264: Antimicrobial Antipsychotics

This Week in Microbiology is brought to you by the American Society for Microbiology at asm.org slash TWIM. you This is TWIM, This Week in Microbiology, episode number 264, recorded on April 28, 2022. I'm Vincent Racaniello, and you're listening to the podcast that explores unseen life on Earth. Joining me today from Charleston, South Carolina, Michael Schmidt. Hello, everyone. Do you have beautiful weather there? It's Chamber of Commerce weather. 75, low humidity. Sun is out. Students are...

playing on the beach. They're playing volleyball outside my office window. It's really quite remarkable. We have a revisiting of winter here. It's 12 degrees Celsius, not Fahrenheit, Celsius. Also joining us from Ann Arbor, Michelle Swanson. Hello, where it's a beautiful 50 degrees Fahrenheit and sunny. Huh. But I heard you got snow recently, right? About a week ago. It only lasted a few hours. Yeah. And from St. Louis, Missouri, Petra Levin.

Hello. It's 70 degrees and overcast, 21 centigrade. Pretty warm. It's Missouri in the spring. It can be 70 or it can be 35. You never know. I hear you. All right.

Well, whether the weather is good or not, we have microbiology for you. And we will start with a snippet from Petra. Okay. So today... The snippet is on a paper that was just published in the Journal of Bacteriology. The title is The Atypical Antipsychotic... Quetiapine Promotes Multiple Antibiotic Resistance in Escherichia Coli. The authors are Yasuhiro Kiono, Lori Elizian, Yu Yu Hu, Canella Aleitis,

June Lone-Moy, Elizabeth Hirsch, Michael Federle, and Stephanie Flowers. And they're from the Department of Pharmacy Practice in the College of Pharmacy at the University of Illinois, Chicago. So this paper is actually, I think, quite interesting and probably relevant to... many of our listeners. It talks about this antipsychotic, which is marketed as Syroquel. I feel like I've seen advertisements on television even for it. It's prescribed to cheat schizophrenia in children and adults over the

age of 13 and bipolar disorder in children and adults over the age of 10. But it's also being used to treat major depressive disorder in combination with other antidepressants. It's typically prescribed long-term. It takes about four to six weeks to even see full effect, and it can be taken for multiple years. It's being increasingly used off-label to treat ADHD and disruptive behaviors. And the estimate is about 1.7% of the U.S. population is currently being treated with...

this particular drug or other atypical antipsychotics. Those are called, again, AAPs.

Why there's an article on an antipsychotic drug in the Journal of Bacteriology is because... These drugs and actually their predecessors, which target dopamine and serotonin receptors, have been associated with antimicrobial activity. And because these drugs are so common... prescribed, they are appearing more and more in wastewater, hospital sewage, and even drinking water. So again, these drugs have...

been observed to have antimicrobial activity, but the mechanism of action is unknown. So they inhibit microbial growth, but nobody really understands why. So in this paper...

which I really like, it's very accessible, Kiono and colleagues set out to systematically characterize the impact of quetiapine on antimicrobial resistance in E. coli. And the experiment is really simple. They took E. coli and they grew it in lysogyny broth, LB, which is just a standard medium, LB plus 10 mg per liter quetiapine, which they call the low dose, or 100.

mg per liter, the high dose of quetiapine, for 43 days. So every day they took cells that had grown in a test tube filled with one of these three. conditions. They put it into a new test tube. They let it grow overnight. And they did that again for 43 days. Why 43 days, you ask? It's because six weeks is the typical trial period for these.

class of drugs, these atypical antipsychotics, these AAPs. And the dosage was based on colonic concentrations found in patients. So basically the dose, low and high, sort of is the range of concentrations found in patients who are... prescribe quetiapine. So this six weeks is really the minimum period somebody would be exposed, far from the maximum. Exactly, far from the maximum. The drugs actually have other side effects, body aches, some other things.

are probably independent of this. And we can actually talk one of the, of these long-term of these drugs. My understanding is also weight gain and we can get to that at the end. But yeah, six weeks is the... kind of typical trial period for these guys. So they want to see what's the minimum amount of, I guess they're saying if you're only prescriptive for six weeks, what's the kind of what can happen? I guess they could do another study later on where they look longer.

Even though they only did this for 43 days, they did see four to eight-fold increases in resistance to ampicillin and tetracycline at both the low and high dose. They didn't see really differences in those two dosings. resistance was stable so if they took them right out of you know after on day 43 and they tested resistance they got to 48 four to eight fold increase in resistance if they took them out of the

after 43 days and they put them into the control, the LB, and let them passage them for three additional days, they found that that resistance was stable. And that's important because it means that...

this resistance that the bacteria are getting is probably due to a stable mutation. They then essentially sequence the genomes of the different isolates. The LB alone, the low dose and high dose of quetiapine. The first thing they found, which is not that surprising, but always gives me pause when thinking about doing this kind of experiment. Even the untreated cells had, I think, 291 mutations. So there are mutations just when you passage bacteria, you get...

different mutations. Many of them have no significant effect, but they're there. So when you do sequencing, you find all these things and how do you sort them out? And I think many bacteriologists have learned that the hard way. You can't keep massaging your strains and expect it to behave the same. Exactly. Exactly. And even like if I compare the...

the lab strains my lab uses versus the lab we got them from, I'm sure that somehow we picked up some mutations along the way. Yeah. And you don't need whole genome sequencing to appreciate it, right? It's better not to do it, actually. When I was a tiny little graduate student, we were taught always, you know, go back to the master stock periodically and get a slug of frozen culture out and restart.

your stock cultures don't continue the same thing just by this serial passage. I mean, this was a well-established practice amongst the old bacteriologists. They, they knew bacteria changed their spot. Oh, yeah. No, the old bacteriologists definitely knew that. And they also knew that. because they would put them in stabs. So they're sort of in a almost stationary phase, not growing, but they're still picking up mutations. And in frozen, we always go back to frozen.

It's not because they're fickle. It's that some of the appendages and things that they need to make for their normal environment are expensive. And so now if you're just growing them on, you know, giving them everything they need, any... any random mutation that comes in will have a growth advantage because it's not spending the energy and the goods to make those appendages or special factors. Exactly.

And since Elio's not here, we can point out he had a colleague who pointed out that LB or lysogyny broth or luria broth, whatever you want to call it, is really pretty lousy. bacteriological media. It's terrible. It's principally starving the poor little suckers. Yeah, it's nitrogen limited.

Yeah. And there's some really beautiful work. I think it's from Dick Dari's lab about, and the cells actually go through, sorry, we're a little off track. They go through dioxin growth because they use up whatever thing they can use best. We talked about those a couple of weeks ago. They use up the carbon and nitrogen sources they could use best, and then they have to switch, and then they have to switch again. And the ideal is if they grow in your culture and they don't switch.

But in LB, because it is terrible, they have to keep switching and using, especially with regard to nitrogen, nitrogen sources that are really not ideal. And yeah, exactly. What would be a better medium to grow in then? Tryptocase soy broth is much, much more predictable for the microbe, and it also cheats in that tryptocase soy broth has a slug of glucose in it. Yeah.

And then they burn the glucose rather than just burning straight up protein to get their energy. We actually use, when we care about these things, we use a defined rich medium. So essentially a defined medium. to which we add nucleotides and amino acids. So essentially they have everything they need all the time and we know exactly what's in it. We could have a whole swim about media. We just...

grow E. coli to grow plasmids. And we always use LB because I was taught to use LB. But I wonder if we should switch. Because, you know, some plasmids don't grow as well. Would it make a difference, you think? Oh, yeah. Oh, yeah. I've done those experiments. You can actually goose more yield. It's all about energy because plasmids cost money. Yeah. But it's curious that that hasn't...

worked its way into the standard lab practice. Yeah. Yeah, no, it wouldn't be that hard to grow things in 2XYT or some of these other. Yeah. The minimal, the rich to find is expensive and odd. very stable. So I wouldn't wreck that for plasmids, but okay. Our digression. A sidebar. So.

In any case, they sequence and they find lots of mutations. And whenever you find that, the big problem is, how do I sort through which of the mutations actually is important for this drug resistance? And there were differences even between, you know, the replicate cultures. Their decision, which I think was a good one, was to choose only those mutations that have been implicated in antimicrobial resistance before.

And they found 14 mutations. Many of them are in a region that alters transcription of antimicrobial resistance-associated genes. And the most interesting one, I think... to me, but also to them, was they had mutations in MARR regulon. So MARR is a transcriptional regulator. It inhibits transcription of genes involved in antimicrobial resistance, including one named MarA. And MarA, the A might give it away, activates transcription of a complex of proteins that's together known as the ACRA.

Toll-C efflux pump. And so these efflux pumps, their job, they sit in on the outside of the cell and their job is basically to pump things that come into the paraplasm. the space between the outer membrane and the inner membrane out and try and keep things the cell doesn't want from entering into the cytoplasm. So they're efflux pumps. And they're involved in resistance to a lot of antimicrobials, including fluoroquinolones, some beta-lactams.

I think tetracycline has its own system. But in any case, this efflux pump, its job is to essentially pump things the bacteria doesn't want away from the bacteria. And quetiapine-exposed cultures had mutations in MAR-R. All of the ones they looked at, so MARR basically inhibits transcription of these guys, all of the cultures that they looked at that were exposed to quetiapine had increased levels of ACRA and Tulsi.

also MarQ. So some other things, ACRA and TOLC, again, being this efflux pump. So they were wondering, like, is this sufficient for this antibiotic resistance caused by the quetiapine? And to look at that, they essentially went to a new strain background, a kind of clean strain that hadn't been cultured in the quetiapine, and they made mutations in MAR-R, so this inhibitor. of these genes that are important for protecting the cell from antibiotics.

And when they did that, they actually saw in the clean strain where they only have one mutation that they know of right in MarR itself, they only saw about a twofold increase in resistance to ampicillin and tetracycline. This is versus the four to eightfold in their original isolates that they'd passage for 43 days. So that says that all those other mutations, hard to know which ones.

probably potentiate this response, that the MARR is important for activating this efflux pump, but probably these other mutations are also contributing. It's just difficult to know which ones and how.

But I think one of the cool things they found is that mutations in MAR-R increased the ability of E. coli to grow in the presence of quetiapine. So basically, when you add quetiapine... the cells don't grow very well. It actually inhibits the growth of the bacteria. So the MARR mutants actually grew better. They grew faster and they reached a higher cell density.

than the wild-type parental strain, especially at high concentrations of quetiapine. And this probably says the reason the cells are... developing these mutations doesn't have to do necessarily with resistance. They're not... be exposed to these antibiotics, that's not why they're growing better. It's because these MARR mutations and probably activation of C-flux pumps helps them get rid of the quetiapine, which is actually inhibiting their growth.

And so it's coincidental that they also become resistant to. ampicillin and tetracycline. So again, they're selecting for a growth advantage because if you can imagine your growing cells in quetiapine and the ones that pick up mutations in Mar-R are going to be able to out-compete or grow better than the ones that don't. and eventually take over the culture. So I thought this was pretty cool. I'm still curious why quetiapine hurts the bacteria or inhibits their growth.

didn't come out, or they haven't looked at that, I guess they could go through and see if those mutants, other mutants, the MarR plus cells and wild type, if they can find mutations that allow. for growth in these guys without activating this efflux pump.

I think the future here is to really understand how treatment with this class of drugs, all these atypical antipsychotics, actually impacts the gut microbiome because if this is happening to E. coli, it's very likely it's happening to other bacteria in the gut. All right.

And whether that has some of the side effects that are associated with long-term use of these drugs is actually due to defects or changes in the gut microbiome, I think would be really interesting and weight gain being one of them. I think it all boils down to how...

this class of antipsychotics impacts the fitness of the gut microbiome because i think what the antipsychotic is doing it's just that By them looking at antibiotic resistance, it was a convenient marker to follow because it's much easier to look for. gain of function than loss of function or trying to define what fitness means in a complex community like the microbiome. But as they point out in their ending paragraph, is these antipsychotic class of medications, we see more severe infections.

in psychiatric and schizophrenic patients. We also see antipsychotics causing more frequent urinary tract infections amongst elderly patients. amongst female patients, and curiously, about Parkinson's disease. And we know that Parkinson's patients... The whole issue with acromantia, which is effectively the gatekeeper microbe in the small intestine that allows things to leak into the system.

It would be really neat to see how acromantia is responding or if the acromantia strain selection that is being selected in Parkinson's patients is actually... a consequence of either their anti-Parkinson's meds or if the patient was on an antipsychotic. I agree. There are a lot of really interesting microbiological questions here.

Even if we just accept that there's a black box, the implications of this is huge. Because these are benefiting a lot of people. And meanwhile, there's this unintended consequence of... excreting, no doubt, antibiotic-resistant microbes. So not only might they be more susceptible to more infections because of disruption of the microbiome or whatever, but those might also be difficult to treat infections.

And then again, there's a lot, there's this compound and these drugs are detectable in wastewater sewage treatment and even tap water. So, you know, whether it kind of like triclosan or kind of inadvertently. essentially select even more antimicrobial resistant, sorry, organisms independent of, you know, the people who are being treated. So not only in a person, but also in the environmental reservoir. Not only in a person, but also in the environment. Exactly. Yep.

Yep. It's a shame because I'm sure many people benefit from these drugs. And yeah, this is bad news. Fascinating. They reference an article in Nature. extensive impact of non-antibiotic drugs on human gut bacteria. They screened 1,000 drugs, non-antibiotics against 40 representative gut bacterial strains and found that 24% of the drugs... inhibit the growth of at least one strain. So this is more common than we'd like.

I know when people are looking for, when companies look for new antibiotics, often they screen libraries of compounds that have known bioactivity. I think I've heard one where they'd found a drug that's used to treat cancer actually was also good at killing bacteria. So once things are bioactive, there's a possibility that... It could also be antimicrobial. So maybe it's not as surprising. But I thought this was really... Let's say you make a new drug.

Do you think the company has the obligation to make sure it's not antimicrobial or they're not going to bother because they don't want to give away their profits, right? This opens up the whole question of antimicrobial stewardship. And should this be a component of the stewardship question? Well, but do the physicians have something else they can reach for to treat patients that benefit from these antipsychotics?

Yeah, they may not. So that's the problem, right? And then you've got to pick your poison. Pick your poison, yep. I mean, I have to think that these drugs encounter antibiotics that are prescribed for many things that they're not necessary for. Like feeding cattle and pigs. Yeah. Pigs, chickens. I feel like these drugs are probably prescribed for the most part for people who really need them and in which they'll be effective.

You can imagine maybe screening people that have been on these drugs and just find out if they're at risk. Yeah, I think that would be, you know, really test and make sure that, you know, that they're... not selecting for too many antibiotic resistant organisms. And also when they do get pneumonia, these or other infections, that would be a great time to actually type.

Instead of just give people ciprofloxacin or something broad spectrum to actually type and see what those infections are sensitive to. So I hope that somebody will... take this data and use it to get money to do studies and find out in the stool of people who are benefiting from these antipsychotics, what is their composition of their gut microbiome and how prevalent is antibiotic resistance.

Oh, it would be a great, that's a great study. And in their family members, right? Right. Because, yeah, we share what lives with us. So one question you may want to ask Daniel. infectious disease doc is in patients that are on antipsychotics who develop a UTI does. he think about drug resistance when he's writing the script for the antibiotic. Interesting question. Yeah. And how, how.

broadly spread is the Mar operon and the efflux pump. Should we expect other gram negatives to also exhibit this? I think it's in most of the enterics. I actually did not do a search that's a good thing to look for. I think it or essentially similar efflux pumps are pretty well conserved.

Yeah. So it might be a different e-flux pump, but the same thing could occur. Exactly. Yeah. Oof. Great paper. Very interesting. Thank you, Patrick. Very interesting. Love it. All right. Now we move on to Michael for a paper.

We're in the medical world today. Today's paper is entitled Bifidobacterium lactis, ProBio M8, Adjuvant Treatment. confers added benefits to patients with coronary artery disease via targeted modulation of the gut, heart, and brain axis. And that paper was published in M Systems and the March-April online edition. And it's by Sun, Ma, Li, Yang, Li, Zhou, Guo, Zhong, Kwok.

Sun and Yong. And it came out of a group of labs in China, principally... the Inner Mongolia Key Laboratory of Dairy Bacteriology and Engineering. in the Inner Mongolian Agriculture University in Hot Hot, Inner Mongolia, China. And the clinical trial was done at the highway. medical center in Wayhai Municipal Hospital. I got my balls mixed up. Wayhai Municipal Hospital and the Chalou College of Medicine.

at the University of Weihai in Shandong, China. So if there's one paper... that you should read, and I'm talking to the TWIM listeners, that's going to substantially change your thoughts on cardiovascular disease and a role. for or of the microbiome and how your diet and other medication... can actually impact the quality of someone's life who has coronary artery disease, then this paper is going to blow your minds. You can...

cut to the chase, and simply look at figure five for the author's model. And the mechanism really illustrates quite nicely how the microbes are actually talking to us. It's akin to they're acting as a mini endocrine system in our gut, and they're sending signals to our brain and other organs that are then responding. So today, as you might imagine, we are dipping our toes into a systematic approach of how microbes may be interacting with their human host.

The fundamental hypothesis that this paper is addressing is whether of the standard of care medications for someone with coronary artery disease, atorvastatin and metoparol. Atorvastatin is a drug that... many of you will know as Lipitor. Its principal mode of action is to lower your circulating cholesterol level and metoporol. is an antihypertensive, also known as a beta blocker. And it's really to address hypertension and cardiac insufficiencies. And our colleagues...

And medicinal biochemistry and cardiology have really developed phenomenal therapeutic strategies for... the amelioration of coronary artery disease ranging from angioplasty to beating heart coronary artery bypass surgery, also known as off the pump surgery. I mean, this is... all pretty remarkable. But we've gone to drug treatments and the drug treatments, namely the statins and metoporol, for the prevention and even the reversal.

of the development of atherosclerotic plaque within our arteries. Now, longtime listeners of TWIM will appreciate that there are... active bidirectional interactions between the gut microbiota and other organs, namely our lung. where on a TWIM episode, we talked about the microbial linkage of a virulent form of helicobacter, that it's very important that you be colonized with it when you're a toddler and its role in preventing asthma.

And then recently we've talked about the brain linkage, where we discuss Parkinson's and, as I mentioned earlier, the role of acromantia. and the importance of different strains of acromantia and how they can impact on the progression of Parkinson's disease. However, today we're going to focus on the organ.

that has been working for us continuously our entire lives without rest, namely our hearts. Now, again, cutting to the chase, the authors are going to stimulate us to think outside of the box. going beyond the comfort level of both microbiologists and our medical colleagues who are responsible for our collective cardiac health. Here, the authors demonstrated through a very simple...

co-administration of a straightforward probiotic powder. They gave two grams a day for 180 days, and they asked a very simple question. Did folks with coronary artery disease get better. And now this is not the first time we've learned about ProBioM8. It made it into the peer-reviewed literature last April, April 2021. when we were all busily reading all the COVID literature that was emerging. But here a group, not this group, but someone else, published something with a very similar title.

to today's paper, namely Bifidobacterium lactis, ProBioNM8, regulates gut microbiota to alleviate Alzheimer's disease in the APP. PS1 mouse model. And that paper was published in the European Journal of Nutrition. And I'll drop that into the show notes for everyone. So as background... for really appreciating how they developed the scientific story of today's paper. We need to think, and I always do this when I read studies that use clinical trial.

to facilitate the development of their conclusions, I always try to ask myself the question, if the outcomes that authors are evaluating really... can address the conclusions they're trying to reach, especially when they dump it into the abstract. So first, what is the standard of care to address? whether or not your coronary artery disease is getting better. Well, first, we always go to a definition. What is coronary artery disease? Well, operationally...

It refers to myocardial dysfunction of the heart, muscle, or organic lesions caused by coronary artery stenosis. And stenosis is nothing more than a fancy word that means the narrowing of the plumbing such that the heart muscle may not be adequately oxygenated as a result of an insufficient blood supply.

that occurs as a consequence of a buildup of plaque that we know if you've ever been to your primary care doctor and they looked at your cholesterol number, they talk to you about plaque and the narrowing of your arteries. and the importance not to do that. And we know it comes from our eating our Western diet that is notoriously high in fats and high in proteins.

That results in cardiovascular disease being a common affliction of the developed world where about 110 million people have cardiovascular disease. So before we get to the micro of this paper, Let's look at how they assess for cardiovascular disease. Here they used a simple questionnaire. They abbreviated as SAQ, which stands for the Seattle Angina Questionnaire.

which has emerged as the most commonly used metric of a disease-specific health status to quantify patient symptoms of angina and to the extent... with which angina affects their functioning and quality of life. And it's important to really appreciate that this Seattle... angina questionnaire is a 19-item self-administered disease-specific patient reported outcome measure. So it could be a little soft in terms of those of us that like...

quantitative data because if you've ever asked your grandparent how they feel, they always tell you they lie. They say they feel fine. They don't give you the full truth. So hopefully. this patient cohort that responded to this questionnaire was... very truthful. The other caveat I'm going to... The study was done blindly, correct? Oh, yes. I'm going to get to that in a second. It's a double-blind, placebo-controlled study that went on for six months.

But I'll drop into the show notes review that's in JAMA Cardiology on the validity of using this instrument to reach a conclusion. So they're using the right tool to answer the question. They also use two other metrics, the self-rating anxiety score and the self-rating depression scale, in addition to two. to quantitative metrics where they can measure something biochemically, like the IL-6 level in a patient or the LDL level in a patient.

And they also measured some liver enzymes and then kidney function by looking at blood urea, nitrogen, and creatine levels. Now, the main objective of their study was to conduct, as Michelle tumble to a randomized double-blind placebo-controlled trial to investigate the synergistic effect of the conventional drug treatment, again, atorvastatin and metoparol. with a probiotic augment, and that's ProBioM8. And they simply were giving each study subject three times 10 to the 10.

organisms in this probiotic powder and the placebo group received a similar product that looked the same tasted the same but was absent the bacteria now So the study was a six month or 180 day randomized double blind. There were 80, uh, cardio. vascular disease patients. They were recruited from a medical clinic at the Weihai Municipal Hospital at the Weihai City, Shandong Province in China, and they were all admitted with a diagnosis.

of coronary artery disease, and were considered for participation. Ultimately, the study is powered by 36 interventions, those individuals who got the probiotic, and 24 subjects that remain to the end of the trial that receive the placebo. The probiotic group... If you go to their first figure, and again, this is open source, the first results the authors offer us found that the adjunctive or giving the probiotic improved.

the coronary artery disease related symptoms. And I really appreciated panel B of that first figure. They lay out the major elements of the Seattle Angina Questionnaire. They look at angina frequency, angina stability, the disease perception by the patient, the physical limitations, and most importantly, treatment satisfaction. And then they looked at depression and anxiety scores through their second metric. And what we see is for the most part, probiotics perform superiorly.

over just the control therapy of the atorvastatin, the mentoporol, which is the standard of care most cardiologists prescribe for patients with coronary artery disease. And what they offer in their presentation of the data is that both the probiotic group and the placebo group had... better angina scores than a control group at day zero where they weren't receiving any treatment than 180 days later. So that means treatment works.

And so the probiotic though, actually was a little bit better, suggesting that the intervention with the... atorvastatin metoporol, both with and without the additional... probiotics could alleviate the clinical symptoms of the coronary artery disease. But what I found fascinating was the treatment of probiotics in conjunction with the conventional drugs led to a

significantly larger magnitude in the individual Seattle scores, namely the angina frequency. Those patients perform better with the probiotic, the angina stability, the physical limitation and the treatment satisfaction. All were increased. Now, this wasn't really a stop the presses increase. It was really only about 1.14 for the angina. frequency and 1.08 over the treatment flow. Do you mean fold? Fold. I mean fold. So one and a half fold. Yes.

So I was glad that the authors didn't over-interpret their data as the sample size was a bit small for my taste at only 36 interventional. And only 24 control subjects. But the trends are still there. I'm a little confused on figure 1B. Okay. Disease perception. was the outlier. There was really not a difference there between the placebo and the probiotic. I think that's your grandparents' response. How do you feel?

That I feel okay. Okay. But when they ask more specific questions, how often do you feel angina? Got it. Thank you. I think that's where they were going. But the one indicator I really found interesting. was the LDL level, the LDL level and the IL-6 level in that particular panel B. If you look at the probiotic group, although they're presenting box plots, the box plot for the probiotic group is down in both cases. And remember,

Coronary arteries disease is really a disease of inflammation. So anytime you see IL-6, it's bad. And in fact... If you've been longtime listeners of TWIM, you've heard Daniel and Vincent talking about IL-6 treatment regimes for the control of COVID. You know, I really liked the IL-6 data and the cholesterol data that the cholesterol levels were down. But again, this is a small, small sample size.

But it's still pretty neat to see that the probiotic supplementation even augmented the antidepressant. anti-depression scores and anti-anxiety effective scores. So it was really pretty neat. When they asked a question, Did liver and kidney functions change as measured by looking for a liver enzyme, serial third transaminase, or looking at the kidney function, looking at BUN or blood urea nitrogen and creatine? levels and white blood cells. It was not appreciably different. So the probiotics...

are not doing anything bad to the liver or the kidneys, and they're not doing anything especially good to the liver or kidneys as well. And as Elio might say, and we should... wish him a happy birthday because his birthday was day before yesterday. And so Elio, if you're listening, happy birthday. We, we miss you today. So as Elio might say, that's pretty much the story, but wait.

This is TWIM. So did they do any really cool microbiology? Well, they did. And boy, was it cool. First, there's a lot of underlying microbiological work behind. how they develop their hypothesis. And for the sake of time, and since this is in the public domain, I'll let you read what the authors point you to. And it basically tells us that... This has been confirmed in animal studies and it's...

Principally, lactobacilli and bifidobacter are good, and they confirmed it in rats. And what do we often hear on TWIV? Rats lie. Monkeys exaggerate. And what do ferrets do, Vincent? Ferrets depends who you talk to, but I say ferrets are not people. All right. Ferrets are not people. So they offer this really great background.

from a human clinical trial where it was shown that the consumption of lactobacillus ruteri for 12 weeks effectively reduced cardiovascular disease risk in obese patients with a reduction in the levels of, again, they're looking at multi-inflammatory biomarkers, things that we can easily measure. with a panel like TNF Alpha, IL-6, and IL-8. And again, this all rolls up to the preliminary evidence.

bears out the data that the clinical trial that they conducted, that where they're simply asking the question, can the administration of exogenous probiotics... be a way to improve the therapeutic effect of the current standard of care, which is lowering cholesterol and dealing with the hypertension. So the rest of the paper.

really gets to mechanism. How might this be happening? And this is where I found that the authors did a really great job. It was very intriguing. And the first figure. They walk, again, this is panel A of figure one. They walk us through how they figured out who's there microbiologically. And what they learned was by adding probiotics, this modulated the patient's gut microbiocomposition as measured by the Shannon Diversity Index, which...

Recall is a common bioinformatic measure that takes into account the number of species present in a particular location. And they were doing stool samples and the relative abundance. And the beta diversity, which recall enables one to evaluate the similarity or dissimilarity of two communities. So you're effectively comparing. the intervention versus the placebo group. And they did that by principle coordinates analysis.

While there were no significant fluctuations observed in the Shannon diversity index for both groups during the intervention or the principal. Ponin analysis, the overall gut microbiota structure between different time points in both groups changed. Now, it was sort of at the... outskirts of a relevant p-value, and that's in figure 2A and figure 2B. But the dramatic changes were observed.

And the abundance, and here's where they gave me a new word to learn, the species level genome bins after pro-bio M8 initiation. So the addition of the probiotic is changing the abundance of certain bacteria. Now, again, the cautionary tale that they offer. is that their sample size of their placebo group is small. And they do offer that, although no significant change was detected in the overall gut microbiome.

diversity in both groups, there were significant and dramatic changes in the observed abundance of some crucial, these bin groups, these species level genome groups. after the probiotic administration. But crucial, we have to be careful about using that because right now they're showing, in a very impressive data set, associations. Yes. But who's doing what?

which has the physiological impact remains to be seen. Yeah, it's really quite remarkable. The third figure. in their paper is just plain wild. And, and this, and since this is an open source M systems. I encourage you to download this paper, though not when you're driving or running. It's really quite remarkable. What we have in the third figure. is a genome-centric metabolic reconstruction of patients with coronary artery disease. And here they look at four relevant...

module groups. So in group one, we have a carbohydrate degradation module. Group two, we have a coronary heart disease module. We have a gut brain metabolism module. And finally, we have an amino acid degradation module. Remember, they're trying to postulate a mechanism of how adding one or two grams of this probiotic to your diet per day can improve your coronary artery disease state.

Now, the upper part of what they call this diagram is a core diagram, shows the distribution of the species-level genome bits that encode these modules across phyla. The lower part of the chord diagram represents the coded modules. The take-home that they're trying to answer was the administration of probiotics. modulating the gut microbiota sufficiently. And their results yielded that 121 of these 138 modules that they evaluated

effectively were encoded by the species-level genome bits. And they again identified modules belonging to 13 phyla distributed mainly from the firmicutes, which... Those of you who don't remember what the firmicutes are, these are your good bacteria. The lactobacilli, the clostridia, the enterococcus, the rheumococcus, the ones that...

have been demonstrated over and over again are good for you. They also found bacteroides or bacteroides, which also harbors flavobacteria, cytophagia, sphingobacteria. They were about at 12%. And then proteobacter, Petra's favorite group, the good old E. coli folks. And they give us a whole bunch of enzymes that are going up and down. In the enzymes that they're looking at, galactose degradation, lactose degradation, and then they look for things like that are going to be...

neurologically remarkable molecules. And these are things like choline degradation and carnitine degradation. And this is in... their CHM group, which stands again for their coronary heartary disease module. And it's really remarkable. found that about 34 of these species genome bits belong to the firmicutes. And the proteobacter were about 41%.

And, you know, it really gets you to think about what's going on. And then they looked at the gut-brain metabolism module, which looks for the short-chain fatty acids in their related modules. and how they're encoded by these highly diverse groups of bacteria. So next up, they asked, did the probiotic powder... modulate the gut microbiota to a point where they made related bioactive compounds. And this takes us to their fourth figure.

If you've ever watched an old episode of Seinfeld, you probably saw the Thanksgiving episode where they talked about tryptophan causing you to fall asleep. So we know that tryptophan from popular TV is a neurologically active molecule simply because Seinfeld told us or taught us that. And so they appreciate. That tryptophan is a bioactive molecule.

And they also looked at the synthesis of GABA metabolism, another neurologically remarkable metabolite. Interestingly, four gut-brain metabolism modules, including cortisol. degradation, another neuroactive compound, acetate, isovalyric acid, and polysaturated fatty acids. were dominated in the pro-bio MA group, which were also characterized by, again, this higher level species genome bid diversity in abundance in their modules.

The GBM module of acetate degradation, which is the bad actor, was highly represented in the placebo group. And, you know, it... When you begin to look at their figures, and it takes some time to go through their figures and to go through their supplemental data. They have more supplemental data than they do have data in the paper. which enables you to really see how they're getting at things. But the most controversial data was related to supplemental table number seven.

And here the authors offered that the probiotic administration modulated the predicted gut microbiome, leading them to offer that comparing metabolites. from the two groups found that the average predicted abundances of these reactive compounds like methylxanthine, cytosine, malinate... and palmitilglycerol increased significantly. in the probiotic receivers, while the C24 carnitine degrees significantly in the same group of subjects. And remember, carnitine is the bad actor.

The fact that it's going down in the probiotic group is good. So this then suggests, doesn't prove, it suggests. that co-administration of the probiotic with conventional drugs can lead to some specific changes in some of the predicted metabolites with reflective differences in the guts.

microbiome's potential to synthesize these compounds. Their figure is clear, but what does it mean? And so here we... have them asking their next question as whether or not the probiotics could modulate serum metabolites. And again, it all rolls up. They're trying to address the mechanism of how do probiotics in the gut repair the heart by communicating with the brain. So this next section addresses the notion. Can we develop an understanding of potential biomarkers?

And everybody loves biomarkers because you can measure them chemically. They're not as soft as a survey where your grandmother may lie to the surveyor asking, so how do you feel? And they look for 25 serum metabolites of which 23 were amino acids, but they looked at two trimethylamine and trimethylamine and oxide. And these are. both metabolites which are proven to be associated with atherosclerosis. And I'll drop into the show notes a paper here talking about how circulating trimethylamine...

Oxide levels are strongly associated with atherosclerosis. And what they found is the ProBioM8 decreased both the serum levels of TMA. especially at day 90, and also the trimethylamine oxide levels were also down. This is really, to me, was the hold the presses. if it's borne out by larger studies at multiple medical centers across the globe. Here, the problem was, is this data point...

only had a total of 41 patients in it. And they were only providing blood at three consecutive time points for analysis of the serum metabolite levels. Still, it's a stop the presses event because we have enough data from the cardio field that shows that these two serum markers are good indicators.

whether or not you're going to have progression of disease. So all of this gets nicely rolled up in their last figure, figure five, where panel A. illustrates the differences in serum metabolomes by describing them through the use of a violin plot, showing the levels of the serum metabolites that were responsive. And if you're unfamiliar with a violin plot, the name offers what they look like. But what they're doing is they're plotting the numeric data.

namely the values that each patient had as a box plot would do, but then they rotate and they have a kernel density plot on each side, which gives that shape. the violin to effectively give you an understanding of how real the data are given the small sample size that they offered to us. And panel B brings it home with this grand unifying mechanistic offering where they offer that the gut microbiome.

is behaving as this virtual endocrine system that is able to effectively regulate distal organs through this metabolism dependent pathway and these biomarkers.

like trimethylamine and trimethylamine oxide, short-chain fatty acids, and then bile acids. So again, the authors condense this all into one sentence at the end of their paper. This study demonstrated that co-administration of ProBioMA, the probiotic we've been talking about, with a conventional regime of statin and antihypertensive.

offered added benefits to patients with cardiovascular disease compared with the standard of care. So it's really, we need... more studies, more clinical trials, but the beneficials of ProBioM8 really were pretty strong. gave me some encouragement that we may be able to really evaluate whether probiotics have any place in our future. So they just gave them...

b lactis nothing else is that correct no they the the powder it's they didn't describe the characterization of the powder i i didn't have time i didn't have time to go back and see if the If the 2020 paper that where ProBioM8 is first described, they offer that it was isolated from a lactating human mother. is where they isolated this strain of bifidobacterium. And whether or not it breeds true is another question. Because I'm looking for it.

And all they sell is B. lactis because I want the reduction in anxiety. I want that. Yes, we all do. I mean, everybody was looking for their cholesterol number to see what their cholesterol number was. But I was impressed to drop the cholesterol numbers. Because you said there's no negative to this. So... But, you know, you can get B-Lactis, but there's nothing sold that's called M8. I don't know what that is. They got it from a company out of China. I did look that up.

And they're not giving that many. They're only giving three times 10 of the 10. That's not that many bacteria and powder. But formally, we don't yet know whether the... bacteria alone could have the effect or it needs to interact with other drugs that they're on. Because they couldn't do bacteria alone because it's standard of care, right? Yes. Right, right. It's not ethical.

What I found challenging about this paper is, well, first of all, just the huge amount of data. Like to their credit, they were very systematic, unbiased. There's just a ton of data and they do statistical analysis. There are associations, and so people can study this and maybe come up with their own working models to go in and test to try to understand who's doing what. But also, it's just hard for me to interpret.

The magnitude difference and then physiologically, it's statistically significant, but is it physiologically significant? And that's what the cardiologist... are going to be asking. And, you know, this is where I think this is a new and emerging field for our infectious disease specialists. I really wish we could have had Daniel on this group to ask him what he...

thinks about this and, you know, whether or not this is a new and emerging discipline for infectious disease folks. These are our doctors who understand bacteria and people, you know, it's, it's really. quite fascinating. I think it's our neurobiology colleagues that we need to bring into the conversation. It was really, I think... This paper is one that

Entering graduate students need to come in and think about, begin to punch holes in how you design the experiments. How do you develop hypotheses around it? This would be a great qualifying exam. foundation paper to think about. Boy, I don't know, Michael. No, everybody's shaking their head. There must be like two dozen different abbreviations in this paper. Oh, yeah.

And then a lot of the parameters, it's just hard for a microbiologist to interpret what their physiological impact is. Yeah, I try to prevent as many abbreviations as possible by... saying the words out loud. Right. So this is not an accessible paper. I will say that. But it is fascinating. So, Michael, my question is, even if you can't get this M8, there definitely are other powders that you can buy. Oh, yeah. Yeah. So are you going to try these or?

I could try these. I get my cholesterol levels checked often enough. So I could ask the question, is it making a difference? So here is... ProBio M8. This is actually in a study, Journal of Dairy Science. They looked at 190 strains from human colostrum samples. And two were identified with better acid and bile salt resistance, right? Because you're taking this orally, right? Right. And one of them was B. lactis probio M8, and the other was L. rhamnosus probio M9.

So they survived the gastric environment. So that's... Yeah, from colostrum. Isn't that some breast milk, right? Breast milk. You got it. The secret magic ingredient that was drummed out of women in the 1950s for the convenience of formula. Because I think if you just take, you know, you can buy B. lactis on Amazon, but I think it'll just get degraded in your stomach. Yeah, that's probably true. Although what's interesting is, yeah, if it's in colostrum, it's obviously.

The target audience for this kind of heart disease is obviously not the under 18 months old audience. But it could begin to seed your gut so that the microbe remains. But then if you're on antipsychotics, they could go away. It could be that not all mothers have this in their breast milk, and so the kids who get it do better over the long time. Who knows, right? I mean, there's definitely diet and exercise components to here.

Oh, no. We all have been to our physicians and they tell us, so have you been exercising? No, I've been writing grants. Very nice. Two provocative papers. Thank you, Michael.

You're welcome. I have to resist buying B-Lactus, okay? Because it won't work. Show notes. That's TWIM's 264. Show notes at microbe.tv slash TWIM. If you have a question or comment, you can send it to twim at microbe.tv. If you enjoy our work, consider supporting us. You can find ways to do that at microbe.tv slash contribute, and your contributions are federal U.S. tax deductible. Michelle Swanson's at the University of Michigan. Thank you, Michelle.

My pleasure. Good to see you all. Michael Schmidt is at the Medical University of South Carolina. Thank you, Michael. Thanks, everyone. And Petra Levins at Washington University in St. Louis. Thanks, Petra. Thank you, everyone. I'm Vincent Racaniello. You can find me at virology.ws. I'd like to thank the American Society for Microbiology for their support of TWIM and Ronald Jenkes 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.