Jpn. J. Infect. Dis., 54 (1), 38-40, 2001

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Laboratory and Epidemiology Communications

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

Kokichi Hamada* and Tomohiro Oshibe

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 serotypes of Salmonella food poisonings in Japan (1,2). We previously reported the genetic variations of the 21 isolates obtained at the food poisonings that occurred from 1997-1999 in Hyogo Prefecture (3) together with detailed analyses of three outbreak cases (4,5). In that study, pulsed-field gel electrophoresis (PFGE) was used to identify six different genotypes, A, A', B, C, D, and E. Fourteen strains belonged to genotype A and three to subtype A' (3). Genotype A was further classified into two subtypes, A-a and A-b, according to the size of the largest band >630 kb; the band in A-a moved more slowly than that in A-b. There were eight A-a subtypes and six A-b subtypes (Figs.1 and 2). Others were classified as genotypes B, C, D, and E (3).

We carried out a similar genotypic analysis of eight new isolates from seven food poisoning cases in Hyogo Prefecture in 2000 (cases 22-28 in Table). Three phage types (PTs) were identified: PT 1, PT 14b, and PT 47. The strains were tested for the 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) (6). All, except two SM-resistant isolates (cases 23 and 24), were sensitive to the 12 antibiotics.

These eight isolates were examined by PFGE using a gene path typing system (Program No.2; Nippon Bio-Rad, Tokyo)(4), and the patterns were compared with those of the previous 21 isolates in 1997-1999. PFGE of BlnI chromosomal DNA digests (Fig.1) revealed the presence of five genotype A (3) (A-a; cases 22, 25, 27,28, and A-b; case 26), and two nondescripts (3) (cases 23 and 24) (Fig. 1A). No correlation was observed between PFGE patterns and phage types (Table) as in our previous observation (3). Figure 1B shows representative PFGE patterns observed among 28 Salmonella from food poisonings in Hyogo Prefecture in 1997-2000.

A dendrogram obtained by molecular analysis software (Finger Printing Plus; Bio-Rad, Hercules, Calif., USA) for the isolates in 1997-2000 is shown in Figure 2. Two large clusters were observed, each consisting of several subclusters. Various genotypically different characteristics including phage type (1-5,7) and PFGE pattern (2-5,7) were observed in studies of a large-scale epidemic in Japan (1,2) as well as in our previous (3-5) and next studies (7).

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 Division, 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CH., 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.
  4. Hamada, K.CTsuji, 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).
  5. Tsuji, H., Shimada, K., HamadaCK. 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.
  6. 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.C53, 174-176.
  7. Oshibe, T. and Hamada, K. (2001): 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. Jpn. J. Infect. Dis., 54, 40-43.


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

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