Colistin in Pig Production: Chemistry, Mechanism of Antibacterial Action, Microbial Resistance Emergence, and One Health Perspectives

1Antibiotics in the polymyxin family include five different chemical compounds (polymyxins A, B, C, D, and E), of which polymyxin B and colistin (also called polymyxin E) are the only two polymyxins used clinically. For humans, two forms of colistin are commercially available: colistin methanesulfonate sodium (CMS) for parenteral use and aerosol therapy; and colistin sulfate (CS) for oral and topical use. Colistin is used in human medicine for the treatment of infections due to multidrug-resistant (MDR) Gram-negative bacteria (GNB) such as Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and carbapenemase-producing Enterobacteriaceae and is used as a last-resort treatment option against these infections. Recently, the World Health Organization (WHO) and several government agencies such as Health Canada have reclassified colistin in the category of very high importance in human medicine. Colistin’s mechanism of action is mainly related to its attachment to the lipopolysaccharides (LPSs) of GNB, leading to membrane-permeability disturbance and cell death.

2Colistin sulfate is the only form of colistin approved in pig production in some countries for the control of Enterobacteriaceae infections, particularly for those caused by Escherichia coli. Since its introduction on the market in the 60s, colistin was used in pig production in several countries with different purposes; therapeutically, prophylactically, and even for growth promotion. Interestingly, in the late 2000s and after decades of colistin use in swine, several studies began reporting a significant resistance rate of Enterobacteriaceae to colistin in pigs. The most common mechanism of colistin resistance in E. coli and Salmonella involves a modification of the lipid A portion of LPS through the addition of phosphoethanolamine (PEtN) and/or a 4-amino-4-deoxy-L-arabinose (L-Ara4N), which reduces its binding to colistin and leads to bacterial resistance. This chromosomal mechanism of colistin resistance is the result of the activation of the two-component systems (TCSs) PhoP/PhoQ and PmrA/PmrB by specific mutations or environmental stimuli leading to an overexpression of LPS-modifying genes. However, several studies have reported the isolation of colistin resistant E. coli strains in the absence of chromosomally encoded mechanisms. At the end of 2015, researchers identified a stable plasmid mediated mcr-1 gene encoded for phosphoethanolamine transferase conferring resistance to colistin in some GNB isolated from food animals, raw meat, and humans in several countries. The discovery of this horizontal mechanism of colistin resistance raised alarm bells about the impact of colistin use on colistin resistance spread in animal production, especially in swine. In fact, the link between pigs and humans in terms of colistin resistant E. coli strain transfer following direct contact has recently been confirmed. These findings have led to a serious fear about the possible loss of colistin effectiveness in the treatment of MDR-GNB in humans. Hence, it is urgent to establish close cooperation between physicians, veterinarians, and countries to ensure judicious use of colistin in both veterinary and human medicine. The application of the One Health concept could be a solution for the management of colistin resistance in the human-pig-environment interface.

3This review is an update on colistin chemistry, its applications and antibacterial mechanism of action, and on Enterobacteriaceae resistance in pigs. We also detail and examine the One Health concept to arrive at proposed guidelines for rational use of colistin in swine and humans and to find ways to prevent bacterial resistance spread in the human-pig-environment interface.

4Please refer to our recent review for rates of colistin resistance in pigs, the possible link between colistin pharmacokinetic/pharmacodynamic (PK/PD), and colistin use and Enterobacteriaceae resistance emergence in swine.

5Colistin (polymyxin E) is a polymyxin antibiotic produced by Paenibacillus polymyxa var colistinus consisting of a cyclic heptapeptide ring with three positively charged amine groups, a tail tripeptide moiety with two positively charged amine groups, and a hydrophobic acyl chain tail (Figure 1;;;;; ). Colistin is an amphipathic molecule, with hydrophobicity mainly attributable to the fatty acyl moiety and hydrophilicity due to the five L-diaminobutyric acid (L-Dab) amino groups. The L-Dab molecules are positively charged in positions 1, 3, 5, 8, and 9 (Figure 1). These amino groups are responsible for the electrostatically interaction between colistin and the lipid A portion of LPS molecules of GNB and play a central role in the bactericidal activity of colistin. The polymyxins family includes five chemically distinct compounds (polymyxins A–E) and only colistin (polymyxin E) and polymyxin B have been used in clinical practice. Polymyxin B and colistin share a similar primary sequence with the only difference being one amino acid in position 6 in which D- phenylalanine in polymyxin B is replaced by D- leucine in colistin (Figure 1;;;;; ).

6Two different forms of colistin are available commercially: CS, which is administered either orally for bowel decontamination or topically as a powder for skin infections, and CMS, which is commonly administered intravenously and used exclusively in human medicine. Both can be delivered by inhalation. CS is the only active ingredient of the polymyxin family and is approved in some countries for the control of Enterobacteriaceae infections in pigs and is used mostly in monotherapy or sometimes in combination with other substances. Researchers found at least thirty different components in commercially available colistin, 13 of which were separated using the isocratic liquid chromatography (LC) method. The two major components of colistin are colistin A (polymyxin E1) and colistin B (polymyxin E2), which differ only in the fatty acid side chain. In fact, colistin A and colistin B are acylated by (S)-6-methyloctanoic acid and (S)-6-methylheptanoic acid, respectively. The proportion of these two major components in commercial products differs between the different pharmaceutical preparations of colistin available on the market. This could be due to the fact that colistin is a natural product produced by fermentation, so its composition can vary considerably between manufacturers. In fact, no pure colistin A and B reference standards are commercially available and no certificates of analysis that include chemical characterization are available in veterinary medicine to adequately establish the purity of the marketed CS formulations. CS is a polypeptide antibiotic with a chemical structure characterized by the presence of multiple peptide bonds documented to predispose CS to chemical and enzymatic degradation. In fact, in pig simulated gastric fluid (SGF), CS led to the formation of degradation products that have a significant antimicrobial activity compared to non-degraded CS.

7Colistin has a narrow antibacterial spectrum with an effect limited to GNB; Gram-positive bacteria do not contain LPS in their cell wall and, as a consequence, are excluded from the spectrum of activity of polymyxins.

8The initial target of colistin is the LPS component of the outer membrane (OM) of GNB. The most documented steps of colistin antibacterial activity are described below.

9Colistin initially binds to LPS and specifically to lipid A, a key component of the LPS, through electrostatic interaction between positively charged Dab residues of colistin and the negatively charged phosphate groups of lipid A. Lipid A plays a crucial role in the control of bacterial permeability.

10Colistin competitively displaces divalent cations calcium (Ca2+) and magnesium (Mg2+) that normally stabilize the LPS and as a consequence the three-dimensional structure of the LPS is altered. In fact, colistin has affinities for LPS that are at least three times higher than those for divalent cations.

11Colistin causes an expansion of the OM monolayer by the insertion of its hydrophobic terminal fatty acyl chain or the D-Leu6 -L-Leu7 segment into the OM.

12Colistin leads to a permeabilization of the OM by the formation of destabilized areas through which colistin will transit the OM via a self-promoted uptake mechanism. This mechanism explains how colistin acts in synergy with conventional antibiotics. In fact, hydrophilic antibiotics such as rifampicin, vancomycin, meropenem, β-lactam, tigecycline, and gentamicin can work synergistically due to this disruption of membrane integrity by colistin.

13Colistin destroys the physical integrity of the phospholipid bilayer of the inner membrane (IM) through membrane thinning by straddling the interface of hydrophilic head groups and fatty acyl chains.

14This leads to IM lysis, leakage of intracellular contents and cell death.

15Colistin also exerts an anti-endotoxin activity because it binds to the lipid A component of LPS. In this way, colistin prevents endotoxin’s ability to induce shock through the release of cytokines such as tumor necrosis factor-alpha (TNF-α) and IL-8.

16It should be stressed here again that colistin’s antibacterial mechanism of action based on membrane lysis death was the most documented explanation for the effectiveness of this antibiotic in the treatment of GNB infections. However, its ultimate mechanism of action is still unknown. Other mechanisms of polymyxin bactericidal activity have been proposed such as a vesicle–vesicle contact pathway and a hydroxyl radical death pathway. The vesicle–vesicle contact antimicrobial mechanism described involves the polymyxin B molecule with a hydrophobic acyl tail that can enter into and cross the OM and induce a lipid exchange between leaflets of the IM and OM; this leads to membrane osmotic instability due to the change in the phospholipid composition, thereby inducing cell lysis. However, this mechanism of action has not been studied with colistin. It has been shown that polymyxin B and colistin exert a rapid antimicrobial activity against the sensitive and multidrug-resistant isolates of A. baumannii and E. coli through hydroxyl radical production by the Fenton reaction, leading to the formation of hydroxyl radicals through the reduction of hydrogen peroxide by ferrous iron (Fe2+). The production of this reactive oxygen species (ROS) might lead to oxidative damage in the bacterial DNA, proteins, and lipids and cause cell death. However, this feature of colistin has not yet been evaluated in clinical practice. Most recently, it was shown that colistin was able to inhibit the vital respiratory enzyme NADH-quinone oxidoreductase in the bacterial IM of GNB. This mechanism was regarded as a secondary mode of action of polymyxins.

17Unlike for human medicine, only a few studies have been conducted in pigs to evaluate the PK of colistin following oral or intramuscular (IM) administration (;;; Table 1). These studies were performed using CS, since this is the only form of colistin approved in swine medicine, and were conducted in healthy pigs. It is reasonable to think that the PK can be different in sick animals. The oral CS PK data in pigs were obtained using either a high-pressure liquid chromatography (HPLC) assay or a LC coupled with the tandem mass spectrometry (HPLC–MS/MS) method. CS PK data in pigs after parenteral administration were obtained using mostly microbiological assays (;; Table 1); these data should be viewed with caution because of the limited sensitivity of this method and the descriptions of the experiment conditions.

18After oral CS administration in pigs and despite the use of a very sensitive analytical method, CS plasma concentrations were very difficult to quantify in healthy pigs. A concurrent oral challenge of pigs with an ETEC: F4 strain did not increase CS intestinal absorption in a subclinical induction model of post-weaning diarrhea (PWD). However, in pigs with clinical PWD following an experimental oral challenge with the ETEC: F4 strain, CS plasma concentrations were higher in the challenged groups compared to the unchallenged one. These studies confirm that colistin is poorly absorbed through pig’s gastrointestinal tract even in infected animals and corroborates the involvement of oral CS administration in exacerbating colistin resistance by exerting selection pressure on pig’s intestinal flora.

19Parenteral CS PK studies in pigs were mainly conducted to study the safety of IM CS administration. The CS intestinal concentrations through the biliary system elimination were not determined following IM administration to assess whether or not colistin exerts a selective pressure on pig’s intestinal microflora after its parenteral administration. There is no available data in the literature concerning the possible renal tubular reabsorption of CS in pigs as previously demonstrated in rats through a carrier-mediated process; if this is the case, it would justify an extension of the colistin withdrawal period in pigs.

20Even though some studies have been able to quantify colistin in the pig’s systemic circulation following its oral administration using very sensitive methods, these concentrations were very low compared to the Maximum Residue Limits (MRLs) for this molecule in pigs, which supports the short withdrawal period of 1–7 days for oral CS in pigs. In fact, the EMEA Committee for Medicinal Products for Veterinary Use (CVMP) has established the MRLs for colistin in swine: 150, 150, 150, and 200 μg/kg in muscle, liver, fat, and kidney, respectively. However, no study has been performed in pigs to assess CS degradation product toxicity, and no screening tests are available in the market to detect these products in pig meat. It was shown that E. coli experimental infection in pigs increased CS intestinal absorption, and authors have claimed that this information should be taken into consideration when determining the CS withdrawal period in pigs. Even with intestinal infection, CS systemic concentrations in pigs remain below MRLs, thus adjusting the withdrawal period after E. coli infection in pigs should be considered for antibiotics that are characterized by high oral bioavailability.

21The potential for the emergence of E. coli resistance in pigs during therapy with CS has been shown following its use at the recommended regimen (100,000 IU/kg/day), as demonstrated previously. In this study, despite a rapid initial reduction in E. coli fecal excretion following CS oral treatment, the emergence of CS resistance among commensal E. coli was observed starting from the 3rd day of treatment. The selection pressure for CS resistant isolates disappeared after 6 days of CS treatment, and CS resistant E. coli strains were isolated 6 days after the last treatment. This is of significant importance in food safety and public health perspective because this means pigs that are treated with CS and given a 1 day withdrawal period as recommended are shipped to slaughter with potential colistin resistant E. coli in their gut. Therefore, applying a microbiological withdrawal time for CS resistant bacteria in addition to the chemical one is of crucial importance to reduce the risk of passage of these bacteria in pig slaughterhouses to humans through the handling of raw meat or the consumption of undercooked meat.

22In order to monitor E. coli colistin resistance in pigs subsequent to the therapeutic use of this antibiotic in the treatment of PWD, our team used MacConkey agar medium supplemented with CS at 2 μg/mL, which represents the breakpoint value. We confirmed that this medium overestimated the number of CS resistant E. coli and that the isolation of putative resistant bacteria on this medium requires confirmation by MIC determination using a Mueller–Hinton broth medium. To overcome this problem related to the absence of a selective medium for the screening of colistin resistant bacteria, developed a screening medium that is able to detect intrinsic and acquired polymyxin-resistant bacteria without the need to confirm resistance isolates by MIC determination. The implementation of this medium will facilitate the monitoring of colistin Enterobacteriaceae resistance in food-producing animals.

23While great advances in colistin research have occurred in the last decade in both human and veterinary medicine, colistin PK/PD data are very limited in pigs. To successfully combat the development and dissemination of bacterial resistance against this antibiotic in swine, we believe that specific CS clinical PK/PD data are of crucial importance (Table 2).

24Furthermore, the recent discovery of a plasmid mediated mcr-1 gene encoding for Enterobacteriaceae colistin resistance in farm animals and in humans has prompted several regulatory agencies such as the European Medicines Agency (EMA) to re-evaluate colistin in farm animals. More data on colistin PK/PD will be essential to ensuring judicious use of colistin in pigs (Table 2).

25It should be stressed here again that the CS commercially available is obtained by a bacterial fermentation process. Consequently, its composition may vary between commercially available CMS products, although no study in veterinary medicine has verified this variability. In addition, the unit of CS dosing in pig production is not standardized; some practitioners use international units whereas others use milligrams per kg of body weight. We believe that the standardization of CS composition and dosage in pigs worldwide is critical to ensuring judicious use of this antibiotic, and it would allow comparison between studies in terms of therapeutic efficacy and resistance rate.

26Only one study has determined the CS concentrations in clinical healthy intestinal tracts of pigs after a single oral administration of this molecule. In this study, colistin concentrations were not detectable in fecal samples, from the duodenum to ileum, after 4 h of its oral administration regardless of doses used (25,000, 50,000, or 100,000 IU/kg). However, CS is usually administrated in swine medicine to treat sick animals at a dose of 50,000 IU/kg body weight every 12 h for 3–5 days, and the intestinal Cmax concentrations of colistin were not determined after a repetitive CS oral treatment to justify the efficacy of this therapeutic regimen in the treatment of pig’s diseases associated with Enterobacteriaceae. The duration of CS oral treatment in pig farms is far longer than 3–5 days as recommended on product monographs. Nevertheless, no study in field conditions has evaluated the impact of CS treatment duration on its clinical efficacy in pigs and on bacterial resistance emergence. Our team showed in experimental conditions that 3 days of CS oral treatment of pigs challenged with an ETEC: F4 strain was enough to treat clinical symptoms of PWD in pigs, and a positive correlation was observed between CS treatment duration and CS selection pressure on commensal E. coli.

27It has previously been demonstrated that antimicrobial activity is related to inoculum size and stage of infection. Specifically, researchers found that antimicrobial activity may be higher for a lower bacterial inoculum, and treating experimental animals at an early stage of infection reduced both the required dose of antimicrobials and the amplification risk of bacterial resistance in the intestine. The impact of the inoculum on the bactericidal activity of colistin has been investigated in vitro for some strains of P. aeruginosa of human origin. In this study, killing of the susceptible population was 23-fold slower for the 109 CFU and sixfold slower for the 108 CFU than for the 106 CFU. These findings require further investigation in pigs to study the efficiency of an early use of CS in the treatment of infections associated with Enterobacteriaceae in swine and to examine the impact of such practice on the resistance amplification risk among pig’s intestinal bacteria and on colistin amounts used at the farm level.

28Numerous in vitro and in vivo studies using colistin and various other antibiotics have provided evidence for increased bacterial killing and decreased emergence of resistance with the use of certain colistin combinations against MDR Gram-negative bacteria. Using colistin with other antimicrobial agents (aztreonam, piperacillin, ceftazidime, imipenem, ampicillin-sulbactam, ciprofloxacin, carbapenems, and rifampicin) is the most used combination treatment in human medicine. Nevertheless, optimal combinations are not defined, and the relative value of a combination may vary between bacterial strains.

29In swine, and despite the use of some combinations of colistin with other antimicrobial agents (Table 3), no study has demonstrated the effectiveness of such association and its role in colistin Enterobacteriaceae resistance occurrence.

30Several susceptibility testing methods are used in pigs to determine colistin MIC against bacterial strains of porcine origin, without specific clinical breakpoints for colistin against Enterobacteriaceae after its oral use in swine medicine. Such information is of crucial importance for identifying the colistin PD index that is predictive of microbiological efficacy and outcome and to establish the quantitative relationship between PK and PD parameters.

31Recently, the plasmid-mediated colistin resistance gene mcr-1 was detected in some Extended-Spectrum-β-Lactamase (ESBL, blaCTX-M) producing E. coli isolates from pigs in Germany and in Vietnam. These findings highlight the importance of the active surveillance of colistin resistance in pigs. The suggested strategies to reduce colistin use in pigs should never be associated with an increase in the use of third and fourth generation fluoroquinolones or cephalosporins or the overall use of antimicrobials on farms as claimed in the last report of the EMA.

32Recommended points of investigation to generate essential PK/PD data for judicious use of colistin in pig production are summarized in Table 2.

33The main indication of colistin in pigs is the treatment of digestive infections caused by Enterobacteriaceae, especially for those caused by E. coli. Colistin is widely used for the control of PWD in piglets in Europe. Some epidemiological surveys have been reported that colistin is sometimes used off-label in pig farms to treat infections other than intestinal diseases such as respiratory disease. Approximately 99% of colistin use in pig production is carried out orally for mass treatment in intensive husbandry systems.

34Colistin sulfate is used therapeutically, prophylactically, and even as a growth promoter in swine in some countries. The CS is not approved in pig production in Canada and in the USA, and this antibiotic is not used as a feed additive for growth promotion in Europe for at least two decades. However, CS is used in Canada, in some cases under veterinarian responsibility, as a last resort option for the treatment of PWD in farms with high rates of resistance to aminoglycosides.

35However, the most common use of colistin in pig production worldwide is oral, metaphylactic use. This practice involves treating all animals belonging to the same pen – animals with clinical symptoms as well as clinically healthy ones. In its last report, the EMA recommended using colistin only for therapy or metaphylaxis purposes in food-producing animals. All indications for prophylactic use of this molecule should be prohibited and indications of colistin should be restricted only for the treatment of enteric infections caused by susceptible non-invasive E. coli.

36Colistin is used in pigs at the dose of 100,000 IU per kg of body weight for three to five consecutive days and divided into two administrations per day. This therapeutic regimen is recommended for colistin veterinary formulations administered in drinking water. However, no recommendation has been made for CS products administered in feed or by an injectable route in pigs. It is important to stress the lack of standardization of therapeutic regimen and its impact on the judicious use of colistin in swine.

37It is difficult to determine the real quantities of colistin used in pig production worldwide because these data vary considerably from one country to another, and sometimes colistin amounts used in pigs in some countries are very high relative to the size of swine herds. Even within the same country, quantities of colistin in pigs vary from one survey to another due to the absence of standardized methods for data collection.

38In vitro and clinical investigations examining synergism of colistin combined with other antimicrobials in human medicine has been investigated recently and reviewed. The ultimate objective of this combination is to overcome the suboptimal exposure and the resistance emergence associated with the use of colistin in monotherapy. Indeed, the combination of colistin with other antibiotics is intended to extend the CS spectrum of activity to cover Gram-positive bacteria and to prevent the emergence of antibiotic resistance. However, a considerable controversy regarding the effectiveness of these combinations to counter the spread of MDR bacteria has been discussed in human medicine. Most recently, showed that colistin and rifampicin combinations were active in vitro against all NDM-1-producing K. pneumoniae strains used in their study. However they claimed that such effectiveness should be further explored in vivo to be considered for clinical use. confirmed that colistin combination therapy should be considered in critically ill patients with MDR Gram-negative pneumonia.

39In swine, CS is typically used in monotherapy for the oral treatment of infections associated with Enterobacteriaceae. However, there are some commercial formulations where CS is associated with others antimicrobial agents, mostly with β-lactam antibiotics such as ampicillin or amoxicillin (Table 3). In fact, it has been shown that the combination of amoxicillin with colistin has a synergy and additive affect in vitro against pathogenic E. coli of avian origin, without antagonism between the two antibiotics. Colistin combinations were used exclusively for the curative therapy of pig bacterial infections (Table 3). Moreover, it has been reported that in the weaning period, colistin was frequently applied in combination therapy with amoxicillin against symptoms of arthritis and/or meningitis and PWD in pigs. Combinations of colistin and amoxicillin plus zinc oxide (ZnO) in the pre-weaning and growing stages in feed were also reported in pigs.

40Given the lack of appropriately conducted randomized controlled clinical trials, reliable data on the efficiency of colistin combination use for the treatment of E. coli in pigs and its impact on bacterial resistance evolution are very limited or non-existent. In a recent study, showed that a combination of CS with bacitracin zinc and chlortetracycline suppressed the increase of tet genes in fecal samples of weaned pigs. In this study, the relative fecal abundances of four tet genes (tetX, tetC, tetL, and tetW) were reduced in pigs treated with a combination of chlortetracycline, bacitracin zinc, and CS compared with the group treated only with chlortetracycline. However, in this study no information was reported regarding the evolution of resistance to colistin following the combination use of these antibiotics.

41With the lack of solid microbiological evidence on the effectiveness and the impact on bacterial resistance evolution of colistin combination therapy in pigs, the CVMP recommended the withdrawal of marketing authorizations for all veterinary formulations containing colistin in combination with other antimicrobial substances.

42Heavy metals such as zinc are widely used in pigs, especially for the control of PWD in combination with colistin, and is incorporated into swine feed at levels of 125–3000 mg/kg of feed. Zinc oxide fed at pharmacological levels reduces diarrhea and mortality and improves growth in pigs. However, there are two major concerns regarding the use of ZnO in swine. On the one hand, there is environmental pollution because of the high levels of supplementation, and on the other there is co-selection and co-resistance where antibiotic resistance genes (ARGs) are located on the same mobile genetic element as ZnO resistance genes. To the best of our knowledge, no study has investigated whether or not resistance genes associated with colistin and heavy metals could be carried on the same mobile genetic element. Such information is crucial since ZnO is among the proposed strategies to reduce colistin quantities used for the control of PWD in pig production.

43In addition to colistin combination therapy used in field conditions (Table 3), there are other combinations with this antibiotic that have been used in several scientific studies to evaluate the efficacy of some colistin alternative substances (Table 4).

44These studies (Table 4) that evaluated the therapeutic efficacy of colistin combination therapy in pigs were carried out in China and focused primarily on clinical effectiveness not the emergence of antimicrobial resistance. Therefore, no information was available concerning the evolution of colistin bacterial resistance subsequent to the use of these combination therapies in swine, and we do not know whether these combinations are used in practice on pig farms in China.

45Owing to an excessive use of colistin in pig production for many decades, several studies conducted with swine reported the isolation of E. coli and Salmonella strains with high percentages of resistance to colistin. In the present review we will detail the mechanisms of resistance to colistin for Salmonella and E. coli, due to the importance of these two bacteria in both swine and human health.

46An initial and essential step in colistin action on GNB is the electrostatic interaction between the positively charged peptide of this antibiotic and the negatively charged lipid A of LPS. Chromosomal resistance to colistin in Salmonella and E. coli is most often mediated by modifications of LPS, which result in alterations in the target and reduced binding of the antimicrobial. Changes in LPS consist in a modification of lipid A with the addition of a 4-amino-4-deoxy-L-arabinose (L-Ara4N) and/or phosphoethanolamine (PEtn). These molecules reduce the net negative charge of LPS and as a consequence increase the resistance to colistin. In Salmonella and E. coli, the biosynthesis of l-Ara4N and/or PEtn is mediated by PmrA/PmrB and PhoP/PhoQ two-component response regulators and sensor kinase systems. In fact, the PhoPQ and PmrAB TCSs in Salmonella and E. coli have been reviewed extensively elsewhere. A brief overview is provided here with a focus on the more recent discoveries.

47PmrB and PhoQ are sensor cytoplasmic membranes activated respectively by high concentrations of Fe3+ and low pH and by low concentrations of Mg2+ and Ca2+ or certain antimicrobial peptides. In colistin resistant Salmonella, apart from an environmental stimuli such as low Mg2+ concentration, a mutation in the PmrA/PmrB and/or PhoP/PhoQ TCS is the major mechanism involved in LPS modification. In Salmonella, PhoPQ further influences lipid A modification by activating the PmrAB system through the activation of PmrD. However, it was proposed in E. coli that the two systems are not coupled because PmrD does not activate the PmrA/PmrB system. This hypothesis was initially justified by a high divergence between the Salmonella and E. coli PmrD proteins. However, it was later found that E. coli PmrB possesses higher phosphatase activity that exceeds the same activity of the Salmonella homolog, and the replacement of the E. coli pmrB gene with the Salmonella homolog was able to render E. coli resistant to polymyxin under PmrD-inducing conditions with low concentrations of Mg2+. Moreover, it has been demonstrated that the sRNA MgrR of E. coli was also involved in the regulation of lipid A modification. Most recently, have shown that in E. coli, another unknown bacterial system activates PmrD under low Mg2+ conditions to promote lipid A modification, even in the absence of PhoPQ.

48Mutations in TCS corresponding to E. coli and Salmonella can cause their constitutive over expression, leading to permanent modification of lipid A by L-Ara4N and PEtN. Recently, various mutations have been identified in both pmrA and pmrB genes of colistin-resistant E. coli isolated from healthy pigs and pigs with intestinal disease (Table 5). Mutations in the PmrAB TCS are mostly involved in the development of resistance to colistin in E. coli.

49For PmrA, mutations mostly occurred in the phosphate acceptor domain, while for PmrB, mutations most commonly occurred in the kinase domain.

50Of note, regardless of the mutation location in PmrA or PmrB genes, there was no association with a difference in MIC of these colistin resistant E. coli strains (Table 5).

51Despite the fact that polymorphism in the PmrAB system has been reported in vitro in Salmonella, did not detect any of the protein polymorphisms of PmrA and PmrB sequences in colistin resistant Salmonella isolates from swine lymph nodes. However, the polymorphism of genes encoding the PhoPQ system in colistin-resistant Salmonella has not been investigated in this study. Recently, an in-depth investigation of these Salmonella isolates showed that 100% of them harbored the plasmid carrying the mcr-1 gene.

52Furthermore, the effects of colistin resistance on virulence and on in vitro and in vivo fitness costs have been extensively studied in other GNB such as A. baumannii and K. pneumoniae. A study of the fitness costs of colistin resistant Salmonella pmrAB mutants in vitro and in a mouse model showed low fitness costs for these strains. Nevertheless, to the best of our knowledge no study has followed the fitness costs of colistin resistant E. coli mutants. Many studies have discussed the factors affecting the fitness cost of colistin resistance, including growth retardation, impaired virulence, increased susceptibility to other antibiotics, and substantially reduced clinical invasiveness. In swine, it has been reported that oral colistin treatment is accompanied by a selection pressure on the colistin resistant E. coli commensal population. Further investigations are required to study the fitness costs of colistin resistant E. coli and Salmonella of porcine origin.

53Before November 2015, several studies in human and in swine medicine confirmed the isolation of E. coli isolates confirmed resistant to colistin without having a mutation in pmrA and/or pmrB genes. The discovery for the first time in early November 2015 in China of a plasmid mediated colistin resistance-1 (MCR-1) protein in Enterobacteriaceae has provided explanation for the other potential colistin resistance mechanisms in GNB. Initially, this plasmid was considered to be a phenomenon relegated to China, however the mcr-1 gene was soon after isolated in several countries on 4 continents: Asia, Africa, Europe, and the Americas.

54Very recently, in June 2016, a novel plasmid-mediated colistin resistance gene, mcr-2, was identified in colistin resistance E. coli isolates from porcine and bovine origin in Belgium. The mcr-2 gene was detected with higher prevalence than of mcr-1 gene among colistin-resistant E. coli of porcine origin.

55MCR-1 and MCR-2 proteins showed 80.65% of identity and are members of the phosphoethanolamine transferase enzyme family that promotes the addition of a phosphoethanolamine group to lipid A, leading to a decreased affinity of colistin for the LPS. In study, the mcr-1 associated plasmid, designated pHNSHP45, is approximately 64 Kb in size and is an IncI2-like plasmid that harbors a predicted 83 open reading frames (ORFs) with a G+C content of 42.7%. The plasmid pHNSHP45 carrying mcr-1 gene was initially isolated in July 2013 from an E. coli strain recovered from a pig farm (Shanghai, China) and showed resistance to most antibiotic families except the carbapenems. Subsequently, mcr-1 has been reported in different plasmid incompatibility groups from different animal species, including IncHI2 (200–290 Kb), pVT553 (62 Kb), IncX4 (30 Kb), and IncP (79 Kb) plasmids in E. coli from broilers poultry, bovine, and swine origin and IncX4 (30 Kb) plasmids in Salmonella from chicken and turkey meat. isolated the mcr-1 gene in pKP81-BE plasmid (91 Kb) from colistin resistant E. coli of porcine origin. The pKP81-BE plasmid showed a G+C content of 44.9% and belonged to IncFII incompatibility type with 4% similarity compared to pHNSHP45. These findings showed that mcr-1 has horizontally transferred to other plasmid types, leading to an increase in its target bacterial range.

56The mcr-2 associated plasmid, designated pKP37-BE, is approximately 35 Kb in size and is an IncX4 incompatibility type, with a G+C content of 41.3%, and did not carry any other resistance genes.

57The mcr-1 gene has been identified in Enterobacteriaceae derived from humans, food, farm animals, vegetables, the environment including water, and even wild migratory bird. The mcr-1 gene has also been identified in several multidrug resistant bacteria such as ESBL producing and carbapenemase-producing E. coli of chicken and swine origin. In colistin resistant E. coli, a co-localization of mcr-1 and blaCTX-M genes on a unique IncHI2-type plasmid was also reported in chickens and in calves. The co-localization of mcr-1 with an ESBL gene on a conjugative plasmid increases the possibility of bacterial resistance to colistin and of broad-spectrum cephalosporins being maintained, even without the use of theses antibiotics in food animals. This finding poses significant challenges for successful clinical treatment of GNB and for resistance control strategies in both veterinary and human medicine. reported for the first time a chromosomally located mcr-1 gene in two colistin resistant E. coli isolated from veal calves. In this study, the mcr-1 gene was associated with the insertion sequence (IS) ISApl1-mcr-1 (or an mcr-1-containing mobile element) located immediately upstream of mcr-1, as also reported in plasmid pHNSHP45. ISApl1 is a member of the IS30 family, which was initially identified in Actinobacillus pleuropneumoniae. The presence of this IS in association with the mcr-1 gene strongly suggests that this gene is able to translocate to the chromosome and to different plasmid backbones – as well as between bacterial strains. Furthermore, the mcr-2 gene was associated with an IS of the IS1595 superfamily.

58In swine, to the best of our knowledge, the plasmid-borne mcr-1 gene has been observed in at least 2 enterobacterial species, E. coli and Salmonella, in ∼12 countries on four different continents. Pig-to-human transmission of MCR-1 colistin resistance has already been reported, raising serious concerns about the consequences of the use of this antibiotic in pig productions on human healthcare.

59In pigs, the mcr-1 gene was isolated mainly from colistin resistant E. coli strains with variable prevalence between countries; China (20.6%), Vietnam (22%), Belgium (13.2%), Brazil (2%), Spain (0.68%), Germany (0.51%), and France (0.50%). Most recently, in the USA pig production, the mcr-1 gene was identified for the first time in a colistin resistant E. coli strain isolated from a pig from South Carolina. In these studies, despite using the same technique (PCR) for mcr-1 gene screening, it is difficult to compare these results between countries because of the lack of data on previous antibiotic treatments in sampled pigs, on the quantities of colistin used at the farm level, on the potential combination of antibiotics with colistin, and on the health status of the pigs. Moreover, there are no published longitudinal studies on pigs that quantify the link between colistin quantities used on farms and the evolution of bacterial resistance against this antibiotic.

60Almost all studies conducted on pigs worldwide to screen mcr-1 gene presence in enterobacterial species reported that colistin resistant strains harboring this gene also showed resistance to one or several classes of antibiotics conventionally used in swine such as: Aminoglycoside, Sulfonamide, Trimethoprim, Tetracycline, Quinolone, Lincosamide, β-lactam, and third generation cephalosporin. This multi-resistance of mcr-1 positive E. coli strains in pigs was associated with the presence of a sul3-containing class 1 integron, In640, in the plasmid’s mediated mcr-1 gene. This integron showed the presence of genes encoding resistance to trimethoprim (dfrA12), aminoglycosides (aadA1a and aadA2), sulfonamides (sul3), and phenicols (cmlA1). Furthermore, IncX4 plasmids have been shown to harbor mcr-1 and mcr-2 genes a swell as ESBL genes.

61In the study of, the mcr-1 gene was screened and detected in three colistin resistant Salmonella strains isolated from 122 lymph nodes and in two colistin resistant E. coli strains isolated from 439 swine fecal samples. This study was the first in swine to demonstrate the existence of a plasmid carrying mcr-1 gene, in addition to a mutation in PmrAB TCS, in two colistin resistant E. coli strains. The coexistence of these two colistin resistance mechanisms in E. coli was not associated with a difference in the MIC of these strains compared to resistant Salmonella strains that expressed only the plasmid carrying mcr-1 gene. It should be stressed here that the mcr-1 gene found in colistin resistant enterobacterial strains of porcine origin was often associated with low levels of resistance; the MICs of 4 or 8 mg/L observed for most isolates are only 2–4 times higher than the EUCAST clinical breakpoint (2 mg/L). reported the isolation of a colistin-susceptible E. coli strain carrying the mcr-1 gene from the fecal sample of a healthy pig. This finding, suggests that mcr-1-positive isolates may be difficult to detect if only the mcr-1 gene is screened in colistin resistant isolates. Further studies are needed to examine the expression of mcr-1 gene in E. coli and to determinate the promoter and the operon responsible for this expression.

62Currently, colistin is an antibiotic widely used in veterinary medicine, particularly in pigs, for the oral treatment of intestinal infections caused by Enterobacteriaceae. In humans, colistin is used for the treatment of infections caused by MDR-GNB and is considered to be a last-resort antibiotic treatment option for carbapenemase-producing Enterobacteriaceae infections. During the last decade, research on colistin experienced a significant increase, especially regarding the mechanism of resistance of colistin and the optimization of its therapeutic regimen using the PK/PD relationship.

63Recently, the mcr-1 gene was isolated from colistin resistant E. coli strains from several farm animals: pigs, piglets, chickens, cattle, and veal calves. A strong similarity was found between the different classes of plasmid carried mcr-1 genes in these animal productions, and the successful gene-plasmid combination was mainly attributed to the presence of ISApl1 upstream in the mcr-1 gene. These findings are in favor of a possible movement of this mobile genetic element between the various animal productions (; Figure 2). In addition to their use in pigs, polymyxins and especially polymyxin B are used in some countries for the treatment of coliform and Pseudomonas mastitis in cows, and this antibiotic is sometime used for this purpose as an extra-label drugs in cattle such as in Canada and in the United States. Intramammary infusions of 1–2 million units of polymyxin B/quarter gave an efficiency for the treatment of cows with severe cases of coliform mastitis. Although, some studies have reported the isolation of colistin resistant E. coli strains harboring the mcr-1 gene from cow with mastitis, the role of polymyxin B, used for the treatment of mastitis, in colistin resistance still unknown. Furthermore, colistin is used outside of North America orally in calves and lambs at a dose of 100.000 IU/kg b.w day divided in two identical doses for three consecutive days for the treatment of gastrointestinal diseases caused by GNB. This use could explain the isolation of bacteria resistant to colistin in calves, even despite the lack of data on an preliminary treatment of these animals with colistin.

64On the other hand, colistin is used in some countries such as China for the control of intestinal infection caused by GNB in chicken, turkeys, rabbits and ducks. Colistin was incorporated into the feed of these animals at the dose of 3.33 mg/kg b.w for turkeys, 3.8 mg/kg b.w for rabbits and chickens, and 20 mg/kg for ducks. Colistin was also used in the drinking water in laying hens at the dose of 3.8 mg/ kg b.w. Furthermore, colistin is widely used in Europe for the oral treatment of E. coli infections in chicken and laying hens at the dose of 75.000 IU/kg b.w day for 3–5 consecutive days in the drinking water. Although, several studies have confirmed the isolation of bacteria resistant to colistin harboring mcr-1 gene from avian origin, however, to the best of our knowledge no scientific study has investigated the resistance of GNB to colistin in turkeys, rabbits, and ducks.

65In addition, the mcr-1 gene was also isolated from wild migratory birds such as the European herring gull (Larus argentatus) in Lithuania and the kelp gulls (Larus dominicanus) in Argentina. The role of these migratory birds in the spread of the mcr-1 gene between continents should not be underestimated.

66The mcr-1 gene was identified in resistant E. coli strains isolated from environmental samples such as river water, chicken feed in trough, and ready-to-eat vegetables. Therefore, the role of animal manure used in the fertilization of agricultural lands in the environmental dissemination of the mcr-1 gene needs to be verified. Several studies have reported the isolation of colistin resistant bacteria from pig manure.

67In addition, the mcr-1 gene was identified in resistant E. coli strains isolated from food samples such as chicken and pork meat, ground beef, and retail meats (chicken, pork, and beef). These foods of animal origin represent a major route of contamination with the mcr-1 gene for slaughterhouse workers and consumers (Figure 2).

68The gene encoding plasmid-mediated colistin resistance, was also identified in resistant E. coli strains isolated from humans with gastroenteritis or wound infections and from asymptomatic people. The mcr-1 gene was isolated from humans from four continents, showing that plasmid-mediated colistin resistance has already spread worldwide.

69It was reported that food animals are the main source of human contamination by the MCR-1 and MCR-2. However isolated the mcr-1 gene in colistin resistant E. coli from five children with ages ranging between 2 and 27 months who did not have pets or a history of animal contact. Moreover, despite the fact that colistin is not approved in animal production in the USA, reported for the first time in the USA, the identification of mcr-1 gene in a colistin resistant E. coli strain cultured from a woman with a urinary tract infection (UTI). However this strain remained susceptible to several other antimicrobial agents. These findings suggest that mcr-1 is already widespread in the environment and transmissible via various routes to humans. Thereby, there is also a potential risk of the transfer of mcr-1 gene from human to animal. However such transfer should be investigated in future studies.

70Most recently, mcr-1-harboring E. coli was isolated from healthy dogs and cats in a pet shop in Guangzhou, China. An interesting finding in this study was that the mcr-1 gene in colistin resistant E. coli was isolated from a worker at this pet shop – and it was the same E. coli strain clonally related to those originating from dogs. This finding is in favor of a possible transmission of mcr-1-harboring E. coli between dogs and humans.

71Polymyxins are used in dogs and cats mostly for topical indications. In fact, polymyxin B is used in the treatment of canine otitis externa, and it showed synergy with miconazole against E. coli and P. aeruginosa. Polymyxin B used also in ophthalmic suspension for the treatment of keratitis in dogs. For the treatment of this ophthalmic disease, polymyxin B is commonly associated with other drugs such as neomycin, and dexamethasone, or chloramphenicol. Furthermore, it was shown that colistin used at the dose of 12. 500 IU/kg IM for 5 days in combination with ampicillin had demonstrated an anti-endotoxic effects in dogs with naturally occurring endotoxic shock. Despite the isolation of E. coli resistant to polymyxins harboring the mcr-1 gene from dogs and cats, it is difficult to determine the role of polymyxin B administered topically in the exacerbation of colistin resistance in dog’s or cat’s intestine.

72Neither the role of waste and contaminants from the pharmaceutical industry nor the role of fish farms has been documented as a source of colistin resistance amplification in the environment. In fact, it has been reported that administration of colistin sulfate with other antibiotics in the diets of fish significantly improved feed conversion and promoted their growth rate. To the best of our knowledge, no study has documented the isolation of colistin resistant E. coli strains or mcr-1 gene from fish.

73Transmission of mcr-1 gene resistance from animals to humans can take place through a variety of routes (Figure 2). Therefore, the management of colistin resistance requires global and coordinated action between the different actors in order to intercept this resistance spread and preserve the efficacy of colistin for the treatment of MDR-GNB in human medicine.

74We believe that the One Health concept is more important than ever to better manage the impact of colistin resistance in human and veterinary medicine. Such a concept needs a global strategy to develop collaborations and interdisciplinary communication between concerned specialists (Figure 3).

75The use of colistin in swine has contributed to the intensification of modern pig productions by assuring successful weaning, higher animal densities, and most likely helped to reduce economic losses caused by E. coli infections such as PWD and edema disease. Economic gains have come at a considerable cost, which is being borne, in particular, by public health and other stakeholders such as the environment and the animals themselves. In fact, the recent discovery of a plasmid-mediated mcr-1 gene encoding for colistin resistance in Enterobacteriaceae has aroused great concern about the possible loss of colistin effectiveness for the treatment of MDR- GNB in humans. Because of the high rate of isolates carrying the mcr-1 gene isolated from animals compared to humans, livestock production has been pinpointed as a reservoir of the mcr-1 determinant, hence the need for rapid action in food animals to prevent the spread of colistin resistance (Figure 3). This section will focus on interventions in swine medicine but is applicable to all animal productions where colistin is used.

76This practice should be banned internationally. In addition to the fact that antimicrobials for growth promotion can generally be purchased without veterinary involvement, low subinhibitory concentrations of antibiotics used to improve animal growth has been shown to promote antibiotic resistance emergence. No recent studies have been able to clearly establish a link between the use of antibiotics as growth promoters and the improvement of animal performance in modern farming conditions with a high level of sanitation.

77This usage is involved in the increase of colistin quantities used in pigs and increases its presence as waste in the environment. Such usage of an antibiotic of very high importance in human medicine should be strictly avoided in swine. Intestinal disease prevention in pigs should be based mainly on livestock preventive management measures (optimal temperature, vaccination, sanitation, housing conditions, applying biosecurity rules, etc.).

78Nevertheless that colistin is a cheap therapeutic strategy with certain efficacy against Enterobacteria associated disease in swine, it has been shown that the oral use of colistin for the treatment of pigs in an experimental PWD model was associated with a pressure selection on E. coli populations. Therefore, the use of colistin as the first therapeutic choice to treat intestinal infections in pigs should be avoided. The therapeutic alternative to colistin should not be an antibiotic belonging to β-lactam family because of the co-localization of mcr-1 and ESBL genes in the same mobile genetic element. In addition, in its very recent advice, the EMA required that the reduction of colistin use in farm animals should not be associated with an increase in the consumption of fluoroquinolones, third- and fourth-generation cephalosporins, or the overall use of antimicrobials.

79Clinical diagnoses of the disease by veterinarians and the isolation of pathogen agent linked with the antibiogram tests to determinate bacterial susceptibility to colistin are essential to justifying its therapeutic use. Isolation from the animal husbandry of bacteria harboring the mcr-1 gene should be considered a strong reason not to use colistin on that farm.

80Moreover, the veterinarian should ensure that colistin prescribed is used in farms only for the treatment of sick pigs as recommended; compliance with label instructions (no underdosing or prolongation of dosing interval, withdrawal period) is of paramount importance. Any deviations from the guideline recommendations must be justified and recorded. In this context, extra-label use of colistin in some countries where this antibiotic is not approved in swine such as in Canada, must take place within a valid veterinarian-client-patient relationship. An analysis of the specific situation at farms and a determination that there are no alternatives to this antibiotic for the treatment of this case is required. Research is very important in order to establish a microbiological withdrawal period that could reduce the risk that pigs sent to slaughter contain colistin resistant bacteria or mcr genes in their gut.

81Veterinarians should ensure that colistin use targets clinical disease, should consider reduction of its use whenever practical, and should direct management and husbandry issues at the same time. Veterinarians should also consider laboratory examination as a routine practice to evaluate the effectiveness of colistin treatment and to monitor the sensitivity of infectious strains on the farm. Educational and awareness campaigns for employers and pig farmers are essential to generate an understanding that can support the veterinarian to withhold colistin. The professional organization of each country should develop clinical-practice guidelines on the judicious use of colistin. Data on colistin usage in food animals are critically important because they provide a basis for the development of national policies and they guide the risk of colistin resistance management and assess the effect of possible interventions. At a minimum, these data should include national use of colistin in kilograms of active ingredient on an annual basis and data should be stratified by animal species. The OIE and WHO recommend collecting the amount of antibiotics in food animals. Finally, the standardization of a data collection method regarding the use of colistin in farms between countries is very important to evaluate the effectiveness of such interventions to manage colistin resistance spread.

82There are many national antimicrobial resistance monitoring and surveillance programs that already exist and are well-established in many countries (; Table 6). Among the principles of the One Health approach is the improved use of existing natural resources and implementation, which includes the monitoring of colistin resistance spread in both human and veterinary medicine. However, regulations and practices vary widely between these surveillance programs and are influenced by the economic and social context of each country.

83Coordination between the various stakeholders is paramount for effective surveillance systems at the country level. In Canada, a new initiative to better manage the dissemination of antimicrobial resistance at the human-animal interface was established by the Public Health Agency of Canada in 2015. The aim of this program, called the Canadian Antimicrobial Resistance Surveillance System (CARSS), is to strengthen the coordination and integration of antimicrobial resistance and antimicrobial use activities and information in Canada and to consolidate surveillance from seven existing systems.

84Governments should fund research to enhance our understanding of environmental and genetics factors that facilitate the development of infectious disease in food animals, and to examine alternative strategies for the use of antibiotics on farms. Financial assistance for farmers in the implementation of sustainable practices and interventions to prevent infections, such as sanitation, housing, improvement of nutritional programs, and immunization, is very important for the reduction of the use of colistin or other antibiotics on farms. In addition, the preparation of guides and educational material for veterinarians and farmers on appropriate disease management and treatment based on the recent results of research is crucial for the responsible use of antimicrobials in farms. Efforts to improve microbiological laboratories are vital to help veterinarians undertake rapid therapeutic action with the most appropriate antibiotic and at an early stage of the disease. Finally, the competent authorities should clearly define guidelines for colistin marketing, sales, and use on farms.

85In addition to the isolation of colistin resistant bacteria from manure, water, migratory birds, and vegetables, the toxicity impact of colistin on the environment is a topic of concern. Indeed, it has been shown that the presence of colistin at therapeutic concentrations in swine farm wastewater was associated with a toxicity against ammonia-oxidizing bacteria (AOB). These AOB are involved in the biodegradation of xenobiotic compounds and in the conversion of ammonia to nitrites in wastewater treatment plants. The ecotoxicity effect of colistin was demonstrated in the earthworm Eisenia fetida; colistin caused significant damage to its intestinal epithelium and caused the induction of stress-related gene expressions.

86In addition, it has been reported that colistin-resistant E. coli were isolated from wild rabbits (Oryctolagus cuniculus) and wild hares (Lepus europaeus) that have not been previously treated with colistin. Consequently, wildlife may represent another potential reservoir of colistin resistance bacteria in the environment that could contaminate humans through contaminated food and water or by direct human and animal contact.

87This section will be devoted to the possible interventions to limit the spread of colistin resistant bacteria and genes in the environment via pig manure (Figure 3).

88It has been estimated that about 75% of the administered antibiotics is not absorbed by animals but is excreted via the feces or urine. This finding is even more pronounced with colistin, which is very poorly absorbed in animal’s gastrointestinal tract. It has also been reported that the frequency of bacteria carrying antimicrobial resistance genes is high in pig manure compared to other farm animals, and a high frequency and concentration of ARGs was detected around swine farms. Therefore, the role of pig manure is not to be underestimated in the dissemination of colistin resistance in the environment. It is crucial to consider reducing the use of antibiotics on farms, especially critically important antimicrobials, in favor of other measures such as the improvement of nutritional programs, housing, and animal immunization.

89Some studies have reported that composting eliminates on average 50–70% of some antimicrobials such as chlortetracycline, monensin, and tylosin and reduces the relative quantities of the blaTEM, sul3, and erm(B) genes in manure. However, to the best of our knowledge, no study has shown the efficacy of this technique in reducing amounts of colistin or mcr genes in pig manure.

90The effectiveness of reducing ARGs in pig manure depends mostly on the method manure is handled; aerobic biofiltration of manure has been reported to reduce erm(X) more effectively than other ARG such as erm(F), erm(B), and tet(G), while mesophilic anaerobic digestion and lagoon storage reduced none of these AR genes. There has been much controversy concerning the efficiency of these biological manure treatments, such as lagoons and composting, in ARG reduction, which is why more research is needed into assessing the effectiveness of swine waste treatment processes in the destruction of resistant bacteria and ARG in pig manure. With the lack of regulation worldwide or international guidelines to control the release of pig manure containing antibiotics, it is difficult to reduce the spread of colistin resistance into the environment by manure land applications.

91Colistin is currently considered to be one of the last-resort antibiotics used for the treatment of infections caused by MDR-GNB in humans. Maintaining the effectiveness of this antibiotic is a challenge for both scientists and physicians. Nevertheless, there are several possible proposals to optimize the use of colistin in human medicine (Figure 3).

92This step is crucial before undertaking a therapeutic intervention using colistin, and screening should be done in both patients with and without prior history of colistin usage. Hospitals should know whether or not their laboratories have the ability and the necessary equipment to perform colistin resistance testing and mcr-1 screening tests among admitted patients who needed colistin as a treatment.

93Hand hygiene plays a crucial role in achieving this goal. Interactive educational programs are important to explain the steps of hand hygiene technique as well as its rationale. Given the coproduction of mcr-1 genes and NDM enzymes by the same colistin resistant isolates, as reported by, we believe that the guide for the control of healthcare-associated infections due to carbapenem-resistant Enterobacteriaceae, published in 2012 by the USA Centers for Disease Control and Prevention (CDC) and updated in 2015 (available from: http://www.cdc.gov/hai/organisms/cre/cre-toolkit/index.html), would be a very good tool to prevent contamination by colistin resistant strains in hospitals. In addition, the identification of a patient carrying isolates that produce mcr-1 gene in association with carbapenemases should be strictly considered a reason for patient isolation.

94Epidemiological studies have described a possible horizontal transmission of a colistin resistant E. coli strains from pigs or from companion animals to humans following close contact. It has been shown that colistin-resistant E. coli was isolated from healthy individuals without prior colistin usage. Better hygiene, particularly hand washing with soap or using alcohol disinfectant after handling animals at a farm, pet shop, or slaughterhouse is obligatory. Also, using gloves during pig or manure handling and taking a shower at the exit of a piggery are mandatory practices that should be enforced. As well, employees must be particularly familiar with hand hygiene techniques and their purpose. Considering that a high percentage of colistin resistant E. coli is isolated from retail meat, consumers should avoid any type of cross contamination between meat and salad or other raw foods.

95In the intensive care unit, colistin is sometimes used orally for selective decontamination of the digestive tract (SDD), mainly to target resistant gram-negative aerobic bacteria, along with a short course of a parenteral broad-spectrum antimicrobial such as cefotaxime (a third generation cephalosporin). This practice has been shown through meta-analysis of randomized control trials to reduce the occurrence of respiratory tract infections, mortality, and overall bloodstream infections in critically ill patients. However, it has been demonstrated that prolonged use of colistin as part of SDD is associated with the emergence of colistin resistance among ESBL producing K. pneumoniae isolates. The long-term effects of colistin use in SDD was singled out as a possible source of colistin resistance amplification, therefore the re-evaluation of this practice is a topic of concern for intensive care units.

96Several in vitro and in mouse model studies have shown that combination of colistin with other antimicrobials such as rifampicin and imipenem may be more effective than colistin monotherapy in the treatment of MDR-GNB. A review of 15 studies involving 55 unique patient cases found that clinical success was lower for colistin monotherapy compared with colistin combination therapy for treatment of infections caused by K. pneumoniae carbapenemases (KPCs) producers. However, another review reported considerable controversy regarding the clinical efficacy of colistin combination therapy during the treatment of MDR-GNB. This interesting therapeutic approach needs to be clinically studied in depth to assess its effectiveness and its impact in MDR-GNB resistance occurrence.

97Colistin is an antibiotic widely used in pigs for the oral control of bacterial infections caused by E. coli and Salmonella. The recent discovery of a plasmid-mediated mcr-1 gene encoding for colistin resistance in Enterobacteriaceae has generated great concern about the possible loss of effectiveness of colistin for the treatment of MDR-GNB in humans. Because of the large amounts of colistin used in food animals and particularly in pigs, pig production has been pointed to as the greatest cause of colistin resistance amplification and spread. Consequently, experts, scientists, and government agencies have called for a reduction of colistin use in pigs and stressed that this antibiotic should be used only for the treatment of diseased animals as a last-resort treatment under strict circumstances. The mcr-1 gene has been isolated on four continents from sources other than food animals, such as the environment and human origins, and some E. coli isolates carrying a plasmid-encoded mcr-1 gene were associated with ESBL or carbapenemases enzymes. This highlights the need for an overarching approach on the judicious use of all antibiotics, especially those of critical importance for human health. The One Health concept is more important than ever to better manage colistin resistance at the human- animal-environment interface through the use of adequate science-based risk management policies that respect interdisciplinary regulations. Finally, we should start thinking beyond colistin therapy in swine and begin evaluating the effectiveness of other alternative strategies against infections caused by Enterobacteriaceae.

98MR conceived and designed the study, wrote the paper; FB designed the study, revised the paper; WT designed the study, revised the paper; AL designed the study, participated in the drafting of the paper.

99The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.