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Author
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Topic: Is bacterial resistance really due to mutation?
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Martin245
Inactive Member
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Message 1 of 27 (198520)
04-12-2005 8:58 AM
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Okay as far as I understand an evolution scientist takes a sample of bacteria in a dish and let it grow. Then they apply some sort of anti-biotics. When some bacteria survive they conclude that some bacteria must have developed resistance due to mutation as they grew. But surely they haven't ruled out the possibility of resistant bacteria being part of the original sample. If that was the case then there was no mutation in this experiment that caused the resistance - resistance existed all along. How do evolutionists actually know that bacterial resistance is due to mutation and that they aren't simply selecting the few resistant bacteria that already existed in the *original* sample? I've looked for an experiment on this on google and found none.
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AdminAsgara
Administrator (Idle past 2324 days) Posts: 2073 From: The Universe Joined: 10-11-2003
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Message 2 of 27 (198801)
04-12-2005 8:40 PM
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Thread moved here from the Proposed New Topics forum.
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crashfrog
Member (Idle past 1488 days) Posts: 19762 From: Silver Spring, MD Joined: 03-20-2003
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Message 3 of 27 (198802)
04-12-2005 8:42 PM
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Reply to: Message 1 by Martin245
04-12-2005 8:58 AM
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An insightful, if common, question
How do evolutionists actually know that bacterial resistance is due to mutation and that they aren't simply selecting the few resistant bacteria that already existed in the *original* sample? As it turns out, and this is often unreported or glossed over when these experiments are communicated to laypeople, they design the experiment so that they can rule out resistant bacteria being part of the original sample. Let me explain. The sort of innoculation experiment you describe is really divided into two phases: phase one is the development of a culture from a single individual (this sort of clonal population is called a "monoculture"), and then in phase two the antibiotic is applied to this population. Bacteria are asexual, so they reproduce by cloning themselves, as you probably know. When you develop a population of bacteria from a single individual, they should all be clones of each other. If, in phase two, some members survive the innoculation and most do not, this reflects variation that can only be due to genetic traits that the founder individual did not possess - in other words, mutations. There's no possibility that the original sample contained resistances, because that original sample was a single individual, and if he had been resistant, then the vast majority of its decendants should have been, as well. Since mutation is the only observed source of genetic and phenotypical variation in a clonal, asexual population, we conclude that mutation was the source of the new resistance. This sort of sampling problem is one scientists have to face; that's part of the reason bacteria are studied so frequently. Their asexuality makes it possible to eliminate the sampling issue by raising a whole population from a single individual. If subsequent members of the population have genes that the founder individual did not, we know that mutation is the only place those new genes could have come from.
| This message is a reply to: | | | Message 1 by Martin245, posted 04-12-2005 8:58 AM | | Martin245 has not replied |
| Replies to this message: | | | Message 5 by Coragyps, posted 04-12-2005 10:31 PM | | crashfrog has not replied |
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JonF
Member (Idle past 189 days) Posts: 6174 Joined: 06-23-2003
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Message 4 of 27 (198805)
04-12-2005 9:08 PM
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Reply to: Message 1 by Martin245
04-12-2005 8:58 AM
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But surely they haven't ruled out the possibility of resistant bacteria being part of the original sample. Gee, I would have said "surely they have ruled out ...". Scientists are invariably curious, and have a marked propensity for digging into things to find out. Crash's reply is right on the money. It's also quite common to identify the exact changes. I hesitate to post the following link; it gives a good list of mutations identified as causing antibiotic resistance in bacteria, but the characterizations of those mutations and the conclusions are a mishmash of non-sequiturs and egregious errors. But I'll post it anyway, as long as it's understood that the conclusions are not to be trusted:
Is Bacterial Resistance to Antibiotics an Appropriate Example of Evolutionary Change?. See also The isoleucyl-tRNA synthetase mutation V588F conferring mupirocin resistance in glycopeptide-intermediate Staphylococcus aureus is not associated with a significant fitness burden (which directly contradicts one of the conclusions of the first link),
Mutations Affecting DNA-Binding Activity of the MexR Repressor of mexR-mexA-mexB-oprM Operon Expression, and
Diversity of -Lactam Resistance-Conferring Amino Acid Substitutions in Penicillin-Binding Protein 3 of Haemophilus influenzae. {Broke the links down into their own "paragraphs" - Adminnemooseus} This message has been edited by Adminnemooseus, 04-12-2005 09:46 PM
| This message is a reply to: | | | Message 1 by Martin245, posted 04-12-2005 8:58 AM | | Martin245 has not replied |
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Coragyps
Member (Idle past 756 days) Posts: 5553 From: Snyder, Texas, USA Joined: 11-12-2002
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Message 5 of 27 (198813)
04-12-2005 10:31 PM
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Reply to: Message 3 by crashfrog
04-12-2005 8:42 PM
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Re: An insightful, if common, question
I hope Crash doesn't mind me adding one important bit he left out, likely because he's been over this ground so much: bacteria have only a single set of genes, not two sets like we sexually reproducing critters. And the have little to no non-coding ("junk") DNA like we eukaryotes have. So there's no way that they can "hide" traits like antibiotic resistance in their genomes - there are no hiding places.
| This message is a reply to: | | | Message 3 by crashfrog, posted 04-12-2005 8:42 PM | | crashfrog has not replied |
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Martin245
Inactive Member
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Message 6 of 27 (198814)
04-12-2005 10:33 PM
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Good answers. I guess the problem I am having is how do they make these monocultures, or more specifically how do they guarantee that the founding bacterium used is non-resistant. How can they find that out without killing it? I have been challenged on this and while I can find methods online which show mutation is random, or methods that show bacterial resistance is not caused by the poison itself, but I cannot find any method that convincingly rules out the resistant variant already being present at the beginning of the experiment. I have just taken it as a given. You are right it must be true, but how do I convince someone of this? The only method I can think of that would seal it would be to take a large number of bacteria and allow them to found seperate large colonies and then poison them. If resistance is caused by mutation you would expect quite a lot of the colonies to have mutated resistant survivors. If instead resistance cannot be aquired by mutation you would expect only a few of the colonies to have resistant survivors. But this is hardly a nice method of determining it. Surely there must be a better way. Please tell me if you know a way.
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NosyNed
Member Posts: 9003 From: Canada Joined: 04-04-2003
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Message 7 of 27 (198815)
04-12-2005 10:41 PM
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Reply to: Message 6 by Martin245
04-12-2005 10:33 PM
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monocultures
The monocultures have been explained as originating with just one bacterial individual. Without mutations (of some sort) the genetics of it's decendants must be the same as that individual. There are two choices regarding the antibiotic resistance of that individual: Either it is resistant or it is not resistant. The above discussion presumed it was not resistant. In which case the expected result without mutations is that all of the monoculture would be killed by a dose of the antibiotic. The other case is that it was resistant. In which case all of the monoculture should NOT die but should all live. If I recall other discussions of this further tests are conducted. For example, after the first antibiotic application one of the survivors is taken and used to produce a monoculture. Now we know that we have started the experiment over again but this time with an individual bacteria that is resistant. The prediction would be that a much larger number of the monoculture will survive the antibiotic. There may be some mutated ones that do not have the resistance. That is what happens. In fact, that is exactly why we now have "super bugs". I don't understand this at all. The number of colonies that have survivors (and the number of survivors in any colony) will depend on how likely the mutation is and whether it is detramental (and to what degree) in the absense of the antibiotic. Apparently (I'm not an expert) many of the mutations supplying antibiotic resistance are not beneficial or neutral in the absense of the anitbiotic. This suggests an obvious strategy: stop using a particular antibiotic for some years and let selection weed out the bacteria resistant to it so it can then be reintroduced. This message has been edited by NosyNed, 04-12-2005 09:45 PM
| This message is a reply to: | | | Message 6 by Martin245, posted 04-12-2005 10:33 PM | | Martin245 has not replied |
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Coragyps
Member (Idle past 756 days) Posts: 5553 From: Snyder, Texas, USA Joined: 11-12-2002
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Message 8 of 27 (198816)
04-12-2005 10:41 PM
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Reply to: Message 6 by Martin245
04-12-2005 10:33 PM
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The only method I can think of that would seal it would be to take a large number of bacteria and allow them to found seperate large colonies and then poison them.
I'm sure that this is precisely what was done in the early days of such experiments - this, and the fact that only a tiny fraction of the poisoned bugs typically survive. Nowadays, they can actually identify the specific DNA change, the protein change it causes, and the monkey wrench that that protein change throws into the works of the antibiotic. I can imagine, too, that the survivors on a Petri dish might be well away from the spot where the great-grandma bacterium was put. That alone would strongly suggest that the resistant strain wasn't there at the point/time of colony start-up.
| This message is a reply to: | | | Message 6 by Martin245, posted 04-12-2005 10:33 PM | | Martin245 has not replied |
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coffee_addict
Member (Idle past 498 days) Posts: 3645 From: Indianapolis, IN Joined: 03-29-2004
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Message 9 of 27 (198823)
04-13-2005 12:05 AM
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Reply to: Message 6 by Martin245
04-12-2005 10:33 PM
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Martin writes: How can they find that out without killing it?
Easy. Only acquire 1 bacterium. Let it go through mitosis and move some sister cells into another petri dish and start pumping in anti-biotics. If all the sister cells of the original bacterium died, then we know for sure that the original bacterium was not anti-biotic resistant. The problem I see is not that you or your friends cannot think of methods for such an experiment. The problem I see is that you and your friends have trouble understanding the significance of how bacterial genes work or what mitosis really means. This is a problem when communicating in layman's terms. The significance and meanings of concepts are lost.
| This message is a reply to: | | | Message 6 by Martin245, posted 04-12-2005 10:33 PM | | Martin245 has not replied |
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crashfrog
Member (Idle past 1488 days) Posts: 19762 From: Silver Spring, MD Joined: 03-20-2003
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Message 10 of 27 (198826)
04-13-2005 12:10 AM
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Reply to: Message 6 by Martin245
04-12-2005 10:33 PM
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I guess the problem I am having is how do they make these monocultures, or more specifically how do they guarantee that the founding bacterium used is non-resistant. Well, if it was, the majority of its decendants would be, and when you cultured and innoculated the petri dishes, you wouldn't see any result. (The few individuals that did die would be replaced by their neighbors, so you wouldn't observe an overall change in population.)
You are right it must be true, but how do I convince someone of this? With logic. If the outcome of the experiment is that all but a few individuals are extinguished by the antibiotic, and all individuals living and dead were the decendants of one single organism, then the only thing that could be different between the living and the dead would be traits that the survivors possessed that the founder did not (otherwise the result of the experiment would be different, as described above), and the only source of those traits beyond a contaminated experiment is mutation. It's the only known possibility, by process of elimination. That's how a good experiment is designed; so that all possibilities except the one you wanted to test are able to be eliminated as an explanation.
| This message is a reply to: | | | Message 6 by Martin245, posted 04-12-2005 10:33 PM | | Martin245 has not replied |
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coffee_addict
Member (Idle past 498 days) Posts: 3645 From: Indianapolis, IN Joined: 03-29-2004
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Message 11 of 27 (198831)
04-13-2005 12:27 AM
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Reply to: Message 6 by Martin245
04-12-2005 10:33 PM
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Actually, going back to your original question, which was how do we know that some kind of change occured, the very fact that you start out with a single bacterium and after it has grown to a culture and at least some of them died as a result of anti-biotics should be enough to show that some kind of change in their genes occured, whether the original bacterium was anti-biotic resistant or not. Sorry for the long sentence. I don't think it's a run-on, though.
| This message is a reply to: | | | Message 6 by Martin245, posted 04-12-2005 10:33 PM | | Martin245 has not replied |
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Wounded King
Member Posts: 4149 From: Cincinnati, Ohio, USA Joined: 04-09-2003
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Message 12 of 27 (198856)
04-13-2005 4:49 AM
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Reply to: Message 6 by Martin245
04-12-2005 10:33 PM
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For a specific example of experimental evolution of antibiotic resistance I would direct you to this paper which I previously referenced in the 'Natural Limitation to Evolutionary Processes' thread.
Evolution of drug resistance in experimental populations of Candida albicans.Cowen LE, Sanglard D, Calabrese D, Sirjusingh C, Anderson JB, Kohn LM. J Bacteriol. 2000 Mar;182(6):1515-22. Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast Candida albicans and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes, CDR1 and CDR2; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway, ERG11; and the major facilitator gene, MDR1. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of C. albicans. I realise that this example is for a yeast rather than a bacteria but the principle is the same. The authors use exactly the sort of clonal expansion Crashfrog has already detailed. They get around the problem you put forward of killing off all the non-resistant cells by using a dose equivalent to the yeast strains Minimium Inhibitory Concentration (MIC). An MIC dose will prevent the growth of a culture but does not neccessarily kill that culture off. The minimum lethal dose to kill off the culture, or Minimum Bacteriocidal Concentration (MBC), may coincide with the MIC for some antibiotics (Bacteriocidals) but not for all (Bacteriostatics). If a dosage less than the MBC is used then you can tell that resistance is present in certain populations as they will have developed elevated MICs. TTFN, WK
| This message is a reply to: | | | Message 6 by Martin245, posted 04-12-2005 10:33 PM | | Martin245 has not replied |
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EZscience
Member (Idle past 5175 days) Posts: 961 From: A wheatfield in Kansas Joined: 04-14-2005
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Message 13 of 27 (207012)
05-11-2005 8:56 AM
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Reply to: Message 7 by NosyNed
04-12-2005 10:41 PM
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Rotation of materials
Ned writes: Apparently (I'm not an expert) many of the mutations supplying antibiotic resistance are not beneficial or neutral in the absense of the anitbiotic. This suggests an obvious strategy: stop using a particular antibiotic for some years and let selection weed out the bacteria resistant to it so it can then be reintroduced. Expert or no, that is quite correct. In fact the strategy you suggest is now employed in a number of applications where resistance is a potenital problem, like pest control. The strategy is one of rotating different materials (drugs or insecticides) to diminish directional selection for resistance to any one. It can work especially well when the mutation causing resistance exacts some cost on the organism, or is less advantageous than its alternative allele in the absence of exposure to the material.
| This message is a reply to: | | | Message 7 by NosyNed, posted 04-12-2005 10:41 PM | | NosyNed has not replied |
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EZscience
Member (Idle past 5175 days) Posts: 961 From: A wheatfield in Kansas Joined: 04-14-2005
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Message 14 of 27 (207013)
05-11-2005 8:57 AM
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Reply to: Message 7 by NosyNed
04-12-2005 10:41 PM
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Sorry - double post. EZ This message has been edited by EZscience, 05-11-2005 08:58 AM
| This message is a reply to: | | | Message 7 by NosyNed, posted 04-12-2005 10:41 PM | | NosyNed has not replied |
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christ_fanatic
Inactive Member
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Message 15 of 27 (244069)
09-16-2005 7:02 AM
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Reply to: Message 1 by Martin245
04-12-2005 8:58 AM
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Viruses and bacteria.
With your thread, I think it should be pointed out that much the same happens with viruses except that viruses may have inherited the resistance to natural drugs, such as penicillin. With bacteria, either they do or don't have it (due to the way they reproduce) and the ones that do survive do so because they have inherited it. Many of the experiments done by scientists to show how mutations can add info have been extremely controversial.
| This message is a reply to: | | | Message 1 by Martin245, posted 04-12-2005 8:58 AM | | Martin245 has not replied |
| Replies to this message: | | | Message 17 by Cal, posted 09-16-2005 7:34 AM | | christ_fanatic has not replied |
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