Antimicrobial Resistance and Molecular Characterization of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated from Ducks in South Korea

Abstract

Ducks are potential carriers of pathogenic bacteria, which are capable of transmitting zoonotic diseases to humans. The global spread of Enterobacteriaceae carrying extended-spectrum β-lactamase (ESBL) genes is a public health concern. This study investigated the prevalence of antimicrobial resistance in Escherichia coli isolated from ducks in Korea and described the molecular characteristics of the ESBLs they produced. A total of 146 E. coli isolates from 404 duck fecal and carcass samples in 85 duck farms were tested for antimicrobial resistance using the broth dilution method and were further characterized using molecular methods. We observed high resistance rates to tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid, ampicillin, and ciprofloxacin. In total, six ceftiofur-resistant isolates (4.1%) were observed, which produced CTX-M-55 (n = 3) or CTX-M-65 β-lactamase (n = 3). All CTX-M-producing E. coli isolates were also resistant to ciprofloxacin, with mutations in the quinolone resistance determining region of GyrA (S83L with or without D87N) and ParC (S80I), and three CTX-M-producing E. coli isolates carried plasmid-mediated quinolone resistance (PMQR) genes, qepA (n = 1), qnrS, and acc(6′)-Ib-cr (n = 2). The transfer of bla _CTX-M genes was observed in one isolate mediated by IncF-family plasmids but not in the co-resistant isolates carrying both bla _CTX-M and PMQR genes. Pulsed-field gel electrophoresis and multilocus sequence typing demonstrated that CTX-M-producing isolates were heterogeneous; however, identical isolates were found in different farms and slaughterhouses. This study presents baseline data on antimicrobial resistance of E. coli derived from duck samples and is the first report of CTX-M-55 and CTX-M-65 β-lactamase-producing E. coli isolated from ducks in Korea. The dissemination of ESBL-producing E. coli poses a potential risk to public health and therefore should be monitored.

Introduction

The emergence of antimicrobial-resistant bacteria among food-producing animals and humans is a public health concern. Antimicrobial-resistant bacteria in animals and animal products may get transferred to humans through the food chain or by direct contact. Investigating antimicrobial resistance in food-producing animals provides important data for risk assessment and management. Escherichia coli, commensal bacteria found in the intestines of animals and humans, have been widely accepted as indicators of antimicrobial-resistant Gram-negative bacteria (FAO/OIE/WHO, 2004), because they are frequently found in a wide range of hosts (Tadesse et al., 2012; Szmolka and Nagy, 2013). The degree of antimicrobial resistance indicator bacteria in the fecal flora of humans and animals is considered to be a good indicator of selective antimicrobial usage.

A variety of antimicrobials have been used to treat and prevent animal diseases, including broad-spectrum cephalosporins used for serious infections in humans and animals (Aarestrup et al., 2008; Smet et al., 2010). Resistance to these antimicrobials has increased in both humans and animals in different countries since late 2000, gaining considerable attention worldwide (Chong et al., 2018). The main mechanism for resistance to cephalosporins is the production of extended-spectrum β-lactamases (ESBLs) or plasmid-encoded AmpC β-lactamases (Smet et al., 2010). Extensive studies surveying and characterizing ESBL-producing bacteria have been conducted in various food-producing animals. These bacteria can colonize in food-producing animals and have been considered as potential sources of human infection through third-generation cephalosporin-resistant bacteria (Lazarus et al., 2015).

The duck industry has been growing recently in Korea, because it has been highlighted as a healthy food (http://kosis.kr). With increases in duck meat consumption in Korea, concerns about food safety have also increased. However, limited information is available on duck food safety despite increasing duck populations worldwide, including China and Korea. Furthermore, resistance to important antimicrobials has been increasing in poultry. Thus, the aims of this study were to provide information about the antimicrobial resistance of E. coli obtained from healthy ducks and their carcasses and to study the molecular characteristics of the ESBLs they produced. This study investigated the antimicrobial resistance of E. coli obtained from healthy ducks and their carcasses and characterized ESBL-producing isolates to provide information on the current antimicrobial resistance situation and inform intervention strategies in the duck industry.

Materials and Methods

Sample collection and bacterial strains

We collected a total of 255 feces samples and 149 carcasses from 4 slaughterhouses that originated from 85 different duck farms in the southern part of Korea from March to July 2016. No more than five fecal and carcass samples were collected from a single farm, and one E. coli strain was isolated from each sample. Samples were processed and E. coli was isolated as described previously (Nam et al., 2010) using eosin methylene blue agar (Becton Dickinson, Sparks, MD) and MacConkey agar plates (BD). Species identification was performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (bioMérieux, Marcy l'Étoile, France).

Antimicrobial susceptibility testing

Antimicrobial susceptibility was assessed by determining minimum inhibitory concentrations (MICs) for 15 antimicrobial agents using the broth microdilution method with a commercially available Sensititre^® panel KRVP4F (TREK Diagnostic Systems, West Sussex, United Kingdom) according to the manufacturer's instructions. The following antimicrobials were tested: ampicillin, amoxicillin/clavulanic acid, cefoxitin, ceftiofur, cephalothin, chloramphenicol, ciprofloxacin, colistin, florfenicol, gentamicin, nalidixic acid, neomycin, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole. The reference strain E. coli ATCC 25922 was used as a quality control when determining MICs. The interpretation of MIC was carried out according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2017). When CLSI breakpoints were not available, the MIC interpretation was carried out according to the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) 2014 (DANMAP, 2014). Multidrug resistance was defined as resistance to three or more antimicrobial subclasses.

After the antimicrobial susceptibility tests, double-disk synergy tests were performed to detect ESBL production by ceftiofur-resistant bacteria using cefotaxime–cefotaxime/clavulanic acid and ceftazidime–ceftazidime/clavulanic acid disks according to CLSI guidelines (CLSI, 2017). Phenotypically ESBL-producing E. coli isolates were further analyzed.

Detection of resistance genes

The presence of the bla _CTX-M gene was screened by polymerase chain reaction (PCR) amplification using group-specific primers for CTX-M-1 and CTX-M-9 families as described previously (Batchelor et al., 2005; Branger et al., 2005). Then, previously described primers were used to amplify and sequence the complete bla _CTX-M genes (Tamang et al., 2013). PCR amplification of the entire bla _TEM and bla _SHV genes was performed as described previously (Rayamajhi et al., 2008). Quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE genes were amplified using previously described primers and protocols (Tamang et al., 2012). Purified PCR products were sequenced with specific primers using an automated ABI Prism 3700 Analyzer (Applied Biosystems, Foster City, CA). The QRDR sequences were compared with those of E. coli strains K-12. The presence of plasmid-mediated quinolone resistance (PMQR), including qnrA, qnrB, qnrC, qnrD, qnrS, acc(6′)-Ib-cr, qepA, oqxA, and oqxB genes, was detected by PCR using specific primers and protocols as described previously (Hong et al., 2009; Tamang et al., 2012). Sequence analysis and comparison with known sequences were performed with the Basic Local Alignment Search Tool (BLAST) programs at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov/BLAST).

Conjugation experiment

Conjugation was performed using a filter-mating protocol, as described previously (Tamang et al., 2014), with sodium azide-resistant E. coli J53 as the recipient strain. In brief, overnight cultures of donor and recipient strains were inoculated with fresh tryptone soy broth and cultured for 4 h at 37°C. The freshly cultured bacteria were mated with a donor-to-recipient ratio of 1:4 and trapped on a membrane filter. The bacteria on the filters were incubated overnight and then suspended in phosphate-buffered saline. Appropriate dilutions of the mixture were transferred to MacConkey agar plates, supplemented with sodium azide (150 μg/mL) and cefotaxime (2 μg/mL), to select putative transconjugants. All selected transconjugants were examined for the presence of β-lactamase genes and tested for antimicrobial susceptibility patterns, as described previously.

Molecular characterization of CTX-M-producing E. coli

Plasmid DNA was extracted using the QuickGene^® plasmid isolation system (FUJIFILM Corporation, Tokyo, Japan) according to the manufacturer's protocol. Replicon typing of the isolated plasmid was performed using the PCR-based replicon typing method with 18 pairs of primers, as previously described (Carattoli et al., 2005). The genetic environment of the bla _CTX-M gene was investigated using PCR and sequencing with the previously described primers. A combination of IS26 (Eckert et al., 2006) or ISEcp1 (Saladin et al., 2002) forward primers and the CTX-M reverse consensus (MA2) primer (Saladin et al., 2002) were used to investigate regions upstream of the bla genes, and the MA1 primer (Saladin et al., 2002), and reverse primers of IS903 or orf477 (Eckert et al., 2006) were used to characterize downstream of the bla genes. Phylogenetic grouping of the E. coli isolates was determined using a triplex PCR for the genes chuA, yjaA, and TSPE4.C2, as described previously (Clermont et al., 2000). Pulsed-field gel electrophoresis (PFGE) was performed using the XbaI enzyme (Takara Bio, Inc., Shiga, Japan), as described previously (Gautom, 1997). PFGE banding profiles were analyzed using Bionumerics software and relatedness was calculated using the unweighted pair-group method with the arithmetic averages algorithm, based on the Dice similarity index. Multilocus sequence typing (MLST) was performed according to the instructions on the E. coli MLST website, and allelic profiles and sequence types were determined using the E. coli MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli).

Results

Antimicrobial resistance

A total of 113 E. coli isolates from 255 fecal samples and 33 E. coli isolates from 149 carcass samples were examined for resistance to 15 antimicrobial agents (Table 1). The most frequently observed resistance in duck feces was to tetracycline (59.3%), followed by resistance to trimethoprim/sulfamethoxazole (51.3%), nalidixic acid (48.7%), and ampicillin (47.8%). Resistance to amoxicillin/clavulanic acid, cefoxitin, and colistin was not observed. In addition, resistance to neomycin and gentamicin was low (<3%). Overall, the resistance rates of feces and carcass samples were similar. However, resistance to cephems was higher in carcasses than in those from fecal samples. Of note, resistance to ciprofloxacin was high (23.0% and 24.2%) in fecal and carcass samples, respectively. Multidrug resistance is given in Table 2. Only 13.7% (20/146) of E. coli isolates were sensitive to all antimicrobials tested. Isolates resistant to one or more antimicrobials showed 54 different resistant patterns (Supplementary Table S1). The majority of isolates from both fecal (54.0%) and carcass (57.6%) samples were resistant to three or more antimicrobial subclasses. Furthermore, a few isolates (4.8%) from both sources were resistant to 10 antimicrobials.

Table 1.

Minimum Inhibitory Concentration Distribution of Escherichia coli (n = 146) Isolated from Duck Fecal and Carcass Samples

Antimicrobial subclass	Antimicrobials	Sample	Distribution (%) of MICs (μg/mL)											MIC₅₀	MIC₉₀	MIC range	Resistance (%)
Antimicrobial subclass	Antimicrobials	Sample	≤0.125	0.25	0.5	1	2	4	8	16	32	64	≥128	MIC₅₀	MIC₉₀	MIC range	Resistance (%)
Aminoglycosides	Gentamycin	Feces				94.7	1.8		0.9			2.7		1	1	1–64	2.7
	Gentamycin	Carcasses				93.9						6.1		1	1	1–64	6.1
	Neomycin	Feces					96.5	0.9			2.7			2	2	2–32	2.7
	Neomycin	Carcasses					97.0	3.0						2	2	2–4	0
	Streptomycin	Feces						4.4	39.8	17.7	7.1	1.8	29.2	16	128	4–128	38.1
	Streptomycin	Carcasses						12.1	42.4	18.2	6.1		21.2	8	128	4–128	27.3
Aminopenicillin	Ampicillin	Feces					32.7	16.8	2.7		0.9	46.9		8	64	2–64	47.8
Aminopenicillin	Ampicillin	Carcasses					27.3	30.3				42.4		4	64	2–64	42.4
β-lactam/β- lactamase inhibitor combinations	Amoxicillin/clavulanic acid	Feces					28.3	23.9	46.0	1.8				4	8	2–16	0
β-lactam/β- lactamase inhibitor combinations	Amoxicillin/clavulanic acid	Carcasses					18.2	45.5	33.3	3.0				4	8	2–16	0
Cephalosporin I	Cephalothin	Feces					5.3	21.2	47.8	19.5	2.7	3.5		8	16	2–64	6.2
Cephalosporin I	Cephalothin	Carcasses						9.1	54.5	24.2	6.1	6.1		8	32	4–64	12.1
Cephalosporin III	Ceftiofur	Feces			96.5				3.5					0.5	0.5	0.5–8	3.5
Cephalosporin III	Ceftiofur	Carcasses			90.9	3.0			6.1					0.5	0.5	0.5–8	6.1
Cephamycin	Cefoxitin	Feces					14.2	71.7	14.2					4	8	2–8	0
Cephamycin	Cefoxitin	Carcasses					12.1	66.7	18.2		3.0			4	8	2–32	3.0
Fluoroquinolone	Ciprofloxacin	Feces	34.5	32.7	6.2	2.7	0.9		8.0	15.0				0.25	16	0.125–16	23.0
Fluoroquinolone	Ciprofloxacin	Carcasses	33.3	21.2	15.2	6.1			18.2	6.1				0.25	8	0.125–16	24.2
Quinolone	Nalidixic acid	Feces					31.0	6.2	10.6	3.5		3.5	45.1	16	128	2–128	48.7
Quinolone	Nalidixic acid	Carcasses					27.3	3.0	6.1	6.1		12.1	45.5	64	128	2–128	57.6
Folate pathway inhibitors	Trimethoprim/sulfamethoxazole	Feces	37.2	7.1	4.4			51.3						4	4	0.125–4	51.3
Folate pathway inhibitors	Trimethoprim/sulfamethoxazole	Carcasses	48.5	9.1				42.4						0.25	4	0.125–4	42.4
Phenicols	Chloramphenicol	Feces					1.8	16.8	44.2	4.4		32.7		8	64	2–64	32.7
	Chloramphenicol	Carcasses						9.1	48.5	9.1		33.3		8	64	4–64	33.3
	Florfenicol	Feces					9.7	52.2	4.4			33.6		4	64	2–64	33.6
	Florfenicol	Carcasses					6.1	48.5	12.1			33.3		4	64	2–64	33.3
Polymyxins	Colistin	Feces					100							2	2	2-2	0
Polymyxins	Colistin	Carcasses					100							2	2	2-2	0
Tetracyclines	Tetracycline	Feces					40.7				6.2	39.8	13.3	64	128	2–128	59.3
Tetracyclines	Tetracycline	Carcasses					54.5				9.1	21.2	15.2	2	128	2–128	45.5

The dilution ranges tested are those contained in the white area. The breakpoints of tested antimicrobial agents are indicated by vertical lines. MIC₅₀ and MIC₉₀ are the concentrations at which 50% and 90% of the isolates were inhibited.

MIC, minimum inhibitory concentration.

Table 2.

Representative Resistance Patterns of Escherichia coli Isolates from Duck Fecal and Carcass Samples

No. of agents	Fecal samples			Carcass samples
No. of agents	No. of isolates (%)	No. of MDR isolates (%)	Most common pattern	No. of isolates (%)	No. of MDR isolates (%)	Most common pattern
0	14 (12.4)	—	—	6 (18.2)	—	—
1	10 (8.8)	—	NAL; TET (n = 4)	5 (15.2)	—	NAL (n = 5)
2	24 (21.2)	—	CIP NAL (n = 7)	3 (9.1)	—	CHL FFC (n = 2)
3	12 (10.6)	8 (7.1)	AMP CHL FFC (n = 3)	4 (12.1)	4 (12.1)	AMP NAL SXT; CIP NAL SXT; NAL SXT TET; STR AMP SXT (n = 1)
4	15 (13.3)	15 (13.3)	STR AMP SXT TET (n = 3)	7 (21.2)	7 (21.2)	AMP CHL FFC TET; CIP NAL SXT TET; STR AMP SXT TET (n = 2)
5	15 (13.3)	15 (13.3)	STR AMP NAL SXT TET (n = 5)	2 (6.1)	2 (6.1)	AMP SXT CHL FFC TET; STR NAL CHL FFC TET (n = 1)
6	9 (8.0)	9 (8.0)	STR AMP CIP NAL SXT TET (n = 4)	1 (3.0)	1 (3.0)	GEN AMP NAL CEP FOX TET (n = 1)
7	5 (4.4)	5 (4.4)	STR AMP NAL SXT CHL FFC TET (n = 2)	1 (3.0)	1 (3.0)	AMP CIP NAL SXT CHL FFC TET (n = 1)
8	4 (3.5)	4 (3.5)	STR AMP CIP NAL SXT CHL FFC TET (n = 3)	1 (3.0)	1 (3.0)	STR AMP CIP NAL SXT CHL FFC TET (n = 1)
9	1 (0.9)	1 (0.9)	STR AMP CIP NAL CEP SXT CHL FFC TET (n = 1)	— (0)	— (0)	—
10	4 (3.5)	4 (3.5)	STR AMP XNL CIP NAL CEP SXT CHL FFC TET (n = 3)	3 (9.1)	3 (9.1)	STR AMP XNL CIP NAL CEP SXT CHL FFC TET (n = 2)
Total	113 (100)	61 (54.0)		33 (100)	19 (57.6)

AMP, ampicillin; CEP, cephalothin; CHL, chloramphenicol; CIP, ciprofloxacin; FFC, florfenicol; FOX, cefoxitin; GEN, gentamicin; MDR, multidrug resistance; NAL, nalidixic acid; STR, streptomycin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline; XNL, ceftiofur.

Molecular characterization of ESBL-producing E. coli

A total of six (4.1%) E. coli resistant to third-generation cephalosporins were detected in samples from three of the slaughterhouses originating from four different farms. All of them produced CTX-M type ESBLs, namely CTX-M-55 (n = 3) and CTX-M-65 (n = 3) (Table 3). All CTX-M-positive isolates were not only β-lactam resistant, but also showed resistance to several antimicrobial classes, such as quinolone, aminopenicillin, folate pathway inhibitor, tetracycline, aminoglycoside, and phenicol. Of note, all CTX-M-producing isolates were also resistant to ciprofloxacin, with mutations in the QRDR of GyrA (S83L with or without D87N) and ParC (S80I), and three of them carried the PMQR genes, qepA (n = 1), qnrS, and acc(6′)-Ib-cr (n = 2).

Table 3.

Characteristics of Third-Generation Cephalosporin-Resistant Escherichia coli Isolates from Duck Fecal and Carcass Samples

Isolates	Farm ID	Slaughter-house ID	Samples	CTX-M type	MICs (μg/mL)		Non-β-lactam resistance pattern	QRDR mutation				PMQR gene	blaCTX-M environment		Phylogenetic grouping	Self-transfer	Plasmid replicon type	Transferred resistance	Pulsotype	MLST
Isolates	Farm ID	Slaughter-house ID	Samples	CTX-M type	XNL	CIP	Non-β-lactam resistance pattern	GyrA	GyrB	ParC	ParE	PMQR gene	Upstream	Downstream	Phylogenetic grouping	Self-transfer	Plasmid replicon type	Transferred resistance	Pulsotype	MLST
07-DF-6	A	J1	Feces	CTX-M-65	>8	16	STR CIP NAL SXT CHL FFC TET	S83L	—	S80I	—	qnrS, acc(6′)-lb-cr	ISEcp1	IS903	A	−			I	ST2179
08-DF-37	B	J2	Feces	CTX-M-55	>8	>16	CIP NAL SXT TET	S83L, D87N	—	S80I	S458A	qepA	ISEcp1	orf477	A	−			II	ST448
08-DF-51	C	J3	Feces	CTX-M-65	>8	16	STR CIP NAL SXT CHL FFC TET	S83L	—	S80I	—	qnrS, acc(6′)-lb-cr	ISEcp1	IS903	A	−			I	ST2179
08-DF-52	C	J3	Feces	CTX-M-65	8	>16	STR CIP NAL SXT CHL FFC TET	S83L, D87N	—	S80I	S458A	—	—	IS903	A	+	F, FIB, FIC	AMP SXT CHL FFC TET	III	ST1431
05-DM-5	D	J1	Carcass	CTX-M-55	8	8	STR CIP NAL SXT CHL FFC TET	S83L, D87N	—	S80I	S458A	—	—	orf477	B1	−			IV	Untypable^a
05-DM-6	D	J1	Carcass	CTX-M-55	8	8	STR CIP NAL SXT CHL FFC TET	S83L, D87N	—	S80I	S458A	—	—	orf477	B1	−			IV	Untypable^a

Untypable, undetermined mdh sequence with one nucleotide substitution in the mdh allele 8 (¹⁶⁵G to ¹⁶⁵A) of ST48.

+, positive; −, negative; AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; FFC, florfenicol; MIC, minimum inhibitory concentration; MLST, multilocus sequence typing; NAL, nalidixic acid; PMQR, plasmid-mediated quinolone resistance; QRDR, quinolone resistance-determining region; STR, streptomycin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline; XNL, ceftiofur.

Transfer of bla _CTX-M genes to the recipient E. coli J53 strain by conjugation was observed in one of the six bla _CTX-M-positive E. coli isolates. In addition to the ESBL phenotype, resistance to non-β-lactam antimicrobials, such as trimethoprim/sulfamethoxazole, chloramphenicol, florfenicol, and tetracycline, also co-transferred. The replicon types of the transferred plasmids were IncF, IncFIB, and IncFIC.

Insertion sequence ISEcp1 elements were present in the upstream region of bla _CTX-M genes in three isolates (50%), but IS26 element was not found in the upstream of bla _CTX-M genes in the studied isolates. orf477 and IS903 were detected in the downstream region of three bla _CTX-M-55 genes and three bla _CTX-M-65 genes, respectively.

Phylogenetic group analysis showed that four isolates (66.7%) and two isolates (33.3%) belonged to phylogenetic group A and B1, respectively. PFGE and MLST demonstrated that CTX-M-producing isolates were heterogeneous (Table 3). PFGE analysis of the six CTX-M-positive E. coli isolates demonstrated four arbitrary (designated I to IV) pulsotypes. Among them, two pairs of isolates, one pair carrying the bla _CTX-M-65 gene from different farms (farms A and C) and another pair carrying the bla _CTX-M-55 gene from farm D, had identical PFGE profiles. MLST analysis demonstrated that the four E. coli strains belonged to ST2179 (n = 2), ST448 (n = 1), and ST1431 (n = 1). The identical MLSTs (ST2179) showed the same PFGE pattern (pattern I). However, two unidentified strains had an untypable mdh sequence, with one nucleotide substitution in mdh allele 8 of ST48.

Discussion

In this study, we observed antimicrobial resistance in E. coli isolates from the feces of healthy ducks and their carcasses and found high resistance to commonly used antimicrobials, such as tetracycline and nalidixic acid. Compared with E. coli from ducks in other countries, the resistance rate to tetracycline was similar in Tanzania (59.3%) (Kissinga et al., 2018) and lower than those of pathogenic E. coli from ducks in China (100%) (Yassin et al., 2017). Resistance to quinolones was also lower than that of pathogenic E. coli in China (95.4% of nalidixic acid and ciprofloxacin) (Yassin et al., 2017). Although the distribution of multidrug-resistant isolates in this study was lower than that of E. coli from ducks in Tanzania (69.3%) and pathogenic E. coli from ducks in China (100%), it was still relatively high in both duck feces (54.0%) and carcasses (57.6%). These variations in antimicrobial resistance among countries might be because of differences in geographical region, types of antimicrobial use, and methods.

The heavy use or misuse of antimicrobials in animals has caused an increase in antimicrobial resistance in E. coli of animal origin. Although data on the use of antimicrobials are not available from national monitoring programs or the duck farms studied, tetracycline, trimethoprim/sulfamethoxazole, and ampicillin are the most frequently administered antimicrobials among food-producing animals (APQA, 2016). In addition, enrofloxacin, which is classified as a fluoroquinolone like ciprofloxacin, is extensively used in the poultry industry in Korea (APQA, 2016). Thus, these antimicrobials might also be commonly used in the duck industry in Korea.

The presence of bacteria resistant to third-generation cephalosporins in animals has been increasing, and this can impact human health directly and indirectly (Smet et al., 2010). In this study, resistance to ceftiofur was detected in both feces (3.5%) and carcasses (6.1%). Although the prevalence of ceftiofur resistance in ducks was lower than that of chicken feces (11.6%) and carcasses (10.7%) collected in the same year in Korea, consumption of duck meats has increased recently, and extensive and strict monitoring of antimicrobial resistance to especially important antimicrobials, such as third-generation cephalosporins, fluoroquinolones, and colistin, are needed urgently.

The production of ESBLs in Enterobacteriaceae is a well-known mechanism for resistance to third-generation cephalosporins. ESBL production has consistently increased in human isolates since the early 1990s and in animal isolates since 2000 (Smet et al., 2010; Zhao and Hu, 2013). In this study, all E. coli resistant to ceftiofur also produced CTX-M type ESBLs, namely CTX-M-55 and CTX-M-65. These CTX-M types were predominant in Chinese food-producing animals including duck (Li et al., 2010; Rao et al., 2014; Xu et al., 2015), but were rare in Korean livestock (Tamang et al., 2013).

Quinolones and β-lactams are among the most common antimicrobials for the treatment of infections caused by pathogenic E. coli, and the presence of E. coli strains that are co-resistant to both quinolone and β-lactam antimicrobials is a serious public health concern (Bajaj et al., 2016). In this study, all the ceftiofur-resistant E. coli isolates were also resistant to fluoroquinolones. All of them carried substitutions in the QRDRs of GyrA (S83L with or without D87N) and ParC (S80I). Moreover, three of them carried the PMQR gene, qepA, qnrS, and acc(6′)-Ib-cr. PMQR genes have often been found to be strongly associated with ESBL genes, and some have been found on the same plasmid (Strahilevitz et al., 2009). In addition, recent reports suggest an increase in the coexistence of PMQR and ESBL genes in the same isolate (Liu et al., 2013; Xu et al., 2015).

The horizontal transfer of these plasmids may promote the dissemination of co-resistant bacteria (Crémet et al., 2011). However, the co-transferability of three bacteria (07-DF-6, 08-DF-37, and 08-DF-51), which had both bla _CTX-M and PMQR genes, to J53 E. coli recipients has not been observed. In only one isolate (08-DF-52) among six E. coli isolates that expressed the CTX-M β-lactamase, transfer of ceftiofur resistance was observed with IncF, IncFIB, and IncFIC replicon-type plasmids. The IncF family of plasmids has a limited host range to Enterobacteriaceae but has some mechanisms to improve plasmid stability (Woodford et al., 2009). Based on this, Bevan et al. (2017) suggested that IncF plasmids are stably maintained in commensal E. coli within the gastrointestinal tract of humans and animals, potentially explaining the global dissemination of ESBLs.

PFGE and MLST analysis demonstrated that CTX-M-producing isolates were heterogeneous; however, isolates with identical PFGE and MLST profiles were found in different farms (A and C) and slaughterhouses (J1 and J3). Two isolates (05-DM-5 and 05-DM-6) had an undetermined mdh sequence with one nucleotide substitution in the mdh allele 8 (¹⁶⁵G to ¹⁶⁵A) of ST48, and next-generation sequencing may help clarify similar cases. Although the MLST was not designed, identical PFGE patterns were observed in the same farm (farm D). These results suggested that CXT-M-producing E. coli might be spread within a farm or between farms.

Conclusions

This report demonstrates the high prevalence of antimicrobial resistance of E. coli from duck feces and carcasses to not only commonly used but also critically important antimicrobials. In addition, both the horizontal transfer of plasmids and the clonal spread of strains associated with quinolone resistance could lead to the dissemination of CTX-M resistance in the duck industry. The emergence of ESBL-producing E. coli from duck farms poses a potential risk to public health and therefore should be continuously monitored, and proper strategies, such as a voluntary ban of third-generation cephalosporins to combat antimicrobial resistance, should be established.

Footnotes

Acknowledgments

This study was supported by a grant from the Animal and Plant Quarantine Agency, Ministry of Agriculture, Food, and Rural affairs, Republic of Korea (N-1543081-2015-24-01).

Disclosure Statement

No competing financial interests exist.

Supplementary Material

Supplementary Table S1

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