Papers by Johannes Kusters
Journal of clinical microbiology, 2015
Whole genome sequencing is becoming a leading technology in typing and epidemiology of microbial ... more Whole genome sequencing is becoming a leading technology in typing and epidemiology of microbial pathogens, but the increase in genomics information necessitates significant investment in bioinformatic resources and expertise, and currently used methodologies struggle with genetically heterogeneous bacteria such as the human gastric pathogen Helicobacter pylori. Here we demonstrate that the alignment-free analysis method Feature Frequency Profiling (FFP) can be used to rapidly construct phylogenetic trees of draft bacterial genome sequences on a standard desktop computer, and that the coupling with in silico genotyping methods gives useful information for comparative and clinical genomics and molecular epidemiology applications. FFP-phylogenetic trees of seven gastric Helicobacter species matched those obtained by analysis of 16S rDNA and ribosomal proteins, and FFP- and core genome single nucleotide polymorphism-based analysis of 63 H. pylori genomes showed again comparable phyloge...
Helicobacter
Gastrointestinal infections with pathogenic Helicobacter species are commonly treated with combin... more Gastrointestinal infections with pathogenic Helicobacter species are commonly treated with combination therapies, which often include amoxicillin. Although this treatment is effective for eradication of Helicobacter pylori, the few existing reports are less clear about antibiotic susceptibility of other Helicobacter species. In this study we have determined the susceptibility of gastric and enterohepatic Helicobacter species to amoxicillin, and have investigated the mechanism of amoxicillin resistance in Helicobacter hepaticus. The minimal inhibitory concentration (MIC) of antimicrobial compounds was determined by E-test and agar/broth dilution assays. The hefA gene of H. hepaticus was inactivated by insertion of a chloramphenicol resistance gene. Transcription was measured by quantitative real-time polymerase chain reaction. Three gastric Helicobacter species (H. pylori, H. mustelae, and H. acinonychis) were susceptible to amoxicillin (MIC < 0.25 mg/L). In contrast, three entero...
Journal of Applied Microbiology, 2001
... 16 Debets-Ossenkopp, YJ, Brinkman, AB, Kuipers, EJ, Vandenbroucke-Grauls, CM, Kusters, JG (19... more ... 16 Debets-Ossenkopp, YJ, Brinkman, AB, Kuipers, EJ, Vandenbroucke-Grauls, CM, Kusters, JG (1998) Explaining the ... 27 Dore, MP, Piana, A., Carta, M., Atzei, A., Are, BM, Mura, I., Massarelli, G., Maida, A., Sepulveda, AR, Graham, DY, Realdi, G. (1998) Amoxycillin resistance is ...
H. pylori is an S-shaped microaerophilic, gram-negative bacterium which colonizes the epithelial ... more H. pylori is an S-shaped microaerophilic, gram-negative bacterium which colonizes the epithelial stomach surface of half the world’s population. The colonisation of H. pylori in human stomachs results in chronic gastritis and sometimes ulcers or gastric cancer. Infection mostly occurs during childhood and unless treated lasts for life. Treatment of H. pylori is relatively complicated and requires antibiotics to which the bacterium is sensitive. Thus a microbiological culture determining antibiotic resistance is a prerequisite for rational antibiotic treatment. Unfortunately, routine clinical practice is often done without such a culture, and hence, the treatment is frequently empirical, not based on antibiotic resistance data. In this chapter we will elaborate on how to isolate and culture this fastidious bacterium.
Molecular immunology, 1989
To study the antigenic structure of the peplomer protein of the avian coronavirus infectious bron... more To study the antigenic structure of the peplomer protein of the avian coronavirus infectious bronchitis virus, fragments from the peplomer gene were generated by restriction-enzyme cleavage or by limited DNase digestion and inserted in the Escherichia coli expression plasmid pEX (Stanley and Luzio, 1984). The antigenicity of the expression products was tested using a number of polyclonal antisera and monoclonal antibodies. The polyclonal antisera recognized different sets of epitopes in the 1162-residue sequence. The N-terminal region of one of the two subunits, S2, was recognized by all polyclonal sera and by two monoclonal antibodies. This clearly immunodominant region contains at least two adjacent or overlapping epitopes, one of which has been localized within 18 residues. The epitopes found as antigenic pEX expression products do not coincide with the regions in the S1 subunit that have been found to contain hypervariable sequences. We suggest that these regions constitute conf...
Gene analysis techniques
Methods to make long and reliable cDNA from viral RNA template have been optimized. The condition... more Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5-5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.
European Journal of Immunology, 1991
Alignment of all available nucleotide sequences of mouse and rat a@ T cell receptor (TcR) variabl... more Alignment of all available nucleotide sequences of mouse and rat a@ T cell receptor (TcR) variable (V) regions revealed the presence of relatively conserved sequences at the 5' end of the V gene segments. Based on these conserved sequences, degenerate primers were developed for use in the polymerase chain reaction (PCR).The degenerate primers developed on the basis of the conserved sequences at the 5' end of rat and mousevgene segments are expected to enable the amplification of all mouse and rat TcR a/@ chain V regions. To test their applicability, the primers were used for the amplification of the V region of the TcR a/P expressed by rat Tcell lines. After amplification, the TcR V regions expressed were cloned and sequenced. The ZlaTcell line was shown to use the sameTcR Vgene segments (V,2 and Vg8.2), as most other experimental allergic encephalomyelitis associated Tcell lines, but had different D and J segments. In spite of these differences at the nucleotide level, a remarkable conservation of the amino acid sequence at the VoDgJp junction was found. Alignment of a large number of human V, and Vg gene segments revealed the presence of similarly conserved sequences. Degenerate primers based on these conserved sequences enabled the amplification of TcR V regions of human Tcell lines. 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 0014-2980/91/0303-0569$3 .SO + .25/0 570 C. P. M. Broeren, G . M. G . M.Vejans,W.Van Eden et al. Eur. J. Immunol. 1991. 21: 569-575
Trends in Microbiology, 1997
Infection and immunity, 1999
The lipopolysaccharide (LPS) of Helicobacter pylori expresses the Lewis x (Lex) and/or Ley antige... more The lipopolysaccharide (LPS) of Helicobacter pylori expresses the Lewis x (Lex) and/or Ley antigen. We have shown previously that H. pylori LPS displays phase variation whereby an Lex-positive strain yields variants with different LPS serotypes, for example, Lex plus Ley or nonfucosylated polylactosamine. H. pylori has two alpha3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes H. pylori LPS phase variation and demonstrate that the on or off status of alpha3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the alpha3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of alpha3-fucosyltransferase knockout mutants. The data also show that t...
Journal of bacteriology, 1995
Clones expressing fragments of the flagellin protein of Salmonella enteritidis were constructed a... more Clones expressing fragments of the flagellin protein of Salmonella enteritidis were constructed and screened with a g,m-specific monoclonal antibody. Results showed that the g,m epitope is localized between amino acids 258 and 348 of the flagellin. The fliC gene, encoding the flagellin of S. enteritidis, was proven to be the only flagellin gene present in S. enteritidis.
Infection and immunity, 1998
Salmonella typhimurium infection of mice is an established model system for studying typhoid feve... more Salmonella typhimurium infection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimurium genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1, 000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimurium genome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonella spp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of...
Helicobacter species DNA has been detected in liver tissue of patients affected by primary biliar... more Helicobacter species DNA has been detected in liver tissue of patients affected by primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). To investigate a potential causative relation between Helicobacter species and PBC/PSC, we compared the presence of Helicobacter species-specific DNA in liver tissue of patients with PBC/PSC (n = 18/n = 13) with those of a control group of patients with various liver diseases with known cause (n = 29). A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from paraffin embedded liver tissue. Control patients had hepatitis-B (n = 9), alcoholic cirrhosis (n = 14), or non-cirrhotic metabolic liver disease (n = 6). There was no significant difference between the incidence of Helicobacter spp.-specific DNA in PBC/PSC (9/31; 29%) and the control group (10/29; 34%). Sequence analysis confirmed Helicobacter spp. DNA. Because Helicobacter spp. DNA can be found in approximately one-third of all samples tested, it is unlikely that PSC and PBC are caused by Helicobacter infection.
Annals of The New York Academy of Sciences, 1995
Journal of Applied Microbiology, 1994
The effect of temperature on the growth rate, protein pattern and fatty acid composition of Yersi... more The effect of temperature on the growth rate, protein pattern and fatty acid composition of Yersinia enterocolitica strain W22703 pYV+, its plasmidless isogenic derivative W22703 pYV-and four recent field isolates was examined.
Infection and Immunity, 2001
Upon contact with host cells, the intracellular pathogen Salmonella enterica serovar Typhimurium ... more Upon contact with host cells, the intracellular pathogen Salmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbi- cidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the pheno- typic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The
Trends in Microbiology, 2004
Proceedings of the National Academy of Sciences, 1994
T-T cell interactions have been proposed in postulated network theories of and autoimmunity. Desp... more T-T cell interactions have been proposed in postulated network theories of and autoimmunity. Despite previous reports ofprotection induced by T-cell receptor (TcR)-derived peptides in experimental autoimuity, no evidence for T-T cell Inter by direct r ti of
PLoS ONE, 2012
Background and Objective: Barrett's esophagus (BE) is characterized by the transition of squamous... more Background and Objective: Barrett's esophagus (BE) is characterized by the transition of squamous epithelium into columnar epithelium with intestinal metaplasia. The increased number and types of immune cells in BE have been indicated to be due to a Th2-type inflammatory process. We tested the alternative hypothesis that the abundance of T-cells in BE is caused by a homing mechanism that is found in the duodenum.
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Papers by Johannes Kusters