Jpn. J. Infect. Dis., 54, 162-165, 2001
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Laboratory and Epidemiology Communications
Phylogenetic Analysis of Genotypic Variations of Escherichia coli O157:H7 Isolates from Sporadic Infections by Using Pulsed-Field Gel Electrophoresis from March 1999 to February 2001 in Hyogo Prefecture
Tomohiro Oshibe, Hidetaka Tsuji and Kokichi Hamada*
Division of Microbiology, Hyogo Prefectural Institute of Public Health, Arata-cho 2-1-29, Hyogo-ku, Kobe 652-0032
Communicated by Takashi Kawamura
(Accepted September 19, 2001)
Entero-hemorragic Escherichia coli (EHEC) is a highly virulent enteric pathogen and, since 1982, it has been found worldwide including the United States, Europe, and Japan (1). E. coli O157:H7 has been the most prevalent serotype among them (1). A large-scale outbreak occurred in 1996 in Sakai City in Osaka Prefecture (2). The EHEC epidemic has continued ever since (3). Even in the early epidemics, its high genetic diversity was revealed by pulsed-field gel electrophoresis (PFGE) (2). We previously reported such genotypic variations in 1997 - 1998 isolates (4). We report here the genetic diversity and phylogenetic analysis of the isolates in 1999 - early 2001. Eighty isolates from human feces of sporadic and small outbreak cases (Table) and one isolate from well water implicated in a familial transmission were analyzed.
The isolates were examined for genes encoding verotoxin (VT) by polymerase chain reaction (PCR) using EVS- (for VT1) and EVT- (for VT2) primers purchased from Takara Shuzo Co. Ltd. (Kyoto) (5). Six isolates were positive for VT1, 41 for VT2, and 34 for the both (Table). Sensitivities to 12 antibiotics, i.e., ABPC (ampicillin), CTX (cefotaxime), KM (kanamycin), GM (gentamicin), SM (streptomycin), TC (tetracycline), TMP (trimethoprim), CPFX (ciplofloxacin), FOM (fosfomycin), CP (chloramphenicol), ST (sulfamethoxazole-trimethoprim), and NA (nalidixic acid) were examined by using Sensi Disk (Nippon Becton Dickinson Co., Ltd., Tokyo) (6). Sixty-two isolates were sensitive to all the antibiotics examined, but the remaining 19 strains were resistant to either ABPC (6 strains), SM (1 strain), ABPC+SM (1 strain), SM+TC (9 strains), or ABPC+SM+TC (2 strains) (Table).
PFGE patterns of the 81 isolates were analyzed by using a gene path typing system (Program No. 5 or No. 23; Nippon Bio-Rad, Tokyo) as reported previously (7). The typical PFGE patterns are shown in Fig. 1. The PFGE patterns of XbaI-digested chromosomal DNA of the 81 isolates and cluster analysis (Finger Printing PLUS, Bio-Rad, Hercules, Calif., USA) of the 72 strains (Fig. 2) revealed a large strain-to-strain or case-to-case variation. The dendrogram indicated the presence of more than ten small clusters. Thus, it was concluded that EHEC O157:H7 epidemics in Hyogo Prefecture in the past 2 years were caused by various EHECs of different genotypes.
The question arises as to why E. coli O157 is so genetically changeable and what advantage this characteristic lends to bacteria. Emergence and rapid spread of this organism can be explained by a high incidence of highly mutable variants which are found among isolates of EHEC and Salmonella enterica; they may cause enhanced genetic variability of a population and accelerate adaptive evolution (8). But, this may not explain the observation that E. coli O157 showed a larger PFGE diversity than did Salmonella Enteritidis (9,10). However, it should be mentioned that variability of PFGE patterns is influenced by the number and mutability of the sites recognized by the restriction enzymes. Therefore, the above mentioned different variability could be more apparent than real.
REFERENCES
*Corresponding author: Fax: +81-78-531-7080
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