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M Simonet

Publications and source records attributed to M Simonet.

At least 19 recordsLinked to original sources

Coordinate involvement of invasin and Yop proteins in a Yersinia pseudotuberculosis-specific class I-restricted cytotoxic T cell-mediated response.

Yersinia pseudotuberculosis is a pathogenic enteric bacteria that evades host cellular immune response and resides extracellularly in vivo. Nevertheless, an important contribution of T cells to defense against Yersinia has been previously established. In this study we demonstrate that Lewis rats infected with virulent strains of Y. pseudotuberculosis, mount a Yersinia-specific, RT1-A-restricted, CD8+ T cell-mediated, cytotoxic response. Sensitization of lymphoblast target cells for cytolysis by Yersinia-specific CTLs required their incubation with live Yersinia and was independent of endocytosis. Although fully virulent Yersinia did not invade those cells, they attached to their surface. In contrast, invasin-deficient strain failed to bind to blast targets or to sensitize them for cytolysis. Furthermore, an intact virulence plasmid was an absolute requirement for Yersinia to sensitize blast targets for cytolysis. Using a series of Y. pseudotuberculosis mutants selectively deficient in virulence plasmid-encoded proteins, we found no evidence for a specific role played by YadA, YopH, YpkA, or YopJ in the sensitization process of blast targets. In contrast, mutations suppressing YopB, YopD, or YopE expression abolished the capacity of Yersinia to sensitize blast targets. These results are consistent with a model in which extracellular Yersinia bound to lymphoblast targets via invasin translocate inside eukaryotic cytosol YopE, which is presented in a class I-restricted fashion to CD8+ cytotoxic T cells. This system could represent a more general mechanism by which bacteria harboring a host cell contact-dependent or type III secretion apparatus trigger a class I-restricted CD8+ T cell response.

Adhesins, Bacterial

Molecular characterization of IS1541 insertions in the genome of Yersinia pestis.

The genome of Yersinia pestis, the causative agent of plague, contains at least 30 copies of an element, designated IS1541, which is structurally related to IS200 (85% identity). One such element is inserted within the chromosomal inv gene (M. Simonet, B. Riot, N. Fortineau, and P. Berche, Infect. Immun. 64:375-379, 1996). We characterized other IS1541 insertions by cloning 14 different Y. pestis 6/69M loci carrying a single copy of this insertion sequence (IS) into Escherichia coli and, for each element, sequencing 250 bp of both flanking regions. In no case was this IS element inserted into large open reading frames; however, in eight cases, it was detected downstream (17 to 139 bp) of genes thought to be transcribed monocistronically or which constituted the last gene of an operon, and in only one case was it detected upstream (37 bp) of the first gene of an operon. Sequence analysis revealed stem-loop structures (deltaG, < -10 kcal) resembling rho-independent transcription terminators in 8 of the 14 insertion sites. These motifs might constitute hot spots for insertion of this IS1541 element within the Y. pestis genome.

Amino Acid Sequence

Nosocomial outbreak of Klebsiella pneumoniae producing SHV-5 extended-spectrum beta-lactamase, originating from a contaminated ultrasonography coupling gel.

Klebsiella pneumoniae resistant to ceftazidime was isolated from six adult women and two neonates hospitalized between July and November 1993 in the Department of Obstetrics and Gynecology of Boucicaut Hospital (Paris, France). The epidemiological investigation revealed a notably short delay (less than 48 h) between admission and contamination of the six adults and peripartum transmission to the neonates. The only environmental source of ceftazidime-resistant K. pneumoniae was the ultrasonography coupling gel used in the emergency room. Phenotypic (biotyping and antibiotyping) and genotypic (plasmid profile and pulsed-field gel electrophoresis) analysis of all the clinical isolates indicated the spread of a single strain. It produced SHV-5 and TEM-1 beta-lactamases, as demonstrated by isoelectric focusing and gene sequencing. The risk of cross-contamination in ultrasonography procedures is usually low and had not been associated so far with bacteria producing an extended-spectrum beta-lactamase (ESBL). Furthermore, this is the first time an epidemic of an SHV-5 ESBL-producing member of the family Enterobacteriaceae has been reported from a French hospital.

Adult

Novel transferable beta-lactam resistance with cephalosporinase characteristics in Salmonella enteritidis.

An isolate of Salmonella enteritidis was found to produce a plasmid-encoded beta-lactamase (DHA-1) that conferred resistance to extended-spectrum cephalosporins and cephamycins. The substrate and inhibition profiles of this enzyme resemble a class C beta-lactamase. This is the first report of a plasmid-mediated cephalosporinase of this class in the Salmonella genus.

Anti-Bacterial Agents

Urease is not involved in the virulence of Yersinia pseudotuberculosis in mice.

A chromosomal locus (ure) involved in the production of urease activity in the bacterial pathogen Yersinia pseudotuberculosis was characterized. The genetic organization of the Y. pseudotuberculosis ure locus closely resembles that of the related ureolytic Yersinia species Y. enterocolitica. This locus encompasses seven open reading frames encoding polypeptides with predicted molecular weights of 10,894 (UreA), 15,820 (UreB), 61,001 (UreC), 25,801 (UreE), 24,551 (UreF), 20,330 (UreG), and 31,308 (UreD). The polypeptides have 85 to 96% identity with the corresponding Ure polypeptides of Y. enterocolitica serotype 0:8. Restriction fragment length polymorphisms of the ure loci from 12 unrelated Y. pseudotuberculosis strains produced by HaeIII and MboI indicate a low level of genetic variability of this locus in this species. The role of urease in the pathogenicity of Y. pseudotuberculosis was studied by constructing an isogenic urease-negative mutant obtained by disruption of structural gene ureB by aphA-3', which encodes kanamycin resistance. Experimental infection of mice with this mutant demonstrates that urease is not essential for Y. pseudotuberculosis virulence. Urease might be required mostly during the saprophytic life of this pathogen.

Amino Acid Sequence

Invasin production by Yersinia pestis is abolished by insertion of an IS200-like element within the inv gene.

The two enteropathogens Yersinia pseudotuberculosis and Yersinia enterocolitica penetrate eukaryotic cells in vitro through invasin, a surface-exposed protein. In contrast, Yersinia pestis, the causative agent of plague, is unable to enter mammalian cell lines, although the inv gene is present on its chromosome. Although 99.3% identical to the inv gene of Y. pseudotuberculosis, the Y. pestis inv gene was disrupted in its central region by a 708-bp IS200-like element. Multiple copies of this insertion sequence element were found within the genome of the plague bacillus.

Adhesins, Bacterial

Evaluation of tuberculosis transmission in a community by 1 year of systematic typing of Mycobacterium tuberculosis clinical isolates.

Interhuman transmission of Mycobacterium tuberculosis was investigated by using molecular typing, including restriction fragment length polymorphism with probes IS6110, DR (direct repeat) and PGRS (polymorphic GC-rich sequence) and a PCR method using the inverted repeat sequences of IS6110 as primers. From 105 patients hospitalized for tuberculosis during a 1-year survey in three hospitals in Paris, France, 111 isolates were collected and analyzed. Eighty-eight patients were infected with genetically different isolates, demonstrating the clonal heterogeneity of M. tuberculosis in these patients originating from various geographical areas. Fourteen patients were infected by strains clustered with identical fingerprints. An epidemiological relatedness was demonstrated for isolates from only seven of these patients. Thus, the typing of isolates from all tuberculous patients in hospitals during 1 year allows the detection of transmission in the general community. This would improve the case findings, thereby further improving the detection of outbreaks.

Adult

Molecular typing of Yersinia pseudotuberculosis by using an IS200-like element.

The IS200-like insertion sequence (IS) is a 708-bp element recently found in Yersinia pestis. Its nucleotide sequence is 85% identical to that of IS200 recovered in most Salmonella enterica isolates. It is also present in multiple copies in Y. pseudotuberculosis. In contrast, this IS is found in some (biotype 1B strains) but not other Y. enterocolitica strains and is absent in the nonpathogenic yersiniae: Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. bercovieri, and Y. mollaretii. The number and locations of the ISs in the Y. pseudotuberculosis genome vary among strains, resulting in a high degree of polymorphism, but IS fingerprints are stable after multiple subcultures of clinical isolates. The discriminative power of IS typing is better than that of ribotyping and almost as good as that of the time-consuming method of pulsotyping. Overall, IS200-like is a useful molecular marker in determining the epidemiology of Y. pseudotuberculosis infections.

Bacterial Typing Techniques

Lack of antibody response to invasin in humans with yersiniosis.

The Yersinia pseudotuberculosis inv gene encodes invasin, a 103-kDa outer membrane protein allowing bacteria to penetrate mammalian cells. This protein is produced in vitro at below 30 degrees C. In this work, we studied the antibody response against invasin in humans suffering from yersiniosis and in mice orally infected with a virulent strain of Y. pseudotuberculosis. Infection with enteropathogenic Yersinia strains did not induce either a systemic or a gut antibody response to invasin. Our results suggest that the inv gene is not expressed in the gut at 37 degrees C and, therefore, that invasin is not present to the immune system when microorganisms multiply in the host tissues.

Adhesins, Bacterial

Immunization with live aroA recombinant Salmonella typhimurium producing invasin inhibits intestinal translocation of Yersinia pseudotuberculosis.

The Yersinia pseudotuberculosis inv gene encodes invasin, a 103-kDa outer membrane protein that allows bacteria to enter mammalian cells. The gene was subcloned into the attenuated aroA mutant of Salmonella typhimurium SL3261. Invasin was produced by the recombinant Salmonella strain and increased the ability of microorganisms to translocate from the intestinal lumen to the mesenteric lymph nodes. Specific antibodies for invasin were detected in sera and intestinal secretions of mice following oral immunization with the live Inv+ Salmonella strain. The immunization strongly inhibited intestinal translocation of Y. pseudotuberculosis when this pathogen was inoculated to mice but failed to prevent Yersinia dissemination from the gut lymphoid tissue.

3-Phosphoshikimate 1-Carboxyvinyltransferase

Investigation of hospital-acquired infections due to Alcaligenes denitrificans subsp. xylosoxydans by DNA restriction fragment length polymorphism.

We demonstrate that DNA restriction fragment length polymorphism determined by pulsed-field gel electrophoresis is very useful in the investigation of the epidemiology of hospital-acquired infections caused by Alcaligenes denitrificans subsp. xylosoxydans. This approach showed that hospital-acquired infections caused by this opportunistic pathogen over a 6-month period in 10 patients hospitalized in an intensive care unit and a surgical unit were not a true outbreak. In addition, this molecular typing method established that the respiratory therapy equipment was the source of the contamination of two patients.

Adult

[Scopulariopsis brevicaulis otomycosis after tympanoplasty].

We report a case of otomycosis due to Scopulariopsis brevicaulis in a patient who has undergone tympanoplasty 3 months before. Mycological finding led us to consider this fungus as the causative agent. Whereas in vitro studies show a better efficiency of azole derivatives, cure was obtained with nystatine after two treatment failures in our patient.

Administration, Topical

[Comparative activity of oral beta-lactam antibiotics against fifty strains of Haemophilus influenzae producing a beta-lactamase according to bacterial inoculum].

Minimal inhibitory concentrations (MICs) of amoxycillin alone and in combination with 2 mg/l or 4 mg/l clavulanic acid, ampicillin alone and in combination with 4 mg/l or 8 mg/l sulbactam, cefuroxime and cefaclor, were determined by the agar dilution method, with bacterial inoculum size ranging from 3 x 10(7) to 3 x 10(9) colony-forming units per ml. As expected, an inoculum effect was observed with cefaclor. In contrast, MICs of amoxycillin in combination with clavulanic acid did not significantly increase with the inoculum size. Ampicillin combined with sulbactam, and cefuroxime were less efficient at the highest bacterial concentration tested.

Anti-Bacterial Agents