Glutamate optimizes enzymatic activity under high hydrostatic pressure in Desulfovibrio species: effects on the ubiquitous thioredoxin system.

In piezophilic microorganisms, enzymes are optimized to perform under high hydrostatic pressure. The two major reported mechanisms responsible for such adaptation in bacterial species are changes in amino acids in the protein structure, favoring their activity and stability under high-pressure conditions, and the possible accumulation of micromolecular co-solutes in the cytoplasm. Recently, the accumulation of glutamate in the cytoplasm of piezophilic Desulfovibrio species has been reported under high-pressure growth conditions. In this study, analysis of the effect of glutamate on the enzymatic activity of the thioredoxin reductase/thioredoxin enzymatic complex of either a piezosensitive or a piezophilic microorganism confirms its role as a protective co-solute. Analysis of the thioredoxin structures suggests an adaptation both to the presence of glutamate and to high hydrostatic pressure in the enzyme from the piezophilic strain. Indeed, the presence of large surface pockets could counterbalance the overall compression that occurs at high hydrostatic pressure to maintain enzymatic activity. A lower isoelectric point and a greater dipolar moment than that of thioredoxin from the piezosensitive strain would allow the protein from the piezophilic strain to compensate for the presence of the charged amino acid glutamate to interact with its partner.

Oblitoma en la cirugía ginecoobstétrica: un problema creciente. Reporte de un caso / Oblitoma in obstetric and gynecologic surgery: a growing problem

Resumen ANTECEDENTES: Los oblitomas, u objetos extraños retenidos en el abdomen posterior a una cirugía, son consecuencia de una iatrogenia que causa morbilidad, dificultad diagnóstica, problemas médico-legales, y complicaciones para las pacientes, el médico y la institución hospitalaria. CASO CLINICO: Paciente de 27 años de edad, con un cuerpo extraño retenido en la cavidad abdominal (bulbo de la cánula de Yankauer), olvidado durante una cesárea de urgencia. El diagnóstico y tratamiento fueron expeditos, con reintervención quirúrgica para extraer el cuerpo extraño, sin complicaciones y con evolución satisfactoria de la paciente. CONCLUSIONES: El estudio actual de los oblitomas u objetos extraños retenidos es un problema creciente, con estadísticas en contra, sobre todo asociadas con elevada frecuencia de cesáreas y alta prevalencia de obesidad materna durante el embarazo, esto debe alertar a los ginecoobstetras a conducirse con más cuidado para evitar este tipo de accidentes.

Abstract BACKGROUND: Oblitomas or retained surgical items (RSI) in the abdominal cavity after surgery are cause of iatrogenic medical problems, that origin high morbidity, difficult diagnosis and medical malpractice claims to may lead complications to patients, physicians included to hospital. CLINICAL CASE: We report an exceptional case, in a 27-year-old women, with a bulb of Yankahuer cannula retained in abdominal cavity, Forgotten during an emergency cesarean section. The diagnosis and management was realized with opportunity, avoiding complications. CONCLUSION: Actually, the study of oblitoma or foreign objects retained that's considered a growing problem, with statistics against it, mainly associated with high frequency of cesarean sections and high prevalence of maternal obesity during pregnancy. In fact, that situation place the gynecologists and obstetricians at a latent risk for this event. It is important to know the predisposing factors for its prevention and to implement institutional programs to reduce complications.

Sec- and Tat-dependent translocation of beta-lactamases across the Escherichia coli inner membrane.

beta-Lactamases represent the major resistance mechanism of gram-negative bacteria against beta-lactam antibiotics. The amino acid sequences of these proteins vary widely, but all are located in the periplasm of bacteria. In this study, we investigated the translocation mechanism of representative beta-lactamases in an Escherichia coli model. N-terminal signal sequence analyses, antibiotic activity assay, and direct measurement of translocation of a green fluorescent protein (GFP) reporter fused to beta-lactamases revealed that most were exported via the Sec pathway. However, the Stenotrophomonas maltophilia L2 beta-lactamase was exported via the E. coli Tat translocase, while the S. maltophilia L1 beta-lactamase was Sec dependent. These results show the possible Tat-dependent translocation of beta-lactamases in the E. coli model system. In addition, the mutation of the cytoskeleton-encoding gene mreB, which may be involved in the spatial organization of penicillin-binding proteins, decreased the MIC of beta-lactams for beta-lactamase-producing E. coli. These findings provide new knowledge about beta-lactamase translocation, a putative new target for addressing beta-lactamase-mediated resistance.

Stx2 subtyping of Shiga toxin-producing Escherichia coli isolated from cattle in France: detection of a new Stx2 subtype and correlation with additional virulence factors.

At least 11 Stx2 variants produced by Shiga toxin-producing Escherichia coli (STEC) isolated from patients and animals have been described. The Stx2 subtyping of STEC isolated from healthy cows positive for stx(2) (n = 104) or stx(2) and stx(1) (n = 63) was investigated. Stx2vh-b, Stx2 (renamed Stx2-EDL933), and Stx2vh-a were the subtypes mostly detected among the bovine isolates (39.5, 39, and 25.5%, respectively). Stx2e was not present, and subtypes included in the Stx2d group (Stx2d-OX3a, Stx2d-O111, and Stx2d-Ount) were found infrequently among the isolates examined (8.5%). A combination of two distinct Stx2 subtypes was observed among 23.5% of the strains. For the first time, a combination of three subtypes (Stx2-EDL933/Stx2vh-b/Stx2d and Stx2vh-a/Stx2vh-b/Stx2d) was detected (3.5% of the isolates). In addition, bovine STEC harboring stx(1) and one or two stx(2) genes appeared highly cytotoxic toward Vero cells. A new Stx2 subtype (Stx2-NV206), present among 14.5% of the isolates, showed high cytotoxicity for Vero cells. Two amino acid residues (Ser-291 and Glu-297) important for the activation of Stx2 by human intestinal mucus were conserved on the Stx2-NV206 A subunit. The gene encoding Ehx enterohemolysin was prominent among STEC harboring stx(2)-EDL933 alone (78%) or a combination of stx(2)-EDL933 and stx(2)vh-b (85%). In addition, Stx2-EDL933 and/or Stx2vh-b subtypes were highly associated with other putative virulence factors such as Stx1 and EspP extracellular serine protease, but not with EAST1 enterotoxin.

Heterogeneity of Shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients, cattle, and food samples in central France.

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx(2)-restriction fragment length polymorphism [RFLP], stx(2) gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx(2)-RFLP and stx(2) variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx(2)-RFLP, stx(2) variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.

[Verotoxin-producing Escherichia coli infections: study of its prevalence in children in the Auvergne region]. / Les infections à Escherichia coli producteurs de vérotoxines: étude de la prévalence chez l'enfant dans la région Auvergne.

Verotoxin producing Escherichia coli (VTEC) have been associated with disease outbreaks of diarrhea hemorrhagic colitis and hemolytic-uremic syndrome in humans. Contamination occurs mainly by ingestion of beef and dairy products, but water and person to person transmission have also been described. Most of the clinical signs are due to the production of Stx1 and/or Stx2 Shiga toxins, also called verotoxins. Other virulence factors include enterohemolysin, and the product of the eae gene, intimin, involved in the attaching and effacing adherence phenotype. The predominant serotype is O157:H7, but VTEC strains of more than one hundred serotypes can cause human disease. In order to determine the prevalence of VTEC infections among children in the central part of France, stool samples from hospitalized children were examined for stx1 and stx2 genes by using a polymerase chain reaction (PCR) technique. From October 1997 to September 1998, 658 stool samples were analysed: among them 19 (3%) were stx-PCR positive. Only 8 children out of 19 had diarrhea, and for 5 of them, an enteric pathogen other than VTEC was isolated. VTEC strains were isolated from 10 samples: most of the isolates did not produce verotoxins at a high level, and they did not belong to serotypes associated with pathogenicity, which might explain the absence of relationship between VTEC isolation and pathogenicity in our study.

Prevalence and characterization of Shiga toxin-producing Escherichia coli isolated from cattle, food, and children during a one-year prospective study in France.

During a 1-year survey of Shiga toxin-producing Escherichia coli (STEC) prevalence in central France, 2,143 samples were investigated by PCR for Shiga toxin-encoding genes. A total of 330 (70%) of 471 fecal samples collected from healthy cattle at the Clermont-Ferrand slaughterhouse, 47 (11%) of 411 beef samples, 60 (10%) of 603 cheese samples, and 19 (3%) of 658 stool specimens from hospitalized children with and without diarrhea were positive for the stx gene(s). A STEC strain was isolated from 34% (162 of 471) of bovine feces, 4% (16 of 411) of beef samples, 1% (5 of 603) of cheese samples, and 1.5% (10 of 658) of stool specimens. Of the 220 STEC strains isolated, 34 (15%) harbored the stx(1) gene, 116 (53%) harbored the stx(2) gene, and 70 (32%) carried both the stx(1) and stx(2) genes. However, 32 (14.5%) were not cytotoxic for Vero cells. The eae gene, found in 12 (5%) of the 220 strains, was significantly associated with the stx(1) gene and with isolates from children. Sequences homologous to ehxA were found in 102 (46%) of the 220 strains. Thirteen serotypes, OX3:H2, O113:H21, O113:H4, OX3:H21, O6:H10, OX178:H19, O171:H2, O46:H38, O172:H21, O22:H16, O91:H10, O91:H21, and O22:H8, accounted for 102 (55%) of 186 typeable isolates, and only one strain (0.5% of the 186 STEC isolates from cattle), belonged to the O157:H7 serotype. We showed that the majority of the STEC isolates from cattle, beef, and cheese were not likely to be pathogenic for humans and that the STEC strains isolated from children in this study were probably not responsible for diarrheal disease. Finally, the strains associated with hemolytic-uremic syndrome in the same geographical area were shown to belong to particular subsets of the STEC population found in the bovine reservoir.

Rapid identification of hyperthermophilic methanococci isolated from deep-sea hydrothermal vents.

16S rDNAs amplified by PCR from 22 hyperthermophilic methanococci isolated from deep-sea hydrothermal vents were compared with those of the six type strains of the genus Methanococcus by RFLP analysis. Restriction fragments obtained with Haelli enabied four of the type species to be distinguished. Restrictions with HhaI, BstUI and MspI were necessary to differentiate Methanococcus jannaschii and Methanococcus fervens. The results indicate that the 16S rDNA PCR-RFLP method provides a rapid and reliable tool for the identification of newly isolated hyperthermophilic Methanococcus spp.

Evidence for an uncommon alpha-actinin protein in Trichomonas vaginalis.

As part of our ongoing project of identification of actin-binding proteins implicated in the cell transition (flagellate to amoeboid/adherent) of Trichomonas vaginalis, we have characterized an alpha-actinin-related protein in this parasite. The protein (P100) has a molecular mass of 100 kDa and an isoelectric point of 5.5. A monoclonal antibody raised against this protein co-localizes with the actin network. P100 gene transcripts are co-expressed with actin throughout the cell cycle. Analysis of the deduced protein sequence reveals three domains: an N-terminal actin-binding region; a central region rich in alpha-helix; and a C-terminal domain with Ca(2+)-binding capacity. Whereas the N- and C-terminal regions are well-conserved as compared to other alpha-actinins, we observe in the central region an atypical distribution of residues in five repeats. The sequence of the repeats does not show any homology with the rod domain of the other alpha-actinins, except for the first repeat which shows some similarity. The four other repeats of T. vaginalis P100 appear to result from a duplication event which is not detectable in the other sequences.