varkila, Christiane Cuny [......] Annalisa Pantosti, Michael Borg, Hajo Grundmann, Manica Mueller-Premru, Barbro Olsson-Liljequist, Andreas Widmer, Stephan Harbarth, Alexander Schweiger, Serhat Unal, Jan A J W Kluytmans

  Emerging infectious diseases. 03/2011; 17(3):502-5.

  To estimate the proportion of methicillin-resistant Staphylococcus aureus (MRSA) isolates from humans that were sequence type (ST) 398, we surveyed 24 laboratories in 17 countries in Europe in 2007.To estimate the proportion of methicillin-resistant Staphylococcus aureus (MRSA) isolates from humans that were sequence type (ST) 398, we surveyed 24 laboratories in 17 countries in Europe in 2007. Livestock-associated MRSA ST398 accounted for only a small proportion of MRSA isolates from humans; most were from the Netherlands, Belgium, Denmark, and Austria.

• Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains.

  Authors: Josefine Betz, Martina Bielaszewska, Andrea Thies, Hans-Ulrich Humpf, Klaus Dreisewerd, Helge Karch, Kwang S Kim, Alexander W Friedrich, Johannes Müthing

  Journal of lipid research. 01/2011; 52(4):618-34.

  Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majorityVascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.

• Population structure of Staphylococcus aureus from remote African Babongo Pygmies.

  Authors: Frieder Schaumburg, Robin Köck, Alexander W Friedrich, Solange Soulanoudjingar, Ulysse Ateba Ngoa, Christof von Eiff, Saadou Issifou, Peter G Kremsner, Mathias Herrmann, Georg Peters, Karsten Becker

  PLoS neglected tropical diseases. 01/2011; 5(5):e1150.

  Pandemic community-acquired methicillin-resistant Staphylococcus aureus isolates (CA-MRSA) predominantly encode the Panton-Valentine leukocidin (PVL), which can be associated with severe infections.Pandemic community-acquired methicillin-resistant Staphylococcus aureus isolates (CA-MRSA) predominantly encode the Panton-Valentine leukocidin (PVL), which can be associated with severe infections. Reports from non-indigenous Sub-Saharan African populations revealed a high prevalence of PVL-positive isolates. The objective of our study was to investigate the S. aureus carriage among a remote indigenous African population and to determine the molecular characteristics of the isolates, particularly those that were PVL-positive. Nasal S. aureus carriage and risk factors of colonization were systematically assessed in remote Gabonese Babongo Pygmies. Susceptibility to antibiotics, possession of toxin-encoding genes (i.e., PVL, enterotoxins, and exfoliative toxins), S. aureus protein A (spa) types and multi-locus sequence types (MLST) were determined for each isolate. The carriage rate was 33%. No MRSA was detected, 61.8% of the isolates were susceptible to penicillin. Genes encoding PVL (55.9%), enterotoxin B (20.6%), exfoliative toxin D (11.7%) and the epidermal cell differentiation inhibitor B (11.7%) were highly prevalent. Thirteen spa types were detected and were associated with 10 STs predominated by ST15, ST30, ST72, ST80, and ST88. The high prevalence of PVL-positive isolates among Babongo Pygmies demands our attention as PVL can be associated with necrotinzing infection and may increase the risk of severe infections in remote Pygmy populations. Many S. aureus isolates from Babongo Pygmies and pandemic CA-MRSA-clones have a common genetic background. Surveillance is needed to control the development of resistance to antibiotic drugs and to assess the impact of the high prevalence of PVL in indigenous populations.

• Normal silica gel and reversed phase thin-layer chromatography coupled with UV spectroscopy and IR-MALDI-o-TOF-MS for the detection of tetracycline antibiotics.

  Authors: Iris Meisen, Sebastian Wisholzer, Jens Soltwisch, Klaus Dreisewerd, Michael Mormann, Johannes Müthing, Helge Karch, Alexander W Friedrich

  Analytical and bioanalytical chemistry. 12/2010; 398(7-8):2821-31.

  Tetracyclines (TCs) form a group of bacteriostatic antibiotics with closely related structures and similar chemical and physicochemical properties. They are widely employed as therapeutics in humanTetracyclines (TCs) form a group of bacteriostatic antibiotics with closely related structures and similar chemical and physicochemical properties. They are widely employed as therapeutics in human and veterinary medicine. Here, we introduce the combination of UV spectroscopic detection of high-performance thin-layer chromatography (HPTLC)-separated TCs with direct analysis on solid phase using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). Normal silica gel phase- and water-wettable hybrid C(18) reversed phase layers (RP-18W) both allowed HPTLC separation and sensitive UV spectroscopic detection followed by MS analysis of distinct TCs bands using the liquid matrix glycerol. The novel approach of direct IR-MALDI-o-TOF-MS analysis resulted in the unequivocal identification of four structurally different TCs employed in this study, and linear calibration curves were produced for analyte amounts from 20 ng to 1 μg. MS analysis of TCs from RP-18W HPTLC plates was found to be superior when compared to the spectra acquired from the silica gel layer. Ionic analytes obtained from the RP-18W surface are mainly detected as protonated species of high abundance accompanied by a reduced formation of adducts as well as background ions arising from the matrix or the stationary phase. This results in decreased complexity of the spectra and enhanced sensitivity of the combinatorial method. An approximate detection limit of 5 ng of individual TCs in mixtures by combining RP-18W HPTLC with IR-MALDI-o-TOF-MS offers a novel timely and cost-efficient method for tracing TCs.

• On the structural diversity of Shiga toxin glycosphingolipid receptors in lymphoid and myeloid cells determined by nanoelectrospray ionization tandem mass spectrometry.

  Authors: Petra Hoffmann, Marcel Hülsewig, Sevim Duvar, Holger Ziehr, Michael Mormann, Jasna Peter-Katalinić, Alexander W Friedrich, Helge Karch, Johannes Müthing

  Rapid communications in mass spectrometry : RCM. 08/2010; 24(15):2295-304.

  Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo-series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Galalpha4Galbeta4Glcbeta1Cer,Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo-series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Galalpha4Galbeta4Glcbeta1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAcbeta3Galalpha4Galbeta4Glcbeta1Cer), respectively, which represent the targets of Stxs on many different cell types. B-cell-derived Raji cells and THP-1 cells of monocytic origin are widely used for the investigation of Stx-mediated cellular response, because Stx is known to cause cell death in both cell lines. Despite their functional importance, the Stx receptors of Raji and THP-1 cells have so far not been investigated. This prompted us to explore the structures of their GSL receptors in detail by means of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) with collision-induced dissociation (CID) in conjunction with Stx1 as well as anti-Gb3Cer and anti-Gb4Cer antibodies. Using the combination of a thin-layer chromatography (TLC) overlay assay and MS(1) and MS(2) analysis we identified Gb3Cer (d18:1, C24:1/C24:0) as the prevalent Stx1-receptor accompanied by less abundant Gb3Cer (d18:1, C16:0) in the neutral GSL fraction of Raji cells. The same Gb3Cer species but with almost equal proportions of the C24:1/C24:0 and C16:0 variants were found in THP-1 cells. In addition, unusual hydroxylated Gb3Cer (d18:1, C24:1/C24:0) and Gb3Cer (d18:1, C26:1) could be identified in trace quantities in both cell lines. As the most obvious difference between Raji and THP-1 cells we observed the expression of Gb4Cer in THP-1 cells, whereas Raji cells failed to express this elongation product of Gb3Cer. Both short- and long-chain fatty acid carrying Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C24:1/C24:0), respectively, were the prevalent Gb4Cer variants. This first report on the differential expression of Gb3Cer and Gb4Cer and their structural diversity in lymphoid and myeloid cell lines supports the hypothesis that such heterogeneities might play a functional role in the molecular assembly of GSLs in membrane organization and cellular signaling of Stx-susceptible cells.

• Neutral glycosphingolipids in human blood: a precise mass spectrometry analysis with special reference to lipoprotein-associated Shiga toxin receptors.

  Authors: Christian H Schweppe, Petra Hoffmann, Jerzy-Roch Nofer, Gottfried Pohlentz, Michael Mormann, Helge Karch, Alexander W Friedrich, Johannes Müthing

  Journal of lipid research. 05/2010; 51(8):2282-94.

  Shiga toxin (Stx)-producing Escherichia coli are the leading cause of hemorrhagic colitis and life-threatening extraintestinal complications in humans. Stx1 and Stx2 are transferred by yet to beShiga toxin (Stx)-producing Escherichia coli are the leading cause of hemorrhagic colitis and life-threatening extraintestinal complications in humans. Stx1 and Stx2 are transferred by yet to be delineated mechanisms from the intestine to the circulation where they injure microvascular endothelial cells. The resulting vascular lesions cause renal failure and brain damage. Because lipoproteins are potential carriers of Stx through the circulation, we investigated human lipoprotein-associated neutral glycosphingolipids (GSLs) with emphasis on high (globotriaosylceramide) and low (globotetraosylceramide) affinity Stx-receptors. TLC overlay employing Stx1, Stx2, and anti-GSL antibodies demonstrated preferential distribution of globo-series GSLs to very low- and low-density lipoproteins compared with minor association with high-density lipoproteins. Electrospray ionization quadrupole time-of-flight mass spectrometry portrayed C24:0/C24:1 and C16:0 as the major fatty acid of the ceramide moieties of Stx-receptors carrying nonvarying d18:1 sphingosine. This structural heterogeneity was also found in precursor lactosylceramide, glucosylceramide, and galactosylceramide, the last showing an exceptionally high degree of hydroxylated C24 fatty acids. Our findings provide the basis for exploring the functional role of lipoprotein-associated Stx-receptors in human blood.

• Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis.

  Authors: Hajo Grundmann, David M Aanensen, Cees C van den Wijngaard, Brian G Spratt, Dag Harmsen, Alexander W Friedrich

  PLoS medicine. 01/2010; 7(1):e1000215.

  Staphylococcus aureus is one of the most important human pathogens and methicillin-resistant variants (MRSAs) are a major cause of hospital and community-acquired infection. We aimed to map theStaphylococcus aureus is one of the most important human pathogens and methicillin-resistant variants (MRSAs) are a major cause of hospital and community-acquired infection. We aimed to map the geographic distribution of the dominant clones that cause invasive infections in Europe. In each country, staphylococcal reference laboratories secured the participation of a sufficient number of hospital laboratories to achieve national geo-demographic representation. Participating laboratories collected successive methicillin-susceptible (MSSA) and MRSA isolates from patients with invasive S. aureus infection using an agreed protocol. All isolates were sent to the respective national reference laboratories and characterised by quality-controlled sequence typing of the variable region of the staphylococcal spa gene (spa typing), and data were uploaded to a central database. Relevant genetic and phenotypic information was assembled for interactive interrogation by a purpose-built Web-based mapping application. Between September 2006 and February 2007, 357 laboratories serving 450 hospitals in 26 countries collected 2,890 MSSA and MRSA isolates from patients with invasive S. aureus infection. A wide geographical distribution of spa types was found with some prevalent in all European countries. MSSA were more diverse than MRSA. Genetic diversity of MRSA differed considerably between countries with dominant MRSA spa types forming distinctive geographical clusters. We provide evidence that a network approach consisting of decentralised typing and visualisation of aggregated data using an interactive mapping tool can provide important information on the dynamics of MRSA populations such as early signalling of emerging strains, cross border spread, and importation by travel. In contrast to MSSA, MRSA spa types have a predominantly regional distribution in Europe. This finding is indicative of the selection and spread of a limited number of clones within health care networks, suggesting that control efforts aimed at interrupting the spread within and between health care institutions may not only be feasible but ultimately successful and should therefore be strongly encouraged.

• Phylogeny and disease association of Shiga toxin-producing Escherichia coli O91.

  Authors: Alexander Mellmann, Angelika Fruth, Alexander W Friedrich, Lothar H Wieler, Dag Harmsen, Dirk Werber, Barbara Middendorf, Martina Bielaszewska, Helge Karch

  Emerging infectious diseases. 09/2009; 15(9):1474-7.

  The diversity and relatedness of 100 Shiga toxin-producing Escherichia coli O91 isolates from different patients were examined by multilocus sequence typing. We identified 10 specific sequence typesThe diversity and relatedness of 100 Shiga toxin-producing Escherichia coli O91 isolates from different patients were examined by multilocus sequence typing. We identified 10 specific sequence types (ST) and 4 distinct clonal groups. ST442 was significantly associated with hemolytic uremic syndrome.

• Shiga Toxin, Cytolethal Distending Toxin and Hemolysin Repertoirs in Clinical Escherichia coli O91 Isolates.

  Authors: Martina Bielaszewska, Franziska Stoewe, Angelika Fruth, Wenlan Zhang, Rita Prager, Jens Brockmeyer, Alexander Mellmann, Helge Karch, Alexander W Friedrich

  Journal of clinical microbiology. 05/2009;

  Shiga toxin (Stx)-producing Escherichia coli (STEC) of serogroup O91 are the most common human pathogenic eae-negative STEC. To facilitate diagnosis and subtyping of these pathogens, we genotypicallyShiga toxin (Stx)-producing Escherichia coli (STEC) of serogroup O91 are the most common human pathogenic eae-negative STEC. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (n = 1), H10 (n = 2), H14 (n = 52) or H21 (n = 20), or were H nontypeable (Hnt; n = 10); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the flagellin subunit-encoding gene fliCH14. Most STEC O91 possesed EHEC-hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx2d-activatable encoding mucus- and elastase-activatable Stx2d was solely present in STEC O91:H21, whereas most strains in the other serotypes possessed stx1. Moreover, only STEC O91:H21 possessed the cdt-V cluster encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes mostly originated in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients stools on enterohemolysin agar and their rapid initial subtyping using fliC genotyping facilitates identification of these emerging pathogens in clinical and epidemiological studies and enables prediction of the risk of a severe clinical outcome.

• Shiga toxins, glycosphingolipid diversity, and endothelial cell injury.

  Authors: Johannes Müthing, Christian H Schweppe, Helge Karch, Alexander W Friedrich

  Thrombosis and haemostasis. 03/2009; 101(2):252-64.

  Shiga toxin (Stx)-producing Escherichia coli (STEC) cause an enteric illness that results in a spectrum of outcomes ranging from asymptomatic carriage to uncomplicated diarrhea, bloody diarrhea, andShiga toxin (Stx)-producing Escherichia coli (STEC) cause an enteric illness that results in a spectrum of outcomes ranging from asymptomatic carriage to uncomplicated diarrhea, bloody diarrhea, and the postdiarrheal haemolytic uremic syndrome (HUS), which leads to renal and other organ microvascular thrombosis. Binding of Stx to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer/CD77) on endothelial cells followed by receptor-mediated endocytosis is the linchpin in STEC-mediated disease. Only GSLs that associate strongly with lipid rafts appear to carry Stxs retrogradely from the plasma membrane through the Golgi apparatus to the endoplasmic reticulum where they are translocated to the cytosol and exert their toxic function. Thus, the biophysical features of the lipid moiety of GSL receptors may influence its incorporation into certain membrane domains and thereby affect toxin destination. Consequently, a detailed structural analysis of Stx-binding GSLs is required to illuminate the molecular causes that may underlie the different Stx susceptibilities of endothelial cells derived from various vascular beds. Solid phase overlay binding assays of thin-layer chromatography (TLC)-separated GSL preparations employing specific antibodies and/or Stxs in conjunction with anti-Stx-antibodies are commonly used for the identification of Stx-binding GSLs. Such GSL-profiling combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) represents a convenient strategy to structurally characterize Stx-receptors from any biological sources such as primary cells, cell lines, or organs. This approach may be helpful to gain insights into Stx-induced impairment of target cells that is suggested to originate at least partly from the structural heterogeneity of the cellular ligands of Stxs.

• Shiga toxin receptor Gb3Cer/CD77: tumor-association and promising therapeutic target in pancreas and colon cancer.

  Authors: Ute Distler, Jamal Souady, Marcel Hülsewig, Irena Drmić-Hofman, Jörg Haier, Alexander W Friedrich, Helge Karch, Norbert Senninger, Klaus Dreisewerd, Stefan Berkenkamp, M Alexander Schmidt, Jasna Peter-Katalinić, Johannes Müthing

  PloS one. 02/2009; 4(8):e6813.

  BACKGROUND: Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cellBACKGROUND: Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas. METHODOLOGY/PRINCIPAL FINDINGS: Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors. CONCLUSIONS/SIGNIFICANCE: Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.

• Comparative molecular analysis substantiates a zoonotic potential of equine Methicillin- resistant Staphylococcus aureus (MRSA).

  Authors: Birgit Walther, Stefan Monecke, Claudia Ruscher, Alexander W Friedrich, Ralf Ehricht, Peter Slickers, Alexandra Soba, Claus-G Wleklinski, Lothar H Wieler, Antina Lübke-Becker

  Journal of clinical microbiology. 01/2009;

  Despite the increasing importance of Methicillin-resistant Staphylococcus aureus (MRSA) in veterinary medicine, knowledge about the epidemiology of these pathogens in horses is still poor. TheDespite the increasing importance of Methicillin-resistant Staphylococcus aureus (MRSA) in veterinary medicine, knowledge about the epidemiology of these pathogens in horses is still poor. The phylogenetic relationship of strains from human and equine origin has been addressed before, usually by analyzing results of common standard classification methods for MRSA. This work intends to go beyond the base line of typing procedures in order to comparatively characterize equine and human MRSA strains of similar phylogenetic background. Additionally to MLST, PFGE, spa typing, SCCmec typing and a PCR for PVL detection, a microarray analysis of altogether 185 structural, virulence-associated and resistance loci was applied. As a result, the clonal complex (CC) 8 was absolutely predominant (16) in 19 investigated equine MSRA strains. Of CC8, 13 strains belonged to sequence type (ST) 254, three others to ST8. This genotype has been isolated from different equine patients in various regions over several years, substantiating the apparent predominance of CC8 STs in MRSA strains of horses worldwide. Beyond that, comparatively investigated human strains of ST254 displayed indistinguishable molecular typing results to those of equine origin. Two further equine strains (ST22, ST1117) showed similarity to ST22 human strains (CC22). One equine strain was of ST398, a genotype recently described to be frequently isolated from specimens of pigs and pig farmers. These data provide evidence for the adaptation to more than one mammalian species of certain MRSA genotypes, reflecting their extended host spectrum.

• Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli.

  Authors: Alexander Mellmann, Martina Bielaszewska, Robin Köck, Alexander W Friedrich, Angelika Fruth, Barbara Middendorf, Dag Harmsen, M Alexander Schmidt, Helge Karch

  Emerging infectious diseases. 09/2008; 14(8):1287-90.

  Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1%Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% of these strains clustered in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection).

• Characterization of clonal relatedness among the natural population of Staphylococcus aureus strains by using spa sequence typing and the BURP (based upon repeat patterns) algorithm.

  Authors: Alexander Mellmann, Thomas Weniger, Christoph Berssenbrügge, Ursula Keckevoet, Alexander W Friedrich, Dag Harmsen, Hajo Grundmann

  Journal of clinical microbiology. 08/2008; 46(8):2805-8.

  We evaluated the BURP (based upon repeat patterns) algorithm, which relies on sequencing of the Staphylococcus aureus protein A gene (spa), for its ability to infer clonal relatedness within aWe evaluated the BURP (based upon repeat patterns) algorithm, which relies on sequencing of the Staphylococcus aureus protein A gene (spa), for its ability to infer clonal relatedness within a population of 110 wild-type strains. BURP clustering of the resulting 66 spa types was highly concordant with multilocus sequence typing (96.5% concordance) and pulsed-field gel electrophoresis (94.9%).

• Shiga toxin-negative attaching and effacing Escherichia coli: distinct clinical associations with bacterial phylogeny and virulence traits and inferred in-host pathogen evolution.

  Authors: Martina Bielaszewska, Barbara Middendorf, Robin Köck, Alexander W Friedrich, Angelika Fruth, Helge Karch, M Alexander Schmidt, Alexander Mellmann

  Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 08/2008; 47(2):208-17.

  BACKGROUND: Attaching and effacing Escherichia coli (AEEC) that lack Shiga toxin genes (stx) and the enteropathogenic E. coli adherence factor (EAF) plasmid (stx-/EAF-) are classified as atypicalBACKGROUND: Attaching and effacing Escherichia coli (AEEC) that lack Shiga toxin genes (stx) and the enteropathogenic E. coli adherence factor (EAF) plasmid (stx-/EAF-) are classified as atypical enteropathogenic E. coli and cause diarrhea worldwide. However, it is unknown whether there are bacterial lineage-specific human disease phenotypes. We compared stx-/EAF- AEEC recovered from patients (mostly children) with bloody and nonbloody diarrhea. METHODS: stx-/EAF- AEEC were isolated using eae colony blot hybridization and were serotyped, tested (by polymerase chain reaction) for putative virulence genes, and analyzed for phylogenetic relationships by use of multilocus sequence typing. RESULTS: During the period 1995-2007, stx-/EAF- AEEC were isolated as the only bacterial pathogens from stool specimens obtained from 18 (15.3%) of 118 patients with bloody diarrhea and from 141 (1.3%) of 10,550 patients with nonbloody diarrhea ([Formula: see text]). All but 1 of 18 strains recovered from patients with bloody diarrhea resembled enterohemorrhagic E. coli (EHEC) on the basis of serotypes, non-stx virulence profiles, and multilocus sequence types. In contrast, most (75.9%) of 141 stx-/EAF- AEEC recovered from patients with nonbloody diarrhea belonged to other serotypes and differed from the former strains phylogenetically and with regard to virulence genes. Three of 18 patients with bloody diarrhea and none of 141 patients with nonbloody diarrhea who shedded stx-/EAF- AEEC developed hemolytic uremic syndrome. CONCLUSIONS: Most stx-/EAF- AEEC associated with bloody diarrhea are plausibly EHEC that lost Shiga toxin during infection (EHEC-LST). To prevent serious complications of such infections, Shiga toxin-independent diagnostic strategies to accurately and rapidly identify such patients should be developed and applied. Multilocus sequence typing has potential to distinguish EHEC-LST from less pathogenic stx-/EAF- AEEC.