Introduction
Colistin, a polymyxin antibiotic, is one of the last effective drugs for the treatment of multidrug-resistant (MDR) Gram-negative infections in human beings. Resistance to colistin used to be considered non-transmissible and most forms are intrinsic properties of bacteria. At the end of 2015, a novel plasmid-mediated colistin resistance gene, mcr-1, was identified as the single determinant to confer polymyxin resistance in Escherichia coli isolates from people and food animals (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016). More alarmingly, mcr-1 could be mobilized among Enterobacteriaceae at a rather high frequency by conjugation and stably persisted (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016). Subsequently, Gram-negative bacteria harboring the transmissible mcr-1-bearing plasmids were reported in five continents, which has caused a significant public health concern and drawn worldwide attention.
The mcr-1 gene exists in the bacterial isolates from both food animals (e.g. pig, poultry, and cattle) and human beings. Extensive usage of colistin in food animals has been proposed as a major driving force for the emergence and transmission of mcr-1 (Rhouma et al., Reference Rhouma, Beaudry and Letellier2016). Thus, recently, use of colistin in animal production has been proposed to be re-evaluated and regulated (Rhouma et al., Reference Rhouma, Beaudry and Letellier2016). In 2016, the Chinese government responded quickly and released an announcement to ban colistin usage as an in-feed growth promoter (Walsh and Wu, Reference Walsh and Wu2016). In the USA, although colistin has never been used in food animals, mcr-1-positive E. coli isolates were still identified in two swine intestinal samples (Meinersmann et al., Reference Meinersmann, Ladely, Plumblee, Cook and Thacker2017). Emergence of colistin resistance in US animal production is still a mystery, but clearly suggests that non-colistin usage risk factors exist and contribute to the persistence, transmission, and emergence of colistin resistance in an animal production system. Addressing this issue is imperative to prevent and control the transmissible colistin resistance in future animal production in the USA and other countries.
In this paper, we briefly reviewed historical status of colistin applications, colistin resistance development as well as recent emergence of mcr-1; additional in-depth information for these topics can be found in several of recent reviews (Li et al., Reference Li, Nation, Turnidge, Milne, Coulthard, Rayner and Paterson2006a; Nation and Li, Reference Nation and Li2009; Yahav et al., Reference Yahav, Farbman, Leibovici and Paul2012; Kempf et al., Reference Kempf, Jouy and Chauvin2016; Rhouma et al., Reference Rhouma, Beaudry and Letellier2016). We also comprehensively summarized recent extensive epidemiological studies focused on the prevalence of mcr gene in food animals, companion animals, and wildlife. In addition, on the basis of published science-based information, we identified and discussed several non-colistin usage risk factors that may contribute to the persistence, transmission, and emergence of colistin resistance in an animal production system.
History and applications of polymyxins
Colistin (also known as ‘polymyxin E’) and polymyxin B are bacteria-derived peptide antibiotics that have been used to treat infections with Gram-negative bacteria (Kwa et al., Reference Kwa, Kasiakou, Tam and Falagas2007). Colistin was discovered in 1949, and was produced by Bacillus polymyxa subspecies colistinus (Koyama et al., Reference Koyama, Kurosasa, Tsuchiya and Takakuta1950; Komura and Kurahashi, Reference Komura and Kurahashi1979). Polymyxins contain a strong positive charge and a hydrophobic acyl side chain, which display a similar mode of action as many antimicrobial peptides (AMPs) which kill bacteria by forming pores in the membrane (Wanty et al., Reference Wanty, Anandan, Piek, Walshe, Ganguly, Carlson, Stubbs, Kahler and Vrielink2013). Due to potential nephrotoxicity and neurotoxicity (Brown et al., Reference Brown, Dorman and Roy1970; Koch-Weser et al., Reference Koch-Weser, Sidel, Federman, Kanarek, Finer and Eaton1970; Nation and Li, Reference Nation and Li2009), polymyxins normally are used for topical human infections, but have not been used as a routine clinical human practice for decades. However, recent emergence of MDR Gram-negative bacteria have made polymyxins regain attention, by becoming the new last line of defense against fatal MDR bacterial infections, because MDR pathogens, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, are still susceptible to polymyxins (Bratu et al., Reference Bratu, Tolaney, Karumudi, Quale, Mooty, Nichani and Landman2005; Yahav et al., Reference Yahav, Farbman, Leibovici and Paul2012).
Two forms of colistin are commercially available for human clinical usage: (1) colistin methanesulfonate sodium (or colistimethate sodium) for parenteral use and aerosol therapy, and (2) colistin sulfate for oral and topical use (Li et al., Reference Li, Nation, Turnidge, Milne, Coulthard, Rayner and Paterson2006a). In aqueous solutions colistin methanesulfonate sodium is spontaneously hydrolyzed to a mixture of partially sulfomethylated derivatives and colistin (Barnett et al., Reference Barnett, Bushby and Wilkinson1964; Li et al., Reference Li, Milne, Nation, Turnidge and Coulthard2003). Colistin methanesulfonate sodium is less toxic than colistin sulfate when administered parenterally (Schwartz et al., Reference Schwartz, Warren, Barkley and Landis1959) and was considered an inactive prodrug of colistin (Bergen et al., Reference Bergen, Li, Rayner and Nation2006). In food animals, colistin sulfate has been frequently used as a veterinary medicine or as an in-feed antibiotic growth promoter, which is reviewed in the section below.
Use of colistin in animal production
Colistin has been extensively used in animal production for prophylactic and therapeutic purposes as well as for growth promotion in some countries, particularly in Asia and Europe (Rhouma et al., Reference Rhouma, Beaudry and Letellier2016). In the USA, colistin is in fact not used in food animals, although colistin has been approved for use by the FDA. Driven by China, the largest user of colistin in agriculture, global demand for colistin in agriculture was estimated to reach 11,942 tons per year by the end of 2015 (Liu, et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016). Colistin has been heavily used as an in-feed antibiotic growth promoter to improve feed efficiency and body weight gain in food animals in Asian countries, which include China, India, Japan, and Vietnam (Kempf et al., Reference Kempf, Jouy and Chauvin2016). In Brazil, colistin is also widely used in animal feed as a growth promoter in livestock, mainly in pigs and poultry (Fernandes et al., Reference Fernandes, Moura, Sartori, Silva, Cunha, Esposito, Lopes, Otutumi, Goncalves, Dropa, Matte, Monte, Landgraf, Francisco, Bueno, de Oliveira Garcia, Knobl, Moreno and Lincopan2016).
As a veterinary medicine, colistin has been used worldwide for decades, especially in swine and veal calves (Catry et al., Reference Catry, Cavaleri, Baptiste, Grave, Grein, Holm, Jukes, Liebana, Navas, Mackay, Magiorakos, Romo, Moulin, Madero, Pomba, Powell, Pyorala, Rantala, Ruzauskas, Sanders, Teale, Threlfall, Torneke, van Duijkeren and Edo2015; Kempf et al., Reference Kempf, Jouy and Chauvin2016; Rhouma et al., Reference Rhouma, Beaudry and Letellier2016). In Korea, colistin was used for prevention and treatment of animal disease and the annual usage of colistin in food animals ranged from 6 to16 tons from 2005 to 2015 (Lim et al., Reference Lim, Kang, Lee, Moon, Lee and Jung2016). In Europe, colistin was used to treat infections caused by Enterobacteriaceae in many animals, such as pigs, broilers, veal and beef cattle, sheep and goats (Catry et al., Reference Catry, Cavaleri, Baptiste, Grave, Grein, Holm, Jukes, Liebana, Navas, Mackay, Magiorakos, Romo, Moulin, Madero, Pomba, Powell, Pyorala, Rantala, Ruzauskas, Sanders, Teale, Threlfall, Torneke, van Duijkeren and Edo2015). In 2011, Germany, Portugal, Italy, and Estonia had higher colistin sales than many other European countries (Irrgang et al., Reference Irrgang, Roschanski, Tenhagen, Grobbel, Skladnikiewicz-Ziemer, Thomas, Roesler and Kaesbohrer2016).
Extensive usage of colistin in food animals has been recognized as a major risk factor for the emergence and transmission of the plasmid-borne colistin resistance gene mcr-1 (Rhouma et al., Reference Rhouma, Beaudry and Letellier2016). Therefore, the usage of colistin in animal production has been proposed to be re-evaluated and regulated (Rhouma et al., Reference Rhouma, Beaudry and Letellier2016). Notably, the Chinese government responded quickly and released an announcement regarding the cessation of colistin as a growth promoter at 7 months after the discovery of mcr-1 (Walsh and Wu, Reference Walsh and Wu2016). However, this ban only prohibits the use of colistin as a feed additive, and does not limit colistin use as a therapeutic agent for disease control in animals, which still raises a concern for the risk of colistin usage in animals (Walsh and Wu, Reference Walsh and Wu2016). Similarly, the Brazilian government also announced ban of colistin usage as a feed additive in food animals on 30 November 2016 (http://www.brasil.gov.br/economia-e-emprego/2016/11/mapa-proibe-uso-de-substancia-antimicrobiana-em-racoes).
Colistin resistance and recent emergence of mcr-1
In some countries, such as China, colistin has been extensively used in animal production, which is consistent with the increasing colistin resistance observed in recent years (Shen et al., Reference Shen, Wang, Shen, Shen and Wu2016; Huang et al., Reference Huang, Yu, Chen, Zhi, Yao, Liu, Wu, Guo, Yi, Zeng and Liu2017). Similarly, a high rate of colistin resistance in Acinetobacter baumannii was reported in Korea recently (Ko et al., Reference Ko, Suh, Kwon, Jung, Park, Kang, Chung, Peck and Song2007). Colistin hetero-resistance also has been widely reported in MDR A. baumannii (Li et al., Reference Li, Rayner, Nation, Owen, Spelman, Tan and Liolios2006b; Hawley et al., Reference Hawley, Murray and Jorgensen2008; Yau et al., Reference Yau, Owen, Poudyal, Bell, Turnidge, Yu, Nation and Li2009), K. pneumoniae (Poudyal et al., Reference Poudyal, Howden, Bell, Gao, Owen, Turnidge, Nation and Li2008; Meletis et al., Reference Meletis, Tzampaz, Sianou, Tzavaras and Sofianou2011) and P. aeruginosa (Bergen et al., Reference Bergen, Forrest, Bulitta, Tsuji, Sidjabat, Paterson, Li and Nation2011). Not surprisingly, given that colistin is an AMP, mechanisms of colistin resistance are similar to those of other AMPs (Ernst et al., Reference Ernst, Guina and Miller2001; Yeaman and Yount, Reference Yeaman and Yount2003; Kraus and Peschel, Reference Kraus and Peschel2006; Peschel and Sahl, Reference Peschel and Sahl2006), which are primarily mediated through non-transmissible modification of cell surface with intrinsic genetic determinants located in the chromosome or in chromosomal mutations (Baron et al., Reference Baron, Hadjadj, Rolain and Olaitan2016; Schwarz and Johnson, Reference Schwarz and Johnson2016).
Mobile polymyxin resistance was not reported until the plasmid-mediated colistin-resistant gene, mcr-1, was recently reported in China (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016). The mcr-1 was identified as the single determinant to confer polymyxin resistance in commensal E. coli from food animals (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016). More alarmingly, the mcr-1 could be mobilized among Enterobacteriaceae at a rather high frequency by conjugation and then stably persisted in transformed bacteria (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016). The MCR-1 has phosphoethanolamine transferase activity, which is involved in lipid A modification, consequently providing protection of E. coli against colistin in an in vivo mouse infection model (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016).
In the past 2 years, mcr-1 has been found in E. coli (Arcilla et al., Reference Arcilla, van Hattem, Matamoros, Melles, Penders, de Jong, Schultsz and Consortium2016; Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016; Olaitan et al., Reference Olaitan, Chabou, Okdah, Morand and Rolain2016), Klebsiella (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016; Stoesser et al., Reference Stoesser, Mathers, Moore, Day and Crook2016), Salmonella (Campos et al., Reference Campos, Cristino, Peixe and Antunes2016; Figueiredo et al., Reference Figueiredo, Card, Nunez, Pomba, Mendonca, Anjum and Da Silva2016; Webb et al., Reference Webb, Granier, Marault, Millemann, den Bakker, Nightingale, Bugarel, Ison, Scott and Loneragan2016), Enterobacter aerogenes (Zeng et al., Reference Zeng, Doi, Patil, Huang and Tian2016; Wang et al., Reference Wang, Tian, Zhang, Shen, Tyrrell, Huang, Zhou, Lei, Li, Doi, Fang, Ren, Zhong, Shen, Zeng, Wang, Liu, Wu, Walsh and Shen2017b), Enterobacter cloacae (Zeng et al., Reference Zeng, Doi, Patil, Huang and Tian2016; Wang et al., Reference Wang, Tian, Zhang, Shen, Tyrrell, Huang, Zhou, Lei, Li, Doi, Fang, Ren, Zhong, Shen, Zeng, Wang, Liu, Wu, Walsh and Shen2017b), Cronobacter sakazakii (Liu et al., Reference Liu, Song, Zou, Hao and Shan2017a), Shigella sonnei (Pham Thanh et al., Reference Pham Thanh, Thanh Tuyen, Nguyen Thi Nguyen, Chung The, Wick, Thwaites, Baker and Holt2016), Kluyvera spp. (Zhao and Zong, Reference Zhao and Zong2016), and Citrobacter spp.(Li et al., Reference Li, Fang, Jiang, Pan, Xia, Liao, Liu and Sun2017; Sennati et al., Reference Sennati, Di Pilato, Riccobono, Di Maggio, Villagran, Pallecchi, Bartoloni, Rossolini and Giani2017). The mcr-1 gene has been reported to exist in different food animals, such as in pigs (Meinersmann et al., Reference Meinersmann, Ladely, Plumblee, Cook and Thacker2017; Roschanski et al., Reference Roschanski, Falgenhauer, Grobbel, Guenther, Kreienbrock, Imirzalioglu and Roesler2017), poultry (Lima Barbieri et al., Reference Lima Barbieri, Nielsen, Wannemuehler, Cavender, Hussein, Yan, Nolan and Logue2017; Monte et al., Reference Monte, Mem, Fernandes, Cerdeira, Esposito, Galvão, Franco, Lincopan and Landgraf2017) and cattle (Huang et al., Reference Huang, Yu, Chen, Zhi, Yao, Liu, Wu, Guo, Yi, Zeng and Liu2017). This gene also has been detected in bacteria from different environment niches and other commodities, such as coastal water (Fernandes et al., Reference Fernandes, Sellera, Esposito, Sabino, Cerdeira and Lincopan2017), rivers and lakes (Zurfuh et al., Reference Zurfuh, Poirel, Nordmann, Nuesch-Inderbinen, Hachler and Stephan2016), hospital sewage (Zhao et al., Reference Zhao, Feng, Lu, McNally and Zong2017), wells (Sun et al., Reference Sun, Bi, Nilsson, Zheng, Berglund, Stalsby Lundborg, Borjesson, Li, Chen, Yin and Nilsson2017c), meats and vegetables (Hasman et al., Reference Hasman, Hammerum, Hansen, Hendriksen, Olesen, Agerso, Zankari, Leekitcharoenphon, Stegger, Kaas, Cavaco, Hansen, Aarestrup and Skov2015; Zurfuh et al., Reference Zurfuh, Poirel, Nordmann, Nuesch-Inderbinen, Hachler and Stephan2016).
In people, mcr-1 was detected in approximately 1% of E. coli and K. pneumoniae isolates (of more than 20,000 isolates) in two recent reports from China (Quan et al., Reference Quan, Li, Chen, Jiang, Zhou, Zhang, Sun, Ruan, Feng, Akova and Yu2017; Wang et al., Reference Wang, Tian, Zhang, Shen, Tyrrell, Huang, Zhou, Lei, Li, Doi, Fang, Ren, Zhong, Shen, Zeng, Wang, Liu, Wu, Walsh and Shen2017b). The mcr-1 gene was found in pathogens from human bloodstream infections (Hasman et al., Reference Hasman, Hammerum, Hansen, Hendriksen, Olesen, Agerso, Zankari, Leekitcharoenphon, Stegger, Kaas, Cavaco, Hansen, Aarestrup and Skov2015; Wang et al., Reference Wang, Sun, Ding, Li, Liu and Feng2017a), in patients after colistin treatment (Beyrouthy et al., Reference Beyrouthy, Robin, Lessene, Lacombat, Dortet, Naas, Ponties and Bonnet2017), in travelers (Arcilla et al., Reference Arcilla, van Hattem, Matamoros, Melles, Penders, de Jong, Schultsz and Consortium2016) and in pilgrims (Leangapichart et al., Reference Leangapichart, Gautret, Brouqui, Mimish, Raoult and Rolain2016). The Centers for Disease Control and Prevention (CDC), the US Food and Drug Administration (FDA), and the US Department of Agriculture (USDA) started tracking mcr-1 prevalence in the USA immediately after the report from China (https://www.cdc.gov/drugresistance/tracking-mcr1.html). Less than 6 months after the first identification of mcr-1 in China, a human bacterial isolate carrying mcr-1 was isolated from a Pennsylvania patient by the Department of Defense, while mcr-1 was identified in two swine intestinal samples that were collected as part of the National Antimicrobial Resistance Monitoring System, a shared project of the USDA, FDA, and CDC (Meinersmann et al., Reference Meinersmann, Ladely, Plumblee, Cook and Thacker2017). To date, there have been 14 human isolates and two animal isolates reported to carry MCR-1 for colistin resistance in the USA (https://www.cdc.gov/drugresistance/tracking-mcr1.html). In 2017, plasmid-mediated colistin-resistant K. pneumoniae were associated with an outbreak in China, leading to six infections and two deaths (Tian et al., Reference Tian, Doi, Shen, Walsh, Wang, Zhang and Huang2017). Thus, the recent emergence of the plasmid-mediated colistin-resistant Enterobacteriaceae in both food animals and in human beings has drawn worldwide attention and fears.
Prevalence of mcr-1 in animals
Recent epidemiological studies of colistin resistance have shown the mcr-1 positive Enterobacteriaceae isolates are highly prevalent in various animal hosts worldwide (Summarized in Table 1). Unregulated usage of colistin in food animals is believed to be the important driving force for the emergence and transmission of the mcr-1 (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016; Rhouma et al., Reference Rhouma, Beaudry and Letellier2016). The following paragraphs summarize findings from recent extensive epidemiological studies for the prevalence of mcr-1 in livestock, companion animals, and wildlife.
Characteristics of mcr-positive isolates from food animals, people and other sources
Livestock
In China, Liu et al. (Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016) first reported 21 and 15% positive rates of mcr-1 in E. coli of pig and retail meat origin, respectively. In a retrospective study on 1611 E. coli isolates of chicken origin, collected from the early 1970s to 2014 in China, only three mcr-1-positive E. coli isolates were identified in the 1980s; for almost two decades afterward, mcr-1 was not detected until in 2004 and 2006 when sporadic occurrences of mcr-1 were observed. The ratio of mcr-1-positive E. coli increased from 5.2% (6/115) in 2009 to 30.0% (15/50) in 2014 (Shen et al., Reference Shen, Wang, Shen, Shen and Wu2016). A similar trend was also observed in another study in China, in which 4438 E. coli isolates of food animal origin were tested for colistin resistance and the presence of mcr-1 (Huang et al., Reference Huang, Yu, Chen, Zhi, Yao, Liu, Wu, Guo, Yi, Zeng and Liu2017). Approximately 16.8% of E. coli isolates from pigs and chickens recovered during 2013–2014 displayed colistin resistance, which is significantly higher than those isolated during 2007–2008 (5.5%) and 2010–2011 (12.4%). The increasing prevalence of mcr-1-harboring E. coli in the past decade (Shen et al., Reference Shen, Wang, Shen, Shen and Wu2016; Huang et al., Reference Huang, Yu, Chen, Zhi, Yao, Liu, Wu, Guo, Yi, Zeng and Liu2017) is coincident with the fact that China started to introduce colistin in the early 2000s and became one of the world's largest users of colistin in agriculture by the end of 2015 (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016). Among the Shiga toxin-producing E. coli isolated from pigs in China, 10.8% (10/93) were mcr-1 positive (Bai et al., Reference Bai, Hurley, Li, Meng, Wang, Fanning and Xiong2016). In Chinese poultry production, Wang et al. (Reference Wang, Zhang, Li, Wu, Yin, Schwarz, Tyrrell, Zheng, Wang, Shen, Liu, Liu, Lei, Li, Zhang, Wu, Zhang, Wu, Walsh and Shen2017c) found that 37 (23.0%) carbapenem-resistant isolates were positive for mcr-1. Yang et al. (Reference Yang, Li, Song, Yang, Jiang, Zhang, Guo, Liu, Wang, Lei, Xiang and Wang2017) reported a 5.11% mcr-1-positive rate in E. coli of chicken origin in 2010–2015. Compared with the extensive surveys focused on E. coli, to date, limited information is available for mcr-1-carrying Salmonella in China. The ST34 Salmonella isolate was reportedly involved in the spread of the mcr-1 in Salmonella enterica from food animals in China (Li et al., Reference Li, Tan, Lin and Feng2016b; Yi et al., Reference Yi, Wang, Gao, Liu, Doi, Wu, Zeng, Liang and Liu2017).
In Japan, a high proportion of colistin-resistant (45%) pathogenic and mcr-1-positive (13%) E. coli were reported in swine production (Kusumoto et al., Reference Kusumoto, Ogura, Gotoh, Iwata, Hayashi and Akiba2016). However, in Japan, the rates of the colistin-resistant and mcr-1-positive E. coli strains isolated from healthy animals were low (1.00 and 0.02%, respectively) (Suzuki et al., Reference Suzuki, Ohnishi, Kawanishi, Akiba and Kuroda2016). Kawanishi et al. (Reference Kawanishi, Abo, Ozawa, Uchiyama, Shirakawa, Suzuki, Shima, Yamashita, Sekizuka, Kato, Kuroda, Koike and Kijima2017) reported that the prevalence of mcr-1 in E. coli from healthy animals actually increased slightly over the years. In Vietnam, a high prevalence (59.4%) of mcr-1 in fecal samples from chickens was observed on farms routinely using colistin, suggesting that emergence and transmission of mcr-1-carrying bacteria were associated with colistin usage in chickens (Trung et al., Reference Trung, Matamoros, Carrique-Mas, Nghia, Nhung, Chieu, Mai, van Rooijen, Campbell, Wagenaar, Hardon, Mai, Hieu, Thwaites, de Jong, Schultsz and Hoa2017). In Korea, although the mcr-1-positive rate in E. coli was low (0.1%), the prevalence of mcr-1 has risen since 2013 (Lim et al., Reference Lim, Kang, Lee, Moon, Lee and Jung2016). The mcr-1-carrying pig isolates were also reported in Laos (Olaitan et al., Reference Olaitan, Chabou, Okdah, Morand and Rolain2016).
In Europe, mcr-1 was reported in Germany (Brennan et al., Reference Brennan, Martins, McCusker, Wang, Alves, Hurley, El Garch, Woehrle, Miossec, McGrath, Srikumar, Wall and Fanning2016; Irrgang et al., Reference Irrgang, Roschanski, Tenhagen, Grobbel, Skladnikiewicz-Ziemer, Thomas, Roesler and Kaesbohrer2016; Guenther et al., Reference Guenther, Falgenhauer, Semmler, Imirzalioglu, Chakraborty, Roesler and Roschanski2017), Belgium (Xavier et al., Reference Xavier, Lammens, Butaye, Goossens and Malhotra-Kumar2016a, Reference Xavier, Lammens, Ruhal, Kumar-Singh, Butaye, Goossens and Malhotra-Kumarb), Spain (Quesada et al., Reference Quesada, Ugarte-Ruiz, Rocio Iglesias, Concepcion Porrero, Martinez, Florez-Cuadrado, Campos, Garcia, Piriz, Luis Saez and Dominguez2016), Italy (Carnevali et al., Reference Carnevali, Morganti, Scaltriti, Bolzoni, Pongolini and Casadei2016), Portugal (Campos et al., Reference Campos, Cristino, Peixe and Antunes2016; Figueiredo et al., Reference Figueiredo, Card, Nunez, Pomba, Mendonca, Anjum and Da Silva2016; Hu et al., Reference Hu, Liu, Lin, Gao and Zhu2016; Tse and Yuen, Reference Tse and Yuen2016), France (Brennan et al., Reference Brennan, Martins, McCusker, Wang, Alves, Hurley, El Garch, Woehrle, Miossec, McGrath, Srikumar, Wall and Fanning2016; El Garch et al., Reference El Garch, Sauget, Hocquet, LeChaudee, Woehrle and Bertrand2017), The Netherlands (Kluytmans-van den Bergh et al., Reference Kluytmans-van den Bergh, Huizinga, Bonten, Bos, De Bruyne, Friedrich, Rossen, Savelkoul and Kluytmans2016; Veldman et al., Reference Veldman, van Essen-Zandbergen, Rapallini, Wit, Heymans, van Pelt and Mevius2016), Estonia (Brauer et al., Reference Brauer, Telling, Laht, Kalmus, Lutsar, Remm, Kisand and Tenson2016), Great Britain (Anjum et al., Reference Anjum, Duggett, AbuOun, Randall, Nunez-Garcia, Ellis, Rogers, Horton, Brena, Williamson, Martelli, Davies and Teale2016), Lithunia, Wallonia and Flanders (Malhotra-Kumar et al., Reference Malhotra-Kumar, Xavier, Das, Lammens, Butaye and Goossens2016). Based on a retrospective survey in Germany, mcr-1 was found in 402 of 10,600 (3.79%) E. coli isolates from livestock and food (Irrgang et al., Reference Irrgang, Roschanski, Tenhagen, Grobbel, Skladnikiewicz-Ziemer, Thomas, Roesler and Kaesbohrer2016). The mcr-1-positive rate ranged between 5.3 and 7.8% in broilers while the highest prevalence of mcr-1 was found in turkeys (>10%) from 2010 to 2014 (Irrgang et al., Reference Irrgang, Roschanski, Tenhagen, Grobbel, Skladnikiewicz-Ziemer, Thomas, Roesler and Kaesbohrer2016). In pigs, mcr-1 was detected with a prevalence of 1.5% in Germany (Irrgang et al., Reference Irrgang, Roschanski, Tenhagen, Grobbel, Skladnikiewicz-Ziemer, Thomas, Roesler and Kaesbohrer2016). In Belgium, a new colistin-resistant gene, mcr-2, was discovered from E. coli in pigs, and the prevalence of mcr-2 in colistin-resistant E. coli (11/53) was higher than that of mcr-1 (7/53) (Xavier et al., Reference Xavier, Lammens, Ruhal, Kumar-Singh, Butaye, Goossens and Malhotra-Kumar2016b).
In the USA, low prevalence of mcr-1 was reported in food animals at slaughter (0.1%) and in swine at slaughter (0.35%) (Meinersmann et al., Reference Meinersmann, Ladely, Plumblee, Cook and Thacker2017). It is interesting that two mcr-1-mediated colistin-resistant E. coli isolates were identified in swine in the USA (Meinersmann et al., Reference Meinersmann, Ladely, Plumblee, Cook and Thacker2017), because colistin has not been used in animals in the USA. Emergence of colistin resistance in US animal production is still a mystery.
In South America, mcr-1-harboring E. coli isolates have been present at least since 2012. In Brazil, mcr-1 was detected in 5% (14/280) and 1.8% (2/113) of E. coli of chicken and pig origin, respectively (Fernandes et al., Reference Fernandes, Moura, Sartori, Silva, Cunha, Esposito, Lopes, Otutumi, Goncalves, Dropa, Matte, Monte, Landgraf, Francisco, Bueno, de Oliveira Garcia, Knobl, Moreno and Lincopan2016); interestingly, some colistin-susceptible E. coli strains were observed to carry the mcr-1 (Fernandes et al., Reference Fernandes, Moura, Sartori, Silva, Cunha, Esposito, Lopes, Otutumi, Goncalves, Dropa, Matte, Monte, Landgraf, Francisco, Bueno, de Oliveira Garcia, Knobl, Moreno and Lincopan2016). In Venezuela, two swine E. coli isolates were mcr-1 positive (Delgado-Blas et al., Reference Delgado-Blas, Ovejero, Abadia-Patino and Gonzalez-Zorn2016).
In South Africa, colistin-resistant Avian-Pathogenic E. coli has significantly increased from an average of 4.5% (from 2008 to 2014) to 13.6% in 2015 (Perreten et al., Reference Perreten, Strauss, Collaud and Gerber2016). Grami et al. (Reference Grami, Mansour, Mehri, Bouallegue, Boujaafar, Madec and Haenni2016) reported a high prevalence (17–83%) of MCR-1 and CTX-M-1-producing E. coli in chicken farms in Tunisia, emphasizing the significant impact of food animal trade on the spread of mcr-1. In Egypt, only one E. coli from a cow was identified to carry mcr-1 (Khalifa et al., Reference Khalifa, Ahmed, Oreiby, Eid, Shimamoto and Shimamoto2016). In another study, three avian-pathogenic E. coli isolates from chickens in Egypt were also reported to be mcr-1 positive (Lima Barbieri et al., Reference Lima Barbieri, Nielsen, Wannemuehler, Cavender, Hussein, Yan, Nolan and Logue2017).
Companion animals
The mcr-1-positive Enterobacteriaceae also have been identified from companion animals. In China, an E. coli strain from a cat was found to carry a mobile IncX3-X4 hybrid plasmid bearing both mcr-1 and bla NDM-5 genes (Sun et al., Reference Sun, Yang, Zhang, Feng, Fang, Xia, Li, Lv, Duan, Liao and Liu2016b). Transmission of E. coli strains harboring mcr-1 between companion animals and human beings was also observed in a recent report in China (Lei et al., Reference Lei, Wang, Schwarz, Walsh, Ou, Wu, Li and Shen2017). In addition, a man who worked in a pet store was reported to carry mcr-1 gene-harboring E. coli (Zhang et al., Reference Zhang, Doi, Huang, Li, Zhong, Zeng, Zhang, Patil and Tian2016). Using real-time polymerase chain reaction (PCR), 7.4% (8/108) of fecal samples from companion animals were mcr-1 positive (Chen et al., Reference Chen, Zhao, Che, Xiong, Xu, Zhang, Lan, Xia, Walsh, Xu, Lu and Li2017).
Wildlife
Recently, mcr-1 was discovered in E. coli isolated from migratory birds in Asia (Mohsin et al., Reference Mohsin, Raza, Roschanski, Schaufler and Guenther2016), Europe (Ruzauskas and Vaskeviciute, Reference Ruzauskas and Vaskeviciute2016) and South America (Liakopoulos et al., Reference Liakopoulos, Mevius, Olsen and Bonnedahl2016). In addition, Magellanic penguins were reported to carry mcr-1-positive E. coli in Brazil (Sellera et al., Reference Sellera, Fernandes, Sartori, Carvalho, Esposito, Nascimento, Dutra, Mamizuka, Perez-Chaparro, McCulloch and Lincopan2017). The lifestyle of wildlife allows them to disseminate pathogenic and resistant microorganisms despite country borders, which may serve as an important risk factor for the spread of mcr-1 (Ruzauskas and Vaskeviciute, Reference Ruzauskas and Vaskeviciute2016).
Genomic features of mcr-1 and mcr-1-bearing plasmids
To date, sequences of the globally prevalent mcr-1 gene from diverse bacterial strains are almost identical. At least six additional variants of the MCR-1 were identified; these variants only differ from MCR-1 by a single amino acid (aa), which include MCR-1.2 (Gln3→Leu) (Di Pilato et al., Reference Di Pilato, Arena, Tascini, Cannatelli, Henrici De Angelis, Fortunato, Giani, Menichetti and Rossolini2016), MCR-1.3 (Ile37→Leu) (Yang et al., Reference Yang, Li, Song, Yang, Jiang, Zhang, Guo, Liu, Wang, Lei, Xiang and Wang2017), MCR-1.4 (Asp439→Asn) (Yin et al., Reference Yin, Li, Shen, Liu, Wang, Shen, Zhang, Walsh, Shen and Wang2017), MCR-1.5 (His451→Tyr) (Yin et al., Reference Yin, Li, Shen, Liu, Wang, Shen, Zhang, Walsh, Shen and Wang2017), MCR-1.6 (Arg535→His) (Lu et al., Reference Lu, Hu, Luo, Zhou, Wang, Du, Li, Xu, Zhu, Xu and Kan2017) and MCR-1.7 (Ala214→Thr) (Yin et al., Reference Yin, Li, Shen, Liu, Wang, Shen, Zhang, Walsh, Shen and Wang2017). The sequences of the MCR-1 identified from recent extensive studies have confirmed the same high aa identity to the first reported MCR-1 (Shen et al., Reference Shen, Wang, Shen, Shen and Wu2016; Lima Barbieri et al., Reference Lima Barbieri, Nielsen, Wannemuehler, Cavender, Hussein, Yan, Nolan and Logue2017; Meinersmann et al., Reference Meinersmann, Ladely, Plumblee, Cook and Thacker2017; Roschanski et al., Reference Roschanski, Falgenhauer, Grobbel, Guenther, Kreienbrock, Imirzalioglu and Roesler2017), including the one located in the contig N009A from a healthy human microbiome (Ye et al., Reference Ye, Li, Li, Gao, Zhang, Wen, Gao, Hu and Feng2016). Strikingly, the three mcr-1 genes identified from E. coli isolates collected in the 1980s are 100% identical to the first reported mcr-1 (Shen et al., Reference Shen, Wang, Shen, Shen and Wu2016).
In contrast to the high-level identity of MCR-1 sequences, the plasmids bearing mcr-1 is fairly heterogeneous (Poirel et al., Reference Poirel, Kieffer and Nordmann2017), such as those belong to IncI2 (Gao et al., Reference Gao, Hu, Li, Sun, Wang, Lin, Ye, Liu, Srinivas, Li, Zhu, Liu, Tian and Feng2016; Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016; Yao et al., Reference Yao, Doi, Zeng, Lv and Liu2016; Meinersmann et al., Reference Meinersmann, Ladely, Plumblee, Cook and Thacker2017), X4 (Hasman et al., Reference Hasman, Hammerum, Hansen, Hendriksen, Olesen, Agerso, Zankari, Leekitcharoenphon, Stegger, Kaas, Cavaco, Hansen, Aarestrup and Skov2015; Gao et al., Reference Gao, Hu, Li, Sun, Wang, Lin, Ye, Liu, Srinivas, Li, Zhu, Liu, Tian and Feng2016; Webb et al., Reference Webb, Granier, Marault, Millemann, den Bakker, Nightingale, Bugarel, Ison, Scott and Loneragan2016), HI2 (Tse and Yuen, Reference Tse and Yuen2016) and P (Webb et al., Reference Webb, Granier, Marault, Millemann, den Bakker, Nightingale, Bugarel, Ison, Scott and Loneragan2016). These findings suggest that mcr-1 might be phylogenetically young and is rapidly spreading through horizontal gene transfer, either via whole plasmid conjugation (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016; Meinersmann et al., Reference Meinersmann, Ladely, Plumblee, Cook and Thacker2017) or possible mcr-1 cassette recombination between different plasmids (Li et al., Reference Li, Yang, Miao, Chavda, Mediavilla, Xie, Feng, Tang, Kreiswirth, Chen and Du2016a). In fact, it was shown that mcr-1, often neighbored by one or two copies of the insertion sequence ISApl1 (IS30 family), forms a 2600-bp cassette containing promoter sequences (Di Pilato et al., Reference Di Pilato, Arena, Tascini, Cannatelli, Henrici De Angelis, Fortunato, Giani, Menichetti and Rossolini2016; Poirel et al., Reference Poirel, Kieffer, Brink, Coetze, Jayol and Nordmann2016). The role of this 2600-bp cassette in the mobilization of the mcr-1 gene was recently confirmed (Poirel et al., Reference Poirel, Kieffer and Nordmann2017).
mcr-like genes
Besides mcr-1-like genes, a plasmid-borne colistin resistance gene mcr-2, which shared 76.7% nucleotide identity to mcr-1, was identified in colistin-resistant E. coli isolates of porcine and bovine origin in Belgium (Xavier et al., Reference Xavier, Lammens, Ruhal, Kumar-Singh, Butaye, Goossens and Malhotra-Kumar2016b). Prevalence of mcr-2 in porcine colistin-resistant E. coli (11/53) in Belgium was reported to be higher than that of mcr-1 (7/53) (Xavier et al., Reference Xavier, Lammens, Ruhal, Kumar-Singh, Butaye, Goossens and Malhotra-Kumar2016b). To date, the mcr-2 was only reported in Belgium; a large survey showed that the mcr-2 was not detected in 436 samples from German pig-fattening farms (Roschanski et al., Reference Roschanski, Falgenhauer, Grobbel, Guenther, Kreienbrock, Imirzalioglu and Roesler2017) and in 1200 Avian Pathogenic Escherichia coli isolates (Lima Barbieri et al., Reference Lima Barbieri, Nielsen, Wannemuehler, Cavender, Hussein, Yan, Nolan and Logue2017).
Another plasmid-borne colistin resistance gene, mcr-3, was discovered in a conjugative plasmid from E. coli of pig origin in China (Yin et al., Reference Yin, Li, Shen, Liu, Wang, Shen, Zhang, Walsh, Shen and Wang2017). The mcr-3 gene displayed 45.0 and 47.0% nucleotide sequence identity to mcr-1 and mcr-2, respectively (Yin et al., Reference Yin, Li, Shen, Liu, Wang, Shen, Zhang, Walsh, Shen and Wang2017).
A recent phylogenetical analysis revealed that MCR-1 is highly homologous to its counterpart PEA lipid A transferase from Paenibacillus spp., which are known producers of polymyxins (Gao et al., Reference Gao, Hu, Li, Sun, Wang, Lin, Ye, Liu, Srinivas, Li, Zhu, Liu, Tian and Feng2016). Interestingly, within the PEA transferase family, the plasmid-borne MCR-1 is closely clustered to the chromosomally encoded LptA from Neisseria spp., suggesting a parallel evolutionary path for MCR-1 and LptA (Gao et al., Reference Gao, Hu, Li, Sun, Wang, Lin, Ye, Liu, Srinivas, Li, Zhu, Liu, Tian and Feng2016). Notably, despite low a sequence identity between MCR-1 and LptA (<30%), expression of LptA in the pBAD24/MG1655 system can confer colistin resistance to the level observed for MCR-1 (Gao et al., Reference Gao, Hu, Li, Sun, Wang, Lin, Ye, Liu, Srinivas, Li, Zhu, Liu, Tian and Feng2016). In addition, Moraxella spp., mainly animal pathogens, were reported as potential reservoirs of mcr-like colistin-resistant genes (Kieffer et al., Reference Kieffer, Nordmann and Poirel2017).
Similar to the findings from the mcr-1-bearing plasmids as summarized above, two IS1595-like insertion sequences were observed to be adjacent to another mobile colistin-resistant gene, mcr-2, in the IncX4 plasmid, pKP37-BE (Xavier et al., Reference Xavier, Lammens, Ruhal, Kumar-Singh, Butaye, Goossens and Malhotra-Kumar2016b). The IS1595-like element (714 bp) carries a transposase gene (654 bp) flanked by two 18-bp inverted repeats (Sun et al., Reference Sun, Xu, Gao, Lin, Wei, Srinivas, Li, Yang, Li, Liao, Liu and Feng2017b). The formation of a circularized intermediate of mcr-2 in the presence of the bracketing ISEc69 elements (Partridge, Reference Partridge2017; Sun et al., Reference Sun, Xu, Gao, Lin, Wei, Srinivas, Li, Yang, Li, Liao, Liu and Feng2017b), indicates the ISEc69 might be involved in the mobilization of mcr-2. These findings further suggest a transpositional mechanism in the spread of mcr at the molecular level.
Risk factors contributing to colistin resistance in animal production systems
Epidemiological studies on mcr-1-mediated colistin and emergence of mcr-like genes in animals suggest that colistin usage in food animals exert a selection pressure and serve as a major risk factor contributing to the emergence and transmission of mcr-1. It is important to mention that colistin is not absorbed by animal gastrointestinal tracts (Guyonnet et al., Reference Guyonnet, Manco, Baduel, Kaltsatos, Aliabadi and Lees2010; Rhouma et al., Reference Rhouma, Beaudry, Theriault, Bergeron, Laurent-Lewandowski, Fairbrother and Letellier2015); thus, accumulation of colistin or its metabolites in manure may significantly increase colistin resistant, mcr-1-harboring bacteria in agricultural ecosystems (Kruse and Sorum, Reference Kruse and Sorum1994; Thanner et al., Reference Thanner, Drissner and Walsh2016). Some studies have suggested a significant impact of livestock trade or the international trade with exotic animals, such as reptiles, on the spread of mcr-1-mediated colistin resistance (Grami et al., Reference Grami, Mansour, Mehri, Bouallegue, Boujaafar, Madec and Haenni2016; Unger et al., Reference Unger, Eisenberg, Prenger-Berninghoff, Leidner, Ludwig, Rothe, Semmler and Ewers2017).
Currently, there is a worldwide trend to limit and even ban colistin usage in animal production. Clearly, limitation of colistin usage is expected to greatly reduce selection pressure, consequently controlling transmissible colistin resistance. However, to simply limit or ban colistin in animal production will not fully solve this serious and challenging issue, because this antibiotic resistance issue can be influenced by complex multi-level factors. One example is that mcr-1-positive E. coli isolates have been identified in two swine intestinal samples in the USA (Meinersmann et al., Reference Meinersmann, Ladely, Plumblee, Cook and Thacker2017), even though colistin has not been used in US animal production. Emergence of the transmissible colistin resistance in US animal production is still a mystery, but clearly suggests there may exist non-colistin usage risk factors contributing to the persistence, transmission and emergence of colistin resistance in an animal production system. Based on published information, here we discuss several potential non-colistin usage risk factors, which may represent several significant knowledge gaps impeding development of an effective mitigation strategy to control colistin resistance.
Co-selection of the mcr-1 with specific clinical antibiotics
Based on published complete genomes of mcr-1 gene-harboring strains, some mcr-1-bearing plasmids were observed to also carry other AR genes (Malhotra-Kumar et al., Reference Malhotra-Kumar, Xavier, Das, Lammens, Butaye and Goossens2016). For example, there are several resistance-encoding genes to trimethoprim (dfrA1), tetracycline (tetA), aminoglycoside (aadA1, aph(6)-Id or strA, and aph(3 ′)-Ib/strB) and sulphonamide (sul1) antibiotics co-residing in the mcr-1-bearing plasmid pKH-457-3-BE (Malhotra-Kumar et al., Reference Malhotra-Kumar, Xavier, Das, Lammens, Butaye and Goossens2016). In one mcr-1-positive E. coli strain, plasmid sequencing identified multiple genes encoding resistance to trimethoprim (dfrA12), tetracycline (tetA), aminoglycoside (aadA3, aph(3′)-IA), phenicol (cmlA1), quinolone (qnrS1, oqxA), lincosamide (lnu(F)), sulphonamide (sul2, sul3) and β-lactam (extended-spectrum β-lactamase bla CTX-M55) antibiotics (Malhotra-Kumar et al., Reference Malhotra-Kumar, Xavier, Das, Lammens, Butaye and Goossens2016). The co-existence of the mcr-1 gene with other AR genes in a single plasmid would enable co-selection of mcr-1 gene by using certain antibiotics, leading to persistence and transmission of the mcr-1 gene-bearing plasmid in the absence of colistin selection pressure. To date, there is not any published information addressing this issue. Unbiased evaluation of this risk factor needs an appropriate animal model system in conjunction with comprehensive epidemiological studies to examine antibiogram, plasmid profile, and complete genome sequences of diverse mcr-1-bearing strains from the animal production system, which is highly warranted in the future.
Factors enhancing horizontal gene transfer
The mcr-1-bearing plasmid can transfer among different enteric bacteria via conjugation with variable frequencies, depending on the donor or recipient strains (Liu et al., Reference Liu, Wang, Walsh, Yi, Zhang, Spencer, Doi, Tian, Dong, Huang, Yu, Gu, Ren, Chen, Lv, He, Zhou, Liang, Liu and Shen2016; Denervaud Tendon et al., Reference Denervaud Tendon, Poirel and Nordmann2017). According to published information (Carnevali et al., Reference Carnevali, Morganti, Scaltriti, Bolzoni, Pongolini and Casadei2016; Poirel et al., Reference Poirel, Kieffer, Brink, Coetze, Jayol and Nordmann2016) and our phylogenetic analysis of diverse mcr-1 positive E. coli strains with pig and chicken origins (unpublished data), mcr-1-harboring strains do not have clonal relationship and mcr-1-bearing plasmids display significant diversity in terms of antibiogram, plasmid profiles and genetic contents. We speculate that any factors that can enhance bacterial conjugation may promote transmission of mcr-1, consequently providing new opportunities for mcr-1 to persist in the host or other niches in the absence of colistin selection pressure. For example, it has been reported that some antibiotics, such as beta-lactams and fluroquinolones, can serve as DNA damaging agents for induction of SOS response, consequently enhancing conjugation efficiency (Barr et al., Reference Barr, Barr, Millar and Lacey1986; Beaber et al., Reference Beaber, Hochhut and Waldor2004). Thus, using such SOS-inducing antibiotics in animals may promote the horizontal transfer of the mcr-1 gene-bearing plasmid. This hypothesis is partly supported by a recent case-control study showing infections caused by mcr-1-positive E. coli were associated with prior use of carbapenems and fluoroquinolones (Wang et al., Reference Wang, Tian, Zhang, Shen, Tyrrell, Huang, Zhou, Lei, Li, Doi, Fang, Ren, Zhong, Shen, Zeng, Wang, Liu, Wu, Walsh and Shen2017b).
Cross-resistance between polymyxins and other AMPs
Natural antimicrobial peptides (AMPs) have been recognized as a novel class of antimicrobials to combat increasing MDR in bacterial pathogens (Hancock and Chapple, Reference Hancock and Chapple1999; Shryock, Reference Shryock2004; Cotter et al., Reference Cotter, Hill and Ross2005; Toke, Reference Toke2005). AMPs are typically cationic, short, amphipathic, and microbicidal peptides that can be found in virtually all species of life (Riley and Wertz, Reference Riley and Wertz2002; Zasloff, Reference Zasloff2002; Brogden et al., Reference Brogden, Ackermann, McCray and Tack2003; Yeaman and Yount, Reference Yeaman and Yount2003; Brogden, Reference Brogden2005; Wehkamp et al., Reference Wehkamp, Schauber and Stange2007). The endogenous AMPs could be derived from both metazoan hosts (e.g. defensins and cathelicidins) and bacteria (e.g. bacteriocins). AMPs display a broad spectrum of antimicrobial activity and have been increasingly recognized as a novel class of antibiotics (peptide antibiotics) to control pathogens (Hancock and Chapple, Reference Hancock and Chapple1999; Shryock, Reference Shryock2004; Cotter et al., Reference Cotter, Hill and Ross2005; Rossi et al., Reference Rossi, Rangasamy, Zhang, Qiu and Wu2008; Sit and Vederas, Reference Sit and Vederas2008). Although bacteria co-evolved with the host innate defense and developed means to curtail the effect of endogenous AMPs such as defensins, cathelicidins and bacteriocins (Ernst et al., Reference Ernst, Guina and Miller2001; Yeaman and Yount, Reference Yeaman and Yount2003; Kraus and Peschel, Reference Kraus and Peschel2006; Peschel and Sahl, Reference Peschel and Sahl2006), bacteria have not developed highly effective AMP resistance mechanisms during millions of years of co-evolution with endogenous AMPs. This intriguing phenomenon is likely due to multiple activities and pleotropic effects of natural AMPs (Peschel and Sahl, Reference Peschel and Sahl2006; Wehkamp et al., Reference Wehkamp, Schauber and Stange2007).
Notably, as a bacterium-derived AMP, polymyxin has been widely and successfully used as model AMP (or AMP surrogate) to study AMP resistance in bacteria, and polymyxin bears some structural resemblance to many other AMPs. Therefore, acquisition of polymyxin resistance has been observed to result in cross-resistance to different types of AMPs (Groisman et al., Reference Groisman, Kayser and Soncini1997; Bengoechea and Skurnik, Reference Bengoechea and Skurnik2000; Gunn et al., Reference Gunn, Ryan, Van Velkinburgh, Ernst and Miller2000; McCoy et al., Reference McCoy, Liu, Falla and Gunn2001; McPhee et al., Reference McPhee, Lewenza and Hancock2003; Campos et al., Reference Campos, Vargas, Regueiro, Llompart, Alberti and Bengoechea2004; Chen et al., Reference Chen, Chuang, Chang, Jeang and Chang2004; Shi et al., Reference Shi, Cromie, Hsu, Turk and Groisman2004; Winfield and Groisman, Reference Winfield and Groisman2004). Based on these extensive AMP studies, the transmissible colistin resistance determinant MCR-1 may also confer a certain level of cross-resistance to some AMPs, raising a serious concern for use or development of AMP-based interventions against bacterial infections. If MCR-1 displays cross-resistance to some AMPs that have been used or are being targeted for developing new antimicrobials, such AMPs may serve as another non-colistin usage factor to promote persistence and transmissibility of mcr-1.
Recently, Dobias et al. (Reference Dobias, Poirel and Nordmann2017) addressed this cross-resistance issue and observed that MCR-1 did not confer cross-resistance to human cathelicidin LL-37, α-defensin 5, and β-defensin 3 in E. coli and K. pneumoniae. We also observed that MCR-1 did not confer cross-resistance to chicken host AMPs in E. coli either (unpublished data). Therefore, the transmissible colistin resistance determinant MCR-1 apparently does not confer cross-resistance to common host defense peptides, which greatly mitigates safety and sustainability concerns for some recent efforts to develop AMP-based intervention, such as using host AMP-inducing compounds as an innovative non-antibiotic approach to control bacterial infections (Sunkara et al., Reference Sunkara, Achanta, Schreiber, Bommineni, Dai, Jiang, Lamont, Lillehoj, Beker, Teeter and Zhang2011, Reference Sunkara, Zeng, Curtis and Zhang2014; van der Does et al., Reference van der Does, Bergman, Agerberth and Lindbom2012). However, to better address this cross-resistance issue, in the future, more extensive studies are needed to examine if MCR-1 can confer cross-resistance to AMPs from various sources, which will provide critical information for risk assessment and management of AMP-based anti-infectives.
Existence and emergence of novel colistin-resistant genes in ecosystem
Recent metagenomics and functional genomics studies have provided compelling evidence that antibiotic resistance genes are widespread; the novel and immensely diverse resistance genes exist in various ecosystems such as the intestinal tracts of people and food animals, agriculture (e.g., animal manure, soil, water and wastewater lagoons), and even ancient soils (Aminov and Mackie, Reference Aminov and Mackie2007; Pehrsson et al., Reference Pehrsson, Forsberg, Gibson, Ahmadi and Dantas2013; Davies, Reference Davies, Atlas and Maloy2014). Functional screening of metagenomic libraries constructed from fecal samples from human beings (Sommer et al., Reference Sommer, Dantas and Church2009), chickens (Zhou et al., Reference Zhou, Wang and Lin2012), pigs (Kazimierczak et al., Reference Kazimierczak, Scott, Kelly and Aminov2009), gulls (Martiny et al., Reference Martiny, Martiny, Weihe, Field and Ellis2011) and dairy cows (Wichmann et al., Reference Wichmann, Udikovic-Kolic, Andrew and Handelsman2014) have demonstrated that majority of antibiotic-resistant genes in gut microbial communities are novel and share low identity (40–60%) with the previously identified resistance genes. These novel antibiotic-resistance genes have potential to emerge if the opportunity arises (Aminov and Mackie, Reference Aminov and Mackie2007; Pehrsson et al., Reference Pehrsson, Forsberg, Gibson, Ahmadi and Dantas2013; Davies, Reference Davies, Atlas and Maloy2014). Based on these metagenomic discoveries, novel colistin-resistance genes may also exist in ecosystems and have potential to emerge if the opportunity arises; this speculation has been partly supported by recent identification of LptA (Gao et al., Reference Gao, Hu, Li, Sun, Wang, Lin, Ye, Liu, Srinivas, Li, Zhu, Liu, Tian and Feng2016) as well as MCR-2 (Xavier et al., Reference Xavier, Lammens, Ruhal, Kumar-Singh, Butaye, Goossens and Malhotra-Kumar2016b) and MCR-3 (Yin et al., Reference Yin, Li, Shen, Liu, Wang, Shen, Zhang, Walsh, Shen and Wang2017) discussed in above sections.
Identification of novel colistin-resistance genes is important for combating the colistin resistance threat, because elucidation of the colistin resistome will greatly facilitate development of molecular diagnostic tools to effectively monitor colistin resistance in agricultural ecosystems. In addition, further in-depth characterization of colistin-resistance genes will improve our understanding of the evolution and molecular basis of colistin resistance, consequently providing insights into new interventions by targeting resistance mechanisms. Finally, identification of novel colistin-resistance genes using a functional cloning approach will complement the modern high-throughput sequencing of metagenomes by greatly improving annotation power. Therefore, identification of a novel colistin resistome from an animal production system using a functional metagenomics screening approach is also highly warranted in the future.
Conclusion
Recent emergence of the transmissible colistin resistance gene, mcr-1, has drawn worldwide attention and fears. Epidemiological studies suggest that use of colistin in animals is a major risk factor for the emergence and transmission of mcr-1. Therefore, there is a worldwide trend to limit colistin usage in animal production. Although the regulated use of colistin would greatly reduce selection pressure, consequently controlling the transmissible colistin resistance in an animal production system, other non-colistin usage risk factors may also exist for colistin resistance development. In this review, in addition to a comprehensive review of epidemiological studies for prevalence of mcr genes in animals, we also summarized published information to support the existence of several non-colistin usage factors that may contribute to the persistence, transmission and emergence of colistin resistance in an animal production system. Comprehensive examination of these non-colistin usage factors using both in vitro and in vivo systems in the future will generate critical information for risk assessment and risk management of transmissible colistin resistance, leading to proactive and effective strategies for mitigation of colistin resistance in animal production system worldwide.
Acknowledgements
This work was supported by University of Tennessee AgResearch and the National Key Research and Development Program of China (2016YFD0501300), the Funds for International Cooperation and Exchange of the National Natural Science Foundation of China (Grant No 31520103918), the Program of Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT13063).
