Nasu H, Iida T, Sugahara T, Yamaichi Y, Park K S, Yokoyama K, Makino K, Shinagawa H, Honda T
Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Clin Microbiol. 2000 Jun;38(6):2156-61. doi: 10.1128/JCM.38.6.2156-2161.2000.
A specific serotype, O3:K6, of Vibrio parahaemolyticus has recently been causing epidemics of gastroenteritis in Southeast Asia, Japan, and North America. To examine whether the new O3:K6 strains possess characteristics that may exacerbate outbreaks, we compared V. parahaemolyticus O3:K6 strains with non-O3:K6 strains using strains isolated from individuals with traveler's diarrhea at Kansai Airport Quarantine Station, Osaka, Japan. All 24 O3:K6 strains possessed a common plasmid, pO3K6 (DNA size, 8,782 bp, with 10 open reading frames [ORFs]). The gene organization of pO3K6 was similar to that of Vf33, a filamentous phage previously described in V. parahaemolyticus. We isolated a phage (phage f237) from the culture supernatant of V. parahaemolyticus O3:K6 strain KXV237, which formed a turbid plaque on an indicator strain. The genome of f237 was single-stranded DNA, and the double-stranded DNA obtained by treatment of the genome with DNA polymerase was identical to that of pO3K6 when analyzed by agarose gel electrophoresis after HindIII digestion. Furthermore, the N-terminal amino acid sequence of the f237 major coat protein was found in ORF4 of pO3K6. Our results showed that pO3K6 is a replicative form of f237. Among the ORFs found in the f237 genome, the sequence of ORF8 had no significant homology to those of any proteins in databases. ORF8 was located on a region corresponding to the distinctive region of Vf33, and its G+C content was apparently lower than that of the remaining DNA sequence of f237. By colony hybridization, ORF8 was detected only in O3:K6 strains isolated since 1996 and was not found in O3:K6 strains isolated before 1996 and clinical V. parahaemolyticus strains other than those of serotype O3:K6. Thus, this study shows that f237 is exclusively associated with recent V. parahaemolyticus O3:K6 strains. The ORF8 gene can be a useful genetic marker for the identification of the recently widespread O3:K6 strains of V. parahaemolyticus.
副溶血性弧菌的一种特定血清型O3:K6最近在东南亚、日本和北美引发了肠胃炎疫情。为了研究新的O3:K6菌株是否具有可能加剧疫情爆发的特征，我们使用从日本大阪关西机场检疫站的旅行者腹泻患者中分离出的菌株，将副溶血性弧菌O3:K6菌株与非O3:K6菌株进行了比较。所有24株O3:K6菌株都携带一个共同的质粒pO3K6（DNA大小为8782 bp，有10个开放阅读框[ORF]）。pO3K6的基因组织与之前在副溶血性弧菌中描述的丝状噬菌体Vf33相似。我们从副溶血性弧菌O3:K6菌株KXV237的培养上清液中分离出一种噬菌体（噬菌体f237），它在指示菌株上形成了浑浊噬菌斑。f237的基因组是单链DNA，用DNA聚合酶处理基因组后得到的双链DNA经HindIII消化后通过琼脂糖凝胶电泳分析，与pO3K6相同。此外，在pO3K6的ORF4中发现了f237主要外壳蛋白的N端氨基酸序列。我们的结果表明pO3K6是f237的复制形式。在f237基因组中发现的ORF中，ORF8的序列与数据库中任何蛋白质的序列均无显著同源性。ORF8位于与Vf33独特区域相对应的区域，其G+C含量明显低于f237其余DNA序列的G+C含量。通过菌落杂交，仅在1996年以来分离出的O3:K6菌株中检测到ORF8，在1996年之前分离出的O3:K6菌株以及血清型O3:K6以外的临床副溶血性弧菌菌株中未发现。因此，本研究表明f237仅与最近的副溶血性弧菌O3:K6菌株相关。ORF8基因可作为鉴定最近广泛传播的副溶血性弧菌O3:K6菌株的有用遗传标记。
