Prospective evaluation of a real-time PCR assay for detection of group B streptococci in vaginal swabs from pregnant women.
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Bacteriological Techniques↗
Biomedical subjects
C Poyart
Publications and source records attributed to C Poyart.
Listeria monocytogenes skin infection with cerebritis and haemophagocytosis syndrome in a bone marrow transplant recipient.
In this report, we describe unusual and unreported manifestations of Listeria monocytogenes infection in a bone marrow transplant recipient, including cutaneous infection with an hamophagocytosis syndrome and cerebritis. L. monocytogenes occurred despite a broad spectrum antibiotherapy. L. monocytogenes was isolated from a skin biopsy. Outcome was favorable with amoxicillin and gentamicin therapy. L. monocytogenes infection should be suspected in patients with cerebritis despite large spectrum antibiotherapy and this report underscores the usefulness of skin biopsies in febrile immunocompromised patients.
Adult↗
[Rapid antigen detection tests for diagnosis of group A streptococcal pharyngitis: comparative evaluation of sensitivity and practicability of 16 in vitro diagnostics medical devices performed in July 2002 by the French health products safety agency (Afssaps) as part of its market control mission].
Within the scope of its health products control mission, the French Health Products Safety Agency (Afssaps) collaborating with two expert's sites, has assessed the 16 tests available on the French market in 2002 for rapid diagnosis of the Streptococcus A tonsillitis. The purpose of this study was to verify the reliability and rapidity of these tests and to give some information to the users about their analytical criteria and practicability characteristics. The analytical study has been performed on a same panel of four reference strains of Streptococcus pyogenes dilutions to determine the limit of detection of all the reagents in the same condition of methodology. The limit of detection has been calculated with the results expressed in colony forming unit by ml (CFU/ml). The practicability study has permitted to analyze the quality of the presentation, the easiness of the final reading and of performing tests. A score has been established for each rapid test. A classification of the analytical sensitivity (limit of detection) and practicability (score) of these 16 devices has been established. The limit of detection of the reagents giving the best results allows the detection of the lowest bacterial concentration of the panel which is 10(5) CFU/ml. Regarding practicability, the results suggest that, the immunochromatographic strip methods have the best score in a view with the use by a non medical laboratory.
Antigens, Bacterial↗
Heme photolysis occurs by ultrafast excited state metal-to-ring charge transfer.
Ultrafast time-resolved resonance Raman spectra of carbonmonoxy hemoglobin (Hb), nitroxy Hb, and deoxy Hb are compared to determine excited state decay mechanisms for both ligated and unligated hemes. Transient absorption and Raman data provide evidence for a sequential photophysical relaxation pathway common to both ligated and unligated forms of Hb* (photolyzed heme), in which the excited state 1Q decays sequentially: 1Q-->Hb*I-->Hb*II-->Hb ground state. Consistent with the observed kinetics, the lifetimes of these states are <50 fs, approximately 300 fs, and approximately 3 ps for 1Q, Hb*I, and Hb*II, respectively. The transient absorption data support the hypothesis that the Hb*I state results from an ultrafast iron-to-porphyrin ring charge transfer process. The Hb*II state arises from porphyrin ring-to-iron back charge transfer to produce a porphyrin ground state configuration a nonequilibrium iron d-orbital population. Equatorial d-pi* back-bonding of the heme iron to the porphyrin during the lifetime of the Hb*II state accounts for the time-resolved resonance Raman shifts on the approximately 3 ps time scale. The proposed photophysical pathway suggests that iron-to-ring charge transfer is the key event in the mechanism of photolysis of diatomic ligands following a porphyrin ring pi-pi* transition.
Heme↗
Contribution of Mn-cofactored superoxide dismutase (SodA) to the virulence of Streptococcus agalactiae.
Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide, which, in turn, is metabolized by catalases and/or peroxidases. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and hence play a role in the pathogenesis of certain bacteria. We previously demonstrated that group B streptococci (GBS) possess a single Mn-cofactored superoxide dismutase (SodA). To analyze the role of this enzyme in the pathogenicity of GBS, we constructed a sodA-disrupted mutant of Streptococcus agalactiae NEM316 by allelic exchange. This mutant was subsequently cis complemented by integration into the chromosome of pAT113/Sp harboring the wild-type sodA gene. The SOD specific activity detected by gel analysis in cell extracts confirmed that active SODs were present in the parental and complemented strains but absent in the sodA mutant. The growth rates of these strains in standing cultures were comparable, but the sodA mutant was extremely susceptible to the oxidative stress generated by addition of paraquat or hydrogen peroxide to the culture medium and exhibited a higher mutation frequency in the presence of rifampin. In mouse bone marrow-derived macrophages, the sodA mutant showed an increased susceptibility to bacterial killing by macrophages. In a mouse infection model, after intravenous injection the survival of the sodA mutant in the blood and the brain was markedly reduced in comparison to that of the parental and complemented strains whereas only minor effects on survival in the liver and the spleen were observed. These results suggest that SodA plays a role in GBS pathogenesis.
Animals↗
Regulation of D-alanyl-lipoteichoic acid biosynthesis in Streptococcus agalactiae involves a novel two-component regulatory system.
The dlt operon of gram-positive bacteria comprises four genes (dltA, dltB, dltC, and dltD) that catalyze the incorporation of D-alanine residues into the lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Streptococcus agalactiae, which, in addition to the dltA to dltD genes, included two regulatory genes, designated dltR and dltS, located upstream of dltA. The dltR gene encodes a 224-amino-acid putative response regulator belonging to the OmpR family of regulatory proteins. The dltS gene codes for a 395-amino-acid putative histidine kinase thought to be involved in the sensing of environmental signals. The dlt operon of S. agalactiae is mainly transcribed from the P(dltR) promoter, which directs synthesis of a 6.5-kb transcript encompassing dltR, dltS, dltA, dltB, dltC, and dltD, and from a weaker promoter, P(dltA), which is located in the 3' extremity of dltS. We demonstrate that P(dltR), but not P(dlA), is activated by DltR in the presence of DltS in D-Ala-deficient LTA mutants resulting from insertional inactivation of the dltA gene, which encodes the cytoplasmic D-alanine-D-alanyl carrier ligase DltA. Expression of the dlt operon does not require DltR and DltS, since the basal activity of P(dltR) is high, being 20-fold that of the constitutive promoter P(aphA-3) which directs synthesis of the kanamycin resistance gene aphA-3 in various gram-positive bacteria. We hypothesize that the role of DltR and DltS in the control of expression of the dlt operon is to maintain the level of D-Ala esters in LTAs at a constant and appropriate value whatever the environmental conditions. The DltA(-) mutant displayed the ability to form clumps in standing culture and exhibited an increased susceptibility to the cationic antimicrobial polypeptide colistin.
Bacterial Proteins↗
Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target.
Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60 gene sequences. Sequence analysis revealed that the staphylococcal sodA genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the sodA gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.
Bacterial Proteins↗
Meningitis due to Streptococcus salivarius.
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Aged↗
Modulation of the oxygen affinity of cobalt-porphyrin by globin.
We have combined two extreme effects which influence the oxygen affinity to obtain a cobalt-based oxygen carrier with an affinity similar to that of human adult hemoglobin (HbA). The goal was to obtain an oxygen transporter with a lower oxidation rate. Exchange of the heme group (Fe-protoporphyrin IX) in Hb with a cobalt-porphyrin leads to a reduction in oxygen affinity by over a factor of 10, an oxygen affinity too low for use as a blood substitute. At the other extreme, certain globin sequences are known to provide a very high oxygen affinity; for example, Hb Ascaris displays an oxygen affinity 1000 times higher than HbA. We demonstrate here that these opposing effects can be additive, yielding an oxygen affinity similar to that of HbA, but with oxygen binding to a cobalt atom. We have tested the effect of substitution of cobalt-porphyrin for heme in normal HbA, sperm whale (SW) Mb (Mb), and high affinity globins for leghemoglobin, two trematode Hbs: Paramphistomum epiclitum (Pe) and Gastrothylax crumenifer (Gc). As for HbA or SW Mb, the transition from heme to cobalt-porphyrin in the trematode Hbs leads to a large decrease in the oxygen affinity, with oxygen partial pressures for half saturation (P(50)) of 5 and 25 mm Hg at 37 degrees C for cobalt-Pe and cobalt-Gc, respectively. A critical parameter for Hb-based blood substitutes is the autoxidation rate; while both metals oxidize to an inactive state, we observed a decrease in the oxidation rate of over an order of magnitude for cobalt versus iron, for similar oxygen affinities. The time constants for autoxidation at 37 degrees C were 250 and 100 h for Pe and Gc, respectively.
Animals↗
Whipple's disease: acquired resistance to trimethoprim-sulfamethoxazole.
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Actinobacteria↗
Characterization of the Tn916-like transposon Tn3872 in a strain of abiotrophia defectiva (Streptococcus defectivus) causing sequential episodes of endocarditis in a child.
Clinical blood isolates from three sequential episodes of endocarditis occurring over a 6-month period in a child with a malformative cardiopathy were investigated. All isolates identified as Abiotrophia defectiva were resistant to erythromycin-clindamycin and to tetracycline-minocycline, due to the presence of sequences homologous to the erythromycin resistance gene ermB and to the tetracycline resistance gene tet(M), respectively. These resistance genes were located on a chromosomally borne composite Tn916-related transposon. These results demonstrate the involvement of conjugative transposons in the dissemination of antibiotic resistance in the genus Abiotrophia.
Anti-Bacterial Agents↗
Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci.
Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 438-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 19 enterococcal type strains (Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus seriolicida, Enterococcus solitarius, and Enterococcus sulfureus). Sequence analysis of the sodA(int) fragments enabled reliable identification of 18 enterococcal species, including E. casseliflavus-E. flavescens and E. gallinarum. The sodA(int) fragments of E. casseliflavus and E. flavescens were almost identical (99.5% sequence identity), which suggests that they should be associated in a single species. Our results confirm that the sodA gene constitutes a more discriminative target sequence than 16S rRNA gene in differentiating closely related bacterial species.
Bacterial Proteins↗
Additive effects of beta chain mutations in low oxygen affinity hemoglobin betaF41Y,K66T.
In order to decrease significantly the oxygen affinity of human hemoglobin, we have associated the mutation betaF41Y with another point mutation also known to decrease the oxygen affinity of Hb. We have synthesized a recombinant Hb (rHb) with two mutations in the beta chains: rHb betaF41Y,K66T. In the absence of 2, 3-diphosphoglycerate, additive effects of the mutations are evident, since the doubly mutated Hb exhibits a larger decrease in oxygen affinity than for the individual single mutations. In the presence of 2,3-diphosphoglycerate, the second mutation did not significantly increase the P(50) value relative to the single mutations. However, the kinetics of CO binding still indicate combined effects on the allosteric equilibrium, as evidenced by more of the slow bimolecular phase characteristic of binding to the deoxy conformation. Dimer-tetramer equilibrium studies indicate an increase in stability of the mutants relative to rHb A; the double mutant rHb betaF41Y, K66T at pH 7.5 showed a K(4,2) value of 0.26 microM. Despite the lower oxygen affinity, the single mutant betaF41Y and double mutant betaF41Y,K66T show only a moderate increase of 20% in the autoxidation rate. These mutations are thus of interest in developing a Hb-based blood substitute.
Escherichia coli↗
Diagnosis and follow-up of Whipple's disease by amplification of the 16S rRNA gene of Tropheryma whippelii.
Amplification of the 16S rRNA gene of Tropheryma whippelii was performed in eight patients with Whipple's disease and 34 control patients to confirm a diagnosis of Whipple's disease and to monitor the course of disease. Polymerase chain reaction (PCR) tests were positive before treatment in 13 of 15 tissue samples from Whipple's disease patients (gut 8/8; lymph nodes 2/2; bone marrow 1/2; peripheral blood 2/3), in contrast to none of 54 tissue samples from controls. PCR tests converted to negative within 4-6 months in six of the Whipple's disease patients undergoing therapy. These results show that PCR is a reliable and useful tool for diagnosis of Whipple's disease and for monitoring bacterial elimination during antibiotic therapy.
Actinobacteria↗
Listeriolysin O-dependent activation of endothelial cells during infection with Listeria monocytogenes: activation of NF-kappa B and upregulation of adhesion molecules and chemokines.
The facultative intracellular bacterium Listeria monocytogenes is an invasive pathogen that crosses the vascular endothelium and disseminates to the placenta and the central nervous system. Its interaction with endothelial cells is crucial for the pathogenesis of listeriosis. By infecting in vitro human umbilical vein endothelial cells (HUVEC) with L. monocytogenes, we found that wild-type bacteria induced the expression of the adhesion molecules (ICAM-1 and E-selectin), chemokine secretion (IL-8 and monocyte chemotactic protein-1) and NF-kappa B nuclear translocation. The activation of HUVEC required viable bacteria and was abolished in prfA-deficient mutants of L. monocytogenes, suggesting that virulence genes are associated with endothelial cell activation. Using a genetic approach with mutants of virulence genes, we found that listeriolysin O (LLO)-deficient mutants inactivated in the hly gene did not induce HUVEC activation, as opposed to mutants inactivated in the other virulence genes. Adhesion molecule expression, chemokine secretion and NF-kappa B activation were fully restored by a strain of Listeria innocua transformed with the hly gene encoding LLO. The relevance in vivo of endothelial cell activation for listerial pathogenesis was investigated in transgenic mice carrying an NF-kappa B-responsive lacZ reporter gene. NF-kappa B activation was visualized by a strong lacZ expression in endothelial cells of capillaries of mice infected with a virulent haemolytic strain, but was not seen in those infected with a non-haemolytic isogenic mutant. Direct evidence that LLO is involved in NF-kappa B activation in transgenic mice was provided by injecting intravenously purified LLO, thus inducing stimulation of NF-kappa B in endothelial cells of blood capillaries. Our results demonstrate that functional listeriolysin O secreted by bacteria contributes as a potent inflammatory stimulus to inducing endothelial cell activation during the infectious process.
Ampicillin↗
In vitro exchange of fluoroquinolone resistance determinants between Streptococcus pneumoniae and viridans streptococci and genomic organization of the parE-parC region in S. mitis.
Transfer of fluoroquinolone (FQ) resistance determinants between Streptococcus pneumoniae and viridans streptococci was explored by transformation in vitro. One-step FQ-resistant parC mutants were selected, and resistance could be transferred from DNA from S. oralis, S. mitis, S. sanguis, and S. constellatus to S. pneumoniae, with frequencies of 10(-3) to <10(-7) in correlation with the homologies of their quinolone resistance determining region sequences (95%, 91%, 85%, and 81%, respectively). Reciprocal transfers of mutated parC from DNA from S. pneumoniae to S. mitis and S. oralis were also observed. Simultaneous transfer of mutated parC and gyrA genes from S. mitis to S. pneumoniae yielded high-level-resistant pneumococcal transformants in one step at low frequencies. The parE-parC region of the type strain S. mitis 103335T had >90% homology with that of S. pneumoniae. The efficient interspecific transfer of quinolone resistance determinants in vitro leads us to anticipate their dissemination in the clinical setting.
Anti-Infective Agents↗
Blind protected specimen brush and bronchoalveolar lavage in ventilated children.
OBJECTIVE: To determine whether nonbronchoscopic protected specimen brush (PSB) and bronchoalveolar lavage (BAL) are contributive for diagnosing ventilator-associated pneumonia in mechanically ventilated children. DESIGN: Prospective study. SETTING: Fifteen-bed pediatric intensive care unit in a university hospital. PATIENTS: A total of 103 mechanically ventilated children, ranging in age from 7 days to 8.8 yrs, most with a high clinical suspicion for bacterial pneumonia. INTERVENTIONS: All the children underwent nonbronchoscopic PSB and BAL. Nonbronchoscopic PSB was performed with a plugged double-sheathed brush and BAL with a double-lumen plugged catheter. Endotracheal secretions and blood cultures were also collected. Open-lung biopsy was performed for any child who died within 7 days after the inclusion in the study, according to the parental consent. MEASUREMENTS AND MAIN RESULTS: The PSB specimens were submitted for bacteriologic quantitative culture (positive threshold, 10(3) colony-forming units [cfu]/mL). The BAL samples were processed for microscopic quantification of the polymorphonuclear cells containing intracellular bacteria (positive threshold, 1%) and quantitative culture (positive threshold, 10(4) cfu/mL). According to diagnostic categories based on clinical, biological, radiologic, and pathologic criteria, 29 children had bacterial pneumonia and 64 did not Ten children were classified as having an uncertain status. Of the 29 children with bacterial pneumonia, 26 (90%) met one of the following three criteria: a) PSB specimen culture, > or =10(3) cfu/mL; b) intracellular bacteria in cells retrieved by BAL, > or =1%; and c) BAL fluid culture, > or =10(4) cfu/mL. In contrast, 56 (88%) of the 64 patients without pneumonia did not. CONCLUSION: The results of this study indicate the following: a) nonbronchoscopic PSB and BAL were feasible in a large population of mechanically ventilated children; b) nonbronchoscopic techniques were contributive for diagnosing ventilator-associated pneumonia in children; and c) a combined diagnostic approach, using nonbronchoscopic PSB and BAL, was superior to using either test alone.
Bacteria↗
CO binding and valency exchange in asymmetric Hb hybrids.
There remains a major controversy concerning the properties of asymmetric hemoglobin hybrids, that is, doubly liganded tetramers consisting of an unliganded dimer and a liganded dimer. Different experimental evidence leads to opposing conclusions. Based on dimer-tetramer equilibrium studies, special "T-like" properties were assigned to this hybrid (species 21), while the other biliganded tetramers were considered as similar to fully liganded Hb [Ackers et al. (1992) Science 255, 54-63]. We report here results for three types of experiment. In the first, the asymmetric hybrids are produced by photodissociating CO ligands from [dimer-CO/dimer-azido-met] hybrids. Since the CO association rates differ by over an order of magnitude for the two allosteric states, the CO kinetics are a sensitive probe of the tetramer conformation. The results show mainly rapid R-like kinetics for CO rebinding to the asymmetric hybrids. The second technique employs a stopped-flow apparatus to obtain a higher percentage and a longer equilibration time of the asymmetric hybrid. In this case, sodium dithionite is used to remove oxygen from a solution containing [dimer-oxy/dimer-azido-met] hybrids. After a fixed delay (but before loss of azide ligands), a second mixing with a buffer equilibrated under CO allows observation of CO binding to species 21. As for the flash measurements, the kinetics show predominantly rapid CO binding, typical of the liganded (R-state) tetramer. The rapid CO binding is not in agreement with the predictions of a T-like conformation for species 21. One possible explanation is that the long incubation times used to study the dimer-tetramer equilibrium do not lead to a stable asymmetric hybrid, but rather a random distribution of oxidized subunits due to electron transfer between the iron atoms of the subunits [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We have repeated these experiments and confirm the valency exchange in a mixture of Hb A and S (or C) parent forms, as evidenced by compensating amounts of oxidation or reduction of the Hb parents.
Binding Sites↗