Publications

4481

ABSTRACT The 16S rRNA gene PCR in the diagnosis of bone and joint infections has not been systematically tested. Five hundred twenty-five bone and joint samples collected from 525 patients were cultured and submitted to 16S rRNA gene PCR detection of bacteria in parallel. The amplicons with mixed sequences were also cloned. When discordant results were observed, culture and PCR were performed once again. Bacteria were detected in 139 of 525 samples. Culture and 16S rRNA gene PCR yielded identical documentation in 475 samples. Discrepancies were linked to 13 false-positive culture results, 5 false-positive PCR results, 9 false-negative PCR results, 16 false-negative culture results, and 7 mixed infections. Cloning and sequencing of 16S rRNA gene amplicons in 6 of 8 patients with mixed infections identified 2 to 8 bacteria per sample. Rarely described human pathogens such as Alcaligenes faecalis , Comamonas terrigena , and 21 anaerobes were characterized. We also detected, by 16S rRNA gene PCR, four previously identified bacteria never reported in human infection, Alkanindiges illinoisensis , dehydroabietic acid-degrading bacterium DhA-73, unidentified Hailaer soda lake bacterium, and uncultured bacterium clone HuCa4. Seven organisms representing new potential species were also detected. PCR followed by cloning and sequencing may help to identify new pathogens involved in mixed bone infection.

Summary Previously uncultured nitrite‐oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira ‐like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira ‐like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 µg ml −1 . The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA‐targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira ‐like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as ‘ Candidatus Nitrospira defluvii’.

ABSTRACTZoothamnium niveumis a giant, colonial marine ciliate from sulfide-rich habitats obligatorily covered with chemoautotrophic, sulfide-oxidizing bacteria which appear as coccoid rods and rods with a series of intermediate shapes. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the ectosymbiont ofZ. niveumbelongs to only one pleomorphic phylotype. TheZ. niveumectosymbiont is only moderately related to previously identified groups of thiotrophic symbionts within theGammaproteobacteria, and shows highest 16S rRNA sequence similarity with the free-living sulfur-oxidizing bacterial strain ODIII6 from shallow-water hydrothermal vents of the Mediterranean Sea (94.5%) and an endosymbiont from a deep-sea hydrothermal vent gastropod of the Indian Ocean Ridge (93.1%). A replacement of this specific ectosymbiont by a variety of other bacteria was observed only for senescent basal parts of the host colonies. The taxonomic status “CandidatusThiobios zoothamnicoli” is proposed for the ectosymbiont ofZ. niveumbased on its ultrastructure, its 16S rRNA gene, the intergenic spacer region, and its partial 23S rRNA gene sequence.

A comparison of the quantification of a specific microbial group in activated sludge by fluorescent in‐situ hybridization, coupled with either direct microscopic counting or flow cytometry, was performed using an enhanced‐biological‐phosphorus‐removal, sequencing‐batch reactor. The population dynamics of Candidatus Accumulibacter phosphatis (Cand. A. phosphatis) was evaluated during two separate runs of the reactor. With the operational conditions used, Cand. A. phosphatis was enriched until a failure in the pH controller eliminated its ecological advantage. As a result, the comparison of quantification techniques included Cand. A. phosphatis concentrations as low as 11% and as high as 96% of the total cells in the samples. The analysis demonstrated that, regardless of the particular limitations of each technique, both provided similar results when the activated‐sludge flocs were easily dispersed. However, when the activated‐sludge samples contained flocs that were difficult to disperse, flow cytometry failed to provide quantitative results.

ABSTRACT Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL . DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum , a novel Ehrlichia sp., Anaplasma centrale , and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis , and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.

A moderately thermophilic, anaerobic bacterium (strain SB91T) was isolated from a freshwater hot spring at Barguzin Valley, Buryatiya, Russia. Cells of strain SB91Twere straight to slightly curved rods, 0·5–0·6 μm in diameter and 3·0–7·0 μm in length. Formation of endospores was not observed. The temperature range for growth was 26–62 °C, with an optimum at 50 °C. The pH range for growth was 5·5–9·5, with an optimum at pH 7·5–8·0. The substrates utilized by strain SB91Tin the presence of 9,10-anthraquinone 2,6-disulfonate included peptone, tryptone, Casamino acids, yeast extract, beef extract, casein hydrolysate, alanine plus glycine, alanine plus proline,l-valine and n-propanol. Carbohydrates were not utilized. Strain SB91Treduced amorphous Fe(III) oxide, Fe(III) citrate, Fe(III) EDTA or Fe(III) nitrilotriacetate with peptone,l-valine or n-propanol as an electron donor. Strain SB91Treduced 9,10-anthraquinone 2,6-disulfonate, thiosulfate, elemental sulfur, fumarate and selenite. Strain SB91Tsurvived after exposure to gamma-radiation at a dose of 5·4 kGy. The G+C content of the DNA of strain SB91Twas 33 mol%. Analysis of the 16S rRNA gene sequence revealed that the isolated organism belonged to cluster XII of the clostridia. On the basis of its physiological properties and the results of phylogenetic analyses, it is proposed that strain SB91Trepresents the sole species of a novel genus,Tepidimicrobium; the nameTepidimicrobium ferriphilumgen. nov., sp. nov. is proposed, with strain SB91T(=DSM 16624T=VKM B-2348T) as the type strain.