Biomedical subjects

Ralf Ehricht

Publications and source records attributed to Ralf Ehricht.

10 recordsLinked to original sources

PubMed2026

Large-scale benchmarking of prokaryotic annotation tools across thousands of species.

BACKGROUND: Genome annotation is an important step in deriving functional meaning from prokaryotic sequencing data, yet systematic evaluations guiding tool selection are lacking. We present the first large-scale investigation of four prominent open-source annotation tools (Prokka, Bakta, EggNOG-mapper, and PGAP) across 156,033 diverse genomes. This includes Escherichia coli strains for baseline performance, thousands of archaea and bacteria genomes, as well as frameshifted and metagenome-assembled genomes. RESULTS: Bakta excels in annotating high-quality bacterial genomes, while PGAP was better for archaeal genomes and challenging bacterial assemblies, including metagenome-assembled, fragmented, or contaminated samples. For Gene Ontology annotation, PGAP consistently provides broader term coverage, whereas EggNOG-mapper offers more terms per feature. CONCLUSIONS: Our findings highlight tool-specific strengths crucial for selecting optimal solutions based on genome quality, taxonomy, and origin (e.g. MAGs). This study provides an evidence-based guide for users and informs future tool development.

Molecular Sequence Annotation↗

PubMed2006

Fast DNA serotyping of Escherichia coli by use of an oligonucleotide microarray.

Classical antibody-based serotyping of Escherichia coli is an important method in diagnostic microbiology for epidemiological purposes, as well as for a rough virulence assessment. However, serotyping is so tedious that its use is restricted to a few reference laboratories. To improve this situation we developed and validated a genetic approach for serotyping based on the microarray technology. The genes encoding the O-antigen flippase (wzx) and the O-antigen polymerase (wzy) were selected as target sequences for the O antigen, whereas fliC and related genes, which code for the flagellar monomer, were chosen as representatives for the H phenotype. Starting with a detailed bioinformatic analysis and oligonucleotide design, an ArrayTube-based assay was established: a fast and robust DNA extraction method was coupled with a site-specific, linear multiplex labeling procedure and hybridization analysis of the biotinylated amplicons. The microarray contained oligonucleotide DNA probes, each in duplicate, representing 24 of the epidemiologically most relevant of the over 180 known O antigens (O antigens 4, 6 to 9, 15, 26, 52, 53, 55, 79, 86, 91, 101, 103, 104, 111, 113, 114, 121, 128, 145, 157, and 172) as well as 47 of the 53 different H antigens (H antigens 1 to 12, 14 to 16, 18 to 21, 23 to 34, 37 to 43, 45, 46, 48, 49, 51 to 54, and 56). Evaluation of the microarray with a set of defined strains representing all O and H serotypes covered revealed that it has a high sensitivity and a high specificity. All of the conventionally typed 24 O groups and all of the 47 H serotypes were correctly identified. Moreover, strains which were nonmotile or nontypeable by previous serotyping assays yielded unequivocal results with the novel ArrayTube assay, which proved to be a valuable alternative to classical serotyping, allowing processing of single colonies within a single working day.

Antigens, Bacterial↗

PubMed2006

Residual DNA in thermostable DNA polymerases - a cause of irritation in diagnostic PCR and microarray assays.

In a validation trial of a DNA microarray test for chlamydiae we repeatedly observed false-positive PCR amplicons from truly negative samples and non-template controls. Various PCR tests, microarray hybridization and DNA sequencing, revealed that residual Escherichia coli DNA from thermostable DNA polymerases was the cause of this cross-reaction. A subsequent survey showed that only five out of 23 commercial polymerases were free of E. coli DNA. When designing generic oligonucleotide sequences for PCR and PCR microarray-based assays one should be aware of such possible internal contamination, particularly when the target organism is E. coli.

Chlamydiaceae↗

PubMed2006

A simple and rapid protein array based method for the simultaneous detection of biowarfare agents.

A protein chip has been developed that allows the simultaneous detection of a multitude of different biowarfare agents. The chip was developed for the ArrayTube platform providing a cheap and easy to handle technology solution that combines a microtube-integrated protein chip with the classical procedure of a sandwich-enzyme-linked immunosorbent assay and signal amplification by streptavidin-poly-horseradish peroxidase. Specific immunoassays for Staphylococcus enterotoxin B, ricin, Venezuelan equine encephalitis virus, St. Louis encephalitis virus, West Nile virus, Yellow fever virus, Orthopox virus species, Francisella tularensis, Yersinia pestis, Brucella melitensis, Burkholderia mallei and Escherichia coli EHEC O157:H7 were developed and optimized. All assays could be completed within 1 to 1 1/2 h and detection levels were demonstrated to be as low as in well established ELISAs. Most interesting, as a result of careful antibody screening and testing, it is currently possible to analyse at least five of the "dirty dozen" agents on one single protein chip in parallel.

Antibodies, Monoclonal↗

PubMed2006

A case of peritonitis caused by Rhizopus microsporus.

We report a case of a 62-year-old female patient who developed peritonitis after receiving a renal transplant. Candida glabrata was detected and treated with voriconazole. As the patient did not improve under therapy, laparotomy was performed. Mould-like plaques were found on the peritoneum. Using culture as well as pan-fungal polymerase chain reaction (PCR) followed by DNA microarray hybridisation of the amplicon, the causative agent was identified as Rhizopus microsporus. Despite aggressive surgical treatment, intravenous therapy with amphotericin B and topical administration of Lavasept (polyhexamethylenbiguanide), the patient died.

Amphotericin B↗

PubMed2006

Use of miniaturized protein arrays for Escherichia coli O serotyping.

Serological typing of Escherichia coli O antigens is a well-established method used for differentiation and identification of O serotypes commonly associated with disease. In this feasibility study, we have developed a novel somatic antibody-based miniaturized microarray chip, using 17 antisera, which can be used to detect bound whole-cell E. coli antigen with its corresponding immobilized antibody, to assess the feasibility of this approach. The chip was tested using the related 17 control strains, and the O types found by the microarray chip showed 100% correlation with the O types found by conventional typing. A blind trial was performed in which 100 E. coli isolates that had been O serotyped previously by the conventional assay were tested by the array approach. Overall, the O serotypes of 88% of isolates were correctly identified by the microarray method. For several isolates, ambiguity of O-type designation by microarray arose due to increased sensitivity of this method, allowing signal intensities of cross-reactions to be quantified. Investigation of discrepancies between conventional and microarray O serotyping indicated that some isolates upon storage had become untypeable and, therefore, gave poor signal intensity when tested by the microarray or retested by conventional means. For all 20 serotype O26 and O157 isolates, the apparent discrepancy in O serotyping was analyzed further by a third independent test, which confirmed the microarray results. Therefore, the use of miniaturized protein arrays increases the speed and efficiency of O serotyping in a cost-effective manner, and these preliminary findings suggest the microarray approach may have a higher accuracy than those of traditional O-serotyping methods.

Antigens, Bacterial↗

PubMed2005

Optimized DNA microarray assay allows detection and genotyping of single PCR-amplifiable target copies.

This study was conducted to determine the detection limit of an optimized DNA microarray assay for detection and species identification of chlamydiae. Examination of dilution series of a plasmid standard carrying the target sequence from Chlamydia trachomatis and genomic DNA of this organism revealed that a single PCR-amplifiable target copy was sufficient to obtain a specific hybridization pattern. This performance renders the test suitable for routine testing of clinical samples.

Chlamydia trachomatis↗

PubMed2005

Use of diagnostic microarrays for determination of virulence gene patterns of Escherichia coli K1, a major cause of neonatal meningitis.

Forty Escherichia coli strains isolated primarily from neonatal meningitis, urinary tract infections and feces were screened for the presence of virulence genes with a newly developed microarray on the array tube format. A total of 32 gene probes specific for extraintestinal as well as intestinal E. coli pathotypes were included. Eighty-eight percent of the analyzed strains were positive for the K1-specific probe on the microarray and could be confirmed with a specific antiserum against the K1 capsular polysaccharide. The gene for the hemin receptor ChuA was predominantly found in 95% of strains. Other virulence genes associated with K1 and related strains were P, S, and F1C fimbriae specific for extraintestinal E. coli, the genes for aerobactin, the alpha-hemolysin and the cytotoxic necrotizing factor. In two strains, the O157-specific catalase gene and the gene for the low-molecular-weight heat-stable toxin AstA were detected, respectively. A total of 19 different virulence gene patterns were observed. No correlation was observed between specific virulence gene patterns and a clinical outcome. The data indicate that virulence genes typical of extraintestinal E. coli are predominantly present in K1 strains. Nevertheless, some of them can carry virulence genes known to be characteristic of intestinal E. coli. The distribution and combination of virulence genes show that K1 isolates constitute a heterogeneous group of E. coli.

Escherichia coli↗

PubMed2005

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria.

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

Anti-Bacterial Agents↗

PubMed2004

DNA microarray-based detection and identification of Chlamydia and Chlamydophila spp.

A microarray hybridization assay for identification of chlamydiae was developed using the ArrayTube platform. The technology is comparatively inexpensive and involves plastic tube-integrated microchips and signal amplification by enzyme-catalyzed silver precipitation. Hybridization probes were designed on the basis of the most variable window approach, which identified species-specific nucleotide polymorphisms in a region of generally high sequence similarity. The selected 26-nt probe sequences were used on two different series of customized microarrays, i.e. combinatorial high-density in situ synthesized arrays and low-density spotted arrays. Target DNA was prepared by consensus PCR amplifying a 1-kbp segment of the ribosomal RNA operon. Unique species-specific hybridization patterns were obtained for all nine species of the family Chlamydiaceae on both microarray types. The present assay proved suitable for unambiguous species identification of chlamydial cell cultures and showed a potential for direct detection of these bacteria from clinical tissue.

Animals↗