Approaches to Mitigation of Antibiotic-containing Agricultural Runoff

Approaches to Mitigation of Antibiotic-containing Agricultural Runoff


[Music] Thank you for the opportunity, and I don’t know if you guys got any snow up there, But, we’re certainly getting it here in Ames today. I am the Associate Professor in Agricultural and Biosystems engineering at Iowa State University. And ah, I’m really pleased to share some of our work on antibiotic resistance and mitigation. We’ve been working on this since I came to Iowa State in 2008 and this is one of the first projects that we started working on, so Amanda asked me to kind of frame this as far as what are some of our biggest questions and some of our most exciting results. so I thought I would start out with the questions and something that’s probably by touched on a little bit today is, How do we monitor resistance in the environment?
So we have done some monitoring of the actual antibiotics. There’s a lot of
challenges with that and so much of our more recent work has actually been
looking at the microbial resistance and so we do a lot of different things. First
of all, we look at the phenotypic resistance. So we’ll plate for fecal
indicator bacteria and then in the cases when we know what antibiotics are being
used, we’ll add those antibiotics to the auger and get a fraction of phenotypic
resistance. We’ve also been doing a lot of qPCR to try and detect certain
resistance genes and mobile genetic elements. So a lot of our work has been
with the swine industry- the pork board- and so we’ve been looking for macrolide
resistant genes and also we’ve been expanding that to some tetracycline
resistant genes. And then we’ve also been collaborating with Dr. Adina Howe, who
does bioinformatics and metagenomic sequencing. And so we have been working
with that to try and expand beyond these kind of key genes that we’ve been
working with to look at the manure- derived or the resistance community. So
how can we track the resistance in the environment especially when we
try to scale up beyond the farm and we have different facilities with different
antibiotic use patterns? How do you really track what that resistance looks
like at a larger scale? Some of the other questions that we have, which I don’t
necessarily have an answer to, is how is the VFD going to impact environmental
resistance? So we’ve got some pre-VFD data and it’s just going to be really
interesting to see if we see impact fairly quickly in reduced resistance or
if it’s more of a lag time like you know I think of phosphorus in the
environment with this legacy effect and what kind of legacy effect might we
see with AMR? And then what is the role of environmental AMR in the human
health picture? I think that’s something that we’d all like to have a better
sense of. So key points for AMR mitigation: We are currently
working on the manure management aspect. So what are some ways that we can treat
the manure before we apply it to the land? So can we mitigate prior to land
application? And we’ve been studying in- field management. So different methods of
applying manure, manure application timing, and then also there’s some new
work starting on edge-of-field management, specifically with prairie
strips. I’m going to talk through the results of some of these studies. So this
is our manure mitigation experiments which have been funded by the Pork Board
and we’re considering a lot of different options to see what kind of reductions
we can see in the phenotypic resistance and then resistant genes. The gene that
we are working with is ermB and so that’s a macrolide resistant gene. So our
manure is coming from the farm where tylosin is used. Up at the top you see I
have ionophore addition and solid separation. And we tested the ionophore
addition just because it’s used in the pit foaming barns that have pit foaming
problems. We’ll add the ionophores to try and reduce the misantigen populations
and so we wanted to see if that had an overall impact. So far it doesn’t seem to have
any impact on the resistance community, and then solid separation is still
pending. And the first figure that I have here is showing the two-phase storage. So
if you look at the left we have no fresh manure added, and on the right is the
scenario where fresh manure is being added on a continuous basis. So the no
fresh manure added would be a situation where there is a pit underneath the
facility and then there is secondary storage. And that would allow the manure
to sit for about six months before it would be land applied. And in this case
you can see that that y-axis is the ermB copy numbers and then the x axis is
the time since the start of the experiment. And when no fresh manure was
added, it looked like in the early populations we were starting to see a
decline over this time period, except for that last data point. But then when you
look at the situation where fresh manure is continually being added to the system,
we continue to see an increase in our ermB copies. Next, we looked at
anaerobic digestion. We considered three different temperatures and the results
are the days for a one-log reduction of tylosin A and tetracycline-resistant
Enterococcus. And so just preliminarily, you can see that there was, at the higher
temperatures, a shorter period for that log 1-log reduction to occur than at
the 25 degrees Celsius. The third option that we considered was centrifugation.
And this is just looking at the Enterococcus tylosin resistance over a
range of different centrifuge times and centrifuge forces and you can see that
over, you know, either a longer period of time or a higher gravity force, that we
were able to see reductions in the resistant populations. Okay so this is
all still lab scale stuff but we tried to we worked with Dr. Andersen who does
manure management here at Iowa State to identify practices that would be
feasible for farmers and ongoing results will be
expanding the qpcr and then to do more of the sequencing. The next slide is
looking at some of our in-field management studies. And these studies have been
conducted at the North Iowa State Northeast research firm and essentially
one acre plot that had either received a manure or a no manure control- and they
have a long history over 30 years of no manure- and then we monitor tile drainage
systems. So we’re really looking at the subsurface transport of these resistant
genes. And the research question is looking at both the crop rotations. So
we’ve looked at a corn and soybean rotation. More recently we’re looking at
corn on corn and how do those resistant genes move to drainage water? And from
this, we can see that in the soil the resistant gene levels returned to that
baseline concentration within about six months to a year after manure
application. So if manure application occurs in the fall when it’s dry
conditions, in some cases, as kind of the climate trend has been in the past
has been dry conditions in the fall, and especially if that manure application
cannot be applied late in the year prior to when soil temperatures are dropping
but before freezing. And then there’s a lot of mitigation that can take place in
that soil and reduction in the resistant gene concentrations before spring when
water starts to move through the system and we start to see stuff moving off
site. We did find that plots receiving manure had greater concentrations of erm-
B and F, so two macrolide resistant genes. And also we’ve demonstrated that the
fecal indicator bacteria do not correlate with these erm genes. So the FIBs
that we’ve used for many, many years to detect the risk to human health
really aren’t correlating with any of the resistant gene concentrations that
we’ve been studying. So this is a prairie strip. So this is new work funded by USDA
to look at prairie strips as a mitigation strategy, so an edge of field
mitigation strategy for AMR. And then this would be really focused on the
surface path waste. So the overland flow, the water
would flow into the prairie strip where the resistance community could be
intercepted, pushed into the soil because prairie strips will increase
infiltration. And then we also know that the microbial community and the prairie
strip is different than an ag growing soil that’s under corn and soybean or corn to
corn rotation. And so we are wanting to test to see what kind of persistence
occurs both in the AG soil and then within the prairie strip community. So
the picture there is of some rainfall simulation experiments that we
conducted this past fall. And then the figure is showing essentially
differences in the microbial community across a gradient starting in the
field and then moving into the prairie strip. So you can see that the green
section- that’s the microbial community- and the corn and the soil that was
planted to corn, there’s kind of a stripped corn interphase. And then as you
move deeper into the prairie strip you can see that the community shifted and
we could see differences there. There was also an old fence line that was you know
clearly had not been in ag production for many years enough. So we
pulled some samples from that site and that community was also completely
different than the corn and the prairie strip. So a lot more work in this area
over the next couple of years. And then the last project that I wanted to share
with you is some of the watershed scale monitoring. So you can see the map of the
watershed. That’s the South Fork of the Iowa River. It’s a large watershed and
each yellow dot is a location of a calf tile and so this is a really heavily
manured watershed. And we partnered with USDA ARS. They have an intensive
monitoring network in this watershed and we were able to get drainage and river
water over many over several years and from multiple locations within the
watershed. And so if you look at the table, on your far left there’s
three surface locations. So those were samples that were collected essentially
from the river and then two locations that were from tile drains. And then the
drainage area that corresponded to each of those catchments or
sub-watersheds the number of samples. And then what I wanted to point out is the
two for ermB and ermF, the limit, the percentage of samples that were
above the limit of quantification. And we always saw greater presence of
resistance genes in the drainage samples than we did in the surface sites. And so
this has been, it’s been really interesting to see that it also, as we
talked a little bit about today, I think it emphasizes the role of the soil in
resistance. If there’s mobility and gene sharing and gene transfer that’s taking
place, a soil environment is a place where that could happen. And that’s
possibly reflected here in these results as we see these higher levels of
resistant genes coming through tile lines and surface. With any other, almost
any other pollutant, you would expect to see higher concentrations in in surface
runoff so in a surface driven system than in a subsurface system. Usually you
see a lot of filtration that takes place as the water moves through the soil
profile and, except for nitrate of course, but in this case where it was very
interesting. Other studies in the same watershed have looked at E. coli, so
just the presence of E. coli, in the watershed and they’re seeing greater
concentrations in the surface sites than in the tile. So more work is looking at
this watershed. We’ve sequenced the samples now and are looking at the
16s and the microbial communities among the surface and the tile drainage
points. That’s all I had. Just wanted to make sure and acknowledge the
collaborators that we work with: Drs. Adina Howe, Dan Andersen, Tom Moorman
and Heather Allen. And Tom and Heather both with the USDA ARS labs here in Ames.
We’ve had grad students and then funding has come from the USDA food safety grant
and a couple other USDA grants as well as the National Pork Board. [music]

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