J. Microbiol. Biotechnol. 2024.

34(5): 1101–1108
https://doi.org/10.4014/jmb.2312.12018

Review

Genetic Characteristics of Extended-Spectrum

Beta-Lactamase-Producing Salmonella
Isolated from Retail Meats in South Korea
Haiseong Kang1, Hansol Kim1, Hyochin Kim1, Ji Hye Jeon1, Seokhwan Kim2, Yongchjun Park1,
and Soon Han Kim1*
1
Food Microbiology Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety
Evaluation, Cheongju 28159, Republic of Korea
2
Food Standard Division, Ministry of Food and Drug Safety, Cheongju 28159, Republic of Korea

Supplementary data for Earlier studies have validated the isolation of extended-spectrum beta-lactamase-producing
this paper are available on- Salmonella (ESBL-Sal) strains from food. While poultry is recognized as a reservoir for Salmonella
line only at http://jmb.or.kr.
contamination, pertinent data regarding ESBL-Sal remains limited. Consequently, the Ministry of
Food and Drug Safety has isolated Salmonella spp. from retail meat and evaluated their antibiotic
susceptibility and genetic characteristics via whole-genome sequencing. To further elucidate these
aspects, this study investigates the prevalence, antibiotic resistance profiles, genomic characteristics,
and homology of ESBL-Sal spp. obtained from livestock-derived products in South Korean retail
outlets. A total of 653 Salmonella spp. were isolated from 1,876 meat samples, including 509 beef,
503 pork, 555 chicken, and 309 duck samples. The prevalence rates of Salmonella were 0.0%, 1.4%,
17.5%, and 28.2% in the beef, pork, chicken, and duck samples, respectively. ESBL-Sal was exclusively
identified in poultry meat, with a prevalence of 1.4% in the chicken samples (8/555) and 0.3% in the
duck samples (1/309). All ESBL-Sal strains carried the blaCTX-M-1 gene and exhibited resistance to
ampicillin, ceftiofur, ceftazidime, nalidixic acid, and tetracycline. Eight ESBL-Sal isolates were
identified as S. Enteritidis with sequence type (ST) 11. The major plasmid replicons of the Enteritidis-
ST11 strains were IncFIB(S) and IncFII(S), carrying antimicrobial resistance genes (β-lactam,
tetracycline, and aminoglycoside) and 166 virulence factor genes. The results of this study provide
valuable insights for the surveillance and monitoring of ESBL-Sal in South Korean food chain.
Keywords: Antimicrobial resistance, extended-spectrum beta-lactamase, Salmonella spp., genetic characteristics,
whole-genome sequencing, prevalence

Introduction
Salmonella is a predominant etiological agent of diarrhea, constituting a pervasive global health concern with
an annual incidence of 1.9 billion cases worldwide [1]. Moreover, its substantial contribution to diarrheal diseases
surpasses that of other enteric pathogens [2]. The main sources of Salmonella infection include contaminated
Received: December 12, 2023
water, eggs, and meats [3]. Contaminated meat is also an important reservoir of antibiotic-resistant genes (ARGs)
Accepted: March 3, 2024
[4]. The dissemination of antibiotic-resistant bacteria (ARB) through the food chain poses a substantial risk as it
First published online: can introduce these pathogens into the human gastrointestinal tract [5]. Therefore, meat contaminated with
March 22, 2024 antibiotic-resistant Salmonella has emerged as a significant threat to human health worldwide.
Various antibiotics have been extensively employed for disease prevention and treatment in the livestock
*Corresponding author industry globally [6]. Notably, fluoroquinolones and cephalosporins have been prominently utilized for treating
E-mail: [email protected] Salmonella infections [7]. Simultaneously, there has been a discernible increase in the application of beta-lactam
antibiotics for treating Salmonella infections [8]. This heightened usage can exert a selective pressure conducive to
pISSN 1017-7825 the proliferation of extended-spectrum beta-lactamase (ESBL) bacteria [9]. Certain ESBL genes, located on
eISSN 1738-8872
plasmids or prophages, facilitate horizontal gene transfer to non-ESBL bacteria [4, 5]. Consequently, food
Copyright © 2024 by the authors.
contaminated with ESBL-producing Salmonella (ESBL-Sal) may serve as a reservoir for the spread of antimicrobial
Licensee KMB. This article is an resistance (AMR) within the local community.
open access article distributed Previous studies have demonstrated that ESBL-Sal strains can be isolated from food samples [10-13]. Although
under the terms and conditions poultry is widely acknowledged as a main source for Salmonella contamination, the available data on ESBL-Sal in
of the Creative Commons this context remains limited [12, 14]. Therefore, to address this gap, the Ministry of Food and Drug Safety (MFDS)
Attribution (CC BY) license. has isolated Salmonella spp. from retail meat samples, assessed their susceptibility to antibiotics, particularly beta-
lactams, and comprehensively characterized their genetics through whole-genome sequencing (WGS). In pursuit

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1102 Kang et al.

of these objectives, the present study was undertaken to determine the antimicrobial prevalence, genetic profiles,
and homology of ESBL-Sal in various meat types in South Korea between 2018 and 2019. The findings of this
study contribute valuable insights for formulating policies to prevent the spread of antibiotic-resistant Salmonella.

Materials and Methods

Sample Collection
Between February 2018 and November 2019, a total of 1,876 meat samples comprising 509 beef, 503 pork, 555
chicken, and 309 duck were collected from retail markets in South Korea. To ensure comprehensive coverage, we
divided South Korea into five zones: Seoul/Gyeonggi-do, Gyeongsang-do, Jeolla-do, Chungcheong-do, and
Gangwon-do (Table 1). All samples were collected and transported under refrigerated conditions.

Isolation and Identification of Salmonella spp.

Salmonella spp. were isolated using an analytical method certified by the MFDS Food Code (https://
www.mfds.go.kr/eng/brd/m_15/view.do?seq=72437). Briefly, 25 g of each sample was homogenized for approximately
30 s with 225 mL of buffered peptone water (Merck, USA) and incubated at 37°C for approximately 24 h.
Subsequently, 0.1 mL of culture was inoculated into 10 mL of Rappaport–Vassiliadis broth (BD, USA), and an
additional 1 mL of culture was inoculated into 10 mL of tetrathionate broth (MBcell, Republic of Korea). Both
inoculated broths were subjected to a secondary incubation for 24 h at 42°C and 37°C, respectively. After
incubation, the secondary culture solution was spread on XLD agar (Oxoid, UK) and brilliant green sulfa agar
(Remel, UK) and incubated at 37°C for 24 h. Next, a typical colony was selected, spread on tryptone soya agar
(Oxoid), and incubated at 37°C for 24 h. Vitek MS (bioMérieux, France) was used to identify the cultured bacteria
following the manufacturer’s instructions.

Antibiotic Susceptibility Test

All strains confirmed as Salmonella spp. were subjected to an antibiotic minimal inhibitory concentration (MIC)
assay using the KRNV5F panel (TREK Diagnostic Systems, USA) following the manufacturer’s instructions. E. coli
ATCC 25922 was used as a reference strain. The MIC assay encompassed a range of antibiotics, including amoxicillin/
clavulanic acid (2/1–32/16 μg/mL), ampicillin (2–64 μg/mL), cefepime (0.25–16 μg/mL), ceftiofur (0.5–8 μg/mL),
cefoxitin (1–32 μg/mL), ceftazidime (1–16 μg/mL), chloramphenicol (2–64 μg/mL), ciprofloxacin (0.12–16 μg/mL),
colistin (2–16 μg/mL), gentamicin (1–64 μg/mL), meropenem (0.25–4 μg/mL), nalidixic acid (2–128 μg/mL),
streptomycin (16–128 μg/mL), sulfisoxazole (16–256 μg/mL), tetracycline (2–128 μg/mL), and trimethoprim/
sulfamethoxazole (0.12/2.38–4/76 μg/mL). The MICs for ceftiofur and streptomycin were interpreted in
accordance with the National Antimicrobial Resistance Monitoring System breakpoints [15], whereas other
antibiotics were interpreted in accordance with the Clinical and Laboratory Standards Institute breakpoints [16].

ESBL Production Test

For cases exhibiting ceftiofur resistance and ceftazidime MIC ≥ 2 μg/mL, the ESBL phenotype was analyzed via
broth microdilution using the ESB1F panel (TREK Diagnostic Systems) following the manufacturer’s instructions.
The ESBL phenotype strain was characterized by an eight-fold reduction in cefotaxime and ceftazidime MICs
when tested in combination with clavulanate, compared to the MICs observed in the absence of clavulanate.

Whole Genome Sequencing and Sequence Analysis

Confirmed ESBL phenotype strains (n = 9) were subjected to WGS at Senigen Inc. (SRepublic of Korea). WGS
was performed to determine serotypes, ARGs, plasmid replicons, multilocus sequence typing (MLST), core
genome MLST (cgMLST), and virulence genes. Briefly, bacterial genomic DNA was extracted using the
UltraClean Microbial DNA Isolation Kit (MoBio Laboratories Inc., USA) following the manufacturer’s
instructions. Sequencing was performed on an Illumina MiSeq desktop sequencer (Illumina Inc., USA). WGS was
performed using 300 bp paired-end sequencing. Raw reads were assembled using the SPAdes genome assembler
version 3.13.0. Contigs less than 200 bp length and 5× sequencing depth were removed. Assembled contigs
maintained an average sequencing depth of 130×.

Nucleotide Sequence Accession Numbers

The raw WGS data have been deposited in GenBank under the BioProject PRJNA1002882, with the following
biosample accession numbers: SAMN36866225 (2018-11), SAMN36866226 (2018-64), SAMN36866227 (2018-
452), SAMN36866228 (2018-800), SAMN36866229 (2018-916), SAMN36866230 (2018-963), SAMN36866231
(2019-258), SAMN36866232 (2019-259), and SAMN36866233 (2019-265).

Table 1. Sample tested in this study.

Meat Region
type Seoul and Gyeonggi-do Gyeongsang-do Chungcheong-do Jeolla-do Gangwon-do Total
Beef 91 137 117 101 63 509
Pork 90 134 124 103 52 503
Chicken 97 194 131 73 60 555
Duck 39 132 61 56 21 309
Total 317 597 433 333 196 1876

J. Microbiol. Biotechnol.
 Genetic features of ESBL-Sal in Korean Retail Meats 1103

Phylogenetic Analysis
Homology among ESBL phenotype strains was compared using MLST and cgMLST. The MLST database
(http://pubmlst.org/database/) referenced seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and
thrA), and MLST 2.0 assigned sequence types to strains. For cgMLST analysis, raw sequence data files were
uploaded to cgMLSTfinder 1.2 at the Center for Genomic Epidemiology [17], and allelic profiles were predicted. A
minimum spanning tree based on the allelic profile of cgMLST was constructed using GrapeTree version 1.5.0.

In Silico Characterization of WGS

ARGs were predicted using ResFinder 4.1, with 90% as the minimum for identity and 60% as the cut-off query
length. Plasmid typing was predicted using Plasmidfinder 2.1, with 95% as the minimum for identity and 60% as
the cut-off query length. Salmonella pathogenicity islands were predicted using SPIFinder 2.0, with 95% as the
minimum for identity and 60% as the minimum for coverage. Salmonella serotypes were predicted using SeqSero
1.2. Virulence factors were identified using the Virulence Factor Database [18], with 90% as the minimum for
identity and 50% as the minimum for coverage.

Statistical Analysis
Chi-square tests on the Epitools website were employed to assess the significance of proportional differences
within the 95% confidence interval scale [19]. Briefly, we compared Salmonella isolates from chicken and duck
samples, specifically examining antibiotic-resistant and antibiotic-sensitive strains.

Results
Prevalence of ESBL-Producing Salmonella in Meat Samples
A total of 191 Salmonella strains were isolated from 1,864 meat samples. The prevalence rates of Salmonella were
28.2%, 17.5%, and 1.4% in the duck, chicken, and pork samples, respectively. No Salmonella strain was isolated
from beef. Furthermore, the prevalence of ESBL-Sal was 1.4% in the chicken samples and 0.3% in the duck samples
(Table 2). Only nine strains were identified as ESBL-producing Salmonella. Thus, poultry meat exhibited a higher
prevalence of Salmonella and ESBL-Sal.

Antimicrobial Resistance Patterns of Salmonella

A chi-square test (p < 0.05) revealed significant differences in AMR between chicken and duck isolates for ten
antibiotics, namely, amoxicillin/clavulanic acid, ampicillin, cefepime, ceftiofur, ceftazidime, nalidixic acid,
streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole (Table 3). Among poultry samples,

Table 2. Prevalence of Salmonella spp. and ESBL-producing Salmonella spp. among retail meats in Korea,
2018-2019.
% (number of positive samples/number of tested samples)
Meat type Salmonella spp. ESBL-producing Salmonella spp.
Beef 0.0 (00/509) 0.0 (0/509)
Pork 1.4 (7/503) 0.0 (0/503)
Chicken 17.5 (97/555) 1.4 (8/555)
Duck 28.2 (87/309) 0.3 (1/309)
Total 10.2 (191/1876) 0.5 (9/1876)

Table 3. Antibiotic resistance profiles of Salmonella spp. isolated from meat samples.
% (number of resistant strains)
Antibiotic Pork Chicken meat Duck meat Subtotal
P-value
(n = 7) (n = 97) (n = 87) (n = 191)
Amoxicillin/clavulanic acid 0.0(0) 9.3(9) 1.1(1) 5.2(10) 0.0355
Ampicillin 14.3(1) 59.8(58) 19.5(17) 39.8(76) <0.0001
Cefepime 0.0(0) 11.3(11) 1.1(1) 6.3(12) 0.0126
Ceftiofur 0.0(0) 22.7(22) 2.3(2) 12.6(24) 0.0001
Cefoxitin 0.0(0) 5.2(5) 1.1(1) 3.1(6) 0.2663
Ceftazidime 0.0(0) 21.6(21) 2.3(2) 12.0(23) 0.0002
Chloramphenicol 0.0(0) 12.4(12) 4.6(4) 8.4(16) 0.1082
Ciprofloxacin 0.0(0) 1.0(1) 3.4(3) 2.1(4) 0.5377
Colistin 0.0(0) 16.5(16) 12.6(11) 14.1(27) 0.5972
Gentamicin 0.0(0) 10.3(10) 2.3(2) 6.3(12) 0.0577
Meropenem 0.0(0) 0.0(0) 0.0(0) 0.0(0) N/A
Nalidixic acid 0.0(0) 88.7(86) 39.1(34) 62.8(120) <0.0001
Streptomycin 28.6(2) 36.1(35) 14.9(13) 26.2(50) 0.002
Sulfisoxazole 14.3(1) 43.3(42) 14.9(13) 29.3(56) <0.0001
Tetracycline 28.6(2) 40.2(39) 17.2(15) 29.3(56) 0.0011
Trimethoprim/Sulfamethoxazole 0.0(0) 13.4(13) 3.4(3) 8.4(16) 0.0331

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the most prevalent resistance to non-beta lactam antibiotics was observed with nalidixic acid and tetracycline.
Salmonella strains isolated from pork were resistant to only four antibiotics: ampicillin, streptomycin,
sulfisoxazole, and tetracycline.

Serotyping and Antimicrobial Resistance of ESBL-Producing Salmonella

The serotypes of the nine isolates were investigated using SeqSero1.2. Among them, eight strains (2018_11,
2018_64, 2018_452, 2018_800, 2018_916, 2018_963, 2019_259, and 2019_265) were predicted to be S. Enteritidis
(Table 4). Notably, all S. Enteritidis strains exhibited resistance to ampicillin, ceftiofur, ceftazidime, nalidixic acid,
and tetracycline. Furthermore, S. Enteritidis demonstrated a resistance rate of 87.5% to cefepime and gentamicin.
The serotype of the 2019_258 strain could not be predicted. This strain was predicted based solely on O-antigen 7,
resulting in an antigenic profile of 7:-:-. The 2019_258 strain was resistant to ampicillin, ceftiofur, ceftazidime,
nalidixic acid, streptomycin, sulfisoxazole, and tetracycline.

Phylogenetic Analysis
The homology among the nine ESBL-Sal isolates was assessed using cgMLSTFinder (version 1.2) and
GrapeTree (version 1.5.0) (Fig. 1). In the minimum spanning tree, these nine ESBL-Sal isolates were divided into
one cluster and one singleton. The singleton, identified as the strain 2019_258 chicken isolate, was assigned
cgST96964 and ST16. The cluster comprised eight strains (2018_11, 2018_64, 2018_452, 2018_800, 2018_916,
2018_963, 2019_259, and 2019_265), all predicted to be cgST58360, ST11, and S. Enteritidis. These cluster strains
were isolated from seven chickens and one duck. Notably, an average of 2,499 allelic differences were observed
between the cluster and singleton.

Table 4. Antimicrobial resistance profiles of ESBL-producing Salmonella spp.

Antibiotics

Trimethoprim/sulfamethoxazole
Amoxicillin/clavulanic acid

Chloramphenicol
Ciprofloxacin

Nalidixic acid
Streptomycin
Sulfisoxazole
Meropenem
Ceftazidime

Tetracycline
Gentamicin
Ampicillin
Cefepime

Cefoxitin
Ceftiofur

Colistin
Source Year Strain ST Serotype

Chicken meat 2018 11 11 Enteritidis

Chicken meat 2018 64 11 Enteritidis
Chicken meat 2018 452 11 Enteritidis
Chicken meat 2018 800 11 Enteritidis
Duck meat 2018 916 11 Enteritidis
Chicken meat 2018 963 11 Enteritidis
Chicken meat 2019 258 16 N/A (7:-:-)
Chicken meat 2019 259 11 Enteritidis
Chicken meat 2019 265 11 Enteritidis

Fig. 1. The minimum spanning tree was based core genome multilocus sequence typing. Nodes colour were
divided; each (A) cgMLST (B) MLST (C) source (D) serotype.

J. Microbiol. Biotechnol.
 Genetic features of ESBL-Sal in Korean Retail Meats 1105

Detection of Antimicrobial Resistance and Plasmid Genes

We identified five distinct plasmid replicon types, namely, IncFIB(S) (reference accession no: FN432031),
IncFII(S) (CP000858), IncQ1 (M28829), IncHI2 (BX664015), and IncHI2A (BX664015) (Table 5). All S.
Enteritidis strains carried IncFIB(S) and IncFII(S). Notably, the 2018_916 strain carried IncQ1 in addition to
IncFIB(S) and IncFII(S). The 2019_258 strain carried three plasmid replicons, namely, IncQ1, IncHI2, and
IncHI2A. Meanwhile, IncHI2 and IncHI2A were observed exclusively in the nine ESBL-Sal strains. Our findings
revealed the presence of beta-lactam, tetracycline, aminoglycoside (five distinct types), and sulfonamide genes
(Table 5). All ESBL-Sal carried blaCTX-M-15, tet(A), and aac(6')-Iaa. Among the cluster strains, seven (excluding the
2018_963 strain) carried acc(3)-lld. Furthermore, the 2018_916 and 2019_258 strains carried five (aac(3)-IId,
aph(3')-Ia, aph(3’)-Ib, aph(6)-Id, and sul2) and three (aph(3'')-Ib, aph(6)-Id, and sul2) ARGs, respectively.

Detection of Pathogenicity Islands and Virulence Factors

All ESBL-Sal strains carried identical pathogenicity island genes. The genes detected included SPI1, SPI2, SPI3,
SPI4, SPI5, SPI9, SPI10, SPI13, SPI14, C63PI, and CS54. A comprehensive assessment of virulence factors revealed

Table 5. Identified antibiotic resistance and plasmid genes profile of ESBL-producing Salmonella spp.
Antimicrobial resistance gene
Beta- Sulphon
Plasmid replicon Tetracycline Aminoglycoside
Serotype
lactam amide
Source
strain

ST

aac(6')-Iaa

aph(3'')-Ib
aac(3)-IId
aph(3')-Ia
IncFIB(S)

blaCTX-M-15

aph(6)-Id
IncFII(S)

IncHI2A
IncHI2
IncQ1

tet(A)

sul2
2018-11 Chicken meat 11 Enteritidis
2018-64 Chicken meat 11 Enteritidis
2018-452 Chicken meat 11 Enteritidis
2018-800 Chicken meat 11 Enteritidis
2018-916 Duck meat 11 Enteritidis
2018-963 Chicken meat 11 Enteritidis
2019-258 Chicken meat 16 N/A (7:-:-)
2019-259 Chicken meat 11 Enteritidis
2019-265 Chicken meat 11 Enteritidis

Table 6. Identified common virulence factor genes of ESBL-producing Salmonella spp.

VF class Virulence factor Related gene
Fimbrial adherence Agf/Csg csgA, csgB, csgD, csgE, csgF, csgG
determinants Bcf bcfA, bcfB, bcfC, bcfD, bcfE, bcfF, bcfG
Fim fimA, fimC, fimD, fimF, fimH, fimI, fimZ
Lpf lpfA, lpfB, lpfC, lpfD, lpfE
Saf safB
Saf safC
Stb stbA, stbB, stbC, stbD, stbE
Std stdA, stdB, stdC
Ste steA, steB, steC, steD, steE, steF
Stf stfA, stfC, stfD, stfE, stfF, stfG
Sth sthA, sthC, sthD, sthE
Sti stiA, stiB, stiC, stiH
Macrophage inducible genes Mig14 mig14
Magnesium uptake Mg2+ transport mgtB, mgtC
Nonfimbrial adherence MisL misL
determinants ShdA shdA
SinH sinH
Regulation PhoPQ phoP, phoQ
Secretion system TTSS (SPI1 encode) hilA, hilC, hilD, iacP, iagB, invA, invC, invE, invF, invG,
invH, invI, invJ, orgB, orgC, prgH, prgI, prgJ, prgK, sicA,
sicP, sipD, spaO, spaP, spaQ, spaR, spaS, sprB
TTSS (SPI2 encode) ssaC, ssaD, ssaE, ssaG, ssaH, ssaJ, ssaK, ssaL, ssaN, ssaO,
ssaP, ssaQ, ssaR, ssaT, ssaU, ssaV, sscA, sscB, sseB, sseC,
sseD, sseE, ssrA
TTSS effectors translocated slrP
via both systems
TTSS1 translocated effectors avrA, sipA, sipB, sipC, sopA, sopB/sigD, sopD, sptP
TTSS2 translocated effectors pipB2, pipB, sifA, sifB, sseF, sseG, sseJ, sseK1, sseL, sspH2

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Table 7. Identified virulence factor genes of ESBL-producing Salmonella spp. except common virulence
factor genes.

2018_452
2018_800
2018_916
2018_963
2019_258
2019_259
2019_265
2018_11
2018_64
Virulence Virulence Related
factor class factors genes

Fimbrial adherence Agf/Csg csgC

determinants Fim fimW
fimY
Pef pefA
pefB
pefC
pefD
Peg pegA
pegB
pegC
pegD
Saf safD
sefC
Stc stcB
stcC
stcD
Sth sthB
Stj stjB
stjC
Stk stkA
stkB
stkC
stkD
stkE
stkF
stkG
Tcf tcfB
tcfC
tcfD
Macrophage inducible Mig5 mig5
genes
Nonfimbrial adherence RatB ratB
determinants
Secretion system TTSS (SPI1 encode) invB
orgA
TTSS (SPI2 encode) ssaI
ssaM
ssaS
ssrB
TTSS1 translocated effectors sopE2
sopE
TTSS2 translocated effectors spiC/ssaB
spvC
spvD
sseI/srfH
sseK2
Serum resistance Rck rck
Stress adaptation SodCI sodCI
Toxin SpvB spvB
Adherence LPS Oantigen
(P. aeruginosa) (Pseudomonas)
Autotransporter EhaB ehaB
Invasion Invasion of brain endothelial cells ibeB
(Ibes) (Escherichia)

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 Genetic features of ESBL-Sal in Korean Retail Meats 1107

182 pertinent genes in the nine ESBL-Sal stains. Among these, 133 were consistently detected (Table 6), whereas
49 exhibited different profiles (Table 7). The identified virulence factor classes included fimbrial adherence
determinants, macrophage-inducible genes, magnesium uptake, non-fimbrial adherence determinants, regulation,
secretion system, serum resistance, stress adaptation, toxin, adherence, autotransporter, and invasion. Among the
nine strains, eight (cgST58360) exhibited comparable virulence factor profiles. This subset of eight strains
(cgST58360) was investigated, focusing on fimbrial adherence determinants (pef, peg, saf, and sth), macrophage-
inducible genes (mig5), nonfimbrial adherence determinants (ratB), secretion system (TTSS (encoded by SPI1),
TTSS (encoded by SPI2), translocated effectors of TTSS1 and TTSS2), serum resistance (rck), stress adaptation
(sodCI), toxin (spvB), and autotransporter (ehaB).

Discussion
In this study, we isolated 97 and 87 Salmonella strains from 555 chicken and 309 duck samples, respectively. The
prevalence of Salmonella was 17.5% in the chicken samples and 28.2% in the duck samples. The chicken isolates
exhibited resistance rates of 22.7% and 21.6% to ceftiofur and ceftazidime, respectively. However, the duck isolates
of Salmonella exhibited resistance rates of 2.3% and 2.3% to ceftiofur and ceftazidime, respectively. The prevalence
of ESBL-Sal was 1.4% in the chicken samples and 0.3% in the duck samples, respectively. Samples were collected
between 2018 and 2019, with a roughly equal distribution of samples collected repeatedly. However, substantial
disparities were observed in the prevalence rate of Salmonella, ESBL-Sal, and 3rd-generation cephalosporin AMR
between the chicken and duck meat samples. The introduction of antibiotics decreased microbial diversity; high
antibiotic selective pressures increased the abundance of ARGs but coincided with a reduction in the diversity of
ARGs [20]. These findings suggest a higher likelihood of Salmonella contamination with a high AMR level in
chicken meat than in duck meat, indicating a potentially greater use of antibiotics in chicken production.
Among the nine ESBL-Sal strains, eight were identified as Enteritidis-ST11 and one as ST16, albeit with an
indeterminate serotype prediction for ST16. Among the Enteritidis-ST11 strains, seven were isolated from the
chicken samples and one from the duck samples. The ST16 strain was isolated exclusively from chicken meat.
ST11 and ST16 were the most common Salmonella strains isolated from chicken meat in South Korea, consistent
with previous studies [21, 22]. Moreover, the cgMLST assay revealed that the Enteritidis-ST11 and ST16 strains
consisted of one cluster and one singleton, respectively. MLST and cgMLST showed similar homologies. All
ESBL-Sal isolates were resistant to ampicillin, ceftiofur, ceftazidime, nalidixic acid, and tetracycline and carried
blaCTX-M-15 belonging to the blaCTX-M-1 group. The Enteritidis-ST11 group exhibited an 87.5% (7/8) resistance rate to
cefepime and gentamicin. Enteritidis carrying the blaCTX-M-1 gene exhibits resistance to ampicillin, ceftiofur,
ceftazidime, nalidixic acid, tetracycline, and gentamicin [21, 23], consistent with our findings. Eight ARGs,
including blaCTX-M-15, were detected in this study. Moreover, S. Enteritidis strains isolated from chickens harbored
comparable ARGs. Consistent with the results outlined earlier, the detection rate of blaCTX-M-15, tet(A), and aac(6')-
laa was 100% (8/8), and that of acc(3)-lld was 87.5% (7/8). However, S. Enteritidis strains isolated from duck
samples harbored four additional ARGs: aph(3')-la, aph(3'')-lb, aph(6)-ld, and sul2. The isolated chicken-derived
ST16 strain harbored blaCTX-M-15, tet(A), aac(6')-laa, aph(3'')-lb, aph(6)-ld, and sul2. All ESBL-Sal strains exhibited
11 identical pathogenicity islands, encompassing SPI1, SPI2, SPI3, SPI4, SPI5, SPI9, SPI10, SPI13, SPI14, C63PI,
and CS54. SPI-1 is associated with target cell invasion during the infection initiation stage, whereas SPI-2 affects a
broad range of systemic infection functions [24]. S. Enteritidis strains contained 166 genes among the 182
virulence factor genes, 94% (156/166) of which were similar. The 2019_258 strain contained 151 of the virulence
factor genes, of which 16 genes were detected exclusively.
In this study, S. Enteritidis was considered a representative serotype from poultry, which may cause ESBL-Sal
infections in humans. The ESBL-Sal strain isolated from poultry meat in most parts of the country exhibited a
specific sequence type and serotype of Enteritidis-ST11. This predominant group displayed one cluster in the
cgMLST analysis. Moreover, these strains exhibited resistance to ampicillin, ceftiofur, ceftazidime, nalidixic acid,
and tetracycline. Genomic characterization revealed the presence of beta-lactam, tetracycline, and aminoglycoside
resistance genes in two plasmid replicons, namely, IncFIB(S) and IncFII(S). WGS analysis revealed 11 identical
pathogenicity islands and 156 identical virulence factor genes.
This study has certain limitations. The study design was limited in the context of the sampling of ESBL-Sal
isolates. Utilizing a Salmonella selective culture medium, we isolated a maximum of one Salmonella spp. per meat
sample. Consequently, this study may differ from studies that used an ESBL-selective culture medium to isolate
ESBL bacteria. Moreover, most Salmonella serotypes could not be determined.
Despite these limitations, this study provides valuable insights into the WGS characteristics of ESBL-Sal in
South Korean retail meat. The use of MLST and cgMLST allowed for homology determination, which could
enable traceback investigations in ESBL-Sal poisoning. Additionally, we identified various major genes, including
those related to AMR, plasmid replicon, identical pathogenicity island, and virulence factor genes. Ultimately, this
research offers valuable information that can aid in preventing the continued spread of ESBL-Sal.
In summary, we investigated the prevalence and genetic characteristics of ESBL-Sal strains isolated from retail
meat in South Korea between 2018 and 2019. The findings suggest that poultry might be the main reservoir for
ESBL-Sal, extending beyond Salmonella spp. The predominant ESBL-Sal type identified was Enteritidis-ST11,
possessing two plasmid replicons and various antibiotic and virulence factor genes. This finding is significant
given previous reports of an outbreak in South Korea involving IncFII plasmids carrying the blaCTX-M-15 gene in
Enteritidis-ST11 isolated from humans [21]. Thus, retail chicken meat may serve as a potential contagion channel
for ESBL-Sal poisoning. Accordingly, continuous monitoring of the poultry industry is essential to prevent the

May 2024  Vol. 34  No. 5

1108 Kang et al.

spread of ESBL-Sal. Moreover, further surveillance of ARBs in the supply chain of livestock products sold at retail
outlets is warranted.

Acknowledgments
This research was supported by grants (Nos. 15161MFDS645 and 20161MFDS009) from the Ministry of Food
and Drug Safety of Korea. The results and conclusions of the study are the sole property of the authors and do not
necessarily represent the views of the Ministry of Food and Drug Safety. We thank the laboratories and center
members for their contribution to the collection of meat samples for the isolation of Salmonella strains used in this
study. We would like to thank Editage (www.editage.co.kr) for English language editing.

Author Contributions
H.S.K., H.K., J.H.J., and S.H.K., designed the research; H.S.K., and Hansol. K. performed the main experiments
and analyses; H.S.K. performed data analyses; H.S.K., and H.K. wrote the manuscript; Y.C.P. and S.H.K.
supervised the study and obtained funding; all authors read and approved the final version of the manuscript.

Abbreviations
ARB, antibiotic-resistant bacteria; ESBL; extended-spectrum beta-lactamase; ESBL-Sal ESBL-producing
Salmonella; AMR, antimicrobial resistance; MFDS; Food and Drug Safety, WGS; whole-genome sequencing;
MIC, minimal inhibitory concentration; ARGs, antibiotic resistance genes; MLST; multilocus sequence typing;
cgMLST, core genome MLST

Conflict of Interest
The authors have no financial conflicts of interest to declare.

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