The Potential for Antibiotic/Biocide
Cross-resistance
DR. LEVY: Thank you very much. Thank you
to the organizers.
I want to say at the outset that the firm
that was mentioned early on is Paracheck [ph]
Pharmaceuticals, and we are not in the business of
making antibacterial consumer products, but I am
president of the Alliance for Prudent Use of
Antibiotics, whose mission is to see that
antibiotics retain their efficacy in the treatment
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of human, animal, and agricultural disease.
[Slide.]
Another point. I have an issue, and those
of you that know me know I have an issue. My issue
is not with the non-residue antibacterials, so I
separate out alcohol, bleaches, and peroxides.
They do their job, they are gone, they don't leave
residues.
So, I have another issue, and that is with
what I heard earlier, that one of the goals for a
consumer product that it persist, and persistence
is, to me, contrary to what I want, because I see
persistence as leading to resistance.
So, at the outset, that is really, if I
didn't say anything more today, this is the message
I want to give.
[Slide.]
Resistance can come to biocides and
antibiotics by a number of means. First, it can be
a target mutation. I will show you an example.
Another is through an efflux system, a pump that
pumps out antibiotics, biocides, organic solvents,
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you name it, and they exist in all bacteria. They
are a real problem in pseudomonas and some of these
gram-negatives, but they are also a problem in
gram-positives.
Finally, there is this co-resistance, the
difference being cross-resistance is the same
mechanism, the pump, the target that will give you
resistance to the biocide and to the antibiotic.
Co-resistance means that they move together, they
are linked on a plasmid or on a transposon.
[Slide.]
The example of a target mutation is
triclosan, and historically, I mean I didn't know
what triclosan was when I was called by a consumer
who said, you know, there is triclosan in all these
products, in toys, and so forth, is that a problem?
You have been preaching about prudent use of
antibiotics. This is an antibacterial.
So, after a few of these calls, I turned
to my associate, Laura McMurray, and I said, Laura,
maybe we should look into this. So, what she did
was to find whether or not resistance was easily
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gotten to triclosan, which had a long literature,
we are talking about decades, but not anything
really dealing with mechanism of action or
mechanism of resistance.
[Slide.]
So, she did a classic genetic experiment.
She put E. coli on a plate with triclosan and
isolated mutants easily, overnight, spontaneous,
and those mutants all were in a single gene, the
fabI, the fatty acid biosynthesis gene I. They had
different mutations, and the mutations correlated
with a different fold in the resistance, or shall
we say, insensitivity to the drug, low, medium, and
high.
Because the enzyme had been crystallized,
one could look and see that, in fact, these
mutations could well be within, and were within,
the substrate binding site.
[Slide.]
The other interesting feature was that the
fabI gene was a homolog or ortholog of an important
gene, a target for isoniazid in tuberculosis, and
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while there has been a discussion as to whether
inhA is or is not the target, I think, at least I
hope, it is now agreed that there are two targets,
a catalase and inhA, and, in fact, this is the
classic or shall we say the real target if you are
going to look at inhibition by an antibacterial
antibiotic.
There is this diazaborine, which Sandoz
was developing it, its target was fabI. We used it
to demonstrate that our mutants were also resistant
to diazaborine. I would like to also say that at
least two companies had decided that the fabI gene
was a good new target for an antibacterial or
antibiotic, I should say, and it turns out that the
mutants that we had isolated to triclosan were
resistant to these newer or shall we say not yet
launched, and probably not going to be launched,
new antibiotics.
[Slide.]
What about the cross-resistance? Is inhA
or triclosan or, shall we say, fabI gene going to
give you resistance to isoniazid?
Well, here is an experiment we published,
which shows that if a mutant MT1, for instance, is
selected by triclosan, we see where the mutation
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is, and then we look and we see that it's 6-fold
more resistant to triclosan and 8-fold more
resistant to isoniazid.
This is in Mycobacterium smegmatis.
If you then go to a mutant that Bill
Jacobs isolated, the MC2651 that was selected in
isoniazid, it has a mutation, and we see that it
has 6-fold resistance to triclosan. Never saw
triclosan before, and a 22-fold resistance to
isoniazid. So, this is what we call
cross-resistance.
[Slide.]
There is another mechanism across
resistance which doesn't deal with the target, and
doesn't really have anything special about it
except that they are protein pumps, they come in
single protein varieties or three protein, a
tripeptide, that is, there is there an inner
membrane, there is a periplasmic, and an outer
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membrane, and these pumps are very good at pumping
out antibiotics where they were first discovered,
and now, more recently, with biocides and other
agents.
[Slide.]
This is a pump, an E. coli, Klebsiella,
Enterobacter, the Enterobacteriaceae. It was
originally described many years ago. It was an
acrogene, resistance gene, only later now
demonstrated to be any flux pump, acrAB.
We have been studying a regulatory gene
called mar, which, in fact, upregulates the acrAB
gene. Now, this efflux gene can be upregulated by
mutation in its promotor, in its represser, or by
upregulating these other outside regulators like
the marA protein or the soxS protein.
So, there is a lot of ways to get this
efflux pump up in the enterobacteriaceae, but it's
not just acrAB, there is an EF, there are many of
these multidrug efflux pumps.
Why is it important? Because look at what
they do. They pump out antibiotics, organic
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solvents, pine oils, bile salts, triclosan,
chloroxylenol. They don't care what they see, they
are going to protect their host, and they have to
think about it then, so are we worried? What are
we worried about?
We are worried because that means that an
antibiotic can select this kind of mutant and make
it resistant to biocides that we may want to use to
protect multiple patients, or we could be using a
biocide which selects a mutant, which now is
resistant to antibiotics, and we are not talking
about just one, we are talking about tetracyclines,
penicillins, fluoroquinolones, chloramphenicol.
[Slide.]
An example of these kinds of biocide
resistance, antibiotic resistance, efflux pumps is
here. There are many others, but I included this
because of the real impact on Pseudomonas
aeruginosa, which is the basis for its multidrug
resistance, the principal basis for its resistance
to all drugs, making it one of the most important
and difficult pathogens to treat in our hospital
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today, we have Pseudomonas, we have Acinobacter,
and we have Stenotrophomonas.
All of these have these pumps that pump
out antibiotics, and it turns out they pump out
biocides. They do it the same way I showed you
with E. coli. Either the pump is already
upregulated, or a regulator of a pump gets mutated,
or an outside regulator pumps it up like the mar.
So, these examples are out there, but they
are dealing with important pathogens, also
pathogens that are opportunistic and pathogens
which really would call commensals. I mean a
Pseudomonas aeruginosa for anyone else is usually
not a problem, so if it's in the household or if
it's in the wake of a use of this, you can really
imagine and see how these kinds of resistant
mutants can be selected.
[Slide.]
What about co-resistance? And there are
lots of examples of these. There are plasmids
which are outside of the chromosome, which are
unique and they replicate by themselves, and they
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carry genes which are supplemental to the host.
In the early days, these plasmids
contained genes, some of which we don't even know
about, some have to do with toxin production, we
are not quite sure why they held on, but we now
know about them because they have resistance to
antibiotics, and they have been picked up actually
over the past four or five decades, probably five
or six decades of antibiotic use.
What is of interest is that one of the
ways that these plasmids accumulate resistance gene
is by an intricate and actually elegant genetic
mechanism, which we call an integron discovered by
Ruth Hall, and this is almost, and for my mind I
look at it like a venus fly trap, something goes
around, there is a gene for resistance, and it kind
of sucks it into the chromosome or into the
plasmid, and now you have two genes for resistance,
then three genes, and interestingly enough, one of
the fundamental early genes for resistance was
their resistance to quaternary ammonium compounds,
the so-called QAC efflux pumps that are present in
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staphylococcal plasmids.
[Slide.]
Here is an example from Dr. Sidhu where he
shows the blaZ gene there, beta-lactamase genes
associated on the same transposon, on the same
plasmid as resistance to quaternary ammonium
compounds.
So, what do you use? Is a Qac going to
select for the plasmid or is it going to be a
beta-lactamase, or is going to be another
antibiotic, because these integrons can have five,
six, seven different antibiotics, and they are all
going to be selected at the same time.
We are talking about population dynamics.
We are talking about selection. We are talking
about inability to use the antibiotic or the
biocide. I am not saying that there is not a place
for biocides, but what I am going to say is that I
don't see that they are needed in the consumer
product.
[Slide.]
We also have learned, and are still
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learning, lessons from antibiotics. We have
learned how we have misused them. We have also
learned how bacteria come back to tell us how we
misused them.
I remind my students that bacteria are not
going to be destroyed. They have been here, they
have seen dinosaurs come and go, and they will be
happy to see us come and go, they are not going to
leave.
So, any attempt to try to sterilize our
home is fraught with failure, and so what we are
seeing is evolution in action, because we start out
with a bug which has decreased susceptibility to a
drug, and it still is susceptible, but it is not
quite as susceptible, but eventually becomes
resistant to that drug.
In the clinical world, we have many
instances, but some of the most relevant recently
are penicillin-resistant Strep pneumo, which get
their resistances by picking up pieces of the
penicillin binding proteins from other bacteria in
the oropharynx, the Strep midas, the Strep
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viridans, which are intrinsically resistant.
So, they accumulate them. They start out
with twice the MIC, but we can still treat them
until they get to high level, and what is the high
level? A totally mosaic penicillin binding
protein, which is not a target for penicillin.
Fluoroquinolone resistance. When
fluoroquinolones came out, you can't get a mutant.
We put it in the laboratory. Interesting
phenomenon. You can't get it in the laboratory,
but we certainly have it out in nature. We have it
in our clinics, and it is multiple mutations in the
target gene and in efflux pumps. Gradually, over
time, like TB, but we never expected it in
something like an E. coli, and we see that in all
the enterobacteriaceae.
The last one, of course, is the
vancomycin- resistant Staph aureus, which began by
becoming chromosomally resistant, lower level,
intermediate, still cause some failures in
treatment until it acquired the vancomycin
resistance transposon on a plasmid from the
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enterococcus.
[Slide.]
So, this early sign of decreased
susceptibility, to me, is a worrisome sign. That
tells me that I am starting these bacteria on the
road to full resistance. This is a study that we
did with Allison, Elaine Larson, and others,
looking at the effect with or without antibacterial
hand soaps on the microbiology of the skin flora.
No statistical difference seen, so start
right out. We saw trends, but no statistical
significance, but what we did see was in the home,
on the hands, a disconcerting finding that there
were Staph aureus, Staph capitis, and other staphs
that had eschewed their susceptibility profiles up
to the 2 and 4, and there have been reports from
Molly Schmidt and others that show that staph is
there, it's moving, more populations of staph are
becoming less susceptible, do we know what it means
by resistance? It may be that the biocide wouldn't
work against these. These studies have not been
done.
[Slide.]
Look at Klebsiella, a problem, a big
problem in our hospitals, and we would love to be
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able to say that we can keep control by using
something like triclosan or other antiseptic or
antibacterial of this type surface in the
hospitals, but look, we had a Klebsiella that was
growing in 32 micrograms/ml of triclosan.
That, to me is not tolerance, that is
resistance, and we are seeing this eschewance, as
long as we see this happening, that's a sign that
full-fledged resistance is on the way.
[Slide.]
This is a paper presented by Fred
Goldstein last year, unexplained, but I think it's
important. He looked at glycopeptides. This is
vancomycin intermediate Staph aureus, so-called
GISA strains. That is that intermediate strain
before it developed and actually acquired the
enterococcal vancomycin resistance transposon.
Looked at 45 of these strains, 24 just
methicillin-resistant staph, 28 MSSA. More than 84
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percent of the French GISA stains had a triclosan
MIC of 0.5 to 2 micrograms, about 100-fold higher
than most MRSA or MSSA. For GISA and other MRSA,
but not MSA, a 2- to 4-fold MIC increase has been
observed for benzalkonium chloride, 2 to 8 for
chlorhexidine, and 6 to 4 for hexachlorophene.
I don't know what it means. I am just
saying that these are the kinds of bacterial
strains that they are facing in French hospitals.
How did that arrive at? Could be vancomycin, could
be the antibacterials, but we have either co- or
cross-resistance in these organisms, and it is
trying to tell us a lesson, that we should be
cautious and we should be concerned, because if, in
fact, we are trying to control a GISA in a
hospital, we may well want to use triclosan or a
QAC.
[Slide.]
A study done in Japan a number of years
ago was somewhat telling to me, and I have
mentioned it, because they took an MRSA, and it
began with a susceptibility MIC benzalkonium
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chloride of 5, and they just raised and picked
mutants at 10, and looked at the dramatic change in
the susceptibility to the penicillins.
The oxacillin goes from 16 to 512, clox
from 0.5 to 256. Unfortunately, the investigators
didn't look at the mechanism, but it does indicate
that you can get, in the selection process, and it
is in the laboratory, cross- or co-resistance to
very valuable antibiotics along with a decreased
susceptibility to a biocide.
I will say if you can do that in
laboratory, it can be done in nature. We had
trouble getting fluoroquinolone resistance in the
laboratory, we have no problem seeing it out there.
It is a question of volume and time.
[Slide.]
Here is another benzalkonium chloride
story, and this is with E. coli, and the circles in
red are those in which the organism had been
adapted to growing in 150 micrograms/ml of
benzalkonium chloride, and you saw cross- or
co-resistance to penicillin, the fluoroquinolone,
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chloramphenicol, tetracycline. They do exist.
[Slide.]
So, in our study, we asked the question:
If you are using an antibacterial soap or a plain
soap, do you have more resistance, less resistance,
the same amount? The answer is the same. You see
the dark bar is higher for antibacterial, but it is
not statistical.
So, someone will say, "Well, there you go.
After a year of use, there is not a problem."
Well, the problem is that, one, the homes
had a lot of antibiotic resistance, and where did
that come from, so we are starting at a high level,
and, two, we learned from antibiotics it doesn't
happen in a year, it can take much longer, but what
we are seeing in the laboratory, what we are seeing
out there in the clinic, the co- and
cross-resistance should tell us that we need to be
careful how we use these drugs.
[Slide.]
So, it comes back to the same story. If I
use penicillin and I use Staph aureus as an
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example, penicillin was used.
[Slide.]
There were penicillinases out there, they
were selected. Methicillin was invented, what
happened? Bugs came up with methicillin
resistance, which was, in fact, a transposable
element.
Then, MRSA, we began to treat with
vancomycin. We got the intermediate, the GISA
strains, like I showed you, the GISA strains, and
with time, VRSA. Now, what is going to stop that
phenomenon occurring if one continues the volume of
use of biocides in the consumer market? Volumes,
volumes.
[Slide.]
So, anyone asks me what is the problem
with antibiotic resistance today? Dr. Levy, does
it come from biocides? I say no, it comes from
misuse of antibiotics, but I don't like this
mounting increase of co-selectors of antibiotic
resistance and the resistance to agents that we
have a place for in the healthcare market by the
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volumes of use in homes casually used by consumers,
and we learned today it makes a big difference how
you use them, and I can tell you, in our look, you
know, if it's a 3- or 4-second wash, that's fine,
and they are left there as a residue, because as I
said before, I am concerned about the persisters.
[Slide.]
So, this is the today. I am happy if we
can keep it this way. I don't want a tomorrow. By
that, I mean where biocides are equal or prominent
contributors to the drug resistance problem, and
from my perspective, with the studies we have done
and what I have seen from other laboratories, I
think this is a concern we should consider.
Thank you.

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DR. PATTEN: I am wondering if there are
other governments, other governmental agencies,
other bodies of scientists in the world who have
taken a look at the benefit-to-risk ratio and made
the decision that it is too risky to incorporate
these biocides in hygienic products.
I don't know if anyone can answer that.
Is it banned anywhere for use in these kinds of
products?
DR. ROGERS: I have looked for information
from other countries, and I haven't found a lot,
but I have found some information that some
countries are having a voluntary ban on products
containing triclosan. I am not sure if Dr. Halden
has any more information on the European countries
or not about whether they have any bans on these
products.
DR. HALDEN: Yes, I think in general the
European Union is a little more concerned about
environmental concentrations of certain chemicals,
and so it has been recognized in the European Union
that concentrations of triclosans are detectable in
various media.
I know in Denmark and I believe in Sweden,
there are initiatives to remove these chemicals
from the market.
I believe that they have been removed from
supermarkets in Great Britain, at least I have read
some reports on triclosan-containing formulation.
There is a risk assessment document
available issued by the U.S. of the Denmark
Environmental Protection Agency that is
downloadable from the Internet. Peculiarly, it is
based a lot on the data generated in the U.S.
because Denmark hasn't done a lot of studies.
So, you will see that they operate on the
same data that we look at here, and they apparently
come to the conclusion that there are certain risks
associated with it.
Since I am on here right now, I have three
more questions or comments maybe regarding the
presentations from the industry. First, I think we
already talked about the concentrations of
triclosan and triclocarban in biosolids, and I
think it was clear now, if you don't have this
information, I can provide it to you, the EPA also
has detected triclosan in milligram per kilogram
concentrations in municipal biosolids, and there is
at least two other studies that I can give you.
Secondly, it was mentioned that the
chemicals degrade, biodegrade. I think we talked
about biosolids, and it was agreed upon that there
isn't much degradation.
Then, it was mentioned that the triclosan
is being degraded in the river very quickly and has
a half-life of only a few hours. This is not
biodegradation. This is a process of
photodegradation. Triclosan has a hydroxl group
that makes is susceptible to photodegradation.
If you have soil, if you have triclosan
bound to a particle, it is not susceptible to
degradation, photodegradation. Biodegradation is
actually very, very slow.
Also, I would like to put on the record
here that there is a paper published in the early
1990s, reporting for two New Jersey wastewater
treatment plants effluent concentrations of 6,000
parts per trillion, so 6 parts per billion of
triclocarban in treated effluent. This is not a
sewage spill, this is a normally operating plant
that was in the early 1990s.
This information is available for you if
you go and do a PubMed search. However, it didn't
enter the EPA robust summary issued by the industry
to the U.S. EPA for the risk assessment that is
currently ongoing for triclocarban. So, it is an
interesting piece of information that should be
figured in.
I don't believe that there is any need for
us to speculate what environmental concentrations
are. Let's just use the data that are out there
and do the measurements that are needed. Thank
you.
DR. WOOD: Did you want to respond to
something?
MR. HOFMANN: Yes, I just wanted to
respond on the question whether there is any ban in
the world. I am Matthias Hofmann from Ciba, and
being the marketing manager, I can assure you that
there is no authority out there in the world known
to me who has banned triclosan in the world.
DR. WOOD: I think somebody said that
there was a voluntary--
MR. HOFMANN: There are some
organizations, let's say, wanting to, like trade
organizations or so, trying to restrict the use of
triclosan in their shops, but there is no authority
out there in the world.
DR. WOOD: Somebody said, I think the
quote was that supermarkets in the UK were not
selling it or something. Is that true?
MR. HOFMANN: That can be the supermarket
chains, but that is not authority itself.
DR. WOOD: Charley.
DR. GANLEY: Can he just stay there for a
minute?
DR. WOOD: Yes.
DR. GANLEY: I have something from the
Internet here, and this is from October 26, 2000,
and it says four Danish Government agencies have
taken the unusual step of issuing a joint statement
advising consumers against the routine use of
antibacterial household and personal hygiene
products. The agencies argue that antibacterials
are unnecessary for domestic use plus potentially
harmful to the environment.
Also, from February 16th, 2001, 6 Finnish
public authorities today urged consumers not to use
certain antibacterial chemicals. Organic
antibacterials are not needed in households, and
their growing use carries a long-term risk of
spreading antibiotic resistance in microbial
populations.
That contradicts what you just said.
MR. HOFMANN: No, I just said authorities
have not banned the substance.
DR. GANLEY: You implied that there were
no voluntary requests by government agencies to not
use these products.
MR. HOFMANN: No, they are not banned in a
way that legislation is made to ban it.
DR. WOOD: So, government agencies have
suggested in other countries that it not be used is
what Charley is saying. Okay.