Let's Talk Micro
149: Talking Microbiome
[Music] Welcome to Let's Talk Micro! Hello dear listeners! Welcome to another episode of Let's Talk Micro! As always, I hope you had a great week, and you can always find Let's Talk Micro on Apple Podcast, Spotify, Amazon Music, Good Thoughts, whatever you listen to podcasts, you can find Let's Talk Micro. As for our social media, I am on Instagram, TikTok and YouTube as Let's Talk Micro, or LinkedIn as Luis Plaza, and on X as Let's Talk Micro 1. I also have an email address, which is Let's Talk Micro at Outlook.com. So either via social media or via email, you can send feedback and topic suggestions. Those are all was welcome and appreciated. So please go ahead and subscribe to the podcast, download episodes, and if the app allows you to do so, please go ahead and leave a review. Thank you so much for the support. As you know, learning about antibiotics is definitely a lot of information, so if you're looking to learn more about antibiotics, please check out www.learnatibiotics.com and the Learn Antibiotics book available on Amazon. These resources include cheat sheets, practice tests, games, and more, and they are being used by thousands of people worldwide and may be helpful for you or your colleagues. So definitely check out these great resources from Dr. Timothy Gothier. A very passionate pharmacist. As usual, I always talk a little bit about the previous episodes, and in this case, it's two of them. As you know, I'm just going to take a minute to talk about this. On Thursdays, we know we normally publish a regular episode, so if you haven't checked the one from last Thursday, that episode, and Grace Loubert, she's the director of the Medical Laboratory Sciences program at the University of Alaska in Anchorage. She joined the podcast, and she was in the previous episode where she talked about her program. In this one, she came in to talk about the research that the MLS students do, which is very atypical, I will say, from your regular MLS programs. You normally work on maybe some sort of project, but this is very detailed research, and of course, she goes over the whole process and the results that they have seen, but the students, you know, they collect samples, they collect scat from moose, bear, and geese, and then they freeze them to the whole procedure, then they thaw them out, and they go ahead and play them, and you know, they screen, and they perform testing, and she talks about the organisms that they have seen, including ESBL, producing organisms. So I think this is very fascinating, and the students come out learning so much, especially about anti-microbials, because they do susceptibility testing on those isolates, so, and as you know, learning about antibiotics, you know, it's so much information, and when we go through a program, we learn some, but we barely touch the surface. So it is great that the students come out learning so much about anti-microbials, so shout out to Grace. Thank you so much for what you're doing. And then the latest episode was a bonus episode that I came out this past Tuesday, and it was about what I liked and the Americans decided from Microbiology event, Microbe 2024, which took place in Atlanta, and you know, I've been going since 2023, and both times, you know, I have recorded a live episode, and basically what I do is, you know, talk about my experience, you know, which talks have been to or I normally attend podcast recordings. I talk about the posters that I like, so it was a great summary, so I hope you check it out. And if you have the opportunity to attend Microbe, which is the largest event from the American Society of Microbiology, and one of the largest events of Microbiology, if not the largest, you know, it's a gathering of and people from all over the world attend, so I definitely invite you to attend this event if you haven't already. So I think, you know, if you're a hardcore microbiologist and you're so into it, you will definitely enjoy it. And of course, you know, one thing that I always love when I go to this type of events is just when I meet people that actually listen to the podcast or they tell me that it has helped them somehow. So I definitely enjoy that, you know, just to know that I'm here, you know, week after week, talking about a topic, and then people are listening, and it is so great when I actually meet those people. So it's just, it's a great feeling and I appreciate so much your support. So I want to do a couple shout outs, shout out to Alexis, who I briefly met before on 2023. But then this time, you know, I had a chance to talk more, shout out. Thank you for listening. And then shout out to Jessica. Jessica is a fellow MLS. And I had a chance to talk to her for a few minutes. And she listens to the podcast. So Jessica, it was so great meeting you. And thank you for listening. Okay, so let's go ahead and jump to today's episode. So today's episode is about the microbiome. And this is something that maybe we see, you know, we might see some publications and we might not be too familiar with it. We definitely hear terms like microbiome, microbiota. And we see published articles out there about how maybe an imbalance of the microbiota and the composition, it can sometimes, you know, it can be correlated with the diseases. And we know that as microbiologists, you know, different different areas of the body, the composition of organisms, it varies. So in this episode, Dr. Crystal Eisenhower, she joins the podcast and, you know, talk about the microbiome. You know, she gives some definitions, specifically, you know, she talks about the human microbiome, what kind of organisms we typically seen, you know, based on different areas, how you can sequence the microbiome, what are some of the applications, and something, you know, she talks about something that's very important, which is, you know, once we do the sequencing and we have the information of the organisms that we have, what do we do with that information, right? As you know, some organisms, you know, are not supposed to be in the body, they are not normal, or sometimes, you know, we have some that are normal in some areas, or maybe you have an abnormal concentration. So I definitely think this was a great episode and a great way to introduce ourselves to the microbiome and those type of terms, you know, organisms and things like that. So it's, it was a great episode, very informative. So I hope you like it. Let's go ahead and listen to it. So those of you that are medical laboratory scientists and microbiologists, and you work on the bench, a lot of times, you know, you definitely know, you know your organisms and you know that some organisms, you know, they're part of the flora, certain areas, and you get quite knowledgeable, knowing, you know, typically what's a pathogen versus what's normal flora, but this is a very interesting topic. And I was on a conference listening to a speaker talk about the microbiome and the microbiota. And sometimes, you know, there's so much information that we might not be too familiar with it. And to be honest, until I started doing a graduate study, that's when I started getting more familiar with it. So very fascinating topic. So I invite her to speak here. And she accepted. So I'm very happy about that. So with us today, we have Dr. Crystal Isanar. Dr. Isanar, welcome to Lestog Microbe. Thank you. It's a pleasure to be here. My pleasure. So maybe let's start with a quick introduction about what you do and anything else that you want to add. Sure. So my career has primarily been focused on biotechnology companies. I've co-founded and led two different biotech companies through to acquisition in my career so far. And currently, I'm working for the last acquire of my former company, LabCorp. So there, I serve as a director for microbiome and specialty infectious diseases. Thank you again. And we're definitely in the laboratory community. We know we're definitely familiar with LabCorp and definitely a big reference lab. So let's start with some basics here. So what is the microbiome? So well, it's not a singular thing. There are many different kinds of microbiomes. There's a microbiome on the desk, on the floor, in your nose, in your belly button. Each environment has its own unique microbiome. And the microbiome, it's the collection of microorganisms. They're metabolic byproducts. It includes bacteria, fungi, parasites, viruses. So it's the whole microbial cosmos that exists in different environments. And each one of those environments, again, is unique. And the microbiome of the nasopharyngeal space is completely different than the microbiome of the intestinal tract. And you can actually tell where the sample originated from by the types of microorganisms that are present in the different spaces. Definitely. And that's something, as we work with cultures, we know based on the source, right? So we're expecting sometimes, you know, we see some organisms. Okay, here, you can expect to see this versus another area where you shouldn't see. So depending on the sample on the source, it's definitely very varied. So maybe, I know, so definitely like it changes, as you said, composition, like from the oral fairings, the intestines, the genital area, maybe can you give some examples of the organisms that we can find for the audience? Sure. So when you're looking at the oral cavity, for example, because it's one of the most diverse microbiomes that is present in a human condition. And my information will be primarily focused on the microbiomes of the human body. And so the oral cavity has hundreds and hundreds of different kinds of bacteria normally found there. It's very diverse, lots of different kinds of lots of different species of bacteria. Things like porphyramonas, which has been linked to gum disease and other things, but also a lot of other bacteria to too many to list. When you look at, say, a fecal sample, again, hundreds of different kinds of bacteria, but slightly less diverse than the oral cavity, interestingly, as far as types of microorganisms. And then there you're looking at things like faciella, bacteria, bacteroides, things of that nature. And then looking at something like the vaginal cavity, there you have very low diversity. You're typically looking at one to a dozen unique microorganisms present in the vaginal space instead of hundreds. And you're looking at things like gardenarella and lactobacillus as kind of the most common things that are found there. So very unique in terms of the types of microorganisms, the species of microorganisms, but also the diversity and the microbial burden. Obviously, a fecal sample has a lot of microbial content. Upwards of 80% of the sample can be comprised of the actual microorganisms. And as a friendial swab, there's very low microbial burden. So when you do a swab there, most of the genetic information you pull out is actually from the human, but the person that was questioned from. So lots of different ways of looking at the differences in microbiomes throughout the body. Yes, definitely. Composition is definitely very, varied. And that's something that we see in the lab with the samples. And I remember when we used to do like fecal, white blood cells on those tools that we put on the slides and they're so concentrated, you see so much bacteria. So it definitely changes from site to site. So you mentioned when I was listening to your talk, there are some diseases that can be linked to an imbalance in the composition of the microbiota. Can you give some examples? Sure, sure. The most overt is infection. So if you have a microorganism growing in high quantities in the wrong part of the body, that's the traditional definition of an infection. So that's the kind of the most drastic perturbation of the microbiome that we typically see. But there's a lot of other dysbiosis. It's not an overt infection. And there's nothing there that's acting as a true pathogen. But when you have the wrong proportion of the microbes or the wrong balance of the microbes, you can get dysbiosis. And that is increasingly being linked to a lot of chronic health conditions, things like some forms of diabetes, cardiovascular disease, Alzheimer's, Parkinson's, all kinds of things are being linked to these perturbations of the microbiome. There's even some connections with the gut microbiome and colon cancers and other types of cancers. So it's really, we're just scratching the surface at understanding, are these dysbiotic states, are they causing the condition or are they result of the condition? So a lot of very interesting work going on and a lot of new discoveries to be found. So I know that I have a couple questions here that are definitely very heavy on the topic. So I know that if as general as you can, or you can give as much information as you can, but I know that they're heavy in information. So as far as what are some microbiome applications? Can you give us some examples? Sure. And so my former company, we were actually using microbiome characterizations to help solve medical issues. So patients who were exhibiting signs of infection, but all the traditional tests were coming back negative or indeterminate. So you can look at the microbiome and see if there's something infectious there. So an example might be say you have a urinary tract sample and there is high levels of DNA virus or something like BK virus, JC virus. They're a normal part of the urinary tract system, but they can grow and then proliferate high levels and cause issues within the body. So things like that are things you can see with looking at the microbiome, things that shouldn't be in the sample type you're looking at. And that's probably the one that's most clinically relevant right now because we understand, no, we shouldn't have tuberculosis circulating in our blood. That's not something we want to see in any healthy individual. There's no normal level of tuberculosis to be found in the human condition. So that's the application that right now has the most direct clinical application in an area that I've worked directly in for a number of years. There's certainly lots of possibilities. And when you look at disruption in the gut microbiome, there's this gut brain access that people talk a lot about. And you can see there's disruptions in the gut microbiome and you can link it to or correlate it to systemic disease. But there's not any clinically validated bodies of work yet being able to use the microbiome characterization to be able to help diagnose those types of more chronic systemic issues. I do believe that it will happen. But we need a lot more data. We need a lot more information. And so that side of microbiome is really more on the discovery side. So discovering these links and connections between the microbes and these chronic conditions that we've sat with them. Well, thank you for that. And so what about some methods used for sequencing? Maybe you can talk a little bit about that? Sure. And there's a couple of different ways that you can characterize a microbiome. All of them require sequencing. So next generation sequencing is the most commonly used. You can, starting with the more simple methods, a lot of groups use 16-S sequencing where they're looking at the 16-S gene or a portion of the 16-S gene that's found in all bacteria and archaea. So you can do basically PCR amplification of that gene or fragments of that gene and then put it through next generation sequencing to differentiate between the different bacteria. It's limited to only bacteria because of that nature. You can do something similar with ITS or 18-S genes and be able to see fungal targets as well. But again, there you're limited to just the fungi. So you're missing parasites. You're missing viruses when you have these approaches. But it depends on the question you're asking. And sometimes you really just want to focus on the bacteria that are present in a sample. So it's fairly straightforward. There's a lot of commercial products out there. Commercial kits you can use to do 16-S sequencing. Mostly it's in the 300-base pair range is what a lot of groups are using as far as amplicon length. One of the other limitations is sometimes because you're only looking at a small fragment of the genetic information for that microbe, you sometimes don't have good resolution to be able to differentiate down to the species or strain level. There's some newer technologies that are looking at complete 16-S sequences. And those have much better resolution as far as getting down to species and strain level. But those are not as commonly in use right now. You can also do kind of a targeted approach where you want to pull out just the viruses or you want to target the specific populations of bacteria. And that's a targeted sequencing approach where you have probes that you use to amplify genetic regions that you know exist in the sample and that you're looking for. And that can be useful to say if you're looking for certain kinds of cancer or things of that nature. That's where it's primarily been used. Or if you have specific microbes that you're only interested in looking at specific microbes, you can use these targeted approaches. One of the more comprehensive methods for doing sequencing is called metagenomic or shotgun sequencing. And this is where you have much more breadth and it's where a lot of groups are focusing on things. It allows you to identify bacteria, DNA viruses, parasites, fungi. So anything that's DNA based, you can identify with this particular kind of method. And basically what you're doing is just like what it sounds. So shotgun approach where you are randomly blasting probes into your DNA sample. And that allows you to get DNA sequence coverage randomly through the sample for theoretically all the microorganisms. And depending on how deep you go and how much, how many sequencing reactions you run, you can sometimes get full length genomes for this for certain, especially smaller things like viruses. But again, it allows you to really take a more broader approach and to be able to see more of the microbes that are present on the sample and not be limited to only bacteria, fungi, or things that you were specifically looking for. Thank you for that. I know that it's, you know, definitely. And I'm sure the audience appreciates it as well. It's a lot of information and we, you know, definitely sequencing is something that we'll hear a lot about. And ultimately a lot of the organisms that we cannot identify in the lab, like we send them and then they end up being identified by sequencing. But we definitely hear some, you know, different terms. So thank you for that clarification. So what is the, what is the ketone? The ketone. So one of the challenges with doing microbial sequencing or microbiome characterization is that there is microbial DNA on almost every surface of the earth in almost every body of water on, you know, the surface of every human. And so it can be very difficult to get an actual true negative control when you're doing this type of work. Even the molecular grade waters that we use in the lab, there are tiny amounts of microbial DNA present in those reagents. In the enzymes we use to perform the sequencing, to perform the, you know, the amplification reactions and the sequencing reactions, those enzymes are often created in industrial bats where they're being synthesized by microorganisms of various sorts, usually bacterial, like thermophilic bacteria. That's where we get our DNA polymerases from. And so those enzymes have a not insignificant amount of residual DNA from the microorganisms that they were synthesized within. And so these things carry through your, your entire process. And so it's almost impossible to get a true negative control running through a sequencing process looking for microbes. So the ketone, I mean, there are a number of publications describing this. There are certain like thermophilic bacteria, water-based bacteria, are very commonly found in the reagents we use to sequence. And so really the best strategy is, you know, obviously using great lab practices to prevent contamination or introduction of any microbes from the environmental service you're working with in or from the people that are handling the sequencing reactions. You know, you can UV irradiate things like water and buffers that don't have any enzymes in them to try to cross link any DNA that may be present in those reagents to try to keep the background as low as possible. But really, you have to run a negative control with every sequencing reaction because you have to know what your "background" is. So the ketone is basically your background level of microbes in a sample. And these 16-S sequencing is very sensitive. The metagenomic sequencing is quite sensitive as well. And so even though there's, there's, you have a negative control, you extract it, you do a DNA quantification, you have, the DNA present there is below the limit of detection. But you still put it through to the library prep process. It's still below the limit of detection. You still get microbial sequencing reads in that, in that well. So it's, it's a challenge. So you, you have to characterize what that, because it can change from depending on what vendors you're using for reagents. You know, if you have, you know, unusual environmental conditions in the space you're working in, you really have to do a negative control with every single reaction so that you know what your, what your background is so that you don't inadvertently include the background microbes on a patient report or some, or interferes with, with how you analyze your data. So that's a lot of information. Yeah. Thank you for that. So, so right. So you, you perform the sequence and then you're looking at the data. And so what questions should we ask ourselves? You know, when interpreting data, I know that you, you know, you gave a great example, with an application that we, you know, seeing something like mycobacterium tuberculosis, that's something that, you know, it's not normal. So what questions should we ask ourselves when we're interpreting data? So I typically work through kind of three high-level logic points. The first is, does this microbe long in the sample I'm looking at? You know, the example of mycobacterium, I had a patient that we worked with a number of years ago that presented with arthritis, chronic, you know, in arthritis, she was diagnosed with Lyme arthritis and she had gone through, I think, two or three rounds of treatment for Lyme disease. And she would get better when she was on the treatment, but then she would, she would relapse as soon as she, she got off of the antibiotics. So in that particular case, her, her doctor sent us a blood sample and we processed it through, we primarily focus on metagenomic sequencing. There were, I think, 270 fragments of DNA that aligned exclusively to mycobacterium tuberculosis and her plasma sample. That's not normal. There's no normal level of mycobacterium tuberculosis in a human sample. So that's an example where, you know, that does not belong. And there's certain things like, like that, or rickettsia, birellia. There's a lot of, you know, zoonotic microbes that do not belong in the human body at any level. So that's kind of what the first tier is. Does this belong in the sample I'm looking at? The second tier down is, is it in the right part of the body? So E. coli is a perfectly normal part of the intestinal tract, but you don't want to see it in, you know, the oral cavity. You don't want to see it in the urinary tract. Those are common causes of infection. So finding a bacteria that is in the wrong part of the body can be another clue on how to interpret that finding. And then the third kind of high level logic point is, is it in the right amount? So again, taking the E. coli example, E. coli is a normal part of the intestinal tract, but it's typically found at less than 1% in abundance in a healthy gastrointestinal tract. If you have a patient that has E. coli at 30% abundance, that's way too high. So, you know, it's, it's clearly growing, you know, out of proportion with a normal, healthy, you know, microbiome state. That's an indication of, of infection. And then all of these things really have to be wrapped around, wrapped up with the information about the case, especially in, and I, most of my work in, again, involves clinical applications. So, you know, the patient history, you know, does this patient have symptoms of the UTI? Do they, you know, do they have, you know, did they go to India or, or someplace where tuberculosis is, you know, more common than you do? Do they have, you know, history of tick or, or insect bite? So, these things can kind of help build the case where you, you find something that doesn't, is typically not found in, in, in a healthy sample. But you, you kind of build the logic and the case around it on, is this causing the disease, the patient's exhibiting? Yeah. And I know, you know, you, you definitely, you know, you gave some cases when you were giving your, your presentation. And I wanted to ask you about, can you talk about the one with the culture negative UTI case that you talked about? Yes. Yeah. We, we worked with a lot of those with my, my former company. And what we know about urinary tract infections is, um, upwards of 50 to 60% of urine cultures are negative for symptomatic patients. And that's a problem because someone who's symptomatic for, for a urinary tract infection, you know, that it's very uncomfortable at a minimum and it can, it can actually be debilitating. And so one of the cases that we, we worked with was a patient who had suffered from recurrent UTIs for over 30 years. So, you know, the majority of her adult life had been spent in, in the state. And so we got a urine sample from the patient, processed it through our metagenetic sequencing, testing. And there was, in this case, it was actually interesting. It was a 90% of her sample was Bifida-Bacterium brevi, which is the primary ingredient in a lot of probiotics. And, but it's not a normal part of the, the urogenital tract. It's not a normal part of the bladder. And so in this case, it had colonized for bladder and had been causing symptoms of a UTI for a very long time. And then we had other cases, we had a pediatric case that was very similar where her urine culture was negative. And they, you know, she was only three years old, so she wasn't quite fully verbal yet in a lot of pain. And fortunately, her mom found my, my former company was able to send us a urine sample. And we identified the bacteria that's not capable of growing in urine culture. And again, the Bifida-Bacterium brevi from the prior case, it won't grow in the standard urine culture. And so you're not going to see it. And so in that case, we were able to identify a bacteria that you just would not be able to identify from urine culture alone. So lots and lots of cases for culture negative UTIs, unfortunately. Well, that's definitely got to be frustrating when, yeah, especially the patient, you know, you have symptoms and you go in and there's nothing growing. So they have, you know, one of the things, you know, not only not all bacteria, you know, our culture, but at the same time, you know, when we talk about urine cultures, we have a more limited set of because of the nature of the organisms that typically are seen in UTIs. But most of them, they do grow in your standard, like, let's say, bulldog or makonki. But there are some limitations to that, whereas when you do other parts of the body, let's say, you know, you have chocolate and other media, like an aerobic media, and then you, based on the organism that you typically see. And so in the urine, you know, the setup is more limited. So there's also the potential to not recover some organisms. Yeah, so I just did elaborate on that point and bring this back to the patient. You know, there are millions of women that are suffering with culture negative UTIs right now. It's it's a huge problem. And it's one of the reasons why I get up and I do what I do every day because I want sequencing type testing to be more commonly available for these cases where people fall into this this this loop because they they go to their doctor and they do a culture for its negative, they send them home. They're they're very resistant to to give antibiotics without a positive test. And just, you know, it's, you know, you you really do need that because we need to be good stewards of a van about a usage. But these patients, they, you know, there's a seven to 10 year cycle for patients that fall into that to get diagnosed. And a lot of times they end up getting diagnosed with something like interstitial cystitis, which is idiopathic. We don't know what causes it. It's just it's inflammation. And and so these patients are basically just sent home told there's nothing wrong with them. It's all in your head. I've had patients who were put in psychiatric wards because they got defiant because their doctor couldn't give them an answer as to why they were sick. But they knew they were sick. And unfortunately it spiraled into a there's nothing wrong with you conversation. So it gets very complicated. And it's very hard for these patients to navigate through when we don't have the right tools for the doctors to use widely available so that they can get answers. Yeah. And that's something that all of us can address on it with, you know, that's something very frustrating when we are not feeling well. And then, you know, we go sick treatment and then they say, you know, there's nothing wrong. So it's just it's a very frustrating feeling and not knowing what's, you know, what's what's wrong with us, what's causing, you know, what we're feeling and it's not, you know, very challenging and frustrating. So I know you you mentioned that any other cases that you have before I jump to the next question. There was another interesting case that we worked. It was a I think he was in his 30s, had been sick and it had had two strokes. And you know, young man shouldn't be having strokes, no family history of strokes. And I think his odyssey was about 10 years to get a final diagnosis. And it turns out he and we received a plasma sample from him and ran it through our system. And we found rickettsia-pilis, which is a bacteria that's normally found in fleas on cats. Turns out he had a cat, the cat had fleas. So both he and the cat had rickettsia-pilis infection from this. But again, it's not something that is one of the standard things that the clinician knows to look for. And so and that's one of the beauties of using tools like this. You don't have to know what you're looking for. You don't have to do a differential diagnosis. You can just run the sample and see what the data says. Well, that one's definitely very interesting. So you mentioned, you know, when I asked you about the applications and you mentioned some that there's still more information, anything else that the future might bring any future applications that involve the microbiome? I do see a role for especially gut microbiome testing to be used for preventive care, where, you know, you go in for your annual checkup. And one of the standard tests they run is they do a gut microbiome assessment and look for subtle changes that could be there that in the future we can definitively link to Alzheimer's or cardiovascular disease and then have preventive care offered much earlier than after symptoms start to appear. So that's where I believe the possibility is there to go there, but we need massive data points to be able to make those kinds of connections and correlations. Well, you know, thank you for that. And as we're coming to the episode is coming to close, is there anything else that you want to add? Well, once again, you know, this has been a very informative and I really enjoy, I enjoy listening to it the first time and I definitely did now again and I hope the audience does too. So I just want to thank you again for taking the time to be on Let's Talk Micro. It's a pleasure. Thanks for having me. I appreciate it. And that, my dear audience, is the end of this episode. I hope you enjoy learning about the microbiome. As always, I enjoy sharing this information with you. Please continue bringing that passion to what you do. It's so important. You do such great work. And thank you so much for support. Thank you for listening. It's always great meeting you and meeting new people. So thank you so much and stay tuned. Great things coming your way. So as always, stay motivated, stay safe and of course, continue talking micro until the next time. Bye. [BLANK_AUDIO]