Jpn. J. Infect. Dis., 54, Ill‑113, 2001

Short Communication

Molecular Analysis Of Salmonella Enteritidis Isolates Resistance toAmpicillin and Streptomycin from Three Outbreaks ofFood Poisonlng ln Shiga Prefecture
WataruMatsune*, Kazuhiko lshikawa. , Ken‑ichi Hayashil , Motohiro Tsuji',Hidemasa Izumlya2 and Haruo Watanabe2
Division of Environmental Health, Otsu Health and Welfare Center ofShiga Prefecture,
Nionohama 4‑4‑5, Otsu, Shiga 520‑0801,
JDivision of Microbiology, Shiga Prefectural Institute of Public Health and Environmental Science,Gotenhama 13‑45, Otsu, Shiga 520‑0834 and2Departmen t of Bacteriology, National Institute oflnfectious Diseases,
Toyama 1‑23‑1, Shinjuku‑ku, To砂0 162‑8640, Japan(Received February 13, 2001. Accepted June 20, 2001)

SUMMARY: In 1998 and 1 999, there were three outbreaks caused by Salmonella Enteritidis in Shiga Prefecture.
One outbreak was suspected to be a dimISe Outbreak, caused by frozen cream puffs that had been sold in chain
stores throughout Shiga Prefecture between the beginnlng Of September and the beginnlng Of October, 1 998.
The other outbreaks occurred in May and in August, 1 999. All isolates of the three outbreaks showed an identicallysIS pattem agalnSt the typlng phage, though this pattem did not confbm to the current scheme, so‑called
RDNC. ln addition all isolates were resistant to ampicillin and streptomycin. However, the pattems of pulsed‑
field gel electrophoresis strongly indicated that the three outbreaks were actually independent.

(1). Consumption of contaminated egg products has been

Thirty were isolatedfrom the three outbreaks, 20 fromoutbreak A, 3from outbreak B, and 7from outbreak C. One
hundred twelve were from sporadic cases in Shiga Prefecture

shown to be a risk factor both in outbreaks and in sporadic

in 1998. The details of the three outbreaks are provided incases (1).

Table I. Patients of outbreak B were residents of Shiga
Prefecture who had traveled to the Kyusyu district and wereinfected there. Outbreak C occurred at the hospital in Shiga
Prefecture. All isolates were identified as SE by the API 20E

Salmonella Enteritidis (SE) has been isolated mostfrequently among Salmonella enterica in Japan since 1 989Serotyping, antimicrobial susceptibilitytests, and bacteQo‑phage typing have been employed to determine the epidem10‑
loglCal association of isolatesfrom patients, or to trace possible

routes oftransmissionfrom animals to humans (213). Recently,

system (BioMerieux S.A., Marcy l'Etoile, France), themolecular methods such as pulsed‑field gel electrophoresis

Kau庁血an serotyping scheme (8),and commercially purchased(PFGE) have proved to be highly discriminatory･ These

antisera (Denka Seiken, Tbkyo).

epidemiologlCal markers have made it possible to identify

Bacteriophage typlng Was perfわrmed by the method ofWard et al. (9).

difhse outbreaks (4‑7).

The antimicrobial susceptibilitytests were carried out using
the Senci‑Disc (BBL Microbiology System, Cockeysville,Md･, USA) and were performed by the Kirby‑Bauer method

Shiga Prefectu托Sufferedfrom three outbreaks caused by SE

in September 1 998 and May and August 1 999, respectively.

We report here a comparative investigation among isolatesof the three outbreaks and sporadic cases in the same period,( 1 0)･ Disks containlng ampicillin, streptomycin, kanamy車,

and reveal that one outbreak was a dimISe Outbreak, and thegentamicin, tetracyc lin, cefazolin, chloramphenlcol, norfloxacln,

three were caused by independent SE strains, respectively.

sulfbmethoxazole and trimethoprlm, and fbsfbmycln Wereused.

One hundred fわrty‑two isolates or SE were examined.

Table 1. Characteristics ofS. Entedtidis isolates rcsistacc to ampicillin and strcptomycin舟om outbreaks

and sporadic patientsoDua.tbCrcOafk

Outbreak A

Scp. /I 998.P!accueJcfncc :.u.Ttbecsrof source phage type

Kindcrgarten

9

Patients'feces

1 1 Frozen cream puffs
Outbreak 良

May / 1 999

Restaurant

3

Outbreak C

Augノ1 999

Hospital

7

Patients'fcces

Sporadic case

Scp.lNov./I 998

32

Paticnts'feces0ct. /1998

Patients'fcccs

1 Patient's feces*Corresponding author: Tel: +8 I‑771522‑6755, Fax: +8 1‑77‑525‑6161

111

RDNRDNRDNR DNRDNl CCCCC

Extraction ofDNA and the conditions fわr PFGE were based

and PFGE. Thirty‑two were identified as RDNC whose lysISon the method ofTerajima et al. (3). Restriction endonucleasepattems were identical to those or the three outbreaks. TheBlnl (Takara, Shiga) was used.

other was identified as phage type (PT) 4. In the PFGE analysis,

resistant to ampicillin and streptomycin. The results of the26 of 32 sporadic RDNC isolates showed pattems identical
tothose of outbreak A; five and one showed pattems with abacteriophagetyping for the isolates were identical, althoughdifference in one and four bands, respectively, compared tothe lysIS pattemS did not confわrm to any current scheme,

those of outbreak A; and the latter PT4 isolate showed pattems

so‑called RDNC (they reacted but did not conform).
The PFGE pattems of isolatesfrom each outbreak differed

quite different from any of the others (Fig. lb). The PFGE

from each other, though those of isolates within each outbreak

could be included in the same subtype as that of outbreak A,

Al1 30 isolates from the three outbreaks were found to be

results suggest that the patterns of the 32 sporadic isolates

were indistinguishable or quite similar to one another (Fig.

according to the criteria ofTenovar et al･ (1 1)･ FurthermQre,la). For example, although five of seven isolates from out‑

most of the 32 isolates were isolated between the beginnlng

break C showed identical PFGE pattems, the pattems of two
isolates were different in two or three bands compared to theof September and the beginnlng OfDecember in 1998, over‑lapping the period of outbreak A (Fig. 2). As such, the periodmajor pattem (Fig. la). These two isolates could be includedof the isolation of the sporadic SE and the sold period of

in a part of outbreak C, according to the criteria ofTenovar etcontaminated frozen cream puffs partly overlapped･ Thus,al. (1 1). Thus, the three ouぬreaks were indicated to be indepen‑

these sporadic cases might have been a part of outbreak A;

dent based on the PFGE results, despite the conflicting resultsthat is, outbreak A can be considered a di飢se outbreakof antimicrobial susceptibilitytests and bacteriophagetypin!･

affectlng the whole of Shiga Prefecture･Thirty‑three of 1 1 2 sporadic isolates were resistant to ampl‑

In combination with antimicrobial susceptibility tests,

cillin and streptomycln, aS Were those of the three outbreaks,

bacteriophage typlng and PFGE appear to be of value inand these 33 were further examined by bacteriophage typlng

epidemiological investigations of SE (3, 12). As fわr phage

M12345M6789101112

M

12345M67(kb )
582‑

485ー

388ー

291ー

194ー

97ー

48.5‑一Fig. 1 a. PFGE patterns ofSE chromosomal DNAfragments digested
with β7円1,Fig. lb. PFGE pattems ofSE chromosomalDNA fragments digested

Lane M: DNA ladder, Lanes 1‑2: isolates from frozen cream puffs,with βJ〃Ⅰ.

3‑5: isolates from patients (outbreak A), 617: isolates from outbreakLane M: DNA ladder, Lane I : isolates from a frozen cream puff,

B, 8‑12: isolates from outbreak C.

2: isolates from sporadic patient (PT4), 3‑7: isolates斤om sporadicpatients (RDNC).

Sale一OSこ0LaqEnN

Jaロ.

Feb.

Mar.

Apr.

May

Jun.

Jul.

Aug.

Sep.

Oct.

Fig･ 2･ Distribution ofS･ Enteritidis isolates from sporadic cases in 1 998･112

Nov.

Dec.

types, isolates with RDNC have shown a recent increase infield gel electrophoresis. Jpn. J. Infect. Dis., 52, 53154.

Japan though PTl and PT4 remain predominant (1 ), suggestl

6. Hamada, K., Tsuji, H. and Shimada, K. (1999): Outbreaks

lng that SE strains with similar characteristlCS tO those in thisof heat stable enterotoxin‑producing Escherichia colt

study are spreading (13). Coordination offield and molecular

O169 in the K.inki district in Japan: epidemiologlCal

epidemiologlCal investlgations will be important in elucidaト

analysis by pulsed‑field gel electrophoresis. Jpn. J. Infect.

lng associations among both outbreaks and sporadic cases.

Dis., 52, 165‑167.
7. TeraJlma, ∫., Izumlya, H., lyoda, S･, Tamura, K. andWatanabe, H. (1999): Detection of multi‑prefectural E.

ACKNOWLEDGMENTS

coli 0 1 57:H7 Outbreak caused by contaminated ikura‑sushi

We would like to thank the laboratories in Shiga Prefecture

lngeStion. Jpn. J. Infect. Dis., 52, 52‑53.

for providing Salmonella Enteritidis isolates. We would also

8. Kaulhan, F. (1972): Serological diagnosis of salmone110‑like to thank Hikone Health and We脆re Center of Shiga

Prefecture for the field epidemiologlCal investlgation. lnsis species. Muksgaard, Copenhagen.
9. Ward, L R., de Sa,J. D. H. andRowe, B. (1987):Aphage‑addition, we thank Laboratory Enteric Pathogens, Public

typlng scheme for Salmonella enteritidis･ Epidemiol.
Infect., 99, 291‑294.

Health Laboratory Service in England fわr supplying the10. National Committee for Clinical Laboratory Standards.

phages for typing Salmonella Enteritidis.

(1997): Perfわrmance standards fわr antimicrobial disk

susceptibility tests･ Approved standard M2‑A4･ National

REFERENCES

Committee fわr Clinical Laboratory Standards, Wayne,

1. National Institute of Infectious Diseases and InfectiousPa.

1 I. Tenover, F. C., Arbeit, R. D., Goerlng, R. V., Mickelsen,
P. A., Murray, B. E., Perslng, D. H. and Swaminathan,Diseases Control Division, Ministry ofHealth and Welfare(2000): Salmonellosis in Japan as of June 2000. Infect.

B. (1 995): Interpreting chromosomal DNA restriction

Agents SuⅣeillance Rep., 21, 162'‑163'.

2･ Rodrigu.e, D･ C･, Tauxe･ R･ V･ and Rowe, B･ (1990):pattems produced by pulsed‑field gel electrophoresis:Internat10nal increase in Salmonella enteritidis: a newcriteria for bacterial strain typlng. J･ Clin. Microbiol･,33, 2233‑2239.

pandemic? Epidemiol. Infect･, 105, 21127･

3. Terajima, J., Nakamura, A. and Watanabe, H. (1998):

1 2. Boonmar, S., Bangtrakulnonth, A., Pomrunangwong, S.,

EpidemiologlCal analysis Of Salmonella enterica Enter‑TeraJlma, J., Watanabe, H., Kaneko, K. and Ogawa, M.

itidis isolates in Japan by phage‑typing and pulsed‑field( 1 998): Epidemiological analysis of Salmonella enteriti‑gel electrophoresisI Epidemiol･ Infect･, 1 20, 223‑229･

dis isolates fiom humans and broiler chickens in Thailand

4. Centers fわr Disease Control and Prevention (1997):by phage typlng and pulsed‑field gel electrophoresis. J.

Clin. Microbio1., 36, 971‑974.

Escherichia coli 01 57:H7 infections associated witheatlng a nationally distributed commercial brand of

13. Noda, H.,Asakawa, Hりlzumiya, H. andKaneko, M. (1998):

frozen ground beef patties and btwgers, Colorado. Morbid.
Mortal. Weekly Rep., 46, 777‑778.
5. Hamada, K., Tsuji, H., Masuda, K. ar)d Uemura, K.

Salmonella strains isolated from patients with sporadic
diarrhea and food poISOnlng due to Salmonella serovarEnteritidis in Yamanashi prefecture.Amu Rep. Yamanashi
lnst. Public Health, 42, 25‑32 (in Japanese).

(1 999): Outbreaks of salmonellosis caused by ingestionof cuttlefish chips: epidemiologlCal analysis by pulsed‑113