Hidden genetic complexity helps bacteria evolve resistance to antibiotics in a variety of unexpected ways WASHINGTON, Sept. 9, 2014 – The ability of disease-causing bacteria to evolve antibiotic resistance poses a growing threat to human health worldwide. Scientists have now discovered that our microscopic enemies may be even more cunning than we anticipated, facilitating rapid evolution through hidden genetic changes in stressed environments and developing resistance to antibiotics in more ways than we expected. The findings are reported in a new article published in the journal Biomicrofluidics, published by the American Physical Society. In the article, researchers from Princeton University in New Jersey report how two similar strains of E. coli they identified rapidly developed similar levels of antibiotic resistance through distinct genetic mutations. The ability to use different approaches to solve the same problem suggests that bacteria can develop a variety of genetic weapons to fight antibiotics, making them more resilient and thus less likely to be destroyed. “Bacteria are smart – they have a lot of hidden ways to fight stress, including reshaping their genomes,” said Princeton biophysicist Robert Austin, who led the research team. Realizing how effective bacteria are at fighting drugs is thought-provoking, Austin said. “It teaches us that we must be more cautious and careful in our use of antibiotics than we are now.” Austin and his colleagues have worked specifically on the theory of developing unique, liquid-filled microstructures to detect bacterial evolution. What they wanted to build were instruments that, in their opinion, better mimicked natural microenvironments. The team used a custom microfluidic instrument that contains about 1,000 connected microhabitats for bacterial populations to grow in. The instrument produces complex food gradients and antibiotics similar to those found in natural bacterial habitats, such as the digestive tract and other internal structures of the human body. “The development of bacterial resistance in complex environments is far more rapid and complex than in test-tube experiments,” Austin said. In previous experiments using microstructure instruments, researchers have learned that some common, wild-type E. coli strains can evolve resistance quickly. Another mutant strain called GASP, which multiplies faster than wild strains in limited nutrients, and the researchers wanted to know if the mutant strain would develop the same type of resistance as wild strains when exposed to the same antibiotics. By sequencing the genomes of wild-type and GASP mutant colonies exposed to the antibiotic ciprofloxacin (Cipro), the researchers found that different genetic variants could lead to similar levels of resistance. For example, two different mutant strains of GASP have emerged: a drug-resistant GASP strain that evolved by “borrowing” DNA fragments from the infecting virus to become a strain that does not require a biofilm to survive external stress. Another drug-resistant GASP strain did not “graft” this way, but eventually developed resistance in several other ways. Viruses usually inject their DNA into bacteria, and this DNA sometimes no longer functions for viral replication. Normally, these DNA fragments are neither helpful nor resistant to bacteria, but under stressful conditions, bacteria can use the new DNA to rapidly evolve drug-resistant mutations. The researchers’ results confirm the diversity and cunning of the ways in which bacteria fight stressful environments, Austin said. He wants to learn more about whether the effective methods we now use to kill bacteria, such as disinfecting surfaces with alcohol, also have their vulnerabilities, and his team plans to test whether bacteria can evolve resistance to alcohol in their apparatus.