1115

Journal of Food Protection, Vol. 83, No. 7, 2020, Pages 1115–1124

https://doi.org/10.4315/JFP-19-483
Copyright Ó, International Association for Food Protection

Research Paper

Characterization of Extended-Spectrum b-Lactamase–Producing

Escherichia coli Isolated from Fresh Produce and Agricultural
Environments in Korea
MIN-HYEOK CHA,1,2 JAE-GEE RYU,3 YOUNG-MIN CHI,4† AND GUN-JO WOO1*†

1Laboratory of Food Safety and Evaluation, Department of Biotechnology, Graduate School, and 4Division of Biotechnology, College of Life Sciences and
Biotechnology, Korea University Seoul, Republic of Korea; 2Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation,
Cheongju, Republic of Korea; and 3Microbial Safety Team, National Academy of Agricultural Science, Rural Development Administration, Wanju,
Republic of Korea

MS 19-483: Received 7 October 2019/Accepted 20 February 2020/Published Online 22 June 2020

ABSTRACT
This study was conducted to characterize Escherichia coli strains and evaluate the spread of antimicrobial resistance among
these strains from fresh produce and farm environments in Korea. We then conducted phenotypic and genetic studies on
antimicrobial-resistant isolates. We determined the genetic epidemiological characteristics of isolates that produced extended-
spectrum b-lactamase (ESBL) and confirmed plasmid transfer in isolates that carried blaCTX-M-type genes. E. coli strains were
isolated from 8 samples of fresh produce and 152 samples from the farm environment collected from May 2014 to June 2016.
Cephalosporin resistance was the most prevalent (61.8%) type of resistance among the isolates. Five ESBL-producing strains
with high genetic homology with E. coli of human or livestock origin were identified. Lateral transfer of plasmids harboring
blaCTX-M-type genes to transconjugants was successful. Two isolates from Chinese cabbage and from water samples collected
from a nearby stream harbored the ISEcp1-blaCTX-M-55-orf477 operon and were confirmed as sequence type 1196 and the same
type of plasmid replicon, suggesting that cross-contamination was highly likely. A high-risk clone of sequence type 69 (clonal
complex 69) isolates was also recovered from the farm environment. This study provides genetic evidence that antimicrobial
resistance factors in E. coli from farm environments originate in the clinic or in livestock, highlighting the fact that good
agricultural practices in farming are important to inhibit the spread of antimicrobial resistance to bacteria on fresh produce.

HIGHLIGHTS

E. coli was isolated from 8 fresh produce and 152 environment samples.

Cephalosporin resistance was the most common type of antimicrobial resistance in E. coli.

Five ESBL-producing E. coli strains were confirmed.

ESBL-producing E. coli had high homology with isolates from humans and livestock.

Horizontal gene transfer was found in plasmids harboring blaCTX-M.

Key words: Antimicrobial resistance; CTX-M–type genes; Escherichia coli; Extended-spectrum b-lactamase; Farm
environment; Fresh produce

Escherichia coli is a commensal bacterium that is part of
the human gastrointestinal microflora and has a symbiotic
relationship with humans (24). However, some E. coli strains
are pathogenic and can cause intestinal or extraintestinal
infections. Increasing attention has been directed toward
antimicrobial resistance in pathogenic strains of E. coli (35).
Antimicrobial resistance in E. coli is a major indicator
of environmental selective pressures, and genetic resistance
determinants are ubiquitously found in agricultural produce,
livestock husbandry, human communities, and the produc-
* Author for correspondence. Tel: þ82-2-3290-3021; Fax: þ82-2-3290-
3633; E-mail: victory8316@naver.com.
† Co-corresponding authors. These authors contributed equally to this
work.
tion and distribution chains of agricultural products (25).
Nationwide monitoring of antimicrobial resistance has been
conducted in clinical areas, livestock environments, and
meat products in many countries. Genes associated with
resistance to new antimicrobials such as carbapenem and
colistin have been confirmed in E. coli strains from both
clinical and livestock samples (23, 28). Extended-spectrum
b-lactamase (ESBL)–producing genes from Enterobacteri-
aceae have also spread worldwide, and CTX-M–type genes
are the most common determinant of ESBL production (19).
Plasmids carrying antimicrobial resistance genes can spread
by horizontal gene transfer. Resistance genes found in
hospitals and on livestock and poultry farms can also be
disseminated to agricultural products (42). Thus, nationwide
1116 CHA ET AL.
monitoring of changes in antimicrobial resistance in fresh
produce and farming environments is important. However,
in some countries, monitoring of E. coli antimicrobial
resistance in agricultural products and farm production
environments remains inadequate.
The aims of this study were to examine the phyloge-
netic distribution of E. coli isolates recovered from fresh
produce and agricultural production environments and to
investigate the dissemination of antimicrobial resistance in
these isolates in the Republic of Korea. We assessed
resistance genes in E. coli isolates and genetically
characterized ESBL-producing strains, confirmed horizontal
transfer of plasmids in the isolates that carried blaCTX-M,
and conducted multilocus sequence typing (MLST) of
isolates to evaluate genetic relatedness.
MATERIALS AND METHODS
Sample collection. In total, 2,191 samples from nine types of
fresh produce (n ¼ 631) and 11 farm environments (n ¼ 1,560)
were collected from 228 farms in Korea between May 2014 and
June 2016. We visited 23 to 28 farms for sampling of various fresh
produce products and collected 8 to 12 samples from each farm.
The types of fresh produce chosen for sampling were selected after
discussion with the Korean Rural Development Administration
based on their rates of consumption. Samples were collected from
paprika (n ¼ 70), tomatoes (n ¼ 67), oriental melons (n ¼ 72),
Chinese cabbage (n ¼ 75), carrots (n ¼ 63), onions (n ¼ 54),
peppers (n ¼ 73), Korean lettuce (n ¼ 91), and lettuce (n ¼ 66).
Farm environment samples included those related to human
activity (feces, n ¼ 114; work gloves, n ¼ 140; and toilet swabs, n
¼ 133), livestock (feces, n ¼ 8; fertilizer or compost, n ¼ 78; and
feed, n ¼ 4), and the environment (swabs of the workplace, n ¼
204; swabs of work tools, n ¼ 229; stream water near the farm, n ¼
205; soil, n ¼ 224; and agricultural surface or ground water that
has direct contact with fresh produce, n ¼ 221). Samples were
transported under refrigerated conditions within 24 h of sampling
to the laboratory for analysis.
Isolation of E. coli. E. coli was isolated and identified as
previously described (21). For samples of fresh produce, work
gloves, fertilizer or compost, feed, stream water near the farm,
soil, and agricultural water, 25 g of each sample was inoculated
into 225 mL of EC medium (BD, Sparks, MD) and cultured at
378C for 24 h. Fecal samples and swabs were first resuspended
and homogenized in 0.85% sterilized saline (25 mL), which was
then used to inoculate 225 mL of EC medium and cultured at 378C
for 24 h. The cultures were spread onto eosin methylene blue agar
(BD) and incubated at 378C for 24 h. The VITEK 2 compact
system (bioMérieux Vitek, Hazelwood, MO) was used to confirm
that each strain was E. coli.
For detection of E. coli O157:H7, 25 g of each sample was
inoculated into 225 mL of modified EC broth (BD) and cultured at
378C for 24 h. MacConkey sorbitol agar plates (BD) were used to
assess lactose fermentation. A latex agglutination test (Oxoid,
Basingstoke, UK) was used to test sorbitol-negative and lactose-
positive colonies for the O157 antigen as previously described
(12).
DNA preparation. Bacterial DNA was prepared using a
genomic DNA extraction kit (MagListo 5M, Bioneer, Daejeon,
Korea) according to the manufacturer’s instructions. The isolated
DNA was used as a template for PCRs for phylogenetic grouping,
J. Food Prot., Vol. 83, No. 7
detection of virulence and antimicrobial resistance genes, and
MLST.
Phylogenetic grouping and detection of enteropathogenic
and extraintestinal pathogenic E. coli virulence genes. The
phylogenetic groups (A, B1, B2, or D) of the E. coli isolates were
determined using a triplex PCR assay as previously described (9).
The isolates were also tested for genes for Shiga toxin (stx1
and stx2), bundle-forming pilus (bfpA), invasion plasmid antigen
(ipaH), enteroaggregative adherence (CVD432), heat-labile toxin
(lt), heat-stable toxin (st), attaching and effacing (eae), diffuse
adherence adhesion (daaD), the major subunit of a putative
chaperone-usher fimbria (yfcV), vacuolating autotransporter toxin
(vat), the yersiniabactin receptor (fyuA), and a heme-binding
protein (chuA) as previously described by Aranda et al. (2), Guion
et al. (14), and Spurbeck et al. (39) (Supplemental Table S1). E.
coli NCCP 13717 (lt), NCCP 13718 (st), NCCP 13720 (stx1),
NCCP 13721 (stx2), NCCP 13715 (eae), and NCCP 13719 (ipaH)
were used as positive controls, and E. coli ATCC 25922 was used
as a negative control. When no strains were available for positive
controls, PCR products were sequenced and aligned using the
NCBI BLAST tool (http://www.ncbi.nlm.nih.gov/BLAST) for
verification of genes.
Antimicrobial susceptibility testing. E. coli isolates were
tested for antimicrobial susceptibility with the disk diffusion
method according to Clinical and Laboratory Standards Institute
(CLSI) guidelines (10). The antimicrobial disks (BD) were
impregnated with ampicillin (10 μg), piperacillin (100 μg),
amoxicillin–clavulanic acid (20 and 10 μg), cefazolin (30 μg),
cephalothin (30 μg), cefamandole (30 μg), cefotaxime (30 μg),
ceftazidime (30 μg), cefepime (30 μg), cefoxitin (30 μg),
imipenem (10 μg), aztreonam (30 μg), amikacin (30 μg),
gentamicin (10 μg), streptomycin (25 μg), tobramycin (10 μg),
trimethoprim-sulfamethoxazole (1.25 and 3.75 μg), chloramphen-
icol (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), and
tetracycline (30 μg). Results are summarized in Table 4. E. coli
ATCC 25922 and Staphylococcus aureus ATCC 29213 were used
as controls for susceptibility testing.
Detection of antimicrobial resistance determinant factors.
All antimicrobial-resistant isolates were assessed with a PCR
assay for the presence of genes for resistance to sulfonamides
(sul1, sul2, and sul3), phenicols (floR and cat), streptomycin (strA,
strB, and aadA), quinolones (aac(6 0 )Ib-cr, qnrA, qnrB1, qnrB4,
qnrD, qnrS, and oqxA), tetracyclines (tet(A), tet(B), tet(C), and tet
(D)), colistin (mcr-1 and mcr-2), and carbapenems (IMP and
NDM) as previously described (15, 16, 20, 25, 31, 33, 44, 45)
(Table S1).
Identification of ESBL-producing strains. All strains that
produced testing plate inhibition zones of ,27 mm in diameter for
cefotaxime and ,20 mm for ceftazidime during susceptibility
testing were further assessed for ESBL production. A phenotype
of ESBL production was identified with a double-disk synergy test
and was further confirmed with the disk diffusion method. Wafer
disks (BD) were impregnated with cefotaxime (30 μg), cefotax-
ime–clavulanic acid (30 and 10 μg), ceftazidime (30 μg), or
ceftazidime–clavulanic acid (30 and 10 μg), and the test was
performed in accordance with CLSI guidelines (10). E. coli
standard strain ATCC 25922 was used as a control.
Plasmid extraction. Plasmid DNA was isolated with a
plasmid mini extraction kit (AccuPrep Nano-Plus, Bioneer) as per
TABLE 1. Isolation rates, distribution of phylogenetic groups, virulence factors, and multidrug resistance of Escherichia coli isolates
E. coli isolation No. (%) of isolates in each Virulence factors No. (%) of isolates
phylogenetic group (no. of isolates)a resistant to antimicrobial classesb
No. of No. of isolates
Sample type samples (prevalence %) A B1 B2 D EPEC ExPEC 0 1 2 3 4 5 6 7 8
J. Food Prot., Vol. 83, No. 7

Fresh produce 631 8 (1.3) 2 2 0 4 bfpA (1), lt (3), st chuA (4) 5 1 1 1

(2) stx2 (1)
Farm environment
Human related 9 8 6 4 stx2 (1), eae (4), yfcV (4), vat (1), 7 7 7 3 2 1
bfpA (3), fyuA (2), chuA
CVD432 (1), lt (10)
(9), st (14)
Feces 114 15 (13.2)
Work gloves 140 6 (4.3)
Toilet swab 133 6 (4.5)
Livestock related 5 2 2 1 stx (2), stx2 (2), yfcV (1), vat (1), 2 1 2 1 1 2 1
eae (3), bfpA fyuA (1), chuA
(1), ipaH (1), lt (3)
(6), st (8)
Feces 8 6 (75.0)
Fertilizer, compost 78 2 (2.6)
Feed 4 2 (50.0)
Environmental 44 28 19 24 stx (3), stx2 (8), yfcV (7), vat (1), 29 31 20 14 11 5 2 2 1
eae (24), bfpA fyuA (2), chuA
(12), ipaH (3), lt (43)
(38), st (77),
daaD (2)
Workplace swab 204 8 (3.9)
Work tool swab 229 10 (4.4)
Stream water 205 54 (26.3)
Soil 224 17 (7.6)
Agricultural water 221 26 (11.8)
Total farm environment 1,560 152 (9.7)
Total 2,191 160 (7.3) 60 40 27 33 38 44 30 19 15 8 3 2 1
(37.5) (25.0) (16.9) (20.6) (23.8) (27.5) (18.8) (11.9) (9.4) (5.0) (1.8) (1.2) (0.6)
ANTIMICROBIAL-RESISTANT E. COLI IN AGRICULTURE

a
EPEC, enteropathogenic E. coli; ExPEC, extraintestinal pathogenic E. coli.
b
Isolates were resistant to antimicrobial agents from zero to eight classes.
1117
1118 CHA ET AL. J. Food Prot., Vol. 83, No. 7

TABLE 2. Antimicrobial resistance patterns among 160 Escherichia coli isolates from fresh produce and the farm environment
No. (%) of isolates

Fresh Human Livestock

Antimicrobial class Antimicrobial agent Total produce related related Environmental

Penicillins Ampicillin 35 (21.9) 2 (25.0) 8 (29.6) 3 (30.0) 22 (19.1)

Piperacillin 33 (20.6) 2 (25.0) 4 (14.8) 2 (20.0) 25 (21.7)
Amoxicillin–clavulanic acid 6 (3.8) 0 1 (3.7) 0 5 (4.3)
Cephems Cefazolin 59 (36.9) 3 (37.5) 7 (25.9) 6 (60.0) 43 (37.4)
Cephalothin 80 (50.0) 5 (62.5) 11 (40.7) 6 (60.0) 58 (50.4)
Cefamandole 39 (24.4) 2 (25.0) 5 (18.5) 2 (20.0) 30 (26.1)
Cefotaxime 43 (26.9) 1 (12.5) 2 (7.4) 5 (50.0) 35 (30.4)
Ceftazidime 8 (5.0) 1 (12.5) 0 0 7 (6.1)
Cefepime 7 (4.4) 1 (12.5) 0 3 (30.0) 3 (2.6)
Cefoxitin 3 (1.9) 0 0 1 (10.0) 2 (1.7)
Penems Imipenem 13 (8.1) 0 1 (3.7) 1 (10.0) 11 (9.6)
Monobactams Aztreonam 14 (8.8) 1 (12.5) 0 3 (30.0) 10 (8.7)
Aminoglycosides Amikacin 5 (3.1) 0 1 (3.7) 1 (10.0) 3 (2.6)
Gentamicin 9 (5.6) 0 1 (3.7) 2 (20.0) 6 (5.2)
Streptomycin 28 (17.5) 0 5 (18.5) 3 (30.0) 20 (17.4)
Tobramycin 11 (6.9) 0 1 (3.7) 2 (20.0) 8 (7.0)
Folate pathway inhibitors Trimethoprim-sulfamethoxazole 12 (7.5) 0 1 (3.7) 1 (10.0) 10 (8.7)
Phenicols Chloramphenicol 13 (8.1) 0 1 (3.7) 2 (20.0) 10 (8.7)
Quinolones Nalidixic acid 19 (11.9) 3 (37.5) 5 (18.5) 1 (10.0) 10 (8.7)
Ciprofloxacin 7 (4.4) 1 (12.5) 0 1 (10.0) 5 (4.3)
Tetracyclines Tetracycline 39 (24.4) 2 (25.0) 4 (14.8) 4 (40.0) 29 (25.2)
Total 160 8 27 10 115

TABLE 3. Distribution of antimicrobial resistance genes from resistant Escherichia coli isolates
No. of isolates

Antimicrobial class Resistance gene Total Fresh produce Human related Livestock related Environmental

b-Lactams (n ¼ 5) TEM 2 0 0 0 2
SHV 0 0 0 0 0
CTX-M 4 1 0 1 2
OXA 0 0 0 0 0
CMY 0 0 0 0 0
Sulfonamides (n ¼ 12) sulI 12 0 2 0 10
sulII 0 0 0 0 0
sulIII 0 0 0 0 0
Phenicols (n ¼ 13) floR 10 0 1 1 8
cat 13 1 1 0 11
Aminoglycosides (n ¼ 42) strA 0 0 0 0 0
strB 16 0 2 3 11
aadA 13 2 2 0 9
Quinolones (n ¼ 21) aac(6 0 )Ib-cr 1 0 0 0 1
qnrA 0 0 0 0 0
qnrB1 6 0 2 0 4
qnrB4 1 0 0 0 1
qnrD 8 1 2 0 5
qnrS 0 0 0 0 0
oqxA 3 0 0 0 3
Tetracyclines (n ¼ 39) tet(A) 39 3 5 3 28
tet(B) 8 1 0 2 5
tet(C) 1 0 0 0 1
tet(D) 10 1 1 1 7
Colistinsa mcr-1 0 0 0 0 0
mcr-2 0 0 0 0 0
Penems (n ¼ 13) IMP 0 0 0 0 0
NDM 0 0 0 0 0
a
Not tested for antimicrobial susceptibility; screening test was performed for all isolates (n ¼ 160).
J. Food Prot., Vol. 83, No. 7 ANTIMICROBIAL-RESISTANT E. COLI IN AGRICULTURE 1119

ST1196

ST1196

AMP, ampicillin; PIP, piperacillin; CFZ, cefazolin; CEP, cephalothin; FAM, cefamandole; CTX, cefotaxime; FEP, cefepime; IPM, imipenem; ATM, aztreonam; GEN, gentamicin; TOB,
tobramycin; SXT, trimethoprim-sulfamethoxazole; NAL, nalidixic acid; CIP, ciprofloxacin; TET, tetracycline; AMK, amikacin; STR, streptomycin; CAZ, ceftazidime. Antimicrobials from the
the manufacturer’s instructions. Extracted plasmids were tested by

MLST

ST224
ST69

ST69
PCR assay for ESBL genes, which included genotyping for
blaTEM and blaCTX-M type, analysis of the blaCTX-M genetic
environment, and replicon typing.

ISEcp1-tnpA-blaCTX-M-14-
Genetic characterization of ESBL-producing E. coli.
blaCTX-M genetic

ISEcp1-blaCTX-M-15-

ISEcp1-blaCTX-M-55-

ISEcp1-blaCTX-M-55-
Strains that were positive for ESBL production were further
environment

assessed for the presence of b-lactamase genes (blaTEM, blaSHV,

blaOXA, blaCTX-M-1 cluster, blaCTX-M-9 cluster, and blaCMY) with a

Not tested
IS903D

multiplex PCR method as previously described (18). PCR products

orf477

orf477

orf477
were resolved and visualized by electrophoresis in 2.0% agarose
gels stained with ethidium bromide (Promega, Madison, WI).
Genotyping was conducted by sequencing positive samples with
TABLE 4. Characteristics and MLST of extended-spectrum b-lactamase–producing Escherichia coli isolates from fresh produce and farm environments

appropriate primers. The blaCTX-M-1 cluster and blaCTX-M-9 cluster

blaTEM-1B
blaCTX-M-14,

blaCTX-M-15

blaCTX-M-55

blaCTX-M-55
bla gene(s)

PCR-positive isolates were amplified with specific primers (27):

blaTEM-1B
blaCTX-M-1 cluster (F) 5 0 -TTCCAGAATAAGGAATCCCATGG-3 0
and (R) 5 0 -TGACCGATTTTAGCCGCCG-3 0 ; blaCTX-M-9 cluster
(F) 5 0 -TGACAAAGAGAGTGCAACGGA-30 and (R) 5 0 -GAAGC-
CAGCACATCGCG-3 0 ; and blaTEM (F) 5 0 -GAAAGGGCCTCGT-
GATACGC-3 0 and (R) 5 0 -TCATCCATAGTTGCCTGACTCC-3 0
[AMP, PIP], [CFZ, CEP, FAM, CTX],
[AMP, PIP], [CFZ, CEP, FAM, CTX,

[AMP, PIP], [CFZ, CEP, FAM, CTX,

[AMP, PIP], [CFZ, CEP, FAM, CTX,

[AMP, PIP], [CFZ, CEP, FAM, CTX,

FEP], ATM, [AMK, GEN, STR],

(Table S1). The genetic environment of blaCTX-M was investigated

Antimicrobial resistance spectruma

FEP], IPM, ATM, [GEN, TOB],

CAZ, FEP], ATM, [NAL, CIP]

CAZ, FEP], ATM, [NAL, CIP]

by PCR and by sequencing regions flanking these genes, as

previously described (11). Sequences were analyzed and compared
SXT, [NAL, CIP], TET

SXT, [NAL, CIP], TET

using the NCBI BLAST tool and SnapGene (www.ncbi.nlm.nih.

gov/BLASTih.gov/BL).

Conjugation and plasmid replicon typing for transferra-

ble plasmids. Conjugation by E. coli isolates that harbor the
blaCTX-M gene was investigated to confirm the transmissibility of
this gene. The experiments were performed with plasmid-free and
NAL

sodium azide–resistant E. coli J53 as the recipient. The broth

mating method was used for conjugation, and the mated cultures
were incubated overnight. Transconjugants were selected on
chuA, eae, lt, bfpA, st

MacConkey agar (BD) plates supplemented with sodium azide

(100 μg/mL; Sigma-Aldrich, St. Louis, MO) and cefotaxime (1
Virulence genes

CVD432, lt bfpA

μg/mL) (40). The conjugation experiments were repeated three

chuA, bfpA, st

fyuA, chuA, st
Not detected

times, and the frequency of gene transfer was calculated and

statistically analyzed. Transconjugants were tested for the
presence of b-lactamase genes and were assessed for antimicrobial
susceptibility as previously described for the wild-type strains.
Transconjugants that harbored b-lactamase genes were assessed
Phylogenetic

for plasmid replicon type using a PCR method with 18 primer

group

pairs as previously described (4).

D

MLST. The E. coli MLST database (http://mlst.warwick.ac.

uk/mlst/dbs/Ecoli) was used for MLST and to analyze genetic
same class are grouped together in square brackets.
Agricultural water

relatedness between ESBL-producing strains (43).

Chinese cabbage
Livestock feces

Working area
Stream water
Origin

RESULTS
Prevalence of E. coli in samples. A total of 160 E. coli
isolates were obtained from 2,191 samples of fresh produce
and samples collected from the agriculture environment
14-109

(Table 1). Eight isolates were obtained from 631 fresh

Farm

15-63

16-16

16-16

16-23

produce samples (1.3% prevalence). Of the eight isolates,

two isolates were from cabbage samples, two were from
Korean lettuce samples, and one each was from tomato,
16RDA-WA-ECO145
16RDA-SW-ECO131
16RDA-VN-ECO130
14RDA-AW-ECO41

15RDA-FL-ECO87

lettuce, paprika, and oriental melon samples. The remaining

152 E. coli isolates were from 1,560 samples (9.7%
Isolate

prevalence) collected from the agricultural environment.

The 115 isolates from environmental samples were from
stream water (n ¼ 54), agricultural water (n ¼ 26), and soil
(n ¼ 17). Few E. coli strains were isolated from sampled
a
1120 CHA ET AL.
working areas (n ¼ 8) and tools (n ¼ 10). A total of 27 E.
coli isolates were obtained from samples associated with
human activity: feces (n ¼ 15, 13.2% of fecal samples),
work gloves (n ¼ 6, 4.3% of glove samples), and swabs of
toilets (n ¼ 6, 4.5% of toilet samples). Ten E. coli isolates
were obtained from samples sourced from livestock
environments: feces (n ¼ 6, 75% of fecal samples), feed
(n ¼ 2, 50% of feed samples), and fertilizer or compost (n ¼
2, 2.6% of fertilizer or compost samples). E. coli O157:H7
was not isolated from any sample.
Distribution of phylogenetic groups and virulence
genes. Most of the 160 isolates were classified as
phylogenetic group A (n ¼ 60, 37.5%), and the rest were
classified as groups B1 (n ¼ 40, 25.0%), B2 (n ¼ 27, 16.9%),
and D (n ¼ 33, 20.6%) (Table 1). Of the eight isolates from
fresh produce samples, half were group D (n ¼ 4), and the
rest were groups A (n ¼ 2) and B1 (n ¼ 2). The isolates from
human-related samples were classified into groups A (n ¼
9), B1 (n ¼ 8), B2 (n ¼ 6), and D (n ¼ 4). E. coli isolates
sourced from livestock were mostly classified into group A
(n ¼ 5), with the remaining isolate in group D. For E. coli
isolates from environmental samples, most were classified
as group A (n ¼ 44) followed by group B1 (n ¼ 28), group D
(n ¼ 24), and group B2 (n ¼ 19).
Of the 160 isolates, 15 did not harbor any entero-
pathogenic or extraintestinal pathogenic genes. In the
isolates that harbored enteropathogenic genes, the viru-
lence factor with the highest prevalence was st (n ¼ 101),
followed by lt (n ¼ 56), eae (n ¼ 31), bfpA (n ¼ 17), and
stx2 (n ¼ 12). Four isolates from fresh produce harbored
enteropathogenic genes, and CVD432, eae, ipaH, daaD,
and stx were not detected in any isolates from fresh
produce samples. The 10 isolates from livestock-related
samples had a relatively high prevalence of st (n ¼ 6, 60%)
and lt (n ¼ 8, 80%) genes.
For E. coli strains that harbored extraintestinal
pathogenic genes, chuA (n ¼ 60) was the most prevalent,
followed by yfcV (n ¼ 12), fyuA (n ¼ 5), and vat (n ¼ 3).
Although the number of E. coli strains isolated from fresh
produce was small, the prevalence of chuA in fresh produce
vegetable samples (50%) was higher than that in other
sample groups and for other genes.
Antimicrobial resistance profiles of E. coli. The
susceptibility of the 160 E. coli isolates to 21 antimicrobial
agents is summarized in Table 2. Of the 160 isolates, 122
(76.3%) were resistant to at least one antimicrobial agent,
and 48 (30.0%) were resistant to at least three classes of
antimicrobial agents, i.e., were multidrug resistant (Table 1).
The most multidrug-resistant isolate (14RDA-AW-ECO41)
was recovered from agricultural water and was resistant to
eight classes of antimicrobials tested (penicillins, cephems,
penems, monobactams, aminoglycosides, folate pathway
inhibitors, quinolones, and tetracyclines). The most multi-
drug-resistant E. coli among the isolates from fresh produce
was from Chinese cabbage (16RDA-VN-ECO130) and was
resistant to only four classes of antimicrobials.
J. Food Prot., Vol. 83, No. 7
Cephalosporin (cephem) resistance was the most
prevalent type of resistance, with 99 isolates resistant to
this class of antimicrobials. Of these, the resistance rates to
cephalothin (n ¼ 80, 50.0%), cefazolin (n ¼ 59, 36.9%),
cefotaxime (n ¼ 43, 26.9%), and cefamandole (n ¼ 39, 24.4)
were high, whereas those to ceftazidime (5.0%), cefepime
(4.4%), and cefoxitin (1.9%) were low. Resistance to
penicillins was the second-most prevalent (n ¼ 48,
30.0%), with relatively high rates of resistance to ampicillin
(n ¼ 35, 21.9%) and piperacillin (n ¼ 33, 20.6%). Resistance
to aminoglycosides (n ¼ 42, 26.3%) was the third-most
prevalent, followed by that to tetracyclines (n ¼ 39, 24.4%).
Isolates from fresh produce samples were resistant to
penicillins, cephalosporins, monobactams, quinolones, and
tetracyclines. Compared with the isolates from human-
related and environmental samples, these isolates had
relatively high resistance rates to cephalosporins and
quinolones. In contrast, the isolates from human-related,
livestock-related, and environmental samples were resis-
tant to eight classes of antimicrobials. No isolates from
fresh produce samples were resistant to penems, amino-
glycosides, trimethoprim-sulfamethoxazole, or chloram-
phenicol. The prevalence of aminoglycoside resistance
was highest among isolates from livestock-related sam-
ples.
Antimicrobial resistance genes. Resistance genes
detected in E. coli isolates are presented in Table 3. For
the three sulfonamide resistance genes tested, only sul1
was detected in resistant isolates (n ¼ 12), and all positive
isolates were human related (n ¼ 2) and environmental (n ¼
10). Thirteen isolates harbored the chloramphenicol
resistance gene (cat), and 10 isolates harbored the
florfenicol resistance gene (floR). However, floR was not
detected in any isolates from fresh produce, and the cat
gene was not detected in any isolates from livestock-
related samples. Sixteen isolates harbored the strB gene,
but the strA gene was not detected. The aadA gene was
detected in 13 isolates. We also detected the plasmid-
mediated quinolone resistance genes qnrB1 (n ¼ 6), qnrB4
(n ¼ 1), qnrD (n ¼ 8), oqxA (n ¼ 3), and aac(6 0 )Ib-cr (n ¼
1) in a total of 19 isolates, but the qnrA and qnrS genes
were not detected.
Of the tetracycline resistance genes, tet(A) (n ¼ 39) was
most prevalent, followed by tet(D) (n ¼ 10), tet(B) (n ¼ 8),
and tet(C) (n ¼ 1). The tet(B) gene was not detected in the
isolates of human origin, and the single isolate harboring the
tet(C) gene was from environmental samples. Most
tetracycline-resistant E. coli isolates harbored only one
tetracycline resistance gene. However, two isolates from
Korean lettuce and agricultural water samples harbored
three tetracycline resistance genes: tet(A), tet(B), and tet(D).
Neither mcr-1 and mcr-2 genes, which confer colistin
resistance, nor IMP and NDM genes, which confer
carbapenem resistance, were detected.
Double-disk synergy test and genetic characteriza-
tion of ESBL-producing E. coli. Of the 160 isolates, 43
had reduced susceptibility or were resistant to ceftazidime
J. Food Prot., Vol. 83, No. 7 ANTIMICROBIAL-RESISTANT E. COLI IN AGRICULTURE 1121

and/or cefotaxime. These 43 isolates were subjected to the

I1, FIA, FIB, FIC

I1, FIA, FIB, FIC
Transferred plasmid

FIA, FIB, HI2, Y

replicon type
double-disk synergy test, and 5 (11.6%) of the isolates were
identified as ESBL producers. Three (60%) of these isolates

FIA, FIB
were recovered from environmental samples (agricultural
water, stream water, and working areas), one was from
livestock feces, and one was from Chinese cabbage.
The five EBSL-producing isolates harbored blaCTX-M-I

Transferred gene
(three isolates) and blaCTX-M-IV (one isolate). For these four

blaCTX-M-14
blaCTX-M-15
blaCTX-M-55
blaCTX-M-55
strains, a PCR assay was used to amplify the blaCTX-M-I and
blaCTX-M-IV genes for sequencing; blaCTX-M-14 (14RDA-
AW-ECO41), bla CTX-M-15 (15RDA-FL-ECO87), and
bla CTX-M-55 (16RDA-VN-ECO130 and 16RDA-SW-
ECO131) were identified (Table 4). The two isolates
production
ESBL

harboring blaCTX-M-55 were from the same farm. Two

þ
þ
þ
þ
isolates (14RDA-AW-ECO41 and 16RDA-WA-ECO145)
harbored blaTEM genes, which were identified as blaTEM-1B
by sequencing.
Conjugation frequency (CFU/mL)

1010
109
109
109

The genetic environment surrounding blaCTX-M was

analyzed, and an insertion sequence, ISEcp1, was detected
3
3
3
3

upstream of blaCTX-M-15 and blaCTX-M-55. ISEcp1-tnpA was

1.08
5.72
1.55
7.56

also present upstream, within the flanking regions of

6
6
6
6

blaCTX-M-14. IS903D was found downstream of blaCTX-M-14,

Recipient for all conjugation experiments was E. coli J53 AZR at 3.30 3 1012 CFU/mL. AZR, sodium azide resistance.
109
108
109
109

and orf477 was detected downstream of blaCTX-M-15 or

blaCTX-M-55.
3
3
3
3
5.76
2.29
3.94
9.19

Plasmid replicon typing and conjugation. Six

replicon types were detected by ESBL genotyping analysis
103
104
103
103

(Table 5). FIB was detected in all five isolates analyzed, and
3
3
3
3

FIA was found in all four isolates that harbored blaCTX-M

Transconjugant

3.56
1.89
5.10
2.49

genes. FIC and I1 were detected in the isolates that harbored

blaCTX-M-55, and Y was detected only in the isolate
6
6
6
6
Level (CFU/mL)a

harboring blaCTX-M-15. All blaCTX-M genes were success-

104
104
104
104

fully transferred during the conjugation experiment. The

3
3
3
3

mean conjugation frequency was 2.29 3 108 to 3.94 3 109

1.90
7.57
1.30
3.03

CFU/mL, with the highest frequency observed for trans-

TABLE 5. Conjugation transfer efficiencies for isolates harboring blaCTX-M

conjugant 87 (2.29 3 108 6 5.72 3 109 CFU/mL),

followed by transconjugants 131 (9.19 3 109 6 7.56 3
1010

1010
1012
1011

109 CFU/mL), 41 (5.76 3 109 6 1.08 3 109 CFU/mL),

Donor

3
3

3
3

and 130 (3.94 3 109 6 1.55 3 109 CFU/mL). Phenotypic

8.50
4.00

5.05
2.00

testing of transconjugants with the double-disk synergy test

confirmed ESBL production. Plasmid replicon typing of
Transferability

transconjugants indicated the presence of blaCTX-M-14 in

FIA and of FIB in transconjugant 41. In transconjugant 87,
þ
þ
þ
þ

we confirmed all plasmid replicon types (FIA, FIB, HI2,

and Y); however, I1 was not found. The presence of all
replicon types from the donor isolates was confirmed in
Transconjugant

transconjugants 130 and 131.

41
87
130
131

MLST. To investigate the genetic relationships among

the five ESBL-producing E. coli isolates, MLST was
performed (Table 4). The E. coli isolate from Chinese
cabbage (16RDA-VN-ECO130) from farm 16-16 and the
16RDA-SW-ECO131
16RDA-VN-ECO130
14RDA-AW-ECO41
15RDA-FL-ECO87

isolate from the stream water near the same farm (16RDA-
SW-ECO131) had the same sequence type, ST1196. Both
Isolate

isolates from agricultural water (14RDA-AW-ECO41) and a

working area (16RDA-WA-ECO145) were identified as
ST69, and the isolate from livestock feces (15RDA-FL-
ECO87) was identified as ST224.
a
1122 CHA ET AL.
DISCUSSION
Several studies have been conducted to evaluate
antimicrobial resistance or pathogenicity in agricultural
products in Korean markets (22, 41). However, few have
been designed to simultaneously evaluate samples of fresh
produce and samples from the production environment of
the same farm. We conducted a nationwide study to monitor
the prevalence of virulence factors and antimicrobial
resistance genes in E. coli isolates obtained from farm
produce and their corresponding agricultural environments
and to determine the association between these virulence
factors and antimicrobial resistance.
Most of the E. coli isolates (.60%) were from
phylogenetic groups A and B1. This finding is consistent
with previous findings regarding E. coli isolates recovered
from meat products (24) and lettuce (34). These data
suggest that livestock-related sources are potential contrib-
utors to the spread of E. coli on fresh produce farms.
However, differences in virulence factors were found
between isolates from fresh produce and those from the
farm environment. In a study of produce from farmers’
markets in Pennsylvania, Scheinberg et al. (36) rarely found
virulence genes in E. coli isolates from fresh produce; stx1,
stx2, and eae were not found in any of the isolates tested;
and no E. coli O157:H7 isolates were found. In Korea, E.
coli O157:H7 has rarely been detected in fresh produce
samples collected directly from farms or from distribution
channels (41). Enterohemorrhagic E. coli infections are rare,
and only a few studies have included information on their
frequency (7). However, the results of the present study on
antimicrobial resistance suggest that the characteristics of E.
coli isolates recovered from the agricultural environment
should be monitored to trace dissemination pathways.
Although the E. coli isolates were not evenly obtained
from each sample group, we observed differences in
antimicrobial resistance between the isolates from fresh
produce and those from environmental samples. We tested
13 antimicrobials against the recovered isolates and found
that the prevalence of resistance was higher in isolates from
human- or livestock-related sources than in isolates from
fresh produce samples. We did not observe resistance to the
nine classes of antimicrobials in all E. coli isolates derived
from fresh produce. These results support those of Thanner
et al. (42), who suggested that antimicrobial resistance
originates in clinical and livestock isolates and is subse-
quently transferred to agricultural products. According to
statistics provided by the Korea Centers for Disease Control
and Prevention (26), b-lactams (penicillins and cephalo-
sporins) and tetracyclines were the most prevalently used
classes of antimicrobials in livestock environments from
2009 to 2018 and were also widely used in clinical trials
from 2014 to 2018 in Korea. Our results revealed relatively
higher resistance to these antimicrobial classes than to other
classes. Therefore, the changes in resistance rates that
accompany usage of antimicrobials in clinical settings or
livestock farming should be monitored across years.
Among the antimicrobial classes examined, resistance
to cephalosporins was the highest. The prevalence of
resistance to first-generation cephalosporins such as cefaz-
J. Food Prot., Vol. 83, No. 7
olin (36.9%) and cephalothin (50.0%) in E. coli isolates
from fresh produce was lower than previously reported
(46.3 and 70.1%, respectively) (21). In 2012, antimicrobial
management policies regulating livestock farming in Korea
banned the use of antimicrobials (including cephalosporins)
as feed additives for growth promotion. Before implemen-
tation of this policy, the use of antimicrobials in animal
farming had been increasing gradually. However, since
implementation of the policy, the total amount of antimi-
crobials used decreased by approximately 30% between
2003 (1,439 tons) and 2017 (1,026 tons). First-generation
cephalosporins are now rarely used on Korean livestock
farms (29, 30). Chantziaras et al. (6) reported a high
correlation between the use of specific antimicrobials and
resistance rates in E. coli isolates recovered from livestock.
This finding supports for the hypothesis that restriction of
total consumption of antimicrobial agents on livestock
farms through government regulation efficiently decreases
the prevalence of resistant microorganisms in farming
environments and in associated fresh produce.
In contrast, because of an increase in usage of ceftiofur
(a third-generation cephalosporin used as parenteral med-
ication), total cephalosporin use for livestock drastically
increased from 2,694 to 11,312 kg during the same time
period. The prevalence of resistance to third- and fourth-
generation cephalosporins (1.9 to 5.0%) is still lower than
resistance to first- or second-generation cephalosporins,
which is consistent with the findings by Kim et al. (17).
However, the trend of increased use of third-generation
cephalosporins on livestock farms could further accelerate
emergence and spread of ESBL-producing E. coli on fresh
produce farms.
Despite overall reduction in antimicrobial resistance, a
major concern is the possibility of spread to other bacteria.
Antimicrobial resistance can be transferred to bacteria in
fresh produce from their cultivation environment, and
outbreaks of foodborne illness have been reported in
various countries (13, 42). In our study, all blaCTX-M-type
ESBL-producing E. coli isolates were able to transfer
plasmids that harbored blaCTX-M resistance genes to the
experimental recipient E. coli J53 isolate. Two isolates that
harbored blaCTX-M-55 genes and were from the same farm
also had the same resistance phenotype and were identified
by sequencing as the same type, ST1196. In clinical and
livestock studies, ST1196 has been found to harbor
plasmids associated with multidrug resistance and other
resistance genes, including fosA3 and a gene for carbape-
nemase (5, 46). An E. coli isolate from a livestock fecal
sample harbored bla CTX-M-15 and was identified by
sequencing as ST224, which has been reported as a major
ESBL E. coli sequence type in livestock (37). Two ESBL
strains that harbored blaTEM-1B were identified as ST69, a
particularly widespread, high-risk clone or clonal complex
(38). One of the isolates also harbored blaCTX-M-14. ST69
has been reported as highly correlated with extraintestinal
infections (3).
b-Lactamase–producing bacteria that harbor blaCTX-M-14,
blaCTX-M-15, and blaCTX-M-55 are prevalent in clinic settings
and livestock production and were detected in this study in the
farm environment. In a comparison between Salmonella
J. Food Prot., Vol. 83, No. 7
enterica and E. coli from clinical and veterinary settings in
Korea, very similar ISEcp1-blaCTX-M-55-orf477 and ISEcp1-
blaCTX-M-15-orf477 structures were observed (19, 40). Direct
detection of an identical ISEcp1-blaCTX-M-55-orf477 genetic
environment in the isolates from Chinese cabbage and from
the environment of the farm on which it was grown provides
strong epidemiological evidence for the dissemination of these
resistance genes. In Japan, the ISEcp1-blaCTX-M-14-IS903D
structure was predominantly present in isolates from hospital
environments (32). ESBL-producing E. coli from Korean
farms are closely associated with clones of resistant isolates
from clinical and veterinary sources and contain mobile
elements such as ISEcp1, tnpA, and IS903D. This finding
strongly suggests that antimicrobial resistance in isolates from
clinical and livestock environments has disseminated to
isolates in farm environments and fresh produce.
The probability of pathogenic microbial dissemination
has been well documented (1), and this study presents
genetic evidence of an association between ESBL-produc-
ing E. coli in stream water and in Chinese cabbage grown
nearby, illustrating the dissemination of antimicrobial
resistance directly from the environment to agricultural
produce. A conjugation experiment also revealed that
blaCTX-M genes could be transferred and highlighted the
possibility that factors associated with ESBL production can
spread from the agricultural environment to the consumer’s
table. Therefore, the effects of human-related, livestock-
related, and environmental factors on the cultivation of
fresh produce should be investigated. Antimicrobial resis-
tance rates in Korea have decreased over time (9). However,
despite an overall decline in the prevalence of resistant
bacteria, determinants or factors of antimicrobial resistance
can still be found in the agricultural environment and thus
can be transmitted laterally.
In conclusion, in the context of the One Health
approach initiated by the World Health Organization, the
results of this study indicate that at the preharvest stage,
good agricultural practices, which include good personal
hygiene and farm environment management practices, are
crucial for preventing contamination of agricultural prod-
ucts by foodborne antimicrobial-resistant pathogens.
ACKNOWLEDGMENTS
We thank Sanigen, Inc. for their assistance with nationwide field
sampling activities. We also express our deep gratitude to the Korean
farmers for their willing assistance with sampling on their private farms.
This study was supported by a grant from the Korea Rural Development
Administration (PJ010500).
SUPPLEMENTAL MATERIAL
Supplemental material associated with this article can be
found online at: https://doi.org/10.4315/JFP-19-483.s1
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