Yotaku Gyobu

Rapid Categorization of Pathogenic Escherichia coli by Multiplex PCR

A one‐shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli (EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli (EPEC); invE for enteroinvasive E. coli (EIEC); elt, estp, and esth for enterotoxigenic E. coli (ETEC); CVD432 and aggR for enteroaggregative E. coli (EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.

emm Typing of Group A Streptococcus Clinical Isolates: Identification of Dominant Types for Throat and Skin Isolates

T and emm types were determined for group A streptococci isolated from patients with various infections during 1990–1999 in Toyama Prefecture, Japan. Out of 906 isolates, 872 isolates were divided into 20 T serotypes, and 34 isoltes were T nontypeable (TNT). T12, T1, and T4 were dominant among 699 throat isolates; on the other hand, T11, T28, TB3264, and TNT were dominant among 80 skin isolates. The emm types of 190 isolates were determined following specific PCR amplification and sequencing of the products. Twenty T serotypes were divided into 34 T type/emm type combinations. Thirty‐four TNT isolates were divided into 14 emm types, in which emm58 was the most common (38%). Among 82 throat isolates randomly selected, predominant T types T12, T1, and T4 isolates were of the respective same numbers in emm type. T11/emm89, T28/emm28, TB3264/emm 13w, and TNT/emm58 were predominant among 80 skin isolates. emm‐type distribution observed in the present study was that usually reported in the western world. To our knowledge, 3 T/emm is a novel combination. These results show that emm typing allows the characterization of group A streptococci from various sources.

Production and Partial Purification of a Fluid‐Accumulating Factor of Non‐O1 Vibrio cholerae

A fluid‐accumulating factor (FAF in the ligated rabbit ileal loop test) from a strain of non‐O1 Vibrio cholerae not producing cholera toxin‐like enterotoxin (CTLT) was partially purified by ammonium sulfate precipitation, gel filtration with Sephadex G‐100, and DEAE cellulose column chromatography. The preparation thus obtained showed collagenolytic, cytolytic, necrotic, and hemorrhagic activities, but was not lethal to mice nor hemolytic to sheep erythrocytes. Desquamation of epithelial cells, inflammatory edema, and hemorrhage were observed in sections of rabbit intestine after inoculation of partially purified FAF (PPFAF). Biological and enzymatic activities of FAF were completely neutralized with anti‐PPFAF rabbit serum. More than 70% of non‐O1 V. cholerae strains from human diarrheal feces produced FAF in the shake culture of heart infusion broth (Difco). A fluid‐accumulating factor immunologically similar to FAF of non‐O1 V. cholerae was also produced by V. mimicus strains isolated from human diarrheal feces. These results indicate that the FAF produced by CTLT‐negative non‐O1 V. cholerae strains is an entity closely related to a cytolytic and hemorrhagic substance or the like, and that this FAF may play a role in the enteropathogenicity of CTLT‐negative strains.

Ecology of Non-O 1 Vibrio cholerae in Toyama Prefecture

The ecology of non‐O 1 Vibrio cholerae and Vibrio mimicus as causes of cholera‐like diarrhea or seafood‐associated gastroenteritis has been investigated in Toyama Prefecture since 1980. The relationship between biological or serological characteristics of the isolates and their enteropathogenicity is discussed.

Studies on the Enteropathogenic Mechanism of Non-O 1 Vibrio cholerae Isolated from the Environment and Fish in Toyama Prefecture

Enteropathogenic mechanisms of non‐O 1 Vibrio cholerae were investigated using strains from the environment and those from fish in Toyama Prefecture.

Response of Ligated Rabbit Ileal Loop to Clostridium perfringens Type C Strains and Their Toxic Filtrates

The vegetative cells and toxic filtrates of Clostridium perfringens type C strains were injected into ligated rabbit ileal loops and the responses were observed. Out of 12 strains examined, 2 strains showed positive reaction in this test, when the vegetative cells were injected. One of these 2 strains was an enterotoxigenic and beta‐toxigenic and the other was beta‐ and delta‐toxigenic but not enterotoxigenic. Culture filtrates containing beta or delta toxin also showed fluid accumulation in the rabbit ileal loop. Histological findings of loops injected with culture filtrates containing beta toxin showed separated and effaced villi, hemorrhage in the mucosa, engorged vessels, inflammatory cell infiltration, and hyperplasia of the lymphoid tissue.

Comparison between the Aged and Children of the Normal Distribution of Pharyngeal and Intestinal Hemolytic Streptococci

A marked difference between the aged and children has been observed in the group distribution of hemolytic streptococci isolated from various clinical specimens. Group B strains from the urine and sputum, and group G from the sputum, pus and exudate have been predominant in the aged, whereas group A strains from the throat swab have dominated in children. The present study was undertaken to clarify the background for such a marked difference by investigating the normal state of pharyngeal and intestinal carriage of hemolytic streptococci both in the aged and children. 1. As to the pharyngeal carriage, quite a contrast was observed between the aged and children. In the former, the rate of carries was low and the predominant groups among the streptococcus isolates were B (Streptococcus agalactiae) and G (identified as Streptococcus equisimilis), while in the latter, the rate was high and group A (Streptococcus pyogenes) strains comprised approximately 75% of the isolates, most of them being from the throat swab. 2. Both the aged and children showed a similar state of streptococcus carriage in the intestine. The rate of carriers was low and the predominant group among the isolates was B in both populations. Although group G strains were occasionally isolated, group A strains were isolated neither in the aged nor in children. These results explain well the difference between the aged and children in the group distribution of the clinical isolates of hemolytic streptococci.

Ecological studies on hemolytic streptococci in aged population

The pattern of group distribution of hemolytic streptococci and the source of specimen, when these organisms were isolated from the clinical specimen of the aged, were quite different from that of streptococcosis among children. The isolation frequency from the upper respiratory tract was rather low in comparison to the other specimen, such as sputum, exudate and urine. Both groups B and G were predominant among isolates from sputum, B more prevailing than G from exudate, and the isolates from the urinary tract were consisted almost solely of group B. These trends have been unchanged in the past 3 years. Normal pharyngeal streptococcus flora among the aged also differ from that of healthy children, the carrier rate being rather low, and groups B and G, instead of A in the case of children, being predominant. This pattern of group distribution, among the isolates of normal pharyngeal streptococci of the aged, seemed to be reflected on that of isolates from clinical specimen, especially those from the sputum. The type distribution of group B isolates from normal throat swabs of the aged resemble that of clinical isolates from the same generation, types Ia and III dominating in both cases.