Yasumoto Suzuki

Characterization of Shiga Toxin-Producing Escherichia coli O26 Strains and Establishment of Selective Isolation Media for These Strains

ABSTRACT We characterized the carbohydrate-fermenting ability of 31 strains of Shiga toxin-producing Escherichia coli (STEC) O26 isolated from diarrhea patients in Aichi Prefecture, Japan, in order to establish selective isolation media for these strains. None of the 31 STEC O26 strains (24 O26:H11, 7 O26:H−) fermented rhamnose, whereas all of the other 108 STEC strains (100 O157, 8 O111) and all of the non-STEC strains except one (i.e., 58 of 59) fermented rhamnose. The great majority of the STEC O26 strains (96.8% [30 of 31]) showed very high resistance to potassium tellurite (MIC ≥ 50 μg/ml), whereas the majority of the non-STEC strains (72.9% [43 of 59]) showed very high sensitivity (MIC ≤ 1.56 μg/ml) to this compound. Accordingly, we developed a rhamnose-MacConkey (RMAC) medium in which the lactose in MacConkey medium was replaced by rhamnose, and cefixime-tellurite-RMAC (CT-RMAC) medium in which potassium tellurite (2.5 mg/liter) and cefixime (0.05 mg/liter) were added to RMAC. All of the STEC O26 strains generated colorless (rhamnose-nonfermented) colonies on both media; the vast majority of selected E. coli strains (95.7% [89 of 93; including 26 STEC O157, 8 STEC O111]), other than STEC O26, generated red colonies on RMAC, and most of the non-STEC strains (84.7% [50 of 59]) did not grow on CT-RMAC. We demonstrate that both the RMAC and the CT-RMAC media can be used for the isolation of STEC O26 and that CT-RMAC has better specificity for the routine isolation for STEC O26 in a laboratory.

Molecular Epidemiology of Salmonella enteritidis. An Outbreak and Sporadic Cases Studied by means of Pulsed-field Gel Electrophoresis

Large outbreaks of diarrhoea due to Salmonella enteritidis in Aichi-ken, Japan, provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. This was performed by comparing the plasmid profile types, phage types, antimicrobial resistance, and the restriction fragment length polymorphisms (RFLPs) by pulsed-field gel electrophoresis (PFGE) of S. enteritidis from outbreaks and sporadic cases. Among the isolates studied, 10 distinctive RFLP types were found with XbaI and four with NotI, while 12 combination types were identified among the 68 isolates from 16 Health Centres in Aichi-ken, Japan. A total of 22 isolates from four outbreaks, however, had the same RFLP and phage types. The RFLP type was subdivided by means of the plasmid profile and phage type. Conversely, plasmid profile and phage type were separated by means of RFLP. This PFGE method may prove useful for subclassifying S. enteritidis and differentiating isolates of the same plasmid profile and phage type.

Serotype-specific antigen ELISA for detection of Chiba virus in stools.

Chiba virus (CV), a Norwalk‐like virus (NLV), was first identified as a cause of oyster‐associated outbreak of gastroenteritis that occurred in Chiba prefecture, Japan, in 1987. An enzyme‐linked immunosorbent assay (ELISA), based on hyperimmune antisera to recombinant baculovirus‐expressed capsid proteins of CV (rCV), was developed to detect CV antigen in stools. No cross‐reactions were observed with other enteric viruses including enteroviruses, rotaviruses, astroviruses, or enteric adenoviruses. The ELISA was used to screen 101 stools collected from 16 oyster‐associated outbreaks of acute gastroenteritis. Twelve stools (11.9%) from seven outbreaks were positive for CV antigen. Ten rCV ELISA‐positive strains were confirmed by RT‐PCR and nucleotide sequencing. ELISA‐positive strains showed 96–100% nucleotide sequence identity to each other, though they were obtained nine years apart. Phylogenetic analysis demonstrated that all ten strains clustered with the prototype CV in genogroup I viruses. We concluded that the antigen ELISA described in this study is highly type‐specific, and that this method should be useful for epidemiological surveys of Chiba virus infections. J. Med. Virol. 62:233–238, 2000.

Pulsed-field gel electrophoretic analysis of Campylobacter jejuni DNA for use in epidemiological studies

The cleavage patterns of the genomic DNA of 42 clinical isolates of Campylobacter jejuni determined by pulsed-field gel electrophoresis (PFGE) were compared with their Lior and TCK serotypes. The fragment patterns of DNA obtained with SalI and SmaI restriction enzymes did not always accord with the corresponding serotypes but strains of the same serotype could be further divided into subtypes by their cleavage patterns. This PFGE method may prove useful for subclassifying C. jejuni.

Discrimination by means of pulsed-field gel electrophoresis between strains of Campylobacter jejuni Lior type 4 derived from sporadic cases and from outbreaks of infection

The cleavage patterns of the genomic DNA in 42 strains of Campylobacter jejuni Lior type 4, obtained from sporadic cases and from outbreaks of infection were analysed by means of pulsed-field gel electrophoresis (PFGE). The cleavage of DNA with SmaI and SalI restriction enzymes showed 16 distinct fragment patterns in 20 sporadic isolates, indicating that they were heterogeneous. On the other hand, the patterns of 22 isolates derived from two outbreaks showed the same unique restriction patterns, respectively. PFGE may therefore prove useful for subclassifying strains of C. jejuni Lior type 4 and for discriminating between strains derived from sporadic cases and those derived from outbreaks of infection.

Restriction fragment length polymorphisms analysis by pulsed-field gel electrophoresis for discrimination of Staphylococcus aureus isolates from foodborne outbreaks.

A number of outbreaks of disease due to Staphylococcus aureus occurring in Aichi-ken, Japan, have provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. Coagulase types, enterotoxin types, phage types, and restriction fragment length polymorphisms (RFLPs) as assessed by pulsed-field gel electrophoresis (PFGE) was performed for S. aureus infections diagnosed in the area of Aichi-ken. Among the 56 isolates of S. aureus from 30 outbreaks, 15 distinctive RFLP types were found by digestion with the restriction enzyme, SmaI. A total of 32 isolates from patients, foodstuffs and cooks on six occasions had the same RFLP types, coagulase types, enterotoxin types and phage types in the same outbreaks. Moreover, the coagulase and phage types could be separated in terms of RFLP. In one outbreak, ten isolates, which were derived from six patients, two foodstuffs and two cooks, had the same coagulase type, enterotoxin type, phage type, and RFLP type. This PFGE method may therefore prove useful for subclassifying S. aureus and differentiating isolates of the same coagulase types and phage types derived from sporadic cases and those derived from foodborne outbreaks.

愛知県における腸炎ビブリオO3: K6血清型による食中毒および散発性下痢症の発生動向 (1988～2001年) と海産魚介類からのO3: K6の検出

Percentage of the outbreaks by O3:K6 Vibrio parahaemolyticus (V. p) in Aichi Prefecture Japan increased from 3% (3/86) for 1988-95 to 75% (33/44) for 1996-2001. The percentage of the sporadic diarrhea cases caused by O3:K6 V. p in a general hospital in Aichi Prefecture also increased from 0% (0/253) to 61% (135/221) during the same periods. Thermostable direct hemolysin (TDH)-positive O3:K6 were isolated from 95% (19/20) of the outbreak incidents and 100% (135/135) of the sporadic cases. Only one TRH (TDH-related hemolysin)-positive O3:K6 was isolated from one outbreak incident. Percentage of the outbreaks by O3:K6 V. p associated with the consumption of boiled shellfishes increased from 5% (6/117) for 1988-95 to 25% (15/59) for 1996-2001, in particular, boiled crabs and squillas associated outbreaks increased from 2% (2/117) to 17% (10/59) and from 2% (2/117) to 10% (6/59), respectively. From 1,548 raw sea foods sampled in the Nagoya Central Wholesale Market in Aichi Prefecture in 1995-99, one TDH-positive O3:K6 was isolated from one live squilla (1/30). Increase in the percentage of outbreaks associated with TDH-positive O3:K6 V. p after 1996 in Aichi Prefecture was revealed to correlate with the increase in the outbreaks associated with consumption of boiled sea foods, especially boiled crabs as well as squillas. Accordingly, it becomes clear that sanitary handling of these boiled foods is important to prevent outbreaks and sporadic cases of diarrhea caused by O3:K6 V. p infection.

Histological and Functional Changes in the Thyroid Glands of Mice Implanted With Hybridomas Secreting Monoclonal Autoantibody Against Mouse Thyroglobulin

Mouse hybridoma cells secreting monoclonal antibody (mAb) against mouse thyroglobulin were established. The implantation of the hybridomas succeeded to induce high titers of circulating mAb against thyroglobulin in sera of mice. By using the implantation of the hybridomas in mice, the effect of autoantibody on the thyroid glands was studied histologically and functionally. In these mice the thyroid follicles were significantly swollen and warped, whereas there was no infiltration of inflammatory cells. The 125I-uptake in their thyroid glands was markedly decreased. There were no functional changes in control mice implanted with non-secreting P3U1 partner cells. Therefore, it was suggested that high titers of anti-thyroglobulin autoantibody could definitely cause the histological and functional damages in the thyroid glands.

Epidemiologic study of Shigella sonnei from sequential outbreaks and sporadic cases using different typing techniques.

We noted that eight outbreaks of Shigella sonnei from an unknown source occurred sequentially in Aichi Prefecture, Japan, between October 1992‐June 1993. For comparative purposes we analyzed 53 outbreak‐related isolates of Shigella sonnei using different subtyping methods and studied the epidemiology of the outbreaks. It appeared from our study that DNA‐based techniques such as plasmid typing and pulsed‐field gel electrophoresis (PFGE) were more useful tools for subtyping Shigella sonnei than colicin typing and the antimicrobial susceptibility test. Moreover, according to PFGE analysis, four genetically related isolates of Shigella sonnei were responsible for the eight sequential outbreaks. To further investigate the epidemiology of outbreaks, 58 sporadic isolates of Shigella sonnei from overseas travelers with shigellosis during the same period were also examined. We found that some sporadic isolates from travelers in Asia were genetically related to those of the outbreak‐related isolates, indicating that genetically related isolates prevailed in Asia during this period, probably because of the extensive movement of people or food.

A new-born baby outbreak of echovirus type 33 infection

OBJECTIVES In 1994, an outbreak of echovirus type 33 (EV33) infection occurred in a maternity hospital in Japan. Nine new-born babies were infected, some presenting symptoms of encephalitis or disseminated intravascular coagulopathy. EV33 was isolated from the faeces or cerebrospinal fluid of all seven of the patients sampled, and serum antibody titres against EV33 were significantly elevated in the convalescence phase sera in all cases. SUBJECTS AND METHODS To study what public health situations EV33 may become a serious pathogen for new born babies, serum EV33 antibody positivity in the general population was examined. Sera were obtained 649 samples before the outbreak, and 344 samples after the outbreak from aged 7 days to 65 years old. RESULTS The average positive rate was 12.0% and the rate increased depending on age. Comparison of positive rates before and after this outbreak showed no increase in any age group. However, the positive rate was found to average only 5.6% in persons aged 16-30 years old, including pregnant women. This low positive rate in young adults would result in a lack or only a low level of antibodies in newborn babies. CONCLUSIONS In conclusion, our findings suggest that EV33 infection in the new-born baby with no or low level of maternal neutralizing antibody may cause serious symptoms.