NOTE Public Health

Emergence of Salmonella enterica Serovar Infantis Harboring IncI1 Plasmid with

blaCTX-M-14 in a Broiler Farm in Japan

Mitsuhiro KAMEYAMA1), Takehisa CHUMA2)*, Tomoki YOKOI2), Junko YABATA1), Kiyoshi TOMINAGA1),
Daisuke MIYASAKO2), Hiroyuki IWATA3) and Karoku OKAMOTO2)

1)Yamaguchi Prefectural Institute of Public Health and Environment, 2–5–67 Aoi, Yamaguchi 753–0821, Japan
2)Laboratory of Veterinary Public Health, Joint Faculty of Veterinary Medicine, Kagoshima University, 1–21–24 Korimoto, Kagoshima
890–0065, Japan
3)Department of Veterinary Medicine, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677–1 Yoshida, Yamaguchi

753–8515, Japan

(Received 28 October 2011/Accepted 20 April 2012/Published online in J-STAGE 16 May 2012)

ABSTRACT. Cefotaxime (CTX)-resistant and -susceptible Salmonella enterica serovar Infantis isolates obtained from broilers raised on a
farm in January 2010 in Japan were characterized to establish their resistance determinants. The CTX-resistant isolates produced CTX-
M-14 extended-spectrum β-lactamase and harbored 2 distinct plasmid of approximately 140- and 95-kb, whereas the CTX-susceptible
isolates harbored one 140-kb plasmid. The 95-kb plasmids were replicon typed as IncI1 carrying the blaCTX-M-14 gene, while the 140-kb
plasmids were IncP and harbored the aphA1, aadA1, tetA, and sul1 genes. Genetic fingerprinting by pulsed-field gel electrophoresis
revealed similar macrorestriction profiles amongst CTX-resistant and susceptible isolates, suggesting a clonal relationship. The presence
of CTX-resistant S. Infantis on a broiler farm has occurred through the acquisition of IncI1 resistance plasmid.
KEY WORDS: β-lactamase, broiler, CTX-M-14, extended-spectrum plasmid replicon typing, Salmonella Infantis.

doi: 10.1292/jvms.11-0488; J. Vet. Med. Sci. 74(9): 1213–1216, 2012

Amongst members of the Enterobacteriaceae fam- Japan in January 2010 were used in this study. The broiler
ily, Escherichia coli (E. coli) and Klebsiella pneumoniae chickens involved in this survey were sampled from 3 dif-
have been implicated as potential producers of extended- ferent flocks raised on the same commercial farm. The iso-
spectrum β-lactamases (ESBLs) which possess hydrolyzing lation of the bacteria was done as follows; approximately 1
activity against third-generation cephalosporins. In recent g of cecal contents was aseptically mixed with 5 ml of steril-
years, several reports have described other bacterial species ized distilled water. Then, 1 ml of suspension was enriched
within the Enterobacteriaceae family capable of producing in 10 ml of tetrathionate broth (Merck KGaA, Darmstadt,
ESBLs. Of these, Salmonella spp. producing ESBLs have Germany) and incubated at 42°C. After 24 hr of incubation,
been detected in salmonellosis cases both in humans [9, 10, a loopful from each of enriched broth was streaked onto
17] and domestic animals [18]. plates of selective deoxycholate hydrogen sulfide lactose
Salmonella enterica serovar Infantis (S. Infantis) is one (DHL) agar (Oxoid Ltd., Basingstoke, Hampshire, UK) and
of the predominant serotypes isolated from broilers in Japan mannitol lysine crystal violet brilliant green (MLCB) agar
[19]. In the late 1990s, S. Infantis isolates harboring ESBLs (Eiken Chemical Co., Ltd., Tokyo, Japan), and incubated
were recovered from hospitalized patients in South America at 37°C for 24 hr. Suspected colonies were selected from
[14]. More recently, S. Infantis producing ESBLs were found each plate and cloned on Mueller-Hinton agar (Oxoid Ltd.),
not only in human patients but also in domestic animals and and the following identification and serotyping of bacteria
commercial meats [3, 5]. Most of these produced the CTX- were performed as described elsewhere [19]. These isolates
M-type ESBLs, indicating the probability that dissemina- were stocked in brain heart infusion broth containing 20%
tion of 3rd-generation cephalosporin-resistant S. Infantis of glycerol at −80°C until use the following tests. Antimi-
associated with various types of β-lactamases is gradually crobial susceptibility testing was performed by employing
expanding world-wide. In this article, we characterized S. the Kirby-Bauer disk diffusion method on Mueller-Hinton
Infantis isolates carrying CTX-M-14 ESBL derived from (Oxoid Ltd.) agar plates using the following antimicrobial
broiler chickens. agents: ampicillin (AMP), cephalothin (CEF), cefotaxime
Ten S. Infantis isolates obtained from 30 broiler cecal (CTX), ceftazidime (CAZ), streptomycin (STR), kanamycin
samples collected at a poultry processing plant in western (KAN), gentamicin (GEN), tetracycline (TET), chloram-
phenicol (CHL), nalidixic acid (NAL), ciprofloxacin (CIP),
tosufloxacin (TFX) and trimethoprim-sulfamethoxazole
* Correspondence to: Chuma, T., Laboratory of Veterinary Public (SXT). The double-disk synergy test using CTX and CAZ
Health, Joint Faculty of Veterinary Medicine, Kagoshima Uni-
disks with or without clavulanic acid disks was performed
versity, 1–21–24 Korimoto, Kagoshima 890–0065, Japan.
according to the new criteria established by the Clinical
e-mail: [email protected]
and Laboratory Standards Institute (CLSI) [2]. The ESBL
©2012 The Japanese Society of Veterinary Science
genes were detected by polymerase chain reaction (PCR)
1214 M. KAMEYAMA ET AL.

Table 1. Resistance profiles and ESBL type of S. Infantis isolates

Source
Isolates Resistance phenotypesa) ESBL type
Date Flock Age of broiler (d)
Y1, Y2, Y3 20-Jan-2010 a 51 STR, KAN, TET, SXT - AMP, CEF, CTX CTX-M-14
Y5, Y6, Y7 20-Jan-2010 a 51 STR, KAN, TET, SXT -
Y4 25-Jan-2010 b 37 STR, KAN, TET, SXT - AMP, CEF, CTX CTX-M-14
Y8, Y9, Y10 25-Jan-2010 c 49 STR, KAN, TET, SXT -
a) STR, streptomycin; KAN, kanamycin; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole; AMP,ampicillin; CEF, cephalo-
thin; CTX, cefotaxime.

Fig. 1. (A) PFGE analysis of S. Infantis genomic DNA digested by BlnI

enzyme. Lane M, Salmonella enterica serovar Braenderup H9812 used as a
size marker; Lane 1, isolate Y1; Lane 2, Y2; Lane 3, Y3; Lane 4, Y4; Lane
5, Y5; Lane 6, Y6; Lane 7, Y7; Lane 8, Y8; Lane 9, Y9; Lane 10, Y10.
Numbers on the left indicate the size of the bands in lane M. (B) Plasmid
profiles of S. Infantis isolates. Lane M, standard Salmonella enterica se-
rovar Cholerasuis (50 kb) and serovar Typhimurium DT 104 (90 kb); Lane
1, isolate Y1; Lane 2, Y2; Lane 3, Y3; Lane 4, Y4; Lane 5, Y5; Lane 6, Y6;
Lane 7, Y7; Lane 8, Y8; Lane 9, Y9; Lane 10, Y10. Numbers on the left
indicate the sizes in lane M and arrow on the right indicate detected band
sizes (approximately).
 CTX-M-14 ESBL IN SALMONELLA INFANTIS 1215

Table 2. MIC of antimicrobials and their resistance genes harbored plasmids of the donor isolates and transconjugants
Plasmid (s) MIC (µg/ml) a)
Strain Size Replicon Resistance gene (s)
AMP CTX KAN STR TET SUL
(kb) type
Donor Y2 95, 140 I1, P >512 256 >512 64 128 >512 blaCTX-M-14, aphA1, aadA1, tetA, sul1
Transconjugant T1 95, 140 I1, P >512 16 >512 32 64 256 blaCTX-M-14, aphA1, aadA1, tetA, sul1
T2 95 I1 >512 32 2 4 1 0.5 blaCTX-M-14

Donor Y6 140 P 4 <0.25 >512 64 128 >512 aphA1, aadA1, tetA, sul1
Transconjugant T3 140 P 4 <0.25 >512 32 32 256 aphA1, aadA1, tetA, sul1
a) AMP, ampicillin; CTX, cefotaxime; KAN, kanamycin; STR, streptomycin; TET, tetracycline; SUL, sulfamethoxazole.

[20, 21]; and obtained amplicons were directly sequenced and sul1 genes. Resistance phenotypes and genes detected
using specific primers [1] with the BigDye Terminator in E. coli transconjugants are summarized in Table 2. T2
v3.1 Ready Reaction Sequencing kit and the ABI 3500 × transconjugants with a 95-kb plasmid showed resistance to
l Automated DNA Sequencer (Applied Biosystems, Foster AMP and CTX; and they also tested positive for the blaCTX-
City, CA, U.S.A.). The sequencing data were compared M-14 gene, suggesting the location of the blaCTX-M-14 to be the
with the published DNA sequences using the BLASTN 95-kb plasmid. Genetic analysis of the T3 transconjugants
database (www.ncbi.nlm.nih.gov). The resistance patterns revealed that resistance traits aphA1, aadA1, tetA, and sul1
of S. Infantis isolates examined are shown in Table 1. All were located on the 140-kb plasmid.
10 S. Infantis isolates were shown resistance to KAN, STR, The PCR-based replicon typing (PBRT) was carried out
TET and SXT, and of those, the 4 isolates (Y1, Y2, Y3 and as previously described [11]. The findings of PBRT demon-
Y4) exhibited resistance to three β-lactam antibiotics; AMP, strated that blaCTX-M-14 was associated with IncI1 plasmid.
CEF and CTX. Preliminary phenotypic double-disk synergy On the other hand, plasmids harboring aphA1, aadA1, tetA,
test results indicated that the 4 CTX resistant isolates were and sul1 were IncP type. Previous reports indicated that
potential ESBL producers. Ultimately, PCR and nucleotide IncI1 plasmids harboring blaCTX-M-14 were detected in Sal-
sequence analysis revealed that the CTX resistant isolates monella enterica serovar Enteritidis (S. Enteritidis) [7] and
carried the blaCTX-M-14 gene. E. coli bacteria [15]. It’s worth noting that several studies
Pulsed-field gel electrophoresis (PFGE) using BlnI re- describing the occurrence of CTX-M-14 ESBL in S. Enteriti-
striction enzyme was performed according to the standard dis isolated from human patients in Spain [17], Hong Kong
PulseNet protocol [16]. The banding patterns obtained were [10] and Japan [9] have been reported since 2003. In Japan,
analyzed using the Molecular Analyst Fingerprinting Plus serovar Enteritidis producing CTX-M-14 enzyme was iso-
software (Bio-Rad Laboratories Inc., Hercules, CA, U.S.A.). lated from chicken meat imported from China in 2004 [13].
The fragment similarity above 94% was observed among the Moreover, a commentary was released in Japan explaining
isolates, suggesting the likelihood of sharing the same origin the occurrence of S. Infantis producing CTX-M-14 ESBL
(Fig. 1A). Isolation of plasmids was conducted using the al- recovered from domestic poultry meat [4].
kaline lysis method as previously described [12]. Analysis of Although the use of cephalosporins including CTX is not
the plasmid profiles demonstrated that 4 isolates (Y1, Y2, Y3 approved for broilers in Japan, we detected CTX-resistant
and Y4) harbored two plasmids of approximately 140 kb and S. Infantis isolates derived from the broiler farm. It is likely
95 kb. The other 6 isolates (Y5, Y6, Y7, Y8, Y9 and Y10) that the blaCTX-M-14 gene was acquired by S. Infantis isolates
carried only one plasmid of approximately 140 kb (Fig. 1B). through interspecies transmission of the potential IncI1
Conjugation experiments were conducted as described resistance plasmid. This hypothesis is supported by early
elsewhere [18] using the isolates Y2 and Y6 as donors and reports which described the existence of CTX-M-14 ESBL
the rifampicin-resistant E. coli DH5α strain as recipient. We producing E. coli from broilers and chicken meats in Japan
observed 3 distinct types of E. coli transconjugants: T1 trans- [6, 8]. The spread of cephalosporin-resistant Salmonella spp.
conjugants contained both 140- and 95-kb plasmids, whereas in poultry via the transmissible resistance plasmids raises
T2 carried 95-kb plasmid and T3 harbored 140-kb plasmid serious veterinary and public health concerns. Thus, continu-
(Table 2). Out of the transconjugants, the T1 and T2 were ous monitoring of members of the Enterobacteriaceae fam-
obtained from Y2 donor, and the T3 were from Y6 donor. ily possessing IncI1 resistance plasmids is required in order
These findings indicate that both 95-kb and 140-kb plasmids to establish the magnitude of the health hazard associated
associated with S. Infantis donor isolates were potentially with these bacteria.
self-transmissible. The minimum inhibitory concentrations
for AMP, CTX, KAN, STR, TET and sulfamethoxazole ACKNOWLEDGMENT. This research was partly supported
(SUL) on E. coli transconjugants were determined by the by a Grant-in-Aid for Scientific Research (No.23580426)
agar dilution method. The PCR assay was performed to con- from the Japan Society for the Promotion of Science.
firm the transmission of the blaCTX-M-14, aphA1, aadA1, tetA,
1216 M. KAMEYAMA ET AL.

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