A Review of Resistance to Polymyxins and Evolving Mobile Colistin Resistance Gene (mcr) among Pathogens of Clinical Significance

The global rise in antibiotic resistance in bacteria poses a major challenge in treating infectious diseases. Polymyxins (e.g., polymyxin B and colistin) are last-resort antibiotics against resistant Gram-negative bacteria, but the effectiveness of polymyxins is decreasing due to widespread resistance among clinical isolates. The aim of this literature review was to decipher the evolving mechanisms of resistance to polymyxins among pathogens of clinical significance. We deciphered the molecular determinants of polymyxin resistance, including distinct intrinsic molecular pathways of resistance as well as evolutionary characteristics of mobile colistin resistance. Among clinical isolates, Acinetobacter stains represent a diversified evolution of resistance, with distinct molecular mechanisms of intrinsic resistance including naxD, lpxACD, and stkR gene deletion. On the other hand, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa are usually resistant via the PhoP-PhoQ and PmrA-PmrB pathways. Molecular evolutionary analysis of mcr genes was undertaken to show relative relatedness across the ten main lineages. Understanding the molecular determinants of resistance to polymyxins may help develop suitable and effective methods for detecting polymyxin resistance determinants and the development of novel antimicrobial molecules.


Introduction
The polymyxin antibiotics colistin and polymyxin B have been recently revitalized as bactericidal drugs due to the increase in bacterial resistance to many commonly used antibiotics [1,2].Polymyxins were originally derived from the bacterium Paenibacillus polymyxa as the products of fermentation in the form of amphipathic lipopeptide molecules [3].Polymyxins were discovered in the 1940s to be cyclic lipodecapeptide antibiotics [4] and recognized for therapeutic use in the 1950s [4,5].Polymyxins contain conserved components that consist of a d-Phe 6 -l-Leu 7 segment, an N-terminal fatty acyl chain separated by cationic residues (l-α-γ-diaminobutyric acid (Dab)), and segments of the polar amino acid threonine (Thr) [6,7].Polymyxins target the negatively charged outer membrane lipopolysaccharides (LPSs) of Gram-negative bacteria [6].
There are five (A to E) types of polymyxins; however, only polymyxin B and polymyxin E (colistin) are available for clinical use.The difference between polymyxin B and E is a result of the replacement of the amino acid D-phenylalanine at the sixth position in polymyxin B with leucine in colistin [6,7].Both polymyxins are heterogeneous assemblies of chemically related molecules that differ from each other at the N-terminus fatty acyl group [8,9].For polymyxin B, the major fatty acyl groups are (S)-6-methyloctanoyl for polymyxin B1 and 6-methylheptanoyl for polymyxin B2, whilst in colistin, the main fatty acyl groups are (S)-6-methyloctanoyl for colistin A and 6-methylheptanoyl for colistin B [8,9].However, the exact proportions of each of these components can vary among different manufacturers or different batches of the same manufacturer [8][9][10].The use of polymyxins was limited partly due to their toxicity.However, polymyxins are now considered to be last resort antibiotics [11], even though bacteria are becoming resistant to polymyxin B and colistin [11,12].
The aim of this literature review was to decipher the evolving mechanisms of resistance to polymyxins among pathogens of clinical significance.A review of the literature was undertaken with the following keywords and Boolean search criteria "(polymyxins) AND (molecular mechanism of resistance) AND (mcr gene) AND (intrinsic resistance) AND (molecular evolution)".Two search engines were used: Scopus and Medline.The articles were restricted to original research articles, reviews, or case reports published in English with full versions available online.After obtaining relevant articles, their references were investigated for any additional articles that were pertinent to the aim of this study.

Rate of Resistance to Polymyxins among Pathogens of Clinical Significance
A constant and ongoing threat to public health is the global emergence of bacteria with multidrug resistance (MDR) and pan-drug resistance (PDR), rendering most or all commercially available antibiotics ineffective [12,13].Among the commonly resistant pathogens are Gram-negative bacteria [12].The World Health Organization (WHO) has identified several bacteria of critical importance due to their increasing resistance to antibiotics, namely, MDR Acinetobacter baumannii, carbapenem-resistant Enterobacteriaceae, and MDR Pseudomonas aeruginosa [12].To treat such pathogens, polymyxins have been considered as last-resort antibiotics [14].However, resistance to polymyxins has been reported to be frequent among clinical isolates of many of these Gram-negative bacteria [15,16].The emergence of mobilized colistin resistance (mcr)-containing plasmids and chromosomally integrated mcr-1 that mediate colistin resistance have generated a significant sense of global public health alarm, leading to concerns about the future effectiveness of colistin [17][18][19][20][21].
The rate of polymyxin resistance varies depending on the bacterial species and geographic location, and the exact rate of polymyxin resistance can only be determined using specific molecular studies or surveillance systems [22][23][24][25].Colistin resistance of A. baumannii was first reported in 1999, and its rate of resistance has gradually increased over the past few decades [26].In 2013, the European Antimicrobial Resistance Surveillance Network (EARS-Net) collected data from 17 countries in Europe and found an average resistance rate of 5%.A retrospective study from French Guiana in 2017 found a similar resistance rate at 4.4% to colistin [27].Another multi-center epidemiological surveillance study, MARATHON, reported a colistin resistance rate of 1.9% in A. baumannii isolates in Russia between 2013 and 2014 [28].However, Italy and Greece reported high rates of colistin resistance A. baumannii isolates, with over 80% resistance [29].A study conducted in South Korea of 265 isolates of Acinetobacter spp.collected from tertiary-care hospitals between 2002 and 2006 reported an overall resistance rate of 27.9% (74/265) and 18.1% (48/265) to colistin and polymyxin B, respectively [30].Another study based on SENTRY data from Korea revealed a high colistin resistance rate of 30.6% in A. baumannii isolates between 2006 and 2009 [15].Furthermore, in Brazil, the resistance rates of A. baumannii were 81.5% in 2016 and 78.5% in 2021.The minimum inhibitory concentrations (MICs) varied from 4 to 64 µg/mL for polymyxin B and from 16 to 128 µg/mL for colistin in 2016, while the colistin MIC was 128 µg/L in 2021 [31,32].
For P. aeruginosa, an increasing trend in resistance has been reported in the EARS-Net surveillance study, with a 1% to 4% rise in colistin resistance in Europe from 2013 to 2016 [33].In 2016, most of the colistin-resistant isolates were from Italy and Greece.A rate of 1-7% colistin resistance has been described in China [34,35].A resistance rate of 11.5% to polymyxins B has been described among P. aeruginosa strains isolated from keratitis in Australia and India [36].
For Klebsiella pneumoniae, an increasing rate of colistin resistance has been observed since the first report of colistin resistance in 2004 [37].There was an increasing rate of colistin resistance in K. pneumoniae in Tunisia from 3.6% in 2002 to 9.7% in 2013 [22].In Europe, the resistance rate of K. pneumoniae to polymyxins increased from 1.1% to 2.2% between 2003 and 2009 [23].The European Antimicrobial Resistance Surveillance Network (EARS-Net) reported that in 2014, the highest resistance rate of 25.8% to polymyxins was found in Greece [38].In this report, colistin resistance was at 29% among all carbapenemresistant K. pneumoniae strains and 3% among carbapenem-susceptible isolates [38].A study conducted in 2018 reported colistin resistance rates of 27% and 43% among carbapenemresistant K. pneumoniae isolates in Dubai and Italy, respectively [39].
According to a study on the global prevalence of colistin resistance, the highest rate of colistin resistance was observed in K. pneumoniae isolates in 2020, with a resistance rate of 12.9% (4 out of 31) [40].In contrast, the colistin resistance rate among K. pneumoniae isolates studied from 2015 to 2019 was 2.9%.The study also found that K. pneumoniae isolates from Thailand had the highest rate of colistin resistance at 19.2%, while South Korea had the lowest prevalence of colistin resistance at 0.8% [40].Reports from India indicated the rates of colistin resistance were 1.3% among a total of 21.3% ICU isolates and 38.4-46.6%among other clinical isolates of K. pneumoniae in 2020 and 2021 [41,42].
Contrary to A. baumannii and K. pneumoniae, colistin resistance is not so common in clinical isolates of Escherichia coli.For example, the colistin resistance rate was 0.2% and 0.9% among clinical and commercial meat specimens, respectively, between 2010 and 2014, and 1.1% to 8.7% in E. coli between 2012 and 2015, respectively, in Taiwan [43].In China, resistance rates to polymyxin B and colistin of 0.3% to 7.3% among clinical isolates of E.coli have been reported in reports from 2013 to 2016 and 2021, respectively [44,45].
A study in 2022 demonstrated that 15% of carbapenem-resistant Enterobacterales isolates in India showed resistance to colistin [46].The geographical distribution of significant polymyxin resistance strains is shown in a geospatial map in Figure 1.
colistin resistance in K. pneumoniae in Tunisia from 3.6% in 2002 to 9.7% in 2013 [22] Europe, the resistance rate of K. pneumoniae to polymyxins increased from 1.1% to 2. between 2003 and 2009 [23].The European Antimicrobial Resistance Surveillance N work (EARS-Net) reported that in 2014, the highest resistance rate of 25.8% to polymyx was found in Greece [38].In this report, colistin resistance was at 29% among all c bapenem-resistant K. pneumoniae strains and 3% among carbapenem-susceptible isola [38].A study conducted in 2018 reported colistin resistance rates of 27% and 43% amo carbapenem-resistant K. pneumoniae isolates in Dubai and Italy, respectively [39].
Data from an epidemiological survey of 25 provinces in China found colistin sistance at 1.4%, 2.9%, 2.4%, and 4% in K. pneumoniae, Enterobacter cloacae, Citrobacter fre dii, and E. coli, respectively, among 1801 carbapenem-resistant Enterobacteriaceae (CR clinical strains between 2012 and 2016 [24]. According to a study on the global prevalence of colistin resistance, the highest r of colistin resistance was observed in K. pneumoniae isolates in 2020, with a resistance r of 12.9% (4 out of 31) [40].In contrast, the colistin resistance rate among K. pneumon isolates studied from 2015 to 2019 was 2.9%.The study also found that K. pneumoniae i lates from Thailand had the highest rate of colistin resistance at 19.2%, while South Ko had the lowest prevalence of colistin resistance at 0.8% [40].Reports from India indica the rates of colistin resistance were 1.3% among a total of 21.3% ICU isolates and 38 46.6% among other clinical isolates of K. pneumoniae in 2020 and 2021 [41,42].
Contrary to A. baumannii and K. pneumoniae, colistin resistance is not so common clinical isolates of Escherichia coli.For example, the colistin resistance rate was 0.2% a 0.9% among clinical and commercial meat specimens, respectively, between 2010 a 2014, and 1.1% to 8.7% in E. coli between 2012 and 2015, respectively, in Taiwan [43] China, resistance rates to polymyxin B and colistin of 0.3% to 7.3% among clinical isola of E.coli have been reported in reports from 2013 to 2016 and 2021, respectively [44,45] A study in 2022 demonstrated that 15% of carbapenem-resistant Enterobacterales i lates in India showed resistance to colistin [46].The geographical distribution of sign cant polymyxin resistance strains is shown in a geospatial map in Figure 1.

Mechanisms of Resistance to Polymyxins in Different Bacteria
Polymyxin-resistant bacteria can cause serious infections and pose a significant threat to public health [7,47].Bacterial cells have evolved various mechanisms to develop resistance toward polymyxins, including modification of the outer membrane, alterations in lipid A, and the use of efflux pumps.Cross-resistance between colistin and polymyxin B has been reported.Two primary mechanisms are responsible for the development of polymyxin resistance: (i) intrinsic mechanisms and (ii) acquired plasmid-mediated mcr-based mechanisms.

Evolving Intrinsic Mechanisms of Resistance to Polymyxins
In the past, resistance to polymyxins was primarily attributed to mutations in chromosomal genes linked to the synthesis of lipopolysaccharides (LPSs) [48].This resistance is commonly associated with two-component systems, often PhoPQ and PmrAB, as well as sets of regulatory genes such as the operon arnBCADTEF (also known as pmrHFIJKLM), crrAB, mgrB, and pmrE [49][50][51][52].
Cationic antibiotics such as polymyxins trigger the loss of cations (Ca 2+ and Mg 2+ ) from the negatively charged outer membrane of Gram-negative bacteria.This stress, as well as a high Fe 3+ concentration and acidic pH, can activate the two-component systems PhoPQ and PmrAB and the arnBCADTEF cascade [14,53,54].PhoPQ and PmrAB induce the synthesis of phosphoethanolamine (PEA) and/or 4-amino-4-deoxy-L-arabinose (L-Ara4N), which are then integrated into outer membrane LPS [53].This addition of PEA or L-Ara4N provides additional cationic groups, leading to modifications in LPSs that neutralize the negative charge on the outer membrane, hindering further binding of colistin [53].Increased expression of arnBCADTEF correlates with polymyxin resistance [55].Mutations in pmrB cause high expression of pmrC, leading to the modification of lipid A with PEA in K. pneumoniae [56].Also, exposure to chlorhexidine is associated with the development of colistin resistance in K. pneumoniae due to a point mutation in pmrB [57].
Furthermore, mgrB is involved in the feedback control of PhoPQ, and thus alterations in mgrB can also contribute to the development of polymyxin resistance.Mutations in crrB that induce crrC expression lead to hyper-expression of the pmrAB system and ultimately the development of resistance [58].Additionally, the regulatory systems of colistin resistance involve vprAB in Vibrio cholerae, and cprRS and parRS in P. aeruginosa, which affect cation peptides in the outer membrane [59,60].Common pathways of polymyxin resistance in E. coli, K. pneumoniae, and P. aeruginosa are shown in Figure 2.

Distinct Mechanisms of Intrinsic Polymyxin Resistance in A. baumannii
A. baumannii exhibits unique mechanisms of colistin resistance.The first mechanism involves the complete loss of lipooligosaccharide, which is caused by mutations in lipopolysaccharide peroxidation (LpX) genes (lpxA, lpxC, lpxD) and vacJ [61], responsible for lipid A synthesis.These mutations lead to permeability defects due to the encoding of acyltransferases, which are key enzymes in lipid A biosynthesis [62][63][64][65][66].The second mechanism involves lipo-oligosaccharide modification by the addition of PEA or the transfer of L-Ara4N to the phosphate groups of lipid A [62,67].There are two distinct pathways for the regulation of this modification.The first pathway involves the pmrAB operon, a two-component system that induces pmrC, which results in an LPS modification [2,61,64,65,68,69].The second mechanism involves the insertion of the IS element ISAba125 into the transcriptional regulator H-NS family to increase the expression of eptA encoding for PEt that synthesizes PEA.This reduces the overall membrane electronegativity and so a reduction in membrane affinity for polymyxins [70,71].Colistin resistance can also result from the loss of OmpW and production of DedA, as well as the expression of eptA in Acinetobacter spp.[72].
that induce crrC expression lead to hyper-expression of the pmrAB system and ultima the development of resistance [58].Additionally, the regulatory systems of colistin sistance involve vprAB in Vibrio cholerae, and cprRS and parRS in P. aeruginosa, which a cation peptides in the outer membrane [59,60].Common pathways of polymyxin sistance in E. coli, K. pneumoniae, and P. aeruginosa are shown in Figure 2. Activation of the two-component systems PhoP-PhoQ and PmrA-PmrB occurs because of sensing the flow of molecules and is represented with small black arrows.The subsequent activation of the arnBCADTEF and pmrCAB operons leads to the synthesis and addition of 4-amino-4-deoxy-Larabinose (L-Ara4N) and phosphoethanolamine (PEA) to lipid A, respectively.The addition of PEA by phosphoethanolamine transferase (PEt) and L-Ara4N by L-Ara4N formyltransferase is denoted with thick black arrows, while PEA addition through mcr genes is represented with a thick blue arrow.Additionally, PhoP-PhoQ activation induces PmrAB through the product of the pmrD gene, which, in turn, activates pmrA to further trigger the arnBCADTEF operon.The PmrB and PhoQ activation is denoted with star-like symbols.Polymyxin resistance is also associated with the inactivation of MgrB, a negative regulator of the PhoP-PhoQ system.Amino acid substitutions in MgrB result in its inactivation, leading to overexpression of the phoP-phoQ operon and subsequent activation of the pmrHFIJKLM operon, which ultimately leads to the production of L-Ara4N.
In 2022, a new two-component system (TCS) named StkSR was discovered in A. baumannii [73].Deletion of stkR significantly increased the expression of pmrA, pmrC, and pmrB, leading to an increase in pmrC transcription and subsequent substitution of lipid A with PEA.There may be a regulatory relationship between the StkSR and the PmrAB systems based on the observed correlation in gene expression [73].Distinct pathways of polymyxin resistance in Acinetobacter spp.are depicted in Figure 3.

Plasmid-Mediated mcr Gene-Based Polymyxins
Mobilized colistin resistance, mcr, genes are mainly associated with bacteria mids.These play an important role in the spread of colistin resistance because o transferability among different strains in different environments [28,44,53,74].
These mcr genes encode phosphoethanolamine-lipid A transferases [75,76] th diate the addition of PEA to the lipid A of an LPS at the 1′ and 4′ positions, cau significant reduction in the overall negative charge on the bacterial outer mem [77,78].This ultimately leads to the loss of binding affinity of an LPS to the cationi myxins and therefore resistance to their action [76,78].

Plasmid-Mediated mcr Gene-Based Polymyxins
Mobilized colistin resistance, mcr, genes are mainly associated with bacterial plasmids.These play an important role in the spread of colistin resistance because of their transferability among different strains in different environments [28,44,53,74].
These mcr genes encode phosphoethanolamine-lipid A transferases [75,76] that mediate the addition of PEA to the lipid A of an LPS at the 1 and 4 positions, causing a significant reduction in the overall negative charge on the bacterial outer membrane [77,78].This ultimately leads to the loss of binding affinity of an LPS to the cationic polymyxins and therefore resistance to their action [76,78].
The mcr-3 variants are very similar to mcr-1 with 45% nucleotide sequence similarity [81] and high protein similarity of 60% to each other [82].To date, mcr-3 has 42 variants, To gauge the phylogenetic relatedness of mcr variants, mcr sequences were retrieved from the NCBI GenBank, and their sequences were aligned using ClustalW.A neighborjoining phylogenetic tree was constructed using MEGA 11 [102] and visualized using iTOLv6 [103] (Figure 4).To gauge the phylogenetic relatedness of mcr variants, mcr sequences were retrieved from the NCBI GenBank, and their sequences were aligned using ClustalW.A neighborjoining phylogenetic tree was constructed using MEGA 11 [102] and visualized using iTOLv6 [103] (Figure 4).
The transmission of mcr genes carrying pathogens could occur from animals to humans via direct contact with food animals and pets [113][114][115].Also, reservoirs for mcr-1-carrying bacteria have been identified in public beaches [116], hospital sewage, wastewater treatment plants [117,118], rivers [115], and water wells in rural areas [119], as well as from houseflies and blowflies [120].Although data from some studies suggests that flies might be intermediate vectors for transmission of mcr-1-containing bacteria between companion animals and humans [121], the exact route for the spread of mcr-1 and the bacteria carrying mcr-1 needs more thorough investigation.
Several species of Enterobacteriaceae possess mcr-1, such as E. coli where the gene is carried on IncI2 and IncX4 plasmids [122], Enterobacter aerogenes on an IncX4 plasmid [123], E. cloacae on an IncFI plasmid [123], Cronobacter sakazakii on an IncB/O plasmid [124], Citrobacter freundii on an IncHI2 plasmid [125], C. braakii on an IncI2-type plasmid, K. pneumoniae on an IncX4 plasmid [126], Salmonella enterica on IncHI2-like plasmids [127], Shigella sonnei on IncHI2like plasmids [128], and Raoultella ornithinolytican on an IncHI2 plasmid [129].Also, mcr-1 variants have been identified in strains co-harboring bla NDM-5 that confers carbapenem resistance to E. coli [108].The mcr-1.1 gene has been found in the chromosome of E. coli and plasmid p16BU137 of K. pneumoniae from environmental isolates in China [76].Further details of recently discovered mcr variants and their respective transposons and plasmids are given in Table 1.In Australia, colistin resistance was reported among poultry isolates of Aeromonas hydrophila, Alcaligenes faecalis, Myroides odoratus, Hafnia paralvei, and Pseudochrobactrum spp.from a chicken processing unit in the state of Victoria [130].Furthermore, mcr-1 was found in association with incompatibility group IncI2 plasmids from isolates in the state of New South Wales (NSW) [131], and mcr-1.1 has been detected in E. coli [132].Similarly, mcr-1.1 and mcr-3 were found among MDR isolates of Salmonella enterica 4 from human and animal sources in NSW [132,133].An evolutionary analysis of multiple drug-resistant Salmonella enterica serovar 4 indicated that the spread of the mcr-3 variant in lineages 1 and 3 was associated with overseas travel to Southeast Asia [84].Lineage 1 included mcr-3.1andbla CTX-M-55 -positive isolates of Salmonella enterica sequence type 34 from Europe and Asia that were resistant to colistin and third-generation cephalosporins [81,84].Whilst mcr-3.2 in lineage 3 was associated with IncHI2 pST3 and IncAC plasmids, wherein the colistin resistance genes were part of dgkA (diacylglycerol kinase) [84,134], which is a small transposable unit associated with IS elements circularized and integrated into Enterobacterales genomes [80].

Evolution of mcr Gene Variants from mcr-1 to mcr-10
In the current study, the phylogeny among mcr variants was determined using Molecular Evolutionary Genetics Analysis (MEGA 11) and is shown in Table 1.This shows the pair-end number of substitutions between mcr-1 and mcr-10, with the number of base differences per site indicated.An estimate of evolutionary divergence between the sequences of mcr-1 and mcr-10.1 was performed using MEGA 11.Overall, the average divergence among mcr ranged from 52 ± 20% for mcr-2 compared to all others to 69 ± 4% for mcr-8.
Moreover, phytogenic analysis of mcr-3 also demonstrated that most occurred and evolved among Aeromonas species.This suggested the origin of mcr-3 was Aeromonas species with gradual evolution and transmission of mcr-3 variants to E. coli and K. pneumoniae, while other mcr gradually evolved among E. coli and K. pneumoniae.Interestingly, after the emergence of mcr-4, the identification of mcr-4.3 in A. baumannii represented a gradual evolution of A. baumannii against colistin with a distinct type of mcr gene in the form of a novel plasmid carrying mcr-4.3[135].
The analysis of evolutionary probabilities in mcr variants used a previously described method [136] using modified evolutionary probabilities (EPs) [137].A user-specified tree topology was analyzed using the maximum likelihood method and the general time reversible model [138].The evolutionary time depths used in the EP calculation can be obtained using the real-time [139] method.This analysis involved using the 10 nucleotide sequences of mcr.Codon positions included the first + second + third plus the noncoding positions.All positions containing gaps and missing data were eliminated (complete deletion option).The results, which represent the number of base differences per site for each mcr variant, are depicted in (Figure 5).The probability of substitution of nucleotides to mcr-1 is demonstrated in Figure 5, which shows that the most likely substitution of adenine was with guanine (12%), of thymine was with cytosine (15%), of cytosine was with thymine (15%), and of guanine was with adenine (11%).The positions of substitution of nucleotides (A, T, G, and C from position 1 to 262 of different sites) for mcr-1 (E. coli strain ZZ1409 KU886144) are shown in Figure 6, respectively.In terms of positioning, cytosine (C) is predominately present at positions 1 to 257, followed by adenine (A) from positions 1 to 253, guanine (G) from positions 1 to 261, and thymine (T) from positions 5 to 261.In terms of probability and position of substitution, guanine was mostly likely to be present at position 27 with a probability of 0.95, and least likely to be present at position 28 with a probability of substitution of 0.007; thymine was most likely to be present at position 30 with a probability of 0.95 and least likely to be present at position 28 with a probability of 0.007; adenine was most likely to be present at position 220 with a probability of 0.94 and least likely to be present at position 27 with a probability of 0.007; cytosine was most likely to be present at position 160 with a probability of 0.93 and least likely to be present at position 262 with a probability of 0.014.

The Processes and Molecular Vehicles Responsible for the Transmission of mcr Variants
Studies have comprehensively analyzed the genetic environments of mcr-carrying genomes using bioinformatics tools such as Geneious R8 [140] and ISfinder software [141] to demonstrate the insertion of mcr variants.The structures of recently reported insertion sequences and the names of their associated transposons are given in Table 2.
Full genome sequencing and analysis for identification of replication origin (oriC) in mcr-1-harboring plasmids from colistin-resistant isolates have identified a novel hybrid IncI2/IncFIB plasmid pGD17-2 [142].Moreover, the co-occurrence of pGD17-2 with plasmids pGD65-3, IncI2, and pGD65-5, IncX4 has been reported in a single drug-resistant isolate (GD65), and this co-occurrence might promote the dissemination of mcr-1 under environmental selection pressure [142].mcr-1 and other clinically significant resistant genes such as extended-spectrum β-lactamase (ESBL) blaCTX-8 and blaCTX-M-1 are related to globally identified sequence types ST10, ST46, and ST1638 in pathogenic strains Studies have comprehensively analyzed the genetic environments of mcr-carrying genomes using bioinformatics tools such as Geneious R8 [140] and ISfinder software [141] to demonstrate the insertion of mcr variants.The structures of recently reported insertion sequences and the names of their associated transposons are given in Table 2.
Full genome sequencing and analysis for identification of replication origin (oriC) in mcr-1-harboring plasmids from colistin-resistant isolates have identified a novel hybrid IncI2/IncFIB plasmid pGD17-2 [142].Moreover, the co-occurrence of pGD17-2 with plasmids pGD65-3, IncI2, and pGD65-5, IncX4 has been reported in a single drug-resistant isolate (GD65), and this co-occurrence might promote the dissemination of mcr-1 under environmental selection pressure [142].mcr-1 and other clinically significant resistant genes such as extended-spectrum β-lactamase (ESBL) blaCTX-8 and blaCTX-M-1 are related to globally identified sequence types ST10, ST46, and ST1638 in pathogenic strains of E. coli responsible for infections in humans and animals [143][144][145].E. coli ST10 stains carrying mcr-1 have been isolated from water at a public beach in the USA where the same ST10 strain had been isolated from an infected migratory Magellanic penguin with pododermatitis [143], suggesting that the ST10 strains carrying mcr-1 can disseminate in the marine environment.E. coli mcr-1-positive environmental isolates have been isolated from German swine farms [146] and in diseased food animals in China [147], Italy, and France [148].A plastidome analysis of mcr-1 of Enterobacterales human isolates suggested that the spread of mcr-1 among commensals such as K. pneumoniae, E. coli, and other clinical isolates could be facilitated by various promiscuous diverse plasmids [149].
Insertion sequences (ISs) or integrons can also facilitate the spread of mcr.An analysis of mcr-1 from various sources using whole genome sequencing supported a single mcr-1 mobilization event in ISApl1-mcr-1-orf -ISApl1 transposon [150].This transposon has been immobilized on different plasmids such as IncI2, IncHI2, and IncX4 [151].Plasmids pGD65-3, IncI2, and pGD65-5, IncX4 contain two insertion sequences, ISEcp1 and ISApl1, that facilitate the mobilization of mcr-1 [142].The insertion sequence ISApl1, which originated in Actinobacillus pleuropneumoniae, is located upstream of mcr-1 in the IncI2-type mcr-1-harboring plasmid Phnshp45 [74,152,153].However, the ISApl1 element is not always found associated with mcr-1 on most IncX4 plasmids [152][153][154].A reason for this may be that the translocation of an mcr-1-pap2 element by integration of an ISApl1 cassette (a member of the IS30 family) [134,152] into plasmids such as pMCR1-IncI2, and pMCR1-IncX4 may induce the formation of circular intermediates by recognizing inverted repeat sequences, which ultimately results in loss of ISApl1 after integration of mcr-1 [134,155,156].
The mcr-2 gene is not associated with ISApl1, but there are two IS1595-like insertion sequences predicted to surround mcr-2 in the IncX4 plasmid pKP37-BE [157].The short IS1595-like element carries a transposase gene flanked by two inverted repeats surrounding mcr-2.This transposase-encoding gene is similar (75% identity) to a fragment found in Moraxella bovoculi strain 58069, which suggests the origin of mcr-2 was from M. bovoculi [155].The occurrence of duplicate target sites adjacent to a spacer sequence suggests that the spacer sequence is the most probable hot site in IncX4 plasmids for integration and transposition of mcr-2 variants [158].Transfer of mcr-2 can occur through IS1595-containing transposons [155,156,158,159].

Methods for Detecting Polymyxin Resistance
As resistance to polymyxins is being reported frequently among different bacterial isolates from humans, animals, and the environment, affordable, accessible, and efficient diagnostic approaches are needed.The phenotypic determination of colistin-resistant isolates can be made by growing on media such as CHROMagar COL-APSE [171], SuperPolymyxin™ [172], and LBJMR [173], as well as using commercial automated MIC-determining instruments such as BD Phoenix, MicroScan, Vitek 2 [174], MICRONAUT-S [175], and Sensititre [176].The rapid polymyxin NP test and its modifications [177], colispot [178] colistin MAC test [179], MIC Test Strip, MICRONAUT-MIC Strip [180], the UMIC System [181], and Sensitest Colistin [176] can also be used [174].Eazyplex SuperBug kit [182] and Taqman/SYBR Green real-time PCR assays have been used for molecular identification of mcr genes that have yielded 100% specificity and sensitivity with a rapid turnaround time (<3 h) [183].More advanced molecular techniques such as multi-loop-mediated isothermal amplification (multi-LAMP) assays can also be used for rapid detection of mcr genes [184].Based on cost, sensitivity and specificity, turnaround time, and the skills required to perform the test, the use of culture media or the Rapid Polymyxin Nordmann-Poirel (RPNP) test are recommended for low-resourced laboratories, while Multiplex PCR or Taqman/SYBR Green real-time PCR assays along with RPNP or novel culture media are applicable for well-resourced laboratories [185,186].
To study the evolution in mcr-positive bacterial strains, different sequencing techniques can be used including Sanger sequencing and the identification of single nucleotide polymorphisms [187] for mutational analysis or identification of new mcrvariant(s) [188].For detailed studies of intrinsic determinants of resistance, whole genome sequencing (WGS) [189], nanopore sequencing, and transposon-directed insertion site sequencing [165] can give insights into the interactions of genetic elements associated with polymyxins resistance.To study coevolution among pairs of mcr or multiple mcr elements within a single bacterial cell, mcr-coevolution assays could be used [165].

Conclusions
Polymyxins are a class of cationic polypeptide antibiotics that are involved in the disruption of LPS in Gram-negative pathogens.Polymyxins have been extensively used to treat infections after their initial approval for clinical use, but their use has been limited due to their nephrotoxicity and neurotoxicity.However, the development of bacterial strains resistant to many other types of antibiotics has led polymyxins to be reconsidered as a lastresort therapeutic option to treat MDR pathogens.This reuse has led to the re-emergence of resistance to polymyxins.Bacterial cells evolve resistance to polymyxins by modifying their LPSs, using antibiotic efflux pumps, or reducing the amount of LPSs produced.Mutations to existing genes or acquisition of mobile genetic elements such as transposons and insertion sequences play a major role in the development of resistance to polymyxins.mcr genes can be major role players in the global spread of colistin resistance because of their high mobility via plasmids.To date, several mcr variants (mcr-1 to mcr-10) have been reported among colistin-resistant pathogens.A detailed understanding of the molecular determinants underlying resistance to polymyxins, and ways of testing for resistance, can help to develop suitable and effective methods for detecting resistance to polymyxins, as well as aiding in the development of novel antimicrobials in the future.Therefore, ongoing research into the molecular determinants of polymyxin resistance is important for the development of effective strategies to combat antibiotic resistance and ensure the continued efficacy of polymyxins and other antimicrobial agents in clinical practice.

Figure 1 .
Figure 1.The global burden of significant polymyxin resistance from 2002 to 2022.

Figure 1 .
Figure 1.The global burden of significant polymyxin resistance from 2002 to 2022.

Figure 2 .
Figure 2. The mechanisms involved in the modification of lipopolysaccharides (LPSs) that contribute to polymyxin resistance in Gram-negative bacteria.In various bacteria such as Salmonella spp., Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, the detection of different stress conditions triggers a response mediated by the histidine kinases PhoQ and PmrB.(The process of gene activation through phosphorylation is represented with dashed arrows, and the outcomes are represented with thick arrows).These stress conditions include the presence of cationic compounds like polymyxins, low concentrations of Mg 2+ and Ca 2+ , acidic pH, and high concentrations of Fe 3+ .Activation of the two-component systems PhoP-PhoQ and PmrA-PmrB occurs because of sensing the flow of molecules and is represented with small black arrows.The subsequent activation of the arnBCADTEF and pmrCAB operons leads to the synthesis and addition of 4-amino-4-deoxy-Larabinose (L-Ara4N) and phosphoethanolamine (PEA) to lipid A, respectively.The addition of PEA by phosphoethanolamine transferase (PEt) and L-Ara4N by L-Ara4N formyltransferase is denoted with thick black arrows, while PEA addition through mcr genes is represented with a thick blue arrow.Additionally, PhoP-PhoQ activation induces PmrAB through the product of the pmrD gene, which, in turn, activates pmrA to further trigger the arnBCADTEF operon.The PmrB and PhoQ activation is denoted with star-like symbols.Polymyxin resistance is also associated with the inactivation of MgrB, a negative regulator of the PhoP-PhoQ system.Amino acid substitutions in MgrB result in its inactivation, leading to overexpression of the phoP-phoQ operon and subsequent activation of the pmrHFIJKLM operon, which ultimately leads to the production of L-Ara4N.

Figure 3 .
Figure 3. Distinct mechanisms of polymyxin resistance in Acinetobacter baumannii attributed ations in either PmrB or the response regulator PmrA, both of which are involved in activa PmrAB two-component system.This activation leads to the upregulation of the pmrCAB ope the naxD gene (as distinct pathways).The upregulation of the pmrCAB operon promotes th tion of PEA to lipid A, while the translation of naxD produces NaxD deacetylase, which is r for galactosamine addition to Lipid A. Another distinct possible pathway leading to PEt o duction in A. baumannii involves ISAbaI insertion element integration upstream of an eptA i Additionally, a mutation in lpxACD causes total loss of LPS Acinetobacter spp., which is a dis pathway of polymyxin resistance.Furthermore, a recently discovered novel pathway of pol resistance in A. baumannii involves the deletion of stkR, which significantly increases the exp of pmrA, pmrC, and pmrB and ultimately increases pmrC transcription and the subsequent su tion of lipid A with PEA.Additionally, an uncommon pathway of Lipid A modification invo mobile colistin resistance gene mcr, which encodes PEt, and the subsequent addition of PEA t

Figure 3 .
Figure 3. Distinct mechanisms of polymyxin resistance in Acinetobacter baumannii attributed to alterations in either PmrB or the response regulator PmrA, both of which are involved in activating the PmrAB two-component system.This activation leads to the upregulation of the pmrCAB operon and the naxD gene (as distinct pathways).The upregulation of the pmrCAB operon promotes the addition of PEA to lipid A, while the translation of naxD produces NaxD deacetylase, which is required for galactosamine addition to Lipid A. Another distinct possible pathway leading to PEt overproduction in A. baumannii involves ISAbaI insertion element integration upstream of an eptA isoform.Additionally, a mutation in lpxACD causes total loss of LPS Acinetobacter spp., which is a distinctive pathway of polymyxin resistance.Furthermore, a recently discovered novel pathway of polymyxin resistance in A. baumannii involves the deletion of stkR, which significantly increases the expression of pmrA, pmrC, and pmrB and ultimately increases pmrC transcription and the subsequent substitution of lipid A with PEA.Additionally, an uncommon pathway of Lipid A modification involves the mobile colistin resistance gene mcr, which encodes PEt, and the subsequent addition of PEA to LPSs.

Figure 4 .
Figure 4.The phylogenetic relationship among the mcr-1 to mcr-10 variants using the neighbor-joining phylogenetic tree using the Kimura parameter with 1000 bootstraps using MEGA10, and visualised using iTOLv5 (Interactive Tree Of Life).

Figure 5 .
Figure 5.The probability of substitution of one base for another base.Substitution patterns and rates were estimated using the general time reversible model [1].The maximum log-likelihood for this computation was 2655.269.This analysis involved all 10 nucleotide sequences of mcr.Codon positions included were 1st + 2nd + 3rd + noncoding.All positions containing gaps and missing data were eliminated (complete deletion option).

Figure 5 .
Figure 5.The probability of substitution of one base for another base.Substitution patterns and rates were estimated using the general time reversible model[1].The maximum log-likelihood for this computation was 2655.269.This analysis involved all 10 nucleotide sequences of mcr.Codon positions included were 1st + 2nd + 3rd + noncoding.All positions containing gaps and missing data were eliminated (complete deletion option).

Figure 5 .
Figure 5.The probability of substitution of one base for another base.Substitution patterns and rates were estimated using the general time reversible model [1].The maximum log-likelihood for this computation was 2655.269.This analysis involved all 10 nucleotide sequences of mcr.Codon positions included were 1st + 2nd + 3rd + noncoding.All positions containing gaps and missing data were eliminated (complete deletion option).

Figure 6 .
Figure 6.Depiction of the evolutionary probabilities of nucleotide substitution with respect to positions 1 to 262 for mcr-1 in Escherichia coli strain ZZ1409 KU886144.

Figure 6 .
Figure 6.Depiction of the evolutionary probabilities of nucleotide substitution with respect to positions 1 to 262 for mcr-1 in Escherichia coli strain ZZ1409 KU886144.The Processes and Molecular Vehicles Responsible for the Transmission of mcr Variants

Table 1 .
The evolutionary divergence among mcr variants (mcr-1 to mcr-10) (a score of 1 indicates no divergence between variants; a score of 0 indicates complete divergence).

Table 2 .
Recently reported insertion sequences and transposon elements associated with mcr genes transmission.