Novel antibiotic discovery tool holds promise to uncover more natural compounds

Philip Abbosh, MD, PhD

Infections originating in a genitourinary source are among the most common hospital and outpatient clinical presentations in urology and non-urology settings. Antibiotic use and overtreatment are well known to cause multidrug-resistant organisms. The lack of new antibiotic therapies exacerbates this problem. Most antibiotics are decades old and were derived from bacterial sources—bacteria are in an arms race to occupy limited ecological niches. The selective pressure applied by this competitive landscape has resulted in bacteria evolving synthetic pathways that yield new antibiotics that kill competitors to make room for the index organism.

Kate Gessner, MD, PhD

Scientists have capitalized on this evolutionary phenomenon to isolate antibiotics—this discovery pathway yielded the famous and serendipitous isolation of penicillin. However, this discovery pathway has been exhausted because most bacteria are not culturable with traditional methods. Once bacteria are removed from their niche, they cease to be able to be cultivated on/in media such as Luria-Bertani, sheep’s blood agar, chocolate agar, etc -the workhorses of modern microbiology labs.

This problem was creatively addressed by Ling et al, who described a simple, productive solution to the bacterial cultivation problem: Grow bacteria in incubators that recapitulate their natural niche. The authors developed a simple tool called iChip. This device is essentially a chamber with a semipermeable membrane on 2 sides and can be buried in dirt (or other natural ecological niches), recapitulating the native environment where bacteria would grow. To isolate individual species and strains, the soil samples are diluted exponentially such that 1 bacterial cell is inoculated into a single chamber. Nutrients, waste products, and other biologically active metabolites diffuse into/out of the chamber and can be isolated with chromatography. Isolated natural products can be screened for biological activity against pathological bacterial species.

This workflow was used to isolate teixobactin, a cell wall synthesis inhibitor that works via a novel mechanism. Teixobactin was isolated from a previously unidentified species of soil-dwelling bacteria named Eleftheria terrae, confirming that iChips can be used to “culture the unculturable.” The authors sequenced the genome of E. terrae, revealing that it belongs to the Betaproteobacteria class and is related to the genus Aquabacterium. The organism’s genome contains an anabolic operon (string of genes that coordinate a physiological task) that synthesizes teixobactin, a peptide. The structure of teixobactin is 11 amino acids long, including 2 nonhuman amino acids. The last 4 amino acids form a ring structure that is generated by enzymes contained in the operon.

The drug was tested for antimicrobial activity and found to be highly potent against gram-positive organisms: Staphylococcus aureus including methicillin-resistant S aureus (MRSA); Enterococcus including vancomycin-resistant Enterococcus; Clostridium difficile; and the causative agents for anthrax and tuberculosis. The compound was nontoxic to cultured mammalian cells at 400 times the minimal inhibitory concentration (MIC) for MRSA (which had the highest MIC of the gram-positive organisms tested). Traditional experiments to identify resistance mechanisms did not generate resistance to teixobactin but did yield cultures resistant to oxacillin (a positive control for the experiment).

Ling et al then sought to identify the bacterial target of teixobactin. It was a potent inhibitor of cell wall biosynthesis and was bacteriolytic/bacteriocidal. To test which step of peptidoglycan biosynthesis was inhibited, they performed biochemical analysis of lysates generated by teixobactin-treated S aureus. They found that there was accumulation of the same peptidoglycan intermediate that accumulates upon treatment with vancomycin. They identified that the target for teixobactin was not an enzyme (as is the case for β-lactams) but a precursor molecule of peptidoglycan called lipid II. Teixobactin traps the lipid II molecule, sequestering it from further synthetic incorporation into the cell wall. This makes resistance highly unlikely, as the bacteria would have to completely reinvent a cell wall synthetic pathway to generate a teixobactin-resistant lipid component. They also found that teixobactin binds to lipid III, another peptidoglycan precursor. Lipid III is important because it sequesters enzymes called autolysins that hydrolyze peptidoglycan. Sequestration of lipid III results in the liberation of autolysins and unregulated hydrolysis of cell wall components and, therefore, bactericidal activity.

Finally, the authors determined whether teixobactin might be used as a drug. Teixobactin was stable in serum incubations, did not break down in microsomal assays, and had a viable circulating half-life in mice. To test its activity, MRSA was injected into the peritoneum of mice, followed by treatment with vancomycin or teixobactin at different doses. This resulted in a 100% survival rate for mice treated with teixobactin doses greater than 0.5 mg/kg but less than a 20% survival rate for mice treated with vancomycin at the same dose. Only single doses were used. Similar activity was seen when injecting MRSA into the thigh for an abscess model.

Where does this leave us? Teixobactin, and more broadly, the iChip approach to novel antibiotic discovery, is an exciting development in infection control. As of this writing, teixobactin is mentioned in 122 PubMed citations, but no publications are clinical trials, and teixobactin is not mentioned in trials listed in ClinicalTrials.gov. However, teixobactin research is being funded by the NIH in the form of a small business initiative research grant to perform key experiments that will validate teixobactin as a drug (toxicity, pharmacokinetic properties, manufacturing optimization), and this discovery platform is as well. It is promising that teixobactin can eventually become a clinically available and useful drug. Still, it also underscores the long path that it takes to translate a lab discovery into a clinical reality, even for urgent needs like infection/sepsis. In any case, iChip seems like a robust tool for developing new antibiotics/antifungals, and it seems likely that the tool could also be applied to the discovery of natural compounds that can be applied to other diseases.

REFERENCE

Ling LL, Schneider T, Peoples AJ, et al. A new antibiotic kills pathogens without detectable resistance. Nature. 2015;517(7535):455-459. doi:10.1038/nature14098