Respectful Insolence

In my last couple of posts on the risks and benefits of ever more sensitive screening tests for various cancers, and in particular breast cancer, I marveled at a a bit of serendipity that had pointed me to a particular old article a mere few days before multiple new papers about breast cancer screening with mammography and MRI were released. It turns out that that’s not the only serendipity that’s been going on lately, as far as blogging goes. For example, there’s been Dr. Michael Egnor, the creationist professor of neurosurgery who’s become the Discovery Institute’s seemingly favorite “authority” on evolution. In the brief time since he’s entered the blogosphere, he’s made a name for himself spouting some amazingly ignorant and easily debunked fallacies about evolution. Perhaps the most jaw-droppingly dumb fallacy that he’s repeated time and time again is that evolution contributes nothing to our understanding of how microbes develop resistance to antibiotics.

I really, really hope that he sees the latest issue of Nature. He’s not going to like it at all, specifically the article by Chait et al. entitled Antibiotic interactions that select against resistance, whose abstract reads:

Multidrug combinations are increasingly important in combating the spread of antibiotic-resistance in bacterial pathogens. On a broader scale, such combinations are also important in understanding microbial ecology and evolution. Although the effects of multidrug combinations on bacterial growth have been studied extensively, relatively little is known about their impact on the differential selection between sensitive and resistant bacterial populations. Normally, the presence of a drug confers an advantage on its resistant mutants in competition with the sensitive wild-type population. Here we show, by using a direct competition assay between doxycycline-resistant and doxycycline-sensitive Escherichia coli, that this differential selection can be inverted in a hyper-antagonistic class of drug combinations. Used in such a combination, a drug can render the combined treatment selective against the drug’s own resistance allele. Further, this inversion of selection seems largely insensitive to the underlying resistance mechanism and occurs, at sublethal concentrations, while maintaining inhibition of the wild type. These seemingly paradoxical results can be rationalized in terms of a simple geometric argument. Our findings demonstrate a previously unappreciated feature of the fitness landscape for the evolution of resistance and point to a trade-off between the effect of drug interactions on absolute potency and the relative competitive selection that they impose on emerging resistant populations.

“Fitness landscape”? They sure don’t mean the lithe women working out at the health club. “Selection”? They ain’t talking about about the variety of coffees at Starbucks. No, they’re talking about evolution and a neat little trick by which evolutionary concepts might be used to overcome antibiotic resistance. I can’t say that I’ve entirely wrapped my brain around it yet, but no matter how you slice it it’s a clever set of experiments.

Before I can try to explain this paper (and it’s actually kind of hard to explain, leading me to hope that I don’t butcher it), a few terms need to be defined. the key trick in this paper is that the investigators looked at the effects of different drug interactions on selection of bacteria. The concepts of drug interaction used here are very familiar to me as a cancer surgeon, because they are often used in reference to combining cancer chemotherapies. In this case, however, they’re simply using them to describe interactions between antibiotics. In general, whenever two drugs are combined, they can have three different interactions. Most commonly (in cancer chemotherapeutics, at least), their effects will be additive, which means that the effects of each individual drug are added together, and that’s what the effect of the combination is. What we as physicians are hoping for is the second form of interaction, which is a synergistic interaction, defined as when the effects of the two drugs are greater than would be expected from adding the effects of each individual drug. in contrast, what we as physicians hope to avoid is the third form of interaction, which, as you might expect, occurs when the two drugs together produce an effect that is less than would be expected from adding the effects of the individual drugs. This last form of interaction is known as an antagonistic interaction. Of this latter form, the worst case is when the combination of the two drugs is actually worse than either of the single drugs themselves, an interaction known as hyper-antagonistic, or suppressive. Working out whether a drug combination is synergistic, additive, or antagonistic requires a detailed set of assays testing the two drugs at numerous different concentrations and a specific mathematical calculation, although it is often easier to look at the shape of a special curves called an isobole, which have characteristic shapes for each interaction.

What the authors did was to look at a model of the competition between two E. coli strains. One strain was wild type, and thus sensitive to tetracycline. The other strain contained a piece of DNA known as the Tn10 transposon, which encodes a protein that pumps tetracycline out of the cell and thus results in resistance to tetracycline. In this case, the resistant strain required about 100-fold higher concentrations of tetracycline to be killed. Normally, in the additive or synergistic situation, resistance to one of the drugs in the two-drug combination leads to an advantage in the two-drug combination as well. However, the researchers hypothesized that in the case of hyperantagonistic, or suppressive, interactions, the opposite might occur. The concept behind this hypothesis is that, although resistance would indeed diminish the burden imposed by one of the drugs, it might also in doing so remove the suppression, resulting in the combined treatment being more effective against the resistant strain than against the wild type. I have to admit here that I’m not sure why one would hypothesize that this might be the case and the authors didn’t really explain very well beforehand why they thought their hypothesis might be true. However, it turns out that their hypothesis might well have merit, as their subsequent experiments demonstrate.

What the investigators did was to test the effect of two antibiotic pairs, tetracycline plus either ciprofloxacin or erythromycin. These combinations were chosen because tetracycline and erythromycin demonstrate synergistic effects against this strain of E. coli, while tetracycline and ciprofloxacin are hyperantagonistic, or suppressive. As expected, for the assays looking at cell survival, resistance to doxycline merely rescaled the the doxycycline scale on the curve upward by about 100-fold. However, when a competition assay was done, to see whether the resistant or the sensitive strains were selected for, the results were very different. Basically, the end point of such a competition assay is the ratio of sensitive to resistant bacteria after a fixed time of selection under the two drug combination at different doses. Not surprisingly, under the synergistic combination, there were no dosage combinations where the sensitive strain ended up being selected for. The resistant strain always came out on top. In contrast, for the suppressive combination (tetracycline plus ciprofloxacin), there was a region on the curve (i.e., a range of dose combinations for the two drugs) in which the sensitive bacterial ended up being selected for, as evidenced by a much larger ratio of the sensitive E. coli to the resistant E. coli remaining at the end of the experiment. This clever strategy ended up selecting against the resistance gene in the resistant strain, leading the resistant strain to be killed more effectively than even the sensitive strain. The authors conclude:

Our data show that in suppressing drug combinations, a drug can be used to exert competitive selection against its own resistance allele. In contrast, synergistic interactions, while increasing absolute potency against both sensitive and resistant strains, also increase relative selection in favour of resistance. These findings point to an inherent tradeoff, where antagonistic combinations, which require a higher dosage and have therefore typically been shunned in clinical therapy, may have the benefit of reducing and even inverting selection for resistance. Although the molecular mechanisms underlying drug interactions may be complex, suppression between antibiotics is not uncommon. Our simple geometrical approximation anticipates a region of competitive selection against resistance in such suppressive drug combinations when the targeted resistance mechanism works specifically (uniaxially) on one of the drugs. Indeed, for doxycycline-ciprofloxacin, a region of drug concentrations permitting the growth of doxycycline- sensitive but not resistant strains appears for three very distinct mechanisms of resistance to tetracyclines…It would therefore be of considerable interest to employ new multidrug screens to search for reciprocally suppressing drug combinations in which each of the drugs suppresses the effect of the other. Such drug combinations may block the two single-step mutational paths to complete resistance by imposing selection against resistance to each of the drugs. We emphasize that our work is limited to sublethal drug concentrations, in a controlled environment in vitro and that any possible therapeutic implications from these findings are beyond its scope. However, we do hope that these findings may suggest avenues of research into new treatment strategies employing antimicrobial combinations with improved selection against resistance.

Indeed, translating this research into drug combinations that might be clinically useful to combat resistant bacteria in real infections in real patients will require a lot of work. It will be difficult, and it might not be translatable to humans, although there is no inherent reason why it shouldn’t be. One problem that I could foresee is that it would be a tricky business to intentionally use antagonistic drug combinations. After all, the sensitive bacteria being treated are still virulent and need to be dealt with., and working out effective dose combinations of two different drugs to produce concentrations in the blood that result in effective selection against the resistant strain will almost certainly be difficult. Even so, the implications of this work could go beyond just antibiotics. It could apply to combinations of chemotherapeutic agents for cancer therapy. Either way, without an understanding of evolution, it would have been impossible even to consider this hypothesis. What was that again that Dr. Egnor said about evolution supposedly being of “no use” in understanding antibiotic resistance?

I’d love to see what Dr. Egnor would say about this study. No doubt he’d lamely try to call it a “tautology” again. (He’s rather one-note that way.) Even so, it’d be fun to see him try.

Comments

  1. #1 Stuart Coleman
    April 9, 2007

    I thought that this was a really cool bit of research, and am quite surprised that it hasn’t really turned up anywhere.

  2. #2 SteveM
    April 9, 2007

    There was a NOVA or similar science show a year or so ago discussing HIV and patients becoming resistant to the anti-virals. It was hypothesized that the HIV was being killed off except for strains resistant to the drugs, so the strategy was to take the patient off the drugs completely for a while and let the “wild” HIV with all its genetic diversity reinvade and compete against the resistant strain hopefully knocking it back down to a low percentage of the population, at which point they would re-introduce the anti-viral drugs wiping out most of the HIV population. I am probably not explaining it very well but the basic point is that the resistant strain was not very “robust” compared to the general population of HIV, it was only when all the other competition was eliminated that it could thrive, so the only way to keep it under control was to let the wild population itself fight it. So in order to get the patient “well” you had to let him get sick for a while.

  3. #3 Joshua
    April 9, 2007

    Wow, that is truly awesome.

    I’m actually thinking that, even if this has no application to clinical treatments, the research might benefit the use of agricultural antibiotics. Given that, in the latter case, antibiotics are used more as a prophylactic than a curative, the risks of using antagonistic treatments would be countered by the benefits of selecting against resistant bacteria (which would cost much more to treat once they became prevalent in the population or animals, as well as liability costs if humans were infected with resistant bacteria through undercooked meat).

  4. #4 TheBrummell
    April 9, 2007

    I have to admit here that I’m not sure why one would hypothesize that this might be the case and the authors didn’t really explain very well beforehand why they thought their hypothesis might be true.

    In my more cynical moments (i.e. most of the time), I view this as “par for the course” at Nature. My experience of reading papers in Nature has often followed the formula:
    Introduction: We are exploring a great idea, that one of us came up with ten years ago. See previous papers.
    Methods: See supplemental online material and our paper published last year. We did some stuff, but we’re going to describe it so vaguely you’ll never figure it out.
    Results: We’re quite proud of Figure 1. Go ahead, look at it. Isn’t it pretty?
    Discussion: Published in Nature, Bitches! We are awesome!

    OK, Nature papers are almost never that bad, but I am sure that the majority of them I’ve read simply refer to previous work rather than explain in situ the introduction or methods. The advantage of Nature‘s tight page limits is you get to read good, concise papers. The disadvantage is you need to read two or three other papers in other journals first in order to understand what’s going on in Nature.

  5. #5 Ethan Romero
    April 9, 2007

    A minor nit-pick: Orac said,

    “In general, whenever two drugs are combined, they can have three different interactions[: antagonism, additivity, and synergism].”

    This isn’t really the case. My argument starts with a need to distinguish biologic interaction (physical interaction at the biochemical level) from statistical interaction (based on an abstract model of interaction).

    Then I would argue that, at the simplest level, biological interaction is either present or absent. When present, biologic interaction is a physical process that either increases or decreases the effect of a drug given certain dosage schedules. Additivity is nonsensical in terms on biologic interactions. If two agents act on unique biochemical mechanisms, then we would expect additive interaction, which is misleading because, in this case, there is no physical interaction.

    Concerning statistical interactions, we must specify a model of interaction–in the case of generalized linear models we must specify the functional form of the interaction term, e.g. +, -, ^2, etc… One might argue that statisticaly significant departure from additivity twords positive infinity implies synergism and that departure twords negative infinity implies antoganism. But, again, this is not really the case. Departure from additivity only implies that the additive model is reasonably excluded. Maybe the “correct” model has regions of synergism and antoganism at certian threshold values. Maybe not. The point is that additivity is a mathematical construct that does not partition all interactions into either antaognism or synergism.

    So, I think that if we are talking about biological interaction we should talk about its absence or presence, then further discussing the nature of that interaction in terms of biology. And, that when discussing departure from the additive model of interaction, we should simply say greater than additive effects or less then additive effects.

  6. #6 S. Rivlin
    April 9, 2007

    TheBrummell,

    I hope your criticism is aimed at Nature, not the authors of the paper.

  7. #7 daedalus2u
    April 9, 2007

    I suspect this will have limited clnical utility because the concentration ranges where these effects occur are quite small. Also, the in vivo environments are much more complex than that in vitro. No doubt there are many natural products of physiology and the immune system that mimic the effects of antibiotics and so cause these effects.

    These were clonal strains. It wouldn’t take much variation in the thresholds for these effects for it to not work on a diverse mixture.

    Many antibiotics are products of other bacteria, and there are always multiple bacteria present in vivo. It would be a bummer to suppress singly resistant E. coli only to amplify multiply resistant P. aeruginosa.

  8. #8 daedalus2u
    April 9, 2007

    Actually, the whole concept of “additivity” is nonsense. Every known biological interaction is non-linear. Maybe if you look at a narrow enough range you can find a peice-wise linear region, but the fundamental mechanism(s) are non-linear.

    The only reason linear models are used is because non-linear models are too complex. That happens to be a pretty good reason, but lets not forget it and somehow delude ourselves that something “linear” is going on.

  9. #9 S. Rivlin
    April 9, 2007

    Ethan Romero,

    Assume a drug (X) that can interact with two separate biochemical mechanisms (A and B) to inhibit two separate processes with similar outcome, each mechanism being affected at a different dose of drug X. Now, assume another drug (Y) that interacts only with one of the mechanism, the one affected by the higher dose of drug X. The only measure of the drugs effect is the degree to which the outcome of the two processes controlled by the two biochemical mechanisms is inhibited. Drug X alone at a low dose inhibits the outcome by 35%; drug Y at low dose inhibits the outcome by 20%; given together at their low dose, the two drugs inhibit the outcome by 70%. Is the combined effect of the two drugs additive or synergistic? if the combined outcome of the two drugs were 55% inhibited, would you call it an additive effect?

  10. #10 Robert M.
    April 9, 2007

    I have to admit here that I’m not sure why one would hypothesize that this might be the case

    The fact that Orac is unsure makes me think I must be missing something. To me, this is seems like one of those things where everyone else in the field hits their head and exclaims “Why didn’t I think of that?”

    What you’re really doing is administering one drug (ciprofloxacin), along with an interfering agent (in this case, tetracycline). The resistant bacteria ignore the interfering agent, so they get the unsuppressed effect of the ciprofloxacin.

    So… what am I missing?

  11. #11 daedalus2u
    April 9, 2007

    This type of behavior is quite general, that is stimulatory at low concentrations leading to inhibition at high concentrations. This behavior is known as hormesis.

    http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Retrieve&list_uids=17107845

  12. #12 Bryn
    April 10, 2007

    Oh, I have a feeling Dr. Egnor won’t come up with much. Instead, he’ll violently wave his hands in the air, repeating, “Chaff, chaff, chaff,chaff…!” over and over thereby emulating “The Little Discovery Institute Engine that Couldn’t.”

    Or he’ll take another lovely page from the DI’s handbook of obfuscation and start screaming about, “There isn’t any new information being created!!!” causing bystanders to go, “Wha….?” while he escapes into the night.

  13. #13 Davis
    April 10, 2007

    I thought that this was a really cool bit of research, and am quite surprised that it hasn’t really turned up anywhere.

    This is in the Science & Technology section of this week’s issue of The Economist, so it’s at least gotten some exposure.

  14. #14 James Collins
    April 10, 2007

    Orac wrote:
    “I’d love to see what Dr. Egnor would say about this study. No doubt he’d lamely try to call it a “tautology” again. (He’s rather one-note that way.) Even so, it’d be fun to see him try.”

    Bro. Orac, Evolution is needless, even a stumbling block to the advancement of science. Any non-evolutionists scientists has NO problem with experimenting and discovering new data in ANY of the sciences. If you think not, please provide an example. All talk and no show is NOT productive.

    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 answersingenesis.org. 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!

  15. #15 Robert M.
    April 10, 2007

    Mr. Collins,

    Part of the problem with engaging creationists is that it’s hard to know where to start.

    Should I explain that the only barrier to constructing a functioning cell is that, until very recently, the kind of technology necessary to manipulate individual atoms and molecules was science fiction?

    Should I talk about the fact that abiogenesis (how life began from not-life) is a separate study from evolution (how life changes over time)? Should I point you to the Miller-Urey experiment and its successors?

    Should I point out that claiming any biologist works without evolution is like claiming that there are lots of computer programmers that don’t believe in transistors?

    Please, instead of listening to answersingenesis.org, contact your local high school or community college, and ask if you can sit in on an introductory biology course. You’ll learn the answers to a lot of your questions, and hopefully learn to recognize the false assumptions in every claim made by organizations like AiG and the Discovery Institute.

  16. #16 Ethan Romero
    April 10, 2007

    S. Rivlin

    Thanks for the reply. To determine whether or not the additive model holds in the case that you mentioned, I would need to know the sample size of the in the hypothetical study. If we assume that there is an infinitely large sample, then I would state the additive model does not hold (the expectation of the additive model with an infinite sample size is the sum of the individual effects). So, no, I would not call it an additive effect. I would say that the additive model does not hold, and that the effects in this case (with infinite n) are greater that would be expected under the additive model. I’m not a medical doctor, but my understanding of the use of “synergism” in medicine is synonymous with any effects that are statistically greater then additive.

    daedalus2u

    You say, “Actually, the whole concept of “additivity” is nonsense.”

    I agree. But I think that it’s important to note that “additivity” is an arbitrary construction in linear models and is nonsense de facto. As you note later, some non-linear systems can be analyzed under the generalized linear models framework to reasonable effect. But, again, as you note, “the fundamental mechanisms are non-linear”. I think that to advance medicine must begin to adopt and apply (if if only as an academic excersice at first) deductive, non-linear models to medical questions. Such models ARE more complex and require some mathematical sophistication to apply, but what is gained (causal, mechanistic understand of the phenomena) is well worth the cost.

  17. #17 daedalus2u
    April 10, 2007

    Mr Collins, stumbling block? Why don’t you remove the forest from your own eyes? Evolutionists are already actively searching for specks that obscure their own vision, and when found, immediately remove them.

    What is pitiable, is the willful blindness required to to ignore the structure that the ToE gives to biology by virtue of the observation of common descent.

    “Science is facts; just as houses are made of stones, so is science made of facts; but a pile of stones is not a house and a collection of facts is not necessarily science.”
    Jules H. Poincare

    Reliable data that contradicted evolution would be front page news in every science journal in the world. Who ever discovered it would be guarenteed a Nobel Prize. Of course, a false statement from a known liar doesn’t constitute “proof”, at least not in scientific circles.

  18. #18 pough
    April 10, 2007

    … Walla …

    Oh, my poor (somewhat) bilingual eyes! The stupid reached new levels! Aidez-moi! Aidez-moi!

  19. #19 Aureola Nominee, FCD
    April 10, 2007

    pough:

    At least he didn’t write “Viola”… creative misspelling is FUN! ;-)

  20. #20 daedalus2u
    April 10, 2007

    Ethan, Unfortunately the level of complexity that is added when the models are non-linear is not modest. They are essentially impossible to solve with more than a few parameters.

    I think that many of the “problems” in physiology and medicine are because of the mistaken notion of linearity. Physiology inherently uses non-linear systems. An example I like to use is anaphylaxis, which some (I think mistakenly) consider to be a “breakdown” of “homeostatic regulation”. Anaphylaxis is not a “breakdown” of “homeostasis”, rather it is a regulated physiological state brought about by the immune system to deal with a life threatening situation. It is an extreme desperate response to an extreme desperate situation. If extreme situations such as anaphylaxis are regulated (that is turned on in seconds), by what basis do we assume that the minor physiology changes are not regulated?

  21. #21 SteveM
    April 10, 2007

    Oh, my poor (somewhat) bilingual eyes! The stupid reached new levels! Aidez-moi! Aidez-moi!

    “Aidez-moi”? Oh for shame. One of the very few French phrases I managed to retain from high school is “M’aidez” for “help me!” [thus the english radio slang “mayday” for “help!”]

  22. #22 pough
    April 10, 2007

    You need to unretain that lesson. “M’Aidez” is not grammatically correct in French, but “aidez-moi” is. Like so many things we English have lifted from other languages, “mayday” has been lifted quite poorly. (Oh, for shame!) I’ve been lead to believe that in times of distress, “au secours” is more likely to be heard, but “aidez-moi” still means “help me.”

    Just for fun, I did some Googling. “Aidez-moi” returns mostly French websites. “M’Aidez” almost exclusively returns English websites. In a Google Fight, “aidez-moi” beats “m’aidez” by 1,340,000 to 238,000. :-p

  23. #23 bharath
    April 11, 2007

    Very interesting read. I got to read it again to get an idea of how this is transferable to practice.

  24. #24 daedalus2u
    April 12, 2007

    Orac, You should look at this paper. There are quite interesting signaling effects of different levels of antibiotics in turning on virulence factors.

    http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17148599&query_hl=27&itool=pubmed_docsum

    Most pathogens are only pathogenic when they are expressing virulence factors, and most of those are regualted by quorum sensing compounds. Look at their figure 2, they show that tetracycline increases the cytotoxicity of P. aeruginosa.

  25. #25 Dave S.
    April 12, 2007

    James Collins writes:

    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.

    The funny part is that for every creationist who says that this would prove evolution for them, another creationist would insist that the same experiment would prove intelligent intervention would obviously be necessary.

    Merde.

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