 Think Tech Away. Civil engagement lives here. And we're back live. We are young talents making way all here on Think Tech Away. I'm Andrea Gabrielli. I'm your host. And every Tuesday we keep an eye on the future with our most brilliant school students as we talk about their science projects. Joining me today is Kyla Kohara from Iolani High School, who carried out a science project regarding the non-tuberculous mycobacteria, which apparently thrive in Hawaii soil. Welcome to the show, Kyla. Thank you. I'm excited to be here. Very nice to have you here. Thank you. So, why do we need to raise awareness on this particular bacterium? What is this bacterium? Well, mycobacteria is, there's a lot of background on it. It's part of a large genus of actinobacteria, which consists of over 190 species, many of which are pathogenic, which means that they can cause diseases in humans and mammals and fish. And two of the common ones, M. leprosy, causes leprosy. M. tuberculosis causes tuberculosis. And everything else in that genus is kind of thrown into this category of NTM, non-tuberculous mycobacteria. And you focused on this particular category for your science project. Why? Well, because we already have a lot of research being done on tuberculosis. Everyone is aware of it. We've all had to get TB tests for work or getting a new job. Right. So, that's really controlled. And I guess the Anson's disease, the leprosy, especially here in Hawaii, we are aware of its effects because of the Kalaupapa. All the people who were affected with this disease were sent in. Father Damien was able to help them. And so we are familiar with this. And so you focused on this particular subject. I'm very curious to learn more about this infection. I believe that your first slide is a schematic of what this infection is about. So, maybe let's have our first slide up. Yeah. Okay. So, what are some of the symptoms? Yeah. So, non-tuberculous mycobacteria can cause pulmonary infections. And they're very similar. They're comparable to that of tuberculosis. Okay. Similar to tuberculosis. But it's a different species. And so it can cause difficulty breathing, chronic coughing, fatigue, weakness. And then it can also just worsen your pre-existing lung conditions. And it just gets progressively more debilitating. So, is this a disease that people who already have lung problems might be more prone to be infected with? Yeah. So, the people who are more susceptible to it are people who have had lung conditions such as bronchitis or also people who are over the age of 65. Oh, okay. People, there are some trends in different ethnicities. Japanese people and Asian Pacific Islanders are one of the people who are at higher risks. Higher risks. Okay. How do you get infected? I believe we have a new slide to describe this. Yeah. So, NTM, they're ubiquitous in the environment. So, you would likely find them in soil. So, it's a bacterium that lives in soil. Yeah. We have it in Hawaii. Yeah, definitely. Okay. And so, we're looking at a picture here. We have a soil with a growing plant, a sprouting plant. But we also have a shower. Yeah. So, Michael Bacteria is hydrophobic. So, while it needs water, it likes to attach the particles and plumbing systems. Oh, okay. So, that's where you would find it. And then you can become infected if you breathe in airborne soil particles or have drinking water that has NTM in it. So, for example, I would get this... I would come in contact with this bacterium if I were, for example, to breathe soil, I suppose, or do you also... And how do you breathe soil, I guess, so we can get the people know? Yeah. Well, it's likely caused by soil being kicked up in the wind or there's just particles floating around and it's likely you just inhale it. Without knowing. What about if you ingest, for example, vegetables that are not well cleaned? So, for example, they have particles of soil on it. Last week, we had a show about this. That's why the question, yeah. That's a good question, actually. I don't think there's much research that's been done on how we are actually getting infected. It's just speculated that that's where infection is being occurred because human-to-human transmission doesn't happen and we only know where it's found in the environment. Right. And talking about this, let's see where we can find it in the environment. So, the geographical distribution of this disease. Yeah. So, this graph is showing the isolation rates of mycobacterium avium, which is a really common species. That's very pathogenic. It's very harmful. So, we're looking at this map in the United States. I'm looking at the highest rate. It seems Florida and Arizona, but also Hawaii. Hawaii is dark in this map. Yeah. So, what I found, the laboratory of clinical infectious diseases actually reported that we have the highest prevalence in the entire United States. We have about 396 cases per 100,000 and that's about 3,000 cases annually of people infected by this. It seems to be quite common. Yeah. And somehow, it's not really well known. Yeah. Okay. So, I'm very interested. Let's learn more about the presence of this disease, this mycobacterium in Hawaii. So, let's have our first next slide up. Okay. So, these are the cases in the Hawaiian Islands. Yeah. So, there's been one study done. It was... The Honda 2016. Yeah. Dr. Jennifer Honda, she's actually been kind of my mentor in this. Oh, okay. She did this study collecting samples, but most of her samples are actually household water samples, so shower heads. Yeah. So, there's kind of a lack of knowledge still in the soil aspect, which is what led me to my interest focusing on that. So, what's your science project about being aware of this lack of information, lack of knowledge, lack of science about this infection? So, I did a lot of research trying to figure out what causes NTM and then where it's found. But the place that research was most lacking in is what kind of interested me. What are the isolation rates of NTM in Hawaii? What does isolation rate mean? That means, so NTM, as we know, is found in the environment, but not all soil particles or not all soil samples would have NTM. So, I wanted to see if there's more concentrated rates of NTM in certain areas around Ahu. Okay. So, basically, you're trying to isolate, so separate this particular bacterium from the other species present that live in the soil. So, let's see some schematics of your experiment. Let's see more about what you actually did as part of the science project. So, let's have our next slide up. So, I decided to research the isolation rates and see if there's any conditions in the environment that cause the higher rates in Hawaii and maybe you can answer why Hawaii is a very high risk area for retaining this lung condition. So, I tried to isolate it from different locations in Ahu and then I predicted that the places with higher acidity, higher temperatures and higher rainfalls would have more NTM. So, the more tropical locations. Okay. I believe we have a map of the study areas. Okay. This is it. These are the samples that you... Yeah. So, those dots are the 11 soil samples I collected around Sancho Ahu. The places I figured would be really high-trafficked. Also, you know, like the Waipio Socket Complex, Manoa Falls hiking areas, places that we really... Get exposed to soil. Yeah. Ah, okay. I see. And so, we have this slide where this is the first goal of your project. We're looking at the second goal here. My second goal is to take the isolation rates, count how much colonies of NTM I can grow per same amount of soil and then take that and compare it to the temperatures and see if there's any correlation between pH, salinity, those factors. So, how do you isolate the bacterium? How do you grow it? Well, I kind of jumped into it. It took protocols given to me from my mentor, Dr. Honda. Right. And so, what you just do is take the soil and turn it into a kind of slurry with water and you plate that onto agar plates so that you can incubate it and grow it up. And you just expose it to detergents or chemicals that might kill non-mycobacterias. This is very interesting. Let's see one more slide where we can see your growing samples, I guess. Okay. So, this is your lab or... Yeah. So, this is actually at my school at Yolani. Yeah. The Yolani school. Yeah. It's kind of broken down into four steps to get the plates in the incubator. I turn it into that soil and water mixture and I work inside the safety cabinet to contain the bacteria. You did this for all of the locations where you collected soil from? Yeah, for example. Yeah. Okay. So, you were able to follow the procedures which your mentor told you? Yeah. They're right? Yeah. I suppose this is a wonderful experience to get to work with. I learned a lot of different scientific procedures, different safety precautions and using different tools. It's really interesting. Yeah. Okay. I'm curious now. Let's see some pictures of these bacteria. Yeah. I have samples of my colonies. This is still your methods, I guess. Okay. These are the... Okay. So, the soil contains tons of bacteria that aren't NTMs. It has bacteria and fungi just living in the soil. All sorts of creatures living in the soil. Yeah. So, I plated that as a control. So, that would show me everything that's living in the soil. And then I repeated the same process, but this time I added a detergent which is CPC. Which is the protocol that's used to isolate NTMs from shower heads and... So, you basically... How do you get rid of the other bacteria in this... So, if you take that soil mix and then add CPC, it's a strong detergent which kills a lot of weaker bacteria. Oh, it kills them. Okay. Yeah. So, they won't grow up on the plates and they won't show up. And mycobacteria is a really hardy cell. So, it's pretty resistant to strong detergents and strong acids. Wow. Okay. So, that can be something to, you know, concern because it seems to be quite resistant to all these chemicals and everything. Yeah. So, I thought it would be pretty simple. Just killing non-NTM bacterias. And I would see, hopefully, NTM grow up on the plates after about three weeks, which is how long we expect it to take. But on my plates, you can see that actually after three days, there's already an overgrowth of bacteria which couldn't possibly be NTMs because of how fast it appeared. But what you say, what you call as the colonies of bacteria, are these sort of brown and greenish dots that we can see in each of the glass samples of this picture? Yeah. So, you see a lot of the same things that are in the control plates on the decontaminated plates, which is not what we're looking for. We're looking for only a sparse amount of growth after about 14 days around. You have to work under certain conditions to grow these bacteria because I suppose a contamination from it would be kind of... Yeah. It was pretty strict. So, I had to make sure I did everything inside a certain room and keep it in one small space where there's an air filter that prevents anything from leaving. Air filters. Okay. That's what you need. And you're watching Young Talent's Making Way here on Think Tech Hawaii. We're talking about these potential dangerous bacteria and we're going to be back for more. We're going to learn more where this bacteria can actually be found here in Hawaii. Stay tuned. Hello, I'm Yukari Kunisue. I'm your host of New Japanese Language Show on Think Tech Hawaii called Konnichiwa Hawaii, broadcasting live every other Monday at 2 p.m. Please join us where we discuss important and useful information for the Japanese language community in Hawaii. The show will be all in Japanese. Hope you can join us every other Monday at 2 p.m. Aloha. And we're back live. We are Young Talent's Making Way here on Think Tech Hawaii. Today we have Kyla Kohara from Iolani High School and we're talking about the potential dangers of this non-tuberculosis Michael bacteria here in Hawaii. So, thank you for being here. Yes. So, today actually we have a special tool. We can join our conversation here live and you can call this number 808-374-214 to join our conversation if you want to ask questions about this bacteria, this disease, this infection with Kyla today. Okay. So, Kyla, before the break we were talking about you were showing pictures of your growing colonies of these bacteria. So, let's see some more pictures. Let's continue to discuss about this and learn more where this bacteria can actually be found. So, my first method of decontaminating non-tiriculous mycobacterias from the soil was kind of ineffective in a way because a lot of non-NTMs were able to survive my decontamination process. And we were talking about the contamination problem issue earlier. Yeah, that's right. So, what did you do to improve on this method? So, I tried another method. I kind of repeated the same procedure but instead of using the agar plate that I had used before I used some that had malachite green which is an antibacterial dye. So, hopefully that would provide a little more pressure on NTMs to grow as opposed to non-NTMs. Is that what happened? Is that what we're looking at? So, that's what you see on the left. Those four plates are actually after about 15 days of growth and absolutely nothing grew. Yeah, I see only the empty plates. Yeah, nothing grew which is a little bit surprising because... So, we have Manoa, Waipahu, Macaulay and Ayia here, these four samples. Yeah. Why did you focus on these specific four locations? Because we showed earlier that you had more. I just repeated the process on a smaller sample just to try to perfect my methods before I went and repeated everything in triplicates so I could have more data. But in the pictures on the right, again Manoa, Waipahu, Macaulay and Ayia samples, I can see that there are some brown spots. So, I guess these are the colonies of the day 15. Yeah, so I tried a third method using sodium hydroxide and oxalic acid. Oh, okay. And these actually provided really promising results. At day 15, there were colonies that looked like NTM, so based on the morphology, you expect them to be these yellow bright round colonies. So, you had three methods to basically grow these bacteria. The first one, it was not strong enough in killing the contaminant bacteria. The second one, I guess it killed everything. Yeah. And so the third one, how do you know that the third one here, we are actually growing these NTM, so this non tuberculosis mycobacteria? Well, we base it off of several factors, one of which is the rate of growth. So, as we expected, it would grow about after two weeks, two to three weeks, and that's when it started appearing. So, that was a good sign. And then we looked at the colors, the size, and just the fact that we did use these detergents that are specific for NTM, it's a pretty good reason to believe. But we wanted to prove even further, identify the species, so then I jumped into a couple more... Let's see our next slide, so we can see... Oh, so you... this is DNA? Yeah. So, it started to get a little bit more technical to identify with positive results. I had to make sure that it was NTM. Yeah, okay. So, it's not just the growth rate, but you really wanted to make sure that's exactly what we had, the NTA, by having DNA analysis. Okay. So, how do you perform DNA analysis in a school lab? I guess this is kind of complicated. Yeah. So, once I have that bacteria on the plates, I took a cotton swab and just basically tried to slice those cell walls so I could release the DNA from the bacteria cells. And then PCR is polymerase chain reaction, which allows me to amplify the DNA and make a bunch of copies that it's... I have enough DNA to send to a lab in sequence. Wow. Okay. So, what did you find out? So, I sent about 11 samples from my previous... the third method that worked best. I sent the 11 samples to the lab and it was actually... Which lab? A large and sequencing lab. So, they do it for you. They take the DNA once you prepare it and make a clean data set. Is that on Oahu or...? No, I shipped it out. Oh, where is it? I'm not sure where it's based, but... Oh, okay, but it's away from Oahu. It's away from here. Oh, so, wow. So, how do you ship? Because it can get, you know, can... He just take it into a little plastic DNA vial and then... Oh, do you have DNA vials that are made on purpose to actually ship? Yeah. Wow, okay. You didn't know you could ship DNA? Yeah. Okay. So, the lab basically sent you the results back? Yeah. So, after waiting about a week, they were able to send me the DNA sequences and then I would take those and put them into a database. It's called GenBank Database, which has archived every researcher's DNA sequences. So, they put a name to it and they put the sequences that they've found out there so that we can compare it to what we find in our own research studies. So, what did you find with respect to the individual samples? Do you have more bacteria than Manola or... So, as it turned out that from my 11 samples, none of them were NTM's, which is really shocking. Oh! Yeah, it was... Initially, when I formed my hypothesis, I kind of had assumed that I would find a lot of NTM's simply because of the high prevalence here. I thought there would be a lot, but as it turns out that there's a lot of complications with the soil compared to the household shower heads, it's a lot easier to extract NTM from those that aren't exposed to as many bacteria. Right. So, a lot of non-NTM bacteria got into my samples and so actually found some bird flus. I found different skin type of infections, which weren't NTM's, but that kind of led me to choosing new research questions, kind of adjusting my focus. So, basically, science is a learning process. You start with an hypothesis, you make an experiment, but then the experiment turns all your hypothesis around. So, what are you going to focus now on? So, I kind of adjusted my question. I kind of realized that before I can even get those isolation rates, I have to perfect the methods. The method of isolation, actually. So, a lot of my research became, you know, that trial and error process of finding the right decontamination detergents, and since we know that there's actually over 190 species of mycobacterias, there's actually different types of resistances. Some are more resistant to CPC, which is what I used. Some will survive that and some will actually be killed by those detergents. Right. And so, we have to keep adjusting our methods specific to Hawaii. Because you don't really know, there is no research published regarding what kind of detergents are good for just keeping the NTM and killing all the rest. Yeah. Okay, so that's very interesting. Now, you presented all this work at the, what is state science and engineering fair, how was this experience? It was a great experience. I got to talk to a lot of judges who gave me a lot of insight on my research. That's right. That's what I would think. Yeah. Because to make more research questions and basically try a better way of making this experiment, you must have got a lot of feedback from. Yeah. So, I kind of explained the same struggles that I've had, the different roadblocks. And they gave me some suggestions such as trying to replicate the exact same procedures that are done in the previous study, as well as swabbing my control plates and seeing if there's any NTM in that. Trying different methods that would help me pose new future questions for future research. Wow. Okay. So, are you planning on carrying out more, you are a 12th grader at Yolani High School. So, next year I guess you will be going to college? Yeah, next year I'm going to be going to college in the University of British Columbia. In Vancouver, Canada. Wow. So, are you going to focus more on this particular subject? Before I leave, yeah. I hope to this summer collect more data and try the different procedures that have been recommended and see if there's any better results in this. Are you going to be working with the same mentor or also other? I'm going to be doing my research still at Yolani and she's been helping me along the way telling me different things I can do. So, yeah, she'll definitely be helping me. Okay, but I was thinking maybe you could find also some potential collaborations with the University of Hawaii and Manoa. Yeah. Actually, throughout my process I was also helped by Dr. Harry and Bibi Davis. They have helped me adjust my presentation, helped me figure out methods of collecting my data such as the pH and the different weather climate data that I needed. So, they've also given me some advice. And we thank Harry and Bibi. Hi, Bibi. And so, now with all this background, so you mentioned you have a variety of experience now on this particular subject. What will you study in college then? So, I'm going into the Faculty of Science at UBC. Yeah. And I'm not entirely sure what my major is going to be, but I'm hoping to either do biochemistry, biochemistry, so you can learn more about this. Yeah. You know, time really flies so we have about one minute left in our conversation. What would you tell our audience today to raise awareness on this particular disease? What should people be aware of? I definitely think people should be aware of where NTM is, where you might be at risk of NTM. And also, the different symptoms that it causes, which is really similar to tuberculosis. So, I want to gain awareness of that as well. This really hazard those symptoms that can cause long problems and everything. Thank you very much, Kyla. Thank you for being here with us today. And so, you've been watching Young Talent's Making Way, only here on Fintech, Hawaii. And next Tuesday, we will be back for more, more Young Talents here. Stay tuned.