Intrinsic characteristics of Min proteins on the cell division of Helicobacter pylori.

Helicobacter pylori divides in the human stomach resulting in persistent infections and causing various disorders. Bacterial cell division is precisely coordinated by many molecules, including FtsZ and Min proteins. However, the role of Min proteins in H. pylori division is poorly understood. We investigated the functional characteristics of Min proteins in wild-type HPK5 and five HPK5-derivative mutants using morphological and genetic approaches. All mutants showed a filamentous shape. However, the bacterial cell growth and viability of three single-gene mutants (minC, minD, minE) were similar to that of the wild-type. The coccoid form number was lowest in the minE-disruptant, indicating that MinE contributes to the coccoid form conversion during the stationary phase. Immunofluorescence microscopic observations showed that FtsZ was dispersedly distributed throughout the bacterial cell irrespective of nucleoid position in only minD-disruptants, indicating that MinD is involved in the nucleoid occlusion system. A chase assay demonstrated that MinC loss suppressed FtsZ-degradation, indicating that FtsZ degrades in a MinC-dependent manner. Molecular interactions between FtsZ and Min proteins were confirmed by immunoprecipitation (IP)-western blotting (WB), suggesting the functional cooperation of these molecules during bacterial cell division. This study describes the intrinsic characteristics of Min proteins and provides new insights into H. pylori cell division.

Leptospira idonii sp. nov., isolated from environmental water.

Strain Eri-1(T) was isolated from a water sample on the campus of Kyushu University, Fukuoka, Japan. The motility and morphology of the isolate were similar to those of members of the genus Leptospira, but the spiral structure of the isolate was sharper under dark-field microscopy. Cells were 10.6 ± 1.3 µm long and 0.2 µm in diameter, with a wavelength of 0.9 µm and an amplitude of 0.4 µm. Strain Eri-1(T) grew in Korthof's medium at both 13 and 30 °C, and also in the presence of 8-azaguanine. 16S rRNA gene-based phylogenetic analysis placed strain Eri-1(T) within the radiation of the genus Leptospira where it formed a unique lineage within the clade of the known saprophytic species of the genus Leptospira. The strain was not pathogenic to hamsters. Strain Eri-1(T) exhibited low levels (11.2-12.6 %) of similarity by DNA-DNA hybridization to the three most closely related species of the genus Leptospira. The DNA G+C content of the genome of strain Eri-1(T) was 42.5 ± 0.1 mol%. These results suggest that strain Eri-1(T) represents a novel species of the genus Leptospira, for which the name Leptospira idonii sp. nov. is proposed. The type strain is Eri-1(T) ( = DSM 26084(T) = JCM 18486(T)).

Molecular typing of Bartonella henselae DNA extracted from human clinical specimens and cat isolates in Japan.

Bartonella henselae is the causative agent of cat scratch disease (CSD). To clarify the population structure and relationship between human and cat strains of B. henselae, 55 specimens isolated in Japan, including 24 B. henselae DNA-positive clinical samples from CSD patients and 31 B. henselae isolates from domestic cats, were characterized by multilocus sequence typing (MLST) and the 16S-23S tRNA-Ala/tRNA-Ile intergenic spacer (S1) sequence, which were used previously for strain typing of B. henselae. Three different sequence types (STs) were identified by MLST, one of which was novel. Fifty-two strains (94.5%), including all strains detected in CSD patients, were assigned to ST-1. Eight S1 genotypes were observed, three of which were novel. The 52 ST-1 strains were classified into seven S1 genotypes, two of which were predominant in both human and cat strains. In addition, 5.5% of the strains (3/55) contained two different intergenic spacer S1 copies. These results indicate that the predominant B. henselae MLST ST-1 in Japan is a significantly genetically diverse population on the basis of the sequence diversity of intergenic spacer S1, and that highly prevalent S1 genotypes among cats are often involved in human infections.

The study of cytopathological aspects induced by human cytomegalovirus infection.

In cytological examination, human cytomegalovirus (HCMV) infection can not be implied unless typical HCMV-infected cells like owl's-eye cells are present. However, such cells are not always observed in HCMV-infection cases. The aim of our study is to establish the cytopathological features induced by HCMV. In vitro transfection and fluorescence in situ hybridization (FISH) were performed on human embryo lung (HEL) cells. Marked cellular aggregation was observed at 6-hr postinfection (hpi). Multinucleated cells, giant cells, and, particularly, small vacuoles were present in the nuclei or cytoplasm before the appearance of inclusion bodies. However, molding and ground glass in nuclei were absent. Cell clusters displayed round cytoplasm, dispersed later, and showed anisocytosis. All features occurred before 48 hpi when the owl's-eye cell appeared. In FISH, the positive signal highlighted viral particles that became predominant and localized in nuclei. These cytological aspects are dependent on viral replication and contribute to the cytological detection of HCMV infection.

[Evaluation of isolation media for the detection of Bartonella henselae--isolation of Bartonella henselae from domestic cats].

Bartonella henselae is a causative agent of cat scratch disease. We preliminarily tested four media for the bacterial growth, including agar plates with sheep, horse or rabbit blood, and chocolate agar. Of these media, rabbit blood and chocolate agar plate were found to be more excellent for the growth than the medium with sheep or horse blood. Blood samples from 60 domestic cats in Yamaguchi Prefecture were then cultured using 7% rabbit blood agar plates and BACTEC9050 (BD), automated blood culture microbial detection system. B. henselae was isolated from six of the 60 (10%) blood samples. Tiny colonies of B. henselae were visible on the agar medium after one week of culture at 35 degrees C in the 5% CO2 atmosphere. BACTEC 9050 detected B. henselae in one of the 10 blood samples and it took two weeks to detect the bacteria automatically, though gram stain failed to show organisms in the blood culture bottle. In conclusion, rabbit blood or chocolate agar and incubation of agar media more than one week and of BACTEC more than two weeks are recommended for the detection of B. henselae.

Evaluation of indirect fluorescence antibody assay for detection of Bartonella clarridgeiae and Seroprevalence of B. clarridgeiae among patients with suspected cat scratch disease.

The possibility of Bartonella clarridgeiae being a causative agent of cat scratch disease (CSD) was investigated by using indirect fluorescence antibody assays with 288 suspected CSD patients. Immunoglobulin G antibody to noncocultivated B. clarridgeiae was suitable only for detection of B. clarridgeiae antibody. Significant cross-reactivity between Bartonella henselae and B. clarridgeiae was noted, and no CSD case caused by B. clarridgeiae was detected.

Degradation process of Mycobacterium leprae cells in infected tissue examined by the freeze-substitution method in electron microscopy.

Mycobacterium leprae cells (strain Thai-53) harvested from infected mouse foot pads were examined by electron microscopy using the freeze-substitution technique. The population of M. leprae cells from the infected tissue consisted of a large number of degraded cells and a few normal cells. These thin sectioned cell profiles could be categorized into four groups depending on the alteration of the membrane structures, and the degradation process is considered to occur in stages, namely from stages 1 to 3. These are the normal cells with an asymmetrical membrane, a seemingly normal cell but with a symmetrical membrane (stage 1), a cell possessing contracted and highly concentrated cytoplasm with a membrane (stage 2), and a cell that has lost its membrane (stage 3). The peptidoglycan layer was found to remain intact in these cell groups.

Comparative studies of the cell structures of Mycobacterium leprae and M. tuberculosis using the electron microscopy freeze-substitution technique.

The cell envelope and cytoplasmic architecture of the Mycobacterium leprae Thai-53 strain were examined using the freeze-substitution technique of electron microscopy and compared with those of the M. tuberculosis H37Rv strain. Both strains had similarly multilayered envelope architectures composed of an electron-translucent layer, a peptidoglycan layer and the plasma membrane, from outside to inside. A comparison of the structures of these two mycobacteria revealed that the M. leprae cell was smaller in size and had a thinner peptidoglycan layer than the M. tuberculosis cell. The cell widths measured on electron micrographs were 0.44 microm for M. tuberculosis and 0.38 microm for M. leprae. The peptidoglycan layer of M. leprae was 4-5 nm, while the corresponding layer of M. tuberculosis was 10-15 nm.

A comparative study and phage typing of silage-making Lactobacillus bacteriophages.

To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus. Phage phiPY4 (group III) infected both L. casei and Lactobacillus rhamnosus. Phage phiPY5 (group IV) specifically infected Lactobacillus casei. Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G + C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli.

Cat scratch disease: analysis of 130 seropositive cases.

To clarify the clinical manifestations of cat scratch disease (CSD), we evaluated a total of 130 seropositive patients with CSD. The patients' ages ranged from 1 to 68 years; 103 (79.2%) were under 18 years of age. CSD occurred predominantly in the fall and winter months. Regional lymphadenopathy was noted in 110 (84.6%) of the cases, and the most common sites were the neck (33%), axillary (27%), and inguinal (18%) regions. One hundred of the patients (77%) had general symptoms, such as fever, headache, and malaise. The clinical manifestations of CSD showed a wide spectrum from typical or classical CSD, with regional lymphadenopathy, to atypical or systemic CSD. Of the 130 cases, 103 (79.2%) were typical CSD and 27 (20.8%) were atypical CSD. Atypical cases of CSD were commonly reported as fever of unknown origin (37.0%), neuroretinitis (22.2%), encephalopathy (14.8%), hepatosplenic granuloma (11.1%), and Parinaud's oculoglandular syndrome (7.4%). Fever of unknown origin or prolonged fever lasting more than 14 days was evident in 27 (20.8%) of the 130 cases in this study. Eleven of the 27 cases lacked lymphadenopathy. Our findings suggest that CSD is not a rare disease in Japan. The indirect fluorescent antibody (IFA) test to detect Bartonella species may provide a prompt diagnosis of CSD and facilitate appropriate therapy.