Phage type and antimicrobial susceptibility of Salmonella enterica serovar Enteritidis from food-producing animals in Japan between 1976 and 2004.

Abstract

A total of 56 isolates of Salmonella enterica serovar Enteritidis, including 38 isolates from poultry, 16 from cattle and two from pigs, collected between 1976 and 2004, were subjected to bacteriophage typing and antimicrobial susceptibility testing. Phage type (PT) 8 was predominant in bovine isolates, whereas PT1 and PT4 were predominant in poultry isolates. Resistance was found for 8 of 11 antimicrobials tested, at the following rates: 46.4% for dihydrostreptomycin followed by ampicillin and oxytetracycline (both 8.9%).

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NEW MICROBIOLOGICA, 31, 555-559, 2008
Phage type and antimicrobial susceptibility
of Salmonella enterica serovar Enteritidis
from food-producing animals in Japan
between 1976 and 2004
Tetsuo Asai1, Kazuki Harada1, Akemi Kojima1, Toshiya Sameshima1, Toshio Takahashi1,
Masato Akiba2, Muneo Nakazawa2, Hidemasa Izumiya3, Jun Terajima3, Haruo Watanbe3
1National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Kokubunji, Tokyo, Japan;
2National Institute of Animal Health, Tukuba, Ibaragi, Japan;
3National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
In Japan, Salmonella enterica serovar Enteritidis
has been the serotype isolated most frequently
from patients suffering from food-borne illness
since 1989 (Infectious Disease Surveillance
Center, 2003). As infections with S. Enteritidis are
closely linked with egg consumption in Japan,
the Enforcement Regulations of the Food
Sanitation Law (Law No. 23 of 1948) were
amended for safe distribution of raw eggs and
liquid egg products in 1998. Although large-scale
outbreaks of food poisoning due to S. Enteritidis-
contaminated egg products have been reduced
(Infectious Disease Surveillance Center, 2003), S.
Corresponding author
Tetsuo Asai
National Veterinary Assay Laboratory
Ministry of Agriculture, Forestry and Fisheries
1-15-1 Tokura, Kokubunji - Tokyo 185-8511, Japan
E-mail: asai-t@nval.go.jp
Enteritidis is still a predominant serovar and ac-
counted for about 50% of all the Salmonella iso-
lates from human sources between 2003 and
2005 (Infectious Disease Surveillance Center,
2006). S. Enteritidis is also a causative agent of
salmonellosis in animals. Salmonellosis caused
by S. Enteritidis in specific domestic animals
such as cattle, sheep, pigs and poultry, is a noti-
fiable disease regulated by the Domestic Animal
Infectious Disease Control Law (Law No. 166 of
1951). Antimicrobial therapy of S. Enteritidis in-
fection is generally needed for systemic infections
in humans and is essential for treatment and con-
trol of salmonellosis in animals. Emergence of
antimicrobial resistance in isolates of S.
Enteritidis is a significant public and animal
health concern. Antimicrobial-resistant isolates
of S. Enteritidis from human and egg products
have been reported in Japan (Murase et al., 2002;
Yamasaki et al., 2007). In the present study, we
characterized phage types and antimicrobial sus-
A total of 56 isolates of Salmonella enterica serovar Enteritidis, including 38 isolates from poultry, 16 from cattle and
two from pigs, collected between 1976 and 2004, were subjected to bacteriophage typing and antimicrobial suscepti-
bility testing. Phage type (PT) 8 was predominant in bovine isolates, whereas PT1 and PT4 were predominant in poul-
try isolates. Resistance was found for 8 of 11 antimicrobials tested, at the following rates: 46.4% for dihydrostrepto-
mycin followed by ampicillin and oxytetracycline (both 8.9%).
KEY WORDS: Salmonella enteritidis, Phage type, Antimicrobial resistance, Food-producing animals
SUMMARY
Received December 31, 2007
Accepted February 19, 2008

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ceptibility of animal isolates over the last three
decades. A total of 56 S. Enteritidis isolates in-
cluding 38 isolates from poultry such as broiler
and layer chickens and geese, 16 isolates from
cattle and 2 isolates from pigs, collected between
1976 and 2004, were subjected to bacteriophage
typing in accordance with the methods of the
Public Health Laboratory Service (PHLS),
London, United Kingdom (Ward et al., 1987). In
cases where a tested strain reacted with some of
the typing phages but did not conform to any of
the schemes the isolate was denoted as RDNC
(“Reacted but did not conform”).
Eighteen phage types of S. Enteritidis were found:
11 phage types were found in poultry isolates,
eight phage types in bovine isolates and two
phage types in porcine isolates. Phage type (PT)
1 and PT4 were predominant in poultry isolates.
PT8 was predominant in bovine isolates, and two
isolates of pig origin were identified as PT8 and
PT5c. The two phage types, PT1 and PT4, were
the most frequently isolated from unpasteurized
liquid egg between 1993 and 1998 (Murase et al.,
2002) and the patients with food poisoning
caused by S. Enteritidis since 1992 (Anon, 1997.
Salmonella, Japan, 1994-1996. IASR, http://idsc.
nih.go.jp/iasr/18/205/tpc205.html) throughout
Japan. During the period from 1997 to 2002, PT6,
PT6a, PT21, PT47 and RDNC were also isolated
from patients in outbreaks of food poisoning
caused by S. Enteritidis (Izumiya et al., 2003).
The present study showed that a variety of phage
types was observed in the isolates from food-pro-
ducing animals, especially poultry, after 1989.
Antimicrobial susceptibility was determined us-
ing an agar dilution method according to the
Clinical and Laboratory Standards Institute (for-
merly, the National Committee for Clinical
Laboratory Standards [NCCLS]) recommenda-
tions (2002). The antimicrobial agents used in
this study were ampicillin (ABPC), cefazolin
(CEZ), dihydrostreptomycin (DSM), kanamycin
(KM), gentamicin (GM), oxytetracycline (OTC),
chloramphenicol (CP), colistin (CL), nalidixic
acid (NA), enrofloxacin (ERFX), and trimetho-
prim (TMP). Staphylococcus aureus ATCC 29213,
Enterococcus faecalis ATCC29212, Escherichia coli
ATCC 25922 and Pseudomonas aeruginosa ATCC
27853 were used as quality control strains. The
minimum inhibitory concentrations (MICs) ex-
cept for those of OTC, DSM and CL were inter-
preted using the recommendations of the NCCLS
(2003); the breakpoints used for DSM, OTC and
CL were in accordance with our previous reports
(Esaki et al., 2004).
Of 56 isolates, 27 (48.2%) were susceptible to all
the antimicrobials tested. Resistance was found
for 8 of the 11 antimicrobials tested at the fol-
lowing rates: 46.4% for DSM, 8.9% for ABPC and
OTC, 7.1% for CP, and 1.8% for KM, GM, CL and
TMP. Even isolates of S. Enteritidis from patients
and chicken food in Japan, the similar result that
a half of isolates were susceptible to antimicro-
bials was reported (Yamasaki et al., 2007).
Compared with previous studies in Korea (Chung
et al., 2004), Spain (Cruchaga et al., 2001) and
southern Brazil (Dias de Oliveria, 2005), fre-
quencies of antimicrobial resistance in S.
Enteritidis were low except for DSM in Japan.
Although tetracycline antibiotics are the most fre-
quently used antibiotic for therapeutic purpose
in food-producing animals (Asai et al., 2007),
streptomycin resistance was most frequently ob-
served in S. Enteritidis in Japan. S. Typhimurium
and Infantis are predominant serovars in
Salmonella isolates from food-producing animals
in Japan (Asai et al., 2006a). High frequencies of
antimicrobial resistance were found in these
serovars in Japan (Asai et al., 2006b; Kawagoe et
al., 2007). Although S. Enteritidis is the leading
serovar in Salmonella food poisoning in Japan,
low frequencies of antimicrobial resistance was
observed in isolates of S. Enteritidis. In Japan,
NA-resistant isolates of S. Enteritidis have fre-
quently been found in imported chicken meats
(Matsumoto et al., 2006). Recently, Izumiya et al.
(2005)reported an
cephalosporin (ESCs)-resistant isolate of S.
Enteritidis from patients in Japan. Although no
isolates of S. Enteritidis resistant to NA, ERFX
and CEZ were found in the food-producing ani-
mals studied, continuous surveillance need to
monitor emergence and prevalence of antimi-
crobial resistance in food-producing animals.
Nine isolates (16.1%) exhibited resistance to two
or more antimicrobials (multi-drug resistance
[MDR]): these included 5 (13.2%) out of 38 poul-
try isolates, 3 (25%) out of 16 bovine isolates, and
one (50%) out of 2 porcine isolates (Table 1). In
Korea between 2000 and 2002, 89.8% of human
isolates, 64.7% of poultry isolates and 13.3% of
pig isolates showed MDR (Chung et al., 2004).
extended-spectrum
556
T. Asai, K. Harada, A. Kojima, T. Sameshima, T. Takahashi, M. Akiba, M. Nakazawa, H. Izumiya, J. Terajima, H. Watanbe

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Although most isolates of S. Typhimurium in cat-
tle (resistance to ABPC, DSM, OTC and either CP
or KM) and pigs (resistance to DSM and OTC)
and S. Infantis (resistance to DSM and OTC) in
broiler chickens showed MDR (Asai et al., 2006b;
Kawagoe et al., 2007), MDR isolates of S.
Enteritidis from food-producing animals re-
mained uncommon in Japan.
Out of the nine MDR isolates, three isolates from
cattle obtained between 1976 and 1982 exhibited
resistance to three to five antimicrobials: two iso-
lates of PT8 exhibited DSM-OTC-CP resistance
(No. 8-3) and ABPC- DSM-OTC-CP resistance
(No. 8-5) and one isolate of PT29a exhibited
ABPC-DSM-KM-OTC-CP resistance. As resistance
to three or more antimicrobials was found in iso-
lates of PT8 from cattle between 1976 and 1982,
all eight isolates of PT8 were subjected to pulsed-
field gel electrophoresis (PFGE) analysis to clar-
ify the relationship between resistant isolates in
1976 to 1982 and susceptible isolates in 2000 to
2004. At first, BlnI digestion was adopted because
of the better discrimination among genotypes of
S. Enteritidis strains, as previously described
(Terajima et al., 1998). PFGE was carried out with
a CHEF-DRIII system (Bio-Rad Laboratories,
Inc., Ca, USA) according to Pulse-net procedures
of the Center of Disease Control and Prevention
(Hunter et al., 2002). The running conditions were
6 V/cm at 14°C for 19 h with pulse times ramped
from 2.2 to 63.8 s. Images were prepared using
Quantity One software (Bio-Rad Laboratories).
BlnI-digested PFGE profiles were classified into
2 types: type 1 contained two susceptible isolates
in 1981 (No. 8-4) and 2003 (No. 8-8), and type 2
contained the remaining six isolates including
two MDR isolates (No. 8-3 and 8-5) with small-
sized bands (less than 33Kbp) (Figure 1a). In fur-
ther work, the PFGE profile of 8-3 digested with
XbaI and BlnI (type 3) could be clearly distin-
Salmonella enteritidis in food-producing animals
557
TABLE 1 - Resistance pattern of S. Enteritidis isolates from food-producing animals during three different
collection periods since 1976.
Resistance patterna
1976~1982
cattle
PT8 others PT8
1989~1999 2000~2004
pig pig cattle
others
poultry cattle poultryTotal%
PT1 PT4 others PT8 others PT5c PT1 PT4 others
ABPC-DSM-KM-OTC-CP11 1,8
ABPC-DSM-OTC-CP11 1,8
DSM-OTC-CP11 1,8
ABPC-DSM11 1,8
ABPC-TMP11 1,8
DSM-OTC22 3,6
DSM-CP11 1,8
DSM-CL11 1,8
ABPC11 1,8
DSM1333116 18 32,1
GM11 1,8
Susceptible211243248 27 48,2
Total4213 10343114 1656
aABPC, ampicillin; DSM, dihydrostreptomycin; KM, kanamycin; OTC, oxytetracycline; CP, chloramphenicol; CL, colistin; TMP, trimethoprim.

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guished from other isolates of type 2 (Figure 1b).
These results showed that, of three types of PFGE
profiles, two types of PT8 strain have been preva-
lent among cattle in the last 2 decades. For plas-
mid curing, 1000-fold diluted cultures of isolate
No. 8-5 were incubated with at 43°C for 20hr.
After 3 consecutive transfers, susceptible clones
were obtained. Plasmid profile tests revealed that
a plasmid (about 130Kbp) was cured in the sus-
ceptible clones. BlnI-digested PFGE pattern of
the clones derived from isolate No. 8-5 was iden-
tical to the isolates of type 2 (Figure 1a). The pres-
ent study could not show genomic differences be-
tween the classic resistant isolates and the recent
susceptible isolates.
ACKNOWLEDGEMENTS
We thank the member of the Livestock Hygiene
Service Centers for providing S. Enteritidis isola-
tes. This work was supported in part by grant in
aid of the Ministry of Health, Labor and Welfare
(H18-Shokuhin-Ippan-003).
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FIGURE 1 - PFGE patterns of PT8 isolates of S. enteri-
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XbaI and BlnI b). Lanes 1, S. enterica serovar
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