Hello, and thank you for listening to the Microbid Key Podcast. Here, we will be
discussing topics in microbial bioinformatics. We hope that we can give you some
insights, tips, and tricks along the way. There is so much information we all
know from working in the field, but nobody really writes it down. There's no
manual, and it's assumed you'll pick it up. We hope to fill in a few of these
gaps. My co-hosts are Dr. Nabil Ali Khan and Professor Andrew Page. Nabil is a
Senior Bioinformatician at the Center for Genomic Pathogen Surveillance,
University of Oxford. And Andrew is the Director of Technical Innovation for
Theogen in Cambridge, UK. I am Dr. Lee Katz, and I am a Senior Bioinformatician
at Centers for Disease Control and Prevention in Atlanta in the United States.
Hello and welcome to the Microbid Key Podcast. We have a special episode today
where we are actually sitting in the same room as a few people from CDC. We have
here today Christine Lee, Sinead Waters, and Megan Mickham from CDC. And
Andrew's on the line. Nabil couldn't be here today, unfortunately. We thought
we'd talk a little bit about outbreaks. And honestly, I've been waiting for them
to be published so we don't get in trouble about talking about insider
government information. So welcome, Christine. You're head of the Live Lab, but
that also includes things like cholera. And Sinead is also in your team, the
bioinformatician, the hero, sorry, hero to us, sorry, you're both heroes. But
this is a bioinformatics podcast, so I have to bring that up. And I don't know,
let's just get into it a little bit. So you work at the CDC, just tell me about
what the Live Lab is and kind of what your day-to-day is, Christine. Yeah, Live
stands for Listeria, Yersinia, Vibrio, and other enterobacterioles. My day-to-
day, depending on what I'm doing, can be, yeah, it's a lot. But we're part of
the National Reference Laboratories and the Enteric Diseases Laboratory Branch.
And I think the most important commitment we have is to our state and local
public health partners here in the United States. And so foodborne illness is a
priority of our division and our branch, we're the Enteric Diseases Laboratory
Branch. What we try to do is understand any kinds of emerging trends and
patterns and help our state and local public health partners characterize these
organisms, especially if they come across some unusual or weird organisms.
Cholera is not endemic here in the United States, but we oftentimes get travel
importation cases and the causative agent of cholera is a special kind of Vibrio
cholerae, which we could get into later. But Vibrio cholerae is present in the
United States domestically, though it's not the kind that we observe in global
settings. And we can talk a little bit more about the global epidemiology of the
cholera strains that in the past couple of years have caused resurgence in areas
where cholera had not been detected for many years. Awesome. And then you have a
bioinformatician in your lab, and that makes you pretty lucky, at least in my
opinion. I was intentional about that. Sine Wolters is here. She's really
talented. I'll let her introduce herself. Thank you, Christine. So, hi. Yes, I'm
Sine Wolters. I am the bioinformatician for the Live Lab. My role, I guess, in
the simplest words, is that I analyze the whole genome sequencing data of the
organisms Listeria, Yersinia, Vibrio, and Enterobacterialis. I also am a
certified CLIA tester, so I get to actually run the wet lab protocols of whole
genome sequencing, so I can sequence DNA of bacteria. That's awesome. I didn't
know that you did CLIA, too. That's actually a big deal. Yeah. Wow. Thanks to
Christine. I get to do a lot of, you know, trying out different things in the
lab, so, wow. So, Sine is an expert about everything whole genome sequencing,
from soup to nuts, and about enterobacteria. CLIA, for those of you who are not
familiar, is a way that we can conduct patient-level reporting, and because it
involves PII, anybody who is involved in CLIA testing undergoes a strict set of,
how do you say, ethics and patient identity sort of protocols to ensure that the
quality of the data is reproducible and of the best quality, and that we keep
patient identifiers private and conduct all of our testing with the utmost
respect, and because these are clinical cases, these patients come to the
hospital really sick and ill, and we want to make sure that we do our very best
to really understand the pathogen that was the causative agent of their illness.
All right. So, I've been really excited to do the actual topic, so I'm going to
jump into it, though. We wanted to talk about cholera today and the Haiti
cholera outbreak, and a little bit of, I guess, a little history for myself is
that I was brought into the live lab originally more than 10 years ago to look
at the Haiti cholera outbreak, so this is sort of a joke question, but a leading
question. Did we already have a Haiti cholera outbreak? Yeah. In 2010, there was
an earthquake in Haiti that really caused devastating environmental damage and
it, how do you say, caused havoc on the water infrastructure systems, and I
think in the aftermath of that came the emergence of a really deadly strain of
Vibrio cholerae that really affected thousands of people for many years, and
Lee, I think you were on the boots on the lab ground doing the analysis for
this, and I think this outbreak in 2010 was the first time whole genome
sequencing was used to solve an outbreak. Am I understanding that correctly? Um,
there might have been some other smaller cases, but this was definitely a very
early high-prominent case, for sure. Can I ask, how did you link or do source
attribution for that? Because I remember there was a big controversy over it.
There was a big controversy. At the time, the opinion of CDC was that we weren't
sure where the source was, but eventually over time, a few years later, WHO
definitely had an opinion, and then CDC eventually had an opinion, and I won't
go too much into that. You're going to get me in trouble, but for me, the
genomics, the bioinformatics is that we did make a phylogeny. We did see some
clustering between Nepalese genomes and Haitian genomes. But then you guys, I'm
going to bring it back to you guys, though. So, how did you do it in the latest
outbreak? What is the latest outbreak, and how did you look at it? So, there was
another devastating natural disaster event in Haiti. End of 2021, it was a
hurricane that blazed through the country and, again, disrupted the water
infrastructure, and some of the sociopolitical dynamics complicated the access
to clean water for the Haitians, which is really unfortunate to hear. And there
was a reemergence of cholera. The country was actually getting ready to put in
an application for cholera-free status through the Global Task Force for Ending
Cholera Control, which is contributed by the WHO, and that requires three years
of no detected clinical cases or no confirmed clinical cases of cholera, and
Haiti was on track to do that. But unfortunately, through natural disasters and
some of these events in-country, cholera reemerged, and it was really
devastating, I think, for the communities because, again, the water
infrastructure was not stable, and a lot of people got really sick. With the
support of our laboratory partners at the National Public Health Laboratory in
Haiti, we were able to help identify some of the strains using whole genome
sequencing and do an analysis based on some of the early cases from the
outbreak. I see Andrew's hand up. Yeah. So, I was just going to ask, were there
new introductions, and could you prove that by genomics, or was it one emergence
of one clone, or was it multiple reemergences of many, many different lineages
within the phylogeny? So, what did it actually look like? Yeah. So, since I
actually did the bioinformatic analysis on this, we received a subset of samples
from the outbreak. One of our colleagues here in the Enteric Diseases Laboratory
Branch, her name is Marianne Ternsik, has had a longstanding relationship with
our Haitian partners and was able to go and deploy to support the response as
part of the laboratory pillar, and was able to coordinate a subset of samples
from multiple regions throughout the country during this outbreak, and Sine can
speak to a little bit about the genomics after the sequencing was conducted.
Yeah. So, we took the whole genome sequencing data, and we used a tool called
Lifesat that Lee actually championed. And so, what Lifesat does, it looks at the
single nucleotide polymorphisms, or the genetic variant.  variation of single
nucleotides in, I guess, the genomic data. And so, using that, we compared the
variation between the new samples that we collected from the reemergence, and we
compared it to historical strains to see if, I guess, the DNA was conserved or
if it changed. And if it changed more, then we would say maybe this is a
reintroduction versus if it stayed relatively the same, it was probably the same
strains. So, what we did find out was that, based on the SNP analysis, they
were…it was about like 0 to 25 SNPs differences between the newer data versus
the historical strains. So, that just tells us that the strains are relatively
similar. So, it was probably…it is related to the historical strains. And then,
Sine, what years did the data include? We had strains from the initial 2010
Haiti outbreak, and I think on your tree, there's another set, subcluster,
right? Yeah. So, yeah, what were the data that you analyzed when you say 0 to 20
or so SNPs different? How was that over time? Yes. So, the collection years of
all the strains ranged from 2010, starting with that first outbreak, all the way
up to 2022. So, some of those years were like 2010, 2011, 2012, 2013, 2014,
2015, 2016, and 2017. So, the 2022 strains were about 0 to 25 SNPs differences
from the 2010. And so, we usually use the threshold of 10 SNPs as being very
closely related. But because the range was from 0 to 25, it was still related,
but not identical or that similar. So, based on that, we know that they
are…these 2022 strains were related to the pre-existing strains. Kalmarai has
obviously got two chromosomes, and how does that impact your pathomatics
analysis? Yeah. So, when you have two chromosomes, it might affect things, but
as long as you have a good reference genome, it doesn't quite change things when
you're running SNP analysis. I would say that the place where we get in trouble
is that there's the superintegron on the second chromosome, the smaller
chromosome of cholera. It's about a megabase and has a lot of transposable
elements. And the cool thing about LiveSat is it will mask those places. It runs
a, quote-unquote, phage-finding program, but really it's just finding anything
that might look like a mobile element or something from a phage. So, those
places are masked, and the SNPs actually won't appear there, so it won't really
break the evolutionary model that we put in there. So, you guys did SNP analysis
on this. That's awesome. Is there something that could have informed you more
that you would have liked to run or is there something else you'd like to run
that might, I don't know, but you have time constraints and stuff, so maybe you
just had to fit everything into one paper at a certain time range. Yes, that's a
good question. So, the re-emergence suggests that this particular strain had
been circulating either in the environment or through the population for more
than a decade, right? And that's an interesting, how do you say, an interesting
characteristic about Vibrio cholerae and how durable it is as a pathogen and how
it can survive in the environment or in reservoirs, right? People could have
been shedding over time and no detections would have been made. What I think
Marco Salemi and Glenn Morris' group were able to show through a paper that was
released at around the same time was evidence from a molecular clock analysis
suggesting that the strain might have been circulating in the environment. They
had a relatively close strain from environmental samples that were related to
the re-emergent strain. And I think that that is a controversial topic, doing
environmental surveillance for cholerae can be, I think there's a lot to unpack
there and I won't go into it too much, but environmental sampling with COVID,
for example, has been shown to be pretty powerful with wastewater surveillance
set by the precedent of polio surveillance in wastewater as well. And I think
that the challenge with Vibrio that's different than polio or SARS-CoV-2, for
example, is that Vibrio live in the water and separating out the natural Vibrio
that live in the environment versus those that cause true pandemics and
epidemics in these huge outbreaks will take a little bit more finessing in terms
of the bioinformatics and then metagenomic piece of it. But I think we're
getting there and I said I wasn't going to make an opinion about that, but I
think that linking to and understanding how these strains can persist and
continue to subtly evolve over time is a compelling question. And I wish that we
had surveillance data that could couple the clinical results that we see. But
again, to answer your question, is there anything that I wish we could have if
we had more time, that would definitely be one of them. That's awesome. So maybe
onto the bioinformatics, did you guys do long-range sequencing on any of the
strains and did you use those as your reference genomes? Not yet, but we use the
type strain, the Haiti cholera strain that Lee had defined as our robust
reference and we have long-range sequencing for that. But we use, I think, the
short-range assemblies for that. I just want to put it in a note, I can't take
by far the credit for that reference genome. Like that was a huge, huge effort
from us and multiple partners, especially Canada. So I can't take credit for
that one, but I appreciate it. Well, if we can thank Canada and every
opportunity that we can get, I'll take it. Yeah. Can I ask a question about drug
resistance and have you been tracking it, AMR, and is there any emergence of any
more or less or, you know, how is that working out? Because every environment is
different. Yep. And Cindy can answer the question about the AMR because she also
did that analysis in this paper. Yeah. So we included the AMR resistance data in
the paper that we published there. I was actually assisted by Jess Chen. She is
one of the more senior bioinformaticians here. We found that really three
strains had a unique AMR profile of, there were numerous genes that were present
versus absent. And so it was really three that were different from the majority
of the samples. So based on the resistance, we know that they are, it's still
the same profile. And so, again, comparing that to the SNP analysis, we know
that the strains are very similar. I don't know much about cholera. So how does
resistance come into cholera? Is it point-based mutations? Is it genes coming
out of plasmids? You know, how does it actually work? Yeah. So they use, and the
paper goes into more details about the methods. So I'll just take a high level,
but ResFinder was used, and the two particular genes that we interrogated were
gyrase A and Parse C. And I see Andrew nodding his head, so that means something
to him, but in any case, but the thing about Vibrio cholerae that I learned from
the legend Cheryl Tarr, who was my predecessor and was really pivotal in
integrating whole genome sequencing for enteric foodborne surveillance here at
the CDC and working with our Canadian partners is that Vibrio cholerae doesn't
change too much over time. It's pretty clonal. And so when we do see
antimicrobial resistance emerging, we don't always attribute it to evolutionary
changes in a specific strain. It's introduced by another strain that's different
that had acquired the resistance possibly elsewhere. Yeah. So that leads into
maybe be a final question just because we're out of time, but so in 2010, the
controversy was, did cholera come from the environment or was it introduced? And
now we have this outbreak a decade later, do you feel like you have that
question answered for this particular outbreak? Like I said, I think if we had
additional data, additional clinical surveillance over time, right? Like Susan,
I mentioned that we included some genomes from 2016, but we don't have any
recent clinical cases reported. And so we don't have a really good benchmark to
say that this had been circulating in the population. So that's an unknown. We
don't have a control for that experiment, if you will. And then environmental
sampling is not something that's routinely done. And so we don't have the data
for that. So I mean, if I want to be true to my scientific inclinations, I would
say we don't have any evidence to suggest either. But we do have some
information from the sequencing data that this is very closely related to the
strains that were circulating from the outbreak in 2010 up through 2016. So
maybe that's also an answer to the previous question, if you could do extra
stuff, maybe just having routine surveillance.  So, the power of routine
surveillance is really important, especially in countries, and if I can mention,
you know, cholera has made a resurgence in many global countries. The WHO has a
nice little dashboard, but we've seen cases and outbreaks in areas where we
thought cholera had been previously eliminated, and there's a lot to be said
about water warming. People in Ontario love to live in warm waters, and they're
thriving, and, you know, there's a lot to be said about the movement, the global
movement of people and things is unprecedented than a decade ago, even. So I
think there's a lot to be said about the changing world, the geopolitical things
that we can't really comment about, but the reality that we live in is totally
different than it was in 2010 in the way that we live our lives and travel and
move, and the way that water is protected is also different when there are
disruptions as well. So, right, what can we do? I think the importance of
surveillance, especially in areas where cholera is about to be eliminated,
that's where tools like whole genome sequencing can be very powerful and
important for really getting at the answer of are we close to elimination or
not. All right, so you guys have been awesome. I think that you learned a lot
about cholera, and you guys are doing a great job. I appreciate it. Thank you so
much for listening to our podcast. If you like this podcast, please subscribe
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Follow us on Twitter at MicroBinfy. This podcast was recorded by the Microbial
Bioinformatics Group. The opinions expressed here are our own and do not
necessarily reflect the views of CDC, Theogen, or the Center for Genomic
Pathogen Surveillance.