A survey of pathogenic genes from diarrhea stool samples obtained in Nara prefecture.

We often experience cases in which no pathogenic organisms are isolated from the stool specimens of patients with diarrhea. There are several reasons for the failure to identify responsible pathogens in such cases, including the use of antibiotics and the presence of both pathogenic strains and nonpathogenic strains. Such cases prompted us to search for diarrheagenic bacteria by the direct detection of bacterial pathogenic genes from stool specimens. In this trial study, target genes were restricted to virulent genes related to Escherichia coli. A survey of diarrhea stool samples in which no virulent agents had yet been detected in clinical laboratories was conducted from May 2004 to March 2005. From four hospitals in Nara Prefecture, a total of 95 refrigerated diarrhea stool specimens with a twice-added volume of 75% glycerol/ phosphate-buffered saline were obtained. For use in polymerase chain reaction (PCR) tests, the DNAs were extracted from the specimens and purified using the QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany). Ready-made primers (Takara Bio, Inc., Tokyo, Japan) were used to test the purified DNA for vt-1, vt-2, lt, st, ipaH, and invE. Using previously reported primers (1), the DNAs were also tested for eae, bfpA, aggR, and astA. The PCRtest-positive specimens were inoculated onto MacConkey agar (Eiken Chemical, Co., Ltd., Tokyo, Japan) and incubated at 37°C for 24 h. Approximately 10 colonies were selected from the incubated agar plates and were tested for the virulent genes. Strains possessing the same virulent genes as those identified in the stool specimens were tested for microbial identification with the Vitek system (bioMerieux SA, Marcy l’Etoile, France) using GNI+ cards. Results from clinical laboratories clarified that virulent bacteria had been detected in 10 specimens. Therefore, 85 specimens were considered to be worthy of further analysis. Of the DNAs of those 85 specimens, none were positive for vt-1, vt-2, st, invE, or bfpA. One specimen was positive for each of lt, ipaH, and eae. In addition, two specimens were positive for aggR, and eight were positive for astA. Inoculation with the stool specimens did not lead to the detection of any bacteria possessing lt, ipaH, or aggR. One strain of E. coli possessing eae was isolated. As regards the eight astA-positive stool specimens, no astA-positive bacteria were detected in three of them, but strains of astA-positive E. coli were isolated from four specimens, and a strain of astApositive Klebsiella was isolated from one specimen. Additionally, the latter strain was identified as Klebsiella planticola using the API-20E biochemical system (bioMerieux SA) and other biochemical tests. The sequences of the PCR products generated from the Klebsiella strain using the primer sets were examined for comparison with the astA gene, which is the open reading frame