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Wyno Academic Journal of Agricultural Sciences
Vol. 2(2), pp. 19-28. Nov. 2014
Available online at http://www.wynoacademicjournals.org/agric_sciences.html
ISSN: 2315-9162
©2014 Wyno Academic Journals
Potential Role of Synthetic Antimicrobial Peptides in Animal Health to Combat Growing
Concerns of Antibiotic Resistance - A Review.
C. P. Bagley.
Tennessee Tech University
Cookeville, TN. USA, 38505-0001.
Email: [email protected]
Tel: 931.372.3218; Fax: 931.372.3150.
Accepted Date: 30th Sept 2014.
ABSTRACT
Antibiotic resistance and particularly multidrug resistant (MDR) pathogens are of increasing concern. WHO (World
Health Organization) wrote in April, 2014 that “this serious threat is no longer a prediction for the future, it is happening
right now in every region of the world and has the potential to affect everyone, of any age, in any country.”
Antimicrobial peptides (AMPs) are a class of naturally occurring compounds found in plants and animals and have been
isolated from living organisms ranging from single-celled microorganisms, to plants, to livestock species, and to humans.
Over 1200 such naturally occurring AMP compounds have been isolated and identified. These compounds are found and
produced by many different tissues, and generally form part of the first line of defenses in organism immune systems.
Additional research indicates that AMPs are also involved with promoting wound repair, the reduction of inflammations,
possibly through the release of prostaglandins, and some are part of the neutrophils and their defense mechanisms against
pathogens. The phagocytic action by neutrophils in fighting infectious agents is a series of biological events which
includes AMPs as a cofactor in fighting infections and in wound repair, with multiple types of AMPs involved. Early
attempts at synthesizing AMPs were unsuccessful due to the expense and the relatively short half-life of the generated
AMPs. Recently, synthetic AMPs of a more simplistic nature have been produced using several different positively
charged amino acid side chains in their formulations, each amino acid attached to a cholic acid backbone that are
inexpensive to manufacture, stable, and better tolerated by the organism. These synthetic AMPs show a range of
activities and specificities for bacteria, viruses, fungi and parasites and the many derivations of these synthetic AMPs are
referred to as Ceragenins or cationic selective antimicrobials (CSAs).
KEY WORDS: Antimicrobial Peptide, Antibiotic Resistance, Ceragenins, Immune System, Neutrophils, Lipid A.
INTRODUCTION: In 1910 there were meat shortages in the U.S. leading to public demand to stabilize meat supplies,
leading to animal concentrations in feedlots to improve constancy of food supply. Concentrating animals in feedlots led
to increased disease problems due to high livestock densities, which led to feeding sub-therapeutic levels of antibiotics
for preventing, rather than treating sick animals. These sub-therapeutic levels of antibiotics led to improved animal
performance and reduction in morbidity and mortality rates. However, this increasing level of sub-therapeutic antibiotic
usage in livestock has caused concerns. Estimates are that in 1952 there were 2 million pounds of antibiotics
manufactured in the U.S., increasing to 50 million pounds in 1998 with 40% of the production being fed to livestock,
with that number increasing to over 80% of all manufactured antibiotics going into livestock feeds in 2013. In the
European Union, antibiotics were banned as a growth agent (sub-therapeutic) in 2006, and banned for prophylactic use in
all animals over signs of overuse. As an example, if the typical Petri dish had a maximum population of bacteria, that
number would be approximately 6 billion CFU (colony forming units). If an antibiotic were applied to the Petri dish and
kills all but one CFU, within 2 days, the Petri dish would be again filled to capacity with 6 billion CFU due to the rapid
rate of reproduction, only these surviving CFU’s may have some level of resistance to the administered antibiotic.
Considering antibiotics have only been marketed for less than 100 years, it is not surprising that issues of resistance and
multidrug resistance (MDR) are rapidly appearing. In his 1945 address for his Nobel Peace Prize lecture, Alexander
Fleming, discoverer of penicillin, argued against the use of sub-therapeutic antibiotic levels:
“The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the
ignorant man may easily under-dose himself and by exposing his microbes to non-lethal quantities of the drug make
them resistant.”
The first known manufactured antibiotic to exhibit pathogen resistance was penicillin, with bacteria able to produce
penicillinase to deactivate the antibiotic within 2 years of its initial released on the market. Since most of the antibiotics
in use today are naturally occurring, it would stand to reason that over the many years of their existence in nature that
20. Agric. Sci.
resistance by pathogens could have occurred separate from the perceived notion that resistance stems from subtherapeutic uses of antibiotics in feedlot animals or sub-therapeutic administration to humans. Likely in the millions of
years of existence of plants and animals and pathogens on Planet Earth, there have been many resistant pathogens that
have been developed, only to have the higher-order plants and animals develop new “natural antibiotics” that protect
them from these pathogens. However, it is perceived that the current wave of antibiotic resistance is caused by subtherapeutic feeding of antibiotics to livestock and to humans and does appear to increase the likelihood of antibiotic
resistance to the current pathogens in livestock that may be passed on to humans causing serious concerns.
Antimicrobial peptides (AMPs) have been a part of the animal and plant immune systems, but their roles are only
recently being understood. AMPs have a completely different mode of action from typical antibiotics in killing
pathogens, killing them on contact rather than affecting pathogen reproduction which, as we have seen, pathogens can
find other mechanisms to become resistant to the antibiotic and successfully reproduce. The lethal mode of action for
AMPs is to disrupt the cell membrane of pathogens leading to increased permeability and the immediate loss of
metabolic processes leading to cellular death. The immune system of an animal is a series of processes to kill pathogens
in the body, with naturally occurring AMPs being that first active line of defense. The second line of the active immune
system when infections occur is generally focused around the delivery via the blood stream of white blood cells (WBC)
and specifically neutrophils; the neutrophils also contain AMPs. That first line of active defense provided by AMPs was
first discovered some 30 years ago, and has since been studied and found in almost every living organism, animal or
plant. However, these peptide compounds manufactured by the living organism to form AMP can be quite complex and
requires energy and time to synthesize them by the host, and are typically found in rather small quantities at the tissue
level. It appears that most AMPs are located in all parts of the living organism that may be susceptible and exposed to
pathogens, but these AMPs are considered to be easily over-whelmed by large infections from bacteria, fungi, and
viruses accessing the body tissues caused by cuts and abrasions. An example of their mode of action would be in the skin
where some AMPs are present, and these first lines of active defense are effective until the skin is broken or cut. At this
point, it is considered AMPs are over-whelmed by the large amounts of pathogens invading the tissues, and the body’s
second line of defense is delivered by the blood stream (WBC) or other mechanism to the damaged or infected site. This
second line of defense against pathogens is the WBC system, which consists of neutrophils, monocytes, eosinophils,
basophils, and lymphocytes, where these lymphocytes are further distinguished as being either T or B cells, with three
different types of T cells, and form the protective mechanism to attempt to combat the pathogens using greater
concentrations. These WBC, and primarily the neutrophils, are delivered by the blood stream to the disrupted part of the
body where pathogens are located as evidenced by the redness and swelling of the impacted area of the body. The WBC
form a complex, multifaceted body defense mechanism, but also leads to increased blood supply, edema, and scarring of
the animal tissue. The role of AMPs initially was not looked upon as being vitally important and thus poorly understood
until recent advances. It now appears AMPs are not only involved with this first line of defense at the exposed tissue
level, but also involved with neutrophils and possibly some of the other WBC components in additional body responses
to pathogens. The increased presence of WBC and neutrophils in infected areas also serves to increase AMPs
concentrations to fight off pathogens. AMPs seemingly have numerous activities related to the mammalian body defense
mechanism, from killing pathogens to initiating the healing process rather than just the first line of bodily defenses as
originally considered. Because of the number of amino acids involved in synthesizing naturally occurring AMPs (12 –
100 amino acids), production of these molecules by the host body is relatively slow and energetically expensive with
levels of AMPs present only in what would be considered “adequate” amounts. The body defense mechanisms attempt to
concentrate AMPs in very specific areas of pathogen infections. Attempts to replicate these AMPs outside the host body
were also problematic due to how slow and expensive the synthesis is, and these compounds undergo proteolysis quickly
in the body environment. Due to the short half-life of these compounds, the commercial manufacture is deemed too
expensive to be useful.
Recently synthetic AMPs have been produced in the lab and are becoming useful in the treatment of animals suffering
cuts and abrasions, mastitis, along with certain pathogenic infections. Veterinarians also report a positive response by
livestock to infections by reducing swelling and inflammation, and faster healing process. Some Veterinarians have
reported substantially improved reductions in post-surgery infections by coating the surgical area and with medical
devices, such as screws and plates, with AMPs. Research indicates that methicillin-resistant Staphylococcus aureus
(MRSA) shows no antibiotic resistance to AMPs and is rapidly killed, making it an excellent choice in these type
situations where artificial devices are placed inside the body, particularly in horses.
The Ceragenins or cationic selective antimicrobials (CSAs) are a relatively simple molecule of cholic acid with one of
several choices of a positively charged amino acid attached. Cholic acid is one of two major bile acids produced by the
liver which is insoluble in water and thus non-polar, similar to Lipid A in the cell wall structure of many pathogens.
Cholic acid is synthesized from cholesterol and these compounds account for approximately 80% of the bile acids
produced by the liver. With the attached positively charged amino acid, this compound yields a compound a bit similar
to common soaps used to wash hands and kill bacteria; a part of the molecule which is non-polar and fat soluble, and
another part of the molecule which is water soluble and with a positive electrical charge allowing the compound to kill
susceptible pathogens on contact due to the attraction to the negative electrically charged cell wall structures.
21. C. P. Bagley
Because of the widespread availability of cholic acid, the compound is stable and relatively inexpensive to manufacture.
These synthetic AMPs come in several different formulations based upon which positively charged amino acid is
attached to the cholic acid. As different amino acids are added to the cholic acid backbone, the ability of the compound
to kill specific bacteria, fungus and viruses and to promote rapid healing can be greater or lesser. Further, most naturally
occurring AMP have a positive charge of +2 to +9, while synthetic AMPs, or CSAs, are typically lower. However, some
of these synthetic formulations are quite lethal and can be very effective in killing pathogens and promoting healing
under certain situations. As more becomes known about the activities of these CSAs, it may be that different
formulations and combinations will be used for different disease and infectious situations along with wound healing,
similar to what is seen in the naturally occurring AMPs.
Some of these CSA compounds have broad spectrum activity against a range of bacteria, fungi and viruses and been seen
to be highly effective in treating animal infections, and to date have been most widely used in horses where they are
often used to treat cuts and abrasions in these animals. However, possibly the greatest economic impact may be in the
treatment of mastitis in dairy cows, a disease condition that has been estimated to cause over $1 billion annually in
economic losses in the U.S. alone. Even of more dramatic impact of these CSAs might be the treatment to purify
drinking water to livestock. Injection systems of CSAs into livestock drinking water to date have been positive in
improving animal performance and reducing morbidity. This may be of most practical usage in the beef cattle industry
in combating “shipping fever” in recently weaned and transported livestock. Commercial poultry production houses with
28,000 birds per house may also see positive benefits to CSA-treated water. The use of Ceragenins or CSAs shows
promise in reducing infections in livestock, speeding recovery times, and studies are also indicating increased animal
performance when Ceragenins are added to the feed or water of livestock species. The product is currently marketed as a
“medical device” and not as an antibiotic, but this designation may change in the future.
ANTIBIOTIC RESISTANCE: Almost every antibiotic in widespread usage is a naturally occurring compound already
found in nature or modified from that original antibiotic, typically identified from soil samples, and isolated for increased
commercial production. Therefore, bacteria, fungi, and parasites have for eternity been attempting to become resistant to
these antibiological compounds produced by higher-order plants and animals. The concerns that antibiotic resistance has
occurred solely due to the sub-therapeutic usage of antibiotics in livestock, or sub-therapeutic use by humans,
particularly in developing countries, seems unfounded. While feeding low levels of antibiotics at sub-therapeutic levels
to both animals and humans may be contributing to antibiotic resistance, there are other methods of pathogens obtaining
resistance. These commercially available antibiotics effective against the more simplistic prokaryotic cells usually
express one of three modes of action in protecting against pathogens: 1) bacterial cell wall synthesis, 2) bacterial protein
synthesis, or 3) bacterial DNA replication and repair. There have been reports of antibiotic resistant pathogens prior to
the widespread production of antibiotics in livestock feeds and for humans that have occurred under natural
environmental circumstances. Two widespread theories as to why we are observing increasing numbers of antibioticresistant pathogens currently is due to the widespread human use of antibiotics in foreign countries where antibiotics are
available over-the-counter and can thus be given at sub-therapeutic levels, and also the widespread use of antibiotics fed
to livestock at sub-therapeutic levels in many countries of the world. There is no doubt that there are antibiotic resistant
pathogens and some of those occurred prior to the commercial production of antibiotics in the 1940’s, and some
pathogens are classified as MDR , with such bacteria as MRSA being one of the most well-known and problematic
bacteria we deal with; but there are certainly others. Interestingly, these MDR and antibiotic-resistant pathogens are
serious problems, but only in certain situations. For instance, MRSA is only a problem when it is found inside the body,
usually as a consequence of a cut or surgery. Oral consumption of MRSA typically is of little consequence, since the
molecule is much too large to cross the gut wall and remain intact and enter the body; it will be broken down by
digestive processes first, which yields the remaining compound pieces ineffective at causing an infection. However, as
these MDR pathogens become more prevalent in the environment, they increase the occurrence of serious infections
from cuts, abrasions, or surgical procedures that may resist typical antibiotic treatments.
The primary antibiotics fed to livestock are classified as either peptides or ionophores. Drugs in these classes include
bambermycin, lasalocid, monensin, salinomycin, virginamycin, and bacitracin. A frequently fed antibiotic to livestock
would be tetracyclines, fed to recently weaned and transported animals plus any group of animals subjected to
confinement feeding circumstances. These recently weaned calves fed sub-therapeutic levels of antibiotics are under
tremendous amounts of stress as they are frequently trucked many hours and mixed with other animals from diverse
locations shortly after weaning, and often times do not eat or drink adequately. The complex of diseases that usually
causes high rates of morbidity and mortality is referred to as “shipping fever,” a complex disease that impacts the
respiratory track of livestock primarily. Tetracyclines are usually fed short periods of time when animals are first
received at their new destinations, but once placed on the feedlot later on in life may be again fed sub-therapeutic levels
of tetracyclines. These tetracyclines account for approximately 41% of all antibiotics fed to livestock, but only 4% of
antibiotics used in humans (Table. 1).
22. Agric. Sci.
Table 1: Use of Antibiotics in Humans and Animals by Classification
Class of Antibiotic
Use in Humans,
Use in Animals,
%
%
Ionophores
0
30
Tetracyclines
4
41
Penicillinsa
87
17
All others
9
11
a
Includes penicillin, sulfa, cephalosporins, flouroquinolones, macrolides, aminoglycosides, and
lincosamides.
The ionophores are another class of antibiotics, and are widely fed to beef cattle and poultry, but not dairy cattle, and not
to humans. Ionophores account for 30% of total antibiotics fed to livestock. In beef cattle, ionophores selectively kill
certain bacteria that result in a shift in the production of volatile fatty acids in the rumen of the ruminant, with the effect
being that lower levels of acetic acid are produced, but higher levels of propionic acid produced, which increases feed
efficiency (less feed for weight gain). This shift in acid production in the rumen also dramatically reduces methane
production by livestock, and methane gas is considered one of the most detrimental “greenhouse gases” in the
environment. Ionophores are not used in dairy cattle, because the shift to propionic acid production in the rumen results
in lowered milk fat production which is undesirable, since acetic acid is a precursor to milk fat and is a major pricing
factor in milk sales. In poultry, these ionophores are effective in reducing coccidia in the gut of poultry in these confined
situations and dramatically reducing the disease situation referred to as coccidiosis . Of some interest, the antibiotic and
ionophore, salinomycin, has been recently shown to be very active against breast cancer stem cells in humans and may
have some utility in battling that cancer.
In the 1950’s the widespread feeding of antibiotics began in livestock and was primarily focused on confinement-fed
animals. In the U.S. in 1952, there were approximately 2 million pounds of antibiotics manufactured, but by 1998 that
amount was increased to 50 million pounds. Of the amount of antibiotics manufactured in 1998, about 50% were
consumed by livestock and 50% to humans. In 2011, it is estimated that 80% of all manufactured antibiotics went to
livestock. In comparison, Germany in 2012 manufactured approximately 5 billion pounds of antibiotics, with
approximately 70% going to animals and 30% to humans. The European Union banned the use of antibiotics in animal
feeds as a growth promoter in 2006. While exact figures are hard to obtain, it is estimated China produces and consumes
more antibiotics than any other country, with about half of those antibiotics fed to livestock and the other half to humans.
India’s manufacturing of antibiotics ranks third in the world.
In the 70 years or so since antibiotics have been manufactured and widely used in human and animal populations, there
is a growing concern about antibiotic resistance, and particularly MDR in some bacteria, such as MRSA. How much the
widespread use of antibiotics in livestock has spurred this resistance, compared to resistance caused by human over-use
and sub-therapeutic use in countries where there is over-the-counter drug availability, or how much is due to correctly
dosing sick people with recommended antibiotic treatments, and how much is due to pathogens finding alternative
biochemical pathways to minimize impacts of other naturally occurring antibiotics is difficult to quantify. Fortunately,
most but not all pathogens that have developed resistance to one antibiotic can still be controlled using different
antibiotics. The notable exception is MRSA, methicillin resistant Staphylococcus aureus bacteria whose evolution allows
it to be resistant to just about every known antibiotic, including Vancomysin which is usually considered as the last and
final possibility for controlling MRSA, at least currently. Therefore, an antibiotic or some other type of compound with
a different mode of action to kill pathogens is an active area of research. And a possible candidate appears to be CSAs
for livestock. The traditional “new” antibiotics are seeing some level of antibiotic resistance at such an early phase in
their testing that there currently appears to be no new antibiotics being readied for the market in the near future due to the
adaptation and mutations of pathogens.
The process of developing new antibiotics is on-going. New antibiotics entering the medical field have limited time
expectancy prior to resistance which will likely occur within months or years. Resistance to penicillin after its release in
the 1940’s was documented within two years. Vancomycin resistance was seen in Vancomycin-resistant enterococci
within 4-6 years in widespread populations. The speed of resistance is simply due to the large number of bacteria
present, which often numbers 1010, coupled with an intrinsic mutation rate of 107 for many bacteria, leading quickly to
mutations. Some of these mutations eventually will confer resistance to an antibiotic, allowing for that strain to have a
competitive advantage over non-resistant bacteria where antibiotics are applied and thus dramatically increase in
numbers as compared to non-antibiotic resistant bacteria
ROLE OF AMPs IN IMMUNIE RESPONSES: Some of the first AMPs to be isolated and studied were from bacteria
where they were found competing with other bacteria for nutrients. These varying AMPs may be very specific or general
in their targeted killing of other bacteria, with these AMP’s in bacteria referred to as bacteriocins. These bacteriocins can
kill other bacteria in the same or in different genera, and can be much more potent compared to their eukaryotic types.
These bacteriocins are classified as either being lantibiotics or non-lanthionine containing, based upon the presence or
absence of the amino acid lanthionine. The bacteriocin produced by Lactococcus lactis is referred to a “nisin” and is one
23. C. P. Bagley
of the most widely studied compounds. This very potent bacteriocin is highly effective against gram-positive bacteria
and has been widely used as a food preservative for over 50 years without incidence of resistance. The bacteria Bacillus
spp. produces the bacteriocin mersacidin which has been shown to kill MRSA as effectively as vancomycin but without
antibiotic resistance. Plants also produce AMPs for protection against bacteria and fungi, but less is known about the
kinds and modes of action. Only peptides with β-sheet globular structure have been identified, with the thionins and
defensins the most widely studied.
The invertebrates have also been widely studied with unique AMPs used in their defense mechanisms. There have been
“Toll-like” receptors, or pathogen recognition receptors, isolated in the fruit fly (Drosophilia melanogaster) which is
being used as a model for studying other invertebrates. These AMPs found in the fruit fly hemolymph are phagocytic and
found in epithelial cells. These can be expressed constitutively in shrimp, oysters, and horseshoe crabs or can be induced
by the presence of pathogens, and specifically fungi, in the fruit fly. Examples of these AMP compounds include the αhelical cecropins and melittin, and the β-hairpin-like peptins tachyplesin and polyphemusins. Some of the most potent
AMPs are produced by the horseshoe crab, with MICs in the range of <2µg/ml. Several of these are also potent against
the HIV virus. The largest group of AMPs in invertebrates is the open-ended cyclic peptides called defensins and can
either be directed toward bacteria or fungi.
The importance of the discovery of naturally occurring AMPs in the immune systems of mammals, fish, vertebrates, and
amphibians was initially underestimated and thought to be only a first barrier to infections, and a line of defense that
could be easily overwhelmed. Concentrations of AMPs vary markedly based on adaptive immune responses and areas of
the body located. As a first line of defense, these AMP concentrations tend to be high in the granules of phagocytic cells
and the crypts of the small intestines. Other sites of significant AMP concentrations would include the mucosal surfaces
of the skin. Over 500 AMPs have been isolated from the surface of amphibian skin glands. With several hundred
naturally occurring AMP compounds isolated and found in all types of mammals and vertebrates, their role is just now
becoming better understood as to how they may impact almost all facets of the immune response, from cuts, swelling,
and wound healing in higher order animals.. There are some similarities between the entire classes of compounds
referred to as AMPs that have been isolated to date, but also large differences. As a class of compounds, AMPs are
relatively small with 12 – 100 amino acids, they all contain a net positive charge of +2 to +9, and they are amphipathic in
structure, meaning hydrophilic (water-loving, polar) and lipophilic (fat-loving, non-polar) portions of the molecule which
is synthesized from cholesterol. These AMPs are found in the tissues and exist in several basic structures, including
peptides with a α-helix structures, as peptides with a β–sheet structure stabilized by disulfide bridges, or as peptides with
extended loop structures. It appears that each of these types of AMPs is found in different parts of the body in varying
concentrations and vary according to organism. There are at least 20 different AMPs on mammalian skin tissue, and
these appear to work synergistically in defending the body against pathogens, and particularly Staphylococcus. Little is
currently known about how these AMPs from higher-order organisms vary in their impact against pathogens as
compared to AMPs produced by prokaryotes and invertebrates and their modes of action, but due to the sheer numbers
involved in animal tissues it must be some type synergism. In mammals, recent data indicates that these different types
of AMPs work synergistically to attempt to form a complete defensive mechanism to protect the organism against
pathogens. With all these differences in structures and configurations, all AMP isolated so far do share a single common
function; they have the capacity to directly kill or inhibit the growth of pathogens.While all these cationic AMPs are said
to be in one of four structural classes, α-helical, β-sheet, loop or extended structures, there is variation. Many of these
AMPs have secondary structures which allow these peptides to react differently under differing circumstances, such as
with different membranes. By changing the shape of the indolicidin at the membrane, the C and N termini can alter its
shape making the molecule more potent in its antimicrobial activity against gram-negative bacteria. There can also be a
disulfide bridge with the addition of cysteine which can decrease protease activity, but not impact its antimicrobial
activity. It is also possible that the introduction of a disulfide bond at the C terminal α-helix of sakacin P can broaden the
activity of an AMP.
Within the broad class of mammalian gene families, two of the most researched AMPs related compounds are the
cathelicidins and the defensins. The cathelicidins have a highly conserved N-terminal region known as the cathelin
domain, containing two disulfide bonds between cysteine residues that allow the molecule the ability to inhibit the
protease cathespin-L. Cathelicidins are found in human neutrophils and stored in those granules and can be concentrated
in relatively high levels as called for in the body immune response to pathogens. These cathelicidins can be secreted
from several epithelial tissue types and they have broad-spectrum antimicrobial activity. With the defensins, there are
approximately 150 of these that have been isolated, many also being stored in the granules of the neutrophils. And
defensins also have a role in antimicrobial activities. These varying types and classifications of AMPs also vary with life
stages. One of the first of these compounds to be studied was a type of cathelicidin that was present in young animals
and was only present for about the first two weeks of life, and gave the neonate protection against Listeria, a grampositive facultative anaerobic bacteria often associated with un-pasteurized milk, but has been found in other foods and
in soil samples. It appears animals possess the unique ability to synthesize specific AMPs to meet the challenge the
pathogens most commonly confronted by tissues based upon the stage and in the correct area to be most effective in
24. Agric. Sci.
preventing infections. These AMPs are typically increased in number and concentrations at specific injury sites
following an injury or infection, and act to both protect the body from invasion by pathogens and to promote the healing
process.
Many of the isolated AMPs have antimicrobial activity against a broad spectrum of microbes, including some, but not all
viruses (but including HIV virus), protozoa, fungi, and both gram-negative and gram-positive bacteria. Obviously
mammalian defense mechanisms utilizing AMPs allow for the most effective AMPs to be located in regions of the
mammalian body where specific pathogens are most routinely encountered. Examples would include some of the α–
defensins being expressed in the gut, where they appear to have optimal activity against the bacterial pathogen
Salmonella, where its infection typically occurs. Many skin infections and reactions are caused by either Staphylococcus
aureus or Pseudomonas aeruginosa, and the AMPs most effective against that pathogen are the β–defensins and this is
where their concentrations have been shown to be greatest. And as expected, it is apparent that specific AMPs are stored
in various parts of the body, and can be transported by neutrophils to specific areas as needed, and that increased
synthesis rates of AMPs occur as needed in the body immune system.
The discovery of AMPs and study of their multifaceted impact on antimicrobial immune defenses and their distribution
has had a significant impact upon our understanding of how the body defends itself against infections and disease
causing pathogens. It is becoming more apparent that AMPs may act in a variety of fashions and can alter molecular
shapes to become more or less pathogenic, related to the situation. This antimicrobial activity was initially considered to
be due to a single action against bacteria, but newer evidence indicates that these antibacterial activities may be
multifaceted. However, this understanding was rather academic initially since it appears the impact of AMPs were rather
simplistic in nature; having a minor role in protecting the body against infections and disease causing agents in only a
primary and limited aspect. Currently over 1200 different AMPs have been identified from a variety of organisms, and
each one seeming to vary in its role and impact in the body defense mechanism and specificity for certain pathogens.
These molecules of 12 to 100 amino acids seem to be present in small quantities in numerous body tissues, and also as
part of the neutrophils as part of the body’s immune defense system, and our knowledge of their impacts in the body
defenses are currently evolving. In general, these amphipathic, positively charged molecules are initially considered to be
attracted by electrostatic forces to the negatively charged headgroups of the cell wall structure of bacteria and other
pathogens. While there likely are several different modes of action, the dependent first step appears to be interaction
between the AMPs and the bacterial cell membrane. This initial attraction appears to be related to the electrostatic
attraction of the positively charged AMP and the negatively charged cell wall. Cell walls of these pathogens are
comprised of amphipathic lipoproteins, comprised of lipopolysaccharides (LPS) in gram-negative bacteria, and several
amphipathic compounds in gram-positive bacteria, including teichoic acids, lipoteichoic acids, and
lysylphophatidylglycerol. While all AMPs are considered to cause membrane permeability, there is debate regarding the
mechanism of action. It appears that one of several activities may take place, including models that call for events
including the formation of a transient channel; dissolution of the cell membrane, or translocation across the membrane.
After accessing the cytoplasmic membrane of the pathogens, AMPs are characterized as either being non- or membrane
acting, interacting with the lipid bilayer and displacing the membrane structure, causing a hole in the membrane,
increasing membrane permeability and the contents of the pathogens leaking out followed by cellular death. Others do
not cause significant membrane permeability but may cause cellular death by targeting other basic cellular activities.
However, there still is a lack of understanding why mammalian AMPs only impact some cells, but not host mammalian,
invertebrate, and plant cells. The cells which appear most resistant to AMPs are the eukaryotes, and the more simplistic
prokaryotes the most susceptible in these higher order organisms. At least part of the reason for these differences seems
to be that eukaryote cells have higher levels of cholesterol, while prokaryotes cell membranes are free of cholesterol. The
theory is that cholesterol presence causes the disruptive effects of AMPs on the cell surface to be much less since
cholesterol is known to cause condensation of phospholipid bilayers. This diminution of activity of AMPs towards
eukaryotic cells is resultant of their failure to be able to penetrate the cytoplasmic membrane and the increased
permeability which would lead to cellular death in the more susceptible prokaryote cells.
Previously it was discussed that AMPs concentrations in body tissues is relatively low, too low to be considered to have
maximum antimicrobial activity. The MIC (Minimum Inhibitory Concentration) of an antibiotic is the amount necessary
to be present to effectively kill pathogens. The concentration of AMPs at the mucosal level are about 2 µg/ml, whereas
the MIC in vitro for killing E. coli is 32 µg/ml, some 16 x higher. However, these AMPs can accumulate at specific sites
in more concentrated amounts, sometimes this occurs due to the delivery of defensins via blood supply to the site of
pathogens and are effective at accumulating in adequate levels to kill pathogens, sometimes in concert with other AMPs.
This increase in blood supply also results in the swelling and inflammation seen at wound sites occurring with the
increased delivery of these neutrophils and their compliments of other immune system compounds. Further, while
concentrations of AMPs may be low, indications are they exert their influences by synergistically working with other
AMPs to bring about desired effects of killing pathogens and promoting wound healing. Healing impacts of AMPs are
seen as defensins gather at the wound bed edge to stimulate tube formation in endothelial tissue and accelerate wound
closure.
25. C. P. Bagley
SYNTHETIC AMPs: While AMPs have been known for 30 years and are routinely considered “natural antibiotics,”
commercial synthesis of these compounds was not feasible due to the complexity of the peptide molecules and the
unstable nature of many of these compounds which underwent proteolysis quickly when placed in contact with tissues.
The impact of AMPs on infections and wound healing are becoming better understood, but their impact has not always
been appreciated since it was originally thought they do not significantly impact immune response to major infections,
cuts and abrasions; their role was more of a weak first-line of defense that could be quickly overwhelmed by the
presence of pathogens, requiring support from WBC/ neutrophils. However, this was due more to a misunderstanding of
AMPs and where they are found and how active they are in the complex immune system. Research showing AMPs are
also part of neutrophils, which are part of the WBC complex and generally considered to be critical to the body in
fighting infections, has led to a more complete knowledge as to the role and scope of AMPs in the body immune system.
However, researchers were at a standstill with this information regarding AMPs because commercial synthesis of exact
copies of these complex peptide molecules was expensive, and these AMPs were quickly broken down in the body. This
changed with producing a rather simple synthetic AMPs and it several derivations that, like naturally produced AMPs,
have a rather specific type of activity against certain types of disease-causing agents; and that impact is to kill them on
contact. A team of scientists at Brigham Young University led by Dr. Paul Savage solved many of these problems by
synthesizing a much less complex, more stable, more easily synthesized molecule known as Ceragenins or cationic
selective antimicrobials (CSAs). These compounds are synthetically produced using a cholic acid backbone, with one of
the various positively charged amino acids attached to it. The CSAs are similar to naturally occurring AMPs in that they
are positively charged and amphipathic in nature. Using a cholic acid backbone allows these compounds to be easily
tolerated by the body, since cholic acid is produced in large quantities daily by the liver as its primary bile salt. These
CSAs are stable and well-tolerated by animal tissues. These compounds can be inexpensively manufactured and are
stable for long periods of time.
These synthetic AMPs, sold under the trade name of PuriShield® (http://www.purishield.com), are also relatively
inexpensive and widely available since they first were approved to come on the market in the U.S in early 2014. Reports
in the field have been positive to date, particularly related to cuts and abrasions in horses, and mastitis treatment in dairy
cows. The treatment of mastitis is a major breakthrough since it is the #1 disease issue in dairy, costing more than $1
billion per year in lost revenue and treatment costs to dairy cows in the U.S alone. The primary but not the only pathogen
causing mastitis is Staphylococcus aureus, which is also a bacterium which has shown a great degree of antibiotic
resistance to multiple antibiotics. Therefore, when mastitis is seen in a dairy cow, the first thing done is to culture a milk
sample to determine what pathogenic bacterium is the causative agent, and then a treatment regimen designed based
upon the bacteria and the “best” antibiotic choice of treatment since Staphylococcus aureus shows a wide resistance to
many antibiotics. With the use of CSAs, since there is no resistance to AMPs, choice of treatment is simplified and can
begin immediately upon detection. However, these CSAs are not approved for mastitis treatment since the research
studies are still underway, but reports are far enough along in the field to show promising results.
Horses are a popular animal, often times used for recreational purposes which have reportedly responded positively to
synthetic AMP treatment. These animals are frequently injured with cuts and abrasions; CSAs seems to be very effective
in reducing infections, reducing swelling and edema, and speeding wound closure and healing. Veterinarians also report
in cases where horses must receive plates, screws, etc. for broken and damaged bones, coating these foreign medical
devices with CSAs have reduced post-infection rates usually caused by Staphylococcus to almost zero. Research has
shown that many problematic pathogens are almost completely killed by administration of CSAs including MRSA, HIV
virus, certain forms of cancer, and Pseudomonas aeruginosa.
Research has previously shown positive production responses to livestock receiving water from cleaner sources, rather
than from ponds, streams, and other sources with possible pathogens present. Poor quality drinking water would seem to
be more of a problem with young and old livestock, and with confinement-fed livestock and the quick spread of
pathogens caused by close contact between animals. Research is underway to evaluate treating livestock drinking water
with CSAs to evaluate impacts on animal performance. Also, reports out in the field report using CSAs on animals with
other conditions, including warts, pinkeye, ringworms, saddle sores, and other conditions caused by bacteria, fungus,
protozoa, and certain viruses also respond positively to CSA treatment. It does appear that CSAs can be beneficial to
livestock in other disease and production situations and these areas still being explored with the thought of reducing
animal morbidity and mortality.
Application of these CSAs seems to reduce swelling and inflammation of cuts and abrasions. It is theorized that the
natural body defense has in place in all exposed tissues, a small amount of AMPs for the first line of defense against
pathogens. Upon a break in the skin, a cut, or an infection, these AMPs at the tissue level are quickly overwhelmed.
Normally, the response by the animal would be to dramatically increase blood flow to the affected and injured part of the
body, and increase swelling and inflammation and also deliver appropriate amounts of neutrophils that also contain
AMPs for combating infection. Personal accounts of treating wounds with CSAs would suggest edema and swelling are
reduced when topical CSAs are administered, hypothesizing that the presence of these synthetic AMPs are in some
method communicated with the body immune system, and there is limited to no edema or swelling response needed to
26. Agric. Sci.
bring in higher concentrations of AMPs as part of the immune response. Like many other areas, this too demands further
study to elucidate the mechanisms of action.
As our use of these synthetic AMPs or CSAs becomes more widespread, our knowledge of their impacts on the animal
immune system will increase. Of interest is how the mammalian body can sense that AMPs, either naturally occurring or
by administration of CSAs, can dramatically reduce swelling by keeping blood flow to normal levels to impacted areas.
This is obviously complex situation and our knowledge of the immune system response in mammals is limited in this
area. Of interest will be determining long-term impacts of the usage of CSAs and if there is any negative feedback
mechanisms that may alter the mammalian body’s normal immune response due to the increased usage of CSAs. As
CSAs come into more widespread uses, further study will need to be conducted to elucidate these possibilities.
SUMMARY
Antibiotic resistance tends to be focused on the current concerns of bacteria become resistant to one or more antibiotics
used in prophylactic care of animals and humans. Most commonly used antibiotics are naturally occurring, so since the
dawn of time there has been a process of antibiotic resistance of pathogens towards higher order plants and animals,
followed by these higher order living systems overcoming these antibiotic resistant pathogens by developing other
immune system responses to pathogens. Antibiotic resistance was known before the widespread manufacturing of
antibiotics, starting with penicillin, but the pace of antibiotic resistance seems to have quickened. There are relatively
few new antibiotics in the testing lines since pathogens adapt and become resistant to these new drugs quickly. The
discovery of AMPs some 30 years ago initially was called a “natural antibiotic” that was originally considered as having
a minor role in the immune system of living, complex organisms and primarily involved in the first line of immune
defense with exposed tissue surfaces. It was initially considered that AMPs could be quickly overwhelmed in disease
situations or when cuts or abrasions led to infected tissue sites. As our understanding of AMP has increased, it has now
been shown that AMPs forms an essential portion of the defensive immune systems in neutrophils in fighting pathogens.
However, these AMPs were too complex, expensive, and short-lived to be manufactured commercially for use in
illnesses and infections. Recently, scientists have developed synthetic AMPs, called Ceragenins or CSAs, which mimic
the action of naturally occurring AMPs and bring about the same anti-pathogenic outcomes as the naturally occurring
AMPs but can be topically applied to affected areas in large quantities. These CSAs are inexpensive to synthesize and
well tolerated by the host animal body. Application of CSAs to cuts and abrasions appears to kill pathogens on contact,
and also reduce swelling and edema that is often caused by the shunting of neutrophils via blood to these tissue sites.
While much research still needs to be conducted, these CSAs appear to be an effective tool in treating and preventing a
number of pathogens livestock may encounter.
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