Isolation of enterotoxigenic Pseudomonas fluorescens from Asia region

我们检测了在印度尼西亚泗水市的水中是否存在荧光假单胞菌（产肠毒素P.fluorescens:ETPF），它产生一种类似产肠毒素大肠杆菌（ETEC）的热不稳定肠毒素（LT）。我们从许多地方获得了环境水或家用水，分离出荧光杆菌，用PCR法检测了LT基因。在大约500株菌株中，我们可以得到1株染色体携带LT样基因的菌株。然后，我们从ETPF中确定了LT样基因的全序列。但与ETEC的LT几乎相同。因此，为了分析其在ETPF中的基因如何从ETEC转移，我们一直在测定ETPF的染色体DNA全序列。此外，我们试图从泗水市150名儿童的腹泻粪便中分离出ETPF。其中，虽然未能分离出志贺氏菌、弧菌、致病性大肠杆菌、弯曲杆菌和ETPF等致病菌，但从6例腹泻便中分离出了兔回肠环试验中引起腹泻的假单胞菌。然后，我们尝试净化毒素，并完成了它的净化。发展中国家儿童腹泻药物种类繁多，20%的腹泻药物尚未确定。因此，本项目分离的假单胞菌可能是发展中国家引起儿童腹泻的病原之一。

We examined whether there was Pseudomonas fluorescens (enterotoxigenic P.fluorescens:ETPF), which produced a toxin like heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC), in water at Surabya city in Indoneshia. From many places, we got environmental or house keeping water, isolated P.fluorescens and detected the gene of LT by PCR procedure.Among about 500 strains, we could get one strain of which chromosome carried the gene like that of LT. Then, we determined the full sequence of LT like gene from ETPF. However, it was almost identical to that of LT of ETEC. Therefore, to order to analyze how its gene in ETPF will be transferred from ETEC, we have been determining the full sequence of chromosomal DNA of ETPF.Moreover, we tried to isolate ETPF from diarrheal stools of 150 children in Surabaya city. Among them, although we could not isolate pathogenic bacteria like Shigellas, Vibrios, pathogenic E.coli, Campylobacters and ETPF, Pseudomonas, which caused diarrhea in rabbit ileal loop test, were isolated from 6 diarrheal stools. Then, we tried to purify the toxin, and finished its purification. There are many kinds of diarrheal agents of children in developing country, 20 % of diarrheal agents are not determined. Therefore, Pseudomonas isolated in this project may be one of the agents causing children diarrhea in developing country.