Use of macrolides for Q fever.
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Coxiella burnetii↗
Biomedical subjects
Didier Raoult
Publications and source records attributed to Didier Raoult.
Culture and antibiotic susceptibility of Bartonella quintana in human erythrocytes.
Bartonella quintana, the agent of trench fever, has recently been implicated in various diseases, in particular, bacteremia and endocarditis in homeless people. The host cell of Bartonella spp. is believed to be the erythrocyte, and in the present study we demonstrate that B. quintana can be cultured in vitro in human erythrocytes. The bacteria were found to be intraerythrocytic by laser confocal microscopy with Bartonella species-specific monoclonal antibodies. Infections with B. quintana decreased the life span of erythrocytes in culture from 8.6 to 4.8 days. In the culture system we found that most of the antibiotics that we tested (doxycycline, fluoroquinolone compounds, and beta-lactams) were not bactericidal. Gentamicin was bactericidal at 4 micro g/ml, as was rifampin, but to a lesser extent. At this concentration, gentamicin has been shown to enter erythrocytes slowly and to reach a peak level of 0.26 micro g/ml after 24 h. At 0.26 micro g/ml, however, we found that gentamicin was not able to kill extracellular B. quintana, even after 96 h of incubation. We hypothesize that erythrocytes may be a reservoir for B. quintana and that the bactericidal activity of gentamicin that we observed occurs mainly when the bacteria emerge from the erythrocytes and are found extracellularly. It would appear that gentamicin should be administered for at least 5 days to cure patients infected with B. quintana.
Anti-Bacterial Agents↗
Wolbachia pipientis growth kinetics and susceptibilities to 13 antibiotics determined by immunofluorescence staining and real-time PCR.
Wolbachia spp. are strict intracellular bacteria that infect a wide range of arthropods and filarial nematodes. Filarial nematodes are important causes of human diseases. There is increasing evidence that Wolbachia spp. influence important functions in the biology of the hosts, specifically, infertility. Preliminary experiments with humans and animals have suggested that antibiotics with activity against Wolbachia may help to treat filariasis. In this study, we determined using a real-time quantitative PCR assay the growth kinetics of a strain of Wolbachia pipientis from a mosquito grown in Aa23 cells. The doubling time was estimated to be 14 h. We then determined the susceptibilities of this strain to 13 antibiotics by two methods: an immunofluorescent-antibody test and a real-time quantitative PCR assay. Both techniques gave similar results. Doxycycline and rifampin were the most effective compounds, with MICs of 0.125 and 0.06 to 0.125 micro g/ml, respectively. Fluoroquinolones were less effective, with MICs of 2 to 4 micro g/ml for ciprofloxacin, 2 micro g/ml for ofloxacin, and 1 micro g/ml for levofloxacin. beta-Lactams (penicillin G, amoxicillin, ceftriaxone) were not effective at concentrations up to 128 micro g/ml. The MIC of erythromycin was >32 micro g/ml, whereas that of telithromycin was 8 micro g/ml. Other antibiotic compounds were bacteriostatic only at high concentrations, including gentamicin, co-trimoxazole, and thiamphenicol. The real-time PCR assay was a convenient and reliable technique for determination of the antibiotic susceptibilities of WOLBACHIA: It may help in the future to simplify antibiotic susceptibility testing of strict intracellular pathogens.
Animals↗
Usefulness of rpoB gene sequencing for identification of Afipia and Bosea species, including a strategy for choosing discriminative partial sequences.
Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability "hot spots" in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.
Afipia↗
History of the ADP/ATP-translocase-encoding gene, a parasitism gene transferred from a Chlamydiales ancestor to plants 1 billion years ago.
Nonmitochondrial ADP/ATP translocase is an energy parasite enzyme. Its encoding gene, tlc, is found only in Rickettsiales, Chlamydiales, and plant and alga plastids. We demonstrate the presence of tlc in Parachlamydia acanthamoebae. This gene shares more similarity with the tlc1 gene of Chlamydiaceae and the tlc of plant and alga plastids than with the tlc2 gene of Chlamydiaceae. Phylogenetic analysis, including all other tlc homologs found in GenBank, showed that tlc was duplicated in a Chlamydiales ancestor before the appearance of multicellular eukaryotes. A time scale, calibrated with seven independent time points obtained from fossil estimates and from the 16S rRNA molecular clock, was congruent with the molecular clock provided by tlc. Plant and alga plastids acquired tlc approximately when Parachlamydiaceae and Chlamydiaceae diverged, at the eucaryotic radiation time, ca. 1 billion years ago.
Base Sequence↗
Western immunoblotting for Bartonella endocarditis.
To differentiate infectious endocarditis (IE) from other Bartonella infections and to identify infecting Bartonella bacteria at the species level on a serological basis, we used Western immunoblotting to test sera from 51 patients with Bartonella IE (of which 27 had previously benefited from species identification by molecular techniques), 11 patients with chronic Bartonella quintana bacteremia, and 10 patients with cat scratch disease. Patients with IE were Western blot positive in 49 of 51 cases, and significant cross-reactivity with three heterologous Bartonella antigens was found in 45 of 49 cases. Sera from bacteremic patients did not react with more than one heterologous antigen, and sera from patients with cat scratch disease gave negative results. Sera reacted only with B. henselae in four cases of IE, including one with a positive PCR result for valve tissue. Western blot and cross-adsorption performed on serum samples from patients with IE (the identity of the causative species having been determined by PCR) were demonstrated to identify efficiently the causative species in all cases. When applied to patients diagnosed on the basis of serological tests only, this technique allowed identification of the causative species in 20 of 22 cases. The results were in accordance with epidemiological features. Six reactive bands of B. quintana (of molecular sizes from 10 to 83 kDa) demonstrated significant association with sera from patients with B. quintana endocarditis. Overall, Western blotting and cross-adsorption made it possible to identify the causative species in 49 of 51 (96%) IE cases.
Antibodies, Bacterial↗
Antigenic classification of Rickettsia felis by using monoclonal and polyclonal antibodies.
Rickettsia felis is a flea-transmitted rickettsia. There is a discrepancy between its reported phylogenic and phenotypic identifications. Following the first report of R. felis, it was considered by tests with serologic reagents to be closely related to another recognized flea-transmitted rickettia, R. typhi. Subsequently, it appeared to be more closely related to spotted fever group (SFG) rickettsiae by genetic analysis. In the present work, R. felis was studied by microimmunofluorescence (MIF) serologic typing and with monoclonal antibodies (MAbs). Mouse polyclonal antisera to R. felis cross-reacted only with SFG rickettsiae. A neighbor-joining analysis based on MIF indicated that R. felis is actually related to SFG rickettsiae antigenically, clustering with R. australis, R. akari, and R. montanensis. A panel of 21 MAbs was raised against a 120-kDa protein antigen or a 17-kDa polypeptide of R. felis. They cross-reacted with most members of the SFG rickettsiae but not with R. prowazekii, R. typhi, or R. canadensis of the typhus group (TG) rickettsiae. Sixty-four MAbs previously generated to seven other ricketttsial species were tested with R. felis. Three MAbs reacted with the 120-kDa antigen and were generated by R. africae, R. conorii, and R. akari, respectively. They exhibited cross-reactivities with R. felis. All our data show that R. felis harbors the antigenic profile of an SFG rickettsia.
Animals↗
Serotyping isolates of Anaplasma phagocytophilum by using monoclonal antibodies.
Ten mouse monoclonal antibodies (MAbs) that react with Anaplasma phagocytophilum (the human granulocytic ehrlichiosis agent) Webster isolates were developed. Seven different isolates of A. phagocytophilum were subtyped with these MAbs. Western blot analysis revealed that these MAbs reacted mainly with 41- to 46-kDa Msp2 proteins. Six MAbs reacted with all isolates. Four other MAbs reacted with human isolates from Wisconsin, but not with human isolates from New York or with animal isolates. Three different serotypes were identified. These features may lead to the development of other specific MAbs in order to provide tools for antigenic characterization of human isolates of A. phagocytophilum.
Anaplasma phagocytophilum↗
Parachlamydia acanthamoebae enters and multiplies within human macrophages and induces their apoptosis [corrected].
Parachlamydia acanthamoebae is an obligately intracellular bacterium that naturally infects free-living amoebae. It is a potential human pathogen and may survive in human macrophages. We studied P. acanthamoebae entry into, and multiplication within, human monocyte-derived macrophages. After 8 h of incubation, 80% of macrophages were infected with a mean of 3.8 P. acanthamoebae organisms per cell. Electron microscopy demonstrated that parachlamydiae were in an intracellular vacuole. After infection with living organisms, the number of parachlamydiae per macrophage increased 4 times from day 0 to day 4, whereas heat-inactivated parachlamydiae were eliminated during the same period. Quantitative PCR confirmed that P. acanthamoebae replicates within macrophages. Transcriptional activity of P. acanthamoeba was detected by reverse transcription-PCR targeting the gene encoding ADP-ATP translocase (tlc). P. acanthamoebae exerted a cytopathic effect on macrophages. When macrophages were infected with living bacteria, their number decreased significantly from day 0 to day 4 due to apoptosis, as shown by annexin-V binding and electron microscopy. This study shows that P. acanthamoebae enters and multiplies within human macrophages before inducing their apoptosis.
Apoptosis↗
Attempted isolation of Nanobacterium sp. microorganisms from upper urinary tract stones.
A single team has reported isolation of nanobacteria in human and bovine blood products, as well as, more recently, kidney stones. This has raised controversy. To confirm the data, we searched for nanobacteria from 10 aseptically removed upper urinary tract (UUT) stones. We used scanning electronic microscopy (SEM) with four stones and culture of stones on either 3T6 fibroblast monolayers or liquid RPMI medium. Detection of nanobacteria was made with a commercially available monoclonal antibody, 16S ribosomal DNA amplification with specific primers, and transmission electronic microscopy (TEM) of inoculated cells. SEM showed nanoparticles in four of four UUT stones similar to those recently described. TEM of inoculated 3T6 cell monolayers has shown transient intracytoplasmic vacuolar formations containing 200- to 500-nm particles in 3 of 10 cell cultures. Gimenez staining, Hoechst staining, and specific monoclonal immunofluorescence failed to reveal nanobacteria. Finally, we could not grow Nanobacterium sp. microorganisms by the techniques described. Although with SEM, we observed nanoparticles morphologically similar to nanobacteria, we failed to isolate Nanobacterium sp. microorganisms in culture and to prove the bacterial nature of these nanoparticles in stones.
Bacteria↗
Searching for Bacillus anthracis in suspect powders: a French experience.
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Bacillus anthracis↗
Comparison of PCR and serology assays for early diagnosis of acute Q fever.
Q fever is a potentially severe disease which can occur in large outbreaks of acute infections and is a possible bioterrorism agent. In order to lessen the delay in diagnosing acute Q fever, we compared LightCycler Nested PCR (LCN-PCR), a rapid nested PCR assay that uses serum sampled early during the disease as a specimen and the LightCycler as a thermal cycler, to serology by indirect immunofluorescence. We used the 20-copy htpAB-associated element as the DNA target. The detection sensitivity of this method was one Coxiella burnetii DNA copy. We applied this method to the first serum samples taken from 100 patients diagnosed in our laboratory as having acute Q fever on the basis of clinical manifestations and serology and to 80 controls. The LCN-PCR had a specificity of 100%. The sensitivity was 26% when no antibodies were detected but only 5% with seropositive patients (P < 10(-2)). The technique was most efficient in the first 2 weeks following the onset of symptoms (P = 0.02), when its sensitivity was 24% compared with 14% for serology. With combined use of LCN-PCR and serology within the first 2 weeks, the sensitivity was significantly increased over that with serology alone (P < 10(-2)). Thus, we propose a strategy for improving the early diagnosis of acute Q fever where LCN-PCR should be performed together with serology in the first 2 weeks of the disease but should be reserved for seronegative patients in the next 2 weeks and not used later than 4 weeks following onset, when serology is highly sensitive.
Adolescent↗
Culture and phenotypic characterization of a Wolbachia pipientis isolate.
The recent isolation of Wolbachia pipientis in the continuous cell line Aa23, established from eggs of a strain of the Asian tiger mosquito Aedes albopictus, allowed us to perform extensive characterization of the isolate. Bacterial growth could be obtained in C6/36, another A. albopictus cell line, at 28 degrees C and in a human embryonic lung fibroblast monolayer at 28 and 37 degrees C, confirming that its host cell range is broader than was initially thought. The bacteria were best visualized by Diff-Quik and May-Grünwald-Giemsa staining. Proteins from 213 to 18 kDa with two major protein bands of 65 and 25 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By Western blotting with specific polyclonal mouse and rabbit antisera, dominant immunoreactive antigens were found at approximately 100, 80, and 30 kDa. The genome size was calculated to be 1,790 +/- 17 kb by pulsed-field gel electrophoresis. The sequence of the citrate synthase gene (gltA) of W. pipientis was determined by gene walking. Its position in the phylogenetic tree constructed with gltA confirmed that found in a phylogenetic tree constructed with 16S rRNA genes and that it belongs in the alpha subgroup of the class Proteobacteria and that it is closely related to but independent from the genera Ehrlichia, Anaplasma, and Neorickettsia.
Animals↗
Gene sequence-based criteria for identification of new rickettsia isolates and description of Rickettsia heilongjiangensis sp. nov.
We propose genetic guidelines for the classification of rickettsial isolates at the genus, group, and species levels by using sequences of the 16S rRNA (rrs) gene and four protein-coding genes, the gltA, ompA, and ompB genes and gene D. To be classified as a member of the genus Rickettsia, an isolate should exhibit degrees of rrs and gltA homology with any of the 20 Rickettsia species studied of >/=98.1 and >/=86.5%, respectively. A member of the typhus group should fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of either Rickettsia typhi or Rickettsia prowazekii of >/=99.4, >/=96.6, >/=92.4, and >/=91.6%, respectively. A member of the spotted fever group should either possess the ompA gene or fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of any member of this group of >/=98.8, >/=92.7, >/=85.8, and >/=82.2%, respectively. The existence of a distinct "ancestral" group should be questioned. To be classified as a new Rickettsia species, an isolate should not exhibit more than one of the following degrees of nucleotide similarity with the most homologous validated species: >/=99.8 and >/=99.9% for the rrs and gltA genes, respectively, and, when amplifiable, >/=98.8, >/=99.2, and >/=99.3% for the ompA and ompB genes and gene D, respectively. By use of our classification scheme, "Rickettsia heilongjiangii" belongs to a new species for which we officially propose the name Rickettsia heilongjiangensis sp. nov.
DNA, Ribosomal↗
Diagnosis of Bartonella endocarditis by a real-time nested PCR assay using serum.
Bartonella endocarditis is a severe disease for which blood cultures frequently remain negative. We tested three PCR assays by using specimens of serum sampled early during the disease from 43 patients diagnosed in our laboratory as having Bartonella endocarditis on the basis of serological, culture, and/or valvular molecular detection. We tested a two-step nested PCR (TSN-PCR), a one-step nested PCR (OSN-PCR) with a regular thermal cycler, and a one-step nested PCR with the LightCycler (LCN-PCR). These assays were performed with primers derived from the riboflavin synthase-encoding gene ribC, never before amplified in our laboratory. Due to contamination of negative controls, the results of the TSN-PCR were not interpretable, and this technique was no longer considered. The LCN-PCR had a specificity of 100% and a sensitivity of 58.1%, higher than those of the OSN-PCR (18.6%; P < 0.01) and prolonged blood culturing (7.1%; P < 0.01). The LCN-PCR results correlated strictly with those of other direct diagnostic tests, when available, and identified the causative species for six patients previously diagnosed on the basis of serological analysis only. The efficacy of the LCN-PCR was not influenced by antibiotics (P = 0.96) but was altered by prolonged storage of serum specimens at -20 degrees C (P = 0.04). Overall, the LCN-PCR is specific and more sensitive than traditional methods (i.e., culturing and/or PCR with EDTA-treated blood). It can easily be applied to the diagnosis of patients with suspected Bartonella endocarditis, especially when only serum is available.
Bartonella↗
Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and other eubacteria in ticks from the Thai-Myanmar border and Vietnam.
A total of 650 ticks, including 13 species from five genera, were collected from animals, from people, or by flagging of the vegetation at sites on the Thai-Myanmar border and in Vietnam. They were tested by PCR to detect DNA of bacteria of the order RICKETTSIALES: Three Anaplasma spp. were detected in ticks collected in Thailand, including (i) Anaplasma sp. strain AnDa465, which was considered a genotype of Anaplasma platys (formerly Ehrlichia platys) and which was obtained from Dermacentor auratus ticks collected from dogs; (ii) Anaplasma sp. strain AnAj360, which was obtained from Amblyomma javanense ticks collected on a pangolin; and (iii) Anaplasma sp. strain AnHl446, which was closely related to Anaplasma bovis and which was detected in Haemaphysalis lagrangei ticks collected from a bear. Three Ehrlichia spp. were identified, including (i) Ehrlichia sp. strain EBm52, which was obtained from Boophilus microplus ticks collected from cattle from Thailand; (ii) Ehrlichia sp. strain EHh324, which was closely related to Ehrlichia chaffeensis and which was detected in Haemaphysalis hystricis ticks collected from wild pigs in Vietnam; and (iii) Ehrlichia sp. strain EHh317, which was closely related to Ehrlichia sp. strain EBm52 and which was also detected in H. hystricis ticks collected from wild pigs in Vietnam. Two Rickettsia spp. were detected in Thailand, including (i) Rickettsia sp. strain RDla420, which was detected in Dermacentor auratus ticks collected from a bear, and (ii) Rickettsia sp. strain RDla440, which was identified from two pools of Dermacentor larvae collected from a wild pig nest. Finally, two bacteria named Eubacterium sp. strain Hw124 and Eubacterium sp. strain Hw191 were identified in Haemaphysalis wellingtoni ticks collected from chicken in Thailand; these strains could belong to a new group of bacteria.
Anaplasma↗
Eubacterium callanderi bacteremia: report of the first case.
Eubacterium callanderi is an environmental anaerobic rod-shaped bacterium first isolated in 1998 from an industrial anaerobic digester. We report on the first clinical isolate of E. callanderi, which was recovered from the blood of a patient with a bladder carcinoma. Identification of the organism was made by cell fatty acid chromatographic analysis and 16S rRNA gene sequencing.
Actinomycetales Infections↗
Corynebacterium freneyi bacteremia.
Corynebacterium freneyi is a recently described alpha-glucosidase-positive species of the genus CORYNEBACTERIUM: To our knowledge, there is no description of human infection due to this species. We report on a case of bacteremia due to C. freneyi following vascular surgery.
Bacteremia↗