Laboratory and Epidemiology Communications
Masumi Taguchi*, Kazuko Seto, Wataru Yamazaki, Teizo Tsukamoto,
Communicated by Yoshichika Arakawa
Infection with salmonellae causes a wide range of symp-
to ESCs have recently been identified in several countries,
nella spp. in Japan. One was regarding CMY-2 β -lactamase-
microbial susceptibility of Salmonella spp. isolates from meat
were found to be positive for the organisms. The most com-
genetical analysis of the two isolates.
CMY-2 β-Lactamase-Producing Salmonella enterica Serovar
Hidemasa Izumiya1 and Haruo Watanabe1
Infantis Isolated from Poultry in Japan
Osaka Prefectural Institute of Public Health, Osaka 537-0025 and
(Accepted March 14, 2006)
toms from self-limiting acute enteritis to systemic sepsis.
and the presence of these strains is considered to be highly
producing S. Newport isolates, and the other a CTX-M-14
specimens of domestic poultry purchased from retail stores
mon serotype was S. Infantis, which was isolated from 21
Antimicrobial susceptibility testing was performed using
1National Institute of Infectious Diseases, Tokyo 162-8640, Japan
*Corresponding author: Mailing address: Osaka Prefectural Insti-
tute of Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka
537-0025, Japan. Tel: +81-6-6972-1321, Fax: +81-6-6972-0772,
Extended-spectrum cephalosporins (ESCs) may be used in
problematic in treating patients (1-3).
β-lactamase-producing S. Enteritidis isolate (4,5).
in Osaka Prefecture, a western part of Japan. Of the 83 speci-
specimens. Two of the serotypes were resistant to ESCs.
treatment of a severe case. Salmonella spp. strains resistant
There have been only two reports of ESC-resistant Salmo-
We have been conducting surveillance regarding the anti-
mens collected from April 2004 to March 2005, 29 (34.9%)
Here we report the results of our detailed bacteriological and
a disk diffusion method, as previously described according
Becton Dickinson Microbiology Systems, Cockeysville, Md.,
seven were resistant to kanamycin (KAN), streptomycin
(AMP), KAN, STR, STX and TET (strain No. 16A-166); one
STR, STX and TET; and one was resistant to NAL, STR,
cephalothin (CEF) and cefoxitin (FOX) were also applied
mL for both strains (Table 1).
to the standards outlined by the Clinical and Laboratory Stan-
USA. MICs were determined with an E-test (AB Biodisk,
(STR), sulfamethoxazole-trimethoprim (STX) and tetracycline
was resistant to AMP, chloramphenicol (CHL), STR and TET
STX and TET (strain No. 16A-158). Since the strains No.
in the MIC tests. Both strains were resistant to CTX at MIC
PCR detection of the blaCMY gene and sequence analysis
dards Institute (formerly, the National Committee for Clini-
Solna, Sweden). All the 21 S. Infantis isolates showed resist-
(TET); six were resistant to KAN, STR and TET; three were
(strain No. 16A-157); one was resistant to AMP, KAN, STR
16A-157 and No. 16A-166 showed intermediate suscep-
16 μg/mL and the resistance was not inhibited by clavulanic
was performed as follows. The blaCMY gene was detected by
cal Laboratory Standards) (6). Disks were purchased from
ance to two or more of the antimicrobials tested. Among them,
resistant to STR and TET; one was resistant to ampicillin
and TET; one was resistant to KAN, nalidixic acid (NAL),
tibility to cefotaxime (CTX) in the disk diffusion method,
acid, while MICs for CEF and FOX were higher than 256 μg/
Jpn. J. Infect. Dis., 59, 2006
PCR using primers described in Zhao et al. (7). Nucleotide
3´) and ampC-3´ (5´-tttttgttaagtgtagatgac-3´). The amplified
tion Kit and ABI PRISM 3100-Avant (Applied Biosystems,
16A-166, a 1-kb fragment was amplified by a PCR method
previously reported for the blaCMY-2 gene in Klebsiella
buffer. Plasmid transconjugation tests were performed using
(Nissui, Tokyo, Japan) containing 20 μg/ml NAL and 5 μg/ml
plasmid was observed in the strain No. 16A-166 (Fig. 1).
donor 16A-166 did not result in any transfer of resistance. In
CEF, FOX, and STR in the transconjugant were quite similar
frequency every year in Japan. The frequency of S. Infantis
affects food hygiene (10). In the year 2005, the serovar was
sequencing was performed as follows. A 1.3-kb DNA frag-
fragment was purified using Microspin Columns (Amersham
Foster City, Calif., USA). The resulting sequences were
for detection of the blaCMY gene (Table 1). In addition, the
S. Infantis strains No. 16A-157 and No. 16A-166 as donors,
CTX, and were used for antimicrobial susceptibility testing,
In the transconjugation test, the conjugation between donor
the 16A-157/158 transconjugant, a plasmid over 90 kb in
to those in the donor strain. In addition, the blaCMY gene was
isolation from domestic poultry has been increasing since the
ranked second following S. Enteritidis (full data are avail-
ment containing the open reading frame of bla was amplified
Biosciences Corp., Piscataway, N. J., USA), and sequenced
searched for homologues in the EMBL database with the
nucleotide sequences of the DNA fragments amplified by
Plasmids were extracted according to the Kado and Liu
and S. Infantis strain No. 16A-158 as a recipient. The result-
plasmid detection, and blaCMY gene detection. The strain No.
16A-157 and recipient 16A-158 transferred antimicrobial
length was detected as in the donor strain (Fig. 1). In anti-
detected in the transconjugants (Table 1).
middle 1970s. Now the organism has become a representa-
able at http://idsc.nih.go.jp/iasr/virus/graph/salm2003.gif). In
by PCR using a primer pair of ampC-5´ (5´-tctgctgctaaatttaaccg-
using a BigDye Terminator Cycle Sequencing Ready Reac-
WU-BLAST program. In both strains of No.16A-157 and No.
primers of ampC-5´ and ampC-3´ were 100% identical to that
method (9), and were separated in a 0.5% agarose gel in TBE
ing transconjugants were selected on trypticase soy agar plates
16A-157 possessed a plasmid over 90 kb in length, while no
resistance at a frequency of 10–4, while conjugation using
microbial susceptibility testing, MICs of AMP, CHL, CTX,
S. Infantis is one of the serovars that are detected in high
tive etiologic agent causing food poisoning and seriously
August 2004, Sakai City in Osaka Prefecture experienced
a large-scale outbreak of food poisoning that involved 366
in Japan, and they were found to possess the blaCMY-2 gene. It
were multi-drug resistant. In addition, the two ESC-resistant
ing blaCMY-2 are usually carried on plasmids (1). The ESC-
and CHL. However, no plasmids were detected in another
has previously been reported that most S. Infantis isolates
isolates were also resistant to AMP, STR, TET, KAN, and
resistance in strain No. 16A-157 appeared to be derived from
resistant strain, No. 16A-166, the resistance of which was not
There has thus far been only one report (2) describing S.
In this study, we identified two isolates of S. Infantis resis-
from poultry show multi-drug resistance in Japan (10), and,
STX (No. 16A-166), and AMP, CHL, STR and TET (No.
a conjugative plasmid. Furthermore, the plasmid of 16A-157
transferred in a transconjugation test. Further study is needed
tant to ESCs, which is the first finding of these resistant strains
accordingly, all 21 isolates of S. Infantis in the present study
16A-157). It is well known that resistance genes includ-
seemed to also have resistance determinants to at least AMP
to elucidate how drug resistance had been acquired in this
Table 1. Characteristics of S. Infantis16A-166, 16A-157, 16A-158 and 16A-157/158 transconjugant
1): Determined by E test.
CTX, cefotaxime; CEF, cephalothin; FOX, cefoxitin; CTL, cefotaxime plus clavulanic acid.
S. Infantis 16A-166
3): NT, not tested.
S. Infantis 16A-157
AMP, ampicillin; CHL, chloramphenicol; STR, streptomycin; TET, tetracycline; KAN, kanamycin; NAL, nalidixic acid; CIP, ciprofloxacin;
S. Infantis 16A-1582)
2): S. Infantis 16A-158: recipient of transconjugation.
1 264 25620.125 0.06444
Fig. 1. Plasmid profiles of S. Infantis 16A-166, 16A-157, 16A-158 and
lambda DNA digested with HindIII; Lane M2, size marker S.
16A-157/158 transconjugant. Lane 1, S. Infantis 16A-166; Lane 2,
Enteritidis L-156 with the size of 60 kb; Lane M3, size marker E.
S. Infantis 16A-157; Lane 3, S. Infantis 16A-158; Lane 4, S.
coli K12 CSH-2 (NR1) with the size of 90 kb.
Infantis16A-157/158 transconjugant; Lane M1, a size marker of
M1 M2 M31234
146 Download full-text
Infantis from a pediatric patient in Honduras that produced
In Japan, six cephalosporins are approved for parenteral
E. coli and S. Infantis strains have become resistant to ESCs
concern, in that they could spread through foods based on
vey of the spread of ESC-resistant Salmonella it would be
This work was supported by a grant from the Ministry of
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However, ESC-resistant E. coli strains were isolated from
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The cephalosporins used in this study were mainly for
not only for humans but also for food-producing animals.
broilers in a nationwide survey (11). The reasons why these
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