[Isolation of Neisseria meningitidis from healthy persons in Japan].

Between September 2000 and March 2003 healthy subjects in 10 prefectures of Japan were investigated to identify carriers of Neisseria meningitidis. Twenty-five N. meningitidis strains were isolated from 5886 throat swab specimens collected from healthy persons, such as students, elderly, and foreigners. Of the 25 carriers, 9 were teenagers, 15 were in their twenties, and only one was in the fifties. The male-female ratio of the carriers was 17 to 8, showing male dominance. The serogroups of the 25 strains were B (9 strains), Y (4 strains) and non-groupable (12 strains). One of the strains was found to be deficient in gamma-glutamyl aminopeptidase activity, which is an identification marker for N. meningitidis.

Prevalence of Salmonella spp. in pet reptiles in Japan.

From November 2000 to July 2002, 112 fecal samples from pet reptiles, including 18 turtles, 71 lizards and 23 snakes, sold at a pet shop were examined for the prevalence of Salmonella spp. in Japan. Salmonella spp. were isolated from 83 (74.1%) of 112 samples, and a total of 112 Salmonella isolates were identified as subspecies I to IV. The majority of isolates (62.5%) belonged to subspecies I and 54 isolates could be identified as any of 28 serovars. The predominant serovars were found to be S. Bardo, S. Newport and S. Panama, which cause human salmonellosis. These results indicate that pet reptiles may be a potential infectious source of human salmonellosis in Japan.

Reliability of the detection of meningococcal gamma-glutamyl transpeptidase as an identification marker for Neisseria meningitidis.

Detection of gamma-glutamyl transpeptidase (GGT; ggt ) activity is one of the useful methods for a specific identification of Neisseria meningitidis. However, we previously happened to isolate a ggt -deficient N. meningitidis strain (NIID113) from a healthy carrier. In this study, in order to re-examine the reliability of the marker, we again investigated the GGT activity of 245 N. meningitidis human isolates and identified two other GGT-defective N. meningitidis isolates besides NIID113. The isolation frequency (1.2%) of ggt mutants among human isolates strongly confirmed the 98.8% reliability of GGT activity as the identification marker for N. meningitidis.

Characterization of Neisseria meningitidis isolates collected from 1974 to 2003 in Japan by multilocus sequence typing.

Analysis of 182 Neisseria meningitidis strains isolated over the past 30 years in Japan by serogroup typing and multilocus sequence typing (MLST) was performed. The serogroups of the 182 Japanese isolates were B (103 isolates), Y (39), W135 (1) and non-groupable (39). By MLST analysis, 65 different sequence types (ST) were identified, 42 of which were not found in the MLST database as of January 2004 and seemed to be unique to Japan. Statistical analysis of the MLST results revealed that, although the Japanese isolates seemed to be genetically divergent, they were classified into six major clonal complexes and other minor complexes. Among these isolates, well-documented ST complexes found worldwide were present, such as ST-23 complex (49 isolates), ST-44 complex (41 isolates) and ST-32 complex (8 isolates). On the other hand, a new clonal complex designated ST-2046 complex (28 isolates), which has not been identified in other countries, was also found, suggesting that this clone was indigenous to Japan. Taken together, it was speculated that meningococcal isolates in Japan comprised heterogeneous clones, which were derived both from clones identified in other countries and clones unique to Japan.

An improved enrichment broth for isolation of Escherichia coli O157, with specific reference to starved cells, from radish sprouts.

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.

[Influence of the conditions of storage and cooking on growth, invasion and survival of Salmonella enteritidis in eggs].

Basal studies for the confirmation of sanitary rules in the kitchen were performed, focusing on preventing an outbreak of food poisoning due to eggs contaminated with Salmonella Enteritidis (SE), using hen and quail eggs. SE did not grow at 5 degrees C but grew markedly at 25 degrees C in eggs. The invasion and growth of SE were marked under very humid conditions regardless of whether the eggshell was damaged. The invasion of SE into egg also occurred when eggs were taken in and out of the refrigerator. Moreover, SE was spread immediately to all non-contaminated eggs when SE-contaminated eggs were cracked into a bowl with non-contaminated eggs. In homemade mayonnaise containing 15% vinegar, sterilization took several hours to occur. On a stainless-steel bowl, SE survived for 2 weeks or more. These findings suggest that it is necessary to pay attention to secondary contamination.

Isolation from a healthy carrier and characterization of a Neisseria meningitidis strain that is deficient in gamma-Glutamyl aminopeptidase activity.

gamma-Glutamyl aminopeptidase (GGT) activity is used as a specific marker for the identification of Neisseria meningitidis. We isolated from a healthy carrier and characterized an N. meningitidis isolate which lacked the activity due to the insertional mutation of the ggt gene, suggesting that naturally occurring N. meningitidis isolates do not always possess GGT activity.

[Shigella boydii strains possessing a new serovar (SM00-27) isolated from diarrhea of overseas travelers in Japan].

Five Shigella strains isolated from stool cultures of five sporadic imported diarrheal cases in Japan during 1999-2001, did not react to any antisera of the established Shigella serovars. These strains had the typical biochemical characteristics of Shigella boydii, and were biochemically identical. All strains were positive in a PCR assay and a cultured-cell invasion test for invasiveness; these indicate that they can cause shigellosis in humans. The results of antigenic analysis revealed that they did not belong to any of the recognized or provisional serovars, and were serologically indistinguishable. Strain SM00-27 is designated as the test strain for this new S. boydii serovar.

Evaluation of sorbitol-salicin MacConkey medium containing cefixime and tellurite (CT-SSMAC medium) for isolation of Escherichia coli O157:H7 from raw vegetables.

The utility of CT-SSMAC medium (sorbitol-salicin MacConkey medium containing cefixime and tellurite) for the isolation of Escherichia coli O157:H7 from raw vegetables was investigated. The colonies of all E. coli O157:H7 and O157:NM strains tested were colorless and beta-galactosidase-positive on CT-SSMAC medium. Furthermore, the number of colorless colonies on the CT-SSMAC medium was less than that on the sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) from several raw vegetable samples. All colorless colonies grown on CT-SSMAC medium from raw vegetable samples were beta-galactosidase-negative. These findings suggest that the CT-SSMAC medium is useful for the isolation of E. coli O157:H7 from raw vegetable samples.

Levels of thermostable direct hemolysin produced by Vibrio parahaemolyticus O3:K6 and other serovars grown anaerobically with the presence of a bile acid.

Twenty-three V. parahaemolyticus strains, including 12 pandemic O3:K6 strains, were examined for their growth and production of thermostable direct hemolysin (TDH) under an anaerobic culture condition with or without presence of a bile acid, taurocholic acid (TCA). Both bacterial growth and TDH production were markedly enhanced by TCA for a majority of the strains, but the scale of the TDH production was disproportionately greater than that of the corresponding growth for 14 strains. Such enhancement was, however, not specific to the pandemic strains.

Genotypic variations of Shiga toxin-converting phages from enterohaemorrhagic Escherichia coli O157: H7 isolates.

Pulsed-field gel electrophoresis (PFGE) analysis revealed that enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains had considerable variations in their genomes. This study investigated whether or not the molecular profile of Shiga toxin (Stx) 1- and Stx2-converting phages isolated from EHEC O157:H7 strains, derived from various sources in the USA and Japan, corresponded to the variations of host strains' genotypes as determined by PFGE. A total of 51 Stx-converting phages including 12 Stx1-converting phages and 37 Stx2-converting phages was isolated from seven USA isolates and 20 Japanese isolates. The average Dice coefficient values showed 44% similarity between phage DNAs in Stx2-converting phages digested with SmaI and 55% in Stx1-converting phages digested with HindIII, indicating considerable variation among phage DNA. In particular, restriction fragment length polymorphism (RFLP) patterns of Stx2-converting phage DNA varied according to the PFGE type of their host strain, which suggests that the phage genomes have altered their genotypic characteristics with those of host genomes. However, there are several exceptions: the RFLP patterns of some Stx2-converting phages were quite similar irrespective of the different genotypes of the host strains, indicating that horizontal transfer of Stx2-converting phage may also occur under some circumstances.