Polymyxin B and polymyxin E (colistin) are cationic cyclic lipodecapeptide antibiotics that disrupt the outer membrane of Gram-negative bacteria by displacing divalent cations Ca2+ and Mg2+ and enabling their interaction with the negatively charged phosphate moieties of lipid A (1), the so-called anchor of the lipopolysaccharide (LPS). This interaction destabilises the LPS, increases the permeability of the bacterial membrane, leads to the leakage of the cytoplasmic content, and eventually kills the bacteria (2). Polymyxins were discovered in 1947 (3) as secondary metabolites isolated from the soil bacterium
Today, polymyxins are used as last-resort agents in human medicine to treat infections which are caused by Gram-negative bacteria resistant to all other available antibiotics (5). Therefore, the emerging resistance to this last-resort treatment is one of the gravest public health concerns, as it leaves no reliable treatment options for patients in intensive care units.
The most prevalent form of polymyxin resistance is through structural modifications of lipid A, a polyanionic segment of the LPS. The two most common modifications include the addition of the cationic sugar 4-amino-L-arabinose (L-Ara4N) and/or phosphoethanolamine (pEtN), as presented in Figure 1. The modified structure of lipid A has a decreased net negative charge, which leads to the repulsion of colistin and protects bacteria from membrane disruption (6, 7). These lipid A structural changes follow distinct mechanisms for plasmid- and chromosome-encoded resistance. Moreover, lipid A structures are species-specific. Polymyxin resistance is intrinsic for some bacterial species such as
The two most common types of lipid A modifications that lead to colistin resistance
Chromosome-encoded resistance is based on the mutation of genes coding for lipid A. The genes which are involved in lipid A biosynthesis are upregulated by the PmrA/PmrB, PhoP/PhoQ, and CrrA/CrrB two-component systems (TCS) or mutations of the
Plasmid-encoded resistance enables horizontal transfer of
Other mechanisms which contribute to polymyxin resistance in synergy with lipid A modification systems include the expression of efflux pumps, capsule formation, and overexpression of the OprH, an outer membrane protein (25, 26).
Regardless of the mechanism, polymyxin resistance raises the greatest global concern in all aspects of the One Health paradigm (an interdisciplinary approach that balances the health of people, animals, and the environment): polymyxin-resistant bacteria have been found in environment-, veterinary medicine-, and human medicine-related bacterial strains (27). Although methods for the detection of lipid A structural modifications have mainly been developed for use in human medicine clinical diagnostics, these principles apply for the detection of environmental and veterinary medicine polymyxin-resistant strains. In addition, drugs which may arise from future research could also be utilised in veterinary medicine. The One Health approach to combat polymyxin resistance will be crucial, as one influences the other. For instance, the first
Although current polymyxin resistance is less frequent than resistance of other classes of antibiotics, we expect an increase in both human and veterinary medicine in the years to come. In this review article we will focus on current potential use of lipid A for clinical diagnostics of resistance and adjuvant therapy.
Currently, the gold standard recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for clinical diagnostics of polymyxin resistance used in clinical microbiology laboratories is minimal inhibitory concentration (MIC) (28). Although these diagnostic methods are the most reliable at the moment, they do not inform about the resistance mechanism involved. In clinical settings, this mechanism is generally interpreted through polymerase chain reaction (PCR) after phenotypic resistance has been established (7). Studies have shown that some bacterial strains, referred to as polymyxin heteroresistant, do not show resistance in MIC susceptibility testing but do acquire lipid A modifications along with the genes for polymyxin resistance (29–35). These strains raise concern over their potential to spread polymyxin resistance in clinics, as they usually are not tested for genetic polymyxin resistance. Yet, information about the exact polymyxin resistance mechanism that a bacterium has developed can help us decide the course of hospital treatment. For instance, a patient infected with a bacterium that has plasmid-encoded polymyxin resistance may require quarantine and more cautious behaviour of clinical staff. This is because plasmids can be transferred horizontally from one bacterial species to another. Bacteria that have colonised a hospital area and clinical staff might receive genes for polymyxin resistance by being in close contact with a patient infected by a bacterium carrying plasmid-encoded polymyxin resistance.
Researchers have been working on developing rapid clinical microbiology diagnostic assays which rely on the mass spectrometry (MS) of lipid A structural modifications. Prior to analysis, these bacterial samples require specific treatment to cleave a ketosidic bond in the LPS to release lipid A.
Clinical microbiology laboratories have increasingly been utilising matrix-assisted laser desorption/ionisation – time of flight – mass spectrometry (MALDI-TOF-MS) to identify microbes based on protein profiling. This includes profiling for lipid A structures to develop assays that rapidly determine mechanisms underlying polymyxin resistance in clinical settings (36–44). Molecules analysed by MALDI-TOF-MS need to be ionised prior to separation. Negative ionisation mode is optimal for lipid A because its phosphate groups can easily be ionised into anionic form. In view of the fact that lipid A structures are species-specific, the findings of MS analysis need to be interpreted carefully and individually. To illustrate the rationale of lipid A analysis for clinical diagnostics, we will show examples of lipid A structural modifications for some bacterial species which can be visualised by MALDI-TOF-MS.
Type | m/z | Proposed structures |
---|---|---|
Native lipid A | 1824 | |
1840 | ||
Native lipid A | 2062 | |
2078 | ||
Chromosome-encoded resistance – L-Ara4N addition | 1971 (1840 + 131) | |
2209 (2078 + 131) | ||
Plasmid-encoded resistance – pEtN addition | 1963 (1840 + 123) | |
2201 (2078 + 123) |
As for
Type | m/z | Proposed structures |
---|---|---|
Native lipid A | 1446 | |
1462 | ||
Chromosome-encoded resistance – L-Ara4N addition | 1577 (1446 + 131) | |
1593 (1462 + 131) | ||
1708 (1446 + 262) | ||
Chromosome-encoded resistance – L-Ara4N addition | 1724 (1462 + 262) |
Native lipid A structures of
Type | m/z | Proposed structure |
---|---|---|
Native lipid A | 1728 | |
Native lipid A | 1910 | |
Chromosome-encoded resistance – pEtN addition | 2033 (1910 + 123) |
The most common
Type | m/z | Proposed structure |
---|---|---|
Native lipid A | 1796 | |
Chromosome-encoded resistance – L-Ara4N addition | 1927 (1796 + 131) | |
Both plasmid- and chromosome-encoded resistance | 1919 (1796 + 123) |
Type | m/z | Proposed structure |
---|---|---|
Native lipid A | 1796 | |
1812 | ||
Native lipid A | 1876 | |
2034 | ||
Native lipid A | 2050 | |
Chromosome-encoded resistance – L-Ara4N addition | 1927 (1796 + 131) | |
Chromosome-encoded resistance – L-Ara4N addition | 2165 (2034 + 131) | |
Plasmid-encoded resistance – pEtN addition | 1919 (1796 + 123) | |
Plasmid-encoded resistance – pEtN addition | 1935 (1812 + 123) | |
2157 (2034 + 123) | ||
Plasmid-encoded resistance – pEtN addition | 2173 (2050 + 123) |
Based on the findings from the Larrouy-Maumus research group at Imperial College London, the MALDIxin test, commercialised by Bruker Corporation under the name MBT Lipid Xtract™ kit (47), shows great promise as indicator of polymyxin resistance to be used in clinical diagnostics.
Currently, polymyxins are the most promising agents for treating infections with extensively drug-resistant (XDR) Gram-negative bacteria. Therefore, we need to foresightedly work on polymyxin adjuvant therapeutics. Adjuvant compounds normally lack inherent antimicrobial activity as single agents, but in combination with a specific antibiotic they potentiate its effects. The idea of polymyxin adjuvant therapy is to reverse polymyxin resistance by inhibiting one of the resistance-driving mechanisms (mechanisms involved in lipid A modifications). Some of the most promising targets include enzymes involved in lipid A modification systems responsible for resistance [e.g. MCR-1, ArnT and diacylglycerol kinase A (DgkA)]. The interactions of colistin with lipid model membranes have been described thoroughly by structural biology studies (48), which have shown how these interactions change when pEtN is added to the phosphate groups of lipid A (49, 50) and how changes in LPS charge renders polymyxins ineffective. Therefore, in theory, for polymyxins to regain their efficacy these changes need to be prevented. In this context, we see another potential application of MALDI-TOF-based polymyxin resistance screening. For instance, analysing lipid A structure would indicate which inhibitor class would be useful for each patient.
MCR-1 is a pEtN transferase encoded by plasmid-borne
As for chromosome-encoded polymyxin resistance, small molecules that downregulate TCS have been found to reverse resistance (69, 70). Another promising target related to chromosome-encoded resistance is ArnT. Ghirga et al. (71) performed a virtual screening for ArnT inhibitors to discover a potent colistin adjuvant BBN149, which can be used as a lead molecule for further development. Quaglio et al. (72) have shown that ent-Beyerane diterpenes inhibit ArnT thanks to their resemblance to L-Ara4N. This means that the structure of these compounds most likely has a similar binding mode as L-Ara4N. Another target which might be of interest is DgkA. As mentioned above, EptA is transferring pEtN to the phosphate groups of lipid A. EptA uses phosphatidylethanoloamine (PE) as its pEtN donor, which results in the formation of diacylglycerol (DAG) as a by-product (10). DAG is quickly phosphorylated by DgkA into phosphatidic acid, a precursor for PE synthesis (73), and DAG accumulation has been shown to inhibit EptA activity (15, 74). All this points to DgkA as a prominent target of polymyxin resistance adjuvant therapy.
Another potential option are eukaryotic kinase inhibitors, which have been found to re-sensitise bacteria resistant to colistin. Although the mechanism is not clear yet, they have been reported to lower the rate of lipid A modifications by L-Ara4N and pEtN (75). Sertraline, an FDA-approved selective serotonin reuptake inhibitor (SSRI) used for the treatment of depression, has been found to revert polymyxin resistance, as it seems to interfere with the
All things considered, there is immense potential for the development of polymyxin adjuvants by targeting lipid A modification systems.