Turn off MathJax
Article Contents

Yu-Ping Cao, Qing-Qing Lin, Wan-Yun He, Jing Wang, Meng-Ying Yi, Lu-Chao Lv, Jun Yang, Jian-Hua Liu, Jian-Ying Guo. Co-selection may explain the unexpectedly high prevalence of plasmid-mediated colistin resistance gene mcr-1 in a Chinese broiler farm. Zoological Research. doi: 10.24272/j.issn.2095-8137.2020.131
Citation: Yu-Ping Cao, Qing-Qing Lin, Wan-Yun He, Jing Wang, Meng-Ying Yi, Lu-Chao Lv, Jun Yang, Jian-Hua Liu, Jian-Ying Guo. Co-selection may explain the unexpectedly high prevalence of plasmid-mediated colistin resistance gene mcr-1 in a Chinese broiler farm. Zoological Research. doi: 10.24272/j.issn.2095-8137.2020.131

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

doi: 10.24272/j.issn.2095-8137.2020.131
Funds:  This work was supported in part by the National Natural Science Foundation of China (31830099; 31902319), International Science and Technology Cooperation Project of Xinjiang Production and Construction Corps(XPCC) (2019BC004), Guangdong Special Support Program Innovation Team (2019BT02N054), and Innovation Team Project of Guangdong University (2019KCXTD001)
More Information
  • Corresponding author: E-mail: jhliu@scau.edu.cnjyguo@scau.edu.cn
  • #Authors contributed equally to this work
  • Received Date: 2020-05-30
  • Accepted Date: 2020-07-21
  • Available Online: 2020-07-31
  • #Authors contributed equally to this work
  • 加载中
  • [1] Barton BM, Harding GP, Zuccarelli AJ. 1995. A general method for detecting and sizing large plasmids. Analytical Biochemistry, 226(2): 235−240. doi:  10.1006/abio.1995.1220
    [2] Casal J, Mateu E, Mejia W, Martin M. 2007. Factors associated with routine mass antimicrobial usage in fattening pig units in a high pig-density area. Veterinary Research, 38(3): 481−492. doi:  10.1051/vetres:2007010
    [3] Elbediwi M, Li Y, Paudyal N, Pan H, Li XL, Xie SH, Rajkovic A, Feng YJ, Fang WH, Rankin SC, Yue M. 2019. Global burden of colistin-resistant bacteria: mobilized colistin resistance genes study (1980-2018). Microorganisms, 7(10): 461. doi:  10.3390/microorganisms7100461
    [4] Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. 2010. Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum β-lactamase producing, Enterobacteriaceae infections: a systematic review. The Lancet Infectious Diseases, 10(1): 43−50. doi:  10.1016/S1473-3099(09)70325-1
    [5] Feng CY, Wen PP, Xu H, Chi XH, Li S, Yu X, Lin XM, Wu SQ, Zheng BW. 2019. Emergence and comparative genomics analysis of extended-spectrum-β-lactamase-producing Escherichia coli carrying mcr-1 in Fennec Fox imported from Sudan to China. mSphere, 4(6): e00732-19. doi:  10.1128/mSphere.00732-19
    [6] Gautom RK. 1997. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram-negative organisms in 1 day. Journal of Clinical Microbiology, 35(11): 2977−2980. doi:  10.1128/JCM.35.11.2977-2980.1997
    [7] Huang X, Yu L, Chen X, Zhi C, Yao X, Liu Y, Wu S, Guo Z, Yi L, Zeng Z, Liu JH. 2017. High prevalence of colistin resistance and mcr-1 Gene in Escherichia coli isolated from food animals in China. Frontiers in Microbiology, 8: 562.
    [8] Lei T, Zhang JM, Jiang FF, He M, Zeng HY, Chen MT, Wu S, Wang J, Ding Y, Wu QP. 2019. First detection of the plasmid-mediated colistin resistance gene mcr-1 in virulent Vibrio parahaemolyticus. International Journal of Food Microbiology, 308: 108290. doi:  10.1016/j.ijfoodmicro.2019.108290
    [9] Lentz SA, De Lima-Morales D, Cuppertino VM, Nunes LDS, Da Motta AS, Zavascki AP, Barth AL, Martins AF. 2016. Letter to the editor: Escherichia coli harbouring mcr-1 gene isolated from poultry not exposed to polymyxins in Brazil. Euro Surveillance, 21(26): 30267. doi:  10.2807/1560-7917.ES.2016.21.26.30267
    [10] Li RC, Xie MM, Zhang JF, Yang ZQ, Liu LZ, Liu XB, Zheng ZW, Chan EWC, Chen S. 2017. Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant. Journal of Antimicrobial Chemotherapy, 72(2): 393−401. doi:  10.1093/jac/dkw411
    [11] Li ZK, Cao YP, Yi LX, Liu JH, Yang QW. 2019. Emergent polymyxin resistance: end of an Era?. Open Forum Infectious Diseases, 6(10): ofz368. doi:  10.1093/ofid/ofz368
    [12] Ling ZR, Yin WJ, Shen ZQ, Wang Y, Shen JZ, Walsh TR. 2020. Epidemiology of mobile colistin resistance genes mcr-1 to mcr-9. Journal of Antimicrobial Chemotherapy. doi:  10.1093/jac/dkaa205.
    [13] Liu LP, He DD, Lv LC, Liu WL, Chen XJ, Zeng ZL, Partridge SR, Liu JH. 2015. blaCTX-M-1/9/1 hybrid genes may have been generated from blaCTX-M-15 on an IncI2 plasmid. Antimicrobial Agents and Chemotherapy, 59(8): 4464−4470. doi:  10.1128/AAC.00501-15
    [14] Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian GB, Dong BL, Huang XH, Yu LF, Gu DX, Ren HW, Chen XJ, Lv LC, He DD, Zhou HW, Liang ZS, Liu JH, Shen JZ. 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. The Lancet Infectious Diseases, 16(2): 161−168. doi:  10.1016/S1473-3099(15)00424-7
    [15] Liu YY, Liu JH. 2018. Monitoring colistin resistance in food animals, an urgent threat. Expert Review of Anti-infective Therapy, 16(6): 443−446. doi:  10.1080/14787210.2018.1481749
    [16] Lv LC, Partridge SR, He LY, Zeng ZL, He DD, Ye JH, Liu JH. 2013. Genetic characterization of IncI2 plasmids carrying blaCTX-M-55 spreading in both pets and food animals in China. Antimicrobial Agents and Chemotherapy, 57(6): 2824−2827. doi:  10.1128/AAC.02155-12
    [17] Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection, 18(3): 268−281. doi:  10.1111/j.1469-0691.2011.03570.x
    [18] Migura-Garcia L, González-López JJ, Martinez-Urtaza J, Aguirre Sánchez JR, Moreno-Mingorance A, Perez De Rozas A, Höfle U, Ramiro Y, Gonzalez-Escalona N. 2020. mcr-colistin resistance genes mobilized by IncX4, IncHI2, and IncI2 plasmids in Escherichia coli of pigs and White Stork in Spain. Frontiers in Microbiology, 10: 3072. doi:  10.3389/fmicb.2019.03072
    [19] Moawad AA, Hotzel H, Neubauer H, Ehricht R, Monecke S, Tomaso H, Hafez HM, Roesler U, El-Adawy H. 2018. Antimicrobial resistance in Enterobacteriaceae from healthy broilers in Egypt: emergence of colistin-resistant and extended-spectrum β-lactamase-producing Escherichia coli. Gut Pathogens, 10(1): 39. doi:  10.1186/s13099-018-0266-5
    [20] Nang SC, Li J, Velkov T. 2019. The rise and spread of mcr plasmid-mediated polymyxin resistance. Critical Reviews in Microbiology, 45(2): 131−161. doi:  10.1080/1040841X.2018.1492902
    [21] Nation RL, Li J. 2009. Colistin in the 21st century. Current Opinion in Infectious Diseases, 22(6): 535−543. doi:  10.1097/QCO.0b013e328332e672
    [22] Perreten V, Strauss C, Collaud A, Gerber D. 2016. Colistin resistance gene mcr-1 in avian-pathogenic Escherichia coli in South Africa. Antimicrobial Agents and Chemotherapy, 60(7): 4414−4415. doi:  10.1128/AAC.00548-16
    [23] Perrin-Guyomard A, Bruneau M, Houée P, Deleurme K, Legrandois P, Poirier C, Soumet C, Sanders P. 2016. Prevalence of mcr-1 in commensal Escherichia coli from French livestock, 2007 to 2014. Euro Surveillance, 21(6). doi:  10.2807/1560-7917.ES.2016.21.6.30135
    [24] Quan JJ, Li X, Chen Y, Jiang Y, Zhou ZH, Zhang HC, Sun L, Ruan Z, Feng Y, Akova M, Yu YS. 2017. Prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae recovered from bloodstream infections in China: a multicentre longitudinal study. The Lancet Infectious Diseases, 17(4): 400−410. doi:  10.1016/S1473-3099(16)30528-X
    [25] Shen ZQ, Wang Y, Shen YB, Shen JZ, Wu CM. 2016. Early emergence of mcr-1 in Escherichia coli from food-producing animals. The Lancet Infectious Diseases, 16(3): 293. doi:  10.1016/S1473-3099(16)00061-X
    [26] Sun J, Zhang HM, Liu YH, Feng YJ. 2018. Towards understanding MCR-like colistin resistance. Trends in Microbiology, 26(9): 794−808. doi:  10.1016/j.tim.2018.02.006
    [27] Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology, 33(9): 2233−2239. doi:  10.1128/JCM.33.9.2233-2239.1995
    [28] Trung NV, Matamoros S, Carrique-Mas JJ, Nghia NH, Nhung NT, Chieu TTB, Mai HH, Van Rooijen W, Campbell J, Wagenaar JA, Hardon A, Mai NTN, Hieu TQ, Thwaites G, De Jong MD, Schultsz C, Hoa NT. 2017. Zoonotic transmission of mcr-1 colistin resistance gene from small-scale poultry farms, Vietnam. Emerging Infectious Diseases, 23(3): 529−532. doi:  10.3201/eid2303.161553
    [29] Wang CC, Feng Y, Liu LN, Wei L, Kang M, Zong ZY. 2020. Identification of novel mobile colistin resistance gene mcr-10. Emerging Microbes & Infections, 9(1): 508−516.
    [30] Wang J, Ma ZB, Zeng ZL, Yang XW, Huang Y, Liu JH. 2017a. The role of wildlife (wild birds) in the global transmission of antimicrobial resistance genes. Zoological Research, 38(2): 55−80. doi:  10.24272/j.issn.2095-8137.2017.003
    [31] Wang Y, Tian GB, Zhang R, Shen YB, Tyrrell JM, Huang X, Zhou HW, Lei L, Li HY, Doi Y, Fang Y, Ren HW, Zhong LL, Shen ZQ, Zeng KJ, Wang SL, Liu JH, Wu CM, Walsh TR, Shen JZ. 2017b. Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive Enterobacteriaceae in patients and healthy adults from China: an epidemiological and clinical study. The Lancet Infectious Diseases, 17(4): 390−399. doi:  10.1016/S1473-3099(16)30527-8
    [32] Wang Y, Zhang RM, Li JY, Wu ZW, Yin WJ, Schwarz S, Tyrrell JM, Zheng YJ, Wang SL, Shen ZQ, Liu ZH, Liu JY, Lei L, Li M, Zhang QD, Wu CM, Zhang QJ, Wu YN, Walsh TR, Shen JZ. 2017c. Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production. Nature Microbiology, 2: 16260. doi:  10.1038/nmicrobiol.2016.260
    [33] Wu RJ, Yi LX, Yu LF, Wang J, Liu YY, Chen XJ, Lv LC, Yang J, Liu JH. 2018. Fitness advantage of mcr-1-bearing IncI2 and IncX4 plasmids in Vitro. Frontiers in Microbiology, 9: 331. doi:  10.3389/fmicb.2018.00331
    [34] Yang YQ, Li YX, Song T, Yang YX, Jiang W, Zhang AY, Guo XY, Liu BH, Wang YX, Lei CW, Xiang R, Wang HN. 2017. Colistin resistance gene mcr-1 and its variant in Escherichia coli isolates from chickens in China. Antimicrob Agents Chemother, 61(5): e01204-16. doi:  10.1128/AAC.01204-16
    [35] Zhang JL, Chen L, Wang JW, Yassin AK, Butaye P, Kelly P, Gong JS, Guo WN, Li J, Li M, Yang F, Feng ZX, Jiang P, Song CL, Wang YY, You JF, Yang Y, Price S, Qi KZ, Kang Y, Wang CM. 2018. Molecular detection of colistin resistance genes (mcr-1, mcr-2 and mcr-3) in nasal/oropharyngeal and anal/cloacal swabs from pigs and poultry. Scientific Reports, 8(1): 3705. doi:  10.1038/s41598-018-22084-4
    [36] Zhi CP, Lv LC, Yu LF, Doi Y, Liu JH. 2016. Dissemination of the mcr-1 colistin resistance gene. The Lancet Infectious Diseases, 16(3): 292−293. doi:  10.1016/S1473-3099(16)00063-3
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(1)  / Tables(1)

Article Metrics

Article views(77) PDF downloads(7) Cited by()

Related
Proportional views

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

doi: 10.24272/j.issn.2095-8137.2020.131
Funds:  This work was supported in part by the National Natural Science Foundation of China (31830099; 31902319), International Science and Technology Cooperation Project of Xinjiang Production and Construction Corps(XPCC) (2019BC004), Guangdong Special Support Program Innovation Team (2019BT02N054), and Innovation Team Project of Guangdong University (2019KCXTD001)
#Authors contributed equally to this work
Yu-Ping Cao, Qing-Qing Lin, Wan-Yun He, Jing Wang, Meng-Ying Yi, Lu-Chao Lv, Jun Yang, Jian-Hua Liu, Jian-Ying Guo. Co-selection may explain the unexpectedly high prevalence of plasmid-mediated colistin resistance gene mcr-1 in a Chinese broiler farm. Zoological Research. doi: 10.24272/j.issn.2095-8137.2020.131
Citation: Yu-Ping Cao, Qing-Qing Lin, Wan-Yun He, Jing Wang, Meng-Ying Yi, Lu-Chao Lv, Jun Yang, Jian-Hua Liu, Jian-Ying Guo. Co-selection may explain the unexpectedly high prevalence of plasmid-mediated colistin resistance gene mcr-1 in a Chinese broiler farm. Zoological Research. doi: 10.24272/j.issn.2095-8137.2020.131
  • 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-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.

    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-1 gene 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 in E. 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-1 in 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-2 to 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 and mcr-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 with E. 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. coli strains 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).

    IsolateaResistance profilebOther resistance genecLocation of mcr-1, sizedGenetic context of mcr-1
    XCLC11AMP, CTX, STR, TET, FFC, CL, FOSblaCTX-M-14,blaCTX-M-64, fosA3IncHI2ISApl1-mcr-1-pap2
    XCLC12AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3IncHI2ISApl1-mcr-1-pap2-ISApl1
    XCLC16AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55IncHI2ISApl1-mcr-1-pap2
    XCLC26AMP, CTX, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3IncHI2ISApl1-mcr-1-pap2
    XCLC37AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3IncHI2ISApl1-mcr-1-pap2
    XCLC31AMP, STR, TET, FFC, CL, CIP-IncHI2ISApl1-mcr-1-pap2
    XCLC33AMP, CTX, GEN, TET, FFC, CL, FOS, CIPblaCTX-M-14IncHI2ISApl1-mcr-1-pap2
    XCLC4AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3IncHI2ISApl1-mcr-1-pap2-ISApl1
    XCLC46AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, blaCTX-M-65, fosA3, floRIncHI2, 244 kbISApl1-mcr-1-pap2
    XCLC52AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3IncHI2ISApl1-mcr-1-pap2-ISApl1
    XCLC54AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, blaCTX-M-65, fosA3,floRIncHI2, 244 kbISApl1-mcr-1-pap2
    XCLC58AMP, CTX, AMK, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3, rmtBIncHI2ISApl1-mcr-1-pap2-ISApl1
    XCLC69AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, blaCTX-M-82b, fosA3,floRIncHI2, 244 kbISApl1-mcr-1-pap2
    XCLC74AMP, CTX, STR, FFC, CL, FOS, CIPfosA3IncHI2ISApl1-mcr-1-pap2-ISApl1
    XCLC75AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3IncHI2ISApl1-mcr-1-pap2-ISApl1
    XCLC78AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-15, fosA3IncHI2ISApl1-mcr-1-pap2
    XCLC82AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIPfosA3IncHI2, 210 kbISApl1-mcr-1-pap2
    XCLC89AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3,floRIncHI2, 244 kbISApl1-mcr-1-pap2
    XCLC28AMP, CTX, AMK, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, blaCTX-M-55, fosA3, rmtBIncHI2, IncI2ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
    XCLC27AMP, CTX, STR, TET, FFC, CL, FOS, CIPfosA3IncHI2, IncI2ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
    XCLC40AMP, CTX, GEM, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3IncHI2, IncI2ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
    XCLC41AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3IncHI2, IncI2ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
    XCLC44AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3IncHI2, IncI2ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
    XCLC55AMP, CAZ, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, blaCTX-M-65, fosA3IncHI2, IncI2ISApl1-mcr-1-pap2-ISApl1(IncHI2),mcr-1-pap2(IncI2)
    XCLC6AMP, CTX, GEN, NEO, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3IncHI2, IncI2ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
    XCLC73AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3IncHI2, IncI2ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
    XCLC8AMP, CAZ, CTX, FOX, GEN, STR, TET, FFC, CL, FOS, CIPfosA3IncHI2, IncI2ISApl1-mcr-1-pap2(IncHI2), mcr-1-pap2(IncI2)
    XCLC35fAMP, CAZ, CTX, FOX, AMK, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, blaCTX-M-55, fosA3, rmtBIncI2, 65 kbmcr-1-pap2
    XCLC5AMP, CTX, AMK, GEN, STR, TET, FFC, CL, FOS, CIPrmtBIncI2, 63 kbmcr-1-pap2
    XCLC76AMP, CAZ, CTX, AMK, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3, rmtBIncI2, 65 kbmcr-1-pap2
    XCLC13AMP, GEN, STR, TET, FFC, CL, FOS, CIPfosA3IncI2, 63 kbISApl1-mcr-1-pap2
    XCLC15AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3IncI2mcr-1-pap2
    XCLC21AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55IncI2, 63 kbmcr-1-pap2
    XCLC2AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3IncI2,63kbmcr-1-pap2
    XCLC20AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-64IncI2, 65 kbmcr-1-pap2
    XCLC24AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-65, fosA3IncI2mcr-1-pap2
    XCLC34AMP, STR, TET, FFC, CL, FOS, CIPfosA3IncI2, 63 kbISApl1-mcr-1-pap2
    XCLC39AMP, CAZ, CTX, NEO, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3IncI2, 63 kbmcr-1-pap2
    XCLC42AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-65, fosA3IncI2, 63 kbmcr-1-pap2
    XCLC45AMP, CTX, TET, FFC, CL, FOS, CIPblaCTX-M-65, fosA3IncI2mcr-1-pap2
    XCLC48AMP, CAZ, CTX, FOX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-24, blaCTX-M-55, fosA3IncI2ISApl1-mcr-1-pap2
    XCLC50AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-24, fosA3IncI2, 63 kbmcr-1-pap2
    XCLC53AMP, STR, TET, FFC, CL, FOS, CIPfosA3IncI2ISApl1-mcr-1-pap2
    XCLC56AMP, CTX, STR, TET, FFC, CL, CIPblaCTX-M-15IncI2mcr-1-pap2
    XCLC60AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-65, fosA3IncI2mcr-1-pap2
    XCLC64AMP, CTX, GEN, NEO, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3IncI2mcr-1-pap2
    XCLC65AMP, CAZ, CTX, GEM, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14, fosA3IncI2mcr-1-pap2
    XCLC71AMP, CAZ, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-55, fosA3IncI2mcr-1-pap2
    XCLC80AMP, CTX, STR, TET, FFC, CL, CIPblaCTX-M-65IncI2, 63 kbmcr-1-pap2
    XCLC81AMP, CTX, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14IncI2mcr-1-pap2
    XCLC83AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIPIncI2mcr-1-pap2
    XCLC92AMP, CTX, STR, TET, FFC, CL, FOS, CIPIncI2mcr-1-pap2
    XCLC85AMP, CTX, GEN, STR, TET, FFC, CL, FOS, CIPblaCTX-M-14IncX4mcr-1-pap2
    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.
    d: Replicon type of plasmid carrying mcr-1 in transconjugant/transformant and approximate size of plasmid are underlined.
    f: Transformant was obtained from this isolate.

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

    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 of 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-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/fosA3 via 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 floR via 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-1 genes 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-1 without 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-1 was 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-pap2 structure 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. coli in 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.

  • The authors declare that they have no competing interests.

  • 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.

Reference (36)

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return