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 QuÃmica 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.


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Orac, since antibiotics are "artificial", it obviously could not be "natural" selection, it was airtifical selection, not evolution at all.

But seriously, what was the time frame over which this occurred?

Please allow me to weigh in respectfully with a small point. I'm not certain of the exact statistics on this, but many, if not most, successful antibiotics are natural products or are derived from them. Penicillin is the most famous example; vancomycin, under discussion here, is a natural product. We know that a wide variety of organisms, including microbes, produce antimicrobial compounds, and that these are mobilized during attack by another orgnaism. For eukaryotes, these comprise what is referred to as innate immunity, which we all have. There is little doubt that these are an important component of interactions between organisms in nature, and highly relevant to the process of natural selection.

I assume the time frame of this experiment is given in the PNAS paper, which anyone can read for him or herself (and which I will read right now).

Not to belabor this, since I suspect most who read this blog are well aware, but just a couple of details I could have mentioned before. Penicillin is a product of a fungus of the genus Penicillium (a eukaryote, like we are); vancomycin is a product of a soil bacterium, Streptomyces orientalis. Plants and animals also produce an extensive repertoire of antimicrobial molecules, quite distinct from antibodies and other components of the adaptive immune system. The range of structures manifested by all these natural antimicrobials is extremely broad, and new ones are being discovered and investigated all the time (for obvious reasons).

"Orac, since antibiotics are "artificial", it obviously could not be "natural" selection, it was airtifical selection, not evolution at all."

Not necessarily. The case could be made that, unless the purpose of giving the antibiotics was to induce the changes in the population, this particular human activity was just another environment that the bacteria were adapting to.

By Scott Simmons (not verified) on 24 May 2007 #permalink

What do "artificial" and "natural" mean, anyway? They're convenient labels, but if we try to make them precise by calling what a human does "artificial", I think we risk establishing an "artificial" dichotomy between humans and everything else. That would seem similar to the aversion some creationists have to common ancestry because they cling fondly to the idea that Man and Nature are separate, with humans being special, made in God's image. Although this isn't any sort of refutation, I dislike the aesthetic of the distinction.

More practically, if human-influenced environments are artificial, rendering selection within them unnatural and therefore not a part of evolution, perhaps the creationists really have won to the extent that there's been no evolution in some time. It's hard to find an environment on Earth that has not to some extent been affected by human activity, but I don't think that prevents us from considering evolution in these environments. Furthermore, other species can also change their environments, so why would we not then consider that also artificial selection?

I'd rather consider "artificial selection" to refer to deliberate attempts to mold a population by selecting desired properties and removing others from the gene pool, as has been practiced with domesticated species for a while now. I don't think I'd want to include human acts that influence selection unintentionally. However, even with artificial selection, I think that we can refer to evolution of the affected population; the selection mechanisms just happen to be a bit unusual.

Since I don't actually work in biology, I may have insufficiently considered the issue. Correction would be welcome from more learned scholars.

The distinction between "artificial" and "natural" selection is used to discredit experiments in evolution. Any laboratory selection is going to be "artificial", and hence has no bearing what so ever on "natural" selection.

I think a way to deal with MRSA, is via destruction of quorum sensing compounds.

This is one of the things that I am working on, inhibition of quorum sensing compounds by the nitrite and NOx produced by biofilms of ammonia oxidizing bacteria. I think that is the mechanism by which they can suppress heterotrophic bacteria in vivo. Oxidation of quorum sensing compounds is a common suppression strategy.

It doesn't kill them, so there is no evolutionary pressure to evolve resistance. Most virulence factors are turned on by quorum sensing, so if you can inhibit quorum sensing, you prevent disease, even if disease causing strains are present.

I think that facilitating natural bacteria which don't cause disease that out compete disease causing strains is a better strategy than trying to kill everything with stronger and stronger antibiotics. They will all evolve resistance eventually, and it leaves the niche open for the first oportunistic organism to fill. Better to keep that niche filled with something benign (like "my" bacteria).

The qualifier is: sometimes you just have to wade in there with classical antibiotics and kill everything. It's all very well to facilitate niche-fillers, but when your immune system is up the creek, can you trust those 'benign' niche fillers to stay benign? Or a febrile, stiff-necked, photophobic child to stay neurologically intact for the time it takes for the niche-fillers to crowd out the rampant meningococcus which is happily sprawling its way across the surface of their brain? And that begs the obvious question: What do you do for areas of the body that are supposed to be rigidly sterile, anyway?

By Justin Moretti (not verified) on 24 May 2007 #permalink

Daedalus -

Wheras I agree that antibiotic use should always be minimised, you have to admit that they have saved a LOT of lives..

By Andrew Dodds (not verified) on 24 May 2007 #permalink

I completely agree that if you have an infection that is causing problems, the bacteria need to be gotten rid of. Yes, antibiotics have saved a lot of lives.

Once you have a serious infection, obviously it is too late to do anything other than use any and every means to suppress them.

Unless you are going to live in a bubble, your environment is not sterile and no part of your body in contact with the external world is sterile. A large part of what keeps pathogenic bacteria at bay is the chemical warfare between our commensals and those pathogens. You knock out the commensals, and the niche is empty, ready for the first organisms that can grow there. That is the reason that many fungal infections follow antibiotic use. The antibiotics knocked out the bacteria that were suppressing the fungi.

There are some benign niche fillers that CANNOT become virulent. The autotrophic bacteria I am working with do not grow on any media used to culture pathogens. They have no virulence factors. They produce no toxins, have no transporters to export them, they have no genes to produce any virulence factors.

The bacteria that are usually associated with probiotic effects are usually lactobacilli. Lactobacilli are normally commensal, but even the strains in yogurt can and have caused liver abcesses. There has not been a single reported case of a "infection" with autotrophic bacteria, likely because it is impossible. Not unlikely, but impossible. Even in immunocompromised individuals.

I have often wondered when there was worry about over prescribing antibiotics were creating resistent strains of bacteria why my oldest kid had to take 4 amoxicillin before he had his teeth cleaned. It seemed like overkill...

But just a month ago that all changed (but not in time for me to reschedule a dental appointment because I forgot about the antibiotics)! Most patients with heart conditions, and that includes my son, do not need antibiotics before dental visits:

Another consideration is what is the concentration of antibiotics in what tissue compartment? The external skin is not usually though of as a "target organ", but that is where a lot of bacteria hang out. The antibiotic dose that might kill stuff systemically might only inhibit (and select for resistance) in a non-target organ.

Why is it that bacteria did not develop antibiotic resistance in wild? Is it simply because of how frequently it encountered antibiotics (which are perhaps ubiquitous due to human use)? If the reason is the way penicillium defended itself, may be there is something in it that we could learn.

"The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin...." Mighty interesting. I suppose one or more of the mutations involved a metabolic pathway implicated in both vanco- and dapto- activity?

Let me ask a possibly dumb question.

If vancomycin comes from a soil bacterium presumably it endowed an advantage on that bacterium over others in the soil. So why not get some isolated soil, stick the vancomycin-producing bacterium in it, add a vancomycin-resistant competitor and wait for our first bacterium to evolve a new antibiotic for us?

Ditto with the penicillin fungus.

For what it's worth from a non-specialist, that sounds like an interesting experiment to me. Might be more difficult with fungi, however, as they have a major disadvantage in this kind of system--they reproduce much more slowly than bacteria. Obviously fungi have evolved some similar competitive capabilities, which Fleming witnessed in his petri dish, but of course there is a difference between observing a pre-existing adaptation and trying to observe its ongoing development. An interesting question is perhaps whether the adaptive mechanism at play in the fungal case would be observable in a closed system, or would the Penicillium, for example, simply be overwhelmed. I would love to be schooled by a mycologist on this.

Evolution not a necessary part of Biology!

If evolutionists want to end the arguments all they have to do is, get their brilliant heads together and assemble a 'simple' living cell. This should be possible, since they certainly have a very great amount of knowledge about what is inside the 'simple' cell.

After all, shouldn't all the combined Intelligence of all the worlds scientist be able the do what chance encounters with random chemicals, without a set of instructions, accomplished about 4 billion years ago,according to the evolutionists, having no intelligence at all available to help them along in their quest to become a living entity. Surely then the evolutionists scientists today should be able to make us a 'simple' cell.

If it weren't so pitiful it would be humorous, that intelligent people have swallowed the evolution mythology.

Beyond doubt, the main reason people believe in evolution is that sources they admire, say it is so. It would pay for these people to do a thorough examination of all the evidence CONTRARY to evolution that is readily available: Try The evolutionists should honestly examine the SUPPOSED evidence 'FOR' evolution for THEMSELVES.

Build us a cell, from scratch, with the required raw material, that is with NO cell material, just the 'raw' stuff, and the argument is over. But if the scientists are unsuccessful, perhaps they should try Mother Earth's recipe, you know, the one they claim worked the first time about 4 billion years ago, so they say. All they need to do is to gather all the chemicals that we know are essential for life, pour them into a large clay pot and stir vigorously for a few billion years, and Walla, LIFE!

Oh, you don't believe the 'original' Mother Earth recipe will work? You are NOT alone, Neither do I, and MILLIONS of others!

By James Collins (not verified) on 01 Jun 2007 #permalink

Beyond doubt, the main reason people believe in evolution is that sources they admire, say it is so. It would pay for these people to do a thorough examination of all the evidence CONTRARY to evolution that is readily available: Try The evolutionists should honestly examine the SUPPOSED evidence 'FOR' evolution for THEMSELVES.

Whenever I see someone citing "Answers in Genesis" as "evidence" against evolution, I know that they have no clue about biology or what they are talking about. Of course, your straw men characterization of evolution and confusing of evolution with abiogenesis made that apparent before you ever mentioned AiG.