POLYMYXINS – ANTIBACTERIAL ACTIVITY, RESISTANCE MECHANISMS AND EPIDEMIOLOGY OF PLASMID MEDIATED RESISTANCE POLIMIKSINI – ANTIBAKTERIJSKA AKTIVNOST, MEHANIZMI REZISTENCIJE I EPIDEMIOLOGIJA REZISTENCIJE POSREDOVANE PLAZMIDOM

The occurrence and spreading of multi-resistant, as well as pan-resistant bacterial isolates, presents a global problem of modern medicine. Narrowed therapeutic options actualized the use of “old” antibiotics such as polymyxins. The use of polymyxins in human medicine has been reduced since the 1960s due to the discovery of safer antibiotics. Modern researches provided a better understanding of their pharmacokinetics and pharmacodynamics, as well as dosing regimens with minimum side effects. However, the increased usage in therapy consequently led to the occurrence of resistance to these “last-line” antibiotics. Most of polymyxin resistant bacterial isolates carried chromosomally mediated resistance, but the discovery of the plasmid mcr-1 gene in 2015 in China changed the paradigm of the origin and spreading of polymyxin resistance. Animals are the main reservoirs of bacteria carrying plasmid with the mcr-1 gene, because of widespread polymyxins application in veterinary medicine and in food industry, as well. Many studies confirmed the transfer of polymyxins resistance genes from animal bacterial isolates to human isolates, as well as between different bacterial species in vivo or in vitro. These findings indicated the need for more detailed epidemiological research and surveillance, as the European Centre for Disease Prevention and Control recommended.

The occurrence and spreading of multi-resistant Gram-negative bacteria, which are resistant to at least one agent in three or more different categories of antibacterial drugs (1), compromised the treatment of even the simplest infections. The development of new antibiotics is necessary, but their introduction into clinical practice is long-term and expensive. One strategy for overcoming this problem lies in the usage of the antibiotics, such as polymyxins. Polymyxins were discovered 70 years ago. Pharmacokinetics (PK) and pharmacodynamics (PD) data were poor, and nephrotoxicity and neurotoxicity, as well as the discovery of safer antibiotics, limited their usage (2). Recent studies enabled better understanding of PK/PD parameters and the optimization of a dosage regimen, resulting in the actualization of polymyxins in therapeutic protocols (3,4). Unfortunately, the increased usage led to the resistance to these "last-line" antibiotics.
The structure and mechanism of action of polymyxins Polymyxins are cyclic, hydrophilic, cationic peptides, very similar to antimicrobial peptides of eukaryotic cells (e.g. defensins). They are natural products of Paenibacillus polymyxa. Among five classes of polymyxins, polymyxin B and polymyxin E (colistin) are the most important in clinical practice. They differ in one amino acid in the peptide ring -phenylalanine in polymyxin B and leucin in polymyxin E. This has a great impact on PD: polymyxin B is applied as active drug, while colistin as inactive, less toxic precursor, colistimethate sodium. Colistimethate sodium converts in aqueous environment, such as body fluids, to its active form. The other form of colistin is colistin sulphate, administered orally for digestive tract decolonization or for topical treatment of skin infections (5).
The cell membrane of Gram-negative bacteria is the target of the action of these antibiotics. Positively charged polymyxins react with phosphate groups of lipid A of negatively charged lipopolysaccharide (LPS), replacing Ca 2+ and Mg 2+ ions. This results in the destabilization of LPS, loss of cell wall integrity, and cell death. Polymyxins, also, neutralize endotoxin effects by binding to LPS released from the bacterial wall after cell death (5).
Polymyxins are bactericidal antibiotics and the antibacterial effect depends on the serum concentration level. The best PK-PD parameter predicting their activity is the area under the concentration-time curve for free drug (fAUC) to minimum inhibitory concentration (MIC), which indicates the importance of maintaining the concentration of the drug in serum over the time. According to the recommendations of the European Medicines Agency (EMA) from 2014, the therapeutic concentration of colistin is 2 µg/ml. Achieving the steady state of the colistin concentration in plasma depends on the proper renal function, because colistimethate sodium is converted into the active drug in kidneys. When renal function decreases, more prodrug is converted into the active form. In healthy individuals only 20-30% of the precursor is converted, but in patients with creatinine clearance below 50 ml/min, the converted portion could be as high as 60-70%, thus reduction in doses are recommended (6,7).
In accordance with the above, achieving the therapeutic concentration of colistin may be difficult in healthy people, but dose increasing could lead to a higher incidence of side effects (e.g. nephrotoxicity and neurotoxicity) (7,9). This can be partially avoided with synergistic combinations of colistin and other antibacterial drugs (hydrophilic antibiotics like carbapenems, rifampicin, glycopeptides and tetracyclines) (3,6,7). Several patients' characteristics, such as renal function, age, and body weight require practically individual dosing in order to achieve the therapeutic concentration and effectiveness of Sažetak Ključne reči: polimiksini, mehanizmi rezistencije, mcr-1 gen, plazmid the treatment. Many studies showed that suboptimal doses led to drug resistance, with minimal post-antibiotic effect, especially in Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Therefore, maintaining optimal polymyxins concentration level in patients' serum is important both in therapy and in order to avoid the development of resistant bacterial clones (2,4,(6)(7)(8).

Mechanisms of resistance to polymyxins
The most common mechanism of resistance to polymyxins in Enterobacteriaceae is chromosomally encoded changes of the binding site of these antibiotics, the lipid A in LPS. The addition of phosphoethanolamine (pEtN) or 4-amino-4-deoxy-L-arabinose (L-Ara-4N) to the lipid A on the membrane surface disables polymyxins activity through the reduction of negative charge. Genes coding enzymes involved in these modifications are located on pmrHFIJFKLM and pmrCAB operons. Their activity is regulated by pmrAB, phoPQ, and mgrB genes, which expression depends on environmental conditions (phagocytosis, low pH, Fe 2+ , Mg 2+ and other ions concentration) (6,(10)(11)(12).
PmrAB two-component system is activated naturally during bacterial phagocytosis by macrophages and enables the survival of the bacterial cell. PmrB protein, as tyrosine kinase, phosphorylates and activates PmrA protein, which increases the transcription of pmrHFIJFKLM and pmrCAB genes responsible for lipid A modifications. The second two-component system, PhoPQ, increases the expression of genes involved in the magnesium transport, lipid A modification and bacterial survival in low pH conditions. Specific mutations in PmrAB and PhoPQ regions lead to their constitutive expression and chromosomally encoded resistance to polymyxins. The mgrB gene is also involved in negative regulation of PhoPQ system, and its inactivation (due to missense or nonsense mutations, insertions, deletions etc.) results in the overexpression of PhoPQ system. One of the most important mutations of the mgrB gene, described in Klebsiella spp., is the transposition of extended-spectrum beta-lactamases (ESBLs) or carbapenemase genes. Selective antibiotic pressure and the acquisition of ESBLs and carbapenemase genes could be responsible for the selection of colistin resistance. The role of efflux pump (AcrAB and KpnEF) and hyperproduction of capsule have been described in some polymyxins resistant strains of Klebsiella pneumoniae. Releasing of anionic capsular polysaccharides from the cell surface captures cationic antimicrobial peptides, such as polymyxins, reducing the amount of antibiotics that reaches the site of action (6,(10)(11)(12).
Mutational alterations of five two-component systems, PmrAB, PhoPQ, ParRS, ColRS and CprRS, have been described in non-fermentative Gram-negative bacteria Pseudomonas aeruginosa. The effect of these mutations is the modification of LPS, efflux pump and outer membrane protein OprH responsible for the stabilization of cell membrane, which could lead to different level of resistance to polymyxins (6,13). Mutations of the pmrAB gene are the main mechanism of resistance to polymyxins in Acinetobacter baumannii. Mutations in lpxA, lpxC or lpxD genes responsible for the lipid A biosynthesis are also very important, because of the complete loss of LPS, but with significant negative impact on fitness and virulence of bacteria (6,13,14).
The occurrence of polymyxins resistance in Enterobacteriaceae was generally related to chromosomally encoded mutations, more often in bacteria exposed to these antibiotics, with vertical transmission of the resistance gene. This paradigm was changed with the discovery of the first plasmid encoded gene responsible for colistin resistance, the mcr-1 gene (11,15). Liu YY et al. discovered the mcr-1 gene located on Escherichia coli plasmid in 2015 (16). The product of this gene belongs to the phosphoethanolamine transferase family, which modifies lipid A, similarly to chromosomally encoded modifications. Despite the fact it is not the most common mechanism of resistance, due to its plasmid location and its great possibility for horizontal transfer, it could become the most important one in the future (10,17).
The discovery of the mcr-1 gene launched the avalanche of researches, and its presence was detected both in human, animal and environmental bacterial isolates (11). Encouraged by these data, Xavier et al. screened colistin resistant, mcr-1 negative animal isolates of Escherichia coli in Belgium, and discovered the mcr-2 gene on IncX4 plasmid. A phylogenetic analysis confirmed a significant diversity between MCR-2 and MCR-1 protein. Interestingly, MCR-2 protein shares considerable similarity with lipid phosphatase, the product of insertion sequence IS1595 gene. This phosphatase has a high degree of similarity with phosphatase of Moraxella spp. strains, intrinsically resistant to polymyxins. This homology indicates the possibility of transmission of resistance genes between different species in conditions of cohabitation in animals, humans or in other environments (21). Kieffera et al. confirmed Moraxella spp. as the reservoir of the mcr-like gene (22).
The epidemiology of resistance to polymyxins The discovery of plasmid mediated colistin resistance had a huge impact on public health due to its potential of global spreading. Many retrospective studies showed the presence of plasmid mediated colistin resistance in Escherichia coli isolates from chicken in China about 30 years ago, when colistin utilization in veterinary medicine first started (30). The polymyxins usage in animals (e.g. livestock industry), which is 600 times higher than in human medicine, was crucial for spreading this resistance (31). The second joint analysis of the European Centre for Disease Prevention and Control (ECDC), the European Food Safety Authority (EFSA), and the EMA for 2013-2015 period showed that the usage of polymyxins, mostly colistin, in the European Union (EU) countries is higher in food-producing animals than in humans, with significant positive association between the occurrence of colistin resistance and its consumption. Similar positive association was detected only among invasive Klebsiella pneumoniae isolates of human origin in 2015 (32).
An evidence of the animal origin of the mcr-1 gene was discovered during the analysis of Escherichia coli urine isolate from an outpatient in Switzerland. Upstream of the mcr-1 gene, ISApI1 insertion sequence was determined, and it was identical to the insertion sequence of Pasteurella multocida, a typical animal pathogen. Additionally, the floR gene was detected in the same isolate, responsible for the resistance to florfenicol, which is used in veterinary medicine only (33).
The latest experimental research has shown that a recombinant plasmid-carrying the mcr-1 gene was successfully transferred to Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, with the introduction of a high level colistin resistance. In Pseudomonas aeruginosa, only moderate changes of susceptibility to colistin were observed (34). This experiment confirms the ability of the horizontal gene transfer between different species.
Up to date, the presence of the mcr-1 gene was confirmed in almost all continents, in different species (Escherichia, Klebsiella, Enterobacter, Cronobacter, Salmonella, Shigella and Kluyvera), isolated from human, animal and food samples (Figure 1) (6,17). During 2014-2015 in the SENTRY Antimicrobial Surveillance Program, colistin-resistant clinical isolates of Escherichia coli and Klebsiella pneumoniae from pacific Asia, Europe, North and South America, showed the incidence of mcr-1 gene of 4,9 % in Escherichia coli, but all Klebsiella pneumoniae isolates were mcr-1 negative (35). The presence of the mcr-1 and the mcr-2 was not detected among colistin resistant Klebsiella pneumoniae isolates of human origin in Serbia (36).

Conclusions
The resistance to polymyxins can compromise application of these drugs in human medicine, therefore, the control of polymyxins consumption is necessary in all spheres. In EU countries, the surveillance of the mcr-1 gene is mandatory (17). The World Health Organization in Global Action Plan for Antimicrobial Resistance recommended the control measures for optimal antimicrobial therapy in both humans and animals (37). Detection and surveillance of the resistant isolates on the local, national, and the global level are strongly recommended, due to global epidemiology threat of the plasmid mediated colistin resistance (6,17).