NZ713792B2 - Antimicrobial compositions and methods for their production - Google Patents
Antimicrobial compositions and methods for their production Download PDFInfo
- Publication number
- NZ713792B2 NZ713792B2 NZ713792A NZ71379214A NZ713792B2 NZ 713792 B2 NZ713792 B2 NZ 713792B2 NZ 713792 A NZ713792 A NZ 713792A NZ 71379214 A NZ71379214 A NZ 71379214A NZ 713792 B2 NZ713792 B2 NZ 713792B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- iodide
- peroxide
- composition
- hydrogen peroxide
- milk
- Prior art date
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Abstract
This invention relates to a method for preparing compositions for preventing or treating microbial infections, compositions suitable for use in such treatments and methods for treatment or prevention of infections. One such composition finds particular use in treating mastitis in ruminants. The composition is administered into the udder of an animal as a highly effective treatment for mastitis, or as a prophylactic therapy, by means of an intra-mammary infusion. The milk produced by the animal, during treatment using the composition and method of the invention, is free of residues, such as antibiotics, antimicrobial agents or antimicrobial proteins, which could affect its suitability for drinking or in the production of milk products, such as cheese or yoghurt. The compositions and methods arc also useful in treating and preventing lung infections; and infections in burns and wounds; and other infections caused by biofilms. The compositions may also be used on medical devices lo prevent infection. osition is administered into the udder of an animal as a highly effective treatment for mastitis, or as a prophylactic therapy, by means of an intra-mammary infusion. The milk produced by the animal, during treatment using the composition and method of the invention, is free of residues, such as antibiotics, antimicrobial agents or antimicrobial proteins, which could affect its suitability for drinking or in the production of milk products, such as cheese or yoghurt. The compositions and methods arc also useful in treating and preventing lung infections; and infections in burns and wounds; and other infections caused by biofilms. The compositions may also be used on medical devices lo prevent infection.
Description
crobial Compositions and Methods for their production
Field of Invention
This invention s to a method for preparing compositions for preventing or treating microbial
infections, compositions suitable for use in such treatments and methods for treatment or
prevention of infections. One such composition finds particular use in treating mastitis in ruminants.
The composition is stered into the udder of an animal as a highly effective treatment for
mastitis, or as a prophylactic therapy, by means of an intra-mammary infusion. The milk produced by
the animal, during treatment using the composition and method of the invention, is free of residues,
such as antibiotics, antimicrobial agents or antimicrobial proteins, which could affect its suitability
for drinking or in the production of milk products, such as cheese or yoghurt. The compositions and
methods are also useful in ng and preventing lung infections; and infections in burns and
wounds; and other infections caused by biofilms. The compositions may also be used on medical
devices to prevent infection.
Background to the invention
Bovine mastitis is the costliest l condition in veterinary husbandry. Mastitis occurs as a result
of a bacterial infection in the udder. There are three elements involved in such infections;
microorganisms, environmental factors, and the cow . Staphylococci and Streptococci are the
cause of c. 90% of infections though others, such as E. coli or monads can also cause
ion. Nutrition, bedding and milking technique all influence the likelihood of infection.
Moreover, the stage of lactation will have an effect (teats can crack and become damaged as the
lactation cycle progresses) and the number of lactations one by an animal will dictate the
likelihood of inflammation (though not necessarily infection). Infection of the udder will lead to a
decreased yield in milk production, and an increase of the Somatic Cell Count (SCC) in milk. SCC
levels are used by dairy s and milk processers as a proxy measure for milk quality and the
presence of bacterial infection. An SCC of >800,000 per ml, d with signs of discomfort (such as
swelling, tenderness of the udder) in an animal, would be considered a al case of mastitis. In
sub-clinical cases, there may be no outward signs of infection in the animal but an ed SCC
Count in milk. Farmers are often paid for their milk based on the SCC counts, with higher quality
milk, classified based on low SCC levels, attracting a premium.
The typical treatment for mastitis involves the use of antibiotic therapies. The use of antibiotics
often precludes the sale of the animal’s milk during and post treatment, for a period of up to 9 days.
The requirement to discard, or at least not sell, milk during antibiotic treatment is a significant
additional economic cost to the milk producer, associated with mastitis. The no-sale requirement is
put in place in order to:
(i) Prevent antibiotic residues from entering the human food chain: Traditional antibiotic therapies
all require a milk discard during and post-treatment for a number of days. This is to ensure that
there are no antibiotics in the food chain. Antibiotics in the food chain will have two important
effects – they increase the possibility of bacterial resistance to the drug by a continued contact with
the drug. Cephalasporins (following an earlier ban of fluoroquinolone use), for example, have
recently been banned for husbandry use in the US because they are such an important class of drug
for human pathogen therapeutics al Register /Vol. 77, No. 4 / Friday, January 6, 2012).
Secondly, the presence of otics can greatly affect the flora of the human gut, inhibiting
important, pro-biotic bacteria, and allowing the eration of potential pathogenic bacteria that
normally do not gain a ‘foot-hold’ in such an environment.
(ii) Ensure bacterial starter cultures for cheese and yogurt production are not inhibited: The
presence of antibiotic residues will inhibit the starter cultures (such as lactobacilli) used for the
manufacturing of both cheeses and s. These es are typically composed of organisms very
sensitive to antibiotics, and thus a long milk discard period is required following antibiotic usage, to
ensure that the starter cultures are not inhibited. The use of a Delvo test at the dairy level is a
relatively simple method for screening for antimicrobial residues in milk. It is based on the use of a
olerant microorganism that is sensitive to antimicrobials, and is considered the ‘Gold Standard’
in dairy practices. Inhibition of the sm tes the presence of antimicrobials in the milk.
Growth of the microorganism indicates that there are no crobials present. Antimicrobialcontaining
milk is discarded, at a large cost to the , as it is not suitable for use in postprocessing.
Various antibiotics are presently sold and all require a milk discard of a certain length.
For example, Tetra Delta requires a milk discard for the two days during treatment and for 5 days
post treatment. Ubro Yellow requires a milk discard for the three days during treatment and for 6
days post treatment. Terrexine requires a milk discard for the one day of treatment and for 6 days
post treatment. No antibiotics are sold that would not e a milk discard. Milk d is viewed
by dairy farmers as the major cost incurred by a case of mastitis.
The use of compounds, other than otics, to treat mastitis, without a withdrawal period, has
been proposed. United States Patent No. 6794181 describes the isolation and use of a ioticclassed
nd named nisin. The compound has antimicrobial ties. Nisin, and similar
compounds (e.g., US Patents 07/317,627; 5950269 and 20110269671) are antimicrobial peptides,
rather than antibiotics, and thus no antibiotic residues would be present in milk. In the absence of
other preservation methods, nisin does not, however, inhibit egative bacteria, yeasts, or
moulds. Consequently, nisin is mainly used as a food preservative and not as a therapeutic, in
combination with other synergistic food preservation s such as low pH and high salt
concentrations. er, nisin residues in milk may also inhibit starter cultures for cheese and
yogurt production and thus ere with downstream sing of milk in a manner similar to
antibiotics. It has been found, for example, that nisin residues in milk could lead to some
interference with cultured dairy products (certain cheeses, yogurts) if a high proportion of animals
are treated at any one time. Nisin use was also shown to elevate the somatic cell count of the
s during treatment, a serious drawback when payment to the farmer is based on SCC .
The acquisition of resistance to nisin and similar molecules by pathogenic bacteria has also been
reported.
In addition to concerns about the development of antibiotic resistance with respect to human
medicine, a major problem in dairy husbandry is antimicrobial resistance in animals, ated with
historical antibiotic usage. Microbial resistance to antibiotic drugs will become prevalent upon
repeated usage, such that any particular antibiotic will become completely ineffective as a y
at some point – this is widely ised as a critical and urgent challenge in both human and
veterinary medicine. Causative organisms for mastitis include the bacteria Escherichia coli,
Staphylococcus aureus, Streptococcus dysgalactiae, agalactiae, Streptococcus uberis, and
Pseudomonas aeruginosa. Of these, S. aureus is a particular problem because of the phenotype of
the microorganism. The bacterial strain is capable of infecting the epithelial lining of the udder, thus
g the antibiotic. Moreover, the organism is generally found growing as a biofilm. In the biofilm
phenotype, microorganisms are much more resilient to the actions of antimicrobials. As such, they
are much more difficult to treat. The antibiotic treatment of S. aureus infections in cows, for
example, often es two phases. Firstly, the antibiotics will cause an initial decrease in SCC,
indicating the killing of the bacteria. However, the antibiotic will not access the cells in the epithelial
lining. Within a short time frame (for example, 2-3 weeks) the infection will re-establish itself as
bacterial cells growing in the lial lining are shed in the udder once more to proliferate in the
nutrient rich environment. This type of case would now be classed as ‘chronic mastitis’. Treatment of
such cases is particularly difficult, as repeated use of the antibiotic results in a bacterial strain that,
due to repeated t with the drug, is highly likely to develop resistance characteristics.
Treatment of these types of cases has a poor chance of success, even using the most potent of
current antibiotic therapies. At this stage, it will normally be economically ible to keep the
animal, and thus the animal would be culled and the farmer would e the animal at a cost of
>€1,000 ($1,300)
The economic costs associated with the occurrence of mastitis thus include: reduction of milk yield,
loss of income due to poorer quality milk produced, narian charge, antibiotic prescription, loss
of income due to withholding of milk and replacement of culled animals.
Microbial Infections
The invention also relates to the prevention and treatment of a number of other bacterial infections.
Drug delivery and resistance to antibiotics is a major problem in the treatment of cystic fibrosis (CF),
tuberculosis (TB) and nias. Antibiotic resistance often occurs as a result of only ibitory
concentrations of the drug reaching the target site, i.e. lungs. Chronic infections will often
result from this situation, seriously impairing the health of the patient. Addition of a composition of
iodide and hydrogen de could act prophylactically on people diagnosed with CF, or could be
used to treat patients that have already developed lung infections. Such a composition, delivered as
a sed spray, or as a physiological saline composition (or indeed using water as a r) has
the potential to be used in hospitals to minimise and l infections, especially those caused by
antibiotic resistant strains of ia. This is a particular risk during surgical procedures, where body
cavities are open to the environment.
Burns/Skin/Mouth
Other infections that are suitable targets for the compositions and methods disclosed herein are
those incurred as a result of burns and open, or chronic, wounds. The advantage of using the
compositions, treatments or methods over present antibiotic treatment regimens would be similar
to those described above in ng CF or TB regarding safety, efficacy, and low risk of bacterial
resistance to the treatment. The present ors have shown that the described itions are
effective in the treatment of biofilm based cells (those attached to a solid stratum). This would be
the bacterial phenotype most noted in the lungs of TB/CF patients, and in open wounds or burns.
The m phenotype confers a generalised tolerance to the bacteria against a wide range of
antibiotics.
The composition of the invention could also serve as a general antibacterial solution, having
us purposes. For example, a mouthwash containing the antibacterial composition could help
prevent the formation of biofilms within the mouth. Likewise, an antibacterial nasal rinse could be
used to help alleviate sinus ms, such as sinusitis or allergic rhinitis. Currently, steroids and
saline nasal washes are used. An antibacterial saline wash would, r, also help to prevent and
combat bacterial colonisation of the nasal cavity.
Medical Devices
The use of the antimicrobial composition of the invention would also hold a number of advantages
in the treatment and prevention of bacterial infections on an implanted medical device. Infections
(in the form of a recalcitrant biofilm) are extremely common in various devices such as catheters.
Antifungal agent
ions can occur as a result of yeast or fungal growth as well as bacteria. As such, a therapeutic
regime capable of exerting antimicrobial activity (as opposed to antibacterial activity, as is the case
with otics) would be of great benefit. To this end, the bed es of compositions are
capable of eradicating a number of fungal strains including, but not limited to: Candida (strains of
fungus are typically bed as opportunistic pathogens. They can cause a variety of skin
conditions, as well as vulvovaginitis and urinary tract infections); Saccharomyces cerevesiae
(commonly known as bakers’ yeast) is an important organism in food tion. It also poses great
problems for the drinks industry, and is a typical organism found on beer lines, and is the cause of
ge spoilage.
The composition of the invention also finds use as an anti-viral agent.
Hydrogen peroxide is used as a high level ectant of surfaces, either alone in aqueous solution,
or in combination with other chemicals. A 3% solution is also used as an antiseptic. These
applications are unsuitable for use in an antimicrobial therapy within the body of a human or an
animal, due to the damage caused by elevated concentrations (0.15% upwards) of peroxide to
mammalian tissue. Furthermore, elevated concentrations of hydrogen peroxide have been shown to
impede healing and lead to scarring of damaged tissue (in, for example wounds and burns) because
it destroys newly formed cells.
Hypoiodite (IO-) has been discussed in terms of being a by-product in processes such as UV
irradiation and as a product of the lactoperoxidase (or other peroxidase) system (EP 2510944 A1,
Patent No. US 2012/0021071 A1, Patent No. US 2012/0128650 A1, Patent No. 5607681).
dite (IO-), produced as a result of the reaction n peroxide, a peroxidase enzyme and
iodide can be bacteriocidal to Gram-negative microorganisms when they are grown in laboratory
media, but the ture teaches that this approach will be ineffective under physiological
conditions as the production of IO- is inhibited by the presence of thiocyanate, at concentrations of
the latter compound normally found in , milk and other physiological settings noff et al.,
1967, J Exp Med 1967 126(6):1063-78; o et al. 2002 Oral Diseases 8, 23-29). IO- is generally
ed as being bacteriostatic (i.e. suppresses the growth only) to ositive organisms as part
of the natural antimicrobial lactoperoxidase system. E. coli and P. aeruginosa are Gram-negative
organisms, while S. aureus and the Streptococci are Gram-positive organisms.
en peroxide itself is antimicrobial at high concentrations, but it is mainly effective against
gram positive bacteria and is regarded as being only bacteriostatic to gram negative bacteria, such as
P. aeruginosa. Moreover, the presence of catalase in bacteria makes solutions of hydrogen peroxide
at a concentration below 3% less effective, even against gram positives . In a similar fashion,
catalases present in tissues can render hydrogen peroxide still less antimicrobial in vivo. For
example, in a al study on S. aureus in blister wounds no reduction in bacterial load was
achieved using 3% hydrogen peroxide. A review of the use of hydrogen peroxide in a variety of
studies on wounds found that “In conclusion, hydrogen peroxide appears not to negatively influence
wound healing, but it is also ineffective in reducing the bacterial count” (Drosouet al., 2003,
Wounds; 15(5). The prior art thus teaches that use of compositions ning less than 3%
hydrogen peroxide for bacteriocidal purposes would be ineffective for in vivo wound applications.
Similar findings are reported with respect to physiological fluids. For example, no peroxide-related
crobial ty was found either when hydrogen peroxide was infused into patients infected
with a variety of organisms. There was no bactericidal activity when hydrogen peroxide was infused
into blood of rabbits ed with peroxide-sensitive E. coli. Moreover, increasing the concentration
of peroxide ex-vivo in rabbit or human blood containing E.coli produced no evidence of bactericidal
ty. The lack of an antimicrobial effect of high concentrations of hydrogen peroxide was directly
uted to the presence of the peroxide-destroying enzyme, catalase.
Many pathogenic bacteria, ing S. aureus and E. coli, express catalase enzymes that inactivate
peroxide. The basis for designing a peroxidase enzyme system as an antimicrobial for use in food
preservation, dentifrices, etc, ore, is that the peroxidase enzyme is required to compete with
the bacteria to convert sub-toxic concentrations of hydrogen peroxide (or other form of de,
such as sodium peroxide) into an antimicrobial agent before the peroxide is d by the
bacteria.
The literature teaches the inherent ance of the use of a peroxidase enzyme to catalyse the
reaction between iodide and peroxide, at concentrations of the latter le that will not be toxic
to mammals. Thus, the peroxidase enzyme (as well as the substrate and peroxide) has been thought
of as essential to allow the l crobial system to work - “In order to be efficient, the three
elements which make up the system need to present simultaneously” - Poutrel et al. (Ann. Rech Vet,
1982, 13 (1), 85-89). Magnusson et al. (J of Biological Chemistry, Vol. 259, No. 22, 1984) describe, in
detail, the central role of lactoperoxidase in transferring an oxygen molecule from the available
peroxide to an ble iodide molecule. They teach that a peroxidase enzyme is essential to
regulate the reactions and that it had a large effect on the tion of not only the major product
IO-, but IOH (hypoiodous acid), I2 e) and other iodous compounds that will also be produced as
a result of the peroxide and iodide reaction.
Iodine has long been used as a method to kill bacteria. The most common found version is that of
polyvinylpyrrilidone, PVP-I, also known as povidone-iodine. It is complexed to aid its stability as
iodine (I2) will y dissipate otherwise. It possesses the distinctive h brown colouration,
and is often used as a l antiseptic during surgeries, or as a teat dip on dairy farms, minimising
chances of mastitis infections transferring. Its limitations are well characterised as it reacts poorly
with organic material, and can be toxic if in the blood stream.
IO- has a relatively short half-life of a few hours, suggesting that it may exert only a transitory static
effect in therapeutic treatments. This is ted by prior art reports describing: (i) poor
effectiveness in killing Gram-positive organisms (‘Due to mainly bacteriostatic effect of the system it
is not possible to disguise poor milk y’, Guidelines for the Preservations of Raw Milk by the use
of the Lactoperoxidase System, CAC/GL 13-1991, WHO “Lactoperoxidase System of Raw Milk
Preservation – Call for data, 2005; Reiter and Harnulv 1984); (ii) transitory bacteriostatic effects
wherein the ia once more start to proliferate after a short delay (Ishido et al., 2011
Milchwissenschaft 66 (1) 2011; Thomas et al., 1994, ion and Immunity, Vol 62, No.2 p 529-535;
Marks et al., 2001 J Appl. Micro. 91, 735-741; Kamau et al., 1990 Appl and Env. Micro. Vol. 56, No. 9;
McLay et al., 2002, Int Jour of Food micro, 73, 1-9); and (iii) inability to ate bacteria growing in
biofilms (Dufour et al., Journal of Food Protection, 67 (2004), pp 1438-1443; Abbeele et al., 1996, Int
Rech Sci Stomotol Odontol 39 (1-2):57-61). This latter information teaches against the use of IO- as a
treatment in bovine mastitis, where biofilm-phenotypes occur regularly. One would expect that the
IO- compound would not be effective at completely killing the pathogens and curing a mastitis
infection, based on published data.
The prior art, however, is incorrect with t to the products of the reaction n low (<0.5%)
concentrations of hydrogen peroxide and iodide, when this reaction takes place in the absence of a
peroxidase . The present inventors have shown that these reaction products are effective at
killing a wide-range of egative and Gram-positive organisms, including those growing in
biofilm mode, and that they provide a highly effective therapy for mastitis, which allows the milk
produced during treatment to be safely used for cheese production and other downstream
processing. Moreover, we disclose that the presence of lactoperoxidase (or any other peroxidase or
any other synergistic agent or compound) is not necessary to te the antimicrobial activity in
milk or other media; and the addition of a peroxidase enzyme did not enhance the crobial
cy of the products produced, nor their active half-life in milk or other media (Result 3). The
short-lived nature of the antimicrobial activity, rather than being a hindrance, acts as a key
advantage in the present ion. This is because we disclose a method to titre the reaction such
that there is no crobial ty remaining within milk when taken from the animal 8-12 hours
after treatment. This is important, as bacterial starter es are used in the production of cheeses
and yogurts, and would be inhibited if there were antimicrobial residues or agents present at that
point.
iocyanite (OSCN-) is produced by a on of thiocyanate and peroxide, which is catalysed
by a peroxidase enzyme. Numerous studies have examined the use of OSCN- as a method of limiting
bacterial growth. In addition, a number of s have described the use of anate in a
peroxidase on (EP 2510944 A1, Patent No. US 2012/0021071 A1, Patent No. US 2012/0128650
A1, Patent No. 1). All the described literature teach the use of a supplemented peroxidase
enzyme to allow the on to proceed. Ishido et al., 2011 described an OSCN- method of inhibiting
bacteria. The enzyme-catalysed system was incapable of eradicating Pseduomonas strains and E. coli
strains, even with a sustained contact time of 48 hours. Other laboratory studies, based on OSCN-,
have described bacteriostatic s, rather than te elimination of the bacteria, often with a
minimal reduction in proliferation time (4 hours or less) in a majority of their isolates; or no
inhibitory effects at all, particularly in mastitis causing pathogens such as E. col. Others have
described the necessary use of immobilized s to produce OSCN-, which is a configuration
unsuited for use in a mastitis therapy. Carlsson and co-workers (Infect. Immunity, 44:581-586, 1984)
further demonstrated that a byproduct of the reaction, SCN-, could potentiate the cytotoxic effects
of the peroxide molecule under specific conditions, such as the presence of excess peroxide. These
findings teach against the use of a OSCN- model as a mastitis therapy for reasons of overall efficacy.
Despite this, the use of an OSCN- model was described as a potential treatment of cystic fibrosis
(Patent No. US 2012/0021071 A1), although no data on animal or human trials were included in that
application. In US 2012/0021071 A1, however, the applicants acknowledge the need for additional
extraneous compounds to enable biofilm removal as OSCN- itself was insufficient for this purpose. A
similar requirement for additional substances is described in US 8263138 B2. Indeed, the results
presented in Table 2 of the present application also teach against use of OSCN- in trying to kill biofilm
cultures in the absence of synergistic compounds.
Surprisingly in the present invention, a composition containing low (<0.5%) concentrations of
hydrogen de and iodide - in the absence of a peroxidase enzyme - is sufficient to kill biofilm
adhered cells, with no ement for any synergistic compounds to be added to aid biofilm
removal.
There are a number of other uses of the anti-microbial composition of the present invention. These
include infections of the mammalian lung. Cystic fibrosis and tuberculosis are two diseases that are,
at present, extremely difficult to treat. Tuberculosis symptoms are caused by infection in the lungs
and require long-term antibiotic treatment. Cystic fibrosis (CF) is a condition wherein the sufferer
cannot regulate the transfer of chloride ions across their membranes, particularly in the lungs. The
condition invariably results in numerous, chronic, lung infections. Antibiotic treatment for either
condition can lead to serious drug resistance, minimising their effectiveness. At present, antibiotics
are delivered through the blood stream intra-venously, or by oral suspension/tablets, or by
inhalation. Drug delivery is a big problem for CF ers as the antibiotic cannot efficiently
transverse the lung ne to where it is required. This leads to problems wherein ance to
the drug, through the introduction of sub-inhibitory concentrations, may become a serious issue.
This makes any further treatment with the drug obsolete.
Burns patients, or patients with open wounds, are extremely susceptible to ial infections,
notably those due to Staphylococcal or Pseudomonad species of bacteria. Treatment of such
infections will invariably be by a regimen of otics, either oral or venously. These may be
given prophylactically, or when ion is apparent. Such use of antibiotics will often lead to
resistance to the drug and an ineffective treatment outcome. We envision a new method of treating
burns patients using the antimicrobial compositions and methods disclosed here, using a bandage or
poultice in conjunction with the described antimicrobial composition(s), that can be swapped or
changed regularaly if required.
In addition, large numbers of antibiotic ents each year are due to medical devices that have
become infected whilst in use by a t. A large number of organisms are responsible for such
ions, ing both ositive and Gram-negative bacteria. Infections, on such items as
urinary or intra-venous catheters, are often the result of the erile installation of such devices.
Over the course of a number of days, any bacterial cells present on the e of the device will
proliferate, leading to the production of biofilms. Such biofilms are extremely difficult to treat with
antibiotics, due to the poor transfer of the drug across to the inner cells of the biofilm mass, leading
often to even greater levels of tolerance of the biofilms to the antibiotic. Infection of the medical
device will often require its removal and replacement, to the fort of the patient. Although the
infection will often be noted a number of days after installation of the l device, it will be
typically incurred as the result of bacteria being present very early in the installation.
Object of the Invention
Disclosed herein is a method and composition for the prevention or treatment of ial
infections. Disclosed herein is a composition, which is capable of killing, as opposed to slowing the
growth of, bacteria, viruses, fungi and yeasts. Disclosed herein is a composition that can kill
antibiotic resistant organisms.
Disclosed herein is a composition for the treatment of both clinical and sub-clinical mastitis, without
the use of antibiotics.
An object of the invention is to provide a treatment for mastitis, which does not require that the
milk be ded during treatment; and/or to provide a clinical solution to mastitis, which would be
easy to administer, and/or to at least provide the public with a useful choice.
Disclosed herein is an cterial pharmaceutical composition suitable for wound care, and killing
bacteria colonised in, or on, the body.
At present, there is no product available that has been shown to be effective at killing mastitis
causing pathogens and curing the infection, while also ng milk produced during treatment to
be immediately processed for use in, for example, cheese and yoghurt production. An additional or
alternative object of the ion is to produce such a ent; and/or that the treatment does
not result in the acquisition of antibiotic resistance in the target pathogens, which provides a major
benefit to veterinary and human medicine.
y of the invention
ccording to the present invention there is provided a method of treating or preventing mastitis in a
ruminant, the method comprising administering an ive amount of a pharmaceutical
composition comprising iodide (I-) and a source of hydrogen peroxide, together with a
pharmaceutically effective carrier or diluent, to a ruminant in need thereof; wherein the
pharmaceutical composition does not e a peroxidase enzyme or neiodine.
Disclosed herein is a ceutical composition comprising iodide (I-) and a source of hydrogen
peroxide, together with a pharmaceutically effective r or diluent. The concentration of
hydrogen de may be less than 1% based on weight/volume or weight/weight. Preferably the
pharmaceutical composition does not include a peroxidase enzyme or starch.
The pharmaceutical ition may comprise a 0.2:1 to 3:1 ratio of iodide to hydrogen peroxide
by . The ratio may be 0.38:1 to 1.52:1 ratio iodide to hydrogen peroxide by weight. The ratio
may be 0.5 : 1 to 2.5 : 1 or 0.75 : 1 to 2.0 : 1.
The ratio may be selected from:-
Between 0.2:1 and 3:1 by weight iodide to en peroxide,
more preferably between 0.38:1 and 1.52:1 by weight iodide to hydrogen peroxide, or more
preferably 0.76:1 by weight iodide to hydrogen peroxide.
The pharmaceutical composition may be d to reach a m concentration of 50 mg
peroxide and 37 mg iodide per litre of milk, during a milking cycle within an animal.
The source of iodide may be sodium iodide, potassium iodide, lithium iodide, caesium ,
hydrogen iodide, rhodium iodide, or other iodide releasing compounds, or a slow-releasing form of
iodide, such as iodates. As used herein, the term ‘iodide’ does not include iodine itself or
povidoneiodine. Iodine would be toxic in blood or in the mammary gland.
The source of hydrogen peroxide may be hydrogen peroxide, or peroxide releasing percarbonates,
de releasing citric acid or n C, or other suitable peroxide releasing compounds, or by
enzymatic means, e.g. by the ion of sugar compounds using an enzymatic pathway. Likewise,
the use of other forms (and thus other form releasing) of peroxide could include sodium peroxide,
lithium peroxide. Furthermore, peroxide salts could be used, including barium oxide, sodium
perborate, and also hydrogen peroxide-urea adduct. Similarly, oxygen releasing pseudo peroxides
could be used, including, but not limited to, superoxides, dioygenals, ozones, and ozonides. Likewise,
the use of organic peroxides may also be of use, and could include, but not d to, peroxy acids,
acyl halides, aliphatic peroxides. There are other le oxidising compounds that may act in a
suitable manner also, such as permanganate in the form of a potassium salt.
When thiocyanate is used in the ition a ratio of between 1 or more preferably between
0.8-1.3:1 of thiocyanate to hydrogen peroxide may be used.
The composition may be adapted for the treatment of mastitic lactating ruminant animals.
The delivery of the peroxide and iodide components may be concurrent, or sequential and/or may
be by means of an intra-mammary device.
The pharmaceutically effective carrier is water, saline, an emulsion, a gel or a hydrogel.
The composition may be adapted for ry by means of a dampened bandage.
The composition may be adapted for use as an antimicrobial nasal rinse, as an antimicrobial mouthwash
, an antifungal wash, as a disinfectant, added to a bandage or poultice for the treatment of
wound or burn infections, or nebulised in the form of a spray for the treatment of bacterial or fungal
infection of the human or animal lung.
Disclosed herein is a medical device coated with the ition as described above, and a bandage
or poultice impregnated with the composition.
Disclosed herein is is a disinfectant composition comprising iodide and a source of en
peroxide.
Disclosed herein is a method of ting or treating an infection comprising administering to a
subject in need of such treatment iodide and a source of hydrogen de, either concurrently or
sequentially.
Here we disclose that hydrogen peroxide at low tration (for example, a 0.003% solution), is
capable of producing an effective antimicrobial therapy, by reaction with a specific concentration of
, in the absence of a peroxidase enzyme or any other synergistic agent. In addition to killing
bacteria growing in planktonic form, such an approach may also be used to eradicate biofilms.
Compositions that contained hydrogen peroxide and iodide at concentrations of between 50-500
mg/L of each nce in water; and at weight ratios n 0.38:1 to a 1.52:1 of iodide to
hydrogen de by weight, and preferably in a ratio of 0.76:1 by weight of iodide to hydrogen
peroxide (for example, 76 mg iodide and 100 mg hydrogen peroxide, with the 76 mg iodide
provided, for example, by addition of 100 mg ium iodide, or atively by addition of 90 mg
sodium iodide, taking into consideration molar differences between potassium with a molecular
weight of 39 and sodium with a molecular weight of 23) can be used:
a) to provide sufficient crobial activity to kill both Gram-negative and Gram-positive
microorganisms;
b) to eliminate bacterial biofilms g on a variety of surfaces;
c) to avoid irritation and damage to mammalian tissue when used in a therapeutic setting;
d) to eliminate mastitis infections when infused into the mammary gland of a cow;
e) to ensure that milk produced during such treatment is free of any antimicrobial activity that
could interfere with the use of milk for cheese, yoghurt or other post-processing;
f) to achieve the aforementioned s without inducing tolerance or resistance
characteristics in a target organism against the treatment itself, or against antibiotics.
The invention relates to the use of a source of antimicrobial activity (produced by a reaction
between hydrogen peroxide and iodide) that can be administered once, or twice, per day for 1-15
days, preferably 1-5 days. Alternatively, an alternative source of unbound oxygen may be supplied
by oxidising compounds such as permanganate.
Likewise, the invention relates to a manner of using an antimicrobial composition containing
hydrogen peroxide and iodide to allow protection of the teat canal tissue in resisting colonisation by
bacteria/killing bacteria on the surface of the teat canal tissue.
In one embodiment, a composition containing hydrogen peroxide, 100 mg (either directly added as
en peroxide; or produced to this concentration by an alternative source of hydrogen
peroxide), and potassium iodide, 100 mg, (a 0.76:1 ratio of iodide to hydrogen de by weight)
would be introduced into the udder environment after the animal is milked, ing crobial
activity, and thus killing any es present. Due to the ve nature of the compounds, they
must be separated (for example, in separate intra-mammary syringes or in a two-chamber syringe)
until their mixture in the udder. This reaction would produce sufficient antimicrobial activity to kill
the infection causing bacteria upon infusion to the empty udder. r to this, the reaction would
have ceased on milking (approximately 10-12 hours post infusion), such that post-processing of the
milk could proceed normally. In another embodiment, the source of peroxide used is sodium
percarbonate with a en peroxide availability of between 20 and 30%. This can be substituted
easily and may provided an extended shelf life.
The invention relates to the use of two consecutive syringes to deliver the treatment.
In an alternative embodiment, the invention s to the use of a multi-barreled syringe to deliver
the treatment to the udder.
At present, teat dips are often used during milkings to help minimise the carriage of bacteria on the
teat surface into the inner udder. Such an action can reduce the instance of mastitis. Iodine based
compounds are often used h these are not effective in the presence of organic material such
as milk). Activated iodide-based compositions are more effective as they will work at lower
concentrations. The presently discussed reaction between iodide and peroxide could be used as a
ation either as an antibacterial teat dip, or as a poultice based antibacterial barrier to an open
wound (or cracked teat canal tissue).
In such a circumstance, the use of a ed composition of hydrogen peroxide and potassium
iodide gel or saline solution could be applied to a poultice or bandage. This ce could be applied
to damaged teat canal tissue or other damaged/undamaged tissues.
Brief ption of the Drawings
Figure 1a and 1b Artificial udder model photograph and tic. Milk was fed through the top
opening by means of a peristaltic pump. Doses were infused and samples were withdrawn by means
of the ‘teat ’ ed at the bottom.
Figure 2 Modified Robbins device, e with 12 polyurethane coupons to allow attachment and
proliferation of the biofilm e. The device was provided with influent media and irrigated,
h the openings at both ends.
Figure 3 Somatic Cell Count (initially, and 21 days after commencement of treatment) of 11 animals
treated for two days, twice a day, with a formulation containing 100 mg potassium iodide and 100
mg hydrogen peroxide.
Examples
1 An extemporaneous composition for daily, or twice daily, treatment is made by means of intramammary
devices; comprised of en peroxide and iodide (a range of 50-1,000 mg of hydrogen
de and a range of 35-700 mg of potassium iodide).
2 An extemporaneous composition for daily, or twice daily, treatment is made by means of intramammary
devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide by weight (76 mg
iodide and 100 mg of hydrogen peroxide)
3 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-
mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (50 mg of hydrogen
peroxide and 38 mg iodide – provided by 50 mg of potassium iodide or 45 mg sodium iodide).
4 An extemporaneous composition for daily, or twice daily, treatment is made by means of intramammary
devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (150 mg of en
peroxide and 114 mg iodide – provided by 150 mg of potassium iodide or 135 mg sodium iodide).
5 An extemporaneous composition for daily, or twice daily, treatment is made by means of intramammary
devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (200 mg of hydrogen
peroxide and 152 mg iodide – provided by 200 mg of potassium iodide or 180 mg sodium iodide).
6 An extemporaneous composition for daily, or twice daily, treatment is made by means of intramammary
devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (300 mg of hydrogen
peroxide and 228 mg iodide – provided by 300 mg of ium iodide or 270 mg sodium iodide).
7 An extemporaneous composition for daily, or twice daily, treatment is made by means of intramammary
devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (400 mg of hydrogen
peroxide and 304 mg iodide – provided by 400 mg of potassium iodide or 360 mg sodium iodide).
8 An extemporaneous composition for daily, or twice daily, treatment is made by means of intra-
mammary devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (500 mg of hydrogen
peroxide and 380 mg iodide – provided by 500 mg of potassium iodide or 450 mg sodium iodide).
9 An extemporaneous composition for daily, or twice daily, treatment is made by means of intramammary
devices; they comprise a 0.76:1 ratio of iodide to hydrogen peroxide (1,000 mg of
hydrogen peroxide and 760 mg iodide – provided by 1,000 mg of potassium iodide or 900 mg of
sodium iodide).
The above itions (1-9) where the peroxide is t as a peroxide releasing compound
such as percarbonates or perhydrates. In such embodiments, the appropriate concentration of
percarbonate can be ated by the available peroxide content (typically 20-30%) of the
percarbonate and the required concentration of peroxide. For example, 1000 mg of sodium
bonate with 30% available hydrogen peroxide would release 300 mg hydrogen peroxide on
dissolving in the environment. Similarly the use of 333 mg percarbonate would release 100 mg
hydrogen peroxide.
11 A composition used to produce a general antibacterial wash, used for example as a teat dip wash,
containing freshly mixed source of hydrogen peroxide and iodide at a range of 10-10,000 mg
hydrogen peroxide and 600 mg .
12 A ition used a general antibacterial wash, for example as a teat dip wash, containing
freshly mixed source of ionic iodide wherein 19-7,600 mg iodide per litre is t (provided by 25-
,000 mg potassium iodide or ,000 mg sodium iodide), and prepared at a ratio of between
0.38-1.52:1 of iodide to hydrogen peroxide.
13 A ition used a general antibacterial wash, for example as a teat dip wash, containing
y mixed source of hydrogen peroxide and iodide wherein 100 mg hydrogen peroxide per litre is
present and 76 mg iodide per litre is present (provided by 100 mg potassium iodide or 90 mg sodium
iodide).
14 A composition used a general cterial wash, for example as a teat dip wash, containing
freshly mixed source of hydrogen peroxide and iodide wherein 200 mg en peroxide per litre is
present and 152 mg iodide per litre is t – provided by 200 mg ium iodide or 180 mg
sodium iodide.
A poultice prepared for wound care where the poultice is dipped into the antibacterial wash
described in examples above (12-14).
16 A compoisition as described above where th source of hydrogen peroxide is additionally, or
alternatively, be provided by a peroxide releasing compound, such as percarbonate, or by enzymatic
means, e.g. by the reduction of sugar compounds, sufficient to produce the required concentration
of hydrogen peroxide. Furthermore, compositions that include an alterantive appropriate oxidising
compound to start the reaction, such as potassium permanganate, are ed.
The source of iodide could additionally, or alternatively, be provided by a list of compounds set out
elsewhere in this text, including, but not limited to sodium iodide, potassium iodide, lithium iodide,
caesium iodide, hydrogen iodide, rhodium iodide, or other iodide releasing compounds, or a slowreleasing
form of iodide, such as a degraded iodate.
Furthermore, iodide could be replaced by a thiocyanate molecule ied in the form of but not
being limited to, for example, sodium or potassium thiocyanate) whilst adhereing to a ratio of 0.2-
:1 or a more preferably 0.8-1.3:1 of anate to hydrogen peroxide) in areas where iodide use
may pose an issue, for example, in people with thyroid iproblems.
This preparation can be administered by the use of consecutive suitable mammary syringes, or
a arreled syringe (preventing premature reaction of iodide and de).
All bed compositions are to be prepared in a pharmaceutically suitable carrier. Such carriers
include, but are not limited to, water, a pH suitable saline, a pH suitable saline including onate
buffer, gel, or hydrogel.
Glucocorticoids from a list including, but not limited to prednisone, prednisilone, cortisol and
hydrocortisone could be supplemented to the composition to aid with local inflammation issues
during infection. For example, supplementation of 10-20 mg prednisolone or prednisone or 30-60
mg hydrocortisone to the antimicrobial composition could be used to counteract infection
inflammation as part of a mastitis treatment.
The application of the composition will dictate the use of carrier. For example, mastitis intramammary
treatment could be prepared in a saline or water, or in a more complex on such as
stearate/oil. A te/oil-based composition would be le in compositions to include the
hydrogen peroxide-releasing sodium percarbonate as it prevents release of peroxide until the
composition is diluted in milk in the mammmary gland.
Furthermore, a hydrogel-based composition could be used to aid application in a wound
andage setting. Such an example is a sodium polyacryalte based hydrogel with a pH adjusted
composition. Moreover, a saline or onate/saline carrier could be used in a ectant setting
or for use as a nasal rinse to relieve sinusitis and accompanying infections.
In on, the use of a temperature sensitive hydrogel containing poultice could be used to
sequester the components such that application of the poultice to tissue at body temperature would
allow the release of antimicrobial activity as the components react.
Pharmacological Results
Result 1
A freshly prepared mixture of hydrogen peroxide and potassium iodide was mixed (100 mg of each,
at a ratio of iodide to en peroxide of 0.76:1 by weight) in 10 ml sterile water. Using a microbroth
dilution technique, the ition was doubly diluted in broth containing the test bacterial
organism using a 96 well plate system. Depending on the sensitivity of the organism, a cut-off point
was reached at a on at which the bacterial culture was no longer able to resist the effects of the
antimicrobial composition. This is characterised as the minimum inhibitory concentration (MIC). The
lower the MIC, the more sensitive the organism.
Table 1. MIC of mastitis g organisms to an antimicrobial composition containing 100 mg
hydrogen peroxide and 100 mg potassium iodide prepared freshly in a 10 ml volume and diluted
accordingly. P. aeruginosa PA-29 exhibited increased tolerance to biocides and ance to
fluoroquinolone classed antibiotics. P. aeruginosa ‘R’ strains are cystic fibrosis clinical isolates that
exhibited nces one or more of amakcin, tobramycin, loxacin, and gentamicin. S. aureus
BH1CC was described ere as an MRSA class organism.
Table 1 shows the sensitivity of a number of mastitis-causing organisms to antimicrobial activity
produced as a result of the reaction between en peroxide and iodide in the absence of a
peroxidase enzyme. As is evident, all strains tested, including the Gram-positive organisms, were
sensitive to the composition. It is evident that, in broth, the use of lactoperoxidase is not required. In
addition, it is evident that the composition is also effective against fungi.
Furthermore, the broths were sub-cultured at various times post testing to assay for cell viability.
This was carried out by sub-culturing 100 microlitres of the treated culture to 10 ml of fresh broth
containing no antimicrobial. This differentiated between cell death and cell stasis (where viable cells
would be detected). No growth was observed after 48, 72 or 96 hours post ent (using
concentrations in the same order of magnitude as those inhibiting them in the MIC test). This
demonstrates that the produced antimicrobial ty is bacteriocidal to both Gram-negative and
Gram-positive organisms, a sing finding.
Result 2
The activity of the antimicrobial composition of the invention (peroxide and iodide) and another
composition containing peroxide and thiocyanate against bacterial cells g in m mode was
established by means of a modified Robbin’s device (MRD). A biofilm of E. coli, S. , P.
aeruginosa, or a mixture of all three was established by inoculating 500 ml LB in a 1 L glass bottle
that was connected to a modified Robbins device. The MRD system consists of 12 sampling ports, to
which medical-grade polyurethane coupons (50 mm2 surface area) were inserted. The culture was
allowed to proliferate at 37°C and the MRD was fed/irrigated with culture at a rate of 0.1 h-1 for at
least 48 hours (a further 100 ml LB were supplemented after the initial 24 hours). The MRD was then
ted with a control or a test solution. A small amount of constant force was used in the
irrigation, such that it took approximately 90 seconds for the complete volume to pass through the
system. The control used was a physiological saline solution. A number of distinct solutions were
tested by irrigating the MRD chamber whilst the coupons were in situ: a ‘1x’ solution containing 0.3 g
l-1 potassium thiocyanate and/or ium , and 0.003% hydrogen peroxide; a ‘5x’ solution
containing 1.5 g l-1 potassium thiocyanate and/or potassium iodide, and 0.015% hydrogen peroxide;
and a ‘20x’ on ning 6 g l-1 potassium thiocyanate and/or potassium iodide, and 0.06%
hydrogen peroxide.
Table 2 Total viable counts (colony g units per coupon) isolated from irrigated/non-irrigated
E. coli biofilm and tested against a thiocyanate-based solution. Values represent the range from
four coupons.
From Table 2 it is clear that 105-6 colony forming units (CFU) were recoverable from the coupons in
the absence of an irrigation step. Likewise, the bacterial viability was not significantly altered when
the system was irrigated with a saline solution. The supplementation of thiocyanate and hydrogen
peroxide to the solution at either 1x or 5x levels did not alter the outcome in cell viability. The use of
a 20x solution did begin to affect cell viability, with a log-reduction in numbers typically noted.
Similar results were noted when repeated with P. nosa and S. aureus biofilm cultures).
Table 3 Total viable counts (colony forming units per coupon) isolated from irrigated/non-irrigated
E. coli biofilm using an iodide-based solution. Values represent the mean from four replicates and
two biological replicates. NG – no viable cells recoverable
The use of iodide instead of peroxide and thiocyanate derived OSCN- had a profound effect on the
outcome of the test (Table 3), against all three organisms. Cell viability was notably lower on use of a
1x iodide-based solution. Typical 100fold reductions in cell viability were noted after the 90
seconds tion (Table 3). A reduction from 106 CFU to 103 CFU was ed for E. coli on using
the iodide at a ‘5x’ level. It should be noted that three of the four s yielded no recoverable
cells at all. A ‘20x’ solution was sufficient to result in a complete lack of viable cell ry (Table 3).
Repetition of the set-up, but using a mixed culture of E. coli ATCC 25922, S. aureus DSM 15676 and
P. aeruginosa NCIMB 10421 resulted in a very similar pattern (Table 4). A ‘1x’ solution was sufficient
to result in a 100-fold reduction in bacterial viability. Both the ‘5x’ and ‘20x’ solutions were sufficient
to result in a complete lack of viable cells recoverable from the coupon surface. ment to the
surface was greater on using a mixed culture and there was some ce to suggest that some
cells were lost on irrigating with the saline solution only. Such a decrease in viability of biofilm
attached cells within such a short space of time (90 seconds) would teach that the /hydrogen
peroxide composition would be very effective as a therapeutic in m centred infections when
involving an in vivo model.
Table 4 Total viable counts (colony g units per coupon) isolated from irrigated/non-irrigated
mixed biofilm of E. coli, P. aeruginosa, and S. aureus cultured together and tested against an
activate-iodide solution. Values represent the mean from four replicates and two ical
replicates. NG – no viable cells recoverable
The data presented in Tables 3 and 4 would strongly indicate that the activity produced by even very
low concentrations of the antimicrobial composition was capable of g biofilm-formed bacterial
cultures of both egative and Gram-positive organisms and even mixed culture consortia. This
result would compare favourably to the use of antibiotics to achieve the same outcome. Many of the
antibiotics would require 1,000-fold ses in concentration to achieve kills of biofilm cells by
comparison to planktonic cells (i.e. free-floating). This was not the case when an iodide/hydrogen
peroxide composition was employed to produce the antimicrobial activity.
We disclose that that hydrogen peroxide, even at low trations (e.g. a 0.003% solution), is
capable of producing an effective antimicrobial action against infectious disease causing organisms,
by reaction with iodide in the absence of a peroxidase enzyme. An increase in contact time (for
example, 10 minutes) would allow 1x solutions to kill the bacterial cultures to the same degree
noted here for er concentrations.
The prior art teaches that even higher concentrations of an antimicrobial agent will be ed to
effectively kill biofilm bacteria. The amount of hydrogen peroxide in the present invention is
effective, however, when used with the correct amount of iodide. The ition of the invention
will provide (a) a greater kill efficiency; (b) effective biofilm eradication and (c) a composition
le for in vivo applications, such as treatment of mastitis or other infections (and doing so
without g irritation to the infection site).
Result 3
Lactoperoxidase has been shown to be present at significant concentrations in bovine milk, up to 30
mg l-1. This level will depend on the health of the animal, breed of the cow etc. and therefore cannot
be depended on to be present at optimal levels for an dual animal treatment. A reliance on
naturally present lactoperoxidase would not allow an optimally designed treatment. To trate
that the described method and composition sed herein does not require endogenous
lactoperoxidase, MICs were carried out in a broth, raw milk, pasteurised milk, and ultra heat treated
(UHT) milk. The lactoperoxidase enzyme is inactivated at elevated temperatures, such as those used
in the pasteurisation process. l pasteurisation processes (72˚C for 15 seconds) reduce activity
of the enzyme by a significant amount (c. 70%), while ent at 80˚C or more (including UHT
treatment at 135˚C) would result in complete destruction of the enzyme. Were lactoperoxidase
present in the milk to be essential to the reaction, the use of low concentrations of iodide and
peroxide in a UHT milk environment should not produce any noted antimicrobial effect.
On testing the composition of the invention in these environments, E. coli was no more tolerant to
the effects in UHT milk than that in broth, raw milk, and pasteurised milk (See Table 5). This clearly
indicates that the composition of the invention is not in any way reliant on the presence of
endogenous or native lactoperoxidase .
Furthermore, the supplementation of lactoperoxidase to the broths of UHT milk concentrations did
not affect the outcome of the minimum inhibitory concentration inations after 24 hours
incubation (Table 5).
Table 5 MICs of E. coli ATCC 25922 to appropriately diluted formulation of 100 mg en
peroxide and 100 mg potassium iodide. Values represent minimum mg/L value of potassium
iodide and hydrogen peroxide required to inhibit growth over 24 hours. LB – lysogeny broth.
Pasteurised milk – 72° C for 15 seconds. UHT milk – 135° C for 3 seconds. LP – lactoperoxidase.
Result 4
In an attempt to induce a nce to an crobial, the micro-broth dilution method was
utilised, with repeated passaging, with E. coli ATCC 25922 and a number of antibiotics, as well as the
composition of the invention containing hydrogen peroxide and potassium iodide. Stock
concentrations of kanamycin, polymyxin B, and levofloxacin (all clinically important and relevant
antibiotics) were used to demonstrate the development of typical resistance characteristics to
antibiotics. ng dilutions of crobial compositions were made using a 96 well plate. The
well contents were inoculated with 104 cfu ml-1 E. coli ATCC 25922 and allowed to incubate. The well
with the highest concentration of antimicrobial that still resulted in growth overnight served as the
inoculum for the next day’s passage. Therefore, for example, the ‘kanamycin-passaged’ strain did
not come into contact with any of the antimicrobials during the test other than kanamycin.
After only 8 passages in the presence of the antibiotics, the organism was completely resistant to the
cin and xin B drugs. After 10 passages, the organism was 8 times more tolerant to the
s of levofloxacin than it was pre-passaging. These results mirror the use of antibiotics in the
environment wherein a bacterium will gain resistance due to repeated re to the antibiotics.
However, during the same experiment, the organism was no more resistant to the effects of the
antimicrobial activity produced by the hydrogen peroxide/iodide composition, after a r
number of passages. This is due to the inherent mode of action wherein there is no single target for
the antimicrobial activity produced by this reaction, acting as is does across a variety of bacterial
proteins. A bacterial cell would require le, concurrent, mutations to gain resistance, whilst
only requiring one mutation in se to traditional antibiotics. This severely limits the ability of
the organism to counter-act the inventions actions. Broad range acting antimicrobials have been
described as unlikely to illicit bacterial resistance, or for development of cross-resistance toward
otics. The antimicrobial composition was also capable of killing a number of antibiotic-resistant
P. aeruginosa bacteria. The developed kanamycin and polymyxin B resistant s, as well as the
levofloxain tolerant mutant, were no more tolerant to the actions of the disclosed composition,
indicating the cross-resistance would not occur. A strain described previously by Mc Cay et al.,
(Microbiology, Vol. 156, No.1 30-38, 2010) as being resistant to fluoroquinolone and tolerant to
benzalkonium chloride biocide was sensitive to the actions of the antimicrobial activity of the
invention (Table 1). se, a number of clinical cystic fibrosis isolated P. aeruginosa strains, with
elevated tolerances to key otics used in the treatment of CF patients, were no more tolerant to
the antimicrobial activity than the P. aeruginosa wild type strain (see Table 1). Further to this, S.
aureus BH1CC (MRSA) is an antibiotic resistant strain (oxacillin MIC > 256 mg/L) ed from
Beaumont hospital, Dublin, Ireland. The strain was no more tolerant to the actions of the reaction
than another Staph. aureus strain (Table 1). This would again indicate that the method and
ition disclosed here would be effective against MRSA isolates.
Result 5
An experimental method was devised to determine the most appropriate concentration of the
composition required to provide antimicrobial action within the udder, but not ting in the milk,
post milking of the animal. A simulated bovine udder was designed, using an amended rubber gas-
tion bag with two inlet channels (see Figure 1). Various formulations, produced freshly by
means of hydrogen peroxide and potassium iodide reactions, were introduced into the bag through
an inlet. To simulate the udder environment, milk was then fed into the udder at the same rate a
typical cow might produce milk, 12 L a day. Samples of the milk were taken routinely at various time
points and the antimicrobial activity of the milk was determined by the spiking of the sample with E.
coli. The spiked milk sample was then incubated and uently tested for the presence of
remaining viable E. coli cells. A lack of viable cells indicated that there was sufficient antimicrobial
activity present to inhibit/kill the E. coli. Conversely, the presence of viable cells ted that the
milk no longer contained sufficient quantities of antimicrobial activity to inhibit the growth of the
bacterium. Using this method, each prospective embodiment was titred, such that activity was
present for at least 5-6 hours post treatment/infusion, but no longer than 10 hours following
treatment (such that activity will be absent when the milk is outside the udder environment). A 10
ml volume containing 100 mg potassium iodide, and 100 mg hydrogen peroxide ed the
desired characteristic (a 0.76:1 ratio of iodide to hydrogen peroxide by ). At peroxide
concentrations less than 50 mg (i.e. a >1.52:1 ratio of iodide to de by weight), there was
insufficient antimicrobial activity over a 4-5 hour period to be an effective treatment. At peroxide
trations greater than 300 mg (a <0.38:1 ratio of iodide to peroxide by weight), antimicrobial
activity persisted for greater than 10 hours, which could be a potential problem for post-processing
of milk. The risk of damage to animal cell tissue also increases with increased de
trations. A ratio of between 0.38-1.52:1 of iodide to en peroxide by weight was
suitable in providing a suitably paced reaction in the simulated cow udder.
Using this method, a suitable ratio of peroxide to iodide was predicted for the treatment various
ruminant s (cows, sheep, etc.). Interestingly, an undiluted 10 ml composition of peroxide and
iodide (200 mg of each at a ratio of 0.76:1 iodide:hydrogen de by weight) was found to have
lost all significant antimicrobial activity within a smaller time frame than useful in a mastitis setting
(though still useful for disinfectant purposes where contact time is much shorter), despite the
elevated initial concentrations. This g confirms that consideration of the cs of the
reactions between hydrogen peroxide and iodide was a uisite for the development of suitable
compositions for therapeutic purposes. It is counter-intuitive that a more concentrated solution of
an antimicrobial compound would be less effective than a more dilute n and that the more
trated solution would lose activity after a shorter period of time, by comparison to the
embodiment containing 100 mg of each nce – which again emphasises the link between
reaction kinetics and efficacy in this case.
Failure to take into account the dynamic volume changes in the udder environment is also a problem
with respect to producing an effective mastitis therapy. For example, a composition (100 mg
potassium iodide and 50 mg hydrogen peroxide – a 1.52:1 ratio by weight of iodide to en
peroxide) was added to a constant, unchanging volume of milk of 1 L. The composition remained
crobially active for >24 hours in this situation, but the same composition recorded
antimicrobial activity for <3 hours during tests in the udder model. Such a result, in the absence of
an appropriate ted udder model method, would teach that the antimicrobial composition was
active for too long (i.e. greater than 10 hours) using this particular ratio and that activity would be
expected long after the milk was taken from the udder. This proved that a simulated udder model,
as disclosed in this application, provided an effective method, which allowed the design of an
appropriate therapeutic for in vivo applications.
Result 6
The correct concentrations of peroxide and iodide are required to ensure an effective therapy for
mastitis and to ensure that the post-processing of milk is not affected. The use of the model udder
system allowed us to develop the correct concentration of composition. If an incorrect
concentration is used the same results will not be observed.
For example, a ing dairy cow was infused with a composition containing both potassium iodide
and hydrogen peroxide (5 ml solution of each, containing 50 mg rting to 38 mg iodide) and
150 mg, respectively – a ratio of 0.25:1 of iodide to peroxide by weight). A 5 ml embodiment was
infused into lactating cow’s udders, twice a day for 2 days. The animals were monitored for changes
in activity, milk yield etc. tion, d milk yield and udder sensitivity was noted in the
animals, indicating that the tion of the antimicrobial activity was insufficient (or too slow) and
that un-utilised peroxide was remaining, which started to irritate or damage the animal tissue. This
experiment used a concentration of 0.1-0.15% peroxide in the udder, which although significantly
lower than the concentrations of peroxide reported to be required in antiseptics, still resulted in
tion in the animals. A treatment for mastitis by use of such an ment would be
unacceptable. Use of this embodiment (or a similar one where the de is present for a
ged period) would also lead to issues in post-processing of milk as well as initial irritation in
the animal. This is because the presence of peroxide would likely inhibit any starter cultures used in
the cheese and yogurt processing stages.
To demonstrate that the use of an alternative and preferred embodiment (containing 100 mg
ium iodide and 100 mg hydrogen peroxide – a 0.76:1 ratio of iodide to peroxide by weight)
would not harm animals, 5 lactating dairy cows were infused with this embodiment, twice a day, for
days. The animals were monitored for changes in activity, milk yield, milk quality, etc during the
period immediately following treatment, and for several months after ent. No signs of
discomfort or changes in udder sensitivity, hardness or temperature were noted following
treatment; and there was no change in the quality (nutritional, chemical or biological) or quantity of
the milk produced by the individual s. This demonstrated that: a) the ition and
treatment was not toxic to the animal at the level described (even after a prolonged, 5 day
treatment); b) there was no ‘bottle neck’ in the reaction to e antimicrobial activity ing in
a prolonged presence of peroxide; and c) that the likely cause of the irritation described r was
the prolonged exposure of the teat canal tissue to excessive levels of peroxide. These specific results
also support the suitability of this embodiment of the therapy for use as a treatment for sub-clinical
mastitis and/or as a prophylactic therapy, given the absence of any negative effects on the cow and
the possibility for continued utilisation of milk produced by an animal undergoing treatment.
Result 7
To demonstrate that the treatment method would not ere with post-processing, milk taken
from healthy animals, infused with a composition containing 100 mg potassium iodide and 100 mg
hydrogen de, was tested using a Delvo test. This is the ‘gold standard’ for g for the
presence of antimicrobials (sensitive to parts per billion level for some antimicrobials and to a broad
range of compounds). Milk produced from healthy cows and from cows with clinical mastitis, which
were treated with the composition, recorded a ‘negative’ result for antimicrobials when tested
during treatment, thus g that the antimicrobial composition was active for a limited period of
time, i.e. between milkings only. ally, this demonstrates the embodiment would not affect postprocessing
of the milk – if there is insufficient antimicrobial activity present immediately post-
milking to inhibit the Delvo test organism, then there is no issue with respect to the inhibition of
starter es.
Result 8
A number of sick animals were identified that were suffering from chronic clinical mastitis due to S.
aureus infections. These animals were non-responsive to previous antibiotic therapies in the months
prior to the trial and demonstrated the typical cyclical SCC increases ated with chronic
mastitis. Eleven of these animals were d with an embodiment of the present invention,
containing 100 mg potassium iodide and 100 mg hydrogen peroxide (0.76:1 by weight). The animals
were treated twice a day, post milking, for two days. The somatic cell count was determined on day
21 post treatment start. This is in line with European nes Agency guideline dates (ensuring
any effect noted is not merely transitory). At day 21, there was no ce of any remaining clinical
infection based on milk SCC counts, udder sensitivity, swelling, ature or other milk quality
indicators, thus trating the therapeutic effectiveness of the method, even in cows chronically
infected with S. aureus (see Figure 3 for SCC count data). This trial demonstrates that the
composition was capable of treating S. aureus infections of a chronic . Crucially, this also
demonstrates that the composition was not harmful to the animals, as may be the case on using
hydrogen peroxide. These animals were due to be culled (low milk yield, non response to numerous
antibiotics, and continuous elevated SCC) but were returned to the herd and were milking normally
for months following ent using the invention. The estimated saving to the farmer due to
sed milk production, no milk discard, and veterinarian fees, and replacement of animals was
>€10,000 ($13,000).
A novel finding from this in vivo study and the in vitro studies ed in this ation using milk
as bacterial growth medium - is the ability of the hydrogen de/iodide model to function in vivo
or in vitro in milk containing relatively elevated levels of thiocyanate. This would not be the case
were a peroxidase enzyme employed to catalyse the reaction betwen hydrogen peroxide and iodide,
based on the prior art.
Klebanoff et al., (J Exp Med 1967 126(6):1063-78) describe the fact that “thiocyanate ions were
inhibitory” to the actions of IO-, produced by the peroxidase-catalysed reaction between hydrogen
peroxide and iodide. The authors of that study believed this was an “apparent paradox” given the
role of anate in the production of OSCN-. Such a finding would further teach that the efficacy
of the peroxidase-catalysed reaction betwen hydrogen peroxide and iodide to produce IO- as an
antimicrobial in the udder environment would be limited, as thiocyanate is present at significant
concentrations in bovine milk (WHO/FAO, 2005). Tenovuo et al. and the articles sed in that
review (Oral Diseases {2002} 8, 23-29) stated that gh an oxidation ts of iodide are much
more potent than thiocyanate oxidation products, the “major problem with iodide in connection with
LP-system is that even relatively small concentrations of salivary thiocyanate abolish the
bacteriocidal effect of the LP/I-/H2O2 systems as SCN- is the preferred ate”. er, it has
been reported that a similar and strong interference n thiocyanate and iodide in an oral
biofilm study of the yeast, Candida albicans ly and vely affects IO- production. The
authors state that “the ability of OI- to affect yeast growth and survival must be questioned in regard
of the other iodine compounds, since clinical trials have shown limited or weak beneficial effects of
other iodine formulations used for in vivo antifungal purposes”. And “The ency of an
iodide/peroxidase system demonstrated in vitro through this investigation is difficult to transfer to
either animal or human. Beside the toxicity of oxidant products on host cells and the immunogenicity
of enzymes isolated for example from bovine milk, the oxidation of iodide is under the l of
thiocyanate, which is not only present in several exocrine secretions (for example in human saliva)
but is also preferentially used as substrate by lactoperoxidase. Indeed, simultaneous incorporation of
both substrates in the same gel provided a decrease of the beneficial effect of 2 mM iodide in the
presence of increasing concentrations of thiocyanate ranging from 0.25 to 4 mM, which correspond
to the normal range of thiocyanate in saliva” (Ahariz and Courtois 2010. Clinical, Cosmetic and
Investigational Dentistry 2010:2 69–78). These authors conclude that peroxidase-generated OI- to
prevent C. albicans biofilm pment would only be useful for treatment of devices in ex vivo
conditions.
The concentrations of thiocyanate in milk are high, relative to the teaching described above, and
they vary widely according to the animal diet. A severe inhibition of IO- tion by thiocyanate
would thus suggest that a therapy based on iodide and hydrogen de would be of no benefit
for the treatment of mastitis.
The results from the described clinical trials of Result 8 carried out by the applicants recorded no
negative impact of innate thiocyanate in milk on the efficacy of the proposed therapy, a highly
surprising finding given the previous cited literature. It is explained by the need to balance the
concentrations of the reactants appropriately, using the method described in the present invention,
and of the critical importance of reaction kinetics to the crobial cy of a therapy based on
the antimicrobial activity produced by the reaction n peroxide and iodide.
Result 9
The simulated udder model method ents a significant advance on the prior art with respect to
the assessment of antimicrobial activity. Results 6 and 8 above describe the use of the therapy with
100 mg potassium iodide and 100 mg hydrogen peroxide (0.76 iodide:1 hydrogen peroxide by
). Use of too low a concentration of components resulted in no irritation or other negative
effects to the animal, but also no significant drop in SCC counts or improvement in infection, during
or post treatment. In vitro ments using traditional minimum inhibitory concentration testing
had, however, ted that the solution would be antimicrobially active for c. 24 hours in a 1 L
volume. The simulated udder model method correctly ted, however, that it would not be
crobial for a sufficient period of time in vivo to result in a sufficient kill of the pathogen. The in
vivo data validated the predictions of the experimental model and demonstrated its importance in
mapping the kinetics of the udder environment. It is now evident that there is a range of
concentrations and ratios that must be achieved in the reaction to be effective for use as a mastitis
treatment, and that the described embodiment in Result 8 achieves this effective concentration for
the treatment of bovine mastitis.
It is evident upon ing these s, that the described hydrogen peroxide/iodide composition
is effective at killing a wide range of organisms (Table 1). It has been shown also that there was a
significant ence in e between using this model, by comparison to one based on the
production of OSCN- with respect to treatment of biofilm cultures of both Gram-positive and Gram-
negative organisms (Tables 2-4). The ratio of the compounds has also been shown to be extremely
important at an in vivo level. Too high a concentration leads to tissue damage or prolonged ty
(affecting rocessing of milk; failure of Delvo test). Too low a concentration or ratio leads to
insufficient activity, minimising the effect of the treatment in killing the t pathogens. The
kinetics of the udder environment, starting from a very low volume and increasing to ~1-4 litres of
milk is also an important factor. Surprisingly, undiluted compositions lost activity much quicker than
diluted compositions. Traditional in vitro models using a constant final volume would teach one to
over-estimate the effectiveness of the hydrogen peroxide/iodide model and would not translate to
success in vivo – this is a feature of the prior art cited in this application. The development, design
and deployment of the novel simulated udder model method disclosed here allowed the proper
evaluation of the disclosed ent and ultimately enabled the development of the novel therapy
for mastitis.
This is the first description of the on between low concentrations of hydrogen peroxide and
iodide as being the basis for a highly efficient bacteriocidal composition against both Gram-negative
and Gram-positive bacteria, independent of lactoperoxidase (or any other peroxidase enzyme)
activity. It is the first to demonstrate that the even low concentrations of the reaction products of
hydrogen peroxide and iodide provide an efficient means of ating bacterial biofilms, even in
the presence of thiocyanate. It is also the first disclosure of the successful use of a hydrogen
peroxide and iodide composition for eutic use for treatment of bovine mastitis, and in a
potential open wound setting by way of a poultice.
This is the first description of an experimental method to characterise the evolving udder
environment in order to allow the development of a mastitis therapy based on hydrogen peroxide
and iodide. We present in vivo data to validate the efficacy of the novel method to produce a safe
ent for bovine mastitis, a method capable of both treating and curing mastitis, while
simultaneously allowing producing of milk that will not affect the post-processing processes, such as
cheese or yoghurt production.
Result 10
To demonstrate the ence n traditional iodine based antimicrobials and the activated IO-
form described herein, MICs were performed in a variety of media, including water, LB broth, and
milk, using E. coli ATCC 25922 as a test organism. As a comparison, PVP-I or povidone iodine, used
ntly in hospital setting as a topical antiseptic, was used as a source of free iodine (I2). Although
the PVP-I is strongly antimicrobial in water and saline, it decomposes extremely quickly in the
presence of organic material t in LB and milk (see Table 6). This limits the cy of PVP-I to
such areas where c material is not present. This is not the case with the present OI-
formations. Organic material does not irreversibly render the on ineffective. Curiously, the use
of a combined hydrogen de and PVP-I in a saline environment resulted in no antimicrobial
activity either, as the PVP-I apparently exerts a negative effect on peroxide antimicrobial activity
(Table 6). Repetition in a broth environment causes the PVP-I to be broken down before it can affect
the peroxide, and the MIC is cal to that of peroxide in the absence of PVP-I. Using the
composition of 100 mg H2O2 and 100 mg KI, MICs of between 20-40 mg L-1 were achieved in each of
the test environments (milk, broth and water), indicating that the presence of organic material did
not inhibit the desired antimicrobial activity
Solution environment MIC (mg L-1)
PVP-I (saline) 1-2
PVP-I (in LB) >250
PVP-I (milk) >250
PVP-I + H2O2 (saline) >250
PVP-I + H2O2 (broth) 20-40
H2O2 (broth) 20-40
Table 6 Activity of and PVP-I in absence and presence of c material
Claims (23)
- Claims 1. A method of treating or preventing mastitis in a ruminant, the method comprising administering an effective amount of a pharmaceutical composition comprising iodide (I-) and a source of hydrogen peroxide, together with a ceutically effective 5 carrier or diluent, to a ruminant in need thereof; wherein the pharmaceutical composition does not include a peroxidase enzyme or povidoneiodine.
- 2. A method according to claim 1, where the concentration of hydrogen peroxide in the composition is less than 1% based on /volume or weight/weight.
- 3. A method according to any claim 1 or 2, wherein the composition comprises a 0.2: 1 10 to 3 : 1 ratio by weight of iodide to hydrogen peroxide.
- 4. A method according to any one of the preceding claims, wherein the composition comprises a 0.38 : 1 to 1.52 : 1 ratio by weight of iodide to hydrogen peroxide.
- 5. A method according to any one of the preceding claims, wherein the composition contains 5-5,000 mg iodide, and is prepared at a ratio of between 0.2 : 1 and 3 : 1 by 15 weight of iodide to hydrogen peroxide.
- 6. A method according to any one of the preceding claims, wherein the composition contains 5-5,000 mg iodide, and is prepared at ratio of between 0.38 : 1 and 1.52 : 1 by weight of iodide to en peroxide.
- 7. A method according to any one of the preceding claims, n the ition 20 ns 5-5,000 mg iodide, and is prepared at a ratio of 0.76 : 1 by weight of iodide to hydrogen peroxide.
- 8. A method according to any one of the preceding claims, n the source of iodide is sodium iodide or potassium iodide, lithium iodide, caesium iodide, hydrogen iodide, rhodium iodide, or a eleasing form of iodide. 25
- 9 A method according to claim 8, wherein the slow-releasing form of iodide is an iodate.
- 10. A method according to any one of the preceding claims, wherein the source of en peroxide is: (i) hydrogen peroxide, 30 (ii) sodium peroxide, (iii) lithium de, (iv) peroxide releasing citric acid or Vitamin C, (v) peroxide salts, (vi) an hydrogen peroxide-urea adduct, (vii) oxygen releasing pseudo peroxides, or (viii) organic peroxides.
- 11. A method according to claim 10, wherein the peroxide salt is barium oxide or sodium 5 perborate.
- 12. A method according to claim 10, wherein the oxygen ing pseudo peroxides are xides, ozones, or ozonides.
- 13. A method according to claim 10, wherein the organic peroxides are peroxy acids or tic peroxides. 10
- 14. A method according to any one of claims 1 to 9, wherein the source of hydrogen de is a peroxide-releasing percarbonate.
- 15. A method ing to claim 14, n the peroxide-releasing percarbonate is sodium or potassium percarbonate, or a slow-releasing form of a peroxide-releasing percarbonate. 15
- 16. A method according to any one of the ing claims, wherein the composition contains 5-5,000 mg iodide, and is prepared at a ratio of n 0.2 : 1 and 1 : 1 by weight of iodide to sodium percarbonate.
- 17. A method as claimed in any one of the preceding claims, wherein the composition further comprises lactoferrin or a orticoid. 20
- 18. A method as claimed in claim 17, wherein the glucocorticoid is predinisolone prednisone, or hydrocortisone.
- 19. The method of any one of the ing claims, wherein the composition is adapted for concurrent, or sequential, delivery of the peroxide and iodide components.
- 20. The method of any one of the preceding claims, wherein the pharmaceutically 25 effective carrier is water, saline, an emulsion, a gel or a hydrogel.
- 21. The method of any one of the preceding claims, wherein the composition is adapted for delivery by means of an mammary device.
- 22. A method according to any one of the preceding claims, wherein the hydrogen peroxide and iodide are provided in a concentrated form in a medical device, suitable 30 for production of a working solution containing between 10-5,000 mg/L hydrogen peroxide and 7-3,800 mg/L .
- 23. A method according to any one of claims 1 to 22 substantially as herein described with reference to any example thereof and with or without refrenece to the accompanying drawings.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP13166137.3A EP2799068A1 (en) | 2013-05-01 | 2013-05-01 | Antimicrobial compositions and methods for their production |
EP13166137.3 | 2013-05-01 | ||
PCT/EP2014/058766 WO2014177590A1 (en) | 2013-05-01 | 2014-04-29 | Antimicrobial compositions and methods for their production |
Publications (2)
Publication Number | Publication Date |
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NZ713792A NZ713792A (en) | 2021-02-26 |
NZ713792B2 true NZ713792B2 (en) | 2021-05-27 |
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