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L Rigottier-Gois

Publications and source records attributed to L Rigottier-Gois.

12 recordsLinked to original sources
PubMed2006

Comparative genomic hybridization analysis of Enterococcus faecalis: identification of genes absent from food strains.

Enterococcus faecalis, a member of the natural microbiota of animal and human intestinal tracts, is also present as a natural contaminant in a variety of fermented foods. Over the last decade, E. faecalis has emerged as a major cause of nosocomial infections. We investigated the genetic diversity in 30 clinical and food isolates, including strains V583 and MMH594, in order to determine whether clinical and food isolates could be distinguished. Data were obtained using comparative genomic hybridization and specific PCR with a total of 202 probes of E. faecalis, selected using the available V583 genome sequence and part of the MMH594 pathogenicity island. The cognate genes encoded mainly exported proteins. Hybridization data were analyzed by a two-component mixture model that estimates the probability of any given gene to be either present or absent in the strains. A total of 78 genes were found to be variable, as they were absent in at least one isolate. Most of the variable genes were clustered in regions that, in the published V583 sequence, related to prophages or mobile genetic elements. The variable genes were distributed in three main groups: (i) genes equally distributed between clinical and dairy food isolates, (ii) genes absent from dairy food-related isolates, and (iii) genes present in MMH594 and V583 strains only. Further analysis of the distribution of the last gene group in 70 other isolates confirmed that six of the probed genes were always absent in dairy food-related isolates, whereas they were detected in clinical and/or commensal isolates. Two of them corresponded to prophages that were not detected in the cognate isolates, thus possibly extending the number of genes absent from dairy food isolates. Genes specifically detected in clinical isolates may prove valuable for the development of new risk assessment markers for food safety studies and for identification of new factors that may contribute to host colonization or infection.

DNA, Bacterial↗
PubMed2005

Reduced diversity of faecal microbiota in Crohn's disease revealed by a metagenomic approach.

BACKGROUND AND AIM: A role for the intestinal microbial community (microbiota) in the onset and chronicity of Crohn's disease (CD) is strongly suspected. However, investigation of such a complex ecosystem is difficult, even with culture independent molecular approaches. METHODS: We used, for the first time, a comprehensive metagenomic approach to investigate the full range of intestinal microbial diversity. We used a fosmid vector to construct two libraries of genomic DNA isolated directly from faecal samples of six healthy donors and six patients with CD. Bacterial diversity was analysed by screening the two DNA libraries, each composed of 25,000 clones, for the 16S rRNA gene by DNA hybridisation. RESULTS: Among 1190 selected clones, we identified 125 non-redundant ribotypes mainly represented by the phyla Bacteroidetes and Firmicutes. Among the Firmicutes, 43 distinct ribotypes were identified in the healthy microbiota, compared with only 13 in CD (p<0.025). Fluorescent in situ hybridisation directly targeting 16S rRNA in faecal samples analysed individually (n=12) confirmed the significant reduction in the proportion of bacteria belonging to this phylum in CD patients (p<0.02). CONCLUSION: The metagenomic approach allowed us to detect a reduced complexity of the bacterial phylum Firmicutes as a signature of the faecal microbiota in patients with CD. It also indicated the presence of new bacterial species.

Adolescent↗
PubMed2003

Alterations of the dominant faecal bacterial groups in patients with Crohn's disease of the colon.

BACKGROUND AND AIMS: The colonic microflora is involved in the pathogenesis of Crohn's disease (CD) but less than 30% of the microflora can be cultured. We investigated potential differences in the faecal microflora between patients with colonic CD in remission (n=9), patients with active colonic CD (n=8), and healthy volunteers (n=16) using culture independent techniques. METHODS: Quantitative dot blot hybridisation with six radiolabelled 16S ribosomal ribonucleic acid (rRNA) targeting oligonucleotide probes was used to measure the proportions of rRNA corresponding to each phylogenetic group. Temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA was used to evaluate dominant species diversity. RESULTS: Enterobacteria were significantly increased in active and quiescent CD. Probe additivity was significantly lower in patients (65 (11)% and 69 (6)% in active CD and quiescent CD) than in healthy controls (99 (7)%). TTGE profiles varied markedly between active and quiescent CD but were stable in healthy conditions. CONCLUSION: The biodiversity of the microflora remains high in patients with CD. Enterobacteria were observed significantly more frequently in CD than in health, and more than 30% of the dominant flora belonged to yet undefined phylogenetic groups.

Adolescent↗
PubMed2001

Fusobacterium prausnitzii and related species represent a dominant group within the human fecal flora.

The human gut microflora plays a key role in nutrition and health. It has been extensively studied by conventional culture techniques. However these methods are difficult, time consuming and their results not always consistent. Furthermore microscopic counts indicate that only 20 to 40% of the total flora can be cultivated. Among the predominant species of the human gut, Fusobacterium prausnitzii was reported either as one of the most frequent and numerous species or was seldom retrieved. We designed and validated a specific rRNA-targeted oligonucleotide probe, called S-*-Fprau-0645-a-A-23, to accurately detect and quantify F. prausnitzii and relatives within the human fecal microflora. The target group accounted for 5.3 +/- 3% of total bacterial 16S rRNA using dot blot hybridization (10 human fecal samples) and 16.5 +/- 7% of cells stained with Dapi using in situ hybridization (10 other human fecal samples). A specific morphology seemed to be typical and dominant: two cells forming an asymmetrical double droplet. This work showed that F. prausnitzii and phylogenetically related species represent a dominant group within the human fecal flora.

Adult↗
PubMed2001

Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia.

The current classification of the rhizobia (root-nodule symbionts) assigns them to six genera. It is strongly influenced by the small subunit (16S, SSU) rRNA molecular phylogeny, but such single-gene phylogenies may not reflect the evolution of the genome as a whole. To test this, parts of the atpD and recA genes have been sequenced for 25 type strains within the alpha-Proteobacteria, representing species in Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Agrobacterium, Phyllobacterium, Mycoplana and Brevundimonas. The current genera Sinorhizobium and Mesorhizobium are well supported by these genes, each forming a distinct phylogenetic clade with unequivocal bootstrap support. There is good support for a Rhizobium clade that includes Agrobacterium tumefaciens, and the very close relationship between Agrobacterium rhizogenes and Rhizobium tropici is confirmed. There is evidence for recombination within the genera Mesorhizobium and Sinorhizobium, but the congruence of the phylogenies at higher levels indicates that the genera are genetically isolated. rRNA provides a reliable distinction between genera, but genetic relationships within a genus may be disturbed by recombination.

Alphaproteobacteria↗
PubMed2000

Simplified procedure for detection of enteric pathogenic viruses in shellfish by RT-PCR.

Epidemiological evidence linking the transmission of enteric viral disease to shellfish has been known for a long time. A variety of methods have been described for the detection of viral contaminants in shellfish using RT-PCR. However, these methods generally include numerous, often fastidious and time consuming steps for virus release from shellfish tissues and viral RNA isolation. A simplified procedure based on the enzymatic liquefaction of shellfish digestive tissues without any mechanical homogenisation step, followed by a simple clarification of the lysate using dichloromethane extraction, was developed. Viral RNA is isolated directly from the shellfish extract by a guanidium thiocyanate-silica extraction method, adapted for the use of a vacuum manifold system. Virus-specific RT-PCR assays were set up for detection of genomic sequences of the predominant viral pathogens, HAV, Astrovirus and Norwalk-like viruses (from genogoups I or II). The specificity of the amplicons is confirmed finally by hybridisation with DIG-labelled specific probes. The overall procedure applied to shellfish samples spiked with HAV particles allowed a detection of 20 pfu of HAV per g of hepatopancreas. In addition, up to 20 samples can be tested within 24 h.

Animals↗
PubMed1998

Organic halogen removal from chlorinated humic ground water and lake water by nitrifying fluidized-bed biomass characterised by electron microscopy and molecular methods.

The dechlorinating and genotoxicity-removing activities of nitrifying fluidized-bed reactor biomass towards chlorinated organic compounds in water were shown at level below 1 ppm. The removal rates of adsorbable organic halogens were 200 micrograms Cl (g VS day)-1 for chlorinated humic ground water and 50 micrograms Cl (g VS day)-1 for chlorinated lake water when studied in batch mode. In a sequenced batch mode the removal rates [micrograms Cl (g VS day)-1] were 2000 from chlorohumus, 1400-1800 from chlorophenols in chlorinated ground water, and 430-720 from chlorohumus in chlorinated lake water. Genotoxicity was removed to a large extent (60%-80%) from the chlorinated waters upon incubation with nitrifying reactor biomass. 2,6-Di-, 2,4,6-tri and 2,3,4,6-tetrachlorophenols competed with chlorinated water organohalogens for dechlorination. The dechlorination of chlorophenols and chlorohumus required no ammonia and was not prevented by inhibitors of ammonia oxidation, nitrapyrin, parathion, sodium diethyldithiocarbamate, or allylthiourea. Electron microscopical inspection of the biomass showed the dominance of clusters of bacteria resembling known nitrifying species, Nitrosomonas, Nitrobacter, and Nitrosospira. This was supported by polymerase chain reaction amplification of the biomass DNA with four different primers, revealing the presence of 16S rDNA sequences assignable to the same species. The most intensive band obtained with the Nitroso4E primer was shown to be closely related to Nitrosomonas europaea by restriction analysis.

Ammonia↗
PubMed1996

Diversity of repC plasmid-replication sequences in Rhizobium leguminosarum.

Homologues of the plasmid replicator gene repC were detected and characterized in a sample of Rhizobium leguminosarum strains. Conserved PCR primers were designed from published sequences of repC; they amplified a fragment of about 750 bp from 39 out of 41 strains tested, and also from several Sinorhizobium strains, including S. meliloti. Restriction endonuclease digestion showed that the PCR product from individual strains, though uniform in size, was often heterogeneous in sequence. PCR products from 24 field isolates of R. leguminosarum from France, Germany and the UK were cloned and partially sequenced from both ends. Phylogenies constructed from the 5' and 3' ends (200 bp each) were largely congruent and demonstrated four clearly defined groups plus several unique strains. Published Agrobacterium repC sequences fall within the phylogeny of R. leguminosarum sequences, though not within any of the four groups. Specific pairs of PCR primers were designed for each of the four groups; 29 out of 41 R. leguminosarum strains gave a PCR product of the expected size with more than one group-specific primer pair. We hypothesize that the sequence groups correspond to incompatibility groups of Rhizobium plasmids.

Amino Acid Sequence↗
PubMed1996

Characterization, distribution, and localization of ISRl2, and insertion sequence element isolated from Rhizobium leguminosarum bv. viciae.

An insertion sequence (IS) element, ISR12, from Rhizobium leguminosarum bv. viciae strain MSDJ4184 was isolated by insertional inactivation of the sacRB gene of pSUP104-sac, which allows positive selection. ISRl2 is 932 bp long, is flanked by 17-bp imperfect terminal inverted repeats, and generated a 3-bp target site duplication. ISRl2 was found to be 63 to 77% homologous to insertion elements of the IS5 group of the IS4 superfamily. A probe incorporating a full-length copy of ISRl2 was used to screen genomic DNAs from a collection of strains and from two field populations of R. leguminosarum to detect and estimate the copy numbers of homologous sequences. Among the collection of 63 strains representing the different species and genera of members of the family Rhizobiaceae, homology to ISRl2 was found within strains belonging to Sinorhizobium meliloti and S. fredii; within four of the six recognized Rhizobium species. R. leguminosarum, R. tropici, R. etli, and R. galegae; and within Rhizobium sp. (Phaseolus) genomic species 2. The apparent copy numbers of ISRl2 varied from one to eight. Among 139 isolates of R. leguminosarum from two field populations, homology to ISRl2 was detected in 91% of the isolates from one site and in 17% from the other. Analysis of the 95 isolates that hybridize to ISRl2 revealed a total of 20 distinct hybridization patterns composed of one to three bands. Probing blots of Eckhardt gels showed that sequences with homology to ISRl2 may be found on plasmids or the chromosome. Analysis of their genomic distribution demonstrated relationships and diversity among the R. leguminosarum isolates tested.

Base Sequence↗
PubMed1996

Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars.

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.

Bacterial Typing Techniques↗
PubMed1994

Fluorescent Pseudomonas species categorized by using polymerase chain reaction (PCR)/restriction fragment analysis of 16S rDNA.

A rapid procedure for the identification of fluorescent pseudomonads, based on the polymerase chain reaction (PCR) and restriction fragment analysis of 16S rDNA genes is described. Thirty-one strains belonging to 10 different Pseudomonas species of the Pseudomonas fluorescens rRNA branch were characterized. Amplified rDNA was digested with 13 different restriction endonucleases. The combined data from restriction analysis enabled the definition of 17 different 16S rDNA genotypes. All type strains belonging to different species were differentiated. The good correlation between grouping obtained using restriction analysis with other molecular classification criteria demonstrates the value of the described method to characterize rapidly fluorescent Pseudomonas strains at the species level.

Bacterial Typing Techniques↗