Jpn. J. Infect. Dis., 54 (1), 40-43, 2001

Laboratory and Epidemiology Communications

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Phylogenetic Analysis of Genotypic Variations of Salmonella Enteritidis Isolates from Sporadic Infections Using Pulsed-Field Gel Electrophoresis from December 1996 to November 2000 in Hyogo Prefecture

Tomohiro Oshibe and Kokichi Hamada*

Division of Microbiology, Hyogo Prefectural Institute of Public Health,
Arata-cho 2-1-29, Hyogo-ku, Kobe 652-0032

Communicated by Shinsaku Imashuku

(Accepted April 5, 2001)

Since 1989, Salmonella serovar Enteritidis has become the most prevalent among the Salmonella serotypes in Japan (1,2). In the previous (3-5) and preceding papers (6) in which were reported 28 food poisoning outbreaks caused by Salmonella Enteritidis from June 1997 to December 2000 in Hyogo Prefecture, we demonstrated, using pulsed-field gel electrophoresis (PFGE), a clear phylogenetic relationship among the causative strains for these food poisonings (6). We report here the genotypic variations and phylogenetic analysis of the total 55 isolates, including 50 from human feces in sporadic infection cases, two from samples from chickens, and three from chicken egg samples (Table).

Twenty-four of the 55 isolates were tested for phage types (PTs), the types being PT1, PT4, PT6, PT6a, PT9b, RDNC (Reaction Does Not Conform), and UT (Untypable). The remaining 31 isolates were not tested (Table). All of the 55 isolates were tested for sensitivity to 12 drugs (ampicillin [ABPC], cefotaxime [CTX], kanamycin [KM], gentamicin [GM], streptomycin [SM], tetracycline [TC], trimethoprim [TMP], ciplofloxacin [CPFX], fosfomycin [FOM], chloramphenicol [CP], sulfamethoxazole-trimethoprim [ST], and nalidixic acid [NA]) by Sensi Disk (Nippon Becton Dickinson Co., Ltd., Tokyo) as reported in a previous paper (7). The majority (31 strains) were sensitive to the drugs，while the remaining 24 strains were resistant to SM, ABPC, TC, and/or GM (Table).

All of the 55 isolates were examined for PFGE patterns using a gene path typing system (Program No. 2; Nippon Bio-Rad，Tokyo) in a manner reported previously (3), except that lysozomal lysates were further treated with 1% sodium laurylsulfate for 1 h at 50℃ for complete lysis. The typical PFGE patterns are shown in Figures１A and 1 B and summarized in Table. The PFGE patterns obtained with BlnI-digested chromosomal DNAs showed 15 different patterns. A dominant pattern was that seen in case 3 (at least 32 cases of the 55 cases) supposedly belonging to genotype A-b (6). There was no correlation between phage types and PFGE patterns (Table). The absence of correlation between PFGE patterns and phage types was previously reported for food poisoning cases (5,6). In summary, out of 55 isolates, 32 cases (about 60%) belonged to A-b (6), and 2 isolates (cases 41 and 49) had a pattern close to A-b. There were one isolate of A-a type (6) (case 25), one isolate similar to D type (5) (case 14), and 19 belonging to other nondescript genotypes (5,6).

Thus, the Salmonella Enteritidis isolates from various sporadic infections showed a great genetic diversity as in the cases of food poisoning outbreaks (5,6). This was clearly demonstrated by cluster analysis (Finger Printing Plus; Bio-Rad, Hercules, Calif., USA) (Fig. 2). The dendrograms indicated the presence of two large clusters, each containing several subclusters as reported for 28 food poisoning cases (6). The sporadic infections of Salmonella Enteritidis in Hyogo Prefecture over the past 4 years appear to have been caused by genotypically different bacteria as in food poisoning cases (6). The data appears to reflect the large-scale epidemic due to Salmonella Enteritidis since 1989 in Japan (1,2) including Hyogo Prefecture (3-6).

The authors are grateful to Drs. Hidemasa Izumiya and Haruo Watanabe, National Institute of Infectious Diseases, Tokyo, for the phage typing of our isolates and for a critical reading of the manuscript. 　

REFERENCES

1. National Institute of Health and Infectious Diseases Control, Ministry of Health and Welfare（1995): Salmonella, Japan, 1992-1994. Infect. Agents Surveillance Rep. 16, 1'-2'.
2. Terajima, J., Nakamura, A. and Watanabe, H. (1998): Epidemiological analysis of Salmonella enterica Enteritidis isolates in Japan by phage-typing and pulsed-field gel electrophoresis. Epidemiol. Infect., 120, 223-229.
3. Hamada, K.，Tsuji, H., Shimada, K. and Hosoda, Y. (1999): Epidemiological analysis of Salmonella serovar Enteritidis isolates from two food poisoning outbreaks in Hyogo Prefecture by pulsed-field gel electrophoresis. Bull. Hyogo Prefect. Inst. Public Health, 34, 113-117 (in Japanese).
4. Tsuji, H., Shimada, K. ,Hamada, K. and Nakajima, H.(2000): Outbreak of Salmonella Enteritidis caused by contaminated buns peddled by a producer using traveling cars in Hyogo and neighboring Prefectures in 1999: an epidemiological study using pulsed-field gel electrophoresis. Jpn. J. Infect. Dis., 53, 23-24.
5. Hamada, K., Tsuji, H., Izumiya, H. and Watanabe ,H. (2000): Comparison by pulsed-field gel electrophoresis of Salmonella Enteritidis genotypes from various food poisoning outbreaks from 1997 to 1999 in Hyogo Prefecture. Jpn. J. Infect. Dis., 53, 25-27.
6. Hamada, K. and Oshibe, T. (2001): Phylogenetic analysis of Salmonella Enteritidis isolates from food poisonings using pulsed-field gel electrophoresis over the period of June 1997 to December 2000 in Hyogo Prefecture. Jpn. J. Inect. Dis., 54, 38-40.
7. Hamada, K., Oshibe, T., Tsuji, H., Yoshida, S. and Aoki, Y. (2000): Outbreaks of heat stable enterotoxin-producing Esherichia coli O169 in the Kinki district in Japan: genotypic comparison by pulsed-field gel electrophoresis of isolates from two outbreaks in 2000 with isolates from four outbreaks in 1997-1998. Jpn. J. Infect. Dis., 53, 174-176.

* Corresponding author: Fax +81-78-531-7080

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