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Britta Leverentz and William
Conway:
Focusing on Phage-Enhanced Food Safety
USDA Agricultural Research Service plant pathologists Britta
Leverentz and William Conway, working out of the ARS Produce
Quality and Safety Laboratory in Beltsville, Maryland, recently
announced the successful conclusion of the first phase of a
study which sought to control foodborne pathogens on fresh-cut
fruits and vegetables through the use of bacteriophages—viruses
that infect and kill bacteria. This first-of-its-kind study was
part of a cooperative agreement with Intralytix, Inc.—a
Baltimore, Maryland-based Corporation which provided known
phages for Salmonella strains as a model. FoodTechSource.com
recently spoke with Leverentz and Conway to better understand
the impact of their work.
FoodTechSource.com: Can you provide an overview of your
research?
Britta Leverentz, Ph.D.: We investigate food safety issues—in
this case the elimination, using biocontrols, of foodborne
pathogens present on the surface of fresh-cut fruits and
vegetables. We have investigated a number of different factors:
the role played by pH, acidity and storage temperature, and the
potential use of bacteriophages, which can target specific
bacteria.
FTS: What are phages and how do they fit into your study?
Leverentz: Phages are naturally occurring viruses that attack
and kill bacteria. They have a protein head and a tail. Inside
the head is their DNA—genetic information packaged in a protein
coat. The phages reproduce by attaching themselves to the
surface of the bacteria, then inserting their DNA. They then
begin to replicate until their numbers are sufficient to rupture
the bacteria, killing it. Then they move on to the next host.
Phages are the focus of our study because they are very specific
and can be used to target specific bacteria. For example, if you
have a phage that is active against salmonella it may not be
active against other bacteria.
William S. Conway, Ph.D.: That is important because many of
the organisms present on fresh fruit are beneficial organisms.
And if, for example, you use a chemical like chlorine to kill
the pathogen, it kills everything—harmful as well as helpful
organisms. But the phage, being more specific, kills the harmful
bacteria while allowing the beneficial organisms to remain on
the surface.
FTS: I’m very curious about this specificity aspect. How
specific are these viruses? Because you can have both good and
bad E. coli in your digestive track as well; can you capture a
phage that can specifically target the harmful E. coli so if it
got into your gut it would leave the beneficial E. coli alone?
Leverentz: Certainly. If it’s a phage that targets E. coli,
it doesn’t necessarily target all strains of E. coli.
Theoretically you should be able to find and isolate a phage
that targets only one particular strain of E. coli.
FTS: Did you attempt to do that in your study? Or were you
just studying phages as a broad, overall tool?
Leverentz: We used phages as a tool here at the USDA. We were
investigating how effective phages can be at eliminating
foodborne pathogens and how the technology might be applied. The
phage itself is supplied by a Baltimore company called
Intralytix. We have a Cooperative Research and Development
Agreement with that company and they isolate the phages.
FTS: So, Intralytix will be taking it to the next step when
you’ve finished with your work?
Leverentz: We will work together on a pilot project to
address issues of applying the technology on a larger scale; we
do expect that Intralytix will commercialize the technology, as
stipulated by the current CRADA between the USDA and the
company.
FTS: How did you proceed with your project? What was the
protocol?
Leverentz: We tried to simulate in-store conditions and
procedures. We cut up fruits and vegetables and contaminated the
samples with salmonella bacteria. Then we applied the phage,
using a variety of different application methods and mixtures,
and stored the produce under a variety of conditions to see if
that had an effect. In recording the results we isolated back
from the tissue to determine the number of bacteria that
remained and also to determine if the phage was still surviving.
FTS: When you say “a variety of mixtures” what do you mean?
Leverentz: What I mean is that we tried a variety of phage
“cocktails.” The phage is specific, but there may be several
different strains of the bacteria present. Also, the bacteria
can mutate. So, we use a phage cocktail that will be effective
against a broad selection of the bacteria we want to target.
FTS: How were these phage cocktails applied to the fruit?
Sprays? Washes? Dipping?
Leverentz: We had small specimens and just used a pipette to
apply a specific amount of liquid to the produce.
Conway: This work really is in its infancy right now. No one
has done this before on fruits and vegetables. This is the first
step. We need to see first if it is going to be effective—which
it seems to be—and then we can develop an effective method for
applying it. Spraying would be the main way to apply it because
we’d need to put a spray somewhere along the line where they
actually process these items. But right now we’re just in the
process of seeing how it works, and how we can tweek it so it
works even better. The next step would be to run some type of
pilot test so it might be better adapted to the needs of
industry.
FTS: Have phages been tested on any other food products?
Conway: Intralytix is presently doing some research on eggs
and poultry, and I heard it was getting close to the commercial
stage. You see, up to this point most foodborne pathogen
outbreaks have involved meats and poultry. But with fresh-cut
produce gaining popularity the potential for outbreaks in this
area is increasing. That’s why we undertook our study.
FTS: Did you only study the effects of phages on fresh-cut
produce? Because produce can get bruised during distribution as
well and I would think those knicks and cuts would allow
pathogens to enter the produce.
Conway: Certainly the main application for this technology
would be fresh-cut fruits and vegetables, because it’s very
difficult for these foodborne pathogens to multiply on the outer
skins and peels of whole fruits and vegetables. On the other
hand, yes, you do get damage during handling, and phage use
could prove beneficial in the distribution phase. But when
produce is cut, that’s when pathogens are provided with a fresh
substrate where they can really multiply and take hold.
FTS: How effective are phages in controlling foodborne
pathogens?
Leverentz: We got a 3- to 5-log reduction in organisms,
depending upon the treatment. On apples we still have a problem
with the acidity because a phage does not function well in an
acidic environment. But on honeydew melons, for example, the pH
is just right and the numbers can drop up to 5-log. That’s the
upper end.
FTS: Are those the numbers you are looking to get?
Leverentz: Yes, the industry standard is a 5-log reduction.
Using chlorine, for example, you get maybe 1.8- to 2.5-log
reduction. So it’s more effective than chlorine.
FTS: Does temperature have any effect?
Leverentz: Phages are more effective at low temperature. They
are effective at higher temperatures as well, but the growth of
the bacteria is also higher at those temperatures.
Conway: Most fresh-cut fruits and vegetables are intended to
be stored at about 5°C, but often they are stored at 10°C, which
is an abusive temperature. So, most of our work was done at
between 5 and 10° because that is what is actually happening in
industry.
FTS: Did you focus solely on salmonella or did you attempt
cocktails that targeted multiple pathogens such as both
salmonella and E. coli?
Leverentz: Right now we are not mixing the different phages.
We could, if we wished, use a cocktail that would target, for
example, Salmonella and E. coli, as well as listeria—and that is
something we will probably try down the line. For this study,
however, we targeted only Salmonella, and we used a cocktail
that included maybe four to six different Salmonella phages. Our
primary reason for this was to study the potential for the
bacteria to develop a resistance. With antibiotics, for example,
bacteria try to develop resistance—and often succeed. But by
using a cocktail, if the bacteria try to develop resistance
against one phage, there are four or five other phages present
which they will not have resistance against. It is much, much
harder for the bacteria to overcome that.
FTS: Have you any suspicion that if you mix the phages for
Salmonella and E. coli, for example, there would be a drop in
effectiveness? Or are you assuming that down the road when you
try that the response will be equally effective?
Leverentz: I don’t see a reason why such a cocktail would not
be effective, because the bacteriophages do not compete. They
are very specific for their target organism. But that’s really
speculation at this point.
FTS: In the area of phage collection, is it easy to get new
phages or phages that respond to new bacterial strains that
might develop? Do researchers just find a phage that works for
the new strain and then just multiply it commercially?
Leverentz: Yes. It’s quite easy to isolate lots of different
phages. Intralytix actually gets most of its phages from the
water in the Baltimore inner harbor and Chesapeake Bay. There
can reportedly be up to 8 million phages in one milliliter of
fresh water. It’s just laborious to find the right one.
Conway: Phages are nothing if not plentiful. I have heard it
reported that if you weighed all the elephants in the world and
all the phages, the phages would outweigh the elephants.
FTS: Is there any reason to use genetic manipulation on these
phages?
Leverentz: I don’t believe there is reason to do any genetic
modification.
FTS: In terms of bringing this technology to market, what
problems or dangers do you foresee?
Leverentz: I don’t foresee dangers. It is a very safe
technique to use. In creating the phage cocktail, the different
strains that are used would probably need to be rotated or
renewed after a while to make sure the bacteria are not gaining
resistance. That would be an ongoing process. There are also
issues with large, industrial-scale production that would need
to be worked out—particularly in regard to the method of
application and storage temperature.
Conway: It also would be something of a long, drawn-out
affair because anytime you have anything to do with food you
need FDA approval and that takes time. But I don’t see a problem
as far as danger to humans. In fact, phages were used to treat
human maladies back in the 1930s before antibiotics became
popular. But now with resistance to antibiotics increasing
somewhat, phage research has found renewed interest.
FTS: How long do you believe it might take?
Conway: To get clearance I honestly couldn’t say whether it
would take months or years.
FTS: Would this fall under the Generally Regarded as Safe
(GRAS) classification? Or since phages do have an effect on
bacteria in the body might you need full-blown FDA approval?
Leverentz: That’s something the agencies responsible would
need to answer. I certainly would consider it as GRAS, but that
is not up to me to say.
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