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Author Topic:   Application of the Scientific Method: Antibiotic Resistance
Taq
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Posts: 7673
Joined: 03-06-2009
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(1)
Message 1 of 20 (691726)
02-22-2013 6:38 PM


In this thread we will be dealing with the subject of how antibiotic resistance appears in bacteria in a specific experimental setup. What I would like to do is have the community come up with hypotheses at each step, and possible ways of testing those hypotheses with experiments. IOW, how can we apply the scientific method as it relates to the emergence of antibiotic resistance. Many of you are already familiar with this subject, so don't give away the punch line too early.

We start with a simple observation in a laboratory setting. I have a standard strain of E. coli that I streak on a plate to get an isolated colony. This is a standard technique that spreads out bacteria so that a single bacterium gives rise to several hundred thousand bacteria. This allows us to start our experiment with a single, founding bacterium that is the single ancestor of the rest of the bacteria used in the experiment. This is an important part of the experiment, so don't forget it.

We use this isolated colony to seed a liquid culture which produces trillions of bacteria from the hundreds of thousands found in the original colony that was itself founded by a single bacterium. We then spread some of that liquid culture onto two agar plates, one containing an antibiotic and the other one without antibiotic. We incubate it overnight and come back the next day to find a handful of colonies growing on the antibiotic plate, each of which was founded by a single bacterium. We compare this antibiotic plate to another that does not contain antibiotic and we find that the no antibiotic plate is overrun with bacteria without any isolated colonies. Obviously, only a handful of the billions of bacteria put on the antibiotic plate were antibiotic resistant.

So how did this antibiotic resistance come about? What are your hypotheses?

Suggested Forums: "Is It Science?" or "Biological Evolution"

Edited by Taq, : No reason given.


Replies to this message:
 Message 12 by bluegenes, posted 02-26-2013 6:33 PM Taq has responded
 Message 14 by Stile, posted 02-27-2013 2:17 PM Taq has responded

  
Adminnemooseus
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Message 2 of 20 (691728)
02-24-2013 10:52 PM


Thread Copied from Proposed New Topics Forum
Thread copied here from the Application of the Scientific Method: Antibiotic Resistance thread in the Proposed New Topics forum.
    
Taq
Member
Posts: 7673
Joined: 03-06-2009
Member Rating: 2.6


Message 3 of 20 (691846)
02-25-2013 3:45 PM


This is peer reviewed
Just as a clarification, I will be drawing from experimental results found in real peer reviewed scientific papers. However, I may not cite them in the early going because I don't want to give the punch line away. Rather, I would like this discussion to be about the process of figuring out how antibiotic resistance emerges in these experiments. So I am asking for a bit of faith on the part of the participants, but I promise to show you the papers once the discussion hits the proper stage. What I hope to do is allow people to understand the thought process and the historical path that science took where it concerns evolution and antibiotic resistance.
Replies to this message:
 Message 4 by Faith, posted 02-25-2013 11:53 PM Taq has responded

  
Faith
Inactive Member


(1)
Message 4 of 20 (691876)
02-25-2013 11:53 PM
Reply to: Message 3 by Taq
02-25-2013 3:45 PM


Re: This is peer reviewed
I almost feel bad that I'm not joining in on this thread since you've invited me so many times, but I just can't relate to bacteria. For one thing they don't have enough junk DNA. Not "one of us" you see.

This particular example you are using here has been discussed before only I do have to say I don't remember the outcome. Supposed to prove that mutations happen on cue just when needed as usual I guess?


This message is a reply to:
 Message 3 by Taq, posted 02-25-2013 3:45 PM Taq has responded

Replies to this message:
 Message 5 by Taq, posted 02-26-2013 11:15 AM Faith has responded

  
Taq
Member
Posts: 7673
Joined: 03-06-2009
Member Rating: 2.6


Message 5 of 20 (691920)
02-26-2013 11:15 AM
Reply to: Message 4 by Faith
02-25-2013 11:53 PM


Re: This is peer reviewed
I almost feel bad that I'm not joining in on this thread since you've invited me so many times, but I just can't relate to bacteria. For one thing they don't have enough junk DNA. Not "one of us" you see.

Bacteria have junk DNA as well. They use the same basic genetic and metabolic systems we do. I really don't see why this is a problem.

This particular example you are using here has been discussed before only I do have to say I don't remember the outcome. Supposed to prove that mutations happen on cue just when needed as usual I guess?

That is certainly one hypothesis we can test. It is also one of the hypotheses that scientists seriously considered when faced with the observations found in the opening post.

So let's develop that idea a little bit. To be "on cue" it would appear to me that the mutations should be a reaction to the presence of antibiotics. That is, we should be able to tie the introduction of antibiotics to the appearance of the mutation. In fact, we don't even have to say that it is a mutation. This could just be a hidden phenotype that is turned on by the presence of the antibiotic.

Does this sound something like the hypothesis you would put forward?


This message is a reply to:
 Message 4 by Faith, posted 02-25-2013 11:53 PM Faith has responded

Replies to this message:
 Message 6 by Faith, posted 02-26-2013 11:45 AM Taq has responded

  
Faith
Inactive Member


Message 6 of 20 (691922)
02-26-2013 11:45 AM
Reply to: Message 5 by Taq
02-26-2013 11:15 AM


Re: This is peer reviewed
Bacteria have junk DNA as well. They use the same basic genetic and metabolic systems we do. I really don't see why this is a problem.

I'm sure I read somewhere maybe Wikipedia that they have a lot less junk DNA than we have, a LOT less. It seems to me that this gives them a huge advantage over us, many more genetic possibilities that couldn't be extrapolated to our situation, maybe even the ability to mutate on demand or something like that. Not to mention that although I know there are "good" bacteria most of them seem to be out to kill us (E COLI???) so that I have a big problem identifying with them. (Half kidding, Taq, but it's also true).

This particular example you are using here has been discussed before only I do have to say I don't remember the outcome. Supposed to prove that mutations happen on cue just when needed as usual I guess?

That is certainly one hypothesis we can test. It is also one of the hypotheses that scientists seriously considered when faced with the observations found in the opening post.

So let's develop that idea a little bit. To be "on cue" it would appear to me that the mutations should be a reaction to the presence of antibiotics. That is, we should be able to tie the introduction of antibiotics to the appearance of the mutation. In fact, we don't even have to say that it is a mutation. This could just be a hidden phenotype that is turned on by the presence of the antibiotic.

Does this sound something like the hypothesis you would put forward?

I just find bacteria to be so alien it is very hard to come up with a hypothesis at all. And here you've got this down to a single cell as the progenitor of all the millions? How on earth does that compare to anything in the multi-celled kingdoms? How could an alternate phenotype be "hidden" in a single cell? In a phenotypically diverse colony, OK, but see I can't even be sure I'm talking about anything that could be compared to a multi-celled creature's situation at all.

I suppose given the description I'd be inclined to think in terms of mutation on cue just as suggested.


This message is a reply to:
 Message 5 by Taq, posted 02-26-2013 11:15 AM Taq has responded

Replies to this message:
 Message 7 by Taq, posted 02-26-2013 12:03 PM Faith has responded
 Message 8 by New Cat's Eye, posted 02-26-2013 12:05 PM Faith has responded

  
Taq
Member
Posts: 7673
Joined: 03-06-2009
Member Rating: 2.6


Message 7 of 20 (691923)
02-26-2013 12:03 PM
Reply to: Message 6 by Faith
02-26-2013 11:45 AM


Re: This is peer reviewed
I'm sure I read somewhere maybe Wikipedia that they have a lot less junk DNA than we have, a LOT less. It seems to me that this gives them a huge advantage over us, many more genetic possibilities that couldn't be extrapolated to our situation, maybe even the ability to mutate on demand or something like that. Not to mention that although I know there are "good" bacteria most of them seem to be out to kill us (E COLI???) so that I have a big problem identifying with them. (Half kidding, Taq, but it's also true).

This is all tangential to the real meat of the thread, so I will just comment briefly. I really don't see how having less junk DNA gives them more genetic possiblities. Their genomes are usually around 2 or 3 million bases compared to our 3 billion. Their gene count is also well below ours.

I just find bacteria to be so alien it is very hard to come up with a hypothesis at all.

Heredity is not alien. It is the same as ours, just with one copy of DNA instead of two. I really don't understand what your protestations are all about.

How could an alternate phenotype be "hidden" in a single cell?

The same way that it is hidden in animal species. Bacteria, just like animals, can change gene expression in response to a changing environment. They can alter their phenotype in response to physical cues just like animals do.

I suppose given the description I'd be inclined to think in terms of mutation on cue just as suggested.

So what we need to do is determine if the mutation conferring antibiotic resistance occurs before or after the bacteria are exposed to antibiotics, correct?


This message is a reply to:
 Message 6 by Faith, posted 02-26-2013 11:45 AM Faith has responded

Replies to this message:
 Message 10 by Faith, posted 02-26-2013 12:27 PM Taq has responded

  
New Cat's Eye
Inactive Member


Message 8 of 20 (691924)
02-26-2013 12:05 PM
Reply to: Message 6 by Faith
02-26-2013 11:45 AM


Re: This is peer reviewed
I just find bacteria to be so alien it is very hard to come up with a hypothesis at all.

Have you considered plants at all? People have been breeding them too. Look what we did with maize becoming corn. That wasn't a loss of genetic diversity. Or how about all the amazing flowers that have been made? There's no way all that stems from some super flower genome that gets chipped away into all the breeds we have today.

And crazily enough, all the plant developments fit perfectly with the Theory of Evolution's mutations and selection mechanism.

It really is correct.


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 Message 6 by Faith, posted 02-26-2013 11:45 AM Faith has responded

Replies to this message:
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Faith
Inactive Member


Message 9 of 20 (691925)
02-26-2013 12:10 PM
Reply to: Message 8 by New Cat's Eye
02-26-2013 12:05 PM


Plants
This is off topic for this thread but if you want to take it over to my thread I'll respond there.
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Faith
Inactive Member


Message 10 of 20 (691928)
02-26-2013 12:27 PM
Reply to: Message 7 by Taq
02-26-2013 12:03 PM


Re: This is peer reviewed
How could an alternate phenotype be "hidden" in a single cell?

The same way that it is hidden in animal species. Bacteria, just like animals, can change gene expression in response to a changing environment. They can alter their phenotype in response to physical cues just like animals do.

Taq, this sounds like a completely other theory of evolution than I'm familiar with.

The WAY I understand animals to "change gene expression in response to a changing environment" is by the reproductive selection of alleles that are best fitted to the new environment which can mean those not fitted have to die out before reproductive age, and all this is supposed to be more or less random. You keep talking as if there were some sort of PURPOSIVE or teleological ability to change the phenotype on demand that I always thought was rejected as a misunderstanding of how evolution works. All the processes are supposed to be random. If nonadaptive types die out before reproduction, then the alleles that are favored by the environment that are reproductively successful will have the opportunity to be expressed in greater and greater numbers until they come to characterize the population.

This is possible in multi-celled sexually reproducing animals that possess the "hidden phenotypes" for such adaptations, probably most often in the form of recessive alleles, perhaps for more than one gene, or perhaps dominant alleles have to work together with other genes to come to expression or something like that. In any case you have to have a situation that allows the adaptive alleles to find opportunity for expression which is usually because the nonadapative individuals aren't reproducing as much.

Isn't something like that the usual idea?

How a single cell could manage such a feat I have no idea. It WOULD have to change in response to the environment in some much more direct way it seems to me.

If you don't jmean to be implying something so directly teleological maybe it would help if you reworded it.

I suppose given the description I'd be inclined to think in terms of mutation on cue just as suggested.

So what we need to do is determine if the mutation conferring antibiotic resistance occurs before or after the bacteria are exposed to antibiotics, correct?

Hm. OK I guess. But even so it's hard to imagine that mutations that save the creature from death just turn up so fortuitously, whether right before or whenever they are "just in time." If before then it wouldn't be in response to the antibiotic of course, but why they should occur at all at such a convenient time is still rather mystifying. It seems to violate the rule of randomness. AND the other "rule" that I thought says that beneficial mutations don't occur all thjat FREQUENTLY.

Edited by Faith, : No reason given.

Edited by Faith, : No reason given.

Edited by Faith, : No reason given.


He who surrenders the first page of his Bible surrenders all. --John William Burgon, Inspiration and Interpretation, Sermon II.

This message is a reply to:
 Message 7 by Taq, posted 02-26-2013 12:03 PM Taq has responded

Replies to this message:
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Taq
Member
Posts: 7673
Joined: 03-06-2009
Member Rating: 2.6


(2)
Message 11 of 20 (691934)
02-26-2013 12:47 PM
Reply to: Message 10 by Faith
02-26-2013 12:27 PM


Re: This is peer reviewed
Taq, this sounds like a completely other theory of evolution than I'm familiar with.

It is called phenotype plasticity, and it is a well understood phenomenon in biology. I'm not trying to sneak anything past you, I promise.

Also, it is entirely possible that the presence of an antibiotic will turn on a resistance gene so that the resistance is not due to a mutation, wouldn't you agree?

The WAY I understand animals to "change gene expression in response to a changing environment" is by the reproductive selection of alleles that are best fitted to the new environment which can mean those not fitted have to die out before reproductive age, and all this is supposed to be more or less random.

This isn't the case at all. Think of how you tan in the summer. This is a change in phenotype as a result of changing gene expression in response to environmental cues. The sun causes DNA damage in your skin cells. The cells respond by upregulating the production of melanin causing a change in your phenotype.

Bacteria can also respond to environmental cues, even including the production of multicellular fruiting bodies in the case of the myxococcus bacterial species.

You keep talking as if there were some sort of PURPOSIVE or teleological ability to change the phenotype on demand that I always thought was rejected as a misunderstanding of how evolution works.

The way in which humans communicate does often indicate a teleologic purpose when in fact there is none. Scientists are as guilty of this as anyone. The fact of the matter is that these are very mechanistic systems without any overarching intelligence to them. Also, each species has a set number of responses to a set number of stimuli, that is if we ignore the claim that mutations increase genetic diversity.

Hm. OK I guess. But even so it's hard to imagine that mutations that save the creature from death just turn up so fortuitously, whether right before or whenever they are "just in time." If before then it wouldn't be in response to the antibiotic of course, but why they should occur at all at such a convenient time is still rather mystifying. It seems to violate the rule of randomness.

In the view of biologists, that is the very definition of randomness. If a mutation occurs when there is no need for it then this is a random mutation with respect to fitness. This is contrasted with non-random mutations which are strongly guided by what the organism needs at that moment. So in our current system, a random mutation is one that occurs in the absence of antibiotics and a guided or non-random mutation is one that occurs only when the organism needs it. Does that sound good?


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bluegenes
Member (Idle past 553 days)
Posts: 3119
From: U.K.
Joined: 01-24-2007


Message 12 of 20 (691974)
02-26-2013 6:33 PM
Reply to: Message 1 by Taq
02-22-2013 6:38 PM


Am I right?
Taq writes:

So how did this antibiotic resistance come about? What are your hypotheses?

My hypothesis is that mutation(s) that occured during the growth of your one organism into trillions are the source of the antibiotic resistance. This mutation (assuming only one's involved) would probably have been near neutral on arrival.

The reasoning is this. If the resistance had existed in the original clone, then virtually all the descendents would have had it, and growth would have occured all over the antibiotic plate. Also, from what I know of bacterial mutation rates, billions of mutations must have taken place in the course of the growth from one into trillions, so there would be considerable variation on the original genome already, as many mutations would be near enough neutral. So, a "hit" (thinking retrospectively) in one or two strains of variants is plausible.

The end result is that a tiny fraction, perhaps one in a few billion, of the organisms have a variation that happens to be useful, which is what you see on your plate.

I don't know the specific paper you're talking about, but that seems to me to be the most plausible hypothesis.

So, If I'm right, the mutation was random in respect to fitness on arrival, at least, so far as its known advantage in relation to the antibiotic is concerned.


This message is a reply to:
 Message 1 by Taq, posted 02-22-2013 6:38 PM Taq has responded

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Taq
Member
Posts: 7673
Joined: 03-06-2009
Member Rating: 2.6


(1)
Message 13 of 20 (691975)
02-26-2013 6:42 PM
Reply to: Message 12 by bluegenes
02-26-2013 6:33 PM


Re: Am I right?
My hypothesis is that mutation(s) that occured during the growth of your one organism into trillions are the source of the antibiotic resistance. This mutation (assuming only one's involved) would probably have been near neutral on arrival.

That is certainly a viable hypothesis worth testing. We would seem to have three possible hypothese to test at this point.

1. Random mutations where the antibiotic resistance is due to a mutation that appears in the absence of antibiotics.

2. Non-random mutations where the antibiotic resistance is due to a mutation that appears in response to the presence of antibiotics and is guided by the specific needs of the organism.

3. The induction of a antibiotic resistance gene that is able to save the bacteria in rare cases. The resistance is innate, but the chances that it works are low.

Before we move on, we should probably give other people a chance to add to these hypotheses or modify them.


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 Message 12 by bluegenes, posted 02-26-2013 6:33 PM bluegenes has not yet responded

  
Stile
Member
Posts: 3392
From: Ontario, Canada
Joined: 12-02-2004
Member Rating: 2.6


Message 14 of 20 (692052)
02-27-2013 2:17 PM
Reply to: Message 1 by Taq
02-22-2013 6:38 PM


Unscientific Question
Taq writes:

We incubate it overnight and come back the next day to find a handful of colonies growing on the antibiotic plate...

I'm not sure how my thoughts should be structured for your experiment, so I'm just going to explain my question/comment.

Regardless of how these colonies developed their antibiotic resistance... they do have it now in some way.
I understand that some may not have had the resistance, and died, and therefore there are pockets of colonies on the antibiotic plate.

But, why didn't the pockets of colonies with antibiotic resistance continue to spread and take over the whole dish? What is preventing them from spreading over the rest of the antibiotic areas if they are, indeed, resistant to the antibiotic?


This message is a reply to:
 Message 1 by Taq, posted 02-22-2013 6:38 PM Taq has responded

Replies to this message:
 Message 15 by Taq, posted 02-27-2013 7:37 PM Stile has acknowledged this reply

    
Taq
Member
Posts: 7673
Joined: 03-06-2009
Member Rating: 2.6


Message 15 of 20 (692116)
02-27-2013 7:37 PM
Reply to: Message 14 by Stile
02-27-2013 2:17 PM


Re: Unscientific Question
But, why didn't the pockets of colonies with antibiotic resistance continue to spread and take over the whole dish?

The short answer is that E. coli don't have legs. They stay right where they are born (or close to it). After some time they use up all of the food in that immediate area causing replication to slow down. This limits the overall size of the colony. Even on plates without antibiotics you will still get small round colonies that look like this:

As an aside, there are bacteria that use "slime" or flagella to spread over a plate, but E. coli lack this ability so they make nice separate colonies if the progenitors are far enough apart.


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