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M Drancourt

Publications and source records attributed to M Drancourt.

At least 37 records · Page 2Linked to original sources
PubMed2001

rpoB sequence analysis of cultured Tropheryma whippelii.

Until recently no isolate of Tropheryma whippelii was available, and therefore genetic studies were limited to those based on PCR amplification of conserved genes. In this study we determined the nucleotide sequence of rpoB (encoding the beta-subunit of RNA polymerase) from a cultured strain of T. whippelii using degenerate consensus PCR and genome walking. The T. whippelii rpoB consists of 3,657 bp with a 50.4% GC content and encodes 1,218 amino acids with a calculated molecular mass of 138 kDa. Comparison of T. whippelii RpoB with other eubacterial RpoB proteins indicated sequence similarity ranging from 57.19 (Mycoplasma pneumoniae) to 74.63% (Mycobacterium tuberculosis). Phylogenetic analysis of T. whippelii based on comparison of its RpoB sequence with sequences available for other bacteria was consistent with that previously derived from the 16S ribosomal DNA (rDNA) sequence, indicating that it belongs to the actinomyces clade. The sequence comparison allowed the design of a primer pair, TwrpoB.F and TwrpoB.R, specific for T. whippelii rpoB. When incorporated into a PCR, this primer pair allowed the detection of T. whippelii rpoB in three of three 16S rDNA PCR-positive biopsy specimens and zero of seven negative controls. rpoB could therefore be targeted in PCR-mediated detection and identification of this emerging bacterial species. This approach has previously been shown useful for the identification of related mycobacteria. This study underscores that a method involving isolation and then propagation of emerging bacteria is a useful way to quickly achieve extensive molecular knowledge of these pathogens.

Actinobacteria↗
PubMed2001

Molecular identification of a Dietzia maris hip prosthesis infection isolate.

Dietzia maris, an environmental actinomycete, has been implicated only once in human disease. We herein report the first D. maris isolate from a bone biopsy specimen in a patient hospitalized for a total hip prosthesis replacement. Cell wall fatty acid analysis and 16S ribosomal DNA gene sequencing were utilized to achieve its definite identification. This case report illustrates the usefulness of such methods for the accurate identification of actinomycetes.

Actinomycetales↗
PubMed2001

Citrate synthase gene sequence: a new tool for phylogenetic analysis and identification of Ehrlichia.

The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versus A. centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99.5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA genes was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.

Animals↗
PubMed2000

Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death.

Medieval Black Death is believed to have killed up to one-third of the Western European population during the 14th century. It was identified as plague at this time, but recently the causative organism was debated because no definitive evidence has been obtained to confirm the role of Yersinia pestis as the agent of plague. We obtained the teeth of a child and two adults from a 14th century grave in France, disrupted them to obtain the pulp, and applied the new "suicide PCR" protocol in which the primers are used only once. There were no positive controls: Neither Yersinia nor Yersinia DNA were introduced in the laboratory. A negative result is followed by a new test using other primers; a positive result is followed by sequencing. The second and third primer pair used, coding for a part of the pla gene, generated amplicons whose sequence confirmed that it was Y. pestis in 1 tooth from the child and 19/19 teeth from the adults. Negative controls were negative. Attempts to detect the putative alternative etiologic agents Bacillus anthracis and Rickettsia prowazekii failed. Suicide PCR avoids any risk of contamination as it uses a single-shot primer-its specificity is absolute. We believe that we can end the controversy: Medieval Black Death was plague.

Adult↗
PubMed2000

Actin mediates Encephalitozoon intestinalis entry into the human enterocyte-like cell line, Caco-2.

Microsporidia are spore-forming obligate intracellular eucaryotes that parasitize eukaryotic cells. Encephalitozoon intestinalis (formerly Septata intestinalis) is a microsporidian species of emerging medical importance, responsible for chronic diarrhoea in immunocompetent patients and enteritis and systemic infections in HIV-1 infected patients. Infection of host enterocytes has been demonstrated in HIV-1-infected patients. However, the mechanisms of entry of E. intestinalis into host enterocytes have not been studied and remain hypothetically based on diacytosis, a model involving the injection of microsporidian sporoplasm through the polar tubule into the host cell. An electron microscopy based study recently challenged this hypothesis. We studied the entry of E. intestinalis into intestinal epithelial cells by infecting the human enterocyte-like cell line Caco-2. Entry was mediated by directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on E. intestinalis uptake, colocalization of E. intestinalis and F-actin and engulfment of E. intestinalis into Caco-2 cell protrusions. Confocal- and electron microscopy observations also suggested that after initial contacts through the posterior pole of the microsporidian spore, the basolateral surface of Caco-2 cells may be the portal of entry for E. intestinalis sporoplasm. Our observations allowed us to propose a new, actin-based model to describe the entry of microsporidia into enterocytes.

Actins↗
PubMed2000

Detection of Coxiella burnetii DNA in dental pulp during experimental bacteremia.

Colonization of dental pulp by blood-borne bacteria in the absence of previous inflammation has been hypothetized but has never been convincingly demonstrated. In order to provide convincing support for this hypothesis we attempted to detect Coxiella burnetii DNA in the dental pulp of bacteremic, intraperitoneally inoculated guinea-pigs by PCR amplification and direct sequencing of two molecular targets. Coxiella burnetii DNA was recovered from 20-50% of the animals depending on the molecular target, from 15-20 days after experimental challenge. These results demonstrated, for the first time, that dental pulp is contaminated by blood-borne bacteria and can be detected by molecular tools.

Animals↗
PubMed2000

Determination of the rpoB gene sequences of Bartonella henselae and Bartonella quintana for phylogenic analysis.

Using the Genome Walker procedure, which allows PCR amplification of genomic DNA using a single gene-specific primer and direct automated sequencing methodology, we obtained the nucleotide sequence of the RNA polymerase beta subunit (rpoB) from Bartonella henselae and Bartonella quintana. A phylogenetic tree constructed from these data and other rpoB sequences available in GenBank is, in part, consistent with those previously derived from 16S rRNA gene sequences and confirms the position of Bartonella within the alpha subdivision of Proteobacteria. In fact, this analysis showed that rpoB data are similar to 16S rRNA data for the alpha, beta and gamma subdivisions of Proteobacteria. In contrast, concerning other bacteria included in our study, the topologies of phylogenetic trees were different. Based on the bootstrap values derived from rpoB phylogenic analysis, we believe that this molecule should contribute to better understanding the evolutionary process.

Amino Acid Sequence↗
PubMed2000

Nosocomial Serratia marcescens infections associated with extrinsic contamination of a liquid nonmedicated soap.

OBJECTIVE: To determine the role of nonmedicated soap as a source of Serratia marcescens nosocomial infections (NIs) in hospital units with endemic S marcescens NI and to examine the mechanisms of soap colonization. SETTING: University-affiliated tertiary-care hospitals. METHODS: A prospective case-control study and an environmental investigation were performed to assess the relationship between S marcescens NIs in hospital units and S marcescens-contaminated soap. Soap-bottle use and handwashing practices were reviewed. Cultures of healthcare workers' (HCWs) hands were obtained before and after hand washing with soap. RESULTS: 5 of 7 hospital units with S marcescens NIs had soap bottles contaminated with S marcescens, compared to 1 of 14 other units (P=.006). After hand washing with an S marcescens-contaminated soap pump, HCWs' hands were 54 times more likely to be contaminated with S marcescens (P<.001). CONCLUSIONS: Extrinsic contamination of a non-medicated liquid soap by S marcescens resulted in handborne transmission of S marcescens NIs by HCWs in our setting. This finding led to the application of strict guidelines for nonmedicated soap use and to the reinforcement of alcoholic hand disinfection.

Case-Control Studies↗
PubMed2000

Skin tolerance and effectiveness of two hand decontamination procedures in everyday hospital use.

BACKGROUND: Hand decontamination is crucial to control nosocomial infections. The utility of hand decontamination is related not only to its antimicrobial effectiveness, but also to its acceptability by hospital staff. OBJECTIVES: We aimed to assess skin tolerance and antimicrobial effects of two widely accepted hand hygiene measures under in-use conditions. METHODS: Fifty-two nurses were randomly assigned for an 8-day period to either an alcohol-based disinfectant or a hand wash with a non-antiseptic soap. At baseline and at the end of the test period, microbiological hand samples were obtained both before and after a hand hygiene procedure, and skin tolerance was assessed using clinical scores and measurement of transepidermal water loss. RESULTS: Self-assessment of skin condition and grade of skin damage worsened significantly more in the group using soap than in the group using alcoholic disinfectant (P = 0.004 and P = 0.01, respectively). The alcohol-based rinse was significantly more effective than liquid soap in removing transient contaminant micro-organisms (P = 0.016). Twenty of 50 hand washes with non-antiseptic soap apparently resulted in bacterial contamination of the hands. At the end of the study, the total bacterial count increased with the increasing number of hand washes in the soap group (P = 0.003), and with the degree of skin damage (P = 0.005) in the antiseptic group. CONCLUSIONS: In everyday hospital practice, alcohol-based disinfectant is more effective and better tolerated than non-antiseptic soap; soap is at risk of spreading contamination; and skin comfort strongly influences the number and the quality of hand hygiene procedures.

Alcohols↗
PubMed2000

16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates.

Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of > or =97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of > or =99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

Animals↗
PubMed2000

rpoB gene analysis as a novel strategy for identification of spirochetes from the genera Borrelia, Treponema, and Leptospira.

Spirochetes are emerging pathogens for which culture and identification are partly unresolved. In fact, 16S rRNA-based sequencing is by far the most widely used PCR methodology that is able to detect such uncultivable pathogens. However, this assay actually has some limitations linked to potential problems of contamination, which hampers diagnosis. To circumvent this, we have devised a simple PCR strategy involving targeting of the gene encoding the RNA polymerase beta subunit (rpoB), a highly conserved enzyme. The complete sequence of the Leptospira biflexa (serovar patoc) rpoB gene was determined and compared with the published sequences for Borrelia burgdorferi and Treponema pallidum. From the resulting analysis, degenerate nucleotide primers were designed and tested for their ability to amplify a portion of the rpoB gene from various spirochetes. Using two different pairs of these primers, we succeeded in obtaining specific rpoB-amplified fragments for all members of the genera Leptospira, Treponema, and Borrelia tested and no other bacteria. Our findings may have significant implications for the development of a new tool for the identification of spirochetes, especially if clinical samples are contaminated or when the infecting strain is uncultivable.

Borrelia↗
PubMed2000

Diagnostic accuracy of protected catheter sampling in ventilator-associated bacterial pneumonia.

The aim of this study was to assess the accuracy of protected telescoping catheter performed using: 1) protected distal aspiration, or 2) protected mini-bronchoalveolar lavage for the diagnosis of ventilator-associated bacterial pneumonia. Twenty-seven patients who died after receiving mechanical ventilation for at least 72 h were included in a comparative prospective post-mortem study. The two microbiological sampling procedures were performed immediately after death. Surgical pneumonectomies and biopsies were performed within 30 min of death at the bedside for histological examination. The results of the two techniques were compared with histological post-mortem lung examination or biopsies. Histological examination of the parenchyma showed signs of pneumonia in 14 cases. Lung tissue culture was positive in nine of these 14 cases. When ventilator-associated bacterial pneumonia was defined by the association of histological signs and positive lung tissue culture the sensitivity was 78% for both sampling techniques, specificity was 86% for mini-bronchoalveolar lavage and 100% for protected distal aspiration (at a threshold of 1 x 10(3) cfu x mL(-1)). Both techniques protected the distal aspiration and mini-bronchoalveolar lavage, and provided good specificity with an acceptable sensitivity for the diagnosis of ventilator-associated bacterial pneumonia.

Aged↗
PubMed1999

[Tropheryma whippelii, an emerging intracellular pathogen causing Whipple disease].

CLINICAL DIAGNOSIS: Whipple's disease is a diffuse disease with a wide variety of clinical manifestations. Differential diagnoses include chronic enteritis, chronic joint disease, chronic meningoencephalitis and prolonged fever with weight loss, chronic uveitis and endocarditis with negative blood culture. MOLECULAR DIAGNOSIS: Histologically the characteristic lesions contain PAS-positive inclusions. PCR of RNAr gene 16S in fresh tissue identifies Tropheryma whippelii and confirms bacteriological diagnosis. AN EMERGING PATHOGEN: Tropheryma whippelii is a recently identified pathogen highly difficult to culture. Since its discovery, it has been a model of emerging pathogens. Clinical observation, microscope studies, molecular and cellular biology, and bacteriology have all contributed to its recent isolation which should lead to the development of indirect diagnostic techniques [abstract corrected]

Actinobacteria↗
PubMed1999

An in-vitro evaluation of the activity of chlorine against environmental and nosocomial isolates of Aeromonas hydrophila.

A chlorine preparation was tested against 34 strains of Aeromonas hydrophila isolated from a hospital water facility and patients with nosocomial infections in a French medical school hospital. In-vitro bactericidal activity was determined by a macro- and micromethod, using specific interfering substances over a range of dilutions and temperatures, after 1 and 5 minute exposure times. A 10(5)-fold reduction in the challenge inoculum was taken as the criterion of efficacy. Results obtained by both methods agreed and indicated a median minimal bacterial concentration of chlorine of from 0.95 ppm without interfering substances to 297 ppm in the presence of interfering substances. These data indicate that Aeromonas hydrophila isolates are relatively resistant to chlorine, thus precluding the use of chlorine alone as a disinfectant for water supplies contaminated with Aeromonas spp.

Aeromonas hydrophila↗