Alexander Mellmann
Research skills
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TechnicalDNA Sequencing
Research interests
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InterestsEvolution
Research experience
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Oct 2007
Research: FBI-Zoo
Zoonotic infections
Other
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LanguagesGerman, English
Publications
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6.79Impact points
Identification of Intermediate in Evolutionary Model of Enterohemorrhagic Escherichia coli O157.
Emerging infectious diseases. 04/2012; 18(4):582-8.
Highly pathogenic enterohemorrhagic Escherichia coli (EHEC) O157 cause a spectrum of clinical signs that include diarrhea, bloody diarrhea, and hemolytic uremic syndrome. The current evolutionary model of EHEC O157:H7/H(-) consists of a stepwise evolution scenario proceeding from O55:H7 to a node (h... [more] Highly pathogenic enterohemorrhagic Escherichia coli (EHEC) O157 cause a spectrum of clinical signs that include diarrhea, bloody diarrhea, and hemolytic uremic syndrome. The current evolutionary model of EHEC O157:H7/H(-) consists of a stepwise evolution scenario proceeding from O55:H7 to a node (hypothetical intermediate) that then branches into sorbitol-fermenting (SF) O157:H(-) and non-SF (NSF) O157:H7. To identify this hypothetical intermediate, we performed single nucleotide polymorphism analysis by sequencing of 92 randomly distributed backbone genomic regions of 40 O157:H7/H(-) isolates. Overall, 111 single nucleotide polymorphisms were identified in 75/92 partial open reading frames after sequencing 51,041 nt/strain. The EHEC O157:H7 strain LSU-61 from deer occupied an intermediate position between O55:H7 and both O157 branches (SF and NSF O157), complementing the stepwise evolutionary model of EHEC O157:H7/H(-). The animal origin of this intermediate emphasizes the value of nonhuman reservoirs in the clarification of the evolution of human pathogens.
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4.80Impact points
Epidemic Escherichia coli O104:H4: Effects of antibiotics on Shiga toxin 2 production and bacteriophage induction.
Antimicrobial agents and chemotherapy. 03/2012;
The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicat... [more] The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated in some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction of stx(2)-harboring bacteriophages, stx(2) transcription and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increased (P < 0.001 to P < 0.05), fosfomycin, gentamicin, and kanamycin insignificantly influenced (P > 0.1), and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased (P ≤ 0.05) stx(2)-phage induction and Stx2 production in outbreak isolates. Ciprofloxacin and chloramphenicol significantly upregulated and downregulated, respectively, stx(2) transcription (P < 0.01); the other antibiotics had insignificant effects (P > 0.1). Meropenem, azithromycin, and rifaximin, which had been used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither induce stx(2)-harboring phages nor increase stx(2) transcription and Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options in patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.
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4.16Impact points
Real-time multiplex polymerase chain reaction for detecting Shiga toxin 2-producing Escherichia coli O104:H4 in human stools.
Journal of clinical microbiology. 02/2012;
A real-time multiplex PCR targeting stx(2), wzy(O104), and fliC(H4) of enterohemorrhagic Escherichia coli (EHEC) O104:H4 correctly determined the presence or absence of these genes in 253 EHEC isolates and 132 patients' stool enrichment cultures. It is a rapid, sensitive and specific tool for de... [more] A real-time multiplex PCR targeting stx(2), wzy(O104), and fliC(H4) of enterohemorrhagic Escherichia coli (EHEC) O104:H4 correctly determined the presence or absence of these genes in 253 EHEC isolates and 132 patients' stool enrichment cultures. It is a rapid, sensitive and specific tool for detecting EHEC O104:H4 in human stools.
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3.93Impact points
Promiscuous Shiga toxin 2e and its intimate relationship to Forssman.
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, neurological disorders, and often fatal outcome. Stx2e-producing strains from the intestine of slaughtered pigs (n=... [more] 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, 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.
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3.69Impact points
Differential Targeting of the E-Cadherin/β-Catenin Complex by Gram-Positive Probiotic Lactobacilli Improves Epithelial Barrier Function.
Applied and environmental microbiology. 12/2011; 78(4):1140-7.
The intestinal ecosystem is balanced by dynamic interactions between resident and incoming microbes, the gastrointestinal barrier, and the mucosal immune system. However, in the context of inflammatory bowel diseases (IBD), where the integrity of the gastrointestinal barrier is compromised, resident... [more] The intestinal ecosystem is balanced by dynamic interactions between resident and incoming microbes, the gastrointestinal barrier, and the mucosal immune system. However, in the context of inflammatory bowel diseases (IBD), where the integrity of the gastrointestinal barrier is compromised, resident microbes contribute to the development and perpetuation of inflammation and disease. Probiotic bacteria have been shown to exert beneficial effects, e.g., enhancing epithelial barrier integrity. However, the mechanisms underlying these beneficial effects are only poorly understood. Here, we comparatively investigated the effects of four probiotic lactobacilli, namely, Lactobacillus acidophilus, L. fermentum, L. gasseri, and L. rhamnosus, in a T84 cell epithelial barrier model. Results of DNA microarray experiments indicating that lactobacilli modulate the regulation of genes encoding in particular adherence junction proteins such as E-cadherin and β-catenin were confirmed by quantitative reverse transcription-PCR (qRT-PCR). Furthermore, we show that epithelial barrier function is modulated by Gram-positive probiotic lactobacilli via their effect on adherence junction protein expression and complex formation. In addition, incubation with lactobacilli differentially influences the phosphorylation of adherence junction proteins and the abundance of protein kinase C (PKC) isoforms such as PKCδ that thereby positively modulates epithelial barrier function. Further insight into the underlying molecular mechanisms triggered by these probiotics might also foster the development of novel strategies for the treatment of gastrointestinal diseases (e.g., IBD).
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4.21Impact points
Comparison of asymptomatic bacteriuria Escherichia coli isolates from healthy individuals versus those from hospital patients shows that long-term bladder colonization selects for attenuated virulence phenotypes.
Infection and immunity. 11/2011; 80(2):668-78.
Asymptomatic bacteriuria (ABU) is a condition where bacteria stably colonize the urinary tract, in a manner closely resembling commensalism at other mucosal sites. The patients carry >10(5) CFU/ml for extended periods of time and rarely develop symptoms. Contrasting the properties of ABU strains ... [more] Asymptomatic bacteriuria (ABU) is a condition where bacteria stably colonize the urinary tract, in a manner closely resembling commensalism at other mucosal sites. The patients carry >10(5) CFU/ml for extended periods of time and rarely develop symptoms. Contrasting the properties of ABU strains to those of uropathogenic isolates causing symptomatic infection is therefore highly relevant to understand mechanisms of bacterial adaptation. The prototype ABU strain Escherichia coli 83972 has a smaller genome than uropathogenic E. coli (UPEC) strains with deletions or point mutations in several virulence genes, suggesting that ABU strains undergo a programmed reductive evolution within human hosts. This study addressed if these observations can be generalized. Strains causing ABU in outpatients or hospitalized patients after catheterization or other invasive procedures were compared to commensal E. coli isolates from the intestinal flora of healthy individuals. Notably, clonal complex 73 (CC73) was a prominent phylogenetic lineage dominated by ABU isolates. ABU isolates from outpatients and hospitalized patients had a similar overall virulence gene repertoire, which distinguished them from many commensals, but typical UPEC virulence genes were less frequently attenuated in hospital strains than in outpatient strains or commensals. The decreased virulence potential of outpatient ABU isolates relative to that of ABU strains from hospitalized patients supports the hypothesis that loss of expression or decay of virulence genes facilitates long-term carriage and adaptation to host environments.
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The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Germany.
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, many countries have also experienced a rising incidence of MRSA infection outside of the health care... [more] 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.
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4.16Impact points
Comparison of multilocus variable-number tandem-repeat analysis and multilocus sequence typing for differentiation of hemolytic-uremic syndrome-associated Escherichia coli (HUSEC) collection strains.
Journal of clinical microbiology. 08/2011; 49(10):3644-6.
Multilocus variable-number tandem-repeat analysis (MLVA) was compared to multilocus sequence typing (MLST) to differentiate hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli strains. Although MLVA--like MLST--was highly discriminatory (index of diversity, 0.988 versus 0.984), a... [more] Multilocus variable-number tandem-repeat analysis (MLVA) was compared to multilocus sequence typing (MLST) to differentiate hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli strains. Although MLVA--like MLST--was highly discriminatory (index of diversity, 0.988 versus 0.984), a low level of concordance demonstrated the limited ability of MLVA to reflect long-term evolutionary events.
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2.95Impact points
Automatic MDCT injectors: hygiene and efficiency of disposable, prefilled, and multidosing roller pump systems in clinical routine.
AJR. American journal of roentgenology. 08/2011; 197(2):W226-32.
This study evaluated three different injection systems with regard to microbiological contamination, time efficiency, and user handling during a clinical routine. A total of 825 patients were included. A double-syringe contrast injector with disposable syringes (system A; n = 150) and one that used ... [more] This study evaluated three different injection systems with regard to microbiological contamination, time efficiency, and user handling during a clinical routine. A total of 825 patients were included. A double-syringe contrast injector with disposable syringes (system A; n = 150) and one that used prefilled syringes (system B; n = 150) were microbiologically analyzed during single use of the syringes in one patient. Moreover, the contamination of a roller pump injector capable of multidosing several patients from a contrast agent container, without the need for prior filling, was determined after being used for an entire day (system C; n = 35 injections/day for 15 days). The hygienic background was guaranteed by taking imprints of the surfaces of devices and the palms of the hands of members of CT staff before the clinical investigation. The time required for assembly of the injection systems and for filling or refilling of each injector system was measured. The handling of the three systems also was subjectively ranked by the technicians. Injection systems A, B, and C remained microbiologically sterile and free of contamination throughout their use in clinical routine. The mean (± SD) time for injection system assembly and installation of syringes and filling did not differ significantly between injection systems A and B (system A, 2.5 ± 1.1 minutes; system B, 1.9 ± 1.3 minutes; p = 0.12), whereas the time for assembly of system C was significantly shorter (0.9 ± 0.6 minutes; p < 0.05 vs system A; p < 0.05 vs system B). In the subjective ranking of injector handling, systems B and C were preferred. Double-syringe injectors used with disposable or prefilled contrast agent syringes, as well as roller pump injectors, ensure hygienic conditions in clinical routine. However, time efficiency and handling are aspects that favor prefilled and roller pump systems.
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15.58Impact points
Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study.
The Lancet infectious diseases. 06/2011; 11(9):671-6.
In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed viru... [more] In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed virulence profiles and relevant phenotypes of outbreak isolates recovered in our laboratory. We analysed stool samples from 80 patients that had been submitted to the National Consulting Laboratory for Haemolytic Uraemic Syndrome in Münster, Germany, between May 23 and June 2, 2011. Isolates were screened with standard PCR for virulence genes of Shiga-toxin-producing E coli and a newly developed multiplex PCR for characteristic features of the outbreak strain (rfb(O104), fliC(H4), stx(2), and terD). Virulence profiles of the isolates were determined with PCR targeting typical virulence genes of Shiga-toxin-producing E coli and of other intestinal pathogenic E coli. We sequenced stx with Sanger sequencing and measured Shiga-toxin production, adherence to epithelial cells, and determined phylogeny and antimicrobial susceptibility. All isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli (stx(2), iha, lpf(O26), lpf(O113)) and enteroaggregative E coli (aggA, aggR, set1, pic, aap) loci and expressed phenotypes that define Shiga-toxin-producing E coli and enteroaggregative E coli, including production of Shiga toxing 2 and aggregative adherence to epithelial cells. Isolates additionally displayed an extended-spectrum β-lactamase phenotype absent in HUSEC041. Augmented adherence of the strain to intestinal epithelium might facilitate systemic absorption of Shiga toxin and could explain the high progression to haemolytic uraemic syndrome. This outbreak demonstrates that blended virulence profiles in enteric pathogens, introduced into susceptible populations, can have extreme consequences for infected people. German Federal Ministry of Education and Research, Network Zoonoses.
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1.52Impact points
Do complement factor H 402Y and C7 M allotypes predispose to (typical) haemolytic uraemic syndrome?
International journal of immunogenetics. 06/2011; 38(5):383-7.
Typical haemolytic uraemic syndrome (HUS) is mainly caused by infections with enterohaemorrhagic Escherichia coli, whereas in atypical, nonbacteria-associated HUS, complement plays a dominant role. Recently, complement has also been shown to be involved in typical HUS. In this study, mostly weakly s... [more] Typical haemolytic uraemic syndrome (HUS) is mainly caused by infections with enterohaemorrhagic Escherichia coli, whereas in atypical, nonbacteria-associated HUS, complement plays a dominant role. Recently, complement has also been shown to be involved in typical HUS. In this study, mostly weakly significant associations with homozygosities of complement allotype C7 M and inversely with factor H 402H were found, suggesting that 402Y and C7 M allotypes predispose to (typical) haemolytic uraemic syndrome.
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4.16Impact points
Characterization of Escherichia coli strains isolated from patients with diarrhea in Sao Paulo, Brazil: identification of intermediate virulence factor profiles by multiplex PCR.
Journal of clinical microbiology. 06/2011; 49(6):2274-8.
Intestinal pathogenic Escherichia coli is a major causative agent of severe diarrhea. In this study the prevalences of different pathotypes among 702 E. coli isolates from Brazilian patients with diarrhea were determined by multiplex PCR. Interestingly, most strains were enteroaggregative E. coli (E... [more] Intestinal pathogenic Escherichia coli is a major causative agent of severe diarrhea. In this study the prevalences of different pathotypes among 702 E. coli isolates from Brazilian patients with diarrhea were determined by multiplex PCR. Interestingly, most strains were enteroaggregative E. coli (EAEC) strains, followed by atypical EPEC (ATEC) strains. Classical enteropathogenic E. coli (EPEC) strains were not detected.
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2.80Impact points
Structures and functions of autotransporter proteins in microbial pathogens.
International journal of medical microbiology : IJMM. 05/2011; 301(6):461-8.
Since their discovery more than 20 years ago the autotransporter protein superfamily has been growing continuously and currently represents the largest protein family in (pathogenic) Gram-negative bacteria. Autotransporter proteins (AT) adhere to a common structural principle and are composed of a C... [more] Since their discovery more than 20 years ago the autotransporter protein superfamily has been growing continuously and currently represents the largest protein family in (pathogenic) Gram-negative bacteria. Autotransporter proteins (AT) adhere to a common structural principle and are composed of a C-terminal β-barrel-shaped 'translocator' domain and an N-terminal 'passenger' domain. The translocator is anchored in the outer membrane and is indispensable for the N-terminal passenger part to traverse the outer membrane. Most if not all AT harbor a chaperone segment that increases protein stability and may be located in the passenger or translocator domain. The passenger mediates the specific virulence function(s) of the particular AT. Accordingly, passenger domains of AT can be quite variable. Interestingly, AT have been identified as the first glycosylated proteins in Gram-negative bacteria. Despite the considerable efforts invested in the characterization of autotransporter biogenesis, various aspects such as the participation of accessory proteins, the fate of the translocator, or the translocation of glycosylated proteins still remain only poorly understood. In addition, recent evidence indicates that the prefix 'auto' might be slightly exaggerated. Here, we will selectively discuss novel insights at various stages of AT biogenesis.
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4.01Impact points
Virulence factors and genotypes of Staphylococcus aureus from infection and carriage in Gabon.
Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 04/2011; 17(10):1507-13.
Staphylococcus aureus isolates from developed countries have been extensively analyzed with respect to their virulence patterns and clonal relatedness but there is only sparse information on the molecular diversity of S. aureus isolates from Africa. In particular, little is known about S. aureus iso... [more] Staphylococcus aureus isolates from developed countries have been extensively analyzed with respect to their virulence patterns and clonal relatedness but there is only sparse information on the molecular diversity of S. aureus isolates from Africa. In particular, little is known about S. aureus isolates from asymptomatic carriers compared with isolates causing infections. From 2008 to 2010, we prospectively collected S. aureus isolates from asymptomatic carriers and infections in Lambaréné, Gabon, Central Africa. For these isolates, we determined major virulence factors, and performed multilocus sequence typing (MLST) and spa typing. Among 163 S. aureus isolates from asymptomatic carriers, we found the MLST clonal complexes (CCs) 5, 6, 7, 8, 9, 15, 25, 30, 45, 88, 101, 121 and 152; 3.7% were methicillin-resistant (MRSA). The clinical isolates were associated with CCs 5, 8, 9, 15, 88, 121 and 152; 11% were MRSA. Sequence types 1 and 88 were significantly associated with infection and sequence type 508 was associated with carriage. Remarkably, there was a high prevalence of Panton-Valentine leukocidin (PVL) -encoding genes both in disease-related isolates (57.4%) and in carrier isolates (40.5%). We found differences in the clonal structure and virulence pattern of Gabonese S. aureus isolates from asymptomatic carriers and infections. Of note, S. aureus isolates from Gabon show a very high prevalence of PVL-encoding genes, which exceeds the rates observed for developed countries.
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5.36Impact points
Chromosomal instability in enterohaemorrhagic Escherichia coli O157:H7: impact on adherence, tellurite resistance and colony phenotype.
Molecular microbiology. 02/2011; 79(4):1024-44.
Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five ... [more] Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five EHEC O157:H7 strains that differentiate into large (L) colonies and small (S) colonies with high and low Tel minimal inhibitory concentrations (MICs) respectively. S colonies (Tel-MICs ≤ 4 µg ml⁻¹) sustained large internal deletions within the Tel(R) OIs via homologous recombination between IS elements and lost ter and iha. Moreover, complete excision of the islands occurred by site-specific recombination between flanking direct repeats. Complete excision of OI 43 and OI 48 occurred in 1.81 × 10⁻³ and 1.97 × 10⁻⁴ cells in culture, respectively; internal deletion of OI 48 was more frequent (9.7 × 10⁻¹ cells). Under iron limitation that promotes iha transcription, iha-negative derivatives adhered less well to human intestinal epithelial cells and grew slower than did their iha-positive counterparts. Experiments utilizing iha deletion and complementation mutants identified Iha as the major factor responsible for these phenotypic differences. Spontaneous deletions affecting Tel(R) OIs contribute to EHEC O157 genome plasticity and might impair virulence and/or fitness.
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4.68Impact points
Transcellular migration of neutrophil granulocytes through the blood-cerebrospinal fluid barrier after infection with Streptococcus suis.
Journal of neuroinflammation. 01/2011; 8:51.
A critical point during the course of bacterial meningitis is the excessive influx of polymorphnuclear neutrophils (PMNs) from the blood into the brain. Both paracellular and transcellular routes of leukocyte transmigration through the blood-brain barrier have been described in CNS diseases so far. ... [more] A critical point during the course of bacterial meningitis is the excessive influx of polymorphnuclear neutrophils (PMNs) from the blood into the brain. Both paracellular and transcellular routes of leukocyte transmigration through the blood-brain barrier have been described in CNS diseases so far. Thus, we investigated the mechanism of PMN transmigration through the blood-CSF barrier under inflammatory conditions. In an "inverted" Transwell culture model of the blood-CSF barrier, the zoonotic agent Streptococcus suis (S. suis) was used to stimulate porcine choroid plexus epithelial cells (PCPECs) specifically from the physiologically relevant basolateral side. Barrier function was analyzed by measuring TEER and TR-dextran-flux, and tight junction morphology was investigated by immunofluorescence. Route and mechanism of PMN transmigration were determined by immunofluorescence, electron microscopy and FACS analysis. Quantitative real time-PCR was used to determine expression levels of ICAM-1 and VCAM-1. Here, we show that the transmigration of PMNs through PCPECs was significantly higher after stimulation with TNFα or infection with S. suis strain 10 compared to its non-encapsulated mutant. Barrier function was not significantly affected by PMN migration alone, but in combination with S. suis infection. Tight junction and cytoskeletal actin reorganisation were also observed after stimulation with S. suis or TNFα. Most strikingly, PMNs preferentially migrated across PCPECs via the transcellular route. Extensive sequential analyses of the PMN transmigration process with Apotome(®)-imaging and electron microscopy revealed that paracellular migrating PMNs stop just before tight junctions. Interestingly, PMNs subsequently appeared to proceed by transcellular migration via funnel-like structures developing from the apical membrane. It is noteworthy that some PMNs contained bacteria during the transmigration process. Flow cytometric and transmigration inhibition studies with integrin-specific antibodies showed that PMN traversal is dependent on CD11b/CD18. Analysis of cell adhesion molecules in PCPECs revealed a significant increase of ICAM-1 and VCAM-1 expression after TNFα and S. suis stimulation. Our data underline the relevance of the blood-CSF barrier as a gate for leukocyte entry into the CNS and suggest a novel transcellular migration step during the pathogenesis of bacterial meningitis.
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4.41Impact points
Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology.
PloS one. 01/2011; 6(7):e22751.
An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with... [more] An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.
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2.80Impact points
Pertussis toxin permeabilization enhances the traversal of Escherichia coli K1, macrophages, and monocytes in a cerebral endothelial barrier model in vitro.
International journal of medical microbiology : IJMM. 11/2010; 301(3):204-12.
The occasionally severe neurological complications following the human respiratory tract infection 'whooping cough' have been attributed to pertussis toxin (PT) expressed by the causative agent Bordetella pertussis. Disruption of the endothelial blood-brain barrier (BBB) by PT might facilita... [more] The occasionally severe neurological complications following the human respiratory tract infection 'whooping cough' have been attributed to pertussis toxin (PT) expressed by the causative agent Bordetella pertussis. Disruption of the endothelial blood-brain barrier (BBB) by PT might facilitate the translocation of immune cells and of hematogenous microbial pathogens. To test this hypothesis, we investigated whether PT enhances the traversal of bacteria employing human brain microvascular endothelial cells (HBMEC) as an in vitro endothelial barrier model. PT incubation significantly increased the translocation of Escherichia coli K1 across the HBMEC barrier. Only intercellular E. coli K1 bacteria could be identified by electron microscopy suggesting paracellular translocation. In addition, the migration of differentiated HL60-derived macrophages and of human monocytic U937 cells through PT-treated HBMEC barriers was also enhanced. In comparison to E. coli C600, E. coli K1 showed prolonged survival in translocated HL60-derived and J774 macrophages as well as in U937 monocytes which suggested a contribution of the 'Trojan horse' mechanism. In summary, our findings demonstrate that the PT-induced permeabilization of endothelial barriers enhances the paracellular transmigration of microbes and immune cells. In vivo, this activity might lower the threshold of bacteremia facilitating secondary cerebral infections and the subsequent development of brain pathologies.
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5.50Impact points
Evolutionary analysis and distribution of type III effector genes in pathogenic Escherichia coli from human, animal and food sources.
Environmental microbiology. 09/2010; 13(2):439-52.
Molecular analysis of Shiga toxin-producing Escherichia coli (STEC) from different sources is considered as a major approach to assess their risk potential. However, only limited data are available about the correlation of evolutionary relationship, the presence of major virulence factor genes and t... [more] Molecular analysis of Shiga toxin-producing Escherichia coli (STEC) from different sources is considered as a major approach to assess their risk potential. However, only limited data are available about the correlation of evolutionary relationship, the presence of major virulence factor genes and the putative risk of an STEC strain for human infection. In this study, we analysed the evolutionary relationship of 136 pathogenic E. coli strains from human, animal and food sources by multi-locus sequence typing (MLST) and molecular subtyping of their Shiga toxin (stx) and intimin (eae) genes. Moreover, the distribution of three type III effector genes, encoded within the locus of enterocyte effacement (LEE), and 16 effector genes, which are encoded outside the LEE, was analysed. One hundred and five strains from different sources harboured 5-15 of the analysed non-LEE-encoded effector genes. In 101 of these strains, the LEE genes eae, map, espF and espG were present simultaneously. Thirty-one isolates deriving mainly from food and patients suffering from haemolytic uraemic syndrome (HUS) were eae-negative and did not carry any of the analysed effector genes. By combination of MLST and virulence gene data, we defined five genetic clusters. Within these clusters a clear-cut affiliation of particular sequence types and the occurrence of certain effector genes was observed. However, in contrast to other studies, a significant correlation between the amount and type of effector genes and the risk to cause HUS could not be demonstrated.
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3.21Impact points
Enterohemorrhagic Escherichia coli O26:H11-Associated Hemolytic Uremic Syndrome: Bacteriology and Clinical Presentation.
Seminars in thrombosis and hemostasis. 09/2010; 36(6):586-93.
Infection by enterohemorrhagic ESCHERICHIA COLI (EHEC) is the most frequent cause of hemolytic uremic syndrome (HUS) in childhood. During a 6-year period, all patients with the clinical diagnosis of HUS were registered in a prospective multicenter study in Austria and Germany. EHEC O26:H11 was the s... [more] Infection by enterohemorrhagic ESCHERICHIA COLI (EHEC) is the most frequent cause of hemolytic uremic syndrome (HUS) in childhood. During a 6-year period, all patients with the clinical diagnosis of HUS were registered in a prospective multicenter study in Austria and Germany. EHEC O26:H11 was the second most frequent detected serotype, accounting for 15.4% of all EHEC isolates. The presence of EHEC O26:H11 was significantly associated with young age at the disease onset ( P < 0.001). Patients infected with this serotype were not different in their clinical presentation than those infected with other serotypes. This study underlines the importance of EHEC serotypes other than O157 in the etiology of HUS and emphasizes the importance of implementation of appropriate diagnostic methods to identify the whole spectrum of EHEC associated with HUS.
Following (5)
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Sebastian Suerbaum
Medizinische Hochschule Hannover -
Johannes Elias
Universität Würzburg -
Anne Sonntag
Kantonspital Aarau AG -
Dag Harmsen
Westfälische Wilhelms-Universität Münster -
Ulrich Vogel
Julius-Maximilians-Universität Würzburg
