Fluoride to the rescue? Meeting the challenge of antibiotic resistant bacteria

Scientists have long been aware of the dangerous overuse of antibiotics and the growing number of antibiotic-resistant microbes that result. While overprescribing antibiotics for medical purposes has worrying implications for human health, so does the increasing presence of antibiotics in the natural environment. These can come from the poor disposal of drugs, but also from the field of biotechnology, which has depended on antibiotics as a screening device in the laboratory.

“In biotechnology, we have long relied on selections of antibiotics and chemicals to kill cells we don’t want to grow,” said Michelle O’Malley, chemical engineer at UC Santa Barbara. “If we have a genetically modified cell and we want to grow only that cell out of a population of cells, we assign it an antibiotic resistance gene. Introducing an antibiotic will kill all cells that are not genetically modified and only allow the ones we want – genetically modified organisms [GMOs]-to survive. However, many organisms have developed ways to bypass our antibiotics, and they are a growing problem both in the biotechnology world and in the natural environment. The issue of antibiotic resistance is a great challenge of our time, the importance of which is growing. “

In addition, GMOs pose a containment problem. “If this GMO got out of the lab and reproduced successfully in the environment, you couldn’t predict what traits it would introduce into the natural biological world,” O’Malley explained. “With the advent of synthetic biology, there is an increasing risk that the things we design in the laboratory will escape and proliferate in ecosystems where they do not belong.

Now, research conducted in O’Malley’s lab and published in the journal Nature’s communications describes a simple method to tackle both the overuse of antibiotics and the containment of GMOs. He calls for replacing antibiotics in the laboratory with fluoride.

O’Malley described fluoride as “a fairly benign chemical that is abundant around the world, including in groundwater”. But, she notes, it’s also toxic to microorganisms, which have developed a gene that encodes a fluoride exporter that protects cells by removing fluoride found in the natural environment.

The article describes a process developed by Justin Yoo, a former student researcher who graduated from O’Malley’s lab. It uses a common technique called homologous recombination to render the gene in a GMO that codes for a fluoride exporter non-functional, so that the cell can no longer produce it. Such a cell would still thrive in the lab, where fluoride-free distilled water is normally used, but if it escaped into the natural environment, it would die as soon as it encountered fluoride, preventing the spread.

Prior to this research, Yoo was collaborating with paper co-author Susanna Seppala, a project scientist in O’Malley’s lab, with the goal of using yeast to characterize the fluoride transport proteins that Seppala had. identified in anaerobic fungi. A first step in this project was for Yoo to eliminate native yeast fluoride transporters.

Shortly after generating the knockout yeast strain, Yoo attended a synthetic biology conference where he heard a talk about a novel biological containment mechanism intended to prevent genetically modified E. coli bacteria from growing. escape from laboratory environments. In this interview, he recalled: “I realized that the knockout strain of yeast that I had generated could potentially act as an effective biological containment platform for the yeast.”

“Basically what Justin did was create a series of DNA instructions that you can give cells that will allow them to survive when fluoride is present,” O’Malley said. “Normally, if I wanted to select a genetically modified cell in the laboratory, I would make a plasmid [a genetic structure in a cell, typically a small circular DNA strand, that can replicate independently of the chromosomes] who had an antibiotic resistance marker so he would survive if an antibiotic was there. Justin replaces that with the gene from these fluoride exporters. “

The method, which O’Malley called “easy fruit to bear – Justin did all of these studies in about a month,” also addresses a simple economic limitation to antibiotic-based cell selection in biotech labs. Besides fueling the increase in resistant bacteria strains, she continued, “from a biotechnological point of view, the process of creating antibiotic resistant organisms is also very expensive. If you were to perform fermentation of ten thousand liters, and it can cost you thousands of dollars per fermentation to add antibiotics, that’s a crazy amount. ”Notably, using low-strength fluoride would only cost about four cents per liter.

Obviously, said Seppala, “we would much prefer to use a chemical like fluoride which is relatively benign, plentiful, and inexpensive, and can be used to do the same thing as obtained with a conventional antibiotic.”

Yoo explained that the role of fluoride transporters was only recently elucidated, in 2013, when this project began. Emerging approaches to the implementation of biological containment have focused on the use of biological parts foreign to the organism of interest, shifting attention to what Yoo described as “brilliant, but complex systems,” while perhaps diverting attention from this simpler approach.