1. Introduction
Before the modern era of antibiotics, bacterial infections such as pneumonia, tuberculosis, and gastrointestinal infections were responsible for high fatality rates worldwide [ 1 ]. Following the introduction of penicillin, the world has witnessed the clinical approval of a plethora of antibiotic classes that have extensively decreased mortality rates, and improved human quality of life [ 2 , 3 ]. Regrettably, the misuse and excessive administration of antibiotics in healthcare and agriculture have led to the selection and proliferation of multi-drug resistant (MDR) strains, including MDR Gram-negative pathogens (e.g., Acinetobacter baumannii , Pseudomonas aeruginosa and K. pneumoniae ) [ 1 , 3 , 4 ]. These MDR Gram-negative bacteria have been recently ranked by the World Health Organization as critical priority pathogens, owing to their prevalence behind most hospital-acquired infections (accounting for 70% since 2015) and the shortage of relevant effective antibiotics to treat them [ 5 ]. Presently in the United States, MDR infections have led to ~35,000 fatalities annually, and it is projected that by 2050, the annual mortality rate of antibiotic resistance (10 million) would supersede even that of cancer (8.2 million) [ 6 , 7 ]. Antibiotic resistance is achieved via genetic mutations and by horizontal gene transfer of resistance genes between bacteria, augmenting the pathogen’s viability in environmental niches previously perilous for survival [ 4 , 8 ]. Resistant pathogens utilize four mechanisms to counter antibiotic action: (i) Active expulsion of antibiotics via the overexpression of membrane efflux pumps, (ii) mutations in membrane porin structure or decreased porin expression to decrease antibiotic uptake, (iii) expression of antibiotic-degradative or inactivating enzymes, and (iv) structural modification of drug targets impeding antibiotic binding [ 9 ].
Exacerbating this troublesome situation is the decline of new antibiotic approvals over the past 30 years, largely because drugs used for treating chronic diseases (i.e., cancer and metabolic diseases) were deemed to have far better profitability compared to antibiotics; the latter exhibit therapeutic activity of short duration, and rapidly become obsolete when resistant pathogens become prevalent in the community over time [ 1 , 3 , 9 ]. Without effective antibiotics, invasive and immunosuppressive treatments would become prohibitive, and healthcare systems would find themselves extensively overwhelmed by the spread of MDR bacteria [ 1 , 4 ]. Hence, while prudent stewardship of antibiotics may mitigate the spread of antibiotic resistance, it remains paramount that we expand our armamentarium of alternative treatment options to compensate for the dwindling efficacies of various last-resort antibiotics, such as polymyxins and carbapenems, which are used to treat persistent Gram-negative infections [ 10 ]. Drug repurposing (also known as drug repositioning) has been receiving heightened attention as an effective strategy to reassess de-risked FDA-approved drugs and/or investigational drugs for new therapeutic purposes [ 11 ]. This serendipitous approach offers lower overall development costs, shorter development timelines, and extremely low safety risks, compared to the de novo discovery of new agents (e.g., antibiotics) [ 12 , 13 ]. For decades, combining conventional antibiotics with non-antibiotics has been successfully used to treat infections caused by problematic Gram-negative and Gram-positive pathogens, tuberculosis, malaria, and viruses [ 14 ].
In this review, we shed light on the progress of the last-resort antibiotics polymyxins and non-antibiotic combinations, in combating the MDR Gram-negative pathogens based on the available literature, highlighting these non-antibiotics as prospective adjuvants. Further perspectives on translating in vitro synergistic polymyxin/non-antibiotic combinations to the same effect in vivo , to yield favorable clinical outcomes, will be discussed as well.
2. Combination Strategies to Overcome Antibiotic Resistance
2.1. Antibiotic-Antibiotic Combination Therapy
Antibiotic–antibiotic combinations have been explored and used as a popular treatment regime, as necessitated by the severity of the blight of antibiotic resistance. The reason behind using combinations is that the general probability of developing resistance against multiple antibiotics simultaneously, is surmised to be minimal; therefore, this enables rapid eradication of resistant pathogens before resistance to either or both drugs used in combination can arise, as the resulting synergistic or additive antibacterial effect of the combined antibiotics exceeds that of either drug alone [ 15 , 16 ]. With different metabolic pathways and/or drug targets inhibited concurrently, it increases the spectrum of coverage and potentially reduces the dosing regimens required for each antibiotic involved [ 17 , 18 ]. One notable antibiotic combination that was approved by the FDA to treat MDR-tuberculosis infections was the bedaquiline, pretomanid and linezolid cocktail [ 19 , 20 ]. This combination exerts synergistic bactericidal activity via the simultaneous disruption of oxidative phosphorylation (bedaquiline), protein synthesis inhibition (linezolid), mycolic acid biogenesis inhibition and/or release of reactive nitric oxide (pretomanid) [ 21 , 22 , 23 ]. However, certain antibiotic combinations can have an unintended antagonistic effect; one antibiotic could indirectly induce resistance against a second antibiotic, and potentially result in super-infections due to the survival of resistant bacteria [ 24 ]. Furthermore, resistance can evolve against both antibiotics concurrently, owing to horizontal gene transfer events [ 25 ]. To illustrate, Vestergaard et. al., observed that the combination of ciprofloxacin and ceftazidime augmented resistance in Pseudomonas aeruginosa compared to either antibiotic used alone. The combination resulted in overexpression of the mexAB–oprM operon; thus, increasing the production of efflux pumps, resulting in increased efflux of drugs out of the bacterial cell, and lowering the intracellular concentration [ 26 ]. Aside from substantially increased monitoring, drug preparation and administration costs associated with antibiotic co-administration, toxic side effects can also be exacerbated following exposure to multiple antibiotics. The latter is exemplified by the combined use of aminoglycosides and cephalosporins, which augment nephrotoxicity [ 27 , 28 ]. Therefore, the development of antibiotic alternatives or the combined use of antibiotics with non-antibiotic drugs that reverse bacterial resistance, is of the utmost importance [ 15 ].
2.2. Drug Repurposing
Drug repurposing is a cost-efficient strategy involving the investigation of existing approved drugs and/or investigational drugs for new therapeutic purposes, such as antimicrobial therapy [ 11 , 29 ]. Through this strategy, failures in drug discovery are mitigated as, for example, certain drugs withdrawn because of safety concerns could be salvaged for treating alternate diseases. One notable example is the teratogen thalidomide, which was successfully repurposed as a first-line antineoplastic for multiple myeloma because its antiangiogenic activity was confirmed to be therapeutically advantageous by destroying blood vessels nourishing malignant tumors—despite being responsible for arrested fetal limb development [ 30 ]. Another drug that is successfully repurposed is cyclosporin A, an immunosuppressant for treating organ transplantation and autoimmune diseases, which displays antibiofilm activity against Mycobacterium tuberculosis , enabling its clinical application as an anti-tuberculosis therapeutic [ 31 , 32 ]. Moreover, as the drug candidates selected for repurposing have their pharmacokinetic/pharmacodynamic profiles established from prior discovery and preclinical stages, they have already passed most of the toxicological and pharmacokinetic safety hurdles, allowing drug developers to bypass approximately 40% of the overall cost of commercializing their candidates [ 33 , 34 ].
Hence, drug repurposing is an attractive option for filling the current antibiotic discovery void [ 35 ]. However, repurposing selected drug candidates as antibacterial agents does bring about its own challenges. To illustrate this point, we would like to emphasize that antibiotics are typically administered at considerably higher dosages than non-antibiotic drugs; therefore, efficacy is determined at doses exceeding the prior effective concentrations for non-infectious diseases that the drug candidate was originally employed for therapeutically, thus posing toxicity risks. In addition, plasma protein binding often results in insufficient free drug concentrations at the infection site, impairing antibacterial activity [ 12 ]. A means to circumvent these drawbacks is combination therapy, of the non-antibiotic drugs of interest with existing antimicrobials, an approach that would prove advantageous due to the dose-sparing impact of synergistic combinations and/or the reversal of bacterial resistance [ 36 ].
2.3. Antibiotic and Non-Antibiotic/Adjuvant Combination Therapy
Certain co-administered non-antibiotic drugs despite being conventionally used to treat non-infectious diseases and possessing little to no in vitro antibacterial activity, remarkably are able to re-sensitize resistant pathogens to antibiotics [ 37 , 38 ]. It is purported that such non-antibiotics, designated adjuvants, potentiate antibiotic activity by interfering with the modes of resistance involving intrinsic mechanisms such as enzyme inhibition, obstructing efflux pumps, increasing membrane permeability, interfering with virulence signaling pathways, and biofilm formation ( Figure 1 ) [ 39 , 40 ]. For these reasons non-antibiotic compounds could be repurposed for use in antibiotic/non-antibiotic combination therapies, to compensate for the severe attrition experienced in the antibiotic pipeline [ 13 , 33 ].
Multiple compounds with adjuvant properties were since discovered and semi-synthetically developed, functioning as efflux pump inhibitors, membrane-permeabilizing peptides and antibiofilm agents (with brief examples outlined in Table 1 ). Nonetheless, the antibiotic/adjuvant combinations that have successfully entered clinical use, to date, are the β-lactam/β-lactamase inhibitor combinations and the siderophore-cephalosporin conjugate cefiderocol ( Table 1 ). β-lactams are popularly prescribed antibiotics that exert bactericidal action by inhibiting peptidoglycan chain cross-linking [ 42 ]. Although the β-lactamase inhibitors themselves do not kill target bacteria, they augment β-lactam efficacy by competitively inhibiting β-lactamases, shielding the β-lactam from degradation [ 43 ]. Similarly, the catechol scaffold of cefiderocol is devoid of intrinsic antibacterial activity but increases cephalosporin uptake via the bacterial membrane iron transport pathways [ 44 ]. Hence, adjuvant strategies display promising potential in circumventing the constant need for new antibiotics to overcome resistance, by acting as decoys that retard the activity of resistance-conferring enzymes.
3. The Time-Honored ‘Magic Bullet’ Polymyxin Lipopeptide Antibiotics Are Rapidly Losing Their Caliber
Polymyxins were discovered in the 1940s and introduced in the clinic as effective antibacterial agents against Gram-negative bacteria. However, their clinical use was reserved in the 1960s owing to their nephrotoxicity and neurotoxicity, and also better tolerated antibiotics from different classes were introduced [ 56 ]. In recent times, the use of polymyxins has experienced a resurgence as a treatment of last resort against difficult-to-treat MDR Gram-negative pathogens; however, alarming reports from clinics showed that there is a sharp upsurge in the polymyxin resistance particularly following polymyxin monotherapy [ 57 , 58 , 59 ]. This would warrant a necessity to develop an innovative strategy (e.g., combining polymyxins with nonantibiotics) to preserve the clinical effectiveness of polymyxin and halt the development of polymyxin resistance [ 60 , 61 ]. In terms of their chemical constitution, polymyxins are non-ribosomal cyclic lipopeptides extracted from the Gram-positive soil bacterium Paenibacillus polymyxa . Fundamentally, polymyxins are comprised of a polycationic ring with a short peptide extension to which a lipophilic chain is attached [ 57 ]. Of the five major polymyxin isoforms (A–E), only two (polymyxin B and polymyxin E aka. colistin) were clinically utilized in the 1950s [ 62 ]. Polymyxin B and colistin share nearly identical structures, only differing by an amino acid at position six in the peptide ring ( Figure 2 A), and as such share cross-resistance [ 57 , 62 ]. Polymyxins bind the lipid A component of LPS via electrostatic attractions, displacing the Ca 2+ and Mg 2+ ions required to stabilize the LPS leaflet. Thus, the polymyxin is anchored in the outer membrane via its lipophilic chain, causing membrane disruption. Subsequent polymyxin-mediated fusion of the inner leaflets of the outer membrane and the outer leaflet of the cytoplasmic membrane, results in osmotic lysis ( Figure 2 B) [ 62 , 63 , 64 ]. Other proposed secondary mechanisms leading to bacterial death include polymyxin inhibition of NADH oxidases involved in cell respiration, and the induction of reactive oxygen species (ROS) production [ 65 ]. However, owing to their membrane-disrupting activities, aside from nephrotoxicity polymyxins also cause neurotoxicity in the host, so their use is unduly restricted. Furthermore, the lack of properly established optimized regimens resulted in suboptimal drug exposure selecting for polymyxin-resistant strains [ 66 ].
The Gram-negative bacterial outer membrane serves as the primary means of resistance against multiple antibiotic classes; any alteration in the outer membrane, such as increased hydrophobicity, mutations in porins, overexpression of efflux pumps, and other factors, significantly impedes antibiotic penetration [ 67 ]. The outer membrane is comprised of a phospholipid-rich inner leaflet, akin to the cytoplasmic inner membrane, and the LPS rich outer leaflet which faces the extracellular environment [ 68 ]. The LPS contains three key domains, namely the O -antigen, 2-keto-3-deoxyoctonoic acid (inner core), and lipid A, which possesses anionic phosphate moieties accentuating the outer membrane’s selective permeability [ 69 ].
The primary resistance mechanisms employed by polymyxin-resistant strains include the synthesis of modified lipid A with anionic charges neutralized, preventing LPS binding by polymyxins. Succeeding events such as the cessation of LPS production, as well as the upregulation of genes expressing phospholipids and other OM components, augment polymyxin resistance [ 63 , 66 ]. Furthermore, a plasmid-borne mobile colistin resistance gene ( mcr-1 ), a gene that was first reported in China, which upon expression culminates in the modification of lipid A with phosphoethanolamine, impairing polymyxin targeting [ 70 ]. The mcr-1 plasmid is readily transferrable via conjugation across Gram-negative species, which results in the global dissemination of mcr-1 , therefore, substantially threatening polymyxin efficacy and raising public health concerns globally [ 71 , 72 ]. Hence, various research groups and our own have investigated combinations of several FDA-approved non-antibiotic drugs and natural products with polymyxins, to evaluate possible synergy against MDR/polymyxin-resistant Gram-negative pathogens and the repurposing potential of these non-antibiotics as polymyxin adjuvants. Instances of non-antibiotic drugs tested in combination with polymyxins are listed in Table 2 .
4. Polymyxin/Non-Antibiotic Combinations
4.1. Antineoplastic Drugs
Our group firstly reported the antibacterial synergetic activity of three mainstream selective estrogen receptor modulators (SERMs), tamoxifen, raloxifene, and toremifene in combination with polymyxin B [ 73 , 74 ]. While conventionally used for breast cancer treatment, some SERMs were reported to exhibit direct antimicrobial properties via interference with cell wall synthesis (e.g., inhibition of wall teichoic acid synthesis in E. faecium and S. aureus by clomiphene) and membrane perforation (e.g., tamoxifen) [ 96 , 97 ]. Based on time-kill assays, polymyxin-resistant P. aeruginosa , Klebsiella pneumoniae , and Acinetobacter baumannii strains were considerably re-sensitized to polymyxin B, in the presence of each SERM (≥2.0-log 10 decrease in bacterial viability relative to either monotherapy at 24 h) [ 74 ]. Metabolomics analysis of the tamoxifen-polymyxin B combination against an MDR polymyxin-resistant cystic fibrosis P. aeruginosa (FADDI-PA006) strain revealed extensive perturbations in the fatty acid and glycerophospholipid synthetic pathways involved in membrane biogenesis; thus implying the combination exerts synergy via concerted damaging effect against the outer membrane [ 73 ].
Mitotane, an antineoplastic drug used for the treatment of adrenal cancers [ 98 ], displayed synergy with polymyxin B against polymyxin-resistant MDR A. baumannii , P. aeruginosa , and K. pneumoniae in vitro. It also prevented the development of polymyxin-resistance (i.e., regrowth) in the polymyxin-susceptible isolates, compared to monotherapy from time-kill assays [ 75 ]. The in vitro synergy was further confirmed in a mouse burn wound model, in which the combination treatment decreased wound infection by a polymyxin-resistant A. baumannii isolate, compared to either polymyxin B or mitotane monotherapy. Scanning electron microscopy and transmission electron microscopy (SEM/TEM) imaging of A. baumannii isolates ATCC™ 17978 and FADDI-AB225 treated with the combination, revealed the formation of aberrant cell clusters suggesting the combination disrupts binary fission. While outer membrane damage was observed in the polymyxin-susceptible ATCC™ 17978 following polymyxin monotherapy, the polymyxin-resistant FADDI-AB225 strain displayed membrane blebbing; thus, it was inferred that polymyxin B perforates the outer membrane sufficiently for the hydrophobic mitotane to diffuse across the outer membrane and exerts antibacterial activity against intracellular targets. Untargeted metabolomics analysis revealed significant disruptions in the TCA cycle and nucleotide metabolism in the metabolome of four A. baumannii isolates treated with the combination [ 76 ].
4.2. Antipsychotic and Antidepressant Agents
Phenothiazines are heterocyclic compounds clinically utilized as antipsychotics, that function as dopamine antagonists [ 99 , 100 ]. Over time, the antimicrobial characteristics of the phenothiazines were serendipitously discovered, such as chlorpromazine which was observed to exert anti-mycobacterial activity and thioridazine which potentiates first-line anti-tuberculosis antibiotics enabling rapid clearance of MDR/XDR (extensively drug-resistant) tuberculosis infections [ 99 , 101 , 102 , 103 ]. The proposed antibacterial mechanisms exerted by phenothiazines include augmenting complete phagocytosis of bacteria by macrophages, inducing abnormalities in binary fission, and inhibiting antibiotic efflux pumps [ 99 , 100 , 101 ]. The three phenothiazines, prochlorperazine, chlorpromazine, and thiethylperazine were investigated by our group [ 78 ]. Among the three, prochlorperazine showed superior synergy with polymyxin B, as evident from the rapid and extensive bactericidal activity (4.0–6.0-log 10 CFU/mL decrease in bacterial inoculum at 4 and 8 h, relative to the untreated control) observed against all tested strains in the time-kill assays. SEM/TEM imaging revealed extensive membrane blebbing, cell shriveling, perforation, and vesicle formation following exposure of the polymyxin-resistant P. aeruginosa strain FADDI-PA070 to the polymyxin B/prochlorperazine combination, reflecting augmented damage to the outer membrane. This was further verified by metabolomics, which reveal considerable perturbations in glycerophospholipid, fatty acid, and LPS biosynthesis, following exposure to the combination. Additional pathways perturbed include acetyl-CoA, arginine, and proline metabolism, suggesting the combination also impairs bacterial respiration, growth, and biofilm formation [ 78 ].
Selective serotonin reuptake inhibitors (SSRI) function by blocking presynaptic axon terminal serotonin transporters, and are largely prescribed for major depressive and anxiety disorders [ 104 ]. Interestingly, various research groups have reported antimicrobial activity from SSRIs, such as femoxetine and paroxetine, that purportedly exert direct antibacterial activity via efflux pump inhibition [ 105 ]. One SSRI in particular, sertraline, was reported by Ayaz et. al., to exert concentration-dependent reversal of resistance towards ciprofloxacin, levofloxacin, norfloxacin, gentamicin, and moxifloxacin in the clinical isolates of S. aureus , E. coli , and P. aeruginosa [ 104 ]. Sertraline was observed to readily synergize with polymyxin B at lower concentrations in time-kill assays (>3.0-log 10 CFU/mL decrease in bacterial viability) against polymyxin-resistant P. aeruginosa , A. baumannii , and K. pneumoniae strains [ 82 ]. Further SEM/TEM and metabolomics studies indicated the combination synergistic killing activity primarily involves the inhibition of amino-sugar, sugar-nucleotide metabolism, glycerophospholipid, and fatty acid metabolism causing disruption of the outer membrane integrity, cell wall synthesis, and respiration [ 82 ].
4.3. Antifungal Drugs
Caspofungin is an antifungal drug that operates by inhibiting the enzyme (1→3)-β-D-glucan synthase, destabilizing fungal cell wall integrity. Caspofungin serves as a therapeutic agent against Aspergillus and Candida fungal infections [ 106 ]. Intriguingly, caspofungin was observed to exert antibacterial activity by inhibiting biofilms [ 80 ]. Further untargeted metabolomics analysis by Hussein et. al. [ 79 ] revealed the synergistic combination of polymyxin B/caspofungin against K. pneumoniae acts by inhibiting multiple interconnected metabolic pathways, including bacterial envelope biosynthesis, the phosphotransferase system (involved in biofilm formation), ATP-binding cassette (ABC) transporter production. Another antifungal, miconazole, possesses broad spectrum fungicidal activity by penetrating the chitin fungal cell wall and permeabilizing the cell membrane to external noxious substances [ 107 ]. This membrane-disrupting ability of miconazole allows it to interfere with bacterial lipid membranes and accounts for its direct antibacterial activity [ 108 ]. The combination of polymyxin B/miconazole was reported to exert synergistic bacterial killing, purportedly through permeabilization of the outer membrane by polymyxin B, which in turn allows miconazole to access the periplasmic space and disrupt the inner cytoplasmic membrane, causing bacterial lysis [ 81 ].
4.4. Antiparasitic Drugs
Closantel exerts an anti-parasitic mechanisms of action via the disruption of oxidative phosphorylation and the inhibition of chitinase activity [ 109 ]. As monotherapy, closantel had no antibacterial effect (minimal inhibitory concentration, MIC ≥ 16 mg/L); however, in combination with polymyxin B, synergy and inhibition of polymyxin resistance against A. baumannii were achieved at therapeutic concentrations [ 88 ]. In the presence of closantel, the polymyxin-resistant A. baumannii strains (polymyxin MIC ≥ 4 mg/L) were considerably re-sensitized to polymyxin B concentrations at 2 mg/L and below, which coincides with the polymyxin MIC susceptibility breakpoints (≤2 mg/L) [ 110 ]. In addition, Domalaon et. al., investigated the combination of colistin/closantel, and with other related anthelmintic salicylanilides oxyclozanide and rafoxanide. The combinations displayed synergy against MDR P. aeruginosa , A. baumannii , K. pneumoniae , E. coli , and E. cloacae , and reversed resistance to colistin apparently via potentiating damage to the outer membrane [ 91 ].
4.5. Natural Products
Cannabinoids are secondary metabolites isolated from the plant Cannabis sativa , the same plant from which the illicit drug marijuana is derived [ 111 ]. Several cannabinoids are behind the psychotropic effects of marijuana; these are synthesized via the alkylation of olivetolic acid, producing cannabigerolic acid, a precursor to most cannabinoids [ 112 ]. Of all the cannabinoids discovered, cannabidiol is the main non-psychoactive ingredient of Cannabis sativa [ 113 ]. Reports of antibacterial activity exerted by cannabidiol date back to the 1950s, with an investigation conducted by Van Klingeren and Ten Ham [ 114 ], noting MICs of cannabidiol within the ranges of 1–5 μg/mL for Gram-positive Staphylococci and Streptococci . Additional investigations carried out by Blaskovich et. al. [ 113 ] revealed consistent MICs (1–4 μg/mL) of cannabidiol against many Gram-positive MDR Staphylococci (methicillin-resistant S. aureus , vancomycin-intermediate S. aureus , vancomycin-resistant S. aureus ), vancomycin-resistant Enterococci ( E. faecalis , E. faecium ), Clostridioides ( C. difficile ), and Streptococci ( S. pyogenes , S. pneumoniae ) strains. Radiolabeled macromolecular synthesis assays in S. aureus RN42200 and bacterial cytological profiling, indicated cannabidiol exerts antibacterial activity via membrane permeabilization, as evident from the marked perturbations in lipid synthesis at sub-MIC levels.
However, cannabidiol was inactive against Gram-negative species tested, with the exceptions of Neisseria and Legionella isolates. Further investigations involving exposure of efflux pump E. coli and P. aeruginosa mutants revealed the inactivity of cannabidiol nonetheless, ruling out efflux pumps as a possible explanation for the cannabinoid’s general ineffectiveness against Gram-negative bacteria. This was also confirmed by Abichabki et. al., who noted the inactivity of cannabidiol against K. pneumoniae , A. baumannii , P. aeruginosa , and E. cloacae even, in the presence of efflux pump inhibitors such as curcumin [ 84 ]. However, cannabidiol was active against an E. coli lpxC cell membrane mutant, indicating that outer membrane LPS deficiency enables penetration of cannabidiol. This conclusion was further supported by the increased susceptibility of lipid A-deficient A. baumannii (ATCC™ 19606R) to cannabidiol (MIC >128 to 0.25 μg/mL), which in turn has an polymyxin MIC (>128 μg/mL) relative to its parent strain (0.25 μg/mL) [ 115 ]. Our group investigated polymyxin B and cannabidiol as another possible antibiotic/adjuvant combination for treating polymyxin-resistant Gram-negative infections [ 83 ]. Broth microdilution tests involving polymyxin B combined with cannabidiol (fixed at 256 μg/mL) against 13 different Gram-negative pathogens (52 strains in total), yielded observable antibacterial activity of the combination against 47 of the 52 strains. Subsequent checkerboard assays combining polymyxin B/cannabidiol verified the synergistic activity, as evident from the low polymyxin B concentrations (≤2 µg/mL, within the EUCAST “susceptible” breakpoint) enabling cannabidiol to exert antibacterial activity at a minimal effective antibiotic concentration (MEAC) ≤4 µg/mL. Moreover, the polymyxin B-cannabidiol combination substantially decreased the viability of four K. pneumoniae strains (>2.0-log 10 CFU/mL decline) relative to polymyxin B monotherapy at several time points, verifying the combination’s synergistic bactericidal activity. Similarly, checkerboard and time-kill assays confirmed synergy against A. baumannii , P. aeruginosa , and K. pneumoniae , both polymyxin-resistant and polymyxin-susceptible isolates alike. Moreover, it was inferred from metabolomics analysis of A. baumannii ATCC™ 19606 that the polymyxin B/cannabidiol combination substantially disrupts cell envelope biogenesis through a time-dependent perturbation of amino–sugar and nucleotide–sugar metabolism, and the pentose phosphate pathway. Consequently, peptidoglycan and LPS synthesis are disrupted owing to decreased levels of core membrane lipid components, phosphatidylethanolamine, phosphatidic acid, sn -glycero-3-phosphoethanolanime, and sn -glycerol 3-phosphate.
Curcumin, a phenolic compound isolated from the plant Curcuma longa , has been traditionally used for gastrointestinal treatments owing to its antioxidant and anti-inflammatory activities [ 116 ]. Intriguingly, this natural product was also reported to exhibit antimicrobial activity; various reported antibacterial mechanisms exerted by curcumin include membrane damage, efflux inhibition, biofilm disruption, and growth inhibition [ 117 , 118 ]. When combined with polymyxin B, considerable synergy was observed against polymyxin-resistant strains (as well as expanding the polymyxin activity spectrum coverage to Gram-positive bacteria) at reduced polymyxin MICs; the mechanisms purported to account for synergy involve outer membrane permeabilization by curcumin, allowing access to the inner cytoplasmic membrane by polymyxin B [ 85 ]. Furthermore, colistin in combination with curcumin was observed to reverse polymyxin-resistance in A. baumannii through outer membrane permeabilization by colistin, facilitating the entry of curcumin which triggers increased ROS production, severely impacting bacterial viability [ 95 ].
Tetrandrine is a bis-benzylisoquinoline alkaloid isolated from the herb Stephania tetrandra [ 119 ]. Although tetrandrine functions pharmacologically as a calcium channel blocker, it was also found to possess anti-inflammatory and antineoplastic properties through the inhibition of pro-inflammatory cytokine production and the induction of apoptosis in tumor cells, respectively [ 120 ]. Interestingly, tetrandrine exhibits antibacterial properties as observed from its ability to inhibit antibiotic efflux from MRSA and Mycobacterium tuberculosis [ 121 , 122 ]. Hence, when tested in combination with colistin against mcr-1 -positive colistin-resistant Salmonella , tetrandrine was noted to enhance colistin activity by enhancing the permeability of the outer membrane through efflux inhibition sufficiently for colistin to act, as well as by disrupting oxidative phosphorylation (ATP production) and down-regulating mcr-1 expression [ 93 ].
4.6. Other Non-Antibiotic Drugs
The cystic fibrosis transmembrane conductance regulator (CFTR) potentiator ivacaftor, commonly used to treat cystic fibrosis patients, exerts apparent antimicrobial activity via weak inhibition of DNA gyrase and topoisomerase IV enzymes, owing to certain structural similarities with quinolones. When combined with polymyxin B against P. aeruginosa , augmented damage against the outer membrane was observed via nitrocefin assay, SEM and TEM [ 87 ]. This was further substantiated by metabolomics analysis which revealed extensive phospholipid and LPS biosynthesis perturbations, following exposure to the polymyxin/ivacaftor combination [ 86 ]. Auranofin, an anti-rheumatic drug for treating arthritis, displays negligible anti-Gram-negative activity [ 123 ]; however, in combining with colistin, it results in extensive cell shrinkage and lysis as observed from SEM/TEM imaging, validating auranofin as a prospective adjuvant. In addition, the colistin/auranofin combination extensively counters colistin resistance in Gram-negative pathogens and substantially improves the survival rate in mouse peritoneal infection models [ 89 ].
Zidovudine is a nucleoside/nucleotide reverse transcriptase inhibitor that acts by incorporating into the elongating viral DNA strand, and slowing down the progression of HIV infection [ 124 ]. Intriguingly, antibacterial activity was also observed from zidovudine, likewise by acting as a DNA-chain terminator and arresting bacterial DNA replication [ 125 ]. When combined with polymyxin B, zidovudine extensively potentiated polymyxin activity against K. pneumoniae , ≥4.0-log 10 CFU/mL in time-kill assay; ≥3.0-log 10 CFU/mL in murine thigh infection model [ 77 ]. Similarly, the colistin/zidovudine combination was observed to reverse colistin resistance in time-kill assay (>3.0-log 10 CFU/mL) with the absence of regrowth in the combination 24 h post-exposure, and displayed in vivo synergy against NDM-1 K. pneumoniae and mcr-1 -positive E. coli in murine peritoneal infection models [ 90 , 126 ].
Melatonin, a hormone secreted by the pineal gland in the brain, functions to regulate circadian rhythms and blood pressure, thus is used medically to treat sleep disorders. Astonishingly, melatonin was reported to exert bacteriostatic (growth-inhibiting) activity, via the perturbation of key metabolic pathways and the expression of genes involved in cell division. Furthermore, the combination of colistin with melatonin substantially permeabilizes the outer membrane and inhibits efflux pump activity, hence re-sensitizing polymyxin-resistant Gram-negative pathogens to polymyxins [ 127 ]. Additional transcriptomics analysis involving mcr-1 -positive E. coli reveals significant disruptions in the expression of genes involved in LPS modifications and efflux pump production, in the presence of the colistin–melatonin combination [ 94 ].
5. Perspectives and Future Directions
This review highlights the promising clinical approach of combining non-antibiotic FDA-approved drugs or natural products with polymyxins, for treating MDR Gram-negative infections. Considering the majority of the FDA-approved non-antibiotic drugs are already well-characterized pharmacokinetically and toxicologically, it would enable them to forgo clinical trials as information from prior safety studies can help guide design of future studies focused on evaluating their applicability as antibacterial adjuvants [ 12 ]. Consequently, our ability to translate these adjuvants in combination with polymyxins to the bedside, could potentially be accelerated due to reduced developmental costs by the leapfrogging of extensive prior preclinical development insights [ 34 , 128 ]. Moreover, as these non-antibiotic drugs do not exert direct inhibitory activity against specific bacterial molecular targets, evolutionary pressure on bacteria to evolve resistance would likely be eschewed [ 38 , 129 ]. Hence, non-antibiotic drugs represent a valuable novel reservoir of anti-infective adjuvants for combination therapy.
In general, the polymyxin/non-antibiotic combinations covered in this review function synergistically by augmenting penetrative damage against the outer membrane causing bacterial lysis. Alternatively, the outer membrane may be permeabilized sufficiently (either by the polymyxin or the adjuvant) for the combination to access the inner membrane, leading to either perforation of the inner membrane (and lysis) or diffusion across the membrane, substantially disrupting vital metabolic pathways (i.e., respiration, DNA replication, cell envelope maintenance) and/or repressing plasmid-mediated polymyxin resistance ( Figure 3 ). In consequence, effective polymyxin concentrations for bacterial killing are decreased, reversing polymyxin resistance, and potentially reducing polymyxin dosing regimens required for the maximum clearance of pathogens. This would improve the polymyxin therapeutic index, since nephrotoxicity and neurotoxicity side effects are likely to be reduced [ 130 ].
In closing, this contemporary review provides detailed information about a potentially central hub for several novel combinations, which could be utilized as promising antibacterial therapies to bridge the antibiotic development gap. In the last decade, this innovative therapeutic approach has emerged as an area of great interest in the anti-infectives field and should be pursued to fruition vigorously.
Acknowledgments
J.L. is an Australian National Health and Medical Research Council (NHMRC) Principal Research Fellow, and T.V. is an Australian NHMRC Industry Career Development Level 2 Research Fellow.
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Author Contributions
Conceptualization, A.K.J.J. and M.H.; A.K.J.J.; investigation, A.K.J.J.; resources, A.K.J.J.; data curation, A.K.J.J.; writing—original draft preparation, M.H.; G.G.R.; J.L.; writing—review and editing, T.V.; supervision. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Not applicable.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Browne K. Chakraborty S. Chen R. Willcox M.D. Black D.S. Walsh W.R. Kumar N. A new era of antibiotics: The clinical potential of antimicrobial peptides Int. J. Mol. Sci. 2020 21 7047 10.3390/ijms21197047 32987946 PMC7582481
2. Piret J. Boivin G. Pandemics throughout history Front. Microbiol. 2021 11 631736 10.3389/fmicb.2020.631736 33584597 PMC7874133
3. Annunziato G. Strategies to Overcome Antimicrobial Resistance (AMR) Making Use of Non-Essential Target Inhibitors: A Review Int. J. Mol. Sci. 2019 20 5844 10.3390/ijms20235844 31766441 PMC6928725
4. Aslam B. Wang W. Arshad M.I. Khurshid M. Muzammil S. Nisar M.A. Alvi R.F. Aslam M.A. Qamar M.U. Salamat M.K.F. Antibiotic resistance: A rundown of a global crisis Infect. Drug Resist 2018 11 1645 1658 30349322 10.2147/IDR.S173867 PMC6188119
5. Venter H. Reversing resistance to counter antimicrobial resistance in the World Health Organisation’s critical priority of most dangerous pathogens Biosci. Rep. 2019 39 BSR20180474 10.1042/BSR20180474 30910848 PMC6465202
6. Dadgostar P. Antimicrobial resistance: Implications and costs Infect. Drug Resist. 2019 12 3903 3910 10.2147/IDR.S234610 31908502 PMC6929930
7. Morris S. Cerceo E. Trends, epidemiology, and management of multi-drug resistant gram-negative bacterial infections in the hospitalized setting Antibiotics 2020 9 196 10.3390/antibiotics9040196 32326058 PMC7235729
8. Miller W.R. Munita J.M. Arias C.A. Mechanisms of antibiotic resistance in enterococci Expert Rev. Anti-Infect. Ther. 2014 12 1221 1236 10.1586/14787210.2014.956092 25199988 PMC4433168
9. Cardoso P. Glossop H. Meikle T.G. Aburto-Medina A. Conn C.E. Sarojini V. Valery C. Molecular engineering of antimicrobial peptides: Microbial targets, peptide motifs and translation opportunities Biophys. Rev. 2021 13 35 69 10.1007/s12551-021-00784-y 33495702 PMC7817352
10. Matlock A. Garcia J.A. Moussavi K. Long B. Liang S.Y. Advances in novel antibiotics to treat multidrug-resistant gram-negative bacterial infections Intern. Emerg. Med. 2021 16 2231 2241 10.1007/s11739-021-02749-1 33956311 PMC8100742
11. Ashburn T.T. Thor K.B. Drug repositioning: Identifying and developing new uses for existing drugs Nat. Rev. Drug Discov. 2004 3 673 683 10.1038/nrd1468 15286734
12. Farha M.A. Brown E.D. Drug repurposing for antimicrobial discovery Nat. Microbiol. 2019 4 565 577 10.1038/s41564-019-0357-1 30833727
13. Jourdan J.-P. Bureau R. Rochais C. Dallemagne P. Drug repositioning: A brief overview J. Pharm. Pharmacol. 2020 72 1145 1151 10.1111/jphp.13273 32301512 PMC7262062
14. Schneider E.K. Reyes-Ortega F. Velkov T. Li J. Antibiotic–non-antibiotic combinations for combating extremely drug-resistant Gram-negative ‘superbugs’ Essays Biochem. 2017 61 115 125 28258235 10.1042/EBC20160058
15. Mulani M.S. Kamble E.E. Kumkar S.N. Tawre M.S. Pardesi K.R. Emerging strategies to combat ESKAPE pathogens in the era of antimicrobial resistance: A review Front. Microbiol. 2019 10 539 10.3389/fmicb.2019.00539 30988669 PMC6452778
16. Ahmed A. Azim A. Gurjar M. Baronia A.K. Current concepts in combination antibiotic therapy for critically ill patients Indian J. Crit. Care Med. 2014 18 310 314 24914260 10.4103/0972-5229.132495 PMC4047693
17. Wang N. Luo J. Deng F. Huang Y. Zhou H. Antibiotic Combination Therapy: A Strategy to Overcome Bacterial Resistance to Aminoglycoside Antibiotics Front. Pharmacol. 2022 13 839808 10.3389/fphar.2022.839808 35281905 PMC8905495
18. Grande V. Kumar A. Optimizing antimicrobial therapy of sepsis and septic shock: Focus on Antibiotic Combination Therapy Semin. Respir. Crit. Care Med. 2015 36 154 166 10.1055/s-0034-1398742 25643278
19. Nimmo C. Naidoo K. O’Donnell M. Treatment of Highly Drug-Resistant Pulmonary Tuberculosis N. Engl. J. Med. 2020 382 2376 10.1056/NEJMc2009939 32521141
20. Conradie F. Diacon A.H. Ngubane N. Howell P. Everitt D. Crook A.M. Mendel C.M. Egizi E. Moreira J. Timm J. Treatment of Highly Drug-Resistant Pulmonary Tuberculosis N. Engl. J. Med. 2020 382 893 902 10.1056/NEJMoa1901814 32130813 PMC6955640
21. Manjunatha U. Boshoff H.I. Barry C.E. The mechanism of action of PA-824: Novel insights from transcriptional profiling Commun. Integr. Biol. 2009 2 215 218 10.4161/cib.2.3.7926 19641733 PMC2717523
22. Zahedi Bialvaei A. Rahbar M. Yousefi M. Asgharzadeh M. Samadi Kafil H. Linezolid: A promising option in the treatment of Gram-positives J. Antimicrob. Chemother. 2017 72 354 364 10.1093/jac/dkw450 27999068
23. Sarathy J.P. Gruber G. Dick T. Re-understanding the mechanisms of action of the anti-mycobacterial drug bedaquiline Antibiotics 2019 8 261 10.3390/antibiotics8040261 31835707 PMC6963887
24. Rybak M.J. McGrath B.J. Combination antimicrobial therapy for bacterial infections Drugs 1996 52 390 405 10.2165/00003495-199652030-00005 8875129
25. Pokrovskaya V. Baasov T. Dual-acting hybrid antibiotics: A promising strategy to combat bacterial resistance Expert Opin. Drug Discov. 2010 5 883 902 10.1517/17460441.2010.508069 22823262
26. Vestergaard M. Paulander W. Marvig R.L. Clasen J. Jochumsen N. Molin S. Jelsbak L. Ingmer H. Folkesson A. Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa Int. J. Antimicrob. Agents 2016 47 48 55 10.1016/j.ijantimicag.2015.09.014 26597931
27. Mannion J.C. Bloch R. Popovich N.G. Cephalosporin-aminoglycoside synergistic nephrotoxicity: Fact or fiction? Drug Intell. Clin. Pharm. 1981 15 248 256 10.1177/106002808101500401 7023896
28. Thiyagarajan D. Das G. Ramesh A. Amphiphilic Cargo-Loaded Nanocarrier Enhances Antibiotic Uptake and Perturbs Efflux: Effective Synergy for Mitigation of Methicillin-Resistant Staphylococcus aureus ChemMedChem 2017 12 1125 1132 10.1002/cmdc.201700260 28618136
29. Strittmatter S.M. Overcoming drug development bottlenecks with repurposing: Old drugs learn new tricks Nat. Med. 2014 20 590 591 10.1038/nm.3595 24901567 PMC4371131
30. Rajkumar S.V. Thalidomide in multiple myeloma Oncology 2000 14 (Suppl. S13) 11 16 11204667
31. Kumar A. Alam A. Grover S. Pandey S. Tripathi D. Kumari M. Rani M. Singh A. Akhter Y. Ehtesham N.Z. Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention npj Biofilms Microbiomes 2019 5 3 10.1038/s41522-018-0075-0 30675370 PMC6333787
32. An Q. Li C. Chen Y. Deng Y. Yang T. Luo Y. Repurposed drug candidates for antituberculosis therapy Eur. J. Med. Chem. 2020 192 112175 10.1016/j.ejmech.2020.112175 32126450
33. Mohammad Sadeghi H. Adeli I. Mousavi T. Daniali M. Nikfar S. Abdollahi M. Drug Repurposing for the Management of Depression: Where Do We Stand Currently? Life 2021 11 774 10.3390/life11080774 34440518 PMC8398872
34. DiMasi J.A. Hansen R.W. Grabowski H.G. The price of innovation: New estimates of drug development costs J. Health Econ. 2003 22 151 185 10.1016/S0167-6296(02)00126-1 12606142
35. Konreddy A.K. Rani G.U. Lee K. Choi Y. Recent drug-repurposing-driven advances in the discovery of novel antibiotics Curr. Med. Chem. 2019 26 5363 5388 10.2174/0929867325666180706101404 29984648
36. Sun W. Sanderson P.E. Zheng W. Drug combination therapy increases successful drug repositioning Drug Discov. Today 2016 21 1189 1195 10.1016/j.drudis.2016.05.015 27240777 PMC4907866
37. Bernal P. Molina-Santiago C. Daddaoua A. Llamas M.A. Antibiotic adjuvants: Identification and clinical use Microb. Biotechnol. 2013 6 445 10.1111/1751-7915.12044 23445397 PMC3918149
38. Melander R.J. Melander C. The challenge of overcoming antibiotic resistance: An adjuvant approach? ACS Infect. Dis. 2017 3 559 563 10.1021/acsinfecdis.7b00071 28548487 PMC5798239
39. Liu Y. Li R. Xiao X. Wang Z. Antibiotic adjuvants: An alternative approach to overcome multi-drug resistant Gram-negative bacteria Crit. Rev. Microbiol. 2019 45 301 314 10.1080/1040841X.2019.1599813 30985240
40. Ejim L. Farha M.A. Falconer S.B. Wildenhain J. Coombes B.K. Tyers M. Brown E.D. Wright G.D. Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy Nat. Chem. Biol. 2011 7 348 350 10.1038/nchembio.559 21516114
41. Gill E.E. Franco O.L. Hancock R.E. Antibiotic adjuvants: Diverse strategies for controlling drug-resistant pathogens Chem. Biol. Drug Des. 2015 85 56 78 10.1111/cbdd.12478 25393203 PMC4279029
42. Nikolaidis I. Favini-Stabile S. Dessen A. Resistance to antibiotics targeted to the bacterial cell wall Protein Sci. 2014 23 243 259 10.1002/pro.2414 24375653 PMC3945833
43. Worthington R.J. Melander C. Overcoming resistance to β-lactam antibiotics J. Org. Chem. 2013 78 4207 4213 10.1021/jo400236f 23530949 PMC3644377
44. Masters C. Selkoe D. Biochemistry of amyloid β-protein and amyloid deposits in Alzheimer disease Cold Spring Harb. Perspect. Med. 2012 2 a006262 10.1101/cshperspect.a006262 22675658 PMC3367542
45. Zhanel G.G. Golden A.R. Zelenitsky S. Wiebe K. Lawrence C.K. Adam H.J. Idowu T. Domalaon R. Schweizer F. Zhanel M.A. Cefiderocol: A Siderophore Cephalosporin with Activity Against Carbapenem-Resistant and Multidrug-Resistant Gram-Negative Bacilli Drugs 2019 79 271 289 10.1007/s40265-019-1055-2 30712199
46. Heo Y.-A. Imipenem/cilastatin/relebactam: A review in Gram-negative bacterial infections Drugs 2021 81 377 388 10.1007/s40265-021-01471-8 33630278 PMC7905759
47. Drawz S.M. Bonomo R.A. Three decades of β-lactamase inhibitors Clin. Microbiol. Rev. 2010 23 160 201 10.1128/CMR.00037-09 20065329 PMC2806661
48. Cochrane S.A. Li X. He S. Yu M. Wu M. Vederas J.C. Synthesis of Tridecaptin–Antibiotic Conjugates with in Vivo Activity against Gram-Negative Bacteria J. Med. Chem. 2015 58 9779 9785 10.1021/acs.jmedchem.5b01578 26636619
49. Van Bambeke F. Pagès J.M. Lee V.J. Inhibitors of bacterial efflux pumps as adjuvants in antibiotic treatments and diagnostic tools for detection of resistance by efflux Recent Pat. Antiinfect. Drug Discov. 2006 1 157 175 10.2174/157489106777452692 18221142
50. Berry L. Domalaon R. Brizuela M. Zhanel G.G. Schweizer F. Polybasic peptide-levofloxacin conjugates potentiate fluoroquinolones and other classes of antibiotics against multidrug-resistant Gram-negative bacteria Medchemcomm 2019 10 517 527 10.1039/C9MD00051H 31057731 PMC6482413
51. Lomovskaya O. Warren M.S. Lee A. Galazzo J. Fronko R. Lee M. Blais J. Cho D. Chamberland S. Renau T. Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: Novel agents for combination therapy Antimicrob. Agents Chemother. 2001 45 105 116 10.1128/AAC.45.1.105-116.2001 11120952 PMC90247
52. Bruhn D.F. Scherman M.S. Liu J. Scherbakov D. Meibohm B. Böttger E.C. Lenaerts A.J. Lee R.E. In vitro and in vivo Evaluation of Synergism between Anti-Tubercular Spectinamides and Non-Classical Tuberculosis Antibiotics Sci. Rep. 2015 5 13985 10.1038/srep13985 26365087 PMC4568539
53. Aboelenin A.M. Hassan R. Abdelmegeed E.S. The effect of EDTA in combination with some antibiotics against clinical isolates of gram negative bacteria in Mansoura, Egypt Microb. Pathog. 2021 154 104840 10.1016/j.micpath.2021.104840 33691177
54. Verderosa A.D. Dhouib R. Fairfull-Smith K.E. Totsika M. Nitroxide Functionalized Antibiotics Are Promising Eradication Agents against Staphylococcus aureus Biofilms Antimicrob. Agents Chemother. 2019 64 e01685-19 10.1128/AAC.01685-19 31636066 PMC7187575
55. Verderosa A.D. Hawas S. Harris J. Totsika M. Fairfull-Smith K.E. Isothiazolone-Nitroxide Hybrids with Activity against Antibiotic-Resistant Staphylococcus aureus Biofilms ACS Omega 2022 7 5300 5310 10.1021/acsomega.1c06433 35187345 PMC8851655
56. Landman D. Georgescu C. Martin D.A. Quale J. Polymyxins revisited Clin. Microbiol. Rev. 2008 21 449 465 18625681 10.1128/CMR.00006-08 PMC2493081
57. Velkov T. Thompson P.E. Nation R.L. Li J. Structure−Activity Relationships of Polymyxin Antibiotics J. Med. Chem. 2010 53 1898 1916 10.1021/jm900999h 19874036 PMC2907661
58. Velkov T. Roberts K.D. Nation R.L. Wang J. Thompson P.E. Li J. Teaching ‘Old’ Polymyxins New Tricks: New-Generation Lipopeptides Targeting Gram-Negative ‘Superbugs’ ACS Chem. Biol. 2014 9 1172 1177 10.1021/cb500080r 24601489 PMC4033650
59. Olaitan A.O. Morand S. Rolain J.-M. Mechanisms of polymyxin resistance: Acquired and intrinsic resistance in bacteria Front. Microbiol. 2014 5 643 10.3389/fmicb.2014.00643 25505462 PMC4244539
60. Bergen P.J. Bulman Z.P. Saju S. Bulitta J.B. Landersdorfer C. Forrest A. Li J. Nation R.L. Tsuji B.T. Polymyxin combinations: Pharmacokinetics and pharmacodynamics for rationale use Pharmacotherapy 2015 35 34 42 10.1002/phar.1537 25630411 PMC5215892
61. Lenhard J.R. Nation R.L. Tsuji B.T. Synergistic combinations of polymyxins Int. J. Antimicrob. Agents 2016 48 607 613 27865626 10.1016/j.ijantimicag.2016.09.014 PMC5237374
62. Ayoub Moubareck C. Polymyxins and Bacterial Membranes: A Review of Antibacterial Activity and Mechanisms of Resistance Membranes 2020 10 181 10.3390/membranes10080181 32784516 PMC7463838
63. Trimble M.J. Mlynárčik P. Kolář M. Hancock R.E. Polymyxin: Alternative Mechanisms of Action and Resistance Cold Spring Harb. Perspect. Med. 2016 6 a025288 10.1101/cshperspect.a025288 27503996 PMC5046685
64. Moore R.A. Bates N.C. Hancock R.E. Interaction of polycationic antibiotics with Pseudomonas aeruginosa lipopolysaccharide and lipid A studied by using dansyl-polymyxin Antimicrob. Agents Chemother. 1986 29 496 500 10.1128/AAC.29.3.496 3013085 PMC180420
65. Nang S.C. Azad M.A.K. Velkov T. Zhou Q. Li J. Rescuing the Last-Line Polymyxins: Achievements and Challenges Pharmacol. Rev. 2021 73 679 728 10.1124/pharmrev.120.000020 33627412 PMC7911091
66. Mohapatra S.S. Dwibedy S.K. Padhy I. Polymyxins, the last-resort antibiotics: Mode of action, resistance emergence, and potential solutions J. Biosci. 2021 46 85 10.1007/s12038-021-00209-8 34475315 PMC8387214
67. Breijyeh Z. Jubeh B. Karaman R. Resistance of Gram-Negative Bacteria to Current Antibacterial Agents and Approaches to Resolve It Molecules 2020 25 1340 10.3390/molecules25061340 32187986 PMC7144564
68. Clifton L.A. Skoda M.W. Daulton E.L. Hughes A.V. Le Brun A.P. Lakey J.H. Holt S.A. Asymmetric phospholipid: Lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic J. R. Soc. Interface 2013 10 20130810 10.1098/rsif.2013.0810 24132206 PMC3808558
69. Velkov T. Roberts K.D. Nation R.L. Thompson P.E. Li J. Pharmacology of polymyxins: New insights into an ‘old’ class of antibiotics Future Microbiol. 2013 8 711 724 10.2217/fmb.13.39 23701329 PMC3852176
70. Liu Y.Y. Wang Y. Walsh T.R. Yi L.X. Zhang R. Spencer J. Doi Y. Tian G. Dong B. Huang X. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: A microbiological and molecular biological study Lancet Infect. Dis. 2016 16 161 168 10.1016/S1473-3099(15)00424-7 26603172
71. Hassan J. Eddine R.Z. Mann D. Li S. Deng X. Saoud I.P. Kassem I.I. The Mobile Colistin Resistance Gene, mcr-1.1, Is Carried on IncX4 Plasmids in Multidrug Resistant E. coli Isolated from Rainbow Trout Aquaculture Microorganisms 2020 8 1636 10.3390/microorganisms8111636 33113918 PMC7690709
72. Gao R. Hu Y. Li Z. Sun J. Wang Q. Lin J. Ye H. Liu F. Srinivas S. Li D. Dissemination and mechanism for the MCR-1 colistin resistance PLoS Pathog. 2016 12 e1005957 10.1371/journal.ppat.1005957 27893854 PMC5125707
73. Hussein M. Han M.-L. Zhu Y. Schneider-Futschik E.K. Hu X. Zhou Q.T. Lin Y.-W. Anderson D. Creek D.J. Hoyer D. Mechanistic insights from global metabolomics studies into synergistic bactericidal effect of a polymyxin B combination with tamoxifen against cystic fibrosis MDR Pseudomonas aeruginosa Comput. Struct. Biotechnol. J. 2018 16 587 599 10.1016/j.csbj.2018.11.001 30546859 PMC6280556
74. Hussein M.H. Schneider E.K. Elliott A.G. Han M. Reyes-Ortega F. Morris F. Blaskovich M.A. Jasim R. Currie B. Mayo M. From breast cancer to antimicrobial: Combating extremely resistant Gram-negative “superbugs” using novel combinations of polymyxin B with selective estrogen receptor modulators Microb. Drug Resist. 2017 23 640 650 10.1089/mdr.2016.0196 27935770
75. Tran T.B. Wang J. Doi Y. Velkov T. Bergen P.J. Li J. Novel polymyxin combination with antineoplastic mitotane improved the bacterial killing against polymyxin-resistant multidrug-resistant gram-negative pathogens Front. Microbiol. 2018 9 721 10.3389/fmicb.2018.00721 29706941 PMC5906568
76. Tran T.B. Bergen P.J. Creek D.J. Velkov T. Li J. Synergistic killing of polymyxin B in combination with the antineoplastic drug mitotane against polymyxin-susceptible and-resistant Acinetobacter baumannii: A metabolomic study Front. Pharmacol. 2018 9 359 10.3389/fphar.2018.00359 29713282 PMC5911485
77. Lin Y.-W. Abdul Rahim N. Zhao J. Han M.-L. Yu H.H. Wickremasinghe H. Chen K. Wang J. Paterson D.L. Zhu Y. Novel polymyxin combination with the antiretroviral zidovudine exerts synergistic killing against NDM-producing multidrug-resistant Klebsiella pneumoniae Antimicrob. Agents Chemother. 2019 63 e02176-18 10.1128/AAC.02176-18 30670431 PMC6437486
78. Hussein M. Hu X. Paulin O.K. Crawford S. Zhou Q.T. Baker M. Schneider-Futschik E.K. Zhu Y. Li J. Velkov T. Polymyxin B combinations with FDA-approved non-antibiotic phenothiazine drugs targeting multi-drug resistance of Gram-negative pathogens Comput. Struct. Biotechnol. J. 2020 18 2247 2258 10.1016/j.csbj.2020.08.008 32952938 PMC7481501
79. Hussein M. Wong L.J. Zhao J. Rees V.E. Allobawi R. Sharma R. Rao G.G. Baker M. Li J. Velkov T. Unique mechanistic insights into pathways associated with the synergistic activity of polymyxin B and caspofungin against multidrug-resistant Klebsiella pneumoniae Comput. Struct. Biotechnol. J. 2022 20 1077 1087 10.1016/j.csbj.2022.02.021 35284046 PMC8897686
80. Fernandes L. Fortes B.N. Lincopan N. Ishida K. Caspofungin and polymyxin B reduce the cell viability and total biomass of mixed biofilms of carbapenem-resistant Pseudomonas aeruginosa and Candida spp Front. Microbiol. 2020 11 573263 10.3389/fmicb.2020.573263 33391197 PMC7772422
81. Pietschmann S. Hoffmann K. Voget M. Pison U. Synergistic effects of miconazole and polymyxin B on microbial pathogens Vet. Res. Commun. 2009 33 489 505 10.1007/s11259-008-9194-z 19085068 PMC2707952
82. Hussein M. Schneider-Futschik E.K. Paulin O.K. Allobawi R. Crawford S. Zhou Q.T. Hanif A. Baker M. Zhu Y. Li J. Effective strategy targeting polymyxin-resistant gram-negative pathogens: Polymyxin B in combination with the selective serotonin reuptake inhibitor sertraline ACS Infect. Dis. 2020 6 1436 1450 10.1021/acsinfecdis.0c00108 32427476
83. Hussein M. Allobawi R. Levou I. Blaskovich M.A. Rao G.G. Li J. Velkov T. Mechanisms Underlying Synergistic Killing of Polymyxin B in Combination with Cannabidiol against Acinetobacter baumannii: A Metabolomic Study Pharmaceutics 2022 14 786 10.3390/pharmaceutics14040786 35456620 PMC9025570
84. Abichabki N. Zacharias L.V. Moreira N.C. Bellissimo-Rodrigues F. Moreira F.L. Benzi J.R. Ogasawara T. Ferreira J.C. Ribeiro C.M. Pavan F.R. Potential cannabidiol (CBD) repurposing as antibacterial and promising therapy of CBD plus polymyxin B (PB) against PB-resistant gram-negative bacilli Sci. Rep. 2022 12 6454 10.1038/s41598-022-10393-8 35440801 PMC9018834
85. Betts J.W. Sharili A.S. La Ragione R.M. Wareham D.W. In Vitro Antibacterial Activity of Curcumin–Polymyxin B Combinations against Multidrug-Resistant Bacteria Associated with Traumatic Wound Infections J. Nat. Prod. 2016 79 1702 1706 10.1021/acs.jnatprod.6b00286 27295561
86. Allobawi R. Ghelani D.P. Schneider-Futschik E.K. Metabolomic description of ivacaftor elevating polymyxin B mediated antibacterial activity in cystic fibrosis Pseudomonas aeruginosa ACS Pharmacol. Transl. Sci. 2020 3 433 443 10.1021/acsptsci.0c00030 32566909 PMC7296539
87. Schneider E.K. Azad M.A. Han M.-L. Zhou Q. Wang J. Huang J.X. Cooper M.A. Doi Y. Baker M.A. Bergen P.J. An “unlikely” pair: The antimicrobial synergy of polymyxin B in combination with the cystic fibrosis transmembrane conductance regulator drugs KALYDECO and ORKAMBI ACS Infect. Dis. 2016 2 478 488 10.1021/acsinfecdis.6b00035 27626100 PMC5300747
88. Tran T.B. Cheah S.E. Yu H.H. Bergen P.J. Nation R.L. Creek D.J. Purcell A. Forrest A. Song J. Velkov T. Anthelmintic closantel enhances bacterial killing of polymyxin B against multidrug-resistant Acinetobacter baumannii J. Antibiot. 2016 69 415 421 10.1038/ja.2015.127 PMC4911330 26669752
89. Feng X. Liu S. Wang Y. Zhang Y. Sun L. Li H. Wang C. Liu Y. Cao B. Synergistic Activity of Colistin Combined With Auranofin Against Colistin-Resistant Gram-Negative Bacteria Front. Microbiol. 2021 12 676414 10.3389/fmicb.2021.676414 34248888 PMC8267823
90. Hu Y. Liu Y. Coates A. Azidothymidine produces synergistic activity in combination with colistin against antibiotic-resistant Enterobacteriaceae Antimicrob. Agents Chemother. 2019 63 e01630-18 10.1128/AAC.01630-18 30373798 PMC6325217
91. Domalaon R. Okunnu O. Zhanel G.G. Schweizer F. Synergistic combinations of anthelmintic salicylanilides oxyclozanide, rafoxanide, and closantel with colistin eradicates multidrug-resistant colistin-resistant Gram-negative bacilli J. Antibiot. 2019 72 605 616 10.1038/s41429-019-0186-8 31028351
92. Ayerbe-Algaba R. Gil-Marqués M.L. Miró-Canturri A. Parra-Millán R. Pachón-Ibáñez M.E. Jiménez-Mejías M.E. Pachón J. Smani Y. The anthelmintic oxyclozanide restores the activity of colistin against colistin-resistant Gram-negative bacilli Int. J. Antimicrob. Agents 2019 54 507 512 10.1016/j.ijantimicag.2019.07.006 31299296
93. Yi K. Liu S. Liu P. Luo X. Zhao J. Yan F. Pan Y. Liu J. Zhai Y. Hu G. Synergistic antibacterial activity of tetrandrine combined with colistin against MCR-mediated colistin-resistant Salmonella Biomed. Pharmacother. 2022 149 112873 10.1016/j.biopha.2022.112873 35349932
94. Liu Y. Jia Y. Yang K. Tong Z. Shi J. Li R. Xiao X. Ren W. Hardeland R. Reiter R.J. Melatonin overcomes MCR-mediated colistin resistance in Gram-negative pathogens Theranostics 2020 10 10697 10.7150/thno.45951 32929375 PMC7482817
95. Kaur A. Sharma P. Capalash N. Curcumin alleviates persistence of Acinetobacter baumannii against colistin Sci. Rep. 2018 8 11029 10.1038/s41598-018-29291-z 30038318 PMC6056455
96. Miró-Canturri A. Ayerbe-Algaba R. Vila-Domínguez A. Jiménez-Mejías M.E. Pachón J. Smani Y. Repurposing of the tamoxifen metabolites to combat infections by multidrug-resistant Gram-negative bacilli Antibiotics 2021 10 336 10.3390/antibiotics10030336 33810067 PMC8004611
97. Al-Janabi A.A.H.S. Repurposing of Tamoxifen Against the Oral Bacteria Turk. J. Pharm. Sci. 2021 18 68 10.4274/tjps.galenos.2019.23500 33634670 PMC7957305
98. Lo Iacono M. Puglisi S. Perotti P. Saba L. Petiti J. Giachino C. Reimondo G. Terzolo M. Molecular mechanisms of mitotane action in adrenocortical cancer based on in vitro studies Cancers 2021 13 5255 10.3390/cancers13215255 34771418 PMC8582505
99. Grimsey E.M. Piddock L.J. Do phenothiazines possess antimicrobial and efflux inhibitory properties? FEMS Microbiol. Rev. 2019 43 577 590 10.1093/femsre/fuz017 31216574
100. Dastidar S.G. Kristiansen J.E. Molnar J. Amaral L. Role of phenothiazines and structurally similar compounds of plant origin in the fight against infections by drug resistant bacteria Antibiotics 2013 2 58 72 10.3390/antibiotics2010058 27029292 PMC4790298
101. Amaral L. Viveiros M. Molnar J. Antimicrobial activity of phenothiazines In Vivo 2004 18 725 732 15646813
102. Amaral L. Viveiros M. Kristiansen J.E. Phenothiazines: Potential alternatives for the management of antibiotic resistant infections of tuberculosis and malaria in developing countries Trop. Med. Int. Health 2001 6 1016 1022 10.1046/j.1365-3156.2001.00804.x 11737839
103. Amaral L. Viveiros M. Thioridazine: A non-antibiotic drug highly effective, in combination with first line anti-tuberculosis drugs, against any form of antibiotic resistance of Mycobacterium tuberculosis due to its multi-mechanisms of action Antibiotics 2017 6 3 10.3390/antibiotics6010003 28098814 PMC5372983
104. Ayaz M. Subhan F. Ahmed J. Khan A.-U. Ullah F. Ullah I. Ali G. Syed N.-I.-H. Hussain S. Sertraline enhances the activity of antimicrobial agents against pathogens of clinical relevance J. Biol. Res. Thessalon. 2015 22 4 10.1186/s40709-015-0028-1 26029671 PMC4449573
105. Hellio C. Pons A.M. Beaupoil C. Bourgougnon N. Le Gal Y. Antibacterial, antifungal and cytotoxic activities of extracts from fish epidermis and epidermal mucus Int. J. Antimicrob. Agents 2002 20 214 219 10.1016/S0924-8579(02)00172-3 12385701
106. Fukushima T. Ueda T. Uchida M. Nakamura T. Action mechanism of idarubicin (4-demethoxydaunorubicin) as compared with daunorubicin in leukemic cells Int. J. Hematol. 1993 57 121 130 8494991
107. Sreedhara Swamy K. Studies on the mechanism of action of miconazole: Effect of miconazole on respiration and cell permeability of Candida albicans Antimicrob. Agents Chemother. 1974 5 420 425 10.1128/AAC.5.4.420 15825399 PMC428986
108. Vanden Bossche H. Marichal P. Willemsens G. Bellens D. Gorrens J. Roels I. Coene M.-C. Le Jeune L. Janssen P. Saperconazole: A selective inhibitor of the cytochrome P-450-dependent ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes Mycoses 1990 33 335 352 10.1111/myc.1990.33.7-8.335 2090934
109. Tabatabaei S.A. Soleimani M. Mansouri M.R. Mirshahi A. Inanlou B. Abrishami M. Pakrah A.R. Masarat H. Closantel; a veterinary drug with potential severe morbidity in humans BMC Ophthalmol. 2016 16 207 10.1186/s12886-016-0387-x 27899086 PMC5129234
110. Li J. Rayner C.R. Nation R.L. Owen R.J. Spelman D. Tan K.E. Liolios L. Heteroresistance to colistin in multidrug-resistant Acinetobacter baumannii Antimicrob. Agents Chemother. 2006 50 2946 2950 10.1128/AAC.00103-06 16940086 PMC1563544
111. Zagzoog A. Mohamed K.A. Kim H.J.J. Kim E.D. Frank C.S. Black T. Jadhav P.D. Holbrook L.A. Laprairie R.B. In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa Sci. Rep. 2020 10 20405 10.1038/s41598-020-77175-y 33230154 PMC7684313
112. Karas J.A. Wong L.J. Paulin O.K. Mazeh A.C. Hussein M.H. Li J. Velkov T. The antimicrobial activity of cannabinoids Antibiotics 2020 9 406 10.3390/antibiotics9070406 32668669 PMC7400265
113. Blaskovich M.A. Kavanagh A.M. Elliott A.G. Zhang B. Ramu S. Amado M. Lowe G.J. Hinton A.O. Pham D.M.T. Zuegg J. The antimicrobial potential of cannabidiol Commun. Biol. 2021 4 7 10.1038/s42003-020-01530-y 33469147 PMC7815910
114. Van Klingeren B. Ten Ham M. Antibacterial activity of Δ9-tetrahydrocannabinol and cannabidiol Antonie Van Leeuwenhoek 1976 42 9 12 10.1007/BF00399444 1085130
115. Moffatt J.H. Harper M. Harrison P. Hale J.D.F. Vinogradov E. Seemann T. Henry R. Crane B. Michael F.S. Cox A.D. Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production Antimicrob. Agents Chemother. 2010 54 4971 4977 10.1128/AAC.00834-10 20855724 PMC2981238
116. Hatcher H. Planalp R. Cho J. Torti F.M. Torti S.V. Curcumin: From ancient medicine to current clinical trials Cell. Mol. Life Sci. 2008 65 1631 1652 10.1007/s00018-008-7452-4 18324353 PMC4686230
117. Dai C. Lin J. Li H. Shen Z. Wang Y. Velkov T. Shen J. The Natural Product Curcumin as an Antibacterial Agent: Current Achievements and Problems Antioxidants 2022 11 459 10.3390/antiox11030459 35326110 PMC8944601
118. Adamczak A. Ożarowski M. Karpiński T.M. Curcumin, a natural antimicrobial agent with strain-specific activity Pharmaceuticals 2020 13 153 10.3390/ph13070153 32708619 PMC7408453
119. Zhang L. Geng Y. Duan W. Wang D. Fu M. Wang X. Ionic liquid-based ultrasound-assisted extraction of fangchinoline and tetrandrine from Stephaniae tetrandrae J. Sep. Sci. 2009 32 3550 3554 10.1002/jssc.200900413 19764054
120. Li D.-G. Wang Z.-R. Lu H.-M. Pharmacology of tetrandrine and its therapeutic use in digestive diseases World J. Gastroenterol. 2001 7 627 10.3748/wjg.v7.i5.627 11819843 PMC4695563
121. Lee Y.-S. Han S.-H. Lee S.-H. Kim Y.-G. Park C.-B. Kang O.-H. Keum J.-H. Kim S.-B. Mun S.-H. Seo Y.-S. The mechanism of antibacterial activity of tetrandrine against Staphylococcus aureus Foodborne Pathog. Dis. 2012 9 686 691 10.1089/fpd.2011.1119 22845553
122. Zhang Z. Yan J. Xu K. Ji Z. Li L. Tetrandrine reverses drug resistance in isoniazid and ethambutol dual drug-resistant Mycobacterium tuberculosis clinical isolates BMC Infect. Dis. 2015 15 153 10.1186/s12879-015-0905-0 25887373 PMC4417324
123. Harbut M.B. Vilchèze C. Luo X. Hensler M.E. Guo H. Yang B. Chatterjee A.K. Nizet V. Jacobs W.R. Jr. Schultz P.G. Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis Proc. Natl. Acad. Sci. USA 2015 112 4453 4458 10.1073/pnas.1504022112 25831516 PMC4394260
124. Mitsuya H. Weinhold K.J. Furman P.A. St Clair M.H. Lehrman S.N. Gallo R.C. Bolognesi D. Barry D.W. Broder S. 3′-Azido-3′-deoxythymidine (BW A509U): An antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro Proc. Natl. Acad. Sci. USA 1985 82 7096 7100 10.1073/pnas.82.20.7096 2413459 PMC391317
125. Doléans-Jordheim A. Bergeron E. Bereyziat F. Ben-Larbi S. Dumitrescu O. Mazoyer M.-A. Morfin F. Dumontet C. Freney J. Jordheim L. Zidovudine (AZT) has a bactericidal effect on enterobacteria and induces genetic modifications in resistant strains Eur. J. Clin. Microbiol. Infect. Dis. 2011 30 1249 1256 10.1007/s10096-011-1220-3 21494911
126. Hu Y. Coates A. Combination Comprising Zidovudine and Polymyxin U.S. Patent No. 9,757,427 12 September 2017
127. He F. Wu X. Zhang Q. Li Y. Ye Y. Li P. Chen S. Peng Y. Hardeland R. Xia Y. Bacteriostatic potential of melatonin: Therapeutic standing and mechanistic insights Front. Immunol. 2021 12 683879 10.3389/fimmu.2021.683879 34135911 PMC8201398
128. Chong C.R. Sullivan D.J. Jr. New uses for old drugs Nature 2007 448 645 646 10.1038/448645a 17687303
129. Harris T.L. Worthington R.J. Hittle L.E. Zurawski D.V. Ernst R.K. Melander C. Small molecule downregulation of PmrAB reverses lipid A modification and breaks colistin resistance ACS Chem. Biol. 2014 9 122 127 10.1021/cb400490k 24131198
130. Hartzell J.D. Neff R. Ake J. Howard R. Olson S. Paolino K. Vishnepolsky M. Weintrob A. Wortmann G. Nephrotoxicity associated with intravenous colistin (colistimethate sodium) treatment at a tertiary care medical center Clin. Infect. Dis. 2009 48 1724 1728 10.1086/599225 19438394