Molecular Epidemiology of Vibrio cholerae O1 Isolated from Sporadic Cholera Cases in Okinawa, Japan

Abstract

In July 1994, 6 cholera cases due to Vibrio cholerae O1 El Tor Ogawa sporadically appeared in Okinawa. All 6 patients had no history of traveling abroad. In the period of this cholera outbreak, a strain of V cholerae O1 El Tor Ogawa was detected from an imported fish at the Naha port quarantine station. The isolates were characterized to clarify whether or not, they belonged to a common clone. Phenotypes were identical except that one strain revealed cured Celebes and the others were original Celebes in kappa phage typing. The restriction fragment patterns of DNA of the isolates hybridized with an enzyme-labeled oligonucleotide probe for cholera toxin gene (ctx) were identical. Randomly amplified polymorphic DNA of the isolates were identical when a primer was used, but 2 patterns were seen when another primer was used. Pulsed-field gel electrophoresis of the chromosomal DNA digested with Notl restriction enzyme showed 3 patterns. The DNA fragment pattern of the strain isolated from the imported fish was different from the clinical isolates. These results suggested that there was no epidemiological relation among the strains of V cholerae O1 isolated during this period.Key words: Vibrio cholerae O1, Okinawa, Genotype, Epidemiology Cholera is not endemic in Japan, however, approximately 100 cases per year have been reported over the past few decades. Although most of the cases are associated with traveling abroad, some cases without any history of traveling abroad have also been seen. In July 1994, 6 cases of cholera patients sporadically appeared in Okinawa, where cholera cases have not been diagnosed for a long time. The patients in Okinawa were infected with Vibrio cholerae O1 El Tor Ogawa, and they had no history of traveling abroad. In spite of an intensive investigation, the vehicle of transmission could not be specified. V cholerae O1 El Tor Inaba has been detected in a river in Naha City but was non-toxigenic (3). Toxigenic V cholerae O1 has never been isolated from the environment of Okinawa. Toxigenic V cholerae occasionally been detected in imported marine products at the Sea Port Quarantine in Japan. Indeed, in Okinawa, toxigenic V cholerae O1 was isolated at the same time of that from the imported fish at the Naha quarantine station. In this study, the V cholerae isolates were phenotypically and genetically compared to examine a possibility that the strains were epidemiologically related. Materials and MethodsBacterial strains. Five strains of V cholerae O1 (OK-1, 2, 3, 4, 5) isolated from 4 patients during illness, and one strain (OK-11) from an imported fish at the Naha port quarantine in July 1994 were used. OK-3 and OK-4 were isolated from the same patient. Other strains of V cholerae O1 stocked in our laboratory were also used when necessary.Phenotypic characterization of the isolates. Phenotypes of the isolates were examined for biochemical behaviors, cholera toxin production, kappa phage production and drug sensitivities. Biochemical behaviors were examined in accordance with the rou...

Gene Analysis of Vibrio cholerae NAGV14 Pilus and Its Distribution

“…This sequence was used for constructing new primers (inv 1 and inv2) applied in inverse PCR (13), using as template self-ligated DNA fragments. Further sequencing was extended by PCR walking (42) using random primer AP 40 (16), and by LA PCR Cloning Kit (TaKaRa Biomedical, Ohtsu, Japan). All primers used in this study are shown in Table 1.…”

“…Two possible starting methionine codons were present at nucleotide position 1 and 10. Therefore, this ORF was composed of 486 bp or 477 bp that encode a polypeptide of 162 aa or 159 aa with a calculated Mr of 16,535 Da or 16,192 Da, which was smaller than the Mr calculated by SDS-PAGE for the purified pilin (20 kDa) (Fig. 2).…”