Jeffrey S. Buguliskis Ph.D. Technical Editor Genetic Engineering & Biotechnology News
Tackling the Inevitability of Resistance One Genome at a Time
“Geology is the study of pressure and time. That’s all it takes really… pressure… and time…,” bellows Morgan Freeman’s character, Red, when foreshadowing his friend Andy Dufresne’s harrowing escape from prison, in the critically acclaimed movie Shawshank Redemption. Oddly enough, this same logic can be applied to antibiotic resistance, although bacterial change occurs exponentially more rapidly than the millennia it can take to create various geological wonders. For microbes, however, the biological pressures that scientists presume underlie the formation of resistance mutations doesn’t come in the form of sedimentation, stalactites, or shifts in plate tectonics, as it does in geology. Rather, the genetic burdens exerted by numerous human developed antibiotic, and drug therapies, as well as natural environmental conditions, are the most significant factors fueling resistance.
While antimicrobial drug resistance is growing at an alarming rate globally, the phenomenon is not new, nor is it an occurrence that only recently has come to pass. In 1940, only slightly a decade after penicillin’s discovery, the first strains of Staphylococcus resistant to the compound were detected. Five years later, in his Nobel lecture concerning his breakthrough discovery, Alexander Fleming prophetically warned those in attendance about the dangers of microbial resistance:
“It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them, and the same thing has occasionally happened in the body…The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant.”
While numerous factors are contributing to this sharp rise in resistance that the world is currently experiencing, chief among them is the overuse of antibiotic compounds. Given their extremely rapid and exponential replication rate (an E. coli bacterium can double itself approximately every 20 minutes), and ability to survive an extensive number of genetic mutations, bacteria have evolved to be the ultimate survivor.
Certainly, long after humans have outlived their time on this planet, microbes will continue to chug along, barely even recognizing our species’ existence. Force the microscopic critters to choose between dying or rapidly adapting by placing them under the chemical squeeze of antibiotic compounds, and they will be all too happy to mutate and return even more virulent. Keep in mind that there are bacterial species (Deinococcus radiodurans) that can withstand a dose of ionizing radiation 3,000 times greater than the dose that would kill a human—due to their extreme ability to repair and adapt to DNA damage that would destroy most life.
Get in Front of It
So how does science keep on top of a such a seemingly formidable, if not tiny, adversary? Obviously, there are multiple approaches, but developing better therapeutics is paramount for most researchers. While compounds that are fast acting, exquisitely lethal, and unique to only pathogenic bacteria would be ideal, science isn’t quite capable of that form of therapy just yet. However, we are well entrenched in the genomic age, and investigators have advanced tools at their disposal which allow them to identify genotypic mutations that rapidly lead to resistance phenotypes.
“Genomic analysis offers an unprecedented window into the origins, functions, and dissemination of antibiotic resistance,” says Michael Gillings, Ph.D., professor in the department of biological sciences at Macquarie University, Sydney, Australia. “We now have the power to understand how antibiotic resistance arises in real time, how resistance genes move between bacterial species, and how bacterial species are spread around the globe. Submissions to globally accessible databases ensure that researchers everywhere can obtain information quickly.”
Gautam Dantas, Ph.D., associate professor in the department of molecular microbiology at Washington University School of Medicine thinks that the simplest impact OMICS is having on the study of resistance is “the ability for us to transcend the culturing barrier. So much of microbiology and the functions encoded in microbiology, such as resistance, are based on ideas of domesticating microbes by bringing them into the lab and studying them deeply. While this method has been very useful, we’ve learned from soil and environmental researchers, as well as the human microbiome project and beyond, that most habitats have rich microbial communities that are not trivial to culture. So genomics sort of gets beyond that by going after the nucleic acids themselves and inferring what the functions might be—and the study of resistance has been expanded because of that approach.”
The influence of genomics on fields like microbial genetics is best viewed through the lens of advanced techniques such as next-generation sequencing (NGS), which has quickly evolved into an affordable method now employed in both research and clinical settings. For microbiologists tasked with monitoring mutations within microbial populations that could lead to resistance, being able to quickly and accurately assess bacterial genotypes is paramount in attemping to stay ahead of drug resistance outbreaks.
Whole-Genome Sequencing (WGS) Gains Appeal
“WGS of bacteria has become a fast and affordable technology that is being adopted by federal agencies (including the FDA, CDC, and USDA) and state health laboratories, and will soon become an integral part of laboratory science worldwide,” explains Patrick McDermott, Ph.D., director of the U.S National Antimicrobial Resistance Monitoring System (NARMS) for enteric bacteria at the U.S. Food & Drug Administration (FDA). “By providing definitive genotype information, WGS offers the highest practical resolution for detecting and characterizing the full complement of resistance determinants, whether acquired exogenously or arising by mutation, including resistance to antibiotics not routinely tested. Sequencing different bacterial species isolated from different sources (humans, animals, foods, and the environment) in the NARMS program will help us understand how resistance emerges and spreads.”
Whole genome-based methodologies such as WGS afford researchers the opportunities to characterize and compare entire microbial genomic sequences to ascertain the specific loci or exact genetic sequence associated resistance. While scientists can be creatures of habit when it comes to certain techniques—often following the “if it isn’t broke don’t fix it” mantra—they are often willing to combine various proven methods together, if they believe it will provide them with greater data output, more sensitivity, or increased speed (often hoping to get all three attributes at once). This is exactly what researchers recently did when studying antibiotic resistance for tularemia, an acute bacterial infection that causes ulceration of the skin, severe pneumonia, and is often lethal.
“We recently applied whole genome-based technologies including DNA microarray and next generation sequencing to characterize an avirulent strain of Francisella tularensis, to the live vaccine strain or LVS, after the strain had been subjected to ciprofloxacin, a commonly used antibiotic to treat bacterial infections,” noted Crystal Jaing, Ph.D., group leader of applied genomics at Lawrence Livermore National Laboratory. “Using this approach, we identified both previously known mutations that confer resistance as well mutations that have not been reported previously. These mutations or markers can be used to develop diagnostic tests to rapidly determine if a bacterium is resistant to antibiotics, and what alternate treatment strategy can be used.”
Genomics is not only reshaping how scientists approach the study of antibiotic resistance, but it is also impacting how governmental agencies are collaborating and investing in future research. When speaking to members of the National Institute of Allergy and Infectious Diseases’ (NIAID) Office of Genomics and Advanced Technologies (OGAT), they told ClinicalOMICS that “NIAID is investing into the application of genomic technologies because we see these as research tools that can be used to understand biological mechanisms that underlie antibiotic resistance, but also powerful high-throughput assays and cost-effective screens that can be readily used to help in the development of new antibiotics and therapeutics.”
The OGAT members provided numerous examples of ongoing collaborations that they believe will pay out big research dividends. For instance, and alliance between the NIAID Genomic Centers for Infectious Diseases and the NIAID Bioinformatics Resources Centers, or Pathosystems Resource Integration Center (PATRIC) is currently using WGS combined with phylogenomics—which uses genomic data to reconstruct evolutionary relationships—along with clinical and geographical data to track the progression of resistance on a global scale across various pathogenic
“This information is being used to understand how pathogens exchange genes, and to develop better diagnostics and therapeutics that take into account this genomic information,” OGAT stated. “These data may also inform surveillance and could ultimately lead to improved stewardship. Understanding the biological mechanisms would enable better responses, however, because of the growing challenge of antibiotic resistance, it is clear that there is a need for new antibiotic drugs.”
Keeping an Eye on the Target
The promise of translation research has always been to transform laboratory techniques into clinical modalities. Genomics transcends the translational path by helping scientists address the needs of patients, from the development of new molecular diagnostics for improving disease prognosis to pharmacogenomic analyses that identify genetic background incompatible with various drug therapies.
“All of us now carry resistance genes in our gut bacteria, and knowing which ones we carry could help to better target antibiotic therapies,” said Dr. Gillings. “I see a time where all patients are subject to routine genome screening both of isolates and their total microbiota to identify resistance genes prior to treatment.”
Dr. Jaing agreed, adding that “it will be really helpful to have rapid point-of-care diagnostic tests to determine someone’s current gut bacterial antibiotic resistance profile before an antibiotic is prescribed.”
Additionally, investigators from multiple disciplines are beginning to team up under the umbrella of systems biology to create novel computational approaches that incorporate multiple omics data (proteomics, metabolomics, transcriptomics, etc.), in order to construct predictive models that help explain human antibiotic resistance.
“As genomics, informatics, and computational technologies continue to be developed and become more ubiquitous in clinical and even personal healthcare, we are looking forward to new opportunities that bolster our mission to support basic and applied research to better understand, treat and, ultimately prevent infectious diseases, and in this case those from antibiotic-resistant bacteria,” OGAT members concluded.
Genomics has opened up avenues of research that could hardly be envisioned when genome mapping projects began so many years ago. However, its service to protecting public health was on many researchers wish lists from the start. Technological advances that are starting to see more frequent use, such as nanopore sequencing and long-read sequencing technology, will only serve to embolden scientists to create more efficient therapies that could one day make concepts like resistance a distant memory. Although, until that day comes, scientist, clinicians, and the public need to stay vigilant and informed to make certain that Alexander Fleming’s prognostication doesn’t become real for every antibiotic therapy we develop.
This article was originally published in the January/February 2017 issue of Clinical OMICs. For more content like this and details on how to get a free subscription to this digital publication, go to www.clinicalomics.com.