Medicine and Evolution, Part 11: Studying the mechanism of multidrug resistance

Our creationist neurosurgeon, Dr. Michael Egnor, isn’t going to like this one bit. No doubt he’ll try to call it “artificial selection” or a “tautology” when he finds out about it, if he doesn’t just ignore it because he it doesn’t fit in with his view that studying evolution is “of no value” in medicine. Too bad, because, via Derek Lowe at In the Pipeline, I’ve found a really cool application of evolutionary biology to the development of antibiotic resistance in response to vancomycin that sheds light on the molecular mechanisms behind the development of antibiotic resistance in Staphylococcus aureus in response to vancomycin.

Bacterial resistance to antibiotics is a very serious problem in medicine. If you want to see the sort of toll that these infections take, you have no farther to look than Mark Chu-Carroll, whose father recently died of a persistent resistant bacterial infection that could not be cleared. For S. aureus, vancomycin has traditionally been the antibiotic of last resort, to be used when the bacteria become resistant to all penicillin and cephalosporin antibiotics. When this happens, we refer to the bacteria involved as methicillin-resistant Staphylococcus aureus (MRSA), named for the antibiotic to which all such strains are all resistant. Recently, resistance has appeared to even vancomycin, jeopardizing its use as an effective last resort antibiotic against this organism, which is usually resistant to multiple antibiotics by this point. Consequently, a lot of effort is going into trying to figure out the molecular mechanisms behind the evolution of vancomycin resistance, about which, surprisingly, little is as yet known. That’s why the following study, published in this week’s Proceedings of the National Academy of Sciences of the United States of America, is so fascinating:

Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing

Michael M. Mwangi *, Shang Wei Wu , Yanjiao Zhou , Krzysztof Sieradzki , Herminia de Lencastre ¶, Paul Richardson ||, David Bruce ||, Edward Rubin ||, Eugene Myers **, Eric D. Siggia *, and Alexander Tomasz
*Physics Department, Cornell University, Ithaca, NY 14850; Center for Studies in Physics and Biology and Laboratory of Microbiology, The Rockefeller University, New York, NY 10021; Department of Microbiology, Tianjin Medical University, Tianjin 300070, People’s Republic of China; ¶Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal; ||United States Department of Energy Joint Genomic Institute, Walnut Creek, CA 94598; and **Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20146

The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

You read right. Mwangi et al sequenced the entire genome of bacteria isolated from the blood of a single patient with endocarditis due to S. aureus. But they did more than that. They sequenced bacterial isolates from the same patient at multiple time points during his clinical course of treatment. They took a lot of care to show that these isolates were all isogenic (i.e., genetically from the same strain) and then looked at the genetic changes that occurred as resistance developed to rifampin, vancomycin, and daptomycin. They found 35 mutations in only 31 loci, a number far smaller than previously described. Previous studies had described 200-500 genetic changes, but were not as well controlled for being isogenic. Key findings include:

  • The first fully antibiotic susceptible blood isolate and the also fully susceptible contact isolate had identically low MICs to the antibiotics tested and carried the same mutation. (Note: MIC= minimum inhibitory concentration, a measure of the concentration that it takes to inhibit bacterial growth. The lower the value, the more susceptible a bacterial strain is to the antibiotic being tested.)
  • Once a mutation appeared in an early blood isolate, it was retained in all subsequent blood isolates, with only one exception. Thus, the genetic changes appeared in a sequential order in parallel with the increasing vancomycin MIC values.
  • The vancomycin susceptibility of the isolates declined gradually in several discrete steps in sequential bacterial isolates.

However, the most important (and amazing) part of the study is that they could correlate genetic changes that occurred with increases in MIC, with specific genetic changes being associated with discrete increases in MIC. This is about as good as it gets for observing evolution in action at the molecular level in order to identify candidate genes that might be responsible for resistance to vancomycin. Moreover, this looked at bacteria isolated from a living patient, a far better model to study than the usual method, which is to expose bacteria growing in culture to progressively higher concentrations of antibiotic in order to produce resistance. This is bacterial evolution in action in response to the selective pressure of antibiotics, examined in detail in a real-world clinical scenario. The main problem with this approach is that there could be patient-specific factors that could be resulting in selection for certain strains that were not controlled for. However, as more data like this is obtained from more patients, it should be possible to determine which mutations are due to selection by the antibiotics and which are due to random drift or patient-specific factors.

One point that Derek brought up about this experiment is how technology-driven it is. Sequencing the entire genomes of even bacteria is time- and labor-intensive, not to mention relatively expensive. A few years ago, it would have been entirely impractical to carry out an experiment like this. A few years from now, it will probably be routine. It is, however, a rather brute force approach to a problem. That’s not necessarily a bad thing, because sometimes brute force approaches are the most straightforward, if not necessarily always the most elegant. Another implication of the increasing use of sequencing techniques like this is that, in the not-too-distant future, it will likely be possible to sequence resistant bacterial isolates from patients in order to identify the specific mutations they have that are responsible for antibiotic resistance and use specific drugs to target these mutations. The bottom line, despite what evolution-dismissing creationists who think way too much of their knowledge and understanding of evolution say, the study of evolution, particularly at the molecular level, is becoming more, not less, important to medical research.