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Researchers all over the world work hard in the discovery of antibiotics, one such researcher is Dr. Kim Lewis, leader of the Lewis Lab at Northeastern University where he is a distinguished professor with expertise in molecular microbiology. Dr. Kim Lewis’ lab consists of the Lewis Lab which studies persister cells responsible for tolerance to antibiotics, uncultured bacteria of the environment and the microbiome. It also includes the antimicrobial discovery center which translates basic discoveries into novel antimicrobial therapies to combat conventional pathogen threats and biowarfare. Working under Dr. Kim Lewis are Dr. Yu Imai and Dr. Kirsten Meyer, who together studied the effects of many potential compounds and their antimicrobial effectiveness in hopes of discovering a viable antibiotic. Dr. Yu Imai started as postdoctoral research associate and became associate research scientist and now works as assistant professor at Shinshu University. He received his bachelor’s degree in the faculty of agriculture, then a master’s degree from the graduate school of agriculture to then receive a doctor of philosophy, PhD, at the Interdisciplinary Graduate School of Science and Technology, all at Shinshu university. Dr. Kirsten Meyer was a postdoctoral research associate in Lewis lab at Northeastern University and is now a postdoctoral fellow at the University of Toronto. She got a bachelor’s degree from Victoria University of Wellington in honours biomedical science, majoring in molecular pharmacology and medicinal chemistry to then go on and achieve a PhD in pharmacology from Johns Hopkins University School of Medicine. Her work now at the University of Toronto involves optimized antimicrobial combinations and their packaging in extracellular vesicles from soil bacteria, towards development of new antimicrobials to counter resistance. Together at Northeastern University, along with the collaborative efforts of other researchers, they worked to find new antibiotics that selectively kill Gram-negative pathogens as an effort against the emergence of antibacterial resistance.

Figure x. The predicted chemical structure of the antibiotic darobactin. Image from Imai et al. 2019.

Nematodes have similar requirements for antibacterials as humans (Tobias et al. 2018). The digestive system of the worms is lined with bacteria similar to E. coli; these bacteria are released by a nematode as it invades an insect larva to feed (Crawford and Clardy 2011). The antimicrobial substances prevent other microorganisms from feeding on the larvae (Tobias et al. 2018). Some of these environmental microorganisms are Gram-negative bacteria (Tambong, 2013). The antibiotics must harm the bacteria without harming the nematode as they are in close proximity (Imai et al. 2019). The fact that the nematode microbiome can kill these bacteria so effectively means there could be a candidate human antibiotic substance produced by one of the commensal bacteria (Imai et al. 2019).

Figure x. The predicted chemical structure of the antibiotic darobactin. Image from Imai et al. 2019.

28 species of bacteria from the nematode microbiome were screened for production of antimicrobial against E. coli (Imai et al. 2019). Very few candidates produced zones of inhibition, the antibacterials are not produced in high enough quantities in vitro (Imai et al. 2019). Concentrated extracts of the bacteria were prepared and an extract from ​​Photorhabdus khanii HGB1456 effectively produced a zone of inhibition and served as a potential antibiotic candidate (Imai et al. 2019). The compound was isolated and identified to have a molar mass of 966.41047, suggesting that the molecular formula would be C47H56O12N11+ (Imai et al. 2019). This compound was checked against a natural products database to ensure that it was novel (Imai et al. 2019). Once it was confirmed to be a new substance, the research group named it darobactin (Imai et al 2019).

Darobactin can effectively inhibit the growth of many Gram-negative bacteria and it has little effect against Gram-positive bacteria (Imai et al. 2019). There is also little activity against the Bacteroides that compose much of our commensal gut microbiome (Imai et al. 2019), Zafar et al. 2021). This led the research group to believe that the compound might inhibit lipopolysaccharide, the main component of the outer membrane of bacteria (Bertani et al. 2019). Tests of darobactin against purified LPS found that there was no inhibition of activity, meaning darobactin does not target LPS directly (Imai et al. 2019). It must target a different component of the outer membrane. A potential candidate would be BamA which is a large, crucial outer membrane protein (Kaur et al. 2021). To identify the target, a ligand-protection thermal proteome analysis was performed, this analysis showed that chaperone proteins were significantly upregulated, whereas outer membrane proteins were decreased (Imai et al. 2019). Mutant E. coli cultured in high quantities of darobactin developed resistance to the drug (Imai et al. 2019). The sequences of the mutant bacteria were observed to determine which mutated genes would confer darobactin resistance, each mutant bacteria had multiple mutations in the bamA which codes for the BamA protein (Imai et al. 2019). This proved that BamA was the likely target of darobactin (Imai et al. 2019).

Further studies were performed to determine how resistance affected the virulence and infectivity of the mutated bacteria (Imai et al. 2019). Mice injected with wild-type E. coli had a mortality rate of 60% within 24 hours of infection, whereas the bacteria injected with the resistant mutants had no deaths within 24h (Imai et al. 2019) The mutations that confer darobactin resistance significantly reduced the virulence of the bacteria (Imai et al. 2019).

Darobactin is an exciting drug candidate that targets the outer membrane protein BamA (Imai et al 2019). If approved for humans, it would be the first drug developed from the microbiome of another animal species (Candanosa, 2022).

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