Publications

4981

ABSTRACT The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria . The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation “ Candidatus Riesia pediculicola” for the louse symbionts.

ABSTRACTTo survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned adps(stands forDNA-bindingprotein fromstarved cells) gene of the symbiotic “CandidatusLegionella jeonii” organism (called the X bacterium) (dpsX) that grows inAmoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented thedpsmutant ofEscherichia coliand conferred resistance to hydrogen peroxide. DpsXproteins purified fromE. colitransformed with thedpsXgene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H2O2-mediated damage. The expression of thedpsXgene in “CandidatusLegionella jeonii” was enhanced when the host amoeba was treated with 2 mM H2O2and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion ofLegionellasp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization ofdpsXexpression in theLegionellasp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.

An intracellular bacterium with the unique ability to enter mitochondria exists in the European vector of Lyme disease, the hard tick Ixodes ricinus. Previous phylogenetic analyses based on 16S rRNA gene sequences suggested that the bacterium formed a divergent lineage within the Rickettsiales (Alphaproteobacteria). Here, we present additional phylogenetic evidence, based on the gyrB gene sequence, that confirms the phylogenetic position of the bacterium. Based on these data, as well as electron microscopy (EM), in situ hybridization and other observations, we propose the name ‘Candidatus Midichloria mitochondrii’ for this bacterium. The symbiont appears to be ubiquitous in females of I. ricinus across the tick's distribution, while lower prevalence is observed in males (44 %). Based on EM and in situ hybridization studies, the presence of ‘Candidatus M. mitochondrii’ in females appears to be restricted to ovarian cells. The bacterium was found to be localized both in the cytoplasm and in the intermembrane space of the mitochondria of ovarian cells. ‘Candidatus M. mitochondrii’ is the first bacterium to be identified that resides within animal mitochondria.

Two Gram-positive bacilli, designated as strains SMC 4352-1T and SMC 4352-2T, were isolated sequentially from the blood of a newborn child with sepsis. They could not be identified by using conventional clinical microbiological methods. 16S rRNA gene sequencing and phylogenetic analysis revealed that both strains belonged to the genus Bacillus but clearly diverged from known Bacillus species. Strain SMC 4352-1T and strain SMC 4352-2T were found to be closely related to Bacillus firmus NCIMB 9366T (98.2 % sequence similarity) and Bacillus cibi JG-30T (97.1 % sequence similarity), respectively. They also displayed low DNA–DNA reassociation values (less than 40 %) with respect to the most closely related Bacillus species. On the basis of their polyphasic characteristics, strain SMC 4352-1T and strain SMC 4352-2T represent two novel species of the genus Bacillus, for which the names Bacillus infantis sp. nov. (type strain SMC 4352-1T=KCCM 90025T=JCM 13438T) and Bacillus idriensis sp. nov. (type strain SMC 4352-2T=KCCM 90024T=JCM 13437T) are proposed.

Abstract:  We recently reported the first documented cases of a new rickettsial disease caused by Rickettsia heilongjiangensis in the Russian Far East (Far Eastern tick‐borne rickettsiosis). Here we report the amplification of DNA of R. heilongjiangensis from both the skin biopsy of an acutely ill patient and the tick removed from him prior to the disease development. The tick has been identified as Haemaphysalis spp. The clinical picture was that of a spotted fever group rickettsiosis and a seroconversion was noted with R. heilongjiangensis antigen . Screening testing of both species of Haemaphysalis ticks inhabiting Russian Far Eastern regions showed that up to 28.13% of H. concinnae and 4.48% of H. japonica douglasii ticks harbor R. heilongjiangensis. It has been concluded that H. concinnae may serve as the main vector for the transmission of R. heilongjiangensis. H. japonica douglasii ticks harbor several varieties of rickettsiae. DNA of “Candidatus Rickettsia tarasevichiae,” previously found in Ixodes persulcatus ticks, was amplified from one male tick. Two sequenced complete gltA genes belong to the novel spotted fever group rickettsial species provisionally called here “Candidatus Rickettsia principis” variants Hjd54 and Hjd61. The rate of infection has been found to be not higher than 1.5%.

Conventional PCR and two real‐time PCR (RTi‐PCR) methods were developed and compared using the primer pairs CQULA03F/CQULA03R and CQULA04F/CQULA04R, and TaqMan probe CQULAP1 designed from a species‐specific sequence of the rplJ/rplL ribosomal protein gene, for diagnosis of citrus huanglongbing (HLB) disease in southern China. The specificity and sensitivity of the three protocols for detecting ‘ Candidatus Liberibacter asiaticus’ in total DNA extracts of midribs collected from infected citrus leaves with symptoms in Guangxi municipality, Jiangxi Province and Zhejiang Province, were tested. Sensitivities using extracted total DNA (measured as copy number, CN per µ L of recombinant plasmid solution) were 439·0 (1·30 × 10 5 CN µ L −1 ), 4·39 (1·30 × 10 3 CN µ L −1 ) and 0·44 fg µ L −1 (1·30 × 10 2 CN µ L −1 ) for conventional PCR, TaqMan and SYBR Green I (SGI) RTi‐PCR, respectively. SGI RTi‐PCR was the most sensitive, but its specificity needed to be confirmed by running a melt‐curve assay. The TaqMan RTi‐PCR assay was rapid and had the greatest specificity. Concerning the correlation of PCR detection results with the various HLB symptoms, uneven mottling of leaves had the highest positive rate (96·50%), indicating that leaf mottling was the most reliable symptom for field surveys. Dynamic analysis results from the TaqMan assays showed that the titre (CN) g −1 citrus tissue of ‘ Ca. L. asiaticus’ was highest between October and December (threshold cycle ( C t ) average = 29·3, CN = 3·35 × 10 7 ) and lowest between March and May ( C t average = 32·0, CN = 5·10 × 10 6 ) in 2004 and 2005. The optimized molecular‐based assays should prove useful for presymptom diagnosis of HLB disease, monitoring and identification of ‘ Ca. L. asiaticus’, and field epidemic regulation.