To prolong and maximize the clinical efficacy of polymyxins and prevent the emergence of antibiotic resistance, novel polymyxin combination therapy against MDR Gram-negative bacteria such as A. baumannii is desperately needed. Although researchers have shown that polymyxins in combination with cannabidiol displayed synergistic killing activity against Gram-negative bacteria, including A. baumannii, they did not perform time-kill assays or investigate the precise mechanisms behind the synergistic killing activity of polymyxins in combination with the cannabidiol against A. baumannii [33,35,36]. To the best of our knowledge, this is the first study to decipher the mechanisms of synergistic killing activity of polymyxins and synthetic cannabidiol combination therapy against A. baumannii using untargeted metabolomics.

The checkerboard and time-kill assays demonstrated synergistic bacterial killing by polymyxins plus cannabidiol treatment against a panel of MDR Gram-negative pathogens (A. baumannii, K. pneumoniae and P. aeruginosa) (Figure 1; Table 1). The metabolomics study showed that polymyxin B–cannabidiol treatment caused a greater perturbation in A. baumannii ATCC 19606 metabolome compared to monotherapies, particularly at the later time exposure (4 h). The most significant findings for integrating pathway enrichment analysis include (i) dysregulation in bacterial cell envelope biogenesis, (ii) inhibition of DNA and RNA metabolism, as reflected by the major perturbation of purine and pyrimidine metabolism, and (iii) perturbation of peptide metabolism.

Polymyxins exert primary antibacterial killing activity by disorganizing the bacterial OM [9,52]. Unsurprisingly, at 1 h, polymyxin B caused a marked suppression, albeit far less compared to that achieved with the combination, in fatty acids, glycerophospholipids metabolism as well as key lipids of OM such as trans-hexadec-2-enoyl-CoA, sn-glycero-3-phosphocholine and sn-glycero-3-phosphoethanolamine (Figure 2A,B). Our findings agreed with previous metabolomics and transcriptomics studies that showed that A. baumannii metabolites and genes involved primarily in bacterial OM biosynthesis and phospholipid trafficking experienced a significant alteration at 1 h polymyxin posttreatment [19,53]. However, the impact from polymyxin B monotherapy on levels of essential bacterial membrane lipids was minimal at 4 h, suggesting that polymyxin resistance may occur within 4 h after polymyxin B monotherapy. The antimicrobial effect of cannabidiol has recently gained great attention; however, the precise mode of action is still unclear [34,35,36,37,38]. It was found that cannabidiol inhibited membrane lipid synthesis of S. aureus [38]. This evidence is in line with our findings that showed the inhibitory impact of cannabidiol on levels of essential bacterial membrane lipids at 1 h, including phosphatidylethanolamine, sn-glycero-3-phosphoethanolamine and 1-hexadecanoyl-2-sn-glycero-3-phosphate (Figure 2A). Nevertheless, this effect disappeared at the later time exposure of 4 h. Promisingly, the combination caused extensive perturbations (largely at 1 h and to a lesser extent at 4 h) in fatty acids and glycerophospholipids (Figure 2A,B). There has been growing interest in fatty acid biosynthesis as a major target for antibiotic development, for instance, the newly discovered fatty acid elongation inhibitors, platensimycin and platencin [54]. A greater reduction in the levels of key bacterial membrane lipids (e.g., phosphatidylethanolamine, 1,2-diacyl-sn-glycerol 3-phosphate (phosphatidic acid), sn-glycero-3-phosphoethanolamine and sn-glycerol 3-phosphate) was observed after the combination treatment at both time points (1 and 4 h) (Figure 2A,B). In Gram-negative bacteria, phosphatidylethanolamine accounts for 70–80% of total membrane lipids, which are synthesized on the cytoplasmic side of the inner membrane [55,56]. It has been found that an alteration in the levels of phosphatidylethanolamine leads to critically compromised cell integrity and ultimately cell death [44]. Sn-glycerol 3-phosphate is an essential precursor required for the formation of membrane phospholipids; it undergoes acylation at the 1-position to form lysophosphatidic acid (LPA), and then a second acylation step produces phosphatidic acid (PA), the principal component in the synthesis of bacterial membrane glycerolipids [57]. Targeting phosphatidic acid synthesis in Gram-negative bacteria might offer a new and effective strategy to develop novel antibacterial therapeutics [46].

Importantly, our study is the first to report that combining polymyxin B with cannabidiol caused a marked suppression in the components of peptidoglycan and LPS biogenesis at 1 h, including UDP-MurNAc-L-Ala-ƴ-D-Glu-m-Dap-D-Ala-D-Ala and D-alanyl-D-alanine (Supplementary Figure S4). This influence may have arisen from inhibition of amino-sugar and nucleotide-sugar metabolism and the pentose phosphate pathway, which are key sources of precursors for peptidoglycan and LPS synthesis. This was manifested by a significant decline in the levels of UDP-GlcNAc, D-sedoheptulose 7-phosphate and Ru5P after combination treatment (Supplementary Figure S4). UDP-GlcNAc plays a pivotal role as an amino sugar donor in several transferase reactions in the biosynthesis of peptidoglycan, the core lipid A components of the LPS, and certain O-antigens of Gram-negative bacteria [58,59]. The synthesis of KDO, an essential residue of the LPS inner core, is initiated by the enzyme D-arabinose-5-phosphate isomerase (API), which regulates the reversible isomerization of D-ribulose 5-phosphate (Ru5P) to D-arabinose-5-phosphate, a precursor of 3-deoxy-D-manno-octulosonate [60]. It is important to note that the combination treatment remained effective in disorganizing the bacterial envelope formation at 4 h, in which more perturbations in peptidoglycan and LPS biosynthesis were most prominent (Figure 3A,B). Intriguingly, the combination treatment caused a profound decrease in the abundance of undecaprenyl phosphate (C55-P), also known as bactoprenol, a sugar carrier lipid that mediates the biosynthesis of bacterial extracellular polysaccharides such as cell wall peptidoglycan and LPS [61,62], at 4 h (Figure 3A,B). It has been proposed that bactoprenol is a potential novel target for new antibacterial agents [63,64].

Consistent with our previous results, it was not unusual that polymyxin B monotherapy caused a significant alteration, though less intense compared to the combination, in the main pathways involved in the cell envelope assembly, including peptidoglycan and LPS biosynthesis at both time points (1 and 4 h) (Figure 3A,B) [17,18,19]. Remarkably, pathway analysis also showed that despite being a non-antibiotic, cannabidiol reduced the KDO and UDP-GlcNAc levels at 4 h (Figure 3B). Hence, taking data from all of the above-mentioned studies together, a possible mechanism of synergistic killing by the polymyxin B–cannabidiol combination is strongly related to the suppression of amino sugar and nucleotide sugar metabolism and PPP and subsequent peptidoglycan and LPS biosynthesis, which eventually leads to bacterial membrane structure deformity.

Apart from its disruptive impact on the bacterial membrane, the combination therapy produced profound changes in the nucleotide pool of A. baumannii ATCC 19606 at both time exposures (1 and 4 h); however, the maximum effect on the nucleotide pool was at 4 h (Figure 4A,B). The perturbed nucleotides were mainly those related to purine and pyrimidine metabolism (e.g., ATP, ADP, UDP and GMP), which ultimately impacted DNA and RNA synthesis. Nucleotide metabolism such as purine metabolism is actively involved in antibiotic efficacy and lethality [65,66]. It has been found that dysregulation in purine metabolism can alter antibiotic lethality; this has been manifested by an increase in the bacterial killing activity of gentamicin following genetic deletion of enzymes involved in purine metabolism [65]. Given the significant alteration of the majority of the nucleotides involved in purine and pyrimidine, it is highly likely that the combination treatment compromised DNA and RNA biosynthesis in A. baumannii. Notably, it has been previously reported that polymyxins were able to dysregulate the nucleotide pool of different Gram-negative pathogens such as A. baumannii, K. pneumoniae and P. aeruginosa [50,64,65]. A recent study using radiolabeled macromolecular synthesis assays revealed the ability of cannabidiol to inhibit DNA, RNA and protein synthesis in S. aureus [38]. In line with these findings, polymyxin B and cannabidiol monotherapies demonstrated significant perturbations, albeit to a lesser extent compared to their combination, in the levels of nucleotides at 1 h, which then faded away at 4 h (Figure 4A,B). In combination, our findings showed polymyxin B and cannabidiol additionally affected peptide metabolism, which most likely led to inhibition of protein synthesis in A. baumannii.

Overall, the novel findings from this study are that polymyxin B monotherapy produces significant changes in multiple cellular metabolic pathways of cell envelope biogenesis, all of which were further disorganized by adding cannabidiol. Furthermore, the combination therapy greatly perturbed the nucleotide and peptide pools, suggesting a unique mechanism (non-bacterial membrane involvement) of synergistic killing for the polymyxin B–cannabidiol treatment. The current findings also showed that cannabidiol affects LPS, DNA, and lipid biosynthesis. The study highlights the importance of elucidating the complex and dynamic interactions of multiple cellular metabolic pathways due to antibiotic–nonantibiotic treatment. Such findings will aid in the understanding of the most likely mechanisms behind the killing synergy and ultimately facilitate future repurposing of cannabidiol as an antimicrobial agent in combination with polymyxins.