The emergence of antibiotic-resistant pathogens has far outpaced the discovery of new antibiotics to combat them. This is partly because antibiotic discovery efforts generally focus on screening culturable environmental microbes (e.g., bacteria from soil) for antimicrobial compounds. However, most environmental microbes cannot be grown in the lab, and thus are useless from a drug discovery standpoint—or are they? Aided by clever culturing techniques, scientists are gaining access to once inaccessible bacteria and, from these microbes, uncovering a spate of new antibiotics.

The Golden Age of Antibiotic Discovery

There was a time when it seemed like antibiotics were being uncovered left and right. This “golden age” of antibiotic discovery took off in the 1940s when Selman Waksman, Ph.D., a Nobel Prize-winning microbiologist, discovered the broad-spectrum antibiotic, streptomycin, from a species of soil-borne actinomycetes. Waksman’s discovery pointed to soil actinomycetes as potential sources of new antibiotics and motivated efforts throughout the pharmaceutical industry to mine the bacteria for promising leads.

These efforts led to the discovery of many of the main classes of antibiotics used today (e.g., aminoglycosides, tetracyclines, β-lactams, etc.). However, in the 1960s, progress tapered off. Soil actinomycetes were tapped out of novel antibiotics that could be uncovered with standard screening methods. Subsequent screens for synthetic antimicrobials were also largely unsuccessful; most synthetic molecules are unable to bypass the bacterial cell membrane (especially the repellent charges and pumps of the outer membrane in gram-negative bacteria), and thus are ineffective.

Since then, progress in antibiotic discovery has been marginal—or, as stated during ASM Microbe 2023 by Kim Lewis, Ph.D., a University Distinguished Professor and Director of the Antimicrobial Discovery Center at Northeastern University, “We are not in a great place to be.”  

All is not lost, though. For Lewis and his colleagues, the key to jump-starting natural product discovery is looking where scientists have never looked before. “One simple proposition is to start screening outside the actinomycetes and see what we can find,” Lewis said. “And if you’re going outside of actinomycetes, why not target uncultured bacteria?” 

Culturing the Unculturable

Only 1% of environmental bacteria can grow on a petri dish, leaving a whopping 99% uncultured. Most of these bacteria cannot be grown in the lab using traditional cultivation techniques; if scientists can’t grow them, they can’t access their potentially useful products. Over the past 20+ years, however, Lewis and his collaborators have developed methods for cultivating uncultivable soil microbes. The ticket, Lewis explained, is to make the microbes feel at home—that is, “trick” the cells into thinking they are growing in their natural environment, where they have access to nutrients and other growth factors. With his colleague, Slava Epstein, Ph.D., Professor of Biology at Northeastern University, Lewis invented what he jokingly referred to as a “very sophisticated device.” 

The device consists of a semi-permeable membrane, dabbed with a mix of agar and environmental cells (i.e., a diluted soil sample), sandwiched between 2 metal washers. The sandwich can be placed in an outdoor sampling site or in a simulated natural environment in the lab. The membrane allows molecules from the environment to diffuse in and out. After incubation for several weeks, bacterial microcolonies populate the membrane and can be isolated. Notably, once a cell population has been established, the bacteria are more apt to grow on a petri dish in the lab (up to 40% growth recovery). Another iteration of the technology, known as the Isolation Chip (ichip), is comprised of hundreds of tiny diffusion chambers that hold approximately 1 bacterial cell each, thus streamlining the process by allowing scientists to both grow and isolate individual bacteria. 

NovoBiotic Pharmaceuticals—a biotechnology company, co-founded by Lewis and Epstein, which focuses on the discovery and development of new drugs from natural sources—has used the diffusion chamber technology to screen soil samples at the industrial scale. The company now has a collection of over 64,000 unculturable bacterial isolates and, from those unusual isolates, has identified several promising antibiotics.   

Drugs from Uncultivable Bugs

The company’s leading antibiotic, teixobactin, was isolated from a previously uncultivated soil bacterium called Eleftheria terrae. Lewis highlighted that the compound shows excellent activity against a slew of gram-positive pathogens, regardless of their antibiotic resistance profile, is nontoxic to eukaryotic cells and, based on current evidence, appears to kill pathogens without detectable resistance. This is likely because teixobactin's targets on the cell membrane (lipid II and lipid III—precursors of peptidoglycan and teichoic acid, respectively) are immutable. That is, they are not proteins (i.e., are not directly coded by genes), and therefore do not acquire genetic mutations that can confer antibiotic resistance. This discovery suggests that “the paradigm that bacteria will always develop resistance to everything is incorrect,” Lewis said.  

Teixobactin’s efficacy is also linked to its unique 2-pronged mechanism. Molecules of teixobactin don’t just bind their targets, which inhibits cell wall synthesis, but also associate to form sheet-like supramolecular structures. “The membrane thins beneath the supramolecular structure,” Lewis explained. “We figured that [this] may disrupt the membrane—and it does.” He highlighted that “teixobactin is giving us a recipe for how to develop safe, membrane-active compounds,” which have remained somewhat elusive, despite scientists’ best efforts to find them. Scientists have since uncovered another antibiotic, clovibactin, that similarly targets lipid II and “zips up into a supramolecular structure,” albeit one that is a bit different from teixobactin.

“The conclusion from these compounds is…[that] nature clearly developed compounds that evolved to avoid resistance,” Lewis said. “And our notion of what is a suitable or druggable target is irrelevant because nature’s oblivious to that [notion].”  

Teixobactin is currently under late-stage preclinical development. Compounds from the NovoBiotic collection that target M. tuberculosis have also been discovered, and the company recently received funding to mine their collection for antifungal drugs to combat the fungal pathogen, Candida auris.  

Taking on the Gram-Negatives

Discovering antibiotics against gram-positive bacteria is notable. However, Lewis acknowledged that there is a paramount need for compounds that target gram-negative pathogens, which are especially concerning from an antimicrobial resistance standpoint (3 out of the 5 pathogens listed as “urgent” antimicrobial resistance threats by the U.S. Centers for Disease Control and Prevention are gram-negative). Yet, when screening soil, the “hit rate” for compounds targeting gram-negative bacteria is 2x lower than for gram-positive. Lewis estimates it would take 100 years to find leads against gram-negative bacteria with the standard soil sampling pipeline.  

To address this, Lewis and his collaborators are narrowing their scope, honing in on bacteria they know have similar requirements for antibiotics as humans (e.g., active against gram-negative bacteria, low toxicity, in vivo efficacy). It turns out, bacteria living in the guts of entomopathogenic nematodes are good candidates. Antimicrobial compounds produced by these gut microbes must have low toxicity against their nematode host, be able to travel through tissues and must work against gram-negative pathogens, which are key competitors in the nematode gut environment. 

So far, this approach has been successful. For example, a screen of nematode gut isolates belonging to the Photorhabdus genus uncovered an antibiotic, darobactin, that is active against prolific gram-negative pathogens (e.g., Pseudomonas aeruginosa, Klebsiella pneumonieae, Acinetobacter baumannii and others) in vitro and in mice, but shows limited activity against gram-positive organisms and other symbionts. Importantly, darobactin targets a complex on the gram-negative bacterial surface (the BAM complex), which overcomes the need to bypass the outer membrane—a formidable barrier to many compounds. Lewis noted that additional Photorhabus-derived compounds are under development. 

Overall, the work of Lewis and his colleagues—from growing uncultivable soil microbes to capitalizing on nematode gut bugs—points to 1 key message: new, effective antibiotics are out there. It’s simply a matter of where (and how) one looks.  

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