Kazunobu Amako

Motility as an intestinal colonization factor for Campylobacter jejuni

The colonization of the intestinal tract of suckling mice by Campylobacter jejuni was examined by orally challenging the mice with a wild-type strain and several nonmotile mutant strains which were isolated after treating the wild-type strain with mutagens. The wild-type strain had colonized the lower portion of the small intestine, the caecum and the colon 2 d after inoculation. Two nonmotile strains, one of which (M8) had lost all the flagellar structure including the filament, the hook and the basal structure, and the other (M1) which had lost only the filament region, were both cleared from the intestinal tract 2 d after challenge. Another nonmotile strain (M14), which had a complete flagellar structure like that of the wild-type strain, did not colonize and was cleared from the intestinal tract like the other nonmotile and nonflagellated strains. One atypically motile strain (M5), which had a shorter flagellar filament than that of the wild-type strain, colonized the intestinal tract only when mice were challenged with a large inoculum. None of the mice challenged with either the wild-type or any of the mutant strains showed signs of illness. We concluded that motility is an important factor in the colonization of the intestinal tract of suckling mice by C. jejuni.

The Release of Outer Membrane Vesicles from the Strains of Enterotoxigenic Escherichia coli

The clinically isolated heat labile enterotoxin (LT)‐producing strains of Escherichia coli can be separated into two groups, namely spontaneous LT‐releasing strain and non‐spontaneous LT‐releasing strain, based on their phenotypes of spontaneous release of LT into the culture medium. The phenotype of spontaneous LT release was observed to correlate closely with the phenotype of the release of numerous small vesicles into the culture medium. Both morphological and biological examinations of the vesicle showed that the vesicle was released from the outer membrane. It can, therefore, be assumed that LT may be released from the cell at the time the vesicles form.

Typing of Staphylococcus aureus colonising human nasal carriers by pulsed-field gel electrophoresis

Colonisation by Staphylococcus aureus in the nares of 120 outpatients and 63 healthy adults was studied for c. 2 years. Two states of carriage of S. aureus were confirmed: persistent carriage and persistent non-carriage. The states of carriage and non-carriage were quite stable and > 60% of the population of any of the study groups were stable non-carriers. The results of typing the strains isolated from the same individuals at different times with DNA fingerprinting by digestion with SmaI enzyme showed that all the stable carriers were persistently infected with the same strain and that changes in the strain seldom occurred.

Construction of a ferritin-deficient mutant of Campylobacter jejuni: contribution of ferritin to iron storage and protection against oxidative stress

The ferritin‐encoding gene (cft) of Campylobacter jejuni was cloned and sequenced. The nucleotide sequence of cft had a 501 bp open reading frame for a protein with 167 amino acids and a predicted molecular mass of 19180 Da, and showed a high similarity to that of Helicobacter pylori and Escherichia coli ferritin genes. To determine the biological function of ferritin in C. jejuni, a ferritin‐deficient mutant was constructed. The growth of ferritin‐deficient strain SNA1 was clearly inhibited under iron deprivation. The ferritin‐deficient mutant was more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These findings demonstrate that ferritin in C. jejuni makes a significant contribution to both iron storage and protection from intracellular iron overload, and resulting iron‐mediated oxidative stress.

Evidence for the presence of a capsule in Vibrio vulnificus.

Vibrio vulnificus strain FCC, isolated from a patient with a wound infection, and reference strain ATCC 27562, were examined by electron microscopy for the presence of capsules. Both strains had a layer heavily stained with ruthenium red. The number of stained cells was high in strain FCC and low in strain ATCC 27562. The proportion of stained cells correlated with virulence against mice and with susceptibility to the bactericidal activity of normal human serum. Rapid freezing and substitution fixation, a mild method, revealed on the cell surface a fibrous layer of relatively low electron density, which we considered to represent a capsule.

Release of the Outer Membrane Vesicles from Vibrio cholerae and Vibrio parahaemolyticus

We found numerous small vesicles released from the cell by thin sectioning of the plate culture of Vibrio cholerae and V. parahaemolyticus fixed with the freeze‐substitution technique. From the broth media of exponentially growing bacteria we could collect the vesicles by the centrifugation but not enough without fixation. The vesicles are encompassed with a membrane structure similar to the outer membrane of these bacteria. The anti‐O (Inaba) serum reacted with the surface of the vesicles and the inside of the vesicle are generally filled with an electron‐dense mass.

Surface Characteristics of Gram-Negative and Gram-Positive Bacteria in an Atomic Force Microscope Image

Bacterial images can be obtained rather easily with an atomic‐force microscope (AFM) in the magnification range of 5,000 to 30,000 times without any pretreatment of the specimens for such observations as chemical fixation, dehydration or staining. The bacterial shapes or the presence of flagella can be clearly recognized in these magnification ranges. In addition, we were also able to distinguish between Gramnegative and Gram‐positive bacteria based on the specific wavy surface appearance of the former. AFM could thus be a useful tool for the identification of bacteria in the resolution range between electron and light microscopy.

Ferritin from the obligate anaerobe Porphyromonas gingivalis: purification, gene cloning and mutant studies

Porphyromonas gingivalis is an obligate anaerobe that utilizes haem, transferrin and haemoglobin efficiently as sources of iron for growth, and has the ability to store haem on its cell surface, resulting in black pigmentation of colonies on blood agar plates. However, little is known about intracellular iron storage in this organism. Ferritin is one of the intracellular iron-storage proteins and may also contribute to the protection of organisms against oxidative stresses generated by intracellular free iron. A ferritin-like protein was purified from P. gingivalis and the encoding gene (ftn) was cloned from chromosomal DNA using information on its amino-terminal amino acid sequence. Comparison of the amino acid sequence deduced from the nucleotide sequence of ftn with those of known ferritins and bacterioferritins identified the protein as a ferritin and positioned it between proteins from the Proteobacteria and Thermotogales. The P. gingivalis ferritin was found to contain non-haem iron, thus confirming its identity. Construction and characterization of a P. gingivalis ferritin-deficient mutant revealed that the ferritin was particularly important for the bacterium to survive under iron-depleted conditions (both haemin and transferrin starvation), indicating that intracellular iron is stored in ferritin regardless of the iron source and that the iron stored in ferritin is utilized under iron-restricted conditions. However, the ferritin appeared not to contribute to protection against oxidative stresses caused by peroxides and atmospheric oxygen.

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 Korthofs 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)).

Purification and characterization of ferritin from Campylobacter jejuni

We purified an iron-containing protein fromCampylobacter jejuni using ultracentrifugation and ionexchange chromatography. Electron microscopy of this protein revealed circular particles with a diameter of 11.5 nm and a central core with a diameter of 5.5 nm. The protein was composed of a single peptide of 21 kDa and did not serologically cross-react with horse spleen ferritin. The UV-visible spectrum of the protein showed no absorption peaks in the visible region, indicating that little or no heme is bound. The ratio of Fe:phosphate ofC. jejuni ferritin was 1.5∶1. From these morphological and chemical examinations, we concluded that theC. jejuni purified protein is a ferritin of the same class as that ofHelicobacter pylori andBacteroides fragilis and differs from the heme-containing bacterioferritin ofEscherichia coli. The 30 N-terminal amino acids were sequenced and were found to resemble the sequences of other ferritins strongly (H. pylori ferritin, 73% identity;B. fragilis ferritin, 50% identity;E. coli gene-165 product, 50% identity), and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 26% identity;Azotobacter vinelandii, 26% identity; horse spleen ferritin 30% identity). Proteins that cross-reacted with antiserum against the ferritin ofC. jejuni were found in otherCampylobacter species and inH. pylori, but not inVibrio, E. coli, orPseudomonas aeruginosa.