Alternative therapeutic strategies to treat antibiotic-resistant pathogens.

Resistance threatens to render antibiotics - which are essential for modern medicine - ineffective, thus posing a threat to human health. The discovery of novel classes of antibiotics able to overcome resistance has been stalled for decades, with the developmental pipeline relying almost entirely on variations of existing chemical scaffolds. Unfortunately, this approach has been unable to keep pace with resistance evolution, necessitating new therapeutic strategies. In this Review, we highlight recent efforts to discover non-traditional antimicrobials, specifically describing the advantages and limitations of antimicrobial peptides and macrocycles, antibodies, bacteriophages and antisense oligonucleotides. These approaches have the potential to stem the tide of resistance by expanding the physicochemical property space and target spectrum occupied by currently approved antibiotics.

Returning to Nature for the Next Generation of Antimicrobial Therapeutics.

Antibiotics found in and inspired by nature are life-saving cures for bacterial infections and have enabled modern medicine. However, the rise in resistance necessitates the discovery and development of novel antibiotics and alternative treatment strategies to prevent the return to a pre-antibiotic era. Once again, nature can serve as a source for new therapies in the form of natural product antibiotics and microbiota-based therapies. Screening of soil bacteria, particularly actinomycetes, identified most of the antibiotics used in the clinic today, but the rediscovery of existing molecules prompted a shift away from natural product discovery. Next-generation sequencing technologies and bioinformatics advances have revealed the untapped metabolic potential harbored within the genomes of environmental microbes. In this review, we first highlight current strategies for mining this untapped chemical space, including approaches to activate silent biosynthetic gene clusters and in situ culturing methods. Next, we describe how using live microbes in microbiota-based therapies can simultaneously leverage many of the diverse antimicrobial mechanisms found in nature to treat disease and the impressive efficacy of fecal microbiome transplantation and bacterial consortia on infection. Nature-provided antibiotics are some of the most important drugs in human history, and new technologies and approaches show that nature will continue to offer valuable inspiration for the next generation of antibacterial therapeutics.

Screening under infection-relevant conditions reveals chemical sensitivity in multidrug resistant invasive non-typhoidal Salmonella (iNTS).

Bloodstream infections caused by invasive, non-typhoidal Salmonella (iNTS) are a major global health concern, particularly in Africa where the pathogenic variant of Salmonella Typhimurium sequence type (ST) 313 is dominant. Unlike S. Typhimurium strains that cause gastroenteritis, iNTS strains cause bloodstream infections and are resistant to multiple first-line antibiotics, thus limiting current treatment options. Here, we developed and implemented multiple small molecule screens under physiological, infection-relevant conditions to reveal chemical sensitivities in ST313 and to identify host-directed therapeutics as entry points to drug discovery to combat the clinical burden of iNTS. Screening ST313 iNTS under host-mimicking growth conditions identified 92 compounds with antimicrobial activity despite inherent multidrug resistance. We characterized the antimicrobial activity of the nucleoside analog 3'-azido-3'-deoxythymidine as an exemplary compound from this screen, which depended on bacterial thymidine kinase activity for antimicrobial activity. In a companion macrophage-based screening platform designed to enrich for host-directed therapeutics, we identified three compounds (amodiaquine, berbamine, and indatraline) as actives that required the presence of host cells for antibacterial activity. These three compounds had antimicrobial activity only in the presence of host cells that significantly inhibited intracellular ST313 iNTS replication in macrophages. This work provides evidence that despite high invasiveness and multidrug resistance, ST313 iNTS remains susceptible to unconventional drug discovery approaches.

Inhibiting fatty acid synthesis overcomes colistin resistance.

Treating multidrug-resistant infections has increasingly relied on last-resort antibiotics, including polymyxins, for example colistin. As polymyxins are given routinely, the prevalence of their resistance is on the rise and increases mortality rates of sepsis patients. The global dissemination of plasmid-borne colistin resistance, driven by the emergence of mcr-1, threatens to diminish the therapeutic utility of polymyxins from an already shrinking antibiotic arsenal. Restoring sensitivity to polymyxins using combination therapy with sensitizing drugs is a promising approach to reviving its clinical utility. Here we describe the ability of the biotin biosynthesis inhibitor, MAC13772, to synergize with colistin exclusively against colistin-resistant bacteria. MAC13772 indirectly disrupts fatty acid synthesis (FAS) and restores sensitivity to the last-resort antibiotic, colistin. Accordingly, we found that combinations of colistin and other FAS inhibitors, cerulenin, triclosan and Debio1452-NH3, had broad potential against both chromosomal and plasmid-mediated colistin resistance in chequerboard and lysis assays. Furthermore, combination therapy with colistin and the clinically relevant FabI inhibitor, Debio1452-NH3, showed efficacy against mcr-1 positive Klebsiella pneumoniae and colistin-resistant Escherichia coli systemic infections in mice. Using chemical genomics, lipidomics and transcriptomics, we explored the mechanism of the interaction. We propose that inhibiting FAS restores colistin sensitivity by depleting lipid synthesis, leading to changes in phospholipid composition. In all, this work reveals a surprising link between FAS and colistin resistance.

The role of bacterial membrane vesicles in antibiotic resistance.

Bacterial survival during antibiotic exposure is a complex and multifaceted phenomenon. On top of antibiotic resistance genes, biofilm formation, and persister tolerance, bacterial membrane vesicles (MVs) provide a layer of protection that has been largely overlooked. MVs are spherical nanoparticles composed of lipid membranes and are common to Gram-positive and Gram-negative bacteria. Although the importance of MVs in bacterial pathogenesis and virulence factor transport has been firmly established, a growing body of work now identifies MVs as key contributors to bacterial survival during antibiotic exposure. Herein, we highlight the ability of MVs to reduce antibiotic efficacy and transmit resistance elements. We also discuss the potential of targeting MV production as an unconventional therapeutic approach.

A genetic platform to investigate the functions of bacterial drug efflux pumps.

Efflux pumps are a serious challenge for the development of antibacterial agents. Overcoming efflux requires an in-depth understanding of efflux pump functions, specificities and the development of inhibitors. However, the complexities of efflux networks have limited such studies. To address these challenges, we generated Efflux KnockOut-35 (EKO-35), a highly susceptible Escherichia coli strain lacking 35 efflux pumps. We demonstrate the use of this strain by constructing an efflux platform comprising EKO-35 strains individually producing efflux pumps forming tripartite complexes with TolC. This platform was profiled against a curated diverse compound collection, which enabled us to define physicochemical properties that contribute to transport. We also show the E. coli drug efflux network is conditionally essential for growth, and that the platform can be used to investigate efflux pump inhibitor specificities and efflux pump interplay. We believe EKO-35 and the efflux platform will have widespread application for the study of drug efflux.

Preclinical Development of Pentamidine Analogs Identifies a Potent and Nontoxic Antibiotic Adjuvant.

The difficulty in treating Gram-negative bacteria can largely be attributed to their highly impermeable outer membrane (OM), which serves as a barrier to many otherwise active antibiotics. This can be overcome with the use of perturbant molecules, which disrupt OM integrity and sensitize Gram-negative bacteria to many clinically available Gram-positive-active antibiotics. Although many new perturbants have been identified in recent years, most of these molecules are impeded by toxicity due to the similarities between pathogen and host cell membranes. For example, our group recently reported the cryptic OM-perturbing activity of the antiprotozoal drug pentamidine. Its development as an antibiotic adjuvant is limited, however, by toxicity concerns. Herein, we took a medicinal chemistry approach to develop novel analogs of pentamidine, aiming to improve its OM activity while reducing its off-target toxicity. We identified the compound P35, which induces OM disruption and potentiates Gram-positive-active antibiotics in Acinetobacter baumannii and Klebsiella pneumoniae. Relative to pentamidine, P35 has reduced mammalian cell cytotoxicity and hERG trafficking inhibition. Additionally, P35 outperforms pentamidine in a murine model of A. baumannii bacteremia. Together, this preclinical analysis supports P35 as a promising lead for further development as an OM perturbant.

Overcoming Acquired and Native Macrolide Resistance with Bicarbonate.

The growing challenge of microbial resistance emphasizes the importance of new antibiotics or reviving strategies for the use of old ones. Macrolide antibiotics are potent bacterial protein synthesis inhibitors with a formidable capacity to treat life-threatening bacterial infections; however, acquired and intrinsic resistance limits their clinical application. In the work presented here, we reveal that bicarbonate is a potent enhancer of the activity of macrolide antibiotics that overcomes both acquired and intrinsic resistance mechanisms. With a focus on azithromycin, a highly prescribed macrolide antibiotic, and using clinically relevant pathogens, we show that physiological concentrations of bicarbonate overcome drug resistance by increasing the intracellular concentration of azithromycin. We demonstrate the potential of bicarbonate as a formulation additive for topical use of azithromycin in treating a murine wound infection caused by Pseudomonas aeruginosa. Further, using a systemic murine model of methicillin-resistant Staphylococcus aureus (MRSA) infection, we demonstrate the potential role of physiological bicarbonate, naturally abundant in the host, to enhance the activity of azithromycin against macrolide-resistant MRSA. In all, our findings suggest that macrolide resistance, observed in the clinical microbiology laboratory using standard culturing techniques, is a poor predictor of efficacy in the clinic and that observed resistance should not necessarily hamper the use of macrolides. Whether as a formulation additive for topical use or as a natural component of host tissues, bicarbonate is a powerful potentiator of macrolides with the capacity to overcome drug resistance in life-threatening bacterial infections.

Outer Membrane Disruption Overcomes Intrinsic, Acquired, and Spontaneous Antibiotic Resistance.

Disruption of the outer membrane (OM) barrier allows for the entry of otherwise inactive antimicrobials into Gram-negative pathogens. Numerous efforts to implement this approach have identified a large number of OM perturbants that sensitize Gram-negative bacteria to many clinically available Gram-positive active antibiotics. However, there is a dearth of investigation into the strengths and limitations of this therapeutic strategy, with an overwhelming focus on characterization of individual potentiator molecules. Herein, we look to explore the utility of exploiting OM perturbation to sensitize Gram-negative pathogens to otherwise inactive antimicrobials. We identify the ability of OM disruption to change the rules of Gram-negative entry, overcome preexisting and spontaneous resistance, and impact biofilm formation. Disruption of the OM expands the threshold of hydrophobicity compatible with Gram-negative activity to include hydrophobic molecules. We demonstrate that while resistance to Gram-positive active antibiotics is surprisingly common in Gram-negative pathogens, OM perturbation overcomes many antibiotic inactivation determinants. Further, we find that OM perturbation reduces the rate of spontaneous resistance to rifampicin and impairs biofilm formation. Together, these data suggest that OM disruption overcomes many of the traditional hurdles encountered during antibiotic treatment and is a high priority approach for further development.IMPORTANCE The spread of antibiotic resistance is an urgent threat to global health that necessitates new therapeutics. Treatments for Gram-negative pathogens are particularly challenging to identify due to the robust outer membrane permeability barrier in these organisms. Recent discovery efforts have attempted to overcome this hurdle by disrupting the outer membrane using chemical perturbants and have yielded several new peptides and small molecules that allow the entry of otherwise inactive antimicrobials. However, a comprehensive investigation into the strengths and limitations of outer membrane perturbants as antibiotic partners is currently lacking. Herein, we interrogate the interaction between outer membrane perturbation and several common impediments to effective antibiotic use. Interestingly, we discover that outer membrane disruption is able to overcome intrinsic, spontaneous, and acquired antibiotic resistance in Gram-negative bacteria, meriting increased attention toward this approach.

Targeting Two-Component Systems Uncovers a Small-Molecule Inhibitor of Salmonella Virulence.

Salmonella serovars are leading causes of gastrointestinal disease and have become increasingly resistant to fluoroquinolone and cephalosporin antibiotics. Overcoming this healthcare crisis requires new approaches in antibiotic discovery and the identification of unique bacterial targets. In this work, we describe a chemical genomics approach to identify inhibitors of Salmonella virulence. From a cell-based, promoter reporter screen of ∼50,000 small molecules, we identified dephostatin as a non-antibiotic compound that inhibits intracellular virulence factors and polymyxin resistance genes. Dephostatin disrupts signaling through both the SsrA-SsrB and PmrB-PmrA two-component regulatory systems and restores sensitivity to the last-resort antibiotic, colistin. Cell-based experiments and mouse models of infection demonstrate that dephostatin attenuates Salmonella virulence in vitro and in vivo, suggesting that perturbing regulatory networks is a promising strategy for the development of anti-infectives.

Uncovering the Hidden Antibiotic Potential of Cannabis.

The spread of antimicrobial resistance continues to be a priority health concern worldwide, necessitating the exploration of alternative therapies. Cannabis sativa has long been known to contain antibacterial cannabinoids, but their potential to address antibiotic resistance has only been superficially investigated. Here, we show that cannabinoids exhibit antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), inhibit its ability to form biofilms, and eradicate preformed biofilms and stationary phase cells persistent to antibiotics. We show that the mechanism of action of cannabigerol is through targeting the cytoplasmic membrane of Gram-positive bacteria and demonstrate in vivo efficacy of cannabigerol in a murine systemic infection model caused by MRSA. We also show that cannabinoids are effective against Gram-negative organisms whose outer membrane is permeabilized, where cannabigerol acts on the inner membrane. Finally, we demonstrate that cannabinoids work in combination with polymyxin B against multidrug resistant Gram-negative pathogens, revealing the broad-spectrum therapeutic potential for cannabinoids.

A Deep Learning Approach to Antibiotic Discovery.

Due to the rapid emergence of antibiotic-resistant bacteria, there is a growing need to discover new antibiotics. To address this challenge, we trained a deep neural network capable of predicting molecules with antibacterial activity. We performed predictions on multiple chemical libraries and discovered a molecule from the Drug Repurposing Hub-halicin-that is structurally divergent from conventional antibiotics and displays bactericidal activity against a wide phylogenetic spectrum of pathogens including Mycobacterium tuberculosis and carbapenem-resistant Enterobacteriaceae. Halicin also effectively treated Clostridioides difficile and pan-resistant Acinetobacter baumannii infections in murine models. Additionally, from a discrete set of 23 empirically tested predictions from >107 million molecules curated from the ZINC15 database, our model identified eight antibacterial compounds that are structurally distant from known antibiotics. This work highlights the utility of deep learning approaches to expand our antibiotic arsenal through the discovery of structurally distinct antibacterial molecules.

Creative targeting of the Gram-negative outer membrane in antibiotic discovery.

The rising threat of multidrug-resistant Gram-negative bacteria is exacerbated by the scarcity of new antibiotics in the development pipeline. Permeability through the outer membrane remains one of the leading hurdles in discovery efforts. However, the essentiality of a robust outer membrane makes itself an intriguing antimicrobial target. Herein, we review drug discovery efforts targeting the outer membrane and the prospective antimicrobial leads identified.

Mimicking the human environment in mice reveals that inhibiting biotin biosynthesis is effective against antibiotic-resistant pathogens.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets1. In Mycobacteria tuberculosis and Francisella tularensis, biotin biosynthesis is a key fitness determinant during infection2-5, making it a high-priority target. However, biotin biosynthesis has been overlooked for priority pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. This can be attributed to the lack of attenuation observed for biotin biosynthesis genes during transposon mutagenesis studies in mouse infection models6-9. Previous studies did not consider the 40-fold higher concentration of biotin in mouse plasma compared to human plasma. Here, we leveraged the unique affinity of streptavidin to develop a mouse infection model with human levels of biotin. Our model suggests that biotin biosynthesis is essential during infection with A. baumannii, K. pneumoniae and P. aeruginosa. Encouragingly, we establish the capacity of our model to uncover in vivo activity for the biotin biosynthesis inhibitor MAC13772. Our model addresses the disconnect in biotin levels between humans and mice, and explains the failure of potent biotin biosynthesis inhibitors in standard mouse infection models.

Overcoming mcr-1 mediated colistin resistance with colistin in combination with other antibiotics.

Plasmid-borne colistin resistance mediated by mcr-1 may contribute to the dissemination of pan-resistant Gram-negative bacteria. Here, we show that mcr-1 confers resistance to colistin-induced lysis and bacterial cell death, but provides minimal protection from the ability of colistin to disrupt the Gram-negative outer membrane. Indeed, for colistin-resistant strains of Enterobacteriaceae expressing plasmid-borne mcr-1, clinically relevant concentrations of colistin potentiate the action of antibiotics that, by themselves, are not active against Gram-negative bacteria. The result is that several antibiotics, in combination with colistin, display growth-inhibition at levels below their corresponding clinical breakpoints. Furthermore, colistin and clarithromycin combination therapy displays efficacy against mcr-1-positive Klebsiella pneumoniae in murine thigh and bacteremia infection models at clinically relevant doses. Altogether, these data suggest that the use of colistin in combination with antibiotics that are typically active against Gram-positive bacteria poses a viable therapeutic alternative for highly drug-resistant Gram-negative pathogens expressing mcr-1.

Silent but deadly: IS200 promotes pathogenicity in Salmonella Typhimurium.

Bacterial transposons were long thought of as selfish mobile genetic elements that propagate at the expense of 'host' bacterium fitness. However, limited transposition can benefit the host organism by promoting DNA rearrangements and facilitating horizontal gene transfer. Here we discuss and provide context for our recently published work which reported the surprising finding that an otherwise dormant transposon, IS200, encodes a regulatory RNA in Salmonella Typhimurium. This previous work identified a trans-acting sRNA that is encoded in the 5'UTR of IS200 transposase mRNA (tnpA). This sRNA represses expression of genes encoded within Salmonella Pathogenicity Island 1 (SPI-1), and accordingly limits invasion into non-phagocytic cells in vitro. We present new data here that shows IS200 elements are important for colonization of the mouse gastrointestinal tract. We discuss our previous and current findings in the context of transposon biology and suggest that otherwise 'silent' transposons may in fact play an important role in controlling host gene expression.

Exploiting the Sensitivity of Nutrient Transporter Deletion Strains in Discovery of Natural Product Antimetabolites.

Actinomycete secondary metabolites are a renowned source of antibacterial chemical scaffolds. Herein, we present a target-specific approach that increases the detection of antimetabolites from natural sources by screening actinomycete-derived extracts against nutrient transporter deletion strains. On the basis of the growth rescue patterns of a collection of 22 Escherichia coli (E. coli) auxotrophic deletion strains representative of the major nutrient biosynthetic pathways, we demonstrate that antimetabolite detection from actinomycete-derived extracts prepared using traditional extraction platforms is masked by nutrient supplementation. In particular, we find poor sensitivity for the detection of antimetabolites targeting vitamin biosynthesis. To circumvent this and as a proof of principle, we exploit the differential activity of actinomycete extracts against E. coli ΔyigM, a biotin transporter deletion strain versus wildtype E. coli. We achieve more than a 100-fold increase in antimetabolite sensitivity using this method and demonstrate a successful bioassay-guided purification of the known biotin antimetabolite, amiclenomycin. Our findings provide a unique solution to uncover the full potential of naturally derived antibiotics.

Pentamidine sensitizes Gram-negative pathogens to antibiotics and overcomes acquired colistin resistance.

The increasing use of polymyxins1 in addition to the dissemination of plasmid-borne colistin resistance threatens to cause a serious breach in our last line of defence against multidrug-resistant Gram-negative pathogens, and heralds the emergence of truly pan-resistant infections. Colistin resistance often arises through covalent modification of lipid A with cationic residues such as phosphoethanolamine-as is mediated by Mcr-1 (ref. 2)-which reduce the affinity of polymyxins for lipopolysaccharide3. Thus, new strategies are needed to address the rapidly diminishing number of treatment options for Gram-negative infections4. The difficulty in eradicating Gram-negative bacteria is largely due to their highly impermeable outer membrane, which serves as a barrier to many otherwise effective antibiotics5. Here, we describe an unconventional screening platform designed to enrich for non-lethal, outer-membrane-active compounds with potential as adjuvants for conventional antibiotics. This approach identified the antiprotozoal drug pentamidine6 as an effective perturbant of the Gram-negative outer membrane through its interaction with lipopolysaccharide. Pentamidine displayed synergy with antibiotics typically restricted to Gram-positive bacteria, yielding effective drug combinations with activity against a wide range of Gram-negative pathogens in vitro, and against systemic Acinetobacter baumannii infections in mice. Notably, the adjuvant activity of pentamidine persisted in polymyxin-resistant bacteria in vitro and in vivo. Overall, pentamidine and its structural analogues represent unexploited molecules for the treatment of Gram-negative infections, particularly those having acquired polymyxin resistance determinants.