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D Harmsen

Publications and source records attributed to D Harmsen.

At least 19 recordsLinked to original sources
PubMed2003

QAlign: quality-based multiple alignments with dynamic phylogenetic analysis.

Integrating different alignment strategies, a layout editor and tools deriving phylogenetic trees in a 'multiple alignment environment' helps to investigate and enhance results of multiple sequence alignment by hand. QAlign combines algorithms for fast progressive and accurate simultaneous multiple alignment with a versatile editor and a dynamic phylogenetic analysis in a convenient graphical user interface.

Algorithms↗
PubMed2001

Fatal gastrointestinal infection with 'Flexispira rappini'-like organisms in a cat.

A 4-month-old male British Blue cat with catarrhal to haemorrhagic enteritis showed massive colonization of the stomach, small intestine and caecum with spiral-shaped bacilli. In the stomach, organisms were located in foveolae and gland lumina and within unaltered and degenerate epithelial cells. Inflammatory infiltration was moderate and T cell dominated. In the intestine, bacilli were found in the gut lumen, berween villi, in crypt lumina and within epithelial cells. Degeneration of crypt epithelial cells as well as crypt dilation and moderate to massive macrophage-dominated infiltration of the mucosa and submucosa were observed. Immunohistochemically, bacilli were positive with an antibody against Helicobacter. Ultrastructurally, the organisms strongly resembled 'Flexispira rappini', a spiral-shaped Helicobacter species known as a normal intestinal colonizer in dogs and mice.

Animals↗
PubMed2001

Diagnostics of neisseriaceae and moraxellaceae by ribosomal DNA sequencing: ribosomal differentiation of medical microorganisms.

Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, and Neisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to Escherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600). Neisseria macacae and Neisseria mucosa subsp. mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, and N. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae and Moraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes.

DNA, Ribosomal↗
PubMed2000

Biocatalytic asymmetric hydroxylation of hydrocarbons with the topsoil-microorganism Bacillus megaterium.

A Bacillus megaterium strain was isolated from topsoil by a selective screening procedure with allylbenzene as a xenobiotic substrate. This strain performed the hydroxylation chemoselectively (no arene oxidation and overoxidized products) and enantioselectively (up to 99% ee) in the benzylic and nonbenzylic positions of a variety of unfunctionalized arylalkanes. Salycilate and phenobarbital, which are potent inducers of cytochrome P-450 activity, changed the regioselectivity of the microbial CH insertion, without an effect on the enantioselectivity. The biotransformation conditions were optimized in regard to product yield and enantioselectivity by variation of the oxygen-gas supply and the time of the substrate addition. The different product distributions (alpha- versus beta-hydroxylated product) that are obtained on induction of cytochrome P-450 enzyme activity demonstrate the involvement of two or more hydroxylating enzymes with distinct regioselectivities in this biotransformation. An oxygen-rebound mechanism is assumed for the cytochrome P-450-type monooxygenase activity, in which steric interactions between the substrate and the enzyme determine the preferred face of the hydroxy-group transfer to the radical intermediate.

Bacillus megaterium↗
PubMed2000

Molecular and phenotypic identification of the yeast pathogen Candida dubliniensis.

Candida dubliniensis is an emerging yeast pathogen generally misclassified as Candida albicans by standard diagnostic procedures. This study examined the efficiency of molecular identification, based on a discriminative PCR test, in a prospective study on the prevalence of C. dubliniensis among 103 oropharyngeal isolates from HIV-infected individuals or transplant recipients, and 30 vaginal isolates. All of the isolates had been classified as C. albicans by standard laboratory procedures. The PCR was evaluated in a blinded fashion against classification achieved by sequencing rDNA. Sequencing results corresponded 100% to the results of the discriminative PCR, indicating the validity of this rapid test. Twenty-one C. dubliniensis isolates were identified, all of them from HIV-infected individuals (prevalence 30%). The internal transcribed spacer regions of the C. dubliniensis isolates were sequenced. Phenotypic features of C. dubliniensis, namely abundant chlamydospore formation, atypical color on CHROMagar, growth defect at 45 degrees C, and colony morphology on Staib agar, were evaluated in a blinded fashion with respect to their discriminative potential, facilitating the design of further epidemiological studies. Carbohydrate assimilation patterns were determined for C. dubliniensis with a novel automated system showing that, in contrast to previous reports, C. dubliniensis is able to utilize D-xylose and trehalose. In evaluating these tests we present a rational approach to identification of the new species and characterization of C. dubliniensis isolates.

AIDS-Related Opportunistic Infections↗
PubMed2000

Differentiation of clinical Mycobacterium tuberculosis complex isolates by gyrB DNA sequence polymorphism analysis.

The discriminatory power of gyrB DNA sequence polymorphisms for differentiation of the species of the Mycobacterium tuberculosis complex (MTBC) was evaluated by sequencing and restriction fragment length polymorphism (RFLP) analysis of a 1,020-bp fragment amplified from clinical isolates of M. tuberculosis, Mycobacterium bovis (pyrazinamide [PZA] resistant as well as PZA susceptible), Mycobacterium africanum subtypes I and II, and Mycobacterium microti types vole and llama. We found sequence polymorphisms in four regions described previously and at one additional position. These differences in the gyrB sequences allow an accurate discrimination of M. bovis, M. microti, and M. africanum subtype I. The PZA-susceptible subtypes of M. bovis shared the M. bovis-specific substitution at position 756 with the PZA-resistant strains, but can be unambiguously differentiated by a characteristic substitution at position 1311. As a drawback, M. tuberculosis and M. africanum subtype II showed an identical gyrB sequence that facilitates discrimination from the other species, but not from each other. A PCR-RFLP technique applying three restriction enzymes could be shown to be a rapid and easy-to-perform tool for the differentiation of the members of the MTBC. Based on these results, we present a clear diagnostic algorithm for the differentiation of species of the MTBC.

Animals↗
PubMed1999

[Trichophyton tonsurans var. sulfureum subvar. perforans in Tinea gladiatorum].

This is the first report on an isolation of Trichophyton tonsurans var.sulfureum subvar.perforans in Germany. In our patient, the strain had caused typical tinea corporis, which was most likely acquired during a wrestling competition. Based on the macroscopic and microscopic morphology and on the physiologic properties occurring under various growth conditions, the identification of Trichophyton tonsurans and the particular characteristics of its var.sulfureum subvar.perforans are described. Partial 18S and 23S nuclear ribosomal RNA sequences and the internal transcribed spacer region I sequences of our strain were completely identical with those of a Trichophyton tonsurans reference strain. The epidemiology of Trichophyton tonsurans var.sulfureum subvar.perforans and a possible association of this variety with tinea in wrestlers (tinea gladiatorum) have not yet been investigated.

Adult↗
PubMed1999

Chronic recurrent multifocal osteomyelitis in children: diagnostic value of histopathology and microbial testing.

Chronic recurrent, unifocal or multifocal osteomyelitis (CRMO), an inflammatory disorder of unknown origin, involves different osseous sites and may be associated with palmoplantar pustulosis. Bacterial cultures of affected tissue were reported negative in nearly all cases. Radiological and magnetic resonance imaging features of CRMO have been described, but differential diagnosis remains difficult, including rheumatic diseases, bacterial osteomyelitis, and malignancy. Although definite diagnosis relies on histopathologic confirmation by biopsy, histopathologic criteria have not been defined. Because CRMO may be treated with nonsteroidal antiinflammatory drugs, but not antibiotics, distinguishing CRMO from bacterial osteomyelitis is of major importance. Histopathologic analysis of 12 patients with CRMO indicated a wide variation of reparative changes of bone, but chronic inflammation could not be found at all sites in the same biopsy. The inflammatory infiltrate was mostly scattered, consisting mainly of lymphocytes, plasma cells, histiocytes, and also few neutrophil granulocytes. Immunohistochemistry showed a predominance of CD3(+), CD45RO(+) T-cells, which were mainly CD8(+). In addition, CD20(+) B cells and CD68(+) macrophages were abundant in each biopsy specimen. Mild lymphocytic and granulocytic infiltrates were also detected in three synovial biopsy specimens obtained from adjacent joints. All bacterial and fungal cultures from native biopsy tissues were negative. Amplification of partial-length 16S ribosomal DNA by polymerase chain reaction (PCR) using broad-range eubacterial primers was below the detection limit in all patients. Because histopathologic features alone may not provide conclusive evidence, CRMO should be included in the differential diagnosis of chronic inflammatory bone lesions in children, and the definite diagnosis should be made by the clinical picture, x-ray studies, bone scan, bacterial culture, and histopathologic analysis in a multidisciplinary approach.

Acquired Hyperostosis Syndrome↗
PubMed1999

Molecular differentiation of dermatophyte fungi.

Our previous studies of the ribosomal DNA variation in dermatophytes have shown that these fungi are monophyletic in origin. However, this approach did not allow us to differentiate all the species defined by classical means. Therefore, we studied the internal transcribed spacer 1 (ITS 1) region of 17 species of the fungal order Onygenales, comprising the pathogenic keratinophilic fungi. Interspecific nucleotide composition and sequence length variation of the ITS 1 region was high, mean identities were as low as 40% and sequence lengths varied from 169 to 293 basepairs. Each established dermatophyte species could be identified. In contrast, the flanking sequences at the 3' end of 18S and the 5.8S rDNA were conserved. Although the value of the ITS 1 region as a phylogenetic tool may be limited because of its high variability, it provides the information necessary to design species-specific probes, or polymerase chain reaction restriction fragment polymorphism systems useful for taxonomic or rapid diagnostic tests.

Arthrodermataceae↗
PubMed1999

Molecular epidemiology of Candida albicans isolates from AIDS and cancer patients using a novel standardized CARE-2 DNA fingerprinting technique.

A total of 277 Candida isolates from various body sites of 149 AIDS and cancer patients treated in four different university clinics in Würzburg, Germany were collected over a period of 27 months and phenotypically and genotypically characterized. The fingerprinting patterns of 194 Candida albicans isolates obtained with the moderately repetitive, C. albicans-specific DNA fragment CARE-2 were digitized and retrospectively compared with a highly accurate computer-assisted standardization method. A total of 168 different genotypic patterns (< 100% identity) could be differentiated using this technique. Although comparative analysis of C. albicans subsets revealed a pronounced tendency of C. albicans isolates from HIV patients to form clusters, the mean genetic variability in HIV and cancer patient isolates was virtually identical. Patients with a specific disease condition or in a certain age group were not found to harbour C. albicans isolates displaying a characteristic "signature genotype". Micro-evolutionary changes were detected by CARE-2 fingerprinting in temporal successive isolates of one patient, but nosocomial transmission of identical isolates between unrelated patients was never seen. Genotyping showed that patient isolates can replace one another; occasionally also species switches were observed. Secreted aspartic protease (SAP) production was not correlated with a specific C. albicans banding pattern; isolates obtained from HIV patients and from an internal control group secreted comparable amounts of SAP. Candida dubliniensis isolates in this study showed an elevated level of SAP production. When used under standardized conditions, CARE-2 fingerprinting is an efficient, reproducible and sensitive technique to characterize C. albicans isolates.

Acquired Immunodeficiency Syndrome↗
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↗