Antimicrobial Resistance Profiles and Molecular Characteristics of Extended-Spectrum β -Lactamase-Producing Salmonella enterica Serovar Typhimurium Isolates from Food Animals During 2010

Abstract

Extended-spectrum β-lactamase (ESBL)-producing Salmonella is emerging as a worldwide public health concern. In this study, we aimed to investigate the antimicrobial resistance profiles and molecular characteristics of ESBL-producing Salmonella enterica serovar Typhimurium (S. Typhimurium). We obtained a total of 995 S. Typhimurium isolates from the feces and carcasses of pigs (n = 678), chickens (n = 202), and cattle (n = 115) during 2010–2021 in Korea. We found that 35 S. Typhimurium isolates (3.5%) showed resistance to ceftiofur: pigs (51.4%, 18/35) and cattle (42.9%, 15/35). All of the ceftiofur-resistant S. Typhimurium isolates demonstrated multidrug resistance. Moreover, ceftiofur-resistant S. Typhimurium isolates displayed significantly higher rates of resistance to chloramphenicol and trimethoprim/sulfamethoxazole than ceftiofur-susceptible S. Typhimurium isolates (p < 0.05). The ceftiofur-resistant S. Typhimurium isolates produced four different CTX-M-type β-lactamase, comprising bla _CTX-M-55 in the majority (51.4%, 18/35), followed by bla _CTX-M-65 (28.6%, 10/35), bla _CTX-M-14 (17.1%, 6/35), and bla _CTX-M-1 (2.9%, 1/35). Among the 35 ceftiofur-resistant S. Typhimurium isolates, 16 bla _CTX-M-55-positive isolates and one bla _CTX-M-1-positive isolate were transferred to recipient Escherichia coli RG488 by conjugation. The predominantly found transposable units were bla _CTX-M-55-orf477 (45.7%, 16/35), followed by bla _CTX-M-65-IS903 (28.6%, 10/35) and bla _CTX-M-14-IS903 (17.1%, 6/35). Ceftiofur-resistant S. Typhimurium represented 19 types, with types P1-19 (22.9%, 8/35) and P12-34 (22.9%, 8/35) making up the majority and being found in most farms nationwide. Sequence types (STs) were different by animal species: ST19 (48.6%, 17/35) and ST34 (42.9%, 15/35) were mostly found STs in pigs and cattle, respectively. These findings showed that food animals, especially pigs and cattle, act as reservoirs of bla _CTX-M-harboring S. Typhimurium that can potentially be spread to humans.

Introduction

Salmonella enterica is an important zoonotic pathogen associated with foodborne illness and a major global public health concern (Sun et al., 2020a). Salmonella infection-related mortality and morbidity significantly cost both developing and wealthy nations (Majowicz et al., 2010; Scallan et al., 2011). In Korea, Salmonella is among the most common causative agents responsible for foodborne diseases (Lee et al., 2016). It has been shown that Salmonella enterica serovar Typhimurium (S. Typhimurium), one of the most prevalent serotypes causing human salmonellosis, which comes from food animals such as chickens, pigs, and cattle, establishes a link between the consumption of Salmonella-contaminated chicken, beef, pork, and dairy products and human salmonellosis (Hur et al., 2012).

Several antimicrobials, including extended-spectrum cephalosporins, are used for the prevention and treatment of infections in food animals and humans. However, the extensive use of these antibacterials has led to the emergence of extended-spectrum β-lactamase (ESBL)-producing Salmonella (Dos Santos et al., 2013). Among the ESBLs, CTX-M-type β-lactamase is the most prevalent, varied, and complex group of enzymes (Castanheira et al., 2021). The bla _CTX-M-1 group and bla _CTX-M-9 group are the most prevalent bla _CTX-M types in Enterobacteriaceae, including Salmonella isolated from food animals worldwide (Bevan et al., 2017; Dos Santos et al., 2013). In recent years, more ESBL-producing S. Typhimurium have been reported globally in food animals, with the most common bla _CTX-M group found in pigs, chickens, and cattle in the United States (Gelalcha and Kerro Dego, 2022), Canada (Bharat et al., 2022), the United Kingdom (Burke et al., 2014), Nigeria (Gideon et al., 2021), and China (Guo et al., 2023). Moreover, several recent studies have identified the presence of bla _CTX-M-55 and bla _CTX-M-65-carrying S. Typhimurium from food animals (Palmeira and Ferreira, 2020; Zhang et al., 2019).

Although S. Typhimurium showed a high resistance rate among the serotypes, resistance to the third-generation cephalosporin in S. Typhimurium was relatively low compared with other serotypes, such as S. Virchow and S. Enteritidis in Korea (Mechesso et al., 2020). However, recently, ceftiofur resistance has increased in pigs and cattle, which could potentially be transmitted to humans through direct contact or the food chain. Systematic exploration on the prevalence and characteristics of antimicrobial-resistant bacteria in food animals is essential to determine and prevent possible hazards to humans. Thus, in this study, we investigated the antimicrobial resistance and characterization of ceftiofur-resistant S. Typhimurium isolated from food-producing animals.

Materials and Methods

Isolation, identification, and serotyping of Salmonella

Salmonella was isolated, identified, and serotyped using the previously mentioned method (Mechesso et al., 2020). S. Typhimurium isolates were collected from 16 laboratories/centers participating in the Korean Veterinary Antimicrobial Resistance Monitoring System between 2010 and 2021 (Table 1). The isolation process involves an initial phase of pre-enrichment in buffered peptone water (Becton Dickinson, CA, USA) at 37°C for 24 h. The samples were thereafter enriched in a modified semisolid Rappaport Vassiliadis medium (Becton Dickinson, CA, USA) and subjected to incubation at 42°C for 24–48 h. The selected colonies were further incubated on CHROMagar (Merck, Darmstadt, Germany) at 37°C for 24 h and were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Biomerieux, Marcy L’Etoile, France). The Salmonella serogroup was determined by polymerase chain reaction (PCR) (Ranieri et al., 2013). Finally, S. Typhimurium was confirmed by agglutination, identifying the Salmonella O and H antigens (phase 1 and phase 2) using the White–Kauffmann–Le Minor technique (Grimont and Weill, 2007). In total, 995 S. Typhimurium isolates were recovered from the feces and carcasses of healthy pigs (n = 475), chickens (n = 173), and cattle (n = 93) and from diseased (diarrheic) pigs (n = 203), chickens (n = 29), and cattle (n = 22) from 650 farms throughout South Korea. One isolate per sample was considered for the following analysis.

Table 1.

Salmonella enterica Serovar Typhimurium Isolates Obtained from Samples of Healthy and Diseased Food-Producing Animals During 2010–2021 in South Korea

Year	Healthy food-producing animals and their carcasses									Diseased Animals
	Cattle			Pigs			Chickens			Cattle		Pigs		Chickens		Total
	No. of slaughterhouse	No. of farms	No. of isolates	No. of slaughterhouse	No. of farms	No. of isolates	No. of slaughterhouse	No. of farms	No. of isolates	No. of farms	No. of isolates	No. of farms	No. of isolates	No. of farms	No. of isolates	^aNo. of slaughterhouse	^aNo. of farms	No. of isolates
2010	2	2	2	10	19	36	4	4	6	4	4	9	18	3	11	16	41	77
2011	1	3	3	2	2	3	1	1	1	0	0	9	11	1	1	4	16	19
2012	5	16	20	13	37	46	3	10	12	7	9	11	22	1	1	21	82	110
2013	6	16	22	13	34	41	5	14	35	1	2	10	16	0	0	24	75	116
2014	3	8	9	13	37	50	8	20	31	1	3	16	36	2	6	24	84	135
2015	2	6	6	11	25	30	4	12	13	2	4	12	27	1	1	17	58	81
2016	3	7	9	8	13	16	1	1	1	0	0	5	5	0	0	12	26	31
2017	4	14	14	14	25	30	2	3	3	0	0	4	6	0	0	20	46	53
2018	2	5	5	19	43	56	3	4	5	0	0	9	24	0	0	24	61	90
2019	2	3	3	15	39	50	4	17	22	0	0	4	4	1	4	21	64	83
2020	0	0	0	9	59	71	7	20	31	0	0	10	22	4	4	16	93	128
2021	0	0	0	10	35	46	6	20	13	0	0	9	12	1	1	16	65	72
Total	30	80	93	137	368	475	48	126	173	15	22	108	203	14	29	75	650	995

Overlaps were counted as once.

Antimicrobial susceptibility testing

The broth microdilution method was used with a commercially available Sensitire^® panel KRNVF (TREK Diagnostic Systems, West Sussex, UK) to determine the minimum inhibitory concentrations (MICs) of the isolates toward 12 antimicrobials. Escherichia coli ATCC 25922 was used as a quality reference strain. The obtained MICs were interpreted according to the (Clinical and Laboratory Standards Institute [CLSI], 2020) guidelines and the (National Antimicrobial Resistance Monitoring System [NARMS], 2021). Resistance to at least three different antimicrobial categories was defined as multidrug resistance (MDR) (Magiorakos et al., 2012). The double-disc synergy test was carried out to detect ESBL producers among ceftiofur-resistant isolates using cefotaxime–cefotaxime/clavulanic acid and ceftazidime–ceftazidime/clavulanic acid discs, according to CLSI guidelines (Clinical and Laboratory Standards Institute [CLSI], 2020).

Detection of resistance gene

The ceftiofur-resistant S. Typhimurium isolates carrying ESBL genes (bla _TEM, bla _SHV, or bla _CTX-M) were determined using a PCR according to a previously delineated method (Na et al., 2020). The list of primers and PCR conditions is listed in Supplementary Table S1.

Conjugation experiment

The conjugation experiment was performed using the filter-mating method with the recipient strain of rifampin-resistant E. coli RG488, as previously described (Na et al., 2020). Briefly, the donor and recipient strains were cultured overnight and subsequently incubated in tryptic soy broth (Becton, Dickinson, MD, USA), followed by culture for 4 h at 37°C. The freshly prepared bacteria were mated using a donor–recipient ratio of 1:4, and bacteria were retained on a membrane filter. After an overnight incubation on tryptic soy agar plates, the bacteria on the filters were suspended in phosphate-buffered saline. The potential transconjugants were selected by transferring the dilution mixture to MacConkey agar plates treated with rifampin (50 µg/mL) and ceftiofur (25 µg/mL). All the selected transconjugants were examined for antimicrobial susceptibility patterns and the presence of β-lactamase (bla) genes.

Molecular characterization of ESBL-producing S. Typhimurium

PCR and Sanger sequencing were performed using the previously mentioned primers and PCR conditions to explore the genomic environment surrounding the bla gene (Eckert et al., 2006; Saladin et al., 2002). In combination with bla gene primers, specific primers for the insertion sequences ISEcp1, IS903, or orf477 were used (Supplementary Table S1).

Multilocus sequence typing (MLST) was carried out to determine the clonal relationship of S. Typhimurium, following the previously mentioned method (Kidgell et al., 2002). In this classical scheme, seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) were amplified and sequenced. Moreover, the allelic profile and sequence types (STs) for S. Typhimurium were detected using web-based MLST databases (https://pubmlst.org/organisms/Salmonella-spp).

Pulsed-field gel electrophoresis (PFGE) was accomplished using the XbaI enzyme (TaKaRa Bio, Inc., Shiga, Japan), as delineated previously (Gautom, 1997). The Bionumerics software was used to evaluate the PFGE band profiles, and relatedness was determined using the unweighted pair-group approach and an algorithm based on arithmetic averages and the Dice similarity index.

Statistical analysis

Rex Software (version 3.0.3, RexSoft Inc., Seoul, South Korea) was used for the statistical analysis. The group comparisons were performed using the chi-square test. The values of p < 0.05 were regarded as statistically significant.

Results

Antimicrobial resistance of S. Typhimurium

Antimicrobial resistance of 995 S. Typhimurium isolates toward 12 antimicrobials is presented in Table 2. A high resistance rate to streptomycin (64%), ampicillin (72.2%), and tetracycline (68.7%) was observed in S. Typhimurium isolates. However, resistance to amoxicillin/clavulanic acid, cefoxitin, ciprofloxacin, and colistin was low, <5%. Among them, 35 isolates (3.5%) showed resistance to ceftiofur, and the majority of these resistant isolates were obtained from pigs (18/35, 51.4%) and cattle (15/35, 42.9%). In addition, we compared the resistance rates of the ceftiofur-resistant group (n = 35) and the ceftiofur-susceptible group (n = 960). It was found that significantly higher proportions of ceftiofur-resistant isolates were resistant to non-β-lactam antimicrobial agents than ceftiofur-susceptible isolates. Chloramphenicol (37.1% vs. 77.1%) and trimethoprim/sulfamethoxazole (20% vs. 40%) resistance rates were significantly higher in the ceftiofur-resistant group than in the ceftiofur-susceptible group (p < 0.05). Among the 995 isolates, most of the isolates (n = 842) were resistant to one or more antimicrobial agents, and the MDR phenotype was identified in 73.6% (732/995) of the isolates (Table 3). In addition, the MDR phenotype was significantly higher in the ceftiofur-resistant group than in the ceftiofur-susceptible group (p < 0.05). Moreover, 65.7% (23/35) of the ceftiofur-resistant isolates were resistant to seven antimicrobial agents. Of note, 18.1% (180/995) of the S. Typhimurium isolates displayed an identical resistance pattern with streptomycin, ampicillin, and tetracycline (Table 3).

Table 2.

Antimicrobial Resistance of S. Typhimurium Isolated from Food-Producing Animals During 2010–2021 in South Korea

Antimicrobial agents	Range tested(μg/mL)	Break points(μg/mL)	Comparison between resistance rates of susceptible isolates and resistant isolates to ceftiofur						p-Value	Total S. Typhimuriumtested (n = 995)
			Ceftiofur-susceptible S. Typhimurium (n = 960)			Ceftiofur-resistant S. Typhimurium (n = 35)				Total S. Typhimuriumtested (n = 995)
			MIC₅₀	MIC₉₀	% Resist. (n)	MIC₅₀	MIC₉₀	% Resist. (n)		MIC₅₀	MIC₉₀	% Resist. (n)
Aminoglycosides
Gentamicin	1–64	≥16	1	32	27.1 (260)	1	16	25.7 (9)	0.9370	1	32	27.0 (269)
Streptomycin	16–128	≥32	128	128	63.6 (611)	128	128	74.3 (26)	0.1256	128	128	64.0 (637)
Aminopenicillin
Ampicillin	2–64	≥32	64	64	71.3 (684)	64	64	100 (35)	0.0002	64	64	72.2 (718)
β-lactam/β-lactamase inhibitor
Amoxicillin/clavulanic acid	2/1–32/16	≥32/16	8	8	1.6 (15)	8	16	0 (0)	0.4627	8	8	1.5 (15)
Cephamycin
Cefoxitin	1–32	≥32	2	8	3.1 (30)	4	8	0 (0)	0.2952	2	8	3.0 (30)
Cephalosporin III
Ceftiofur	0.5–8	≥8	1	2	0 (0)	8	8	100 (35)	<0.001	1	2	5.5 (55)
Fluoroquinolone
Ciprofloxacin	0.12–16	≥1	0.12	0.5	4.6 (44)	0.5	0.5	0 (0)	0.2016	0.12	0.5	4.4 (44)
Quinolone
Nalidixic acid	2–128	≥32	4	128	34.5 (331)	16	128	48.6 (17)	0.0622	4	128	35.0 (348)
Polymyxins
Colistin	2–16	≥4	2	2	0.7 (7)	2	2	0 (0)	0.6173	2	2	0.7 (7)
Folate pathway inhibitors
Trimethoprim/sulfamethoxazol	0.12/2.38–4/76	≥4/76	0.12	4	20.0 (192)	0.25	4	40.0 (14)	0.0028	0.12	4	20.7 (206)
Phenicols
Chloramphenicol	2–64	≥32	8	64	37.1 (356)	64	64	77.1 (27)	<0.001	8	64	38.5 (383)
Tetracyclines
Tetracycline	2–128	≥16	128	128	68.6 (659)	128	128	71.4 (25)	0.5458	128	128	68.7 (684)
MDR isolates					72.7 (698)			100 (35)	0.0004			73.6 (732)

MIC₅₀ and MIC₉₀ are the concentrations (μg/mL) at which 50% and 90% of the isolates were inhibited, respectively.

MIC, minimum inhibitory concentration; MDR, multidrug resistance.

Table 3.

Multidrug Resistance and Representative Resistance Patterns of S. Typhimurium Isolated from Food-Producing Animals During 2010–2021 in South Korea

Resistance patterns	% of Resistant isolates (n)			Most common pattern (No. of isolates)
Resistance patterns	Ceftiofur-susceptible S. Typhimurium(n = 960)	Ceftiofur-resistant S. Typhimurium(n = 35)	Total(n = 995)	Most common pattern (No. of isolates)
No resistance	15.9 (153)	0 (0)	15.4 (153)	(n = 153)
Resistant to 1 agent	2.1 (20)	0 (0)	2.0 (20)	NAL (n = 8)
Resistant to 2 agents	9.3 (89)	0 (0)	9.0 (89)	STR AMP (n = 37)
Resistant to 3 agents	35.7 (343)	20.6 (7)	35.2 (350)	STR AMP TET (n = 180)
Resistant to 4 agents	12.5 (120)	2.9 (1)	12.2 (121)	STR AMP NAL TET (n = 27)
Resistant to 5 agents	14.7 (141)	11.8 (4)	14.6 (145)	STR AMP SXT CHL TET (n = 28)
Resistant to 7 agents	4.7 (45)	65.7 (23)	6.7 (67)	GEN STR AMP NAL SXT CHL TET (n = 24)
Resistant to 8 agents	4.9 (47)	0 (0)	4.9 (47)	GEN STR AMP CIP NAL SXT CHL TET (n = 23)STR AMP AMC FOX NAL SXT CHL TET (n = 4)
Resistant to 9 agents	0.1 (1)	0 (0)	0.1 (1)	STR AMP AMC FOX XNL NAL SXT CHL TET (n = 1)
Resistant to 10 agents	0.1 (1)	0 (0)	0.1 (1)	GEN STR AMP FOX XNL CIP NAL SXT CHL TET (n = 1)
MDR isolates	72.7 (698)	100 (35)	73.6 (732)

AMC, amoxicillin/clavulanic acid; AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; COL, colistin; FOX, cefoxitin; GEN, gentamicin; MDR, multidrug resistance; NAL, nalidixic acid; STR, streptomycin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline; XNL, ceftiofur.

Molecular characteristics of ceftiofur-resistant S. Typhimurium

A total of 35 ceftiofur-resistant S. Typhimurium strains were detected in pigs (18 isolates), cattle (15 isolates), and chickens (2 isolates) from 20 farms located in 5 provinces (Table 4). All ceftiofur-resistant S. Typhimurium isolates carried β-lactamases, such as bla _CTX-M-55 (n = 18), bla _CTX-M-65 (n = 10), bla _CTX-M-14 (n = 6), and bla _CTX-M-1 (n = 1), whereas none of the isolates produced bla _TEM or bla _SHV. Moreover, the occurrence of these CTX-M type β-lactamases was varied by animal species, with the highest proportion of bla _CTX-M-55 (94.4%, 17/18) in pigs and all the bla _CTX-M-65 and 66.7% (4/6) of bla _CTX-M-14 in cattle isolates. The prevalence of β-lactamases-producing S. Typhimurium isolates also varied over the years. Before 2017, bla _CTX-M-14 was predominant, bla _CTX-M-65 in 2017, and bla _CTX-M-55 in 2018.

Table 4.

Characteristics of bla _CTX-M-Carrying S. Typhimurium Isolates

Isolates	Source	Year	Province	Farm ID	MIC (μg/mL) ofXNL	bla_CTX-M gene	Resistance pattern(non-β-lactams)	Transfer	bla_CTX-M geneenvironment		PFGE-ST	Serotype
Isolates	Source	Year	Province	Farm ID	MIC (μg/mL) ofXNL	bla_CTX-M gene	Resistance pattern(non-β-lactams)	Transfer	Upstream	Downstream	PFGE-ST	Serotype
V10-008	Diseased pig	2011	Jeju	JJ-1	>8	bla _CTX-M-14	STR, NAL, COL, CHL, TET	−	ISEcp1	IS903	P4-19	ST
V04-005	Healthy chicken	2013	Chungnam	CN-1	>8	bla _CTX-M-14	STR, SXT, CHL, TET	−	—	IS903	P14-34	ST
V04-016	Chicken carcass	2015	Gyeonggi	GG-1	>8	bla _CTX-M-1	STR, SXT, CHL	+	ISEcp1	orf477	P10-36	ST
V13-002	Cattle carcass	2016	Gyeongbuk	GB-1	>8	bla _CTX-M-14	STR, SXT, CHL, TET	−	—	IS903	P13-34	ST.V
V13-003	Cattle carcass	2016	Gyeongbuk	GB-2	>8	bla _CTX-M-14	STR, SXT, CHL, TET	−	—	IS903	P13-34	ST.V
V13-004	Cattle carcass	2016	Gyeongbuk	GB-2	>8	bla _CTX-M-14	STR, SXT, CHL, SXT	−	—	IS903	P13-34	ST.V
V13-005	Cattle carcass	2016	Gyeongbuk	GB-2	>8	bla _CTX-M-14	STR, SXT, CHL, TET	−	—	IS903	P13-34	ST.V
V16-001	Healthy cattle	2016	Chungnam	CN-2	>8	bla _CTX-M-55	STR, CHL, TET	+	—	orf477	P15-34	ST.V
V02-002	Cattle carcass	2017	Gyeonggi	GG-2	>8	bla _CTX-M-65	GEN, STR, SXT, CHL, TET	−	—	IS903	P12-34	ST.V
V02-003	Cattle carcass	2017	Gyeonggi	GG-3	>8	bla _CTX-M-65	GEN, STR, SXT, CHL, TET	−	—	IS903	P12-34	ST.V
V02-004	Cattle carcass	2017	Gyeonggi	GG-4	>8	bla _CTX-M-65	STR, SXT, CHL	−	—	IS903	P11-34	ST.V
V02-005	Cattle carcass	2017	Gyeonggi	GG-5	>8	bla _CTX-M-65	GEN, STR, SXT, CHL, TET	−	—	IS903	P12-34	ST.V
V02-006	Cattle carcass	2017	Gyeonggi	GG-6	>8	bla _CTX-M-65	STR, SXT, CHL,	−	—	IS903	P11-34	ST.V
V02-007	Cattle carcass	2017	Gyeonggi	GG-7	>8	bla _CTX-M-65	GEN, STR, SXT, CHL, TET	−	—	IS903	P12-34	ST.V
V02-008	Cattle carcass	2017	Jeonbuk	JB-1	>8	bla _CTX-M-65	GEN, STR, CHL, TET	−	—	IS903	P12-34	ST.V
V02-009	Cattle carcass	2017	Chungnam	CN-3	>8	bla _CTX-M-65	GEN, STR, SXT, CHL, TET	−	—	IS903	P12-34	ST.V
V02-010	Cattle carcass	2017	Chungnam	CN-4	>8	bla _CTX-M-65	GEN, STR, SXT, CHL, TET	−	—	IS903	P12-34	ST.V
V02-011	Cattle carcass	2017	Gyeonggi	GG-8	>8	bla _CTX-M-65	GEN, STR, SXT, CHL, TET	−	—	IS903	P12-34	ST.V
V06-001	Healthy pig	2018	Jeonbuk	JB-2	>8	bla _CTX-M-55	CHL, TET	+	—	orf477	Not done	ST
V01-003	Healthy pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	STR, CHL, NAL, TET	+	—	orf477	P5-19	ST
V01-004	Healthy pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	CHL, NAL	+	—	orf477	P1-19	ST
V01-006	Healthy pig	2018	Chungnam	CN-6	>8	bla _CTX-M-55	STR, CHL, NAL, TET	+	—	orf477	P1-19	ST
V01-014	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	STR, CHL, NAL, TET	+	—	—	P6-19	ST
V01-015	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	NAL	+	—	orf477	P16-19	ST
V01-016	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	NAL	+	—	orf477	P1-19	ST
V01-017	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	NAL	−	—	—	P1-19	ST
V01-019	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	STR, CHL, NAL, TET	+	—	orf477	P1-19	ST
V01-020	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	NAL	+	—	orf477	P1-19	ST
V01-021	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	STR, CHL, NAL, TET	+	—	orf477	P2-19	ST
V01-022	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	STR, CHL, NAL, TET	+	—	orf477	P9-19	ST
V01-023	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	STR, CHL, NAL, TET	+	—	orf477	P3-19	ST
V01-024	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	NAL	+	—	orf477	P1-19	ST
V01-025	Diseased pig	2018	Chungnam	CN-5	>8	bla _CTX-M-55	NAL	+	—	orf477	P8-19	ST.V
V01-009	Diseased pig	2018	Chungnam	CN-7	>8	bla _CTX-M-55	STR, CHL, NAL, TET	+	—	orf477	P1-19	ST
V01-011	Diseased pig	2018	Chungnam	CN-7	>8	bla _CTX-M-55	STR, CHL, NAL, TET	+	—	orf477	P7-19	ST

Underline: transferred resistance.

CHL, chloramphenicol; COL, colistin; GEN, gentamicin; NAL, nalidixic acid; PFGE-ST, pulsed-field gel electrophoresis−sequence typing; ST, Salmonella Typhimurium; STR, streptomycin; ST.V, monophasic S. Typhimurium variant (I,4,[5],12:i:-); SXT, trimethoprim/sulfamethoxazole; TET, tetracycline; XNL, ceftiofur.

The transfer of ceftiofur resistance was different among bla _CTX-M types. All bla _CTX-M-55-carrying isolates were transferred except one; however, none of the bla _CTX-M-65- or bla _CTX-M-14-carrying isolates were transferred. In addition, streptomycin, chloramphenicol, and/or tetracycline resistance were suggested to be transferred with bla _CTX-M. Moreover, the bla _CTX-M environments differed by CTX-M types. IS903 was detected in bla _CTX-M-14 (6 isolates) and bla _CTX-M-65 (10 isolates), orf477 was detected in bla _CTX-M-55 (16 isolates) downstream, whereas ISEcp1 was detected in one bla _CTX-M-14 and one bla _CTX-M-1 upstream.

Molecular typing by PFGE and MLST revealed a total of 16 PFGE types (P1–P16) and three STs (ST19, ST34, and ST36) in 35 ceftiofur-resistant isolates (Table 4, Supplementary Fig. S1). By combining the two methods, we found 19 different types. Among them, P1-19 type (22.9%, 8/35) and P12-34 type (22.9%, 8/35) were predominantly detected. P1-19 type was obtained from pigs in three farms in one province, whereas P12-34 type was obtained from cattle in eight farms in three provinces. Interestingly, the P1-19 type was predominantly detected in 2018, while the P12-34 type was mainly detected in 2017. Moreover, the two most commonly found STs were ST19 (48.6%, 17/35) and ST34 (42.9%, 15/35) in pigs and cattle, respectively. In addition, among the 35 S. Typhimurium isolates, 45.7% were identified as monophasic S. Typhimurium variants (I,4,[5],12:i:-) (Table 4).

Discussion

Our findings showed that ESBL-producing S. Typhimurium was detected mainly in pigs and cattle. bla _CTX-M-55 and bla _CTX-M-65 were predominant in pigs and cattle, respectively. Furthermore, specific clones, ST19 and ST34, and transposable elements, orf477 and IS903, were prevalently identified in ceftiofur-resistant S. Typhimurium.

Salmonella isolates from food animals have frequently been shown to be resistant to β-lactam antibacterials globally. However, the prevalence of the third-generation cephalosporin resistance was different among serotypes. In our previous studies, a high third-generation cephalosporin resistance (63.8%) was observed in S. Virchow (Na et al., 2020) but a low (3.7%) in S. Albany (Ali et al., 2023). In S. Typhimurium, ceftiofur resistance was found in 3.5% (35/995); among them, 51.4% (18/35) isolates obtained from pigs, 42.9% (15/35) from cattle, and 5.7% (2/35) from chickens. Previous investigation demonstrated high extended-spectrum cephalosporin resistance in S. Typhimurium isolated from pigs in Spain (91.8%) (Galán-Relaño et al., 2022) and Nigeria (100%) (Ugwu et al., 2015). Similarly, S. Typhimurium isolates from cattle in Malaysia (27.6%) (Benacer et al., 2010) and the USA (78%) (Basbas et al., 2021) showed high resistance to extended-spectrum cephalosporin. In contrast, a relatively small portion of the cephalosporin-resistant S. Typhimurium isolates was found in chickens in China (4.99%) (Ke et al., 2014) and the USA (16%) (F. et al., 2018).

In this study, it has been observed that ceftiofur-resistant S. Typhimurium demonstrated a significantly higher resistance rate to other non-β-lactam antimicrobials (especially folate pathway inhibitors and phenicols) than ceftiofur-susceptible S. Typhimurium isolates, which is consistent with a recent observation in Salmonella Virchow isolated from food animals in Korea (Na et al., 2020). Moreover, ceftiofur-resistant S. Typhimurium isolates demonstrated MDR, concurring with the previous report that all the ceftiofur-resistant S. Enteritidis isolated from food animals exhibited MDR phenotypes (Mechesso et al., 2022). In addition, consistent with previous studies, our investigation showed that Salmonella isolates obtained from humans and food animals revealed common resistant patterns with streptomycin, ampicillin, and tetracycline (Rao et al., 2020; Wang et al., 2019).

We identified four different types of bla _CTX-M genes in the ceftiofur-resistant S. Typhimurium isolates; among them, bla _CTX-M-55 comprised the majority (51.4%, 18/35), followed by bla _CTX-M-65 (28.6%, 10/35) and bla _CTX-M-14 (17.1%, 6/35), while bla _CTX-M-1 was detected in one isolate. In addition, the prevalence of these CTX-M-type β-lactamases differed by animal species, with 94.4% of bla _CTX-M-55 in pigs and all the bla _CTX-M-65 and 66.7% of bla _CTX-M-14 detected in cattle isolates. Although bla _CTX-M-55 was once a less prevalent gene detected in Salmonella isolated from humans and animals, recently, bla _CTX-M-55-carrying Salmonella from both humans and pigs has been regularly identified throughout the world, including in Korea (Kim et al., 2017), China (Zhang et al., 2019), Cambodia (Nadimpalli et al., 2019), Switzerland (Gallati et al., 2013), and Denmark (Torpdahl et al., 2017). Similarly, bla _CTX-M-65-carrying Salmonella has also increased in food animals, including cattle (Palmeira and Ferreira, 2020). In addition, bla _CTX-M-14 is one of the more frequently detected bla _CTX-M variants in Enterobacteriaceae worldwide (Seiffert et al., 2013). Salmonella strains isolated from food animals and humans share common genetic traits (Fey et al., 2000; Lim et al., 2011). As a result, the emergence and transmission of these bla _CTX-M genes may reduce the efficacy of β-lactam antibiotics used to treat human salmonellosis.

In this investigation, of the 35 CTX-M-type β-lactamase-producing S. Typhimurium isolates, 17 bla _CTX-M-55 and one bla _CTX-M-1 genes were transferred to the recipient E. coli RG488. On the other hand, none of the bla _CTX-M-65-carrying isolates were transmitted to E. coli RG488 recipients. A previous study found that 8.3% of Salmonella isolated from food-producing animals could transfer the bla _CTX-M-55 gene to recipients by conjugation (Zhang et al., 2019). However, the low transfer rate of the bla _CTX-M genes may be related to their chromosomal position, though more research is required in this regard (Hamamoto and Hirai, 2019).

The mobile genetic elements play a crucial role in the accumulation, interaction, and maintenance of antimicrobial resistance by facilitating the mobilization and dispersion of antimicrobial resistance genes (Chenghao et al., 2023). In our study, bla _CTX-M-55-orf477 transposable units were predominantly detected (16 isolates), followed by bla _CTX-M-65-IS903 (10 isolates) and bla _CTX-M-14-IS903 (6 isolates). It has been shown that the orf477 transposable element located downstream plays an important role in the dissemination and expression of bla _CTX-M genes (Kim et al., 2017). In addition, IS903 transposable units can aid in the transfer of bla _CTX-M among transmissible plasmids as well as from plasmids to the chromosomes of host bacteria (Yang et al., 2020).

We suggested that the bla _CTX-M-55 gene was cotransferred with streptomycin, chloramphenicol, and/or tetracycline resistance, concurring with the previous study that showed S. Typhimurium isolates cotransferred these antimicrobial resistances with the bla genes (Wang et al., 2019). Moreover, Protonotariou et al. (2022) studied the horizontal transmission of the bla _CTX-M-55 and strA (genes for streptomycin) and tetA (genes for tetracycline) in S. Typhimurium in Greece. These findings in our current study suggest that high resistance to streptomycin, chloramphenicol, and tetracycline might be associated with bla _CTX-M-55-carrying plasmids in ceftiofur-resistant S. Typhimurium.

In this study, a total of 19 types by combination of PFGE patterns and MLST were detected in 35 ceftiofur-resistant S. Typhimurium isolates, with two types (P1-19 and P12-34 type 22.9%, respectively) making up 45.8%. Moreover, P1-19 was predominantly identified in 2018, and P12-34 was mainly detected in 2017, possibly because of specific clones spreading in one province. However, these clones were widely found in pigs and cattle at different farms and provinces, indicating their spreading among the livestock in Korea, consistent with the previous report (Na et al., 2020). The previous studies showed the clonal spread of multidrug-resistant Salmonella spp. in both humans and animals globally (Tang et al., 2023; J. et al., 2015).

According to the MLST data, a total of three different STs (ST19, ST34, and ST36) were detected in this study. Among them, ST19 was the most common genotype identified in pigs, while ST34 was the most frequent genotype recovered in cattle. Previous studies showed that the multidrug-resistant S. Typhimurium ST19 clone has been identified in humans and pigs (Antunes et al., 2011; Yu-Ping et al., 2018). In addition, this clone has been associated with human salmonellosis (Chen et al., 2022). Moreover, the multidrug-resistant S. Typhimurium ST34 clone has been distributed worldwide, posing a critical health threat to humans (Biswas et al., 2019; Sun et al., 2020b). Consuming contaminated food-producing animal meats and their products and exposing the meat processing instruments are likely responsible for transferring this infection to humans (Carrasco et al., 2012).

Conclusion

Increasing ceftiofur-resistant S. Typhimurium in cattle and pigs could be a serious public health concern. Our data showed that the occurrence of bla _CTX-M-55 and bla _CTX-M-65 producing isolates might spread from food-producing animals to humans through the food chain. Especially, transferable bla _CTX-M-55 encoding plasmid could play an important role in the transmission among different bacterial species. These results suggest that prevention measures such as restriction use for prevention and mandatory testing for treatment use of the third-generation cephalosporins are urgently needed in food animals in Korea.

Footnotes

Authors’ Contributions

S.-K.L. and S.-H.N. developed the concept of the study. S.-H.N., B.-Y.M., T.-S.K., H.Y.K., and S.-K.L. designed the experiments. H.-S.K., S.-J.K., Y.-E.H., M.S.A., H.Y.K., and Y.-J.H. performed the experiments and collected and analyzed the data. M.S.A. wrote the original draft. S.K.L., S.-H.N., B.-Y.M., M.S.A., and S.S.Y. revised the article. All authors read and approved the final version of the article for publication.

Disclosure Statement

The authors declare that there are no competing interests.

Funding Information

This research was supported by the Animal and Plant Quarantine Agency, Ministry of Agriculture, Food, and Rural Affairs, Republic of Korea (Grant number N-1543081-2017-24-01). The sponsor had no involvement in planning the study, gathering, analyzing, and interpreting the data, writing the report, or selecting to submit the article for publication.

Supplementary Material

Supplementary Figure S1

Supplementary Table S1

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Supplementary Material

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Antimicrobial Resistance Profiles and Molecular Characteristics of Extended-Spectrum β -Lactamase-Producing Salmonella enterica Serovar Typhimurium Isolates from Food Animals During 2010–2021 in South Korea

Abstract

Introduction

Materials and Methods

Isolation, identification, and serotyping of Salmonella

Antimicrobial susceptibility testing

Detection of resistance gene

Conjugation experiment

Molecular characterization of ESBL-producing S. Typhimurium

Statistical analysis

Results

Antimicrobial resistance of S. Typhimurium

Molecular characteristics of ceftiofur-resistant S. Typhimurium

Discussion

Conclusion

Footnotes

Authors’ Contributions

Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material