351: Resistance Reboot

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This is Twim this week in microbiology episode 351 recorded on March 5th, 2026. I'm Vincent

Drakenello and you're listening to the podcast that explores unseen life on Earth. Joining

me today from Michigan, Michelle Swanson. This is Michigan, but I don't know where. Yeah, I'm in

Northern Michigan up near Crystal Lake, like Michigan. Yeah, I could tell that the wood is not your

normal. Yes, yes. A little more rustic. A log cabin. Look also joining us from Charleston,

South Carolina, Michael Schmidt. The measles capital of the United States were almost at a thousand

cases and our great fear is spring break begins for many of our colleges next week and we're

fearful that their parents are going to take them all to church and they're going to get measles

when they come back. There's a lot of anti-vex sentiment there. In the upstate, that's where all

the cases are concentrated and they seem to be associated with group gatherings principally when

they go to church. Have you had any in Charleston? We've had a couple of isolated cases, but

none that have ticked on the map. They've been principally around the colleges. We haven't seen

any in Charleston, but we've had some imports from that enter our hospital. Also joining us from

Tacoma, Washington, Mark Martin. Always glad to be here. Tacoma. How is Tacoma?

I really like it here. I like it better than Seattle. This is not a popular point of view,

but the reason is it isn't as cramped because I was just up in Seattle yesterday trying to find

parking. Cramped come here to New York. I'll give you cramped. Sure. It's all relative, right?

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Okay, this is something unusual. We're going to talk about microbes today. So I will start with a

snippet, which is in science advances. Bacteria Fage loaded micro needle patches were targeted

and minimally disruptive foodborne pathogen decontamination. This is all about food folks. We have two

co-first authors, Kansha Prasad and Shadman Khan and three corresponding authors, Carlos Felipe,

Zainab, Hussein, Nidust and Tohid, Dedar. Where are they from? Well, they're from McMaster,

University, Caltech, University of Guelph. And I think that does it.

Foodborne illness. You will hear this again from Mark Martin.

Major global crisis, 600 million annual cases causing seven and a half percent of all annual

deaths globally. Isn't that incredible? By eating tainted food and something we do every day is

eat. We eat. We cannot not eat. So things like E. Coli, O157, H7 and Salmonella and Tarrika,

you know, you find these they cause they cause food recalls and so forth. And this is because we

have a huge global food supply, right? And your hamburger may be coming from cows all over the

place. Who knows where it's coming from? So this is microbial contamination, of course, and it

results in food recalls and what a particularly heinous culprit is ready to eat products, right?

Ready to eat. So that means it's even warm, right? So you go to the supermarket, you go buy a

chicken sitting there under the hot lamps. And it's actually quite tasty, but this could be a problem.

I've never eaten those. Gosh, I would not do that. So what one thing you could do is you could go

upstream. You could treat the animals with antibacterials, right? But many food products are contaminated

later, right? After the food is out of the animal. And so you need to do something else. And these

include many things have been tried, you know, additives, antibacterial additives, chemical compounds,

essential oils, and so forth. You feed radiation. But these are not ideal because they're not

specific, they're not stable. And they also disrupt the organoleptic properties of the food. Now

I had to look that up. Same here. It's like how it looks and feels and tastes to you. It's all about

mouthfeel. Yeah, it's all of those qualities that go into making a food pleasurable. All right,

so they're going to try and find an alternative. And here they're using bacteria phages,

which have already been used to eliminate bacterial contamination in various food products.

And they have approval for food applications. And they don't affect the products organoleptic

properties. Isn't that interesting? Phages don't taste like anything, apparently.

It's because they're so small. There's not a lot of them in there. But you know, you can put a grain

of pepper in there and taste it. Yeah, but that's much bigger than a phage. Anyway, um, phages have

been incorporated into approaches like edible films, hydrogeal-based packaging composites,

and phage suspensions, aerosolized phage sprays. There's a Listeria spray that contains a Listeria

phage, which is used to decontaminate, cook meat preparation surfaces, and so forth.

However, all these have more issues as well, right? They're not stable. You have to redesign the

packages. A lot of volume of phage needed. But the most, or the biggest problem they say is poor

delivery within the three-dimensional food matrix. Or you have to get inside the pork chop,

not just on the surface, right? Or the three pounds of ground beef that you've just purchased.

That's right. So that's what they're doing with here. And they're looking at micro needles,

which we know we've already explored micro needles as delivery vehicles for vaccines.

They're little square patches with needles on them. They're saturated with vaccine,

and they can be put on your skin and held in place with a bandaid. So no needle needed,

and they're being tested for a lot of vaccines. Here, they're going to make micro needle patches.

They're going to saturate them with phages and put it on food of various types, as you will see.

In the end, it looks good. I just have a few questions, though, but we'll save that for later.

So they say, okay, this is microneedle platform good. So they're going to test it on raw beef and

ready to eat chicken. And they've found that this outperforms flat patches, which don't have

micro needles on it. And so then they first look at four different materials to make these

micro needles, right? And they're all good for food, according to the FDA, right? Generally,

regarded as safe. So grass, grass, polymethyl methacrylate, polyvinyl alcohol,

and polydiamethylsiloxane. And also gelatin, they're going to try. So they are going to try

making micro needles of all these compounds. And so basically the micro needle is going to pierce

the meat. And you're not going to put anything but the phage into the meat, right? And it also

introduces micro plastics to our diet because the majority of those materials are plastic.

Yeah, that's right. Well, we have a lot of micro plastics in us already, Michael.

All right. Yes. You know, I was listening to a guy in Florida complaining that, you know,

forced vaccination is bad and that he says, I don't want any corporate chemicals injected into

me, but what do we have here, right? Corporate chemical. Anyway, they explore the different

materials for making the micro needles. They check their mechanical properties, right?

The gelatin is going to imagine is a little bit floppy and it doesn't go into the meat well.

They look at the effect of temperature. They look at the effect of pH, what they call

penetrative strength. They do agarose discs to see how far the needles can go in. And also the

absorption capacity of the different micro needle candidates. And if you have access to this paper,

you can see nice pictures. The needles are 2.9 millimeters high and at the base, 1.4 millimeters

in diameters. All right. And then they show you putting them on to beef and chicken. And

we're going to also put it on peaches and mushrooms and cheese. Mushrooms. That's right.

Okay. So the PMMA micro needles had the best mechanical properties, but they

looked at all of them for food applications. And they selected 5 solid moisture rich products.

Mushrooms, peaches, fish, ready to eat chicken and cheese for testing. And they did

penetration studies. They also want to know if you could reuse the same patch because that

would be cheaper to do that. They used different forces to push them in, right? To see how well

they did. And they end up saying PMMA is the best. It has high performance in penetration and

reusability. And Vincent, before we get into the other details, I just wanted to give them a

shout out. They did this very systematic comparison as Vincent just described, but they had color

coded each of the four types of micro needles. And they've used those same colors consistently

in all their data figures. And they also provide little schematics of what they're doing,

what the micro needles look like, along with their data. So they're really pulling us into the

laboratory bench next to them. And we can kind of quickly see how these four different micro needles

behave. And are they cost efficient as well?

You know, friends, a million years ago, I worked in a food science lab. And this is very reminiscent.

We were using bacterial polymers for things. And it was very much as you described the people who

knew about the, I'm going to call it the engineering of a food item would come in. And we do

rheology and all these different things. So, you know, food sciences is not simple at all.

Yeah. Food is not simple. All right. Next, they did some more studies with food. And for example,

they wanted to know if you tumble the peaches, which is apparently, you know, part of the processing

thing for peaches. And it has these patches on what's going to happen. And, and again, they

compare all the different polymers. They put them in matrix fluids in the form of fish saline

and chicken purge. I don't know what chicken purge is. Do you guys know what chicken purge?

I guess you do. I don't even know. Let's be some fluid that comes out of the chicken.

Yes, you do. And they, uh, they say, okay, some of these have better penetration than others. And

then the last thing they did was, uh, in cheese and the cheese is wrapped in plastic and vacuum sealed.

And they want to see how these microneedals do when they're vacuum sealed, uh, to the cheese.

They also did a stability study. They put them in the products for 24 hours and then assessed all

of this penetration. And so overall PMMA wins. High performance across all the candidates and

always, they, so they selected this as the material for their bacteria, phage loading platform.

Up to now, no phages have been harmed in this study. Okay. It's just, it's just microneedal patches.

But they, they say that, you know, this microneedal design PMMA can give you strong applicability across

different food products without substantial adjustments. But we could tune it if we have to.

Right? It's a different kinds of foods and so forth.

Okay. So then they put phages on this, on this, uh, microneedals phages that, in fact,

E. coli and salmonella and tarika. So very specific, uh, phages. And they, first, they take

these microneedal patches and they put them on, uh, lawns of bacteria on agar plates and they see

clearing, right? The shows it's still working. They stored them for four weeks at four degrees.

They were still active. And then they contaminate meat with E. coli.

So they put 10 to the five CFUs high and low, 10 to the two CFUs. And then they put the microneedal

patch right on top of where they contaminated it and it lowers over a hundred fold.

And then they did it with, with cooked chicken. They did the same thing. And it also reduces

contamination substantially. Uh, they go from 10 to the six to 10 to the two CFU. And remember,

you need to eat 10 to the eighth, um, to get sick, uh, claustridia in order to get chicken gravy

food poisoning. So the fact that they're reducing at such a significant level will not only

increase the shelf life of the product, but it'll also lessen the likelihood that you'll end up

with food poisoning. Oh, so by the way, there are regulatory thresholds of contamination.

It doesn't have to be zero, right? No. So you can have 10 colony forming units per gram in

chicken, basically. And so they brought it down to that level. So that would be acceptable.

And then they mixed the two phages together and showed that they work on, on material that is

combined contaminated with both bacteria. So it looks like that's it. That's the data. It looks

like it works. The only question I have is and I look through, I could not find the answer to this.

So they take a steak and then they cut a piece of it out like how many grams do they cut out?

Um, uh, 200 gram size pieces. So that's pretty small or 200 gram, right? So what if you put,

what do you have to do? Put multiple patches? You have to cover the whole steak with this. It's

like a punch biopsy for steak because they in these 200 gram pieces, they achieve reduction

because they put the bacteria in it. They put the phage on top in the micro needle and then

they homogenize the whole thing and measure CFUs, right? So the phage is spreading through the meat,

which is very interesting. But what about laterally? You know, steaks can be large. And so do you

have to put a lot of micro needle patches to cover it or they don't actually address that? I don't

know, but it works. And Vincent, can you clarify? They're, um, talk about incorporating this into

the packaging, which would be great because there's shelf stable for four weeks in the cold.

But then when you take them food out to begin to prepare it, does the micro patch come with the

food? Yeah, I think it does. If it's packaged. So you can imagine a steak right in one of those

trays. And usually there's this, um, this, this, this absorbent material underneath. So maybe

that would have this on top of it. So the bottom of the steak would be covered with, but then when

you take out the food, you leave the patch behind. Yeah, you leave the patch behind. Yeah.

Even if it's in the food, uh, the PMMA melts at 130 degrees C. I see. So you, if you don't like

well done steaks, you don't have to worry. But if you want a black and blue steak where you basically

get a rare steak that doesn't heat up all that much, you're going to be eating plastic.

Because it melts out. But if you look at it and probably sets on. If you look at figure four F,

and I know not everyone has the paper, but they have this big steak. And then the tiny patches

in one part of the steak, and I can't imagine the phage diffuse entirely throughout the whole steak. Do

you? I just don't see that. Well, then micro needles will be gone by then because if the

phage is going to diffuse, it has to detest from the micro needle. Yeah. Well, I don't know. They

don't talk about it. They didn't ask me the distal part of the steak. Okay.

That's what I want to know. Yeah. But maybe, yeah, it is a principle that it works and you can

lower the contamination. But let's see how it's executed in practice. So help me think about

how I should feel about ingesting these phage. And what will they do to my the E. coli and my gut,

the good, the good E. coli? Hmm. Are the phage sufficiently specific that they'll just get the

O157, but not all my good microbes. So that they implied, yeah. Most of the most of the phage in

your gut already misogynized. And so you're not going to get a true phage infection. And last,

the phage can actually co-infect with the pro phage. And that's hard to do. Right. And so that'll

then activate the phages. Well, but you're not going to cook the peaches. You won't necessarily

cook the cheese. Yeah. Michelle, I really do believe that if depending on which ones that they

use for this, it's fine because people used to use phages before immunology made monoclonals,

they would use them to identify different strains. And absolutely, there are phages that are

specific for OH17 and also ones for K12, MG1655, and so on. So I guess it's we're back to the,

you need to measure twice, cut once school to be very, very careful about that. About which phage

you use. Yeah. Yeah. And I mean, we have issues right now in society with how we're not being as

careful as we might be in a number of ways. And this is a good example where you'd have to be

really cautious. But it's probably better than dosing everything with antibiotics. Well,

yeah, that's sure. Right. And I'd rather have some phage running around than, you know, 0157 or

salmonella. So the author works at campbell backter. Yeah. Remember campbell backter

infects more people than salmonella and shagella combined. And causes a bloody diarrhea,

painful anti diarrhea. Yeah. The authors conclude it's tunability, food safe nature,

cost effectiveness and translatability into commercial food production application,

make it well suited for applications within the food industry. So we'll see if this makes it.

Someday you might buy a piece of food and you see this spiky mat. And that's it phages.

You know, Vincent, I had a friend of Todd at Oberlin College and there used to be, I don't think

people do it anymore, a really common lab. I did it as an undergrad when dinosaurs ruled the

earth where you bring in a sample of hamburger, you vortex it up and do serial delusions.

And of course, you've got tons and tons of coliforms on maconkey plates or EMB plates.

But my friend at Oberlin went to one of these and we use the expression here, booji,

but we're just going to go ahead and say boutique. These boutique butcheries where they, I guess,

they massage the cows and they treat them very carefully. And they didn't get any contamination

in that hamburger. And that is the way that for good or for ill, I'm not doing uptints and

clear with you the way that we process food. You know, no, if you spill the gut, you know,

everything's everywhere. That's a good point. And you know, the the halal preparation of meat

is much gentler and I've had it in a taste way better than, yeah, regular meat because you don't

get the stress response in the animals, right? And all the chemokines and cytokines being made,

they gently killed them. And so maybe there's something to that. That's a great point.

But you know, the high volume, high profit industry is never going to change its way sadly.

And that differentially affects people on the lower economic strata. That's the issue too that,

you know, students are always saying there needs to be more social impact in your classes. Well,

this is a good example. You know, this reminds me of RFK's recent proclamation. He said,

you know, people need to eat more meat. And someone said, well, you know, meat is expensive,

not everyone can afford it. So what did you say? Buy the cheap cuts. Buy liver.

Well, again, I really, really appreciate it how systematic they were. And it's an ambitious

project. I like it. Yeah. Okay. Now we will take a short drive with Mark Martin.

Well, I really short. It's sort of phagey, isn't it? CRISPR came from faith. Well, yeah. And

there is some. And this paper is, is to me, it was old home week because I used to do a lot of

work in graduate school with broad host range plasmids and conjugal natures of them and all the

rest. And this reminds me of the old joke about Joshua Letterberg's papers and bacterial genetics

that they had. And I quote, a rabbinical complexity because there are aspects of that in this particular

paper. And if you work with it all the time, it's nice and clear. But I want folks to understand

the power of what they're talking about. This paper is from MPG antimicrobials and resistance

published on February 2nd of this year. And the title is a conjugal gene drive-like system

efficiently suppresses antibiotic resistance in a bacterial population. And I was instantly nervous.

And, and this has come Ethan Beers group. And it's a caruole, steward,

already a car, washabah, a mire, and of course Ethan Beer. And I really recommend folks look at

the website as a video about the use of gene drive that might be worth your time. And there is

distribution. I'm sorry. You're absolutely right. You know, I always mess up. It's you, you make me

nervous, Michelle. That's what it is. You have to blame yourself. Okay, it's my fault.

University of California at San Diego, a place I used to live in in in La Jolla and is is just

lovely, although very expensive. So in any event with this kind of paper, it's important to

remember there are lots of acronyms and ideas. And I just want to hit them really quickly.

I'm going to call this the dramatist persona of this story. And AR in their context they use

for antibiotic resistance. And we've all seen it as AMR pro AG stands for pro-cariotic

active genetics. You'll see what I mean. CRISPR, of course, our good friend CRISPR is clustered

regularly. Interspersed short palindromic repeats. And I'm delighted by HBD, which is not happy

birthday, but homology based deletion. And they also spent some time talking about tooled friends

of mine, which would be recay, the general recombinase in bacteria, and it does a lot of repair work,

and the lambda red system, which is used a lot in recombineering. And all of these things are

pretty important. So the first thing that the authors do is they talk about the threat of

increasingly resistant disease causing bacteria. They quote 1.27 million deaths per year now,

and predicted to reach 10 million deaths per year by 2050. And this is because of the rise again

of antibiotic resistant bacteria. Two problems. First is the evolution of the matter, how bacteria

become resistant to antibiotics by one of four different mechanisms. And of course, horizontal

gene transfer. It's one thing for a bacterium to change the shape of its RNA polymerase to avoid

rifimicin. It's another thing entirely for them to share that information with their bacteria.

So this is an increasing problem that's been talked about on to him many times. And it's important

that we remember it is a crisis. And could I add some of our farming practices have contributed to this.

Policies are now raining this in, but at 1.70% of the antibiotics consumed globally were by

farm animals. Yes. Not just to treat them, although that is one reason, but also at low doses,

it promotes more rapid weight gain, and therefore cheaper meat. Yes. So, and then the one health

concept recalled that if we're using antibiotics in meat, and then you've got of course manure,

and it can get into the soil, it can get into the water, it can easily get them to humans. So

our farming practices absolutely impact our health.

I couldn't agree more, and we all see it all the time. It's one thing to have a little garden

patch when you're growing up as my father insisted that I have, but doing it at scale, which is what

we do is the real problem. And again, when I tell my students, more to your point, Michelle, that

the primary use of antibiotics is actually agricultural. They're shocked.

Including fruit, fruit trees, right? That kind of thing. So we do need new strategies to deal

with this, and there are lots of different ideas for it. And the first thing I want to introduce,

and I want to just get this out of the way, is the concept of gene drive, which I'm used to in

eukaryotic genetics. Hey Vincent, do you know what gene drive is? I do. I do. It's a gene that will

drive itself through a population, because there's some selection for it, and you can have it carry

something else along and do what you want to be done. I couldn't agree more. That's really

well put. Thank you. And that's kind of the issue that we work with. A lot of people are nervous

about it, and I'm sure I'm going to come to that with some of you. It's very different. It's very

different from a hard drive. Very different. But you know, we microbiologists have been using gene

drive ever since we made selective media. I mean, that's effectively what drives a lot of the

fundamental bacteriology. And then the molecular biologists took it one step further by adding

antibiotic resistance traits to keep their plasmids inside the host. So selecting bacteria with an

antibiotic that's present on a plasmid is a form of gene drive. Yeah. Or think about the issue with

Wolbachia, where it affects the insects that it inhabits so that only their progeny carry Wolbachia.

Right. And a lot of folks have debated this, and you know, folks who have to deal with dengue,

they don't have much of a problem with it, but it's an issue. And again, this is something

Ethan Beer works with a lot. And I'll put a link in the show notes to a video he did.

What is the German group Kürzegatz? I'm saying that incorrectly, but they do wonderful animated

videos. Kyrgyzats. Thank you. They do wonderful animated videos. So it's certainly possible

to do lots of things that are completely inexpensive to try and blunt the drive of antimicrobial

resistance. And this is something that Michelle had just mentioned too. But it is possible nowadays

to do gene editing. In fact, it's often taught in many undergraduate laboratories. I used to

use Lambda Red to make specific changes. You have to basically, you're using a phage-based,

originally a phage-based recombination system to replace a wild-type copy of a gene with a mutant

version that you constructed. And then CRISPR, as most people have read, this is something that can

be used in many ways to alter what genes are. But the problem is, recombinering is fine. CRISPR

is fine. But the authors make this very clear, but it needs to be emphasized because this is

stuff I used to work on in experiments, including ones that I did in graduate school. Plasmids

can transfer themselves to 50% or more of a recipient population. And that's in the absence of

selection. Just as there is a problem with the spreading antibiotic resistance genes,

can this same principle use to fight the spread of antibiotic resistance? And I mean, that's

something that makes, I don't know if you're, if you're all feeling a little nervous right now.

Are you being haunted by Jeff Goldblum in Jurassic Park saying life finds a way, right?

Life finds a way, yeah. So the plan that they have here was, could they use a CRISPR-like system,

originally, yes, from streptococcus pyogenes and synthetic guide DNA to kind of learn how to

remove or otherwise destroy antibiotic resistance in a particular population? And they want it to

kind of amplify itself to move through the population. So even high copy number plasmids with

antibiotic resistance genes can be precisely inactivated. And I was unaware that a lot of folks that

worked on this would use CRISPR to basically break genes rather than replace things.

So imagine if you will, you're a patient in a hospital bed that has an antibiotic resistant infection.

You then take this gene drive system out of the pharmacy and you administer to the patient and

you literally transform the infection from a drug resistant creature for which we have no drugs

to now an infection that is susceptible to the drug and effectively give the patient an opportunity

to recover. As long as you... That's the practical application. Just take Michelle's

tack from earlier as long as you don't destroy your own microbiome, right? Absolutely.

See, I'm spaszing out with jazz hands because I have seen experiments not work the way I wanted

them to over and over again in my career. And I am fortunate in that nothing I have ever done

has been involved with human health. So this is good. So this idea of...

Very, very happy. We're very happy that you should be.

Because I have great ideas that probably aren't a good idea if we try them. But I think they're

interesting. So in any event, what they decided to do, and I shouldn't say they, what the authors who

did a lot of work on this, we like it to this, they wanted to put this kind of system onto a broad

host-range plasma. Now, this is something I want to make very clear because I spent a lot of time

with this. The bacteria that I worked with in graduate school and then later were non-ecoly,

undemesticated type bacteria, and you had to find the proper systems to study them. And standard

plasmids that we use all of the time in the lab don't work in them. And the larger plasmids that

are broad-host-range, well, they're a lot as you'll discover. And they did a wonderful job in

this paper kind of modifying it. So the idea would be because these broad-host-range plasmids can

be transferred, and we used the word, used to use the word promiscuously, but we probably shouldn't.

Let's just say that they can go to a wide variety of bacteria. You're on-host-range.

So there's a difference between broad-host-range and transferability, Michael, because there are

ones that are both, again, I'm sorry about the use of this word, promiscuously in terms of the

bacteria that they can replicate in and to which they can transfer to, which is a very interesting

idea, because they're different from one another. And so what they want to do is see if they could put

this kind of system onto a broad-host-range, broad transmissibility plasmid, and then that would

spread. And this would be an interesting idea. I want to emphasize, again, under many conditions,

conjugation of these large plasmids can be easily half of the population. And remember,

that was done in the absence of any kind of selection. Now, this is a big problem when it comes

to antibiotic resistance because the same plasmids can pick up drug resistance genes.

Some of these broad-host-range plasmids can carry 7, 8, 9 antibiotic resistance genes,

so a bacterium that receives it, bam, is resistant to all of those antibiotics. But this is a

little bit different. They want to do the reverse. And so what they wanted to do is put this system

that they'd worked before onto that kind of plasmid, and they wanted to target it against

ampacillin resistance, the beta-lactamase gene, as a model system. This is because, and I love

the fact that the gene name is blah, makes me happy. But blah is so well-known and so well-studied,

you had all kinds of information that you need it. And to make a long story short, and we'll get

to the details, they were able to show that using this approach, they could wipe out antibiotic

resistance in a recipient population by certainly five orders of magnitude. And because of this

transmissibility and the amplifying nature of the process, they were a bit worried, so they

actually did a reverse experiment, and I'll get to these figures in a moment where they could show

they could recover the things that they've taken out. Now, why they would do this? And I like the

commentary here, and I would have written something similar to it. You have to worry when you turn

this kind of thing loose. So you want to be able to show that you have control of the process,

and that if something happens that you don't want, you can fix it. Again, I'm glad I don't work in

this field. So if you go to the figure one, you can see how they created the plasmid, and they go

through at least six paragraphs of the iterative nature of doing so. And having tried to build these

plasmids, I'm here to tell you, this is years of work. I can smell it. Because these are low copy

plasmids, generally speaking, and they're hard to work with, and it just makes you frustrated.

So they call it a pro-ag plasmid, and that just stands as I said at the beginning for pro-cariotic

active genetics. That's a funny thing. What they mean by it is it's just going to be a donor of

this editing function. And so they also have a target plasmid. And the target plasmid basically

has constitutive production of the signal guide RNA that CRISPR needs to alter things. But to get

back to the pro-ag plasmid, it has broad host range, broad transmissibility. And they found out the

hard way that you have to have both the lambda-red and cast-9 genes in a single transcriptional unit

driven by an Arabinose promoter. Now this is good because that's a strong promoter, and it turns

on very strongly and off very strongly. So a lot of folks use it. And so in figure one, you can see

what they put it together. Again, the big plasmid, the pro-ag plasmid, is going to be the donor.

The recipient has the ampicillin resistance, and the guide RNA being constitutively made.

Now what they do, and you have to look carefully at figures C. What they're showing is that when

they use a particular drug called Gentamysin, that lets you know how many bacteria, whether or not

their amp resistance are there. An amp resistance shows you how many of them have that intact

ampicillin resistance gene. And if you look at that carefully, and I think they did about as good

as can be done in making this clear, but it's important to sit with that figure a while,

you're able to knock ampicillin resistance down. In solid dots, in this case, the ampicillin

resistant results, open dots are the Gentamysin resistant results, and you activate with a

rabinose. So you look with and without a rabinose, solid or open dots. And what they have done at the

bottom of C is they use three different types of donor plasmids because they are different in terms

of their transmissibility and their copy number. They were trying to find the right thing to use.

But regardless of that, if you look at the statistical charts, you'll notice it appears to be

working pretty well. Does everyone see that? Now, I look at this and I'm thinking and I went

right to materials and methods and read about it because I do not things like this, but I certainly

count a lot of colonies on plates. So this is good. So in figure two, they made the next generation

of it. Now, the next generation donor plasmid, what they call their Pro-AG plasmid, has a

constitutively expressed guide RNA, has the lambda red and cast nine before, and the target is ampicillin

resistance. And because they have to shuffle a number of different drug resistance markers to show

who's who, and it turns into kind of alphabet soup and I apologize for this, but it's really true.

When you work with these plasmids in particular, often you don't have every possible drug resistance

gene you'd like to work on. So in figure two, streptomycin resistance shows the plasmid

without ampicillin resistance can survive because you'll notice streptomycin resistance is on the

target in that case. And you check out the conjugation, which is the second part of this figure.

And I actually had used both of these plasmids a lot in grad school. And I'll sit here and swear

at PBBR1 because it was my bane for about a year and a half. The RK2 plasmids were more forgiving.

So when you get to part C, you can see exactly what you need to see, that it's using both the crisper

approach, which is where you're trying to put something in the middle of ampicillin resistance.

And what they're going to call the happy birthday approach, the homologous deletion approach,

where you actually kind of get rid of it. And they had some unintentional effects with this in

figure two, where the target plasmid went after some promoters, the HBD and the target plasmid

went after some promoters not involved with ampicillin resistance, but removed it anyway. Once again,

unintended events. So how do you feel about figure two?

I want you to explain what this conjugation machinery is.

Yeah. So I used to say that in graduate school. And so it makes me feel good that other people

use this expression. There are a series of different gene products that are carried by these broad

host range plasmids. And one of them is for something like a pillus. It doesn't have to be a

pillus, by the way, because when you think pillus, you think it's a hollow tube and DNA travels

down it. No, no, no, anchoring point. And you basically deassemble the pillus. It brings them close.

And that's what the ORET region is about. I like to tell people that ORET, the origin of transfer,

is where you break the circle of a plasmid and then it's able to enter the recipient cell.

What's important to note here is that again, it's really, really, really high efficiency naturally.

And most of the time when we look at it, we just look at plasmids moving. But Vincent,

if any of these plasmids can even transiently associate with a chromosome, they can move chromosomes

around. And that's called an HFR. It is indeed. Okay. Okay, so this allows the bacteria to move

the plasmids to other bacteria independent of cell division. Yes, absolutely, absolutely.

Sparkling drive through the population. Yeah. So this is the driver you're saying, this

conjugation machinery, this is what's driving it through the population. Because it's moving into

other cells as it's going. Right. So there's no selection in this case for drive. Not at this point,

just like when I used to do the experiments with conjugation, I would mix the donor and recipient

and then I would resuspend and I would play it on selective media. The transfer took place before

I ever did that. Okay, got it. And it's fascinating. And you would dope the multiplicity of infection

so that you would have more donor than recipient so that you would ensure the efficiency. And

for our listeners, you need to appreciate that this is extremely efficient in the sense that

you can move about 100,000 base pairs in under two minutes.

There are a lot of things in the bacterial chromosomes or we don't like a lot of the what they're

called as genomic islands are better to call them. They used to be called pathogenesis islands,

which sounds like a game show. But when you think about it, some of these are like quite large.

And we don't know exactly how they moved. But I suspect something like you just mentioned,

Michael, is responsible for that. Yeah. And Ilio had a phrase he referred to them as fitness islands.

Yeah, that's right. Because that was what was driving the the stability of those accessory

genes, if you will, out of the core genome.

Per the recipient became more fit. Yes.

Work it out, replicate, populate the gene drive.

And I basically believe that almost everything is mosaic of DNA. It's received from other organisms.

But that's an extreme position that I don't ask you to take.

So I want to talk a little bit with you about figure four, because I want to watch time carefully.

And this is what it is. As we talk about the gene drive problem,

as we talk about some of the unintentional effects that happened. Now this is just laboratory work,

but it's important to study it. What if you made a mistake? Can you fix it?

Some unintended consequence. Yeah.

Better. Yeah. As God, who was the fellow who used to paint all the time? Bob Ross,

you can call those happy little genomic accidents, right? But with that in mind, could they actually

repair a change that they made? And I thought this was really quite clever. What they did is they

took the ampicillin resistance gene and they put GFP in the middle. And then they were going to see

if they could remove things with CRISPR or HPD, right? And GFP is green fluorescent protein.

So it's a visual mark for them to follow the impact. You have an easy phenotype.

And it's funny, because I would have shown photos of it because it's delightful.

They also say that carrying GFP on multi-copy plasmids is deleterious. And I haven't done the fitness

studies, but I do know that if you overexpress GFP, the cells are not happy. I know that.

So to get back to what figure four did, and again, they do a lot of very intricate studies to show

this, but what they show in figure four, and you can see this. So they have something that's

ampicillin sensitive and GFP plus. And then they have something they call a mitigation plasmid,

which is working exactly like we talked about, only the goal of it due to the signal DNA is to see if

they can excise the GFP precisely and restore ampicillin resistance, and they can't.

And then they went further than that to put some of those components into bacteria

phage and see if they could do the same thing that way.

Bacteria phage. Bacteria phage here replacing the conjugal machinery.

Right. And that's something, and you know, even, yeah, I actually will be honest with you.

I wasn't clear that because they, I think that they, when they said the replace, they weren't using

a replica on inside the phage. I know they totally replaced the phage DNA, which you certainly

can do. And then create these. I'm just going to call them taxi cabs of information because they are.

But it's a generalized transducing partner. Right. Yeah.

As opposed to a specialized transducing particle where you have remnants of the ORET

going along with it. Exactly. So so the idea here is, is I believe they didn't say it as abruptly

as I am, is that they didn't want gene drive to be part of that. They just wanted to see if they

could fix a population by using a large number of bacteria phages. Check me on that, Michelle. How did I do?

Yeah. She's not in her head, folks.

We don't we don't have videos. So right. So this is actually really good. And what's important

about this is to talk about it a little bit. Is it perfect? No. Is it remarkable? Yes.

Is it teaching all of us about how we can engineer bacteria using natural systems in that bacteria

and reverse that approach? Yes. They actually suggest the authors that it could be used to

excise fitness islands and an honor of alio as well as inactivate antibiotic resistance genes.

And further, they suggest perhaps in recurrent long-term infections, this could be used in patients.

An example would be Vibrio. If they have the phage that encodes Vibrio, the collar at

oxygen, then you get the fused watery diarrhea. It spreads like very quickly through a population.

So if you could just go in and knock out that cholera toxin phage gene.

Yeah. Yeah. And you know, obviously when you talk about this kind of experiment,

people go to the sci-fi channel in the way that they think about it. What could go wrong?

But I would argue that every patient is an incubator and evolution is occurring inside every patient

as well. And just as the folks who want to use Wolbachia to reduce mosquito's ability to

transmit dengue, and some folks are worried about the people who face dengue all the time have

very little issue with that. I'm not saying yay or nay, I am saying it's something that we need

to have a discussion about, I think. Well, the Wolbachia experiments are not transgenic. And so

people accept those. Yeah. There are transgenic gene drive experiments that have been

designed for mosquitoes, but they have not yet released those because people don't like transgenics.

That's the problem. So there's a, and this is basically a transgenic, I would say.

Yeah. I would, I would say that when I did conjugations, Vincent, I found and a lot of other

other people, I wouldn't be the first to have done this. Most people did conjugations in liquid

as they described it. And in fact, we did them on filters or on dry plates, and we got much

better transfer. Honestly, 75% in my hands, at least with some of the RP4 RK2-style plasmids.

And so when that comes to its use clinically, which I know is a million miles away, but there are

animal models and insect models that could be used to study this, which I hope they do,

I do know that you get a lot of surface attachment of certain types of pathogenic organisms.

And I wonder how conjugation works under that kind of modality.

We know it works really well, hence the antibiotic resistance problem we're having.

But I would not start with the patient, but take a couple steps back and let's go to the

root of the problem, as we talked in Vincent's paper, industrial farming, spread of antibiotic

resistance. They have these lagoons that are going to be filled with microbes that are likely

antibiotic resistant. So you could imagine industrial farms building into the waste management

practices. They already apply a gene drive like this to clear out the antibiotic resistance.

Yeah, they say exactly that in a discussion, right? The use of antibiotics and animal husband

and fish farms. Also, it's another one, especially fish farms. You can put these in to clear out

the resistance genes periodically, because they're going to return, right?

Well, it reminds me when people would say, well, let's use phage to clean up, and you're talking

about the pig, I don't know when expression is for the pig lagoons or fish farms, which again,

are filthy. And you'll get things that are resistant to those phages very quickly, but then you

get another phage. Yeah. So where could go wrong? Well, there's there may be minority bacteria

where there's a polymorphism in the blog gene. And it is not cut out by CRISPR by Cas9, right?

And those bacteria will now have a fitness advantage and they will overgrow and they're still

resistant. So I would like to see you have to reengineer. Yeah, I would like to see experiments to

look at that frequency, right? And that kind of parallels the problem we have like with the

COVID vaccines. We need to keep adjusting the antigen because the virus is evolving. But

of course, the RNA virus has changed a lot more frequently than DNA containing bacteria,

but they still change, right? Right, right, right. And so I don't know, maybe this is not going to

work at all because of that. I just don't know. I think it's a great experiment to do, and I would

be anxious to because it's the kind of thing that is up my alley. This is the kind of work that I

know how to do because when you are looking for a drug-resistant organism in a population of

sensitive ones, that works really well. Yeah. You can detect things that are very rare that way.

I don't know how many changes, base changes you need in the guide RNA sequence to make it not work.

That's a good question. Someone has to have done that, don't you think? I'm going to look it up right

now. Well, Mark, I really appreciate it. You're selecting this paper because as I was reading through

the methods, I was just having flashbacks to learning all the basic concepts of bacterial genetics,

homologous recombination, transformation, double strand, big break repair by wrecking,

antibiotic resistance, sensitivity, the origin of replication of plasmids, whether they're high

copy or post-range restrictive, and then more modern techniques like CRISPR R and lambda red

recombineering and then applying it to this major public health challenge. No, I thought it was

a very interesting paper and I've followed Dr. Beers lab work for quite a while, so I was delighted

to see that. He has a prior paper where they worked out some of the aspects of this, but the idea

of going after a population is fascinating to me. They do to their credit, they're very rigorous,

they look at a lot of different details and variables, but they have clear color coded schematics

with each figure. They try to walk us through it, but it is, it's only four figures, but it's 21

different panels and many of them have multiple graphs, so the concepts are really elegant.

It will take you some time to go through it. So one or two base changes are enough

to prevent short-guide RNA binding. It's got to happen then. Of course, it's going to happen.

Yeah, it's going to happen a lot. So in the population, especially with antibiotic resistance,

I mean, these can be non-coding changes, obviously, right? They can be through third basin,

so readily happening. Okay. So and like the case in figure two, where they had that very unusual

thing where they had homologous deletion between two promoter regions for the antibiotic resistance

genes that have nothing to do with ampacillin resistance was interesting, too. Yeah.

The other issue is off-target effects, right? We really look at that because they're only

asking in a microbial resistance, but they're going to be off-target effects. But again,

it's proof of principle. And now this is our first question. We're just pointing out all the

problems that we can see. Yep. That's why they give us the big bucks, right? We show.

Well, into their credit, they discuss a number of things that the next iteration could incorporate

and to fine tune this even further. You certainly can get these types of plasmids because I've done

it into pseudomonas. And so it would be pretty trivial to show this in say an animal model,

either murine, or even like things like nematodes, just to show what could happen if the gene

drive took place under those conditions. And I'm quite sure they're thinking about that.

So Michael, they use a lot of antibiotics in fish farms? Oh, yeah, really. That I didn't know.

And you know, that's the whole issue with farm raised salmon versus wild caught salmon.

In addition, you're effectively eating a cow when you eat farm raised salmon because they're

growing on corn just like cows. It's tricky, though, because we've got to balance

delivering food at a low price. It's all about farm to table. And you know, the poor egg

industry is struggling to put food on our tables as our planet earth continues to grow.

Shrimp farming is every bit as problematic. I hate that word, but it applies. Again,

they just dump antibiotics in those. And I like me some shrimp. So that's a big deal to me.

And when I was working with the copper folks in Chile, they do open water seabass farm raising.

And we were looking at antimicrobial copper nets because the dolphins would scare the seabass

and they would hit the net. And if it was made out of traditional chicken wire, the galvanized

wire would corrode and they could break the net and the fish would get out. And they were

similarly feeding the fish in order to reduce the farm to table time, even in open water or fishing.

But there you had the solution to pollution as the ocean was taking care of the waste rather

than a lagoon or something. But they then, the moral of the story is because they were so crowded

in these cages, they developed a viral infection. And they got the Salmon Anemia virus.

And that started in Norway with the salmon farming and then it somehow got to Chile

in South America with the seabass industry. Crouting. It's crowding.

Well, there you have it. Two very early days approaches to different problems. We'll see

where they go. I mean, they're both fascinating and experimental. We'll see.

An ambitious. An ambitious is good. Well, at the same time, teaching you some basic microbiology

of how stuff works. Who knew? All right. That's Twim 351. The show notes are at micro.tv slash

Twim. You can send us comments or questions to Twim at micro.tv. If you enjoy these programs,

we'd love your support micro.tv slash contribute. Our guests today,

the Twim adjacent Mark Martin from the University of Puget Sound in Tacoma, Washington. Thank you,

Mark. You're very welcome. Michelle Swanson's at the University of Michigan. Thank you, Michelle.

Thank you. And we're at basketball school now, by the way. Go blue.

Oh, you no longer a football school. Not a not-sense Indian football. Our basketball teams

are fantastic. Both the men and women. Indiana, huh? Wow. Michael Schmitz at the Medical

University of South Carolina. Thank you, Michael. Thanks, everyone. I'm Vincent Racken Yellow.

You can find me at micro.tv. I'd like to thank the American Society for microbiology for their

support of Twim and Ronald Jankies for the music. This episode of Twim was edited by Ray Ortega.

Thanks for listening, everyone. We'll see you next time on this week in microbiology.