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D Harmsen

Publications and source records attributed to D Harmsen.

35 records · Page 2Linked to original sources
PubMed1999

Standardized molecular typing.

Molecular typing methods are useful tools in molecular mycology. The results of these biotyping procedures may help to identify pathogenic strains in order to detect sources of nosocomial infection and for the investigation of epidemiological relationships. With respect to the facultative pathogen, Candida albicans, various methods such as pulse-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP), DNA fingerprinting methods and hybridization with repetitive DNA elements have been described as useful tools in molecular epidemiology. The previously described hybridization method with the Candida albicans specific CARE-2 probe and subsequent rehybridization with a molecular size marker is a standardized reproducible typing method for comparison of results obtained in different laboratories. In a larger epidemiological study conducted at the University Hospital of Würzburg analysing clinical C. albicans isolates, we were able to describe relationships between sequential patient isolates. These findings demonstrate that standardized molecular typing methods are a powerful tool in molecular mycology studies.

DNA Fingerprinting↗
PubMed1998

Development of rRNA-targeted PCR and in situ hybridization with fluorescently labelled oligonucleotides for detection of Yersinia species.

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of Yersinia species by PCR and in situ hybridization, respectively. As few as 10(2) Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic Yersinia species (in particular Y. pestis and Y. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestis and Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifying Y. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.

Animals↗
PubMed1997

A fatty acid alpha-ketol, a product of the plant lipoxygenase pathway, is oxidized to 3(Z)-dodecendioic acid by a bacterial monooxygenase.

The fatty acid alpha-ketol 13-hydroxy-12-oxo-9(Z)-octadecenoic acid (methyl ester) was incubated with a bacterial culture isolated from soil. The bacteria (tentatively identified as Ralstonia sp.) exhibited strong monooxygenase activity growing on 2-tridecanone as sole source of carbon. They catalyzed a Baeyer-Villiger type of oxidation and converted the alpha-ketol to 3(Z)-dodecendioic acid. 3(Z)-Dodecendioic acid was isolated from the incubation mixture and identified by comparison with an authentic reference compound. These findings offer both a physiological role for alpha-ketol fatty acids in plant lipid hydroperoxide metabolism and new insights into an alternative biosynthetic pathway leading to traumatic acid (2(E)-dodecendioic acid).

Bacterial Proteins↗
PubMed1997

Invasion and destruction of mucosal plasma cells by Tropheryma whippelii.

Whipple's disease is a poorly understood systemic disorder associated with the bacillus, Tropheryma whippelii. An early stage of Whipple's disease is studied by using electron microscopy (ELMI) and immunohistochemistry. The diagnosis was confirmed by polymerase chain reaction-amplification and sequencing of the 16S ribosomal-RNA of the bacterium. By using ELMI, Tropheryma whippelii was found in plasma cells and macrophages in the jejunal mucosa. The immunoglobulin (Ig)A-positive plasma cells were focally destructed and their number significantly reduced. However, the bacilli in the plasma cells were morphologically intact. In contrast, the macrophages showed no signs of cell destruction, but contained bacilli in various stages of disintegration. A cytopathic effect of Tropheryma whippelii to IgA plasma cells may be the reason for the commonly found plasma cell reduction in the small intestine mucosa and an important pathogenic mechanism contributing to the evasion of the bacilli from local immune response.

Actinobacteria↗
PubMed1997

Standardized molecular typing of Candida albicans strains.

A method is presented for the standardization of Candida albicans DNA fingerprinting, which is based on Southern hybridization of EcoRI-digested chromosomal DNA with the moderately repetitive DNA element CARE-2 and the subsequent rehybridization of the blots with a molecular size marker also included in each DNA sample. This method resulted in extremely precise alignment of all strain-specific CARE-2 hybridization patterns, even when analysed on different gels, and will enhance the accuracy of genetic relationship determinations in epidemiological studies including large numbers of strains.

Candida albicans↗
PubMed1996

An unusual case of refractory Campylobacter jejuni infection in a patient with X-linked agammaglobulinemia: successful combined therapy with maternal plasma and ciprofloxacin.

An unusual hippurate-negative strain of Campylobacter jejuni caused a chronic refractory infection in a patient with X-linked agammaglobulinemia; this infection persisted for > 2 years despite therapy with various antibiotics and immunoglobulins (Igs). To characterize the defense status of this patient, several in vitro studies, including those with T cells and polymorphonuclear leukocytes (PMNLs), were performed. T cell responses specific for C. jejuni were only weak in this patient. Chemiluminescence and bacterial killing studies with PMNLs revealed that the bactericidal activity of PMNLs against Campylobacter was enhanced more vigorously by maternal serum than by commercial Ig preparations. On the basis of these results, combined treatment with ciprofloxacin and maternal plasma was initiated, and the C. jejuni infection was rapidly cured. This case report shows that in vitro immunologic assays may be useful for characterizing immune functions of patients with chronic or refractory C. jejuni infections, thus leading to individual treatment strategies.

Adoptive Transfer↗
PubMed1996

Specific detection of Candida albicans and Candida tropicalis by fluorescent in situ hybridization with an 18S rRNA-targeted oligonucleotide probe.

In situ hybridization of whole cells with rRNA-targeted, fluorescently labelled oligonucleotide probes is a powerful method to specifically detect microorganisms in their natural habitat without cultivation and subsequent identification by phenotypic characterization. To examine the use of this method for the specific detection of pathogenic Candida species, we have designed an oligonucleotide probe which binds to the 18S rRNA of C. albicans and C. tropicalis, the two most important pathogenic Candida species, and differentiates them from other clinically relevant species. After establishing suitable hybridization conditions, we confirmed the specificity of our probe O20 in RNA dot blot hybridizations with a series of reference strains and clinical isolates of medically important Candida species. All C. albicans and C. tropicalis strains hybridized with the probe, whereas all strains of C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. kefyr and C. lusitaniae did not. When we used the fluorescently labelled probe O20 to specifically detect single cells of the two target species by in situ hybridization, both C. albicans and C. tropicalis reacted strongly with the probe and could be clearly differentiated from C. krusei and C. parapsilosis, although the latter organism contains only two nucleotide mismatches in the probe target region. This discrimination capacity was also seen when mixed suspensions of C. albicans and C. parapsilosis were hybridized with the probe. After infection of a human endothelial cell line with C. albicans and C. krusei, C. albicans cells adhering to the endothelial cells were easily distinguishable from the C. krusei cells by fluorescent in situ hybridization with probe O20. In addition, germ tubes and hyphae of C. albicans were also efficiently labelled. The application of fluorescently labelled rRNA-targeted oligonucleotide probes therefore appears to be a valuable tool for the specific detection and identification of different members of the genus Candida, which does not require any cultivation.

Candida↗
PubMed1995

Genotyping of Shiga-like toxin genes in non-O157 Escherichia coli strains associated with haemolytic uraemic syndrome.

The pheno- and genotypes of Shiga-like toxins (SLTs) in non-O157 Escherichia coli strains from patients with haemolytic uraemic syndrome were determined. The clinical isolates investigated were from Italy and Germany and belonged to serotypes O22:H8, O26:H-, O26:H11, O91:H-, O111:H- and O128:H-; one isolate was non-typable. SLT genotypes were analysed by complete nucleotide sequence analysis of the B-subunit genes. The results showed that 14 strains possessed slt-I alone, two contained slt-II alone and five isolates harboured both slt-I and slt-II genes. In only two strains were slt-II-related genes found, together with either slt-I or slt-II. These findings indicate that variants of SLT-II are rarely found in non-O157 E. coli isolates from patients with haemolytic uraemic syndrome. Polymerase chain reaction (PCR) with Taq cycle sequencing was found to be a suitable method for classification of slt genotypes.

Bacterial Toxins↗
PubMed1995

Clonal relatedness of Shiga-like toxin-producing Escherichia coli O101 strains of human and porcine origin.

Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) O101 has recently been associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans. In this study, SLTEC O101 strains from humans and pigs were characterized for clonal relatedness by nucleotide sequence analysis of their slt genes, DNA finger-printing of genomic DNA, and determination of virulence factors. The slt genes of five E. coli O101 strains were cloned and sequenced. For all strains, the deduced amino acid sequences of the B subunits were identical to those of the SLT-IIe present in the classical SLTEC O139 strains that cause edema disease in pigs. The A subunit revealed more than 99% homology to that of SLT-IIe. DNA fingerprinting revealed a high degree of genetic relatedness between the human and porcine O101 isolates. None of the O101 strains investigated had virulence factors frequently found in porcine (F107 fimbriae or heat-stable or heat-labile enterotoxins) or human SLTEC strains (eaeA or enterohemorrhagic E. coli hemolysin). The absence of virulence factors typical of SLT-I- and SLT-II-producing E. Coli together with the presence of SLT-IIe, a toxin previously seen only in porcine E. coli, suggests a new pathogenic mechanism for E. coli O101 infection of humans. For diagnostic purposes, we recommend the use of PCR primers and DNA probes complementary to slt-IIe to correctly identify such strains and to further evaluate their role in human diseases.

Amino Acid Sequence↗
PubMed1994

The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function.

The iron-repressible outer membrane protein FyuA of Yersinia enterocolitica operates as a receptor with dual function: (i) as a receptor for the Y. pestis bacteriocin pesticin, and (ii) as a receptor for yersiniabactin, a siderophore that is produced by mouse-virulent Y. enterocolitica strains of biogroup IB. Cloning of the FyuA-encoding gene was achieved by mobilization of a genomic cosmid library of the pesticin-sensitive and mouse-virulent Y. enterocolitica O:8 strain WA into the pesticin-resistant WA fyuA mutant and subsequent in vivo selection of transconjugants for the ability to survive and multiply in mice (phenotype mouse virulence). The reisolated transconjugants which survived in mice for 3 d harboured a unique cosmid and phenotypically were pesticin sensitive. From this cosmid a 2650 bp SalI-PstI fragment conferring pesticin sensitivity was subcloned. Sequencing of this DNA fragment revealed a single open reading frame of 2022 bp, which encodes a deduced polypeptide of 673 amino acids with a predicted molecular mass of 73,677 Da. Cleavage of a putative signal sequence composed of 22 amino acids should lead to a mature protein of 651 amino acids with a molecular mass of 71,368 Da. The open reading frame is preceded by a sequence which shares homology with the postulated consensus Fur iron-repressor protein-binding site. FyuA shows homology to other iron-regulated TonB-dependent outer membrane proteins with receptor functions (e.g. BtuB, CirA, FepA, IutA, FhuA, FoxA, FcuA). On the basis of multiple alignment of amino acid sequences of FyuA and other TonB-dependent receptors, a phylogenetic tree was constructed, demonstrating that FyuA probably belongs to the citrate subfamily or represents a new subfamily of TonB-dependent receptors. Moreover, by complementation of the WA fyuA mutant by the cloned fyuA gene, yersiniabactin uptake and mouse virulence were restored. These studies demonstrate that the cloned pesticin/yersiniabactin receptor FyuA of Y. enterocolitica has the typical features of iron-regulated TonB-dependent outer membrane receptors for siderophores and bacteriocins and is required for mouse virulence.

Amino Acid Sequence↗
PubMed1994

[Detection by molecular biology techniques of diphtheria toxin production in corynebacteria].

The ability to secrete a potent protein-toxin called diphtheria toxin (DT) is of outstanding importance for the outcome of diphtheria epidemics. Rapid molecular-biological screening methods are shown that can improve the differentiation between toxinogenic and non-toxinogenic Corynebacterium diphtheriae strains. The recently reported infection with an endemic, putative toxinogenic Corynebacterium diphtheriae strain in Lower Saxony is discussed.

Corynebacterium diphtheriae↗
PubMed1990

Characterization of a nucleic acid probe for the diagnosis of human coronavirus 229E infections.

A cDNA copy of the HCV229E nucleocapsid protein gene was isolated and characterized. Sequence analysis predicts a nucleocapsid polypeptide of 389 amino acids with a molecular weight (mol. wt.) of 43,450. Single strand RNA probes derived from the cDNA copy hybridize specifically to HCV229E RNA and approximately 50 pg of intracellular viral RNA can be readily detected. The application of nucleic acid hybridization as a routine procedure for the diagnosis of HCV229E infection is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Phylogeny and dating of some pathogenic keratinophilic fungi using small subunit ribosomal RNA.

The phylogenetic relationship of representative members of the genera Trichophyton, Microsporum and Epidermophyton was studied. 844 base pairs were compared after sequencing the nuclear 18S ribosomal RNA gene spanning the most variable area within the central 600 bases of this gene. The dermatophytes, which are monophyletic in origin, could be classified most effectively when placed as a subgroup in the Onygenales, Ascomycotina. The calculated radiation time was about 50 million years ago. The early cenozoic adaptive explosion of mammals, which can serve as a host for the keratinophilic fungi, provides corroboration for proximate co-evolution.

Arthrodermataceae↗
PubMedDate not supplied

Prevalence of enteropathogenic bacteria in surgically treated chronic inflammatory bowel disease.

BACKGROUND/AIMS: In order to assess the potential impact of bacterial eradication on recurrence rates, the prevalence of various enteropathogenic bacteria and toxins in chronic inflammatory bowel diseases (CIBD) was prospectively examined. METHODOLOGY: Stool, sera and gut tissue samples from a total of 59 patients (33 males, 26 females; mean age: 42 years +/- 14; 21 Crohn's disease, 14 ulcerative colitis, 24 controls) were examined for the presence of enteropathogenic bacteria by culture, immunoblotting and PCR. RESULTS: Conventional cultures failed to detect obligate pathogenic bacteria. By PCR, mycobacteria were found in 85% of all groups, with mycobacterium paratuberculosis not detected. Yersinia species were observed in 63% of patients with Crohn's disease, in 46% of patients with ulcerative colitis, and in 36% of the control patients. Pathogenic E. coli were identified in stool samples of three patients with ulcerative colitis (21%) by amplifying the EAE-gene, one of whom exhibited shiga-like-toxin as well. CONCLUSIONS: We concluded that mycobacteria do not play a causative role in CIBD. Yersinia species seem to persist in intestinal tissue in CIBD patients without adequate immune response and might, thus, contribute to tissue destruction. E. coli infections contribute to the disease process in a small group of patients with ulcerative colitis and their eradication might eliminate the need for immediate surgical intervention.

Adult↗