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121（ 1 ）〈Original Article〉

Contamination rate of broad-spectrum cephalosporinresistant Enterobacterales isolated fromdomestic and imported retail chicken meat in Tokyoand Kanagawa, Japan
Yuya Hasunuma, Ph.D.1,2, Nozomi Hiyoshi1, Masaya Tanaka2 andYoshikazu Tokuoka, Ph.D.1,2
1

2

Graduate School of Engineering, Toin University of Yokohama
Department of Medical Technology, Faculty of Biomedical Engineering,Toin University of Yokohama(Received for publication September 10, 2021)

Extended-spectrum cephalosporin-resistant Enterobacterales are among the mostprevalent antimicrobial-resistant organisms in Gram-negative bacteria, and theirspread in the veterinary ﬁeld is of global concern. We determined the contamination
rate of extended-spectrum cephalosporin-resistant Enterobacterales in domestic andimported retail chickens in Japan, and examined their antimicrobial resistance andmolecular characteristics. Fifty-three domestic and thirty-nine imported retailchickens were purchased, and antimicrobial susceptibility, β-lactamase encoding
genes, sequence type, and incompatibility type of plasmids were assessed in theisolates. The contamination rates of cefotaxime-resistant Enterobacterales isolated
from domestic and imported retail chickens were 60.4% and 87.2%, respectively (p＜ 0.05). Approximately 50% of isolates harbored the blaCTX-M gene, with blaCTX-M-1
and blaCTX-M-2 groups predominating domestic and imported samples, respectively.
The gene for plasmid-mediated AmpC was detected in 34% and 33% of isolates
from domestic and imported samples, respectively. Isolates possessing blaCIT with
IncK plasmids were found in the isolates from both sample groups. Additionally, ourresults indicated that the IncK plasmid had the potential for transconjugation with
blaCIT. Escherichia coli sequence type (ST) 57 and ST117 were predominant,followed by ST38. The ﬁndings of this study suggest that the high contamination
Corresponding author: Yuya Hasunuma, Graduate School of Engineering, Toin University of Yokohama, 1614 Kurogane-cho,Aoba-ku, Yokohama, Kanagawa 225–8503, Japan, Tel.: ＋81–45–972–5881; Fax: ＋81–45–974–5269, E-mail: hyuya@toin.
ac.jp

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rate of drug-resistant bacteria in retail chicken meat leads to frequent exposure tothese pathogens by the general public in Tokyo and Kanagawa, Japan.
Key words: Escherichia coli, extended-spectrum β-lactamase, plasmid-mediated AmpC
β-lactamase, plasmid incompatibility type K

Introduction
Extended-spectrum cephalosporin-resistant Enterobacterales are the most prevalent antimicrobial-resistant organisms among Gram-negative bacteria, spreading to both patients and healthy
individuals as well as amimals, including companion animals, livestock, and environments1). In
agricultural and veterinary settings, antibiotics are frequently employed as pharmaceuticals foranimals, as growth promoters for livestock. This use may contribute to the spreading of broadspectrum cephalosporin-resistant Enterobacterales in livestock farming environments and theemergence of the bacterial strains carrying genetic determinants of cephalosporin resistance, suchas Extended-Spectrum β-Lactamases (ESBLs) and plasmid-mediated AmpC (pAmpC) genes. The
selection of antimicrobial resistant organisms in livestock and its dissemination in the farms directly impact the contamination of their retail meat. The contamination of ESBLs-producing organisms in retail chicken meat has been investigated in several countries2–5).
ESBLs and AmpC β-lactamase-producing Escherichia coli were detected in poultry between
1999 and 2007 in Japan6, 7). Hiki et al. demonstrated that the restriction of ceftiofur use at thehatchery level has reduced the percentage of broad-spectrum cephalosporin-resistant E. coli isolates from broilers since 2012, when the oﬀ-label use of ceftiofur in conjunction with in ovo vaccination or vaccination of newly hatched chicks was voluntarily discontinued in Japan8). However, the inﬂuence at the retail level on the contamination rate of extended-spectrum
cephalosporin-resistant Enterobacterales in domestic chickens has not been evaluated. Additionally, Japan imports chicken from Brazil, the US, Thailand, and China (https://www.alic.go.jp/
joho-c/joho05_000073.html). Given that antibiotic use in livestock production processes varies
between countries, the molecular epidemiology of the extended-spectrum cephalosporin-resistantEnterobacterales between Japan and each country is likely to diﬀer. The harboring and spread of
ESBL encoding genes in Enterobacterales is depends on the host organismʼs clone, plasmid incompatibility types, and other factors. However, there are few reports on the diﬀerences between
isolates from domestic and imported retail chicken meat in these epidemiological and characteristic data.
Considering the possibility that contaminated retail meat may be a source of food-borne antimicrobial resistance, it is important to regularly monitor antibitoic resistance to protect publichealth. In this study, we assessed the contamination rate of extended-spectrum cephalosporin-re-Dec. 2023

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123（ 3 ）sistant Enterobacterales in domestic and imported retail chicken, and examined their antimicrobial resistance proﬁles and genotypic characteristics, such as β-lactamase encoding genes, plasmid incompatibility typing, and clonal relationships.

Materials and Methods
1) Isolation from samples, identiﬁcation, and antimicrobial susceptibility test
Domestic (53 products) and imported (39 products) retail fresh chicken meat samples (34, 3,
and 2 samples from Brazil, Thailand, and the US, respectively) were randomly purchased (January 2016–August 2019) from various markets in Tokyo metropolitan and Kanagawa prefecture,
Japan. Samples were purchased as a maximum of two diﬀerent products per market. Ten gramsof each retail meat sample were homogenized and were incubated for 18–24 hr in Brain-Heart Infusion culture broth (BD, Japan) supplemented with 2 mg/L cefotaxime (CTX) (Sigma, Japan).
Cultured samples were spread on MacConkey agar (Eiken Chemical, Japan) containing 2 mg/L
CTX, incubated at 37°C for 18–24 hr. After the incubation, four to six colonies suspected to beEnterobacterales were randomly picked up from each meat sample and were subjected to further
analysis. Enterobacterales isolates were identiﬁed by API 20E (bioMerieux, France).
Antimicrobial susceptibility testing was carried out using agar plate dilution methods including ampicillin (ABPC) (Wako, Japan), cefazolin (CEZ) (Sigma, Japan), cefmetazole (CMZ)(Sigma, Japan), ceftazidime (CAZ) (Sigma, Japan), CTX, imipenem (IPM) (Wako, Japan), gentamycin (GM) (Wako, Japan), amikacin (AMK) (Wako, Japan), and ciproﬂoxacin (CPFX) (Wako,
Japan). The concentration ranges of ABPC and AMK were 4–128 mg/L, and those of the remainders were 0.5–16 mg/L. The quality control strain was E. coli ATCC 25922. Breakpoints were determined according to the recommendations of the Clinical and Laboratory Standards Institute9).
An ESBL conﬁrmation was performed by a double-disk synergy test using CTX (BD, Japan),
CTX/clavulanate (Eiken Chemical, Japan), CAZ (BD, Japan), and CAZ/clavulanate (Eiken
Chemical, Japan).
2) DNA extraction and detection of β-lactamase encoding genes
A DNA template was obtained using the DNeasy Blood & Tissue Kit (QIAGEN, Germany).
The presence of ESBL and pAmpC β-lactamase encoding genes were identiﬁed through multiplex PCR ampliﬁcation of blaCTX-M, blaTEM, and blaSHV genes, and six main groups (blaACC,
blaCIT, blaDHA, blaEBC, blaFOX, and blaMOX) of pAmpC-type genes, as described previously10, 11). In
addition, if the strain tested positive for the blaCTX-M gene, the CTX-M groups (-1, -2, -8/25, and-9) genes of the strain were classiﬁed, as described previously12). PCR was performed with Takara Ex taq (Takara Bio, Japan) to detect these genes. All PCR amplicons were veriﬁed by gelelectrophoresis on 1.8% SeaKem LE agarose (Takara Bio, Japan).

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3) Multi-locus sequence typing and phylogenetic grouping of E. coli strains
Multi-locus sequence typing (MLST) was performed in accordance with the Achtman
scheme using seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA)13). The
MLST procedure, including allelic type and sequence type (ST) assignment methods, was conducted according to the protocol described on the website (https://enterobase.warwick.ac.uk/species/ecoli/allele_st_search). A total of 49 E. coli strains, randomly selected from isolates, were
tested. Among the novel STs found in this study, the single-locus variants of known ST complexes were considered to belong to the previously identiﬁed ST complexes. E. coli isolates werealso analyzed to determine four major phylogenetic groups (A, B1, B2, and D) using a triplex
PCR assay of chuA, yjaA, and tspE4.C2, as described previously14).
4) Plasmid incompatibility typing
PCR-based replicon typing was performed on all strains, as described previously15). Eighteen
primer pairs targeting the FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F,
and FII replicons were used in multiplex and simplex PCR reactions.
5) Transconjugation testing
The transmissibility test was performed the 19 IncK and blaCIT-positive isolates. An E. coli
DH5α strain that was resistant to rifampicin (generated by our laboratory) was used as the recipient. The donor-to-recipient ratio was 1 : 4, and broth mating was incubated at 37°C for 20 hr.
Transconjugants were selected on modiﬁed Drigalski agar (Eiken Chemical, Japan) supplemented
with 50 mg/L rifampicin (Wako, Japan), and 2 mg/L CTX. The transconjugants obtained were
subjected to antimicrobial susceptibility testing as used CMZ, CAZ, CPFX, and GM. Additionally, the presence of bla genes and the typing including plasmid incompatibility in the transconjugants ware conﬁrmed by PCR.
6) Statistical analysis
The contamination rate of the sample and antimicrobial resistance rate of the isolates werecompared between domestic and imported retail chicken using the chi-square test and Fisherʼsexact test. A p-value of ＜ 0.05 was considered to be a statistically signiﬁcant. All statistical calculations were performed using R (version 3.2.1) and R Studio (version 1.0.153).

Results
1) Contamination rate and bacterial characterization of isolates
The contamination rates of CTX-resistant Enterobacterales isolated from domestic and imported retail chicken meat were 60.4% (32/53) and 87.2% (34/39), respectively (p ＜ 0.05). ADec. 2023

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total of 41 and 64 isolates were obtained, with an average of 1.28 and 1.88 isolates per sample
from domestic and imported samples, respectively. Of the 105 isolates, 92 were identiﬁed as E.
coli, and the remaining isolates were identiﬁed as 8 species, as shown in Table 1. There were 24
and 41 positive strain isolates from domestic and imported samples, respectively, in the ESBL
screening test. Ninety-two E. coli isolates were further analyzed for distribution of CTX-M andpAmpC type β-lactamase encoding genes, MLST, phylogenetic grouping, and plasmid incompatibility typing.
The resistance rate of antibiotics among the isolates from domestic and imported samples
were 100% vs. 100% for ABPC, 35.6% vs. 35.9% for CMZ (p ＝ 1), 100% vs. 100% for CTX,
61.0% vs. 45.3% for CAZ (p ＝ 0.1299), 8.8% vs. 11.9% for CFPM (p ＝ 1), 0% vs. 0% for IPM,
29.3% vs. 31.3% for CPFX (p ＝ 1), 19.5% vs. 54.7% for GM (p ＜ 0.05), and 0% vs. 0% for
AMK, as shown in Table 2.
Table 1. Contamination rates and bacterial isolates identiﬁed from eachsampleDomestic

Imported

60.4%(32/53)

87.2%(34/39)Number of isolates from each sample

41

64

Distribution for identiﬁed isolates
Escherichia coli
Escherichia fergusonii
Enterobacter cloacae
Enterobacter intermedius
Citrobacter freundii
Klebsiella pneumoniae
Proteus mirabilis
Salmonella sp.
Serratia liquefaciens

37
—
1
—
—
1
—
1
1

55
1
—
2
1
—
4
1
—

Number of ESBL screening test-positive isolates

24

41

Contamination rate
(Number of contaminated samples/Total sample)—：The em dash represents no isolate collected.

Table 2. Comparison of antimicrobial susceptibility proﬁling of 105
isolates between domestic and imported samples
Susceptible (%)
Ampicillin
Cefmetazole
Cefotaxime
Ceftazidime
Cefepime
Imipenem
Ciproﬂoxacin
Gentamicin
AmikacinNon-susceptible (%)

Domestic

Imported

Domestic

Imported

0 (0)26 (63.4)
0 (0)16 (30.0)31 (91.2)
41 (100)
29 (91.2)
33 (80.5)
41 (100)0 (0)41 (64.1)0 (0)35 (54.7)
57 (89.1)
64 (100)
44 (88.1)
29 (45.3)
64 (100)

41 (100)
15 (36.6)
41 (100)
25 (70.0)
3 (8.8)0 (0)12 (8.8)
8 (19.5)0 (0)64 (100)
23 (35.9)
64 (100)
29 (45.3)
7 (10.9)0 (0)20 (11.9)
35 (54.7)
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2) Distribution of β-lactamase encoding genes detected in isolates
Table 3A shows the number of bla genes detected in isolates. Approximately 50% of isolates
from domestic and imported chickens possessed the blaCTX-M type ESBL genes, and blaTEM andblaSHV were detected in 40 isolates and one isolate, respectively. It is worth noting that blaTEM andblaSHV were included in non-ESBL. In the classiﬁcation of the blaCTX-M grouping, the proportionof blaCTX-M-1 group genes was higher in the domestic (13/41, 32%) than in the imported samples(3/64, 5%) (p ＜ 0.05). The genes of pAmpC were detected at a rate of 34% and 33% in isolates
from domestic and imported samples, respectively, and were classiﬁed as belonging to the blaCIT
type. The distribution of isolates possessing two or more β-lactamase is shown in Table 3B. Two
isolates harboring blaCTX-M and blaCIT were isolated from imported samples and identiﬁed as E.
coli and Proteus mirabilis.
3) Multi-locus sequence typing and phylogenetic grouping of E. coli
Of the 49 strains of E. coli, 34 strains were examined to determine sequence types. The remaining 15 strains had two or more diﬀerent alleles in the housekeeping genes. As a result, E.
coli ST57 and ST117 were the most common sequence types with 6 and 6 strains, respectively,
followed by E. coli ST38, and ST770 (Fig. 1A). All the strains assigned to these sequence types
were isolated from imported samples. On the other hand, the sequence types of E. coli isolates
from domestic samples were diverse, and categorized into 15 types of STs.
Fig. 1B indicates the relationship between the STs and the β-lactamases determined in E. coli
isolates. The isolates harboring the blaCTX-M-1 group of ESBL genes—which were predominantly
detected in isolates from domestic samples—were classiﬁed into ST10, ST69, ST355, andTable 3. Distribution of β-lactamase genotypes of 105 isolates between domestic and imported
samplesAB

β-lactamases genes

Number of positive isolates (%)
Domestic

Imported

ESBL
blaCTX-M-1 group
blaCTX-M-2 group
blaCTX-M-8 group
blaCTX-M-9 group
blaTEM a
blaSHV a

13 (32)4 (10)0 (0)7 (17)
20 (49)
0 (0)

3 (5)
28 (44)1 (3)0 (0)20 (31)
1 (2)

pAmpC
blaCIT

14 (34)21 (33)41

64

Total isolates

β-lactamases genes

Number of positive isolates
Domestic

Imported

ESBL
blaCTX-M-1 group ＋ blaTEM a
blaCTX-M-2 group ＋ blaTEM a
blaCTX-M-9 group ＋ blaTEM a

10
1
4

3
9
0

ESBL ＋ pAmpC
blaCTX-M-2 group ＋ blaCIT
blaTEM a ＋ blaCIT

0
5

2b
4b

A: The number of isolates harbored single β-lactamase gene detections from each strain, B: The number of isolates harbored
two or more combined β-lactamase gene detections from each strain.
a: blaTEM and blaSHV were included in non-ESBL, b: One isolate harbored blaCTX-M-2, blaTEM, and blaCIT.

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127（ 7 ）Fig. 1. Distribution of Multi-locus Sequence Typing (MLST) of E. coli Isolates

All graphs show MLST with (A): sample source, (B): determinate β-lactamase encoding genes, and (C): phylogenetic grouping.

ST770. Then, the isolates harboring the blaCIT type of pAmpC genes were extensively classiﬁed
into 10 sequence types. E. coli ST38, possessing β-lactamase, included only the blaCIT type of
pAmpC gene.
The ST, except for ST38, was recognized in each phylogenetic group, as shown in Fig. 1C.
In both domestic and imported samples, the dominant phylogenetic group of E. coli isolates wasgroup D, followed by group A. In the domestic sample, the proportion of group D isolates wasover 50%, whereas in the imported samples, the proportions of group A and group D of the isolates were almost equal at 40%.
4) Plasmid incompatibility typing and β-lactamase in E. coli
The distribution of the plasmid Inc type in E. coli was shown in Table 4. The plasmid type
IncFIB was the most dominant among all β-lactamase groups possessing isolates. The types of
IncHI1, IncN, IncP, and IncA/C were found only in isolates from domestic samples. On the other
hand, there were no Inc types found in isolates from imported samples. In comparison with

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Table 4. Number of positive isolates including plasmid incompatibility type and β-lactamase genes
Number of isolates distributed for each replicon type from domestic and imported samples

β-lactamase
genes

Domesticor Imported

HI1

HI2

I1-Iγ

X

L/M

N

FIA

FIB

W

Y

P

FIC

A/C

T

FIIAs

Frep

K

B/O

blaCTX-M-1
group

Domestic
Imported

—
—

—
—

4
0

—
—

—
—

3
0

1
0

9
2

—
—

—
—

1
0

—
—

—
—

—
—

—
—

11
2

1
0

4
0

blaCTX-M-2
group

Domestic
Imported

—
—

—
—

2
0

—
—

—
—

—
—

1
0

2
23

—
—

—
—

—
—

—
—

—
—

—
—

—
—

4
21

—
—

0
3

blaCTX-M-8
group

Domestic
Imported

—
—

—
—

0
1

—
—

—
—

—
—

—
—

0
1

—
—

—
—

—
—

—
—

—
—

—
—

—
—

1
0

—
—

—
—

blaCTX-M-9
group

Domestic
Imported

—
—

—
—

—
—

—
—

—
—

—
—

2
0

7
0

—
—

—
—

—
—

1
0

—
—

—
—

—
—

6
0

—
—

1
0

blaCIT

Domestic
Imported

1
0

—
—

5
3

—
—

—
—

—
—

2
2

10
16

—
—

2
0

1
0

0
1

3
0

—
—

—
—

10
11

9
10

5
5

—：The em dash represents no isolate collected.

β-lactamase types, the diversity of the Inc type in isolates harboring blaCTX-M was higher than thatin isolates harboring pAmpC genes. The isolates involved in IncN were found only in those harboring blaCTX-M, which was classiﬁed as the blaCTX-M-1 group. On the other hand, IncFIB and
IncK were simultaneously found in isolates harboring the blaCIT type pAmpC gene from both domestic and imported samples.
5) Ability of Transconjugation of IncK plasmid in E. coli
The IncK-positive and blaCIT-positive E. coli isolates from domestic and imported samples
demonstrated transtcojugation ability in 8/9 (88.9%) and 9/10 (90.0%) cases, respectively. The
antimicrobial susceptibility of transconjugants was exhibited to be resistant for CMZ and CAZ.
The β-lactamases genes transferred in the transconjugants were all detected as blaCIT. Additionally, plasmid incompatibility was detected IncK in all isolates. The frequency of the transconjugation did not diﬀer between isolates from domestic (3.07×10−3−2.78×10−8) and imported (1.77
×10−4−5.34×10−8) samples (Table 5).

Discussion
In the present study, we aimed to investigate the prevalence and molecular epidemiology ofextended-spectrum cephalosporin-resistant Enterobacterales in retail chicken meat in the Tokyo
metropolitan and Kanagawa prefecture regions of Japan. The contamination rate of the strain was
found to between 60–90% in both domestic and imported retail chickens, higher than previously
reported16). Such diﬀerences in contamination rates may be due to diﬀerences in years, regions,and sampling sizes of the products examined. Hiki et al. reported a decrease in the cephalosporin
resistance rate in E. coli isolated from healthy broilers at farms after a voluntary withdrawal ofceftiofur8). However, both the study by Kawamura et al. and our own study indicated that the de-Dec. 2023

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Table 5. The ability of transconjugation for 19 IncK and blaCIT-positive E. coli isolates
StrainaAntimicrobial susceptibility b(Donor/Tc)

β-lactamase genes c

Plasmid inc typing

Frequency d

CMZ

CAZ

CPFX

GM

Donor

Tc

Donor

Tc

D-1
D-2
D-3
D-4
D-5
D-6
D-7
D-8
D-9

I/I/S
S/R
R/R
I/R
I/R
I/R
I/R
I/I

I/R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R

S/S/S
S/S
S/S
R/S
S/S
S/S
S/S
S/S

S/S/S
S/S
S/S
S/S
R/S
R/R
R/R
R/I

CIT
CIT
CIT T
CIT T
CIT T
CIT T
CIT
CIT
CIT

—
CIT
CIT
CIT
CIT T
CIT
CIT
CIT
CIT

A/C K
I1-Iγ FIB Y K
I1-Iγ FIA FIB F K
HI1 FIB F K
FIB Y F K
FIB A/C F K
FIB F K B/O
P FIB F K B/O
I1-Iγ FIB F K B/O—
I1-Iγ FIB Y K
K
K
K
K
K B/O
P K B/O
I1-Iγ K B/O—
2.78×10−8
6.67×10−5
2.93×10−4
3.74×10−5
4.20×10−7
3.69×10−3
3.07×10−3
1.36×10−6

I-1
I-2
I-3
I-4
I-5
I-6
I-7
I-8
I-9
I-10

I/S
R/R
S/R
I/R
I/R
I/S
I/S
I/I/R
I/R

I/R
R/R
I/R
R/R
I/R
I/R
I/R
R/R/R
R/R

S/S
R/S
R/S
R/S
S/S
S/S
S/S
R/S/S
S/S

R/S
R/S
S/S
S/S
S/S
S/S
S/S
R/R/R
S/R

CIT
CIT T
CIT
CIT
CIT
CIT
CIT
CIT T
CIT
CIT

CIT
CIT
CIT
CIT
CIT
CIT
CIT
—
CIT
CIT

FIB F K
I1-Iγ FIB F K
I1-Iγ FIB K
FIB F K
K
FIB K
FIB K
FIA FIB F K
FIB F K B/O
FIB F K B/O

K
K
K
K
K
K
K
—
K B/O
FIB K B/O

1.84×10−6
1.77×10−4
4.15×10−4
7.92×10−5
3.67×10−6
4.73×10−7
5.34×10−8—
1.68×10−7
2.93×10−6

a: Tested strains from domestic samples (D), and from imported samples (I).
b: S, susceptible; I, intermediate; R, resistant. CMZ, cefmetazole; CAZ, ceftazidime; CPFX, ciproﬂoxacin; GEN, gentamicin.
The Clinical and Laboratory Standards Institute breakpoints were used to interpret the susceptibility results.
c: CIT, blaCIT (One of the plasmid-mediated AmpC group, including blaCMY-2, etc.); T, blaTEM.
d: Frequency was calculated as the number of colonies of transconjugant divided by the recipient. —： The em dash represents
no obtained the transconjugant.

crease in resistance rate at both the broiler and farm environment was not accompanied by a decrease in the contamination rate of extended-spectrum cephalosporin-resistant bacteria in retailmeat16). This suggests that the prevalence of retail chicken meat contaminated with drug-resistantbacteria is strongly inﬂuenced not only by the colonization of the bacteria in the poultry intestine,
but also by contamination at the slaughterhouse and other factors. Furthermore, our results indicate that the proportion of the extended-spectrum cephalosporins resistance mechanism mediatedby ESBL and pAmpC β-lactamases among Enterobacterales isolates was similar between domestic and imported retail chicken meat in the Tokyo metropolitan and Kanagawa prefecture regionsof Japan.
Initially, there was a marked diﬀerence in the proportion of blaCTX-M group classiﬁcation between domestic and imported samples. Kameyama et al. conducted an investigation into the epidemiology of broad-spectrum cephalosporin-resistant E. coli from various broiler farms andfound that the blaCTX-M-1 group, which comprised of blaCTX-M-1, blaCTX-M-15, and blaCTX-M-55, wasdominant in Japan17). Additionally, the dissemination of CTX-M-15 ESBL- and CTX-M-55
ESBL-producing E. coli has been reported through surveillance on farms in Europe and Southeast Asia18, 19). Notably, the blaCTX-M-55 ESBL gene is frequently detected in livestock, farms, and130（ 10 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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meat production in Southeast Asia and East Asia20–22). Therefore, it is expected that the types of
blaCTX-M-1 group ESBL genes present among the isolates from domestic samples in this study will
be designated as blaCTX-M-1, blaCTX-M-15, and blaCTX-M-55. However, this is unclear in this study because we did not determine as far as sequencing the blaCTX-M gene. From the point of view of incompatibility typing, isolates possessing blaCTX-M, which is associated with the dominant CTXresistant factor, generally carry the IncFIB type plasmid. IncI, IncN, and IncFIA were identiﬁed
only in the isolates from domestic samples (Table 4). These varieties of Inc types were consistent
with the blaCTX-M coding plasmid found in the isolates from patients in Japan23). On the other
hand, of course, it is diﬃcult to refer to a direct relationship between meat and patient derived
blaCTX-M based on incompatibility type concordance alone, and analysis of the surrounding structure of the bla genes and comparison with clinical strains is necessary even for the isolates.
In this study, the majority of retail chicken meat was sourced from Brazil, and the ESBL
coding genes carried by the isolates were predominantly from the blaCTX-M-2 and blaCTX-M-8
groups, with high GM and CPFX resistance rates, which is in accordance with previous studies16).
It is reported that blaCTX-M-2 and blaCTX-M-8 encoding integrons possessing Enterobacterales havespread in Brazil24). Moreover, Norizuki et al. also demonstrated that the blaCTX-M-8-possessing E.
coli was also detected in healthy humans, and the region surrounding blaCTX-M-8 was similar to thecomponent genes found in isolates from imported retail meat samples25). These ﬁndings strongly
suggest that antimicrobial resistant genes and/or cassettes may transfer blaCTX-M-8 from contaminated bacteria in retail meat to intestinal bacteria in human through ingestion. If the isolates inthis study also harbor GM or CPFX resistance genes within their integron structure, there is aconcern that exposure to imported retail chicken meat may lead to dissemination of multidrug-resistant strains to the general public. Indeed, fecal carriage of ESBL-producing Enterobacterales
in healthy adults is on the rise26). Sequence types 38, 57, and 117 of E. coli contained blaCTX-M-2
group ESBL genes, indicating that the E. coli of these sequence types harboring blaCTX-M-2 groupwere colonized in foreign broiler farms. In particularly, E. coli ST38 and ST117 isolates were associated with humans27–28). In light of this, attention should not only be paid to the resistance gene
cassette, but also to the direct transfer of the strain as a factor in the entry of resistance genes inmeat into the human intestinal tract.
Secondly, IncK-positive strains in both samples simultaneously carried blaCIT. Although it
has been reported that E. coli isolates possessing IncK with pAmpC β-lactamase were present in
the surveillance of Japanese broilers, the report did not provide detailed information about thecharacteristics of the strains because it only obtained a few isolates carrying IncK with thepAmpC β-lactamase encoding gene. Thus, this present report is the ﬁrst to document the epidemiological isolation of E. coli carrying IncK with blaCIT from retail chicken meat in Japan. Furthermore, we also demonstrated that the plasmid classiﬁed IncK has a high potential for transconjugation (17/19, 89.5%) with blaCIT (Table 5). Indeed, Hiki et al. found that 41.9% (13/31) ofDec. 2023

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131（ 11 ）transconjugants possessed blaCMY-2 carrying plasmids29). However, they have not clariﬁed even
the Inc typing of the plasmid possessed by the isolates. On the other hand, Seiﬀert et al. determined the complete sequence of the IncK plasmid in E. coli harboring blaCMY-2/4 and reported thatthe isolates from poultry, chicken and humans share the plasmid in Switzerland30). Consequently,
in this study, the diversity of E. coli sequence types carrying blaCIT may be due to the fact that the
IncK plasmid has transconjugation ability and its horizontal transfer occurs from one strain to another sequence type strain. Unfortunately, the reason why strains harboring blaCMY-2 are not so
widespread among healthy humans and patients in Japan is currently unknown.
Our study has several limitations. First, the locations where retail chicken meat was purchased were regionally restricted. Second, it is unclear whether the imported chicken meat samples were processed at a poultry slaughterhouse or supermarket. Then, if the imported chickenmeats collected in this study was processed domestically, it cannot be ruled out that the isolates
on the samples may be contaminants from other sources. However, the high contamination of thestrain isolated from retail chicken meat in Japan raise concerns about antimicrobial resistance andthe need for continued surveillance of antimicrobial resistance. Additionally, it is anticipated thatthe potential inﬂuence of human intestinal colonization through ingestion of retail meat will bedirectly eﬀective against community dwellers, as the retail meat samples in this study were available to consumers in the community. Indeed, in exploring the possibility of transfer of resistancegenes from retail meat, continuous surveillance of broad-spectrum cephalosporin-resistant Enterobacterales in the healthy carriers is clearly necessary.

Conclusion
In this study, retail chicken meats was found to be highly contaminated with extended-spectrum
cephalosporin-resistant Enterobacterales, with a higher contamination rate in imported samples.
The proportion of β-lactamase genes in isolates was comparable between ESBL and pAmpC encoding genes. However, the proportion of blaCTX-M group genes harbored the isolates diﬀered between domestic and imported samples, with blaCTX-M-1 group and blaCTX-M-2 group being predominant in domestic and imported samples, respectively. The pAmpC gene, classiﬁed as blaCIT, wasdetected in approximately 35% of isolates. The IncK plasmid was found to have the ability totransconjugate in a high percentage of diﬀerent ST isolates. This high-level of drug-resistant bacterial contamination of retail chicken meat presents a risk of frequent contact with the generalpublic in Tokyo and Kanagawa, Japan.

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Dec. 2023

Declarations
Availability of data and materials
All datasets on which the conclusions of the manuscript rely are presented in the paper.
Competing interests
We have no conﬂict of interest to declare.
Authorʼs contribution
Hasunuma, Y designed the experiments and wrote the manuscript. Hiyoshi, N. carried out
experiments, among which she mainly led the plasmid transconjugation and molecular epidemiological analysis. Tanaka, M. collected strains and conducted antimicrobial susceptibility testing.
Tokuoka, Y is organizing our research. All of the authors read and approved the ﬁnal manuscript.

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