Characterization of mcr-1-Harboring Plasmids from Pan Drug-Resistant Escherichia coli Strains Isolated from Retail Raw Chicken in South Korea

Abstract

A number of studies from different countries have characterized mcr-1-harboring plasmids isolated from food; however, nothing has been reported about it in South Korea. In this study, we report the characterization of mcr-1 plasmids from pan drug-resistant (PDR) Escherichia coli strains isolated from retail food in the country. Colistin-resistant E. coli strains were isolated from retail raw chicken, and PCR was carried out to detect the mcr-1 gene. Whole genome sequencing of the mcr-1-positive strains was performed for further characterization. The results of whole genome sequencing revealed that all mcr-1 plasmids belonged to the IncI2 type. In addition to the mcr-1 plasmids, all of the isolates also carried additional plasmids possessing multiple antibiotic resistance genes, and the PDR was mediated by resistant plasmids except for fluoroquinolone resistance resulting from mutations in gyrA and parC. Interestingly, the mcr-1 plasmids were transferred by conjugation to other pathogenic strains including enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), Salmonella, and Klebsiella at the frequencies of 10⁻³−10⁻⁶, 10⁻²−10⁻⁵, 10⁻⁴−10⁻⁵, 10⁻⁴−10⁻⁶, and 10⁻⁵−10⁻⁶, respectively. The results showed that mcr-1 plasmids can be easily transmitted to pathogenic bacteria by conjugation.

1. Introduction

The emergence of multidrug-resistant (MDR) pathogens, such as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., or recently Enterobacteriaceae) and the lack of effective antimicrobials are a serious issue in public health [1]. Colistin is one of the last-resort antibiotics to treat MDR Gram-negative bacteria, and its binding to the lipid A moiety of lipopolysaccharide (LPS) destabilizes the outer membrane and results in cell death [2]. Colistin resistance is mainly associated with the modification of LPS, such as phosphoethanolamine modification of lipid A [3,4,5], and causes serous clinical problems in the control of Gram-negative pathogens in ESKAPE [6,7,8].

Since the first discovery of the mobilized colistin resistance (mcr)-1 gene on plasmids in Escherichia coli by Liu et al. in China, 2016 [4], E. coli strains harboring mcr-1 have been reported in many countries throughout America, Asia, and Europe [4,9,10] and have been isolated from various sources, such as animals, humans, environmental samples, and food [10,11,12]. In South Korea, mcr-1-positive E. coli strains have been isolated in livestock and humans [13,14]. However, there have been no studies about mcr-1-positive E. coli from retail food in the country.

Several recent reports also suggested that the spread of mcr-1 to multidrug-resistant bacteria can contribute to the development of the pan drug-resistant (PDR) phenotype, since mcr-1 on plasmids can be easily disseminated [3,15]. The increasing number of PDR bacteria, including E. coli, is considered a threat to public health [16]. E. coli is a major cause of human diseases, such as urinary tract infections, sepsis, and pneumonia [17]. Therefore, the emergence of PDR E. coli isolates, particularly those harboring extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes, have aggravated the public health burdens of antibiotic resistance.

A number of studies have shown that retail chicken is a major reservoir of disseminating antibiotic-resistant E. coli to humans [9,18,19]. In this study, we aimed at isolating mcr-1-positive E. coli strains from retail chicken in South Korea, characterizing the DNA sequence of the mcr-1 plasmids, and determining the frequencies of conjugational transfer of mcr-1 plasmids to other Gram-negative pathogens.

2. Materials and Methods

2.1. Bacterial Strains and Culture Methods

Three mcr-1-positive E. coli strains (JSMCR1, FORC81 and FORC82) were isolated from retail raw chicken in South Korea in our previous study [20]. The mcr-1-positive E. coli strains, E. coli ATCC 43889 (Enterohemorrhagic E. coli; EHEC), E. coli NCCP 14039 (Enteroaggregative E. coli; EAEC), enterotoxigenic E. coli (ETEC, a laboratory collection), Salmonella enetrica serovar Typhimurium SL1344, and K. pneumoniae (a laboratory collection) were cultured on Luria–Bertani (LB) media at 37 °C. The pathogenic E. coli strains (EHEC, ETEC, EAEC), Salmonella Typhimurium, and Klebsiella were used as recipient strains in the conjugation assay.

2.2. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing was performed with a broth dilution method as described previously [21,22] with ten antibiotics, including ampicillin, cephalothin, tetracycline, chloramphenicol, ciprofloxacin, kanamycin, gentamicin, streptomycin, polymyxin B, and colistin. E. coli ATCC 25922 was used as the quality control strain.

2.3. Conjugation Assay

For the selection of transconjugants, we first obtained spontaneous mutants of streptomycin-resistant recipient strains by culturing them on LB media supplemented with streptomycin. Donor and recipient cells were prepared by transferring 1% inoculum from overnight cultures into fresh LB broth, followed by incubation at 37 °C for 4 h with constant shaking. E. coli was conjugated with recipient cells at a ratio of 1:1. Cells were pelleted by centrifugation, washed twice with 10 mM MgSO₄, and resuspended in 50 µL of MgSO₄. The mixture of donor and recipient cells were spread on LB agars supplemented with streptomycin (2 µg/mL) and colistin (4 µg/mL). Transconjugants were confirmed with PCR using mcr-1-specific primers [4] and the recipient strains. Conjugation frequencies were calculated as the number of transconjugants per recipient cell.

2.4. Whole-Genome Sequencing

Whole-genome sequencing and assembly were performed commercially at ChunLab Inc. (Seoul, South Korea). The whole genome of E. coli JSMCR1, FORC81 and FORC82 was sequenced using PacBio RS II (Pacific Biosciences, Menlo Park, CA, USA). The genome sequences were annotated using the online Rapid Annotation Subsequencing Technology (RAST) and CLC Main Workbench 3.6.1 (CLC bio, Aarhus, Denmark), and deposited in the GenBank database with accession numbers CP030152-CP030157 (JSMCR1), CP029057-CP029061 (FORC81), and CP026641-CP026644 (FORC82).

3. Results and Discussion

3.1. Whole-Genome Sequencing of mcr-1-Positive E. coli Strains

Three mcr-1-positive E. coli strains (JSMCR1, FORC81 and FORC82) were isolated from retail chicken in South Korea. The results of whole genome sequencing revealed that the three mcr-1-postive E. coli strains possessed multiple plasmids and some of the plasmids harbored a number of antibiotic resistance genes (

Table 1. Plasmids present in E. coli isolates harboring mcr-1.

E. coli Strain	Plasmid	Size (bp)	GenBank Accession No.	Inc Group	Resistance Genes
JSMCR1	pJSMCR1_1	152,677	CP030153	IncFIB, IncFII	aph(3′)-Ia, aac(3)-IId, blaCTX-M-65, fosA3
	pJSMCR1_2	134,064	CP030154	p0111	aadA1, blaOXA-10, qnrS1, floR, cmlA1, arr-2, tet(A), dfrA14
	pJSMCR1_3	109,689	CP030155	IncI1	-
	pJSMCR1_4	61,828	CP030156	IncI2	mcr-1
	pJSMCR1_5	29,589	CP030157	IncX4	-
FORC81	pFORC81_1	253,947	CP029058	IncI1, IncFII	aadA1, aadA2, aac(3)-IId, blaTEM-1B, qnrS1, floR, cmlA1, sul3, tet(A), dfrA12
	pFORC81_2	61,917	CP029059	IncI2	mcr-1
	pFORC81_3	38,749	CP029060	IncX1	-
	pFORC81_4	32,945	CP029061	IncI1	blaTEM-1B, floR
FORC82	pFORC82_1	250,778	CP026642	IncHI2A, IncHI2, IncN	aadA1, aph(3′’)-Ib, aph(6)-Id, blaCTX-M-65, blaOXA-10, qnrS1, mph(A), floR, cmlA1, arr-2, sul2, tet(M), tet(A), dfrA14
	pFORC82_2	101,404	CP026643	IncFIC, IncFIB	-
	pFORC82_3	65,206	CP026644	IncI2	mcr-1

and Figure S1). The three mcr-1-harboring plasmids belonged to the IncI2 type and possessed the genetic elements for bacterial conjugation (Table 1). The three mcr-1-harboring plasmids were similar to pHNSHP45 (accession no. KP347127), the first mcr-1-harboring plasmid isolated in China; pJSMCR1_4 (96% query coverage, 100% max nucleotide identity), pFORC81_2 (89% query coverage, 99% max nucleotide identity), and pFORC82_3 (97% query coverage, 99% max nucleotide identity). Unlike pHNSHP45, however, insertion sequences were not found in the plasmids (Figure 1). The sequences of the mcr-1-harboring plasmids were similar to the IncI2-type mcr-1-harboring plasmids, which were isolated from livestock and humans in Korea [13,14]. This is also consistent with a previous extensive analysis revealing that IncI2 is predominant in mcr-1-harboring plasmids in Asia, whereas IncHI2 plasmids are predominant in Europe [23].

3.2. Antimicrobial Susceptibility Profiles and Other Resistance Genes

All of the mcr-1-positive E. coli strains were highly resistant to most of the tested antibiotics belonging to different classes (

Table 2. Minimum inhibitory concentrations (MICs) of mcr-1-positive E. coli strains and their transconjugants.

Strain	Origin a	mcr-1 Gene b	MIC c (μg/mL)
			AMP	CEF	TET	CHL	CIP	KAN	GEN	STR	POL	COL
E. coli JSMCR1	WT	+	>64	>64	128	>64	>8	>32	>64	64	8	8
E. coli FORC81	WT	+	>64	64	>128	>64	>8	8	>64	8	8	8
E. coli FORC82	WT	+	>64	>64	128	64	2	4	2	2	8	8
EHEC (E. coli ATCC 43889)	WT	-	≤0.5	≤0.5	≤0.0628	≤0.5	≤0.0039	≤0.25	≤0.5	>128	≤0.25	≤0.25
pJSMCR1/EHEC	TC	+	≤0.5	≤0.5	≤0.0628	≤0.5	≤0.0039	≤0.25	≤0.5	>128	4	4
pFORC81/EHEC	TC	+	≤0.5	≤0.5	≤0.0628	≤0.5	≤0.0039	≤0.25	≤0.5	>128	4	4
pFORC82/EHEC	TC	+	≤0.5	≤0.5	≤0.0628	≤0.5	≤0.0039	≤0.25	≤0.5	>128	4	4
ETEC (isolate)	WT	-	>64	8	32	64	0.0312	>32	>64	>128	4	2
pJSMCR1/ETEC	TC	+	>64	8	32	64	0.0312	>32	>64	>128	8	8
pFORC81/ETEC	TC	+	>64	8	32	64	0.0312	>32	>64	>128	8	8
pFORC82/ETEC	TC	+	>64	8	32	64	0.0312	>32	>64	>128	8	8
EAEC (E. coli NCCP 14039)	WT	-	>64	16	>128	4	0.0312	8	2	>128	4	2
pJSMCR1/EAEC	TC	+	>64	16	>128	4	0.0312	8	2	>128	8	4
pFORC81/EAEC	TC	+	>64	16	>128	4	0.0312	8	2	>128	8	8
pFORC82/EAEC	TC	+	>64	16	>128	4	0.0312	8	2	>128	8	8
S. Typhimurium SL1344	WT	-	2	2	0.5	4	0.0156	4	2	>128	4	4
pJSMCR1/SL1344	TC	+	2	2	0.5	4	0.0156	4	2	>128	8	8
pFORC81/SL1344	TC	+	2	2	0.5	4	0.0156	4	2	>128	8	8
pFORC82/SL1344	TC	+	2	2	0.5	4	0.0156	4	2	>128	8	8
Klebsiella (isolate)	WT	-	>64	16	>128	>64	>16	>32	1	>128	4	4
pJSMCR1/Klebsiella	TC	+	>64	16	>128	>64	>16	>32	1	>128	8	16
pFORC81/Klebsiella	TC	+	>64	16	>128	>64	>16	>32	1	>128	8	16
pFORC82/Klebsiella	TC	+	>64	16	>128	>64	>16	>32	1	>128	32	16

^a WT: wild type, TC: Transconjugant. ^b Presence (+) or absence (-) of mcr-1, based on PCR and confirmed by sequencing. ^c AMP: ampicillin, CEF: cephalothin, TET: tetracycline, CHL: chloramphenicol, CIP: ciprofloxacin, KAN: Kanamycin; GEN: gentamicin; STR: streptomycin; POL: polymyxin B; COL: colistin.

). In particular, E. coli JSMCR1 was resistant to all the antibiotics tested in this study. Based on the results of whole genome sequencing, all isolates carried a few plasmids with different replicon types and multiple other antibiotic resistance genes conferring resistance to several different antibiotic classes (Table 1 and Figure S1). Whole-genome sequencing discovered point mutations in gyrA and parC, which confer resistance to fluoroquinolones; however, other antibiotic resistance genes were not found in the chromosome of the three strains, suggesting that pan drug resistance is primarily mediated by resistance plasmids in the strains.

3.3. Conjugation Assay

The conjugation experiments were performed with pathogenic E. coli strains (EHEC, ETEC, EAEC), Salmonella Typhimurium, and Klebsiella as the recipient strains. The results of the antimicrobial susceptibility test showed that only the minimum inhibitory concentrations (MICs) of polymyxin B and colistin were increased in transconjugants from two-fold to 16-fold compared to their parental strains (Table 2). However, other plasmids harboring antibiotic resistance genes, which were simultaneously present in the mcr-1-postive strains, were not transferred to the recipient strains under the experimental settings based on PCR testing with primers specific to the plasmids. These results indicated that mcr-1 plasmids are highly transmissible compared to other resistance plasmids; this presumably enables mcr-1 plasmids to be disseminated from E. coli to Gram-negative bacteria [4,24].

To confirm this, we determined the conjugational frequency of mcr-1 plasmids and found that the transmission rates of the mcr-1 plasmids were 10⁻³–10⁻⁶ in EHEC, 10⁻²–10⁻⁵ in ETEC, 10⁻⁴–10⁻⁵ in EAEC, 10⁻⁴–10⁻⁶ in Salmonella, and 10⁻⁵–10⁻⁶ in Klebsiella (Figure 2). Conjugation frequencies varied depending on the recipient strain. For instance, the conjugation frequencies of pFORC81-2 were as high as 3 × 10⁻³ in ETEC and as low as 2 × 10⁻⁶ in EHEC (Figure 2). The frequencies of conjugational transfer of the mcr-1 plasmids to E. coli strains were similar to previous studies [25]. Furthermore, in this study, we demonstrated that mcr-1 plasmids were transmitted to other Gram-negative bacteria, such as Salmonella and Klebsiella, at the frequencies comparable to those observed in E. coli (Figure 2).

4. Conclusions

In this study, we isolated and characterized three mcr-1 plasmids from PDR E. coli strains from retail raw chicken in South Korea. Although the number of isolated plasmids was small, the findings of this study are important because this is the first report about mcr-1 plasmids originating from retail food in the country. The whole genome sequencing of the PDR E. coli strains showed that all the genetic determinants for antibiotic resistance were associated with plasmids, except for fluoroquinolone resistance caused by point mutations in gyrA and parC. The mcr-1 plasmids were highly transferrable to pathogenic E. coli strains, Salmonella, and Klebsiella. This may allow colistin resistance to easily spread in the food supply system.