Co-selection may explain the unexpectedly high prevalence of plasmid-mediated colistin resistance gene mcr-1 in a Chinese broiler farm.

The rise of the plasmid-encoded colistin resistance gene mcr-1 is a major concern globally. Here, during a routine surveillance, an unexpectedly high prevalence of Escherichia coli with reduced susceptibility to colistin (69.9%) was observed in a Chinese broiler farm. Fifty-three (63.9%) E. coli isolates were positive for mcr-1. All identified mcr-1-positive E. coli (MCREC) were multidrug resistant and carried other clinically significant resistance genes. Furthermore, the mcr-1 genes were mainly located on the IncI2 and IncHI2 plasmids. Conjugation experiments unraveled the co-transfer of mcr-1 with other antibiotic resistance genes (blaCTX-M-55, blaCTX-M-14, floR, and fosA3) via the IncI2 (n=3) and IncHI2 (n=4) plasmids. The stable genetic context mcr-1-pap2 was common in the IncI2 plasmids, whereas ISApl1-mcr-1-pap2-ISApl1 was mainly found in the IncHI2 plasmids. The dominance of mcr-1-bearing IncI2 and IncHI2 plasmids and co-selection of mcr-1 with other antimicrobial resistance genes might contribute to the exceptionally high prevalence of mcr-1 in this broiler farm. Our results emphasized the importance of appropriate antibiotic use in animal production.

DEAR EDITOR,

The rise of the plasmid-encoded colistin resistance gene mcr-1 is a major concern globally. Here, during a routine surveillance, an unexpectedly high prevalence of Escherichia coli with reduced susceptibility to colistin (69.9%) was observed in a Chinese broiler farm. Fifty-three (63.9%)E. coli isolates were positive for mcr-1. All identified mcr-1-positive E. coli (MCREC) were multidrug resistant and carried other clinically significant resistance genes. Furthermore, the mcr-1 genes were mainly located on the IncI2 and IncHI2 plasmids. Conjugation experiments unraveled the co-transfer of mcr-1 with other antibiotic resistance genes (blaCTX-M-55, blaCTX-M-14, floR, and fosA3) via the IncI2 (n=3) and IncHI2 (n=4) plasmids. The stable genetic context mcr-1-pap2 was common in the IncI2 plasmids, whereas ISApl1-mcr-1-pap2-ISApl1 was mainly found in the IncHI2 plasmids. The dominance of mcr-1-bearing IncI2 and IncHI2 plasmids and co-selection of mcr-1with other antimicrobial resistance genes might contribute to the exceptionally high prevalence of mcr-1 in this broiler farm. Our results emphasized the importance of appropriate antibiotic use in animal production.

Multidrug resistant (MDR) bacteria have become a major public health concern. Colistin, the silver bullet against infections caused by MDR bacteria, was reintroduced into human clinics and hailed as an antibiotic of last resort (Nation & Li, 2009). In animal production, colistin was heavily used as a growth promoter (Casal et al., 2007), which inevitably led to colistin resistance. Since the first detection of the mobile colistin resistance gene mcr-1 in 2015, the prevalence of colistin resistance has become worrisome (Liu et al., 2016). The mcr-1gene encodes phosphoethanolamine transferase MCR-1 for the modification of lipid A, which reduces the negative charge of bacterial outer membranes and causes colistin resistance (Li et al., 2019). Primarily, mcr-1 is found inE. coli, as well as several other Enterobacteriaceae species and Vibrio parahaemolyticus (Lei et al., 2019; Nang et al., 2019). Various studies have reported on the existence of mcr-1in humans, animals, plants, and the environment (Liu & Liu, 2018; Nang et al., 2019; Wang et al., 2017a). In addition, an increasing number of mcr variants (e.g., mcr-2to mcr-10) have been identified in Enterobacteriaceae (Ling et al., 2020; Wang et al., 2020). The wide distribution of mcr-1 is usually mediated by mobile genetic elements, with the IncI2, IncX4, and IncHI2 plasmids considered as the main culprits (Liu & Liu, 2018; Sun et al., 2018). Generally, the occurrence of colistin resistance andmcr-1 among Enterobacteriaceae isolates from humans (0.1%–8.8%) is lower than that from livestock (0.9%–76.9%) (Liu & Liu, 2018; Liu et al., 2016; Quan et al., 2017; Wang et al., 2017b). For avian species, the detection rate of Enterobacteriaceae carrying mcr-1 is generally below 30% (Lentz et al., 2016; Moawad et al., 2018; Perrin-Guyomard et al., 2016; Shen et al., 2016; Trung et al., 2017). In China, the prevalence of mcr-1 and colistin resistance in E. coli from avians (~10%) is generally lower than that from swine (~30%) (Huang et al., 2017; Yang et al., 2017; Zhang et al., 2018). However, during routine surveillance of antimicrobial resistance in E. coli from food animals, an unexpectedly high prevalence (69.9%) of reduced susceptibility to colistin was found in E. coli from a Chinese broiler farm in 2013. Therefore, in the current study, we investigated the potential mechanism behind this phenomenon.

In July 2013, a total of 100 fresh fecal samples (~2 g per sample) were randomly collected from 100 broilers (27 days old) on a farm in eastern China. Bacterial recovery was conducted by incubating the samples in 3 mL of Luria Broth for 16–24 h. Then, 2 μL of bacterial solution was inoculated into MacConkey agar plates, from which non-duplicate colonies withE. coli morphology were selected and identified using MALDI-TOF MS (Shimadzu-Biotech Corp., Japan). Minimum inhibitory concentrations (MICs) of 14 antibiotics against E. coli isolates were evaluated using agar dilution. The results were interpreted according to the interpretative criteria recommended by CLSI (M100-S30) (ampicillin, cefotaxime, gentamicin, amikacin, fosfomycin, and ciprofloxacin) (Clinical and Laboratory Standards Institute, 2020) and epidemiological cut-off (ECOFF) values recommended by EUCAST (colistin, florfenicol, and neomycin) (http://www.eucast.org). Identification of MDR E. coli was confirmed after the bacteria showed resistance to at least three agents from different antimicrobial categories (Magiorakos et al., 2012). Polymerase chain reaction (PCR) amplification and Sanger sequencing were used to screen resistance genes, including mcr-1, blaCTX-M (β-lactamase genes),fosA3(fosfomycin resistance gene), and rmtB(aminoglycoside resistance gene), as well as plasmids (IncHI2, IncI2, IncI1, IncX4, and IncFII) in the E. coli strains with the primers listed in Table S1.

In total, 83 E. colistrains were recovered from the broiler farm. Overall, 58 (69.9%) strains showed reduced susceptibility (MIC ≥ 2 mg/L) to colistin, among which 53 (63.9%) were positive for mcr-1 (MCREC) (Table 1). The reason why the other five mcr-1-negative strains showed reduced susceptibility to colistin remains to be studied. Also, 55 (66.3%) strains showed resistance (MIC ≥ 4 mg/L) to colistin. The high prevalence of colistin resistance and circulation of mcr-1 among the E. coli collected from this broiler farm was unexpected, as the occurrence of MCREC in avian farms is usually low, e.g., 10% in China (Yang et al., 2017), 8% in Egypt (Moawad et al., 2018), 2% in South Africa (Perreten et al., 2016), and 2% in France (Perrin-Guyomard et al., 2016). The exceptionally high detection rate of MCREC (63.9%) in the current study is worrying as distribution of mcr-1 along the broiler industry chain is possible (Wang et al., 2017c).

Table 1

Antibiotic resistance profiles, resistance genes, and genetic backgrounds and locations of mcr-1 in 53 E. coli isolates

Isolatea	Resistance profileb	Other resistance genec	Location of mcr-1, sized	Genetic context of mcr-1
a: Isolates from which mcr-1 gene was transferred to recipient by conjugation or transformation are underlined. b: AMP: Ampicillin; CAZ: Ceftazidime; CTX: Cefotaxime; FOX: Cefoxitin; AMK: Amikacin; GEN: Gentamicin; NEO: Neomycin; STR: Streptomycin; TET: Tetracycline; FFC: Florfenicol; CL: Colistin; FOS: Fosfomycin; CIP: Ciprofloxacin. Resistance phenotypes transferred to recipient by conjugation are underlined. c: Genes co-transferred with mcr-1 by conjugation or transformation as determined by PCR are underlined. –: Not available d: Replicon type of plasmid carrying mcr-1 in transconjugant/transformant and approximate size of plasmid are underlined. e: Transformant was obtained from this isolate.
XCLC11	AMP, CTX, STR, TET, FFC, CL, FOS	blaCTX-M-14,blaCTX-M-64, fosA3	IncHI2	ISApl1-mcr-1-pap2
XCLC12	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, fosA3	IncHI2	ISApl1-mcr-1-pap2-ISApl1
XCLC16	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55	IncHI2	ISApl1-mcr-1-pap2
XCLC26	AMP, CTX, TET, FFC, CL, FOS, CIP	blaCTX-M-14, fosA3	IncHI2	ISApl1-mcr-1-pap2
XCLC37	AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, fosA3	IncHI2	ISApl1-mcr-1-pap2
XCLC31	AMP, STR, TET, FFC, CL, CIP	–	IncHI2	ISApl1-mcr-1-pap2
XCLC33	AMP, CTX, GEN, TET, FFC, CL, FOS, CIP	blaCTX-M-14	IncHI2	ISApl1-mcr-1-pap2
XCLC4	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, fosA3	IncHI2	ISApl1-mcr-1-pap2-ISApl1
XCLC46	AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	bla CTX-M-14, blaCTX-M-65, fosA3, floR	IncHI2, ~244 kb	ISApl1-mcr-1-pap2
XCLC52	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, fosA3	IncHI2	ISApl1-mcr-1-pap2-ISApl1
XCLC54	AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	bla CTX-M-14, blaCTX-M-65, fosA3,floR	IncHI2, ~244 kb	ISApl1-mcr-1-pap2
XCLC58	AMP, CTX, AMK, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, fosA3, rmtB	IncHI2	ISApl1-mcr-1-pap2-ISApl1
XCLC69	AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	bla CTX-M-14, blaCTX-M-82b, fosA3,floR	IncHI2, ~244 kb	ISApl1-mcr-1-pap2
XCLC74	AMP, CTX, STR, FFC, CL, FOS, CIP	fosA3	IncHI2	ISApl1-mcr-1-pap2-ISApl1
XCLC75	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, fosA3	IncHI2	ISApl1-mcr-1-pap2-ISApl1
XCLC78	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-15, fosA3	IncHI2	ISApl1-mcr-1-pap2
XCLC82	AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	fosA3	IncHI2, ~210 kb	ISApl1-mcr-1-pap2
XCLC89	AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	bla CTX-M-14, fosA3 ,floR	IncHI2, ~244 kb	ISApl1-mcr-1-pap2
XCLC28	AMP, CTX, AMK, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, blaCTX-M-55, fosA3, rmtB	IncHI2, IncI2	ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
XCLC27	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	fosA3	IncHI2, IncI2	ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
XCLC40	AMP, CTX, GEM, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, fosA3	IncHI2, IncI2	ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
XCLC41	AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, fosA3	IncHI2, IncI2	ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
XCLC44	AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, fosA3	IncHI2, IncI2	ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
XCLC55	AMP, CAZ, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, blaCTX-M-65, fosA3	IncHI2, IncI2	ISApl1-mcr-1-pap2-ISApl1(IncHI2),mcr-1-pap2(IncI2)
XCLC6	AMP, CTX, GEN, NEO, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, fosA3	IncHI2, IncI2	ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
XCLC73	AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, fosA3	IncHI2, IncI2	ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
XCLC8	AMP, CAZ, CTX, FOX, GEN, STR, TET, FFC, CL, FOS, CIP	fosA3	IncHI2, IncI2	ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
XCLC35e	AMP, CAZ, CTX, FOX, AMK, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, bla CTX-M-55, fosA3, rmtB	IncI2, ~65 kb	mcr-1-pap2
XCLC5	AMP, CTX, AMK, GEN, STR, TET, FFC, CL, FOS, CIP	rmtB	IncI2, ~63 kb	mcr-1-pap2
XCLC76	AMP, CAZ, CTX, AMK, GEN, STR, TET, FFC, CL, FOS, CIP	bla CTX-M-55, fosA3, rmtB	IncI2, ~65 kb	mcr-1-pap2
XCLC13	AMP, GEN, STR, TET, FFC, CL, FOS, CIP	fosA3	IncI2, ~63 kb	ISApl1-mcr-1-pap2
XCLC15	AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, fosA3	IncI2	mcr-1-pap2
XCLC21	AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55	IncI2, ~63 kb	mcr-1-pap2
XCLC2	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, fosA3	IncI2, ~63kb	mcr-1-pap2
XCLC20	AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	bla CTX-M-64	IncI2, ~65 kb	mcr-1-pap2
XCLC24	AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-65, fosA3	IncI2	mcr-1-pap2
XCLC34	AMP, STR, TET, FFC, CL, FOS, CIP	fosA3	IncI2, ~63 kb	ISApl1-mcr-1-pap2
XCLC39	AMP, CAZ, CTX, NEO, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, fosA3	IncI2, ~63 kb	mcr-1-pap2
XCLC42	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-65, fosA3	IncI2, ~63 kb	mcr-1-pap2
XCLC45	AMP, CTX, TET, FFC, CL, FOS, CIP	blaCTX-M-65, fosA3	IncI2	mcr-1-pap2
XCLC48	AMP, CAZ, CTX, FOX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-24, blaCTX-M-55, fosA3	IncI2	ISApl1-mcr-1-pap2
XCLC50	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-24, fosA3	IncI2, ~63 kb	mcr-1-pap2
XCLC53	AMP, STR, TET, FFC, CL, FOS, CIP	fosA3	IncI2	ISApl1-mcr-1-pap2
XCLC56	AMP, CTX, STR, TET, FFC, CL, CIP	blaCTX-M-15	IncI2	mcr-1-pap2
XCLC60	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-65, fosA3	IncI2	mcr-1-pap2
XCLC64	AMP, CTX, GEN, NEO, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, fosA3	IncI2	mcr-1-pap2
XCLC65	AMP, CAZ, CTX, GEM, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14, fosA3	IncI2	mcr-1-pap2
XCLC71	AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-55, fosA3	IncI2	mcr-1-pap2
XCLC80	AMP, CTX, STR, TET, FFC, CL, CIP	blaCTX-M-65	IncI2, ~63 kb	mcr-1-pap2
XCLC81	AMP, CTX, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14	IncI2	mcr-1-pap2
XCLC83	AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIP		IncI2	mcr-1-pap2
XCLC92	AMP, CTX, STR, TET, FFC, CL, FOS, CIP		IncI2	mcr-1-pap2
XCLC85	AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIP	blaCTX-M-14	IncX4	mcr-1-pap2

All 53 MCREC showed the MDR phenotype as well as very high resistance rates to tetracycline (100%), ampicillin (100%), florfenicol (98.1%), cefotaxime (92.5%), and fosfomycin (94.3%) (Supplementary Figure S1A). Of note, PCR revealed that the MCREC carried various resistance genes with clinical significance, including fosA3 (n=41, 80.7%), blaCTX-M (n=41, 80.7%), and rmtB (n=5, 4.2%) (Figure S1b and Table 1). The blaCTX-M variants included blaCTX-M-14 (n=19), blaCTX-M-55 (n=16), blaCTX-M-65 (n=8), and blaCTX-M-64 (n=2). High frequencies of the IncHI2 (47%) and IncI2 (48%) plasmids were also observed (Supplementary Figure S1B). The high occurrence of resistance and resistance genes to third generation cephalosporines, which are used in frontline therapy, and to fosfomycin, which is effective against infection by MDR Enterobacteriaceae (Falagas et al., 2010), among these MCREC is alarming. Though the usage of colistin in this broiler farm is not clear, the high prevalence of antimicrobial resistance among E. coli might result from the heavy usage of multiple antibiotics in broilers as ceftiofur, enrofloxacin, and florfenicol are routinely used in this farm (data not shown).

To elucidate the mechanism mediating the spread of mcr-1 in the studied farm, we first investigated vertical transfer of mcr-1 by evaluating the clonal relationships among MCREC with pulsed-field gel electrophoresis (PFGE) on a CHEF-MAPPER System (Bio-Rad, USA), as described previously (Gautom, 1997). Specifically, total DNA was digested by the XbaI enzyme (TaKaRa Bio Inc., Japan) and embedded in low-melting-point agarose (Bio-Rad, USA). The electrophoretic conditions were: initial switch time, 2.16 s; final switch time, 63.8 s; run time, 19 h; angle, 120°; gradient, 6.0 V/cm; temperature, 14 °C; ramping factor, linear. BioNumerics (Applied Maths, Belgium) was used to analyze the results, with the unweighted pair group method, arithmetic mean, and dice similarity index. The results were interpreted according to previous criteria (Tenover et al., 1995). PFGE was successfully performed on 45 MCREC isolates with the XbaI enzyme, with the remaining eight isolates not typable. Twenty-eight different XbaI PFGE patterns were identified (Figure 1), indicating that most MCREC were clonally unrelated.

Figure 1

PFGE pattern of mcr-1-positive E. coli

The horizontal mobility of mcr-1 was also investigated via conjugation using streptomycin-resistant E. coli C600 as the recipient (Wu et al., 2018). Twenty-seven isolates were randomly included in the conjugation. Using E. coli DH5α as the recipient, chemical transformation was performed on strains that failed in the conjugation assay. For the selection of transconjugants/transformants, colistin, cefotaxime, trimethoprim/sulfamethoxazole, and florfenicol were used. Subsequently, the transconjugants and transformants were subjected to PCR to confirm the existence of mcr-1 and co-transfer of other resistance genes (blaCTX-M-1G, blaCTX-M-9G, fosA3, and rmtB) with mcr-1. S1-nuclease PFGE was performed to confirm the single plasmids within the transconjugants/transformants, and to evaluate their sizes (Barton et al., 1995). The antibiotic resistance profiles of transconjugants and transformants were also determined. Plasmid replicon typing was performed with PCR and Sanger sequencing using the primers listed in Supplementary Table S1. In addition, the locations and genetic contexts of mcr-1 in all MCREC isolates were analyzed by PCR mapping with primers targeting the region of the plasmid backbone and mcr-1 (Supplementary Table S2).

Seventeen mcr-1-positive plasmids were successfully transferred from their hosts via conjugation (n=16) or transformation (n=1) (Table 1). S1-PFGE showed that only one plasmid carrying mcr-1 was transferred to the recipients and mcr-1 was located on the IncI2 plasmids with sizes varying from ~63 to ~65 kb (n=12) or IncHI2 plasmids with sizes ranging from ~210 to 244 kb (n=5) (Table 1). Of note, PCR revealed the co-transfer of mcr-1 with blaCTX-M-64/blaCTX-M-55 via IncI2 plasmids (n=3, 25%), and with blaCTX-M-14/floR/fosA3via IncHI2 plasmids (n=4, 80%) (Table 1). The co-transferred resistance genes were able to confer relevant antibiotic resistance to the recipients (E. coli C600 and DH5α). Feng et al. (2019) also reported the co-transfer of blaCTX-M-64 with mcr-1 via IncI2 plasmids in E. coli from an imported wild fox in China. In addition, fosA3 and floR are frequently co-transferred with mcr-1 via IncHI2 plasmids (Li et al., 2017; Zhi et al., 2016). These results are of concern because β-lactams (ceftiofur) and florfenicol routinely consumed in animals may select MCR-1-producing plasmids co-harboring blaCTX-M and/or floRvia co-selection, and further aggravate the distribution and persistence of mcr-1 in this broiler farm. Thus, we should not underestimate the risk that mcr-1 may spread via a similar mechanism.

The PCR mapping results revealed that nine isolates simultaneously carried mcr-1-positive IncI2 and IncHI2 plasmids (Table 1). All 62 (53+9) mcr-1genes were located in the IncI2, IncHI2, and IncX4 plasmids, with IncI2 dominating the host profile (Table 1), in agreement with other findings (Elbediwi et al., 2019; Migura-Garcia et al., 2020; Sun et al., 2018; Wu et al., 2018). IncI2 plasmids have also been reported as the vectors of blaCTX-M genes, e.g.,blaCTX-M-55 and blaCTX-M-64 (Liu et al., 2015; Lv et al., 2013). The dominance of IncI2 (55%) may result from the low fitness cost of mcr-1-positive IncI2 plasmids compared with IncHI2 and IncX4 plasmids (Wu et al., 2018). Of the 62 mcr-1 genes, three different genetic structures were detected, including mcr-1without ISApl1 (mcr-1-pap2) (n=31), mcr-1 with ISApl1 upstream (ISApl1-mcr-1-pap2) (n=24), and mcr-1 embedded in the complete transposon Tn6330(ISApl1-mcr-1-pap2-ISApl1) (n=7). In addition, the frequency of these genetic contexts in IncHI2 and IncI2 plasmids was varied. In IncI2 plasmids,mcr-1-pap2 was the most common (n=30), whereas the remaining four plasmids encoded ISApl1-mcr-1-pap2. In IncHI2 plasmids, all mcr-1 genes were flanked by ISApI1 upstream, and the complete transposon Tn6330 was present in seven isolates. Generally, mcr-1was translocated into plasmid backbones via transposon Tn6330(ISApl1-mcr-1-pap2-ISApl1). Following translocation, loss of ISApl1 would disrupt the structure of transposon and stabilize mcr-1 (Sun et al., 2018). Thus, the presence of the stable mcr-1-pap2structure in the IncI2 plasmids may also contribute to the circulation of mcr-1 in this broiler farm.

In conclusion, this study reported on an unusually high prevalence of mcr-1-positive E. coliin a Chinese broiler farm, which may result from the co-existence of mcr-1 with other resistance genes in the same plasmid or strain. Our findings emphasize the importance of appropriate antibiotic use in animal production as the misuse and abuse of antibiotics could facilitate the co-selection of mcr-1.

Supplementary data to this article can be found online.

Click here for additional data file.

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS’ CONTRIBUTIONS

J.G. and J.H.L. conceived the research. Q.L., W.H., M.Y., J.W., Y.C., and L.L. collected the data. J.H.L., Q.L., Y.C., J.W., J.G., and J.Y. analyzed and interpreted the data. Y.C. drafted the manuscript. J.H.L., J.W., and J.G. revised the report. All authors read and approved the final version of the manuscript.