Jpn. J. Infect. Dis., 56, 77-79, 2003
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Laboratory and Epidemiology Communications
A New Clonal Line of Salmonella Saintpaul Having Emerged and Prevailed since 1999 in Aichi, Japan
Mami Hata*, Masahiro Suzuki, Masakado Matsumoto, Masao Takahashi, Mitsugu Yamazaki, Reiji Hiramatsu, Hironori Matsui, Kenji Sakae, Yasumoto Suzuki and Yutaka Miyazaki
Department of Microbiology, Aichi Prefectural Institute of Public Health, Nagoya 462-8576
Communicated by Yutaka Miyazaki
(Accepted May 9, 2003)
*Corresponding author: Mailing address: Department of Microbiology, Aichi Prefectural Institute of Public Health, Nagare 7-6, Tsuji-Machi, Kita-ku, Nagoya 462-8576, Japan. Tel: +81-52-910-5669, Fax: +81-52-913-3641, E-mail: mami_hata@pref.aichi.lg.jp
Though having long been a rare Salmonella serotype in Japan, Salmonella enterica serotype Saintpaul (antigenic formula, 4:eh:1,2) has become one of the most frequent serotypes throughout Japan including Aichi Prefecture since 1999. In the list of 15 most common Salmonella serovars from human sources in Japan (National Institute of Infectious Diseases website http://idsc.nih.go.jp in 2002), Salmonella Saintpaul first appeared in 1997 as ranking 9th, and since 1999, its rank has risen every year, i.e.; 1999: 9th, 2000: 7th, 2001: 5th, 2002: 4th. In 2001, Shiga Prefecture experienced a food poisoning outbreak caused by this serotype (1). A nationwide food poisoning outbreak in Germany in 1993 involved multiple Salmonella serotypes including Saintpaul (2).
The epidemiology of this serotype in humans is not well known and the reason this serotype has recently become predominant is unknown.
The present study examines the clonal relationship among apparently epidemiologically unrelated Saintpaul serotyopes isolated in Aichi Prefecture in 1981-2002. We performed pulsed-field gel electrophoresis (PFGE) analysis of BlnI- and XbaI-DNA digests, antibiotic sensitivity tests, and plasmid profiling on 80 such isolates. Five were obtained from patients in sporadic cases and the remaining 75 from healthy carriers. Among these 80 strains, 21 strains were isolated in 1981-1998, and the remaining 59 in 1999-2002.
PFGE typing of the Saintpaul serotype was very difficult due to degradation of DNA during the procedure as in the case of many Clostridium difficile or Pseudomonas aeruginosa strains. Recently, Corkill and others reported that degradation of the DNA of such bacteria was prevented by the addition of 50 mM thiourea in the gel buffer (3,4). The addition of thiourea in the running buffer was effective in our analysis as well. PFGE of DNA digests was performed using Pulsaphor (Pharmacia Biotech AB, Uppsala, Sweden) through 1% agarose in TBE buffer. The pulse times were increased from 5 to 40 s for 20 h.
Strains isolated in 1999-2002 exhibited similar PFGE patterns (Figs. 1A and 1B; representative patterns of 2001-2002 isolates are shown). In contrast, strains isolated before 1999 exhibited diverse PFGE patterns (Figs. 1C and 1D).
Inspection of PFGE patterns of BlnI-digests and those of XbaI-digests identified 20 patterns among 21 strains isolated before 1999, i.e., only one pattern was shared by two isolates, and other patterns were unique to the isolates. Meanwhile, the PFGE patterns of isolates after 1999 were largely similar; we identified 27 non-identical but similar patterns for 59 isolates. The 21 isolates before 1999 and 27 isolates after 1999 (1999 inclusive) with representative PFGE patterns were subjected to cluster analysis by Fingerprinting II software (Bio-Rad Laborotories, Hercules, Calif., USA).
All of the strains except one, i.e., 58/59, isolated after 1999 (1999 inclusive) were grouped into one, cluster I (Fig. 2). Sub-cluster A with over 77% similarity was the majority (91%) of cluster I. Several isolates were identical in the cluster analysis (sub-sub-clusters a, b, c in Fig. 2). All isolates before 1999, except one (98-298), belonged to the clusters other than the cluster I, and shared less than 48% similarity with those in the cluster I. Isolates later than 1999 were probably clonal in origin.
In Japan, the most prevalent serotype in human salmonellosis is Enteritidis (5) and is often associated with contaminated eggs. As in the case of nationwide food poisoning in 1999 attributed to cuttlefish contaminated by S. Oranienburg and S. Chester (6), the surge in the prevalence of Saintpaul serotype could be brought about consumption of a particular contaminated food by a large population.
We tested several other typing methods for their applicability
to epidemiological investigation of Saintpaul serotypes. As shown
below, none of them worked as well as PFGE.
(1) Ribotyping with the 16S rRNA gene probe using several enzymes
such as EcoRI, SmaI, PvuII, and PstI
revealed only one or two band differences among the strain examined,
suggesting poor discrimination.
(2) Most serotypes contain a 708-bp long, Salmonella-specific
insertion element IS200, which can be applicable for molecular
epidemiology (7). S. Saintpaul lacked this IS element.
(3) Plasmids were detected in seven out of 80 strains (Fig.
3). Five were isolated before 1999; no particular plasmid
pattern predominated. The plasmid profiles of two isolates were
similar (lanes 6: 99-318 and 7: 00-162)). They were isolated after
1999 and shared an 87% similarity in PFGE (Fig.
2). As plasmids were detected only in a minority, 5/21 before
1999 and 2/59 after 1999, plasmid profiling was not of much value
for the Saintpaul serotype.
(4) We examined patterns of antimicrobiotic sensitivity for ampicillin,
cefaloridine, kanamycin, streptomycin, tetracycline, minocycline,
fosfomycin, chloramphenicol, colistin, nalidixic acid, pipemidic
acid, and norfloxacin by using Antibiotic Disks (KB disc, Eiken
Chemical Co., Tokyo). Among 80 isolates, only two showed resistance
to drugs. One isolate (00-162) was resistant to tetracycline and
minocycline, and the other (94-158) to streptomycin, tetracycline,
and minocycline. Both strains harbored plasmids (Fig. 3, lanes
4 and 7).
REFERENCES
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