Sabine Chapelle

Compilation of small ribosomal subunit RNA structures

The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk.

Development of a Multiplex PCR for the Detection of asa1, gelE, cylA, esp, and hyl Genes in Enterococci and Survey for Virulence Determinants among European Hospital Isolates of Enterococcus faecium

ABSTRACT A multiplex PCR for the simultaneous detection of five virulence genes (asa1, gelE, cylA, esp, and hyl) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher (P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher (P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent (P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific (esp-positive) VREF clones in Italy and one center-specific (hyl-positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1, gelE, cylA, esp, and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp-positive VREF clones (in Italy) and hyl-positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

Structure of the large ribosomal subunit RNA of Phytophthora megasperma, and phylogeny of the oomycetes

The 5.8S and 28S rRNA sequences of the oomycete Phytophthora megasperma were determined in order to study the secondary structure of these molecules and to assess the phylogenetic position of the oomycetes among the eukaryotes. Preliminary results point to an affiliation between the oomycetes, dinoflagellates and ciliates, a cluster which seems related to the fungi. In the course of this work, we developed a set of primers which allow sequencing and PCR amplification of eukaryotic large ribosomal subunit RNA genes of a wide range of phylogenetically distant organisms.

Evolutionary Relationships Among Higher Fungi Inferred from Small Ribosomal Subunit RNA Sequence Analysis

The primary structure of the small ribosomal subunit RNA. (SSU rRNA) was determined for 13 species belonging to 10 ascomycete families and for the basidiomycetous anamorphic yeast Rhodotorula glutinis. The sequences were fitted into an alignment of all hitherto published complete or nearly complete eukaryotic small subunit rRNA sequences. The evolutionary relationships within the fungi were examined by construction of a tree from 87 SSU rRNA sequences, corresponding to 71 different species, by means of a distance matrix method and bootstrap analysis. It confirms the early divergence of the zygomycetes and the classical division of the higher fungi into basidiomycetes and ascomycetes. The basidiomycetes are divided into true basidiomycetes and ustomycetes. Within the ascomycetes, the major subdivisions hemiascomycetes and euascomycetes can be recognized. However, Schizosaccharomyces pombe does not belong to the cluster of the hemiascomycetes, to which it is assigned in classical taxonomic schemes, but forms a distinct lineage. Among the euascomycetes, the plectomycetes and the pyrenomycetes can be distinguished. Within the hemiascomycetes, the polyphyly of genera like Pichia or Candida and of families like the Dipodascaceae and the Saccharomycetaceae can be observed.

A variable 23S rDNA region is a useful discriminating target for genus-specific and species-specific PCR amplification in Arcobacter species.

Summary A genus-specific PCR assay was created for the detection of all Arcobacter strains and three speciesspecific PCR assays were developed for the discrimination among A. cryaerophilus, A. butzleri and A. skirrowii . The PCR assays were based on a target sequence which comprises the most variable areas of 23S rDNA. The specificity of the developed PCR assays was extensively tested on an assortment of 54 Arcobacter and 71 phylogenetically related reference strains. All primer sets were further evaluated on a large number of field strains isolated from different sources in various geographical locations.

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence forCampylobacter jejuni subsp.jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.

Genotypic Diversity, Antimicrobial Resistance, and Virulence Factors of Human Isolates and Probiotic Cultures Constituting Two Intraspecific Groups of Enterococcus faecium Isolates

ABSTRACT The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.

Macrolide- and telithromycin-resistant Streptococcus pyogenes, Belgium, 1999-2003.

We found a 13% macrolide resistance in 3,866 Streptococcus pyogenes isolated from tonsillopharyngitis patients; 59% macrolide-resistant isolates were distributed in 5 clones, suggesting the importance of both resistance gene transfer and clonal dissemination in the spread of these organisms. We also report one of the largest collections of telithromycin-resistant isolates.

Specific detection of Campylobacter concisus by PCR amplification of 23S rDNA areas.

The phenotypic detection of Campylobacter concisus, a species of considerable genomic and phenotypic heterogeneity, has proven to be rather tedious in the past. Although alternative methods like DNA:DNA hybridization, immunotyping or whole-cell protein electrophoresis are valuable for the specific detection of C. concisus, they are too laborious to be performed in routine settings. Hence a simple Campylobacter concisus-specific PCR assay was developed, based on a target sequence which comprises the most variable areas of 23S rDNA. The PCR assay was successfully evaluated on a broad selection of C. concisus strains and phylogenetically related bacteria.

Species-specific detection of campylobacters important in veterinary medicine by PCR amplification of 23S rDNA areas

Summary A species-specific PCR assay was developed for the detection of campylobacters important in veterinary medicine, including Campylobacter fetus, Campylobacter hyointestinalis and Campylobacter mucosalis . Specific primers were also developed for the non-pathogenic Campylobacter sputorum . The PCR assays were based on a target sequence which comprises the most variable areas of 23S rDNA. The developed PCR assays were evaluated on a broad selection of Campylobacter strains and phylogenetically related bacteria. Each of the four primer pairs allowed the selective detection of all the strains belonging to the species Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter mucosalis and Campylobacter sputorum .