Masaaki Iwaki

Epigallocatechin gallate and gallocatechin gallate in green tea catechins inhibit extracellular release of Vero toxin from enterohemorrhagic Escherichia coli O157:H7.

We studied the effects of six catechin derivatives (catechin, epigallocatechin, epicatechin, epicatechin gallate, epigallocatechin gallate (EGCg) and gallocatechin gallate (GCg)) in green tea on the production and extracellular release of Vero toxins (VTs) from enterohemorrhagic Escherichia coli (EHEC) cultured at 37 degrees C for 24 h. EGCg and GCg in the culture medium markedly inhibited extracellular VTs release from EHEC cells into the culture supernatant fluid at concentrations of 0.05 mg/ml or higher, as estimated by both the reversed passive latex agglutination assay and cytotoxic assay using Vero cells. Production and extracellular release of maltose binding protein, a periplasmic protein, into the culture supernatant were also inhibited by EGCg and GCg, indicating that their inhibitory effect on release from periplasm into the outer milieu is not specific to VTs, but general to the proteins accumulated in EHEC periplasm.

Novel Corynebacterium diphtheriae in domestic cats.

Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae.

Analysis of Bordetella pertussis isolates collected in Japan before and after introduction of acellular pertussis vaccines

Because of recent concern that whole-cell pertussis vaccination can drive antigenic divergence of circulating isolates of Bordetella pertussis, we compared 12 clinical isolates of B. pertussis collected in Japan, the first country to introduce acellular pertussis vaccines, with the vaccine strain. We used pulsed-field gel electrophoresis, sequencing of ptx and prn genes and expression of fimbriae. Most of the isolates collected before or after introduction of acellular vaccine possess similar restriction patterns. They contain ptx genes and prn alleles similar to the vaccine strain and to European isolates collected before the introduction of vaccination. Two recently collected isolates exhibiting a different pulsed-field gel electrophoresis pattern possess ptxS1 and prn alleles similar to the alleles harbored by European isolates circulating currently. Our preliminary results suggest that, if acellular pertussis vaccine-induced antigenic divergence exists, it is likely to be a slow or rare process.

Corynebacterium ulcerans 0102 carries the gene encoding diphtheria toxin on a prophage different from the C. diphtheriae NCTC 13129 prophage

BackgroundCorynebacterium ulcerans can cause a diphtheria-like illness, especially when the bacterium is lysogenized with a tox gene-carrying bacteriophage that produces diphtheria toxin. Acquisition of toxigenicity upon phage lysogenization is a common feature of C. ulcerans and C. diphtheriae. However, because of a lack of C. ulcerans genome information, a detailed comparison of prophages has not been possible between these two clinically important and closely related bacterial species.ResultsWe determined the whole genome sequence of the toxigenic C. ulcerans 0102 isolated in Japan. The genomic sequence showed a striking similarity with that of Corynebacterium pseudotuberculosis and, to a lesser extent, with that of C. diphtheriae. The 0102 genome contained three distinct prophages. One of these, ΦCULC0102-I, was a tox-positive prophage containing genes in the same structural order as for tox-positive C. diphtheriae prophages. However, the primary structures of the individual genes involved in the phage machinery showed little homology between the two counterparts.ConclusionTaken together, these results suggest that the tox-positive prophage in this strain of C. ulcerans has a distinct origin from that of C. diphtheriae NCTC 13129.

Distribution of pertussis antibodies among different age groups in Japan

We examined the distribution of antibody levels against pertussis toxin (PT) and filamentous hemagglutinin (FHA) among a healthy Japanese population aged from 0 to 77 years. Levels of both antibodies in 1108 serum samples collected in 1994 from nine prefectures were assayed using polystyrene ball ELISA. The ratio of individuals positive (>or=10 ELISA U/ml) for anti-PT and anti-FHA antibodies at ages ranging from 0 to 3 years increased rapidly with the increase in the population vaccinated over three times with acellular pertussis (aP) vaccine. However, the ratio of those positive for anti-PT antibody tended to decrease until 6-8 years of age and to increase again from 9 to 19 years among the vaccinated population, although the ratio of individuals positive for anti-FHA antibody remained constant at 80-100% in children and adolescents over 3 years old. Moreover, positivity for anti-PT antibody was high (>or=50 ELISA U/ml) in some serum samples collected from adolescents and young adults, suggesting recent symptomatic or asymptomatic infection with circulating Bordetella pertussis. On the other hand, 50-60% of infants below 12 months of age was below the detection limit (1.0 ELISA U/ml) for anti-PT and anti-FHA antibodies, and most early infants were not vaccinated for pertussis. Since intermittent circulation of B. pertussis remains among the Japanese population, complete vaccination with aP vaccine for all infants should be highly recommended to prevent pertussis.

Two Japanese Corynebacterium ulcerans isolates from the same hospital: ribotype, toxigenicity and serum antitoxin titre

Two toxigenic Corynebacterium ulcerans isolates recovered from pharyngeal swabs of two patients from the same hospital in Japan during 2001-2002 were characterized by PFGE and ribotyping. Toxin production in different culture media was examined and serological analysis of patient sera was performed. The two isolates could not be distinguished by PFGE; however, their ribotypes were distinguishable. One of the isolates could represent a novel ribotype. Analysis of toxin production in different culture media demonstrated that the two isolates produced varying amounts of the diphtheria toxin. Serological analysis showed a greater than sevenfold increase in the serum antitoxin titre during the course of infection in one patient.

Prevalence of Corynebacterium ulcerans in dogs in Osaka, Japan

Diphtheria-like human illness caused by Corynebacterium ulcerans is an emerging threat in developed countries, with incidence sometimes higher than that of diphtheria caused by Corynebacterium diphtheriae. Companion animals are considered a potential source of human infections. In order to determine the prevalence of C. ulcerans among dogs, we performed a screening for the bacterium in 583 dogs in the custody of the Osaka Prefectural government. Forty-four dogs (7.5 %) were positive for the bacterium, although they did not show any clinical symptoms. All bacterial isolates showed resistance or decreased sensitivity to clindamycin, and some showed decreased sensitivity to levofloxacin. Comparative analysis of isolates using PFGE, toxin gene typing and antibiotic sensitivities suggests that transmission between asymptomatic dogs might have occurred.

Genome Organization and Pathogenicity of Corynebacterium diphtheriae C7(−) and PW8 Strains

ABSTRACT Corynebacterium diphtheriae is the causative agent of diphtheria. In 2003, the complete genomic nucleotide sequence of an isolate (NCTC13129) from a large outbreak in the former Soviet Union was published, in which the presence of 13 putative pathogenicity islands (PAIs) was demonstrated. In contrast, earlier work on diphtheria mainly employed the C7(−) strain for genetic analysis; therefore, current knowledge of the molecular genetics of the bacterium is limited to that strain. However, genomic information on the NCTC13129 strain has scarcely been compared to strain C7(−). Another important C. diphtheriae strain is Park-Williams no. 8 (PW8), which has been the only major strain used in toxoid vaccine production and for which genomic information also is not available. Here, we show by comparative genomic hybridization that at least 37 regions from the reference genome, including 11 of the 13 PAIs, are considered to be absent in the C7(−) genome. Despite this, the C7(−) strain still retained signs of pathogenicity, showing a degree of adhesion to Detroit 562 cells, as well as the formation of and persistence in abscesses in animal skin comparable to that of the NCTC13129 strain. In contrast, the PW8 strain, suggested to lack 14 genomic regions, including 3 PAIs, exhibited more reduced signs of pathogenicity. These results, together with great diversity in the presence of the 37 genomic regions among various C. diphtheriae strains shown by PCR analyses, suggest great heterogeneity of this pathogen, not only in genome organization, but also in pathogenicity.

Cytotoxic effects of aflatoxin B1 and its association with cellular components in chicken embryo primary cultured cells.

We have examined the cytotoxicity and cellular incorporation of aflatoxin B1 (AFB1) in several types of established and primary cultured cells. The inhibition of DNA synthesis by AFB1 at 1 microgram/ml was about 0-30% in the established cell lines, including human hepatic cells. In chicken primary hepatocytes, however, DNA synthesis as well as RNA and protein syntheses were strongly inhibited by much lower concentrations of AFB1, e.g., 0.1 microgram/ml. In contrast, chicken primary fibroblasts showed almost no significant response to the toxin. Microsomal cytochrome P-450 activities in hepatic tissues were 10-20-fold higher than those in fibroblastic tissues. The amount of [3H]AFB1 incorporated into acid-insoluble materials in the primary hepatocytes was also 10-100-fold more than that in the primary fibroblasts. However, a significant amount of AFB1, which was enough to induce cytotoxic effects on the primary hepatocytes, could be incorporated into the primary fibroblasts when the concentrations of AFB1 were increased. Characterization of the AFB1-associated cellular components showed that most of them were DNA, RNA, and proteins in the primary hepatocytes, while in the primary fibroblasts a large portion of the incorporated AFB1 was recovered from lipid fractions. In addition, the selective binding of [3H]AFB1 to several proteins was observed only in the primary hepatocytes. The possible role of the AFB1-binding proteins are also discussed.

Genetic Characterization and Comparison of Clostridium botulinum Isolates from Botulism Cases in Japan between 2006 and 2011

ABSTRACT Genetic characterization was performed for 10 group I Clostridium botulinum strains isolated from botulism cases in Japan between 2006 and 2011. Of these, 1 was type A, 2 were type B, and 7 were type A(B) {carrying a silent bont/B [bont/(B)] gene} serotype strains, based on botulinum neurotoxin (BoNT) production. The type A strain harbored the subtype A1 BoNT gene (bont/A1), which is associated with the ha gene cluster. The type B strains carried bont/B5 or bont/B6 subtype genes. The type A(B) strains carried bont/A1 identical to that of type A(B) strain NCTC2916. However, bont/(B) genes in these strains showed single-nucleotide polymorphisms (SNPs) among strains. SNPs at 2 nucleotide positions of bont/(B) enabled classification of the type A(B) strains into 3 groups. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) also provided consistent separation results. In addition, the type A(B) strains were separated into 2 lineages based on their plasmid profiles. One lineage carried a small plasmid (5.9 kb), and another harbored 21-kb plasmids. To obtain more detailed genetic information about the 10 strains, we sequenced their genomes and compared them with 13 group I C. botulinum genomes in a database using whole-genome SNP analysis. This analysis provided high-resolution strain discrimination and enabled us to generate a refined phylogenetic tree that provides effective traceability of botulism cases, as well as bioterrorism materials. In the phylogenetic tree, the subtype B6 strains, Okayama2011 and Osaka05, were distantly separated from the other strains, indicating genomic divergence of subtype B6 strains among group I strains.