Makoto Saito

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.

Detection of Clostridium perfringens enterotoxin gene by the polymerase chain reaction amplification procedure

The polymerase chain reaction (PCR) procedure was evaluated to see if it is a simple and rapid method to detect Clostridium perfringens enterotoxin gene. The method, involving the use of two synthesised primers and gene amplification by the PCR procedure, detects a DNA fragment of 364 base pairs of C. perfringens enterotoxin gene by gel electrophoresis. The enterotoxin gene of strains was detected by use of purified chromosomal DNA. The supernatant of sporulating cultures in a sporulating medium was able to be used as template DNA. Template DNA can be obtained by merely culturing the strain in DS medium, a sporulating medium, for 48 h at 37 degrees C. All C. perfringens strains showing positive results in the PCR procedure were demonstrated to produce enterotoxin by a conventional method and all strains showing negative results were enterotoxin negative. To detect the enterotoxin gene in stool specimens by the PCR procedure, the specimen was heat-treated for 10 min at 90 degrees C and cultured in a sporulating medium, the supernatant of which was used as template DNA. From the stool specimens showing positive results in the PCR procedure by this method, enterotoxigenic C. perfringens was isolated from the heat-treated specimens. Thus, it is possible to detect enterotoxigenic C. perfringens in stools without isolation of the organism.

Adenoviral vector–mediated gene transfer of IL-13Rα2 chain followed by IL-13 cytotoxin treatment offers potent targeted therapy for cytotoxin-resistant cancers

Previous studies demonstrated that IL‐13Rα2 chain–overexpressing cancer cells were highly sensitive to IL‐13 cytotoxin (IL13‐PE38QQR) and could be targeted by cytotoxin treatment. However, the majority of human tumors do not express high levels of IL‐13Rα2 chain. To expand the IL‐13 cytotoxin–mediated cancer targeting therapy, we combined cytotoxin treatment with gene transfer of IL‐13Rα2 chain. We constructed a recombinant adenoviral vector carrying the human IL‐13Rα2 gene (Ad‐IL‐13Rα2), which expresses high levels of IL‐13Rα2 chain on infected cells. Human cancer cell lines A549 and HOS, which originally show no IL‐13Rα2 expression and little sensitivity to IL‐13 cytotoxin, were effectively converted to become sensitive to this cytotoxin after Ad‐IL‐13Rα2 infection. The CC50 of IL‐13 cytotoxin for Ad‐IL‐13Rα2‐infected A549 cells was <10 ng/ml, whereas the CC50 for uninfected or control vector‐infected cells was >500 ng/ml. We also examined the antitumor activity of IL‐13 cytotoxin in an established xenograft model of cytotoxin‐resistant human lung tumor. Only a single i.t. injection of Ad‐IL‐13Rα2 markedly enhanced the sensitivity of established tumors to IL‐13 cytotoxin treatment; furthermore, this antitumor effect was significantly sustained for more than 1 month after the last treatment with IL‐13 cytotoxin. Taken together, these results suggest the combination of adenoviral vector–mediated IL‐13Rα2 gene transfer and IL‐13 cytotoxin administration can be an effective targeting approach for several types of IL‐13 cytotoxin–resistant cancers which show no or little expression of IL‐13Rα2 chain.

Cholera toxin producibility by Vibrio cholerae isolated during the cholera outbreak in the NTT Nagoya Hall

An outbreak of cholera occurred among guests of the NTT Nagoya Hall in September 1989. Clinical findings showed that all but one were symptomatic infections out of 44 bacteriologically confirmed cases. In relation to the high incidence of symptomatic infections, we examined cholera toxin (CT) producibility of the isolated V. cholerae. 1. Strains of the NTT case produced 16-256 (mean 130) ng of CT per ml in CAYE-L medium at 30 degrees C and 32-256 (mean 142) ng of CT per ml by Polymyxin B treatment. But strains of past case produced 8-256 (mean 34), 8-128 (mean 44) ng of CT per ml, respectively. Strains of the NTT case produced a larger amount of CT than that of the past cases. 2. Strains of the NTT case produced 512-4096 (mean 2100) ng of CT per ml in CAYE-L medium at 37 degrees C and 1024-2048 (mean 1600) ng of CT per ml by Polymyxin B treatment. But strains of the past case produced 8-64 (mean 25), 8-128 (mean 45) ng of CT per ml, respectively. Strains of the NTT case produced a larger an amount of CT than them of past case. We observed the same phenomenon in AKI medium at 37 degrees C. The yield of CT in CAYE-L medium was greater at 37 degrees C than 30 degrees C. 3. Strains of NTT case grew faster than that of the past case in CAYE-L medium at 37 degrees C. But the growth rate was the same as both strains in AKI and CAYE media.(ABSTRACT TRUNCATED AT 250 WORDS)