A composition for inhibiting biofilm formation and treating biofilm-related disorders.

Abstract

The present disclosure relates to thiosulfur-containing compositions, particularly propyl-propane thiosulfonate (PTSO) and propyl-propane thiosulfinate (PTS). Such compositions are useful for treating infections and reducing or degrading biofilms both in vivo and in vitro. In particular, such compositions are useful in treating biofilm-associated disorders, including, but not limited to, mastitis, digital dermatitis, and chronic wound infections.

Description

[Technical Field]

[0001] This disclosure relates to thiosulfur-containing compositions, particularly propyl-propane thiosulfonate (PTSO) and propyl-propane-thiosulfinate (PTS). Such compositions are useful for treating infections and reducing or degrading biofilms both in vivo and in vitro. In particular, such compositions are useful in treating biofilm-related disorders, including but not limited to mastitis, digital dermatitis, and chronic wound infections. [Background technology]

[0002] The health of the udder plays a crucial role in dairy animals from both a health and wellness perspective and an economic one. Mammary gland infections (known as mastitis) in dairy animals, such as dairy cows, have a significant economic impact on dairy farms worldwide. The annual global loss is estimated at €4 to €5 billion. In mastitis, the udder is unable to produce an effective defense response against microbial invasion. Several factors are known to disrupt the balance at the udder level, which can impair the dairy animal's ability to kill the microorganisms that cause mastitis. As a result, the host's response mechanisms may not be able to produce an efficient defense response to eliminate the invading pathogens, leading to bacterial colonization of the udder and the development of clinical or asymptomatic mastitis.

[0003] Bacterial colonization, particularly the formation of bacterial reservoirs in the udders of dairy cows, generally leads to chronic infections that are difficult to manage and can persist despite certain treatment strategies, with or without antibiotics. Key factors associated with the persistence of bacterial infections in the udders of dairy cows include epithelial adhesion, biofilm formation, and bacterial susceptibility to phagocytosis.

[0004] Several strategies, such as antibiotic treatment and vaccination, are used to control or prevent mastitis in dairy cows and to mitigate the clinical and economic impacts of the disease. However, most treatments and strategies have little to no effect on improving the disease. Several reasons can be considered for the lack of effectiveness. Firstly, although many pathogenic factors have been suggested as potential antigens for single-component vaccines, experimental studies have demonstrated that inducing immunity against a single factor is not sufficient to provide robust protection against the bacteria that cause mastitis in dairy cows. Secondly, bacterial antigens have low immunogenicity and require appropriate adjuvants. Thirdly, a major challenge in controlling mastitis is finding effective antibiotics that can reach the bacterial pool in the udder, for example, as a result of bacterial biofilm formation.

[0005] Microorganisms, such as bacteria, do not necessarily need to form biofilms, but if they can adhere to, for example, epithelial cells, they are much more likely to survive within the host. Adhesion is an active process involving a series of attachments and detachments, resulting in biofilm formation, which involves significant genetic and subsequent physiological changes in the microorganism, leading to a loss of susceptibility to virtually all classes of antibiotics. Thus, the management of bacterial mammary infections is becoming increasingly difficult due to the emergence and spread of antibiotic-resistant bacterial pathogens. In some cases, low doses of antibiotics can even enhance biofilm formation, suggesting a natural defense mechanism of bacteria in avoiding the lethal effects of antibiotics. Due to this complex and problematic situation, mastitis remains one of the most important diseases in dairy cows, despite recent advances in improving overall mammary health.

[0006] In addition, when cattle are frequently or continuously treated with antibiotics, the antibiotics and their degradation products are also found in their manure. Fertilizers containing the antibiotics and their degradation products are applied to the soil. The presence of these antibiotics has been shown to affect the diversity of bacteria in the soil. This is an undesirable impact on the environment. Alternative compositions for treating infections in cattle would prevent the spread of antibiotics through the environment and restore the soil microbiome.

[0007] While descriptions of bacterial biofilms can be found in scientific literature long before, the significance of biofilms became known in 1982 when Costelton observed that Staphylococcus aureus formed biofilms on the leads of cardiac pacemakers. Subsequent research and clinical observations revealed that bacterial biofilms can be found on implants and catheters, prosthetics, and other transplanted biomaterials. More importantly, it was observed that microbial biofilms can also form on biological surfaces in human and animal tissues, such as the periodontal mucosa in the oral cavity (plaque), the sinuses (chronic sinusitis), the inner ear (otitis media), blood vessels and heart valves (endocarditis), the alveolar surface (polypulmonary disease), or the biliary tract and bladder.

[0008] The first stage of biofilm formation involves the attachment of cells to a surface. In the second stage, cell proliferation occurs, resulting in the formation of a mature structure with many cell layers. A mucous layer is also formed, which further protects the bacteria. See, for example, Melchior et al. Veterinary Journal 2006 171:398-407. When a critical mass is reached, the outermost cell layer of the biofilm may release "planktonic organisms." These organisms may further form colonies on other surfaces. Biofilms can form on a variety of surfaces, such as living tissues, implantable medical devices, piping in industrial or drinking water systems, or the aforementioned various surfaces encompassing natural water systems. As will be understood by those skilled in the art, not all infections lead to biofilm formation.

[0009] Research over the past 20 years has revealed that collective biofilm formation is facilitated by a bacterial communication system called quorum sensing (QS). QS is generated by small chemical molecules (so-called auto-inducers, AI) that are permanently excreted into their environment by bacteria. These signaling molecules (e.g., oligopeptides (AIP) or N-acetylhomoserine lactone (AHL)) are recognized and monitored by other bacteria in their vicinity via specific receptors. When a certain density of AI is reached (quorum), bacterial cells collectively alter gene expression and either produce pathogenic factors to attack somatic cells or activate metabolic pathways to form a biofilm on the tissue surface. Biofilm formation initially involves the formation of an extracellular matrix consisting of large polymers, primarily polysaccharides, which, as it matures, is stabilized by proteins and lipids, resulting in a three-dimensional structure.

[0010] Once a biofilm infection is established, eradicating it is extremely difficult. Mature biofilms will intermittently release planktonic cells, which can lead to chronic infections with intermittent exacerbations. While antibiotics or the host's immune response may resolve the symptoms caused by planktonic cells, the mature biofilm may persist.

[0011] Microbial cells under the protection of a biofilm are often more resistant to antibiotics and the body's natural immune response. Dormant bacteria are metabolically inactive and therefore do not express typical targets of many antibiotics, such as bacterial cell wall components (targets of β-lactam antibiotics, e.g., penicillins, cephalosporins, and vancomycins), and rapid proteins (targets of aminoglycosides, tetracyclines, macrolides, and linezolids) and DNA (fluoroquinolones and rifampicin) or folic acid (sulfonamides, aminopyrimidines (e.g., trimethoprim)). Therefore, antibiotic treatment alone is generally insufficient to eradicate biofilm infections (Wu et al. Int J Oral Sci. 2015). See also Mar;7(1):1~7). While antibiotics may be effective against dispersed (floating) bacteria, it is difficult to reach the minimum antibiotic concentration required to eradicate microorganisms within a biofilm. Various in vitro experiments have demonstrated that bacteria growing in biofilms are 10 to 1,000 times more resistant to various antimicrobial agents compared to floating bacteria of the same strain (see, for example, Amorena et al. 1999 J. Antimicrob. Chemother. 44:43~55; Ceri et al., 1999 J. Clin. Microbiol. 37:1771~1776; and Olson et al., 2002 Can. J. Vet. Res. 66:86~92). Olsen et al. outline various mechanisms of antibiotic resistance and tolerance induced by biofilms (Eur J Clin Microbiol Infect Dis). 2015 34:877~886).

[0012] Bovine mastitis has been reported in studies using field strains of Staphylococcus aureus (Melchior, Gaastra & Fink-Gremmels, J.Vet.Med. 2006 53:326~332). MIC50, minimum bacterial concentration (BMIC), and minimal biofilm eradicating concentration (MBEC) were measured for seven strains isolated from cows infected with mastitis. The antibiotics tested included those commonly used in the treatment of bovine mastitis, such as penicillin, amoxicillin, cloxacillin, cephalothin, cefoperazone, cefquinome, cloxacillin / penicillin combinations, lincomycin, pyrimicin, tyrosine, neomycin, gentamicin, trimethoprim / sulfamethoxazole, florfenicol, and danofloxacin. For all antibiotics tested, the difference between MIC (micromicrobial intake) and MBEC (biofilm eradication concentration) was more than 256 times, and in many cases more than 2048 times.

[0013] There is also evidence that antibiotics may stimulate biofilm formation. For example, some antibiotics (e.g., tetracycline, quinopristin-dalfopristin, and erythromycin) promote bacterial adhesion by affecting genes in bacteria (e.g., ICA It can stimulate the expression of the gene (see Melchior et al., above). Interestingly, among Staphylococcus aureus mastitis isolates... ICA The prevalence of this gene has been shown to be high (reviewed by Melchior et al., see above). These results support the hypothesis that mammary gland infections are associated with biofilm formation.

[0014] Biofilms can also contain dormant bacteria. Biofilms use many mechanisms to evade the host immune response, including the activation of regulators / suppressors that affect the activity of immune cells and the aforementioned immune response that acts as a physical barrier against immune cells (Gonzalez Pathog Dis. 2018 Apr, 76(3)), but they can break out of dormancy and become active. Generally, the immune system acts only on active bacteria, and therefore dormant bacteria can escape the individual's immune system. Dormant bacteria leave the biofilm and quickly become active and harm the host.

[0015] Fungal biofilms are also known to be more resistant to antifungal agents than free-floating cells (see, for example, Fanning and Mitchell PLOS Pathog 2012 8:e1002585 for an overview). Therefore, compounds with antimicrobial effects are not always suitable for treating or preventing biofilms.

[0016] In mature biofilms, the dormancy phase is utilized by downregulation (gene shift) of primary metabolism. Dormancy involves biofilm bacteria almost completely suppressing the expression of typical antibiotic targets, such as protein and DNA synthesis, as well as cell wall reconstruction. Subsequently, biofilms are essentially insensitive to antibiotics and are often more than 1000 times more resistant to them than free-floating bacteria. Furthermore, the higher cell density observed in biofilms significantly increases the possibility of horizontal gene transfer, which increases the likelihood of the emergence of strains with increased resistance or altered pathogenicity profiles. The clinical outcome is phenotypic resistance to common (even modern) antibiotics.

[0017] In such a protective biofilm environment, bacterial survival time is extended. Furthermore, the self-constructed, inactive, polysaccharide-rich matrix is ​​non-immunogenic, protecting bacteria embedded in the biofilm from recognition (via PAMPS - molecular patterns associated with pathogens) and phagocytosis by host immune cells.

[0018] Biofilm formation can have serious adverse effects in healthcare, industrial, and natural environments. In particular, biofilm-associated infections (i.e., biofilm-associated disorders) are a serious problem in both humans and animals. Such disorders can be characterized by recurrent acute episodes and chronic inflammatory responses accompanied by resistance to antimicrobial therapy and / or host defenses. Biofilms in wounds delay tissue repair, resulting in chronic wounds. It is now suggested that biofilm infections account for up to 80% of all human microbial infections (Bartell JA et al., 20 Evolutionary highways to persistent bacterial infection. Nature Communications (2019) 10:629 and Sharma et al. 2019 Antimicrobial Resistance and Infection Control 8:76). Jamal et al. reported that the National Institutes of Health showed that 65% of microbial infections and 80% of chronic infections are associated with biofilm formation (Journal of Chinese Medical Association 81:7~11). Biofilms and biofilm-related disorders are widely discussed in the literature; see, for example, Sharma et al. 2019 Antimicrobial Resistance and Infection Control 8:76; Roy et al. 2018 Virulence 9:522~554; and Jamal et al. 2018 Journal of the Chinese Medical Association 81:7~11. Vestby et al. outline bacterial biofilms and their role in disease (Antibiotics (Basel). 2020 Feb;9(2):59).An inclusive overview of biofilm infections was presented in 2014 at the European Clinical Society of Microbiology and Infectious Disease (see also David Lebeaux et al. Microbiol. Mol. Biol. Rev. 2014; doi:10.1128 / MMBR.00013~14).

[0019] For example, Pseudomonas aeruginosa, an organism that causes nosocomial infections, forms biofilms on various surfaces such as the lung tissue of cystic fibrosis, contact lenses, and catheter lines. Pseudomonas aeruginosa growing as a biofilm has also been found in chronic wounds and may cause healing disorders of the wounds. Biofilms, particularly Pseudomonas aeruginosa biofilms, also cause chronic infections in respiratory diseases such as bronchiectasis, chronic obstructive pulmonary disease, and chronic rhinosinusitis. Biofilms formed on medical devices function as reservoirs of bacteria that may be discharged into the body and cause chronic systemic infections. Candida albicans (yeast) is the most common fungal biofilm found in hospitals, but it is very difficult to treat and does not respond well to typical antifungal treatments.

[0020] Pioneering research on bacterial biofilm formation has been conducted, focusing on Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) due to their involvement in recurrent mastitis and complex wound infections in dairy cows. In addition, biofilm formation by Streptococcus ssp, avian pathogenic E. coli (APEC), and Campylobacter jejuni has attracted attention because these bacterial species are relevant to public health. Subsequently, other important animal pathogens, such as Actinobacillus pleuropneumoniae (severe lung infections in pigs that can be fatal), Escherichia coli (local and systemic infections and septicemia, which are often lethal in poultry), skin and intestinal diseases in dogs and cats, and wound infections and endometritis in horses, have been recognized as biofilm infections. Since biofilm formation is a common property of almost all microorganisms, this list is not exclusive.

Summary of the Invention

Problems to be Solved by the Invention

[0021] Therefore, there is a need for alternative treatments for biofilm-related disorders.

Means for Solving the Problems

[0022] The present disclosure provides the following preferred embodiments.

[0023] 1. A compound according to formula I below, or a composition comprising a compound according to formula I, for use in the treatment of biofilm-related disorders, wherein preferably the composition substantially does not have diallyl thiosulfinate

Chemical formula

[0024] 2. The compound or composition for use according to Embodiment 1, wherein the compound is propylpropanethiosulfonate (PTSO).

[0025] 3. For use as described in Embodiment 1, the compound or the composition, wherein the composition further comprises a compound according to the following formula II, preferably the compound according to formula II is propylpropanechosulfinate (PTS). [ka] Here, R 3 and R 4 independently selected from optionally substituted linear or branched alkyl, optionally substituted linear or branched alkenyl, optionally substituted linear or branched alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl, wherein formula II is not as shown below. [ka]

[0026] 4. Compounds according to the following formula II, for use in the treatment of biofilm-related disorders, wherein the compound is preferably propylpropanethosulfinate (PTS), or compositions comprising a compound according to the formula II, wherein the composition is substantially free of diallylthiosulfinate. [ka] Here, R 3 and R 4Each of these is independently selected from optionally substituted linear or branched alkyls, optionally substituted linear or branched alkenyls, optionally substituted linear or branched alkynyls, optionally substituted aryls, optionally substituted cycloalkyls, and optionally substituted heterocycloalkyls, provided that formula II is not one of the following. [ka]

[0027] 5. A compound or composition according to any one of Embodiments 1 to 4, wherein the composition further comprises a compound having the following formula III: [ka] Here, n is 1, 2, or 3, and R 1 and R 2 Each of the following is independently selected from optionally substituted linear or branched alkyls, optionally substituted linear or branched alkenyls, optionally substituted linear or branched alkynyls, optionally substituted aryls, optionally substituted cycloalkyls, and optionally substituted heterocycloalkyls, provided that formula III is not as follows. [ka]

[0028] 6. The compound or composition according to any one of Embodiments 1 to 5, wherein the use further comprises the administration of an antimicrobial agent, preferably an antifungal agent or an antibiotic selected from among antimicrobial agents.

[0029] 7. The compound or composition according to any one of Embodiments 1 to 6, wherein the use further comprises the administration of an anti-inflammatory agent.

[0030] 8. The compound or composition according to any one of Embodiments 1 to 7, wherein the treatment is for reducing the formation or growth of a biofilm and / or for degrading or reducing a biofilm.

[0031] 9. The compound or composition according to any one of Embodiments 1 to 8, wherein the biofilm-related disorder is a chronic infection and / or a persistent infection.

[0032] 10. The compound or composition according to any one of Embodiments 1 to 9, wherein the biofilm-related disorder is digitoritis or chronic wound infection.

[0033] 11. The compound or composition according to any one of Embodiments 1 to 10, wherein the biofilm-related disorder is a mammary gland infection, preferably mastitis.

[0034] 12. The compound or composition according to any one of Embodiments 1 to 11, wherein the treatment is for mammals.

[0035] 13. The compound or composition according to any one of Embodiments 1 to 12, wherein the treatment is for a ruminant, preferably a cattle.

[0036] 14. The compound or composition according to any one of Embodiments 1 to 13, wherein the composition substantially does not contain diallylthiosulfinate.

[0037] 15. The compound or composition according to any one of Embodiments 1 to 14, wherein the biofilm comprises bacteria, yeast, fungi, microalgae, or a combination thereof.

[0038] 16. A method for treating a biofilm-related disorder in an organism, comprising administering to an organism in need of such treatment a composition comprising a compound according to the following formula I, wherein preferably the compound is propylpropanethiosulfonate (PTSO), and the composition substantially contains diallylthiosulfinate. [ka] Here, R1 and R 2 are each independently selected from optionally substituted straight-chain or branched alkyl, optionally substituted straight-chain or branched alkenyl, optionally substituted straight-chain or branched alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl.

[0039] 17. An article having a surface at least partially coated with a composition comprising a compound according to the following formula I, wherein preferably the article is a cleaning product, a medical instrument or a surgical instrument, and the composition substantially does not have diallyl thiosulfinate, said article

Chemical formula

[0040] 18. An in vitro method for preventing or reducing the formation or growth of a biofilm on a surface or for degrading or reducing a biofilm on a surface, said method comprising applying a composition to the surface to prevent or reduce the formation or growth of a biofilm on the surface or to degrade or reduce a biofilm on the surface, said composition comprising a compound according to the following formula I, wherein the composition substantially does not have diallyl thiosulfinate, said method

Chemical formula

[0041] 19. A composition comprising a compound according to the following formula I, wherein the composition is a pharmaceutical composition or a functional food, wherein the composition substantially does not contain diallylthiosulfinate. [ka] Here, R 1 and R 2 These are independently selected from optionally substituted linear or branched alkyls, optionally substituted linear or branched alkenyls, optionally substituted linear or branched alkynyls, optionally substituted aryls, optionally substituted cycloalkyls, and optionally substituted heterocycloalkyls.

[0042] 20. An article, method, or composition according to any one of embodiments 16 to 19, wherein the compound is propylpropanethiosulfonate (PTSO).

[0043] 21. The article, method, or composition according to any one of Embodiments 16 to 20, wherein the composition further comprises an antimicrobial agent, preferably selected from antifungal agents or antibiotics.

[0044] 22. The article, method, or composition according to any one of embodiments 16 to 21, wherein the composition further comprises an anti-inflammatory agent.

[0045] 23. Article, method, or composition according to any one of Embodiments 16 to 22, wherein the composition further comprises a compound according to the following formula II and / or the composition further comprises a compound having the following formula III. [ka] Here, R 3 and R 4 Each of these is independently selected from optionally substituted linear or branched alkyls, optionally substituted linear or branched alkenyls, optionally substituted linear or branched alkynyls, optionally substituted aryls, optionally substituted cycloalkyls, and optionally substituted heterocycloalkyls, provided that formula II is not one of the following. [ka] [ka] Here, n is 1, 2, or 3, and R 1 and R 2 Each of the following is independently selected from optionally substituted linear or branched alkyls, optionally substituted linear or branched alkenyls, optionally substituted linear or branched alkynyls, optionally substituted aryls, optionally substituted cycloalkyls, and optionally substituted heterocycloalkyls, provided that formula III is not as follows. [ka] [Brief explanation of the drawing]

[0046] [Figure 1A] Figures 1A and 1B show the procedure used to measure the biofilm eradication effect of QS1 and QS2 in well measurements of a microtiter plate. Figure 1A shows the method used to measure biofilm in a microtiter plate. [Figure 1B] Figures 1A and 1B show the procedure used to measure the biofilm eradication effect of QS1 and QS2 in well measurements of microtiter plates. Figure 1B shows the method for measuring biofilm formation of pegs. [Figure 2A]Figure 2A shows the effects of QS1 and QS2 on late biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus under rich medium conditions. Left figure: effect on late biofilm formation in wells; Right figure: effect on late biofilm formation in PEGs. Significance levels are indicated by asterisks (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001). [Figure 2B] Figure 2B shows the effects of QS1 and QS2 on late-stage biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus under minimal medium conditions. Left figure: effect on late-stage biofilm formation in wells; Right figure: effect on late-stage biofilm formation in PEGs. Significance levels are indicated by asterisks (*: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001). [Figure 3A] Figure 3A shows the biofilm released directly from the udder after milking. [Figure 3B] Figure 3B shows the biofilm obtained after sieving post-milk milk from cows with mastitis, 1 to 3 days after treatment with PTSO / PTS tablets. [Figure 4] Figure 4 shows the progression of infection (straight arrows) and healing (dotted arrows). [Modes for carrying out the invention]

[0047] This disclosure provides compounds useful for reducing, degrading, and / or preventing biofilm formation. The compounds described herein have surprisingly been found to degrade biofilms containing highly heterogeneous microbial populations (see, e.g., Example 2) and biofilms containing known human pathogens (see, e.g., Examples 3 and 4). This disclosure further provides that the compounds are active against biofilms both in vitro and in vivo and can be administered both systemically (e.g., orally) and topically (see, e.g., Examples 5–8).

[0048] This disclosure provides a thiosulfonate according to the following formula I. [ka] Here, R 1 and R 2 These are independently selected from optionally substituted linear or branched alkyls, optionally substituted linear or branched alkenyls, optionally substituted linear or branched alkynyls, optionally substituted aryls, optionally substituted cycloalkyls, and optionally substituted heterocycloalkyls.

[0049] In some embodiments, the present disclosure provides thiosulfinates according to the following formula II. [ka] Here, R 3 and R 4 independently selected from optionally substituted linear or branched alkyl, optionally substituted linear or branched alkenyl, optionally substituted linear or branched alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl, wherein formula II is not as shown below. [ka]

[0050] In some embodiments, the present disclosure provides compounds having the following formula III. [ka] Here, n is 1, 2, or 3, and R 1 and R 2 Each of the following is independently selected from optionally substituted linear or branched alkyls, optionally substituted linear or branched alkenyls, optionally substituted linear or branched alkynyls, optionally substituted aryls, optionally substituted cycloalkyls, and optionally substituted heterocycloalkyls, provided that formula III is not as follows. [ka]

[0051] Compounds having formula I, formula II, or formula III are referred to herein as “compound,” “therapeutic compound,” or “therapeutic thiosulfur compound.” The compound includes salts of formula I, formula II, or formula III. Preferably, the compound has formula I or formula II. Compounds having formula I or formula II can be used together with compounds having formula III. More preferably, the compound has formula I. Preferably, the compound of formula I is propylpropanethiosulfonate (PTSO). PTSO has the following structure. [ka]

[0052] Preferably, the compound of formula II is propylpropanechosulfinate (PTS). PTS has the following structure. [ka]

[0053] Propylpropanthiosulfonate (PTSO) and propylpropanthiosulfinate (PTS) are natural compounds found in plants belonging to the Allium family. Examples include Allium sativum (garlic), Allium cepa (onion), Allium ampeloprasum (leek), Allium schoenoprasum (chives), and Allium chinense (shallot). These compounds can be extracted from natural sources or produced synthetically. Both compounds, PTS and PTSO, are also commercially available.

[0054] In some embodiments, the compound is obtained from natural sources, such as plants. The compound can be extracted from plant material in a variety of ways. The appropriate method depends on the chemical properties of the compound. For example, the extraction may begin with a non-polar solvent, followed by solvents of increasing polarity. Alternatively, the compound from plants can be extracted in alcohol. Such an extract may contain, for example, about 80% PTSO and about 20% PTS. In preferred embodiments, the composition disclosed herein contains PTSO and PTS in a weight ratio of at least 3:1, preferably about 4:1. While we do not wish to be bound by theory, this disclosure presents that the anti-biofilm activity is primarily due to PTSO.

[0055] Garlic extract has been previously described for use in dietary supplements. Garlic is a herb cultivated worldwide. It is related to onions, leeks, and chives. Garlic extract, particularly the compound allicin, has been studied for its potential to treat various types of diseases. It is most commonly used for conditions related to the heart and blood system. These conditions include high blood pressure, high levels of cholesterol or other fats in the blood, and arteriosclerosis.

[0056] For example, garlic is a tablet containing garlic extract. It is described as a special dosage form, namely an enteric-coated tablet, and is made from garlic powder dried at a controlled temperature. This coating protects the tablet as it passes through the stomach and dissolves in the small intestine, releasing its contents. Garlic is presented as a protector against high serum cholesterol levels and the risk of heart disease. In addition, European Patent Application Publication EP2110128 discloses the use of compounds derived from the Allium family as natural additives in animal feed as an alternative to antibiotics.

[0057] In contrast to garlic extracts, which contain numerous different compounds, PTSO is a trace component, and the compositions provided herein preferably contain PTSO at a higher concentration than natural garlic extracts. In addition, natural garlic extracts contain numerous different substances, each with different (sometimes undesirable) effects, and the compositions provided herein are useful for providing PTSO without the undesirable side effects of other components in garlic extracts. Furthermore, compositions rich in PTSO allow for higher doses.

[0058] For example, garlic extract has a strong odor. When animals are given such garlic extract, these odors can be found in animal products, such as milk and eggs. Consumers may perceive products with these odors as spoiled, and such odors can also affect the taste of such products. In practice, animal products collected while using garlic extract should usually be discarded. Ingestion of garlic extract by humans can cause body odor and bad breath.

[0059] In contrast, the therapeutic compounds disclosed herein preferably do not have the negative effects of the garlic extract described above. For example, in the case of farm animals, this has had a significant commercial advantage in that the resulting animal products (e.g., milk and eggs) are suitable for consumers. In some embodiments, the disclosure provides a method comprising administering the compositions disclosed herein to an individual in need, wherein the treatment does not result in the presence of body odor, bad breath, or abnormal odor in bodily fluids (e.g., milk), or, in the case of birds, abnormal odor in eggs, or at least an abnormal odor less than that of the garlic extract.

[0060] This disclosure provides compositions comprising compounds disclosed herein, particularly compositions comprising compounds having formula I and / or formula II. Preferably, such compositions are substantially free of diallylthiosulfinate, which is commonly known as allicin. Allicin is an organosulfur compound. When fresh garlic is chopped or crushed, an enzyme called alliinase converts alliin into allicin, which is responsible for the aroma of fresh garlic. The resulting allicin is unstable and rapidly converts into a range of other sulfur-containing compounds, such as diallyl disulfide. One clove of garlic contains about 5 mg to about 18 mg of allicin.

[0061] As used herein, “substantially free” means a composition containing less than 5% by weight of diallylthiosulfinate. In some embodiments, the composition contains less than 1% by weight of didiallylthiosulfinate, preferably less than 0.5% by weight of diallylthiosulfinate. In some embodiments, the composition contains a weight ratio of therapeutic thiosulfur compounds (e.g., PTSO and PTS) to diallylthiosulfinate of at least 10:1, more preferably at least 100:1.

[0062] The compositions of this disclosure are also preferably substantially free of diallyl disulfide. In some embodiments, the composition comprises less than 1% by weight of diallyl thiosulfinate, preferably less than 0.5% by weight of diallyl thiosulfinate. In some embodiments, the composition comprises a weight ratio of a compound having formula I and / or formula II (e.g., PTSO and PTS) to diallyl disulfide of at least 10:1, more preferably at least 100:1.

[0063] In some embodiments, compositions are provided in which at least 50% by weight, preferably at least 90% by weight, of the active ingredient is a compound according to Formula I, Formula II, or Formula III as disclosed herein. In some embodiments, compositions are provided in which the sole active ingredient is a compound according to Formula I, Formula II, or Formula III and optionally comprises further antimicrobial and / or anti-inflammatory agents. In some embodiments, compositions are provided in which at least 50% by weight, preferably at least 90% by weight, of the active ingredient is a compound according to Formula I or Formula II as disclosed herein. In some embodiments, compositions are provided in which only the active ingredient is a compound according to Formula I or Formula II and optionally comprises further antimicrobial and / or anti-inflammatory agents.

[0064] The compounds and compositions containing the same disclosed herein are useful in the treatment or prevention of infectious diseases. For example, specific uses include the treatment or prevention of respiratory infections, intestinal infections, breast infections, udder infections in animals, skin infections, bladder infections, ear infections, systemic infections, joint infections, and cerebral infections.

[0065] As used herein, “infection” means, for example, a pathogenic infection that can lead to disease. In particular, such infections are bacterial or fungal infections. In a preferred embodiment, the infection is a bacterial infection. Bacteria and fungi are found almost everywhere and exist in a great diversity of forms. Most are harmless and are actually essential to life on Earth, as well as essential to the health of plants, animals, and humans. For example, the microbiome in the intestines of humans and animals, where bacteria and fungi live in symbiosis with hosts, is the so-called gut flora. Bacteria also exist naturally on the skin, and they form part of the immune system. Another example is soil biology, which is largely composed of bacteria and fungi. Some bacteria and fungi can cause pathogenic infections, for example, in animals or humans. These pathological infections can lead to illness and disease in the infected individual.

[0066] As used herein, “treatment of infection” means reducing the severity and / or duration of an infection, and / or reducing the severity and / or duration of symptoms from the infection. Preferably, such treatment results in the recovery of the individual’s health, preferably with fewer or shorter duration of disease symptoms. As used herein, “prevention of infection” means preventing or delaying the onset of an infection or one or more symptoms associated with an infection.

[0067] Several microorganisms, such as bacteria, microalgae, and fungi, can form biofilms. The compounds disclosed herein are also useful in preventing or reducing the formation or growth of biofilms and / or for the degradation or reduction of biofilms. Preferably, the compounds and compositions containing them are useful in treating or preventing biofilm-related disorders.

[0068] The term "biofilm" was first used in technical and environmental microbiology to describe a community of attached bacteria and other microorganisms that adhere to natural or artificial surfaces. The formation of a microbial biofilm is initiated by the colonization of bacteria on a surface where bacteria adhere and produce a slimy film composed of organic polymers. This primary bacterial film attracts other microorganisms, such as algae and protozoa, fungi and protozoa, resulting in the formation of visible multi-species biofilms. Such three-dimensional biofilms are ubiquitous in nature and are found on all surfaces in contact with water. A public health concern is microbial biofilms in municipal and household water pipelines and equipment. The efficacy of compounds according to formulas I, II, and III in such multi-species environmental biofilms is demonstrated in Examples 2 and 3.

[0069] As used herein, the term “biofilm” means a collection of microorganisms concentrated at an interface (usually solid / liquid) and typically surrounded by an extracellular polymeric mucous matrix. Biofilms can form on living or non-living surfaces and are found in natural, industrial, and hospital environments. Biofilms can contain various types of microorganisms, such as bacteria, archaea, protozoa, fungi, and algae, and preferably such biofilms contain bacteria, microalgae (e.g., Prototheca spp.) or fungi.

[0070] Where used herein, “treatment of biofilm-related disorder” is also referred herein to “disorder associated with biofilm,” and the treatment results in a reduction in the severity and / or duration of the disorder, and / or the severity and / or duration of symptoms from the disorder, particularly symptoms of infection, preferably resulting in the individual having fewer or shorter disease symptoms.

[0071] As used herein, “prevention or reduction of biofilm formation or growth” means the prevention, delay, or reduction of biofilm formation or growth. As will be understood by those skilled in the art, such reduction of biofilm formation or growth may slow the growth of biofilm compared to the growth of untreated biofilm. Preferably, the composition is useful for reducing biofilm formation or growth. As used herein, “decomposition or reduction of biofilm” means either partial or complete removal of biofilm. As will be understood by those skilled in the art, after such treatment, planktonic bacteria may still be present.

[0072] The compounds disclosed herein may disrupt the structure of biofilms, such as extracellular mucus matrices. In some embodiments, the compounds are useful for inhibiting cell adhesion. In particular, the compounds may prevent adhesion (without killing bacteria) of all cell types encountered in microbial biofilms, especially free living microbes, to static or living surfaces.

[0073] While we do not wish to be bound by theory, this disclosure suggests that the therapeutic compounds disclosed herein may exert some of their effects by influencing quorum sensing. Quorum sensing (QS) signaling plays a crucial role in regulation, such as the control of the expression of bacterial pathogenicity factors. QS is involved in the accumulation of signaling molecules in the surrounding environment, enabling a single cell to sense bacterial density and signaling molecules, thus allowing for coordinated responses in the bacterial population as a whole. These intercellular communication systems regulate various bacterial functions, such as motility, pathogenicity, spore formation, antibiotic production, DNA exchange, and the development of more complex multicellular structures, such as biofilms. Therefore, interference with the QS signaling system may offer novel strategies for combating persistent (chronic) bacterial infections. The ability of certain substances, such as naturally occurring compounds with quorum quenching (QQ) capabilities, can be used as anti-adhesion compounds and compounds that inhibit biofilm formation.

[0074] The compounds disclosed herein are particularly useful for treating biofilm-related disorders, characterized by chronic and / or persistent infections. While persistent and chronic infections are often used interchangeably, they are based on different mechanisms. Persistent infections are usually suppressed by immune defenses, but can be activated when such immune defenses weaken. Persistent infections are often asymptomatic and only become clinically apparent when immune defenses fail to control the pathogen. Although persistent infections are often asymptomatic, those skilled in the art are well aware of means for detecting such persistent infections, including, for example, detecting microorganisms from patient samples (e.g., blood or urine). In chronic infections, the pathogen remains in a group of cells / part of a tissue (e.g., joint or lung tissue). Patients always have symptoms of the disease, but these may be milder than those in the acute phase of the infection.

[0075] The most prominent example of biofilm disease remains bovine mastitis. Bovine mastitis is a clinical term for infection of the mammary glands of cattle, which can be caused by multiple pathogens, the most common of which are Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactia, Streptococcus dysgalactiae, as well as Serratia marescens, and other facultative pathogenic Enterobacteriaceae and Prototheca spp., the latter of which are considered new pathogens causing aggressive and incurable mastitis in many parts of the world. The present invention encompasses treating biofilms containing such microorganisms with the compounds and compositions disclosed herein.

[0076] The examples described herein describe the remarkable efficacy of PTSO in various field studies in cattle, demonstrating its broad activity in biofilm infections. Another notable example of a biofilm infection is chronic, non-healing wound infection. Dermatitis digitorum, a disease associated with known biofilms in cattle, serves as an example of such a delayed wound healing process and was successfully treated with PTSO (see Example 7). In some embodiments, wounds treated with the compounds of the present invention include, for example, Staphylococcus aureus; Streptococcus; Gram-negative bacteria, such as Treponema species, Escherichia coli, Yersiania pestis, Pseudomonas aeruginosa; or yeasts / fungi, such as Candida spp. (Candida albicans), Cladosporidium herbarum, Trichosporon, Rhodosporidium, and Malassezia.

[0077] It is now generally recognized that microorganisms associated with biofilms cause numerous infections, such as endocarditis, osteomyelitis, sinusitis, urinary tract infections, chronic prostatitis, periodontitis, chronic lung infections in patients with cystic fibrosis, middle ear infections, and various hospital-acquired infections, particularly those associated with all known indwelling devices (catheters, implants). The burden of biofilm diseases is significant and a major concern in healthcare.

[0078] While it is now known that almost all bacterial species can form biofilms under stress conditions, several bacterial and fungal species are currently attracting considerable attention in clinical practice. This is because, even when the non-biofilm, floating forms of the same species and strains show excellent susceptibility to common therapeutic agents (antibiotics and antifungal / fungicides), the associated infections are almost completely resistant to treatment.

[0079] Examples of pathogens that pose a major clinical concern due to treatment resistance associated with biofilms are listed below: Aspergillus fumigatus - Pulmonary aspergillosis (a fungal disease); Burkholderia cepacia - a co-infection of pulmonary cystic fibrosis; Candida spp. (yeast) - mucosal surface of the gastrointestinal and urogenital tract; Gardnerella vaginalis (urogenital tract); Escherichia coli (multi-organ disease and sepsis); Pseudomonas aeruginosa (infections of multiple organs and the lungs, including cystic fibrosis); Staphylococcus aureus (infection of multiple tissues and wounds, hospital-acquired infections); Staphylococcus epidermidis (infects multiple tissues and wounds); Stenotrophomonas maltophilia (chronic respiratory disease). Treatment of such biofilm-related disorders and microorganisms associated with biofilms is included in the present invention.

[0080] Microbial biofilms are formed not only by bacteria but also by other microorganisms, particularly pathogenic fungi (Aspergillus fumigatus, the main cause of several Aspergillus-related lung diseases) and yeasts (Candida species), which colonize the mucosal surfaces of the gastrointestinal and urogenital tracts. The most common implant-related biofilms are formed by Staphylococcus aureus (MSSA and MRSA), Candida albicans, Pseudomonas aeruginosa, Klebsiella pneumonia, and Enterococcus faecalis. In addition, microalgae, such as Prototheca species, can form biofilms and are a cause of disease in humans and animals (protothecosis).

[0081] In some embodiments, the biofilm contains Treponema spp., Yersiania pestis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Serratia marescens, Trueperella pyogenes, Mannheimia haemolytica, Pasteurella multocida, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, and Haemophilus influenzae. This includes bacteria selected from one or more of the following: influenza, Legionella neumophila, Fusobacterium necrophorum, Corynebacterium pseudotuberculosis, Streptococcus spp., Porphyromonas gingivalis, Pseudomonas aeruginosa, Enterococcus faecalis, Neisseria gonorrhoeae, Escherichia coli, Salmonella enteritidis, and Pseudomonas aeruginosa. In some embodiments, the biofilm contains genera such as Absidia, Actinomyces, Aspergillus, Botrytis, Candida, Centrospora, Cephalosporium, and Ceratocystis.), Chaetoconidium spp., Chaetomium spp., Cladosporium spp., Colletotrichum spp., Conidiobolus spp., Fulvia spp., Fusarium spp., Geotrichum spp., Guignardia spp., Helminthosporium spp., Histoplasma spp., Lecythophora spp., Malassezia spp., Nectria spp., Nocardia spp.), Oospora spp., Ophiobolus spp., Paecilomyces spp., Paracoccidioides brasiliensis, Penicillium spp., Phymatotrichum spp., Phytophthora spp., Pythium spp., Piedraia hortai, Rhizoctonia spp., Rhizopus spp., Rhodosporidium spp., Saccharomyces (spp.), Scerotium spp., Sclerotinia spp., Torulopsosis spp., and Trichophyton spp.This includes fungi selected from the following. Many medically important fungi, such as Candida, Aspergillus, Cryptococcus, Trichosporon, Coccidioides, and Pneumocystis, are known to form biofilms. In some embodiments, the biofilm includes microalgae, such as the genus Prototheca.

[0082] In one embodiment, the bacterial infection or biofilm-associated disorder is caused by Gram-negative bacteria. In one embodiment, the bacterial infection or biofilm-associated disorder is caused by multidrug-resistant bacteria. In one embodiment, the bacterial infection is a methicillin-resistant Staphylococcus aureus (MRSA)-associated infection or a Staphylococcus epidermidis (e.g., MRSE)-associated infection.

[0083] In a preferred embodiment, the biofilm-causing bacterium is Escherichia coli, and preferably, the biofilm infection is a recurrent urinary tract infection, a catheter-associated urinary tract infection, or a biliary tract infection.

[0084] In a preferred embodiment, the biofilm-causing bacterium is Pseudomonas aeruginosa, and preferably, the biofilm infection is cystic fibrosis pulmonary infection, chronic wound infection, catheter-associated urinary tract infection, chronic rhinosinusitis, chronic otitis media, bronchiectasis, chronic obstructive pulmonary disease, or contact lens-associated keratitis.

[0085] In a preferred embodiment, the biofilm-causing bacteria is Staphylococcus aureus, and preferably, the biofilm infection is chronic osteomyelitis, chronic rhinosinusitis, endocarditis, chronic otitis media, or (orthopedic) implants.

[0086] In a preferred embodiment, the biofilm-causing bacterium is Staphylococcus epidermidis, and preferably, the biofilm infection is a central venous catheter, an orthopedic implant, or chronic osteomyelitis.

[0087] In a preferred embodiment, the biofilm-causing bacteria is Streptococcus pneumoniae, and preferably, the biofilm infection is a nasopharyngeal infection, chronic rhinosinusitis, chronic otitis media, or an infection in chronic obstructive pulmonary disease.

[0088] In a preferred embodiment, the biofilm-causing bacteria is Streptococcus pyogenes, and preferably, the biofilm infection is an infection of the oral cavity and nasopharynx, or recurrent tonsillitis.

[0089] The clinical signs of biofilm infections are known to healthcare professionals (see, for example, Table 1 in Wu et al. Int J Oral Sci. 2015 Mar;7(1):1-7). Such biofilm disorders can lead to chronic infections. The determination of acute and chronic infections is also known to healthcare professionals. For example, according to the Mayo Clinic, more than four occurrences of yeast infections in a year indicate the presence of a chronic yeast infection, while more than two occurrences of bladder infections in a six-month period indicate the presence of a chronic bladder infection (also known as a recurrent urinary tract infection).

[0090] The most common method for treating bacterial pathological infections is the use of antibiotics. Current antibiotics primarily function through growth-dependent mechanisms and target rapidly dividing bacteria. However, non-replicating or slow-growing bacteria (e.g., dormant cells, biofilms) exhibit high levels of antibiotic resistance and / or tolerance, contributing to persistent and recurrent infections. The compounds disclosed herein are suitable for use in infections or biofilms involving antibiotic-resistant bacteria, antibiotic-tolerant bacteria, and antibiotic-persistent bacteria. The compounds disclosed herein are also suitable as a second-line treatment, or rather for individuals who have not responded to previous treatment (e.g., antimicrobial treatment), or for individuals whose disorder has recurred, for example, within one year or six months.

[0091] This disclosure further provides compounds and compositions comprising the same disclosed herein for treating any ailments induced by or associated with biofilms. Ailments induced by or associated with biofilms are well known to those skilled in the art. In particular, such ailments are biofilm-associated infections. Ailments suitable for treatment include, for example, bacterial prostatitis, bacterial vaginosis, biliary tract infections, chronic sinusitis, chronic lung disease, dental caries, endocarditis, kidney stones, laryngitis, lung infections in cystic fibrosis, gingivitis, mastitis, middle ear infections, hospital-acquired (bloodstream) infections, obstructive pulmonary disease, osteomyelitis, otitis media, periodontitis, pneumoniae, prostatitis, sinusitis, tonsillitis, tuberculosis, urinary tract infections, and wound infections. For example, Mycoplasma Bovis is known to cause mammary gland and joint infections in animals. Biofilm-related disorders encompass disorders caused by biofilms formed on indwelling devices (e.g., medical implants, catheters, etc.). Generally, such disorders are treated by removing / replacing the implant. In preferred embodiments, the disorder is mastitis. In some embodiments, the disorder is not mastitis. In some embodiments, the treatment is not for inflammatory bowel disease, and not in particular colitis.

[0092] The compounds and compositions disclosed herein are also useful for treating and preventing infections of implantable medical devices, such as artificial joints and heart valves, as further disclosed herein.

[0093] The compounds of the present invention have been shown to be effective in bovine udder. This indicates that the compounds can cross the blood-milk barrier, which is formed by low-permeability tight junctions between mammary epithelial cells and prevents milk leakage. Crossing the tight barrier between blood flow and milk indicates that the compounds can also cross the blood-brain barrier and treat brain infections, and cross the intestinal epithelium to enable systemic treatment of biofilm-related disorders. Data from examples also demonstrate in vivo safety and efficacy, including pharmacokinetic properties that make the compounds suitable for therapeutic use.

[0094] This disclosure further provides compounds and compositions comprising the same disclosed herein for preventing or reducing inflammation in response to bacterial infection or biofilm. Inflammation is part of a complex biological response of body tissues to harmful stimuli, such as pathogens, and is a defense response involving immune cells and molecular mediators. The function of inflammation is to eliminate pathogens.

[0095] In a preferred embodiment, treatment of an individual with the compounds disclosed herein or compositions containing the same prevents or reduces clinical inflammation in animals, preferably cattle. Preferably, the treatment prevents or reduces (clinical) inflammation of the mammary gland. In another embodiment, treatment of an individual with the compounds and compositions containing the same prevents or reduces (clinical) inflammation in humans. For example, the treatment prevents or reduces skin inflammation, preferably preventing eczema.

[0096] While we do not wish to be bound by theory, treatment of organisms with the compounds disclosed herein or compositions containing them reduces the formation or growth of biofilms and / or causes the degradation or reduction of biofilms. Therefore, the organisms no longer respond to inflammatory responses in the presence of pathogens. In other words, the clearance of biofilms and pathogens within these biofilms reduces inflammatory responses and prevents clinical inflammation.

[0097] During the treatment of biofilms, microorganisms (e.g., bacteria and fungi) are released from the biofilm. In some cases, the individual's immune system reacts to the active microorganisms. This can result in inflammation of the tissue. Activated immune cells and inflammatory responses can also damage tissues, such as mammary glands. Therefore, suppressing the inflammatory response can prevent or reduce damage to such tissues. For example, mammary gland damage will be reduced, and cow's milk production will recover more quickly.

[0098] Furthermore, once tissue is damaged and the inflammation subsides, the body begins to repair itself. The remaining macrophages stimulate the formation of new blood vessels. These macrophages also ensure the attraction of fibroblasts. These fibroblasts ultimately lead to the formation of granulation tissue. For example, in the case of dairy cows, scar tissue may form in place of milk-producing tissue. Therefore, the cow's milk yield may decrease compared to before the inflammation.

[0099] In addition to the compounds described herein, further anti-inflammatory agents may be administered to suppress the inflammatory response and reduce tissue damage, such as tissue damage in the mammary glands. In preferred embodiments, the treatments (both therapeutic and preventive) disclosed herein further include the administration of anti-inflammatory agents. Anti-inflammatory agents include, for example, non-steroidal anti-inflammatory drugs (Cox / Lox inhibitors), such as ibuprofen, paracetamol, aspirin, diclofenac, ketoprofen, tolmetin, etodolac, and fenoprofen. Natural anti-inflammatory agents, such as curcumin, ginger, spirulina, cayenne, cinnamon, clove, sage, rosemary, black pepper, natural aspirin, Boswelia, Sanguinaria, and / or green tea may also be used. In some embodiments, the methods and uses disclosed herein include combination therapy of the therapeutic thiosulfur compounds disclosed herein with anti-inflammatory agents. These compounds may be administered together or separately. In some embodiments, compositions are provided that include an anti-inflammatory agent along with a therapeutic thiosulfur compound disclosed herein.

[0100] In some embodiments, the method preferably involves administering a composition containing the compounds disclosed herein to an individual in need for the purpose of preventing or reducing the formation or growth of a biofilm, degrading or reducing a biofilm, and / or treating or preventing a bacterial or fungal infection. In some embodiments, the composition may be administered to an individual for the treatment (e.g., a therapeutic agent) or prevention (e.g., a prophylactic agent) of a disease or disorder or infection. In some embodiments, the individual has or is at risk of developing a biofilm-associated infection.

[0101] The composition can be administered to any individual, particularly an animal. Preferably, the animal is a ruminant (e.g., cattle and goats), more preferably a cattle. In some embodiments, the animal is not a cattle. Preferably, the animal is a non-ruminant, such as a monogastric animal, rodent, non-human primate, pig, horse, dog, cat, or bird. In some embodiments, the animal is a human. In some embodiments, the animal is a non-human animal. In some embodiments, the animal is not an aquatic animal, such as a fish, mollusk, and crustacean. Preferably, the animal is a mammal or a bird.

[0102] While we do not wish to be bound by theory, this disclosure provides that the compositions disclosed herein may have beneficial effects after a single dose. In preferred embodiments, the effect is achieved by providing a single oral dose of the compositions disclosed herein. Such an oral dose may be, for example, a tablet that provides a sustained release of the compound disclosed herein.

[0103] This disclosure also provides multiple doses. For example, the composition may be provided more than once a day, daily, weekly, or monthly. In an exemplary embodiment, the composition may be provided once a day for a week, or until symptoms are relieved.

[0104] The actual dosage levels of the pharmaceutical formulations described herein may vary to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage level depends on a variety of factors, including the activity of a particular compound, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or substances used in combination, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical field. A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition.

[0105] In some embodiments, ruminants, particularly cattle, are provided with at least 1 gram, preferably at least 2 grams, and more preferably at least 5 grams of the compound disclosed herein. In some embodiments, ruminants, particularly cattle, are provided with at least 3 g of PTSO. Compositions containing the compound for ruminants are preferably provided as oral tablets.

[0106] It is apparent to those skilled in the art that smaller amounts of the compound can be administered to smaller ruminants, such as goats. This example describes the administration of a tablet containing 3.84 g of PTSO and 0.96 g of PTS to a cattle. Since the average body weight of a cattle is approximately 650 kg, this corresponds to a dose of approximately 5.9 mg / kg of PTSO and 1.5 mg / kg of PTS. Those skilled in the art are aware that smaller animals have a higher metabolic rate and therefore require a larger drug dose on a body weight basis. Dose conversions between animals, and between humans and animals, have been reviewed in Nair and Jacob (J Basic Clin Pharm. March 2016-May 2016;7(2):27~31) and Holliday et al. (1967 The Relation of Metabolic Rate to Body Weight and Organ Size. A Review. Pediat. Res.1:185~195).

[0107] In some embodiments of the methods and uses disclosed herein, at least 5 mg / day of the compound disclosed herein is provided to a human (e.g., by oral administration). Preferably, at least 10 mg / day of the compound is provided. In some embodiments, PTSO is provided to a human in doses ranging from 0.1 mg / kg to 100 mg / kg. Such amounts of the compound are particularly useful when the compound is provided systemically (e.g., orally). Those skilled in the art will recognize that smaller amounts may be used when administered topically (e.g., on the skin, gums, or wounds). The compositions disclosed herein are preferably provided for at least one week, or until symptoms are relieved. Such compositions may be provided several times (e.g., once a week, once a month, twice a year, etc.), but the preventive and therapeutic effects are observed after a single use.

[0108] In some embodiments, compositions are provided comprising the compounds disclosed herein together with one or more additional agents, such as antibiotics (e.g., antibacterial agents, antiviral agents, antifungal agents), anti-inflammatory agents, antipyretics, and analgesics.

[0109] In some embodiments, the compounds disclosed herein are used in conjunction with antimicrobial agents, such as antifungal agents or antibiotics. While we do not wish to be bound by theory, this disclosure provides that the compounds disclosed herein target biofilms. The antimicrobial agent then exerts its effect on remaining suspended cells and microbial cells within the disrupted biofilm. As those skilled in the art will understand, the combination of an antimicrobial agent with the compounds described herein can reduce the dosage and / or frequency of the antimicrobial agent.

[0110] Exemplary antimicrobial agents that may be used in the combination therapy include antifungal agents such as miconazole, ketoconazole, econazole, terbinafine, cyclopirox, tolnaftate, sertaconazole, sulconazole, amphotericin b, chlorooxylenol, cryoquinol, butenafine, naftifine, nystatin, and clotrimazole. Exemplary antibiotics include penicillins, tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulfonamides, glycopeptides, aminoglycosides, and carbapenems.

[0111] This disclosure provides compositions comprising the compounds disclosed herein (in particular, compounds of formula I) together with an antimicrobial agent. As those skilled in the art will understand, the compounds and antimicrobial agents may also be provided separately. In some embodiments, the compounds and antimicrobial therapy overlap. In some embodiments, treatment with the compounds of the present invention precedes antimicrobial therapy.

[0112] In some embodiments, the compositions disclosed herein are provided as or within foods or functional foods. As used herein, “functional foods” means foods prepared to satisfy not only nutritional properties but also specific functions, such as improving health and reducing the risk of disease. Such functional foods may also be referred to as dietary supplements or (animal) food additives. For this purpose, biologically active compounds, such as minerals, vitamins, fatty acids, beneficial bacteria, dietary fiber, and antioxidants, are added thereto. Such foods may be in any form suitable for oral administration, for example, in the form of liquids, gels, powders, pills, tablets, or gel capsules.

[0113] The functional food may also contain animal digest, for example, any material resulting from the chemical and / or enzymatic hydrolysis of clean and undegraded animal tissue. The functional food may also contain dried brewer's yeast, for example, a dried inert agent that is a by-product of the brewing industry. Animal digest and dried brewer's yeast have been shown to enhance the palatability of the functional food. When present in the functional food, animal digest constitutes about 10% to about 90% of the functional food, and dried brewer's yeast constitutes about 1% to about 30% of the functional food.

[0114] In exemplary embodiments, the present disclosure provides compositions comprising pectin, yucca, vitamin E, calcium carbonate, probiotics, plant extracts, herbs, and oregano. Preferably, the composition comprises about 1 to about 5 grams of therapeutic thiosulfur compounds. The composition is suitable as tablets, particularly for the treatment of cattle. As described in these embodiments, the composition is suitable for reducing the cell count in milk and for treating mastitis.

[0115] In some embodiments, the Disclosure provides compositions comprising a therapeutic thiosulfur compound disclosed herein, together with at least one pharmaceutically acceptable carrier, diluent, and / or additive (see, for example, Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor), Mack Publishing Company, April 1997). As used herein, the term “pharmaceutically acceptable” means those compositions, or combinations of drugs, materials, or compositions, and / or their dosage forms, which are within the bounds of appropriate medical judgment and suitable for use in contact with human and animal tissues in proportion to a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic reactions, or other problems or complications. Furthermore, the term “pharmaceutically acceptable diluent or carrier” means pharmaceutically acceptable materials, such as liquid or solid fillers, diluents, additives, solvents, or encapsulating materials, such as those encapsulating materials involved in the transport or delivery of peptides from one organ or part of the body to another organ or part of the body.

[0116] The pharmaceutical composition may be administered by any suitable route and form. As will be understood by those skilled in the art, the route and / or form of administration will vary depending on the desired outcome. The pharmaceutical composition may be formulated according to the usual procedures for any route, e.g., parenteral, topical (including ocular), oral, sublingual, transdermal, or inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intracoronary, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcapsular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Preferred routes are oral or topical administration.

[0117] The composition may be in any suitable form, e.g., liquid, semi-solid, and solid dosage forms. The composition may be in the form of tablets, capsules, powders, granules, lozenges, creams, or liquid formulations (especially liquid formulations for administration to the skin or eyes), e.g., sterile parenteral solutions or suspensions, or in the form of sprays, aerosols, or other conventional inhalation methods. The pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical (including topical, cheek, and sublingual), rectal, vaginal, and / or parenteral administration. In certain embodiments, the composition is a topical composition in the form of a cream, gel, ointment, lotion, foam, suspension, spray, aerosol, or powder aerosol. The composition is particularly useful for administration to the skin. Preferred compositions also include oral care compositions, e.g., toothpaste, toothpaste, tooth gel, subgingival gel, mouth rinse / mouthwash, artificial saliva, denture products, mouth sprays, lozenges, oral tablets, and chewing gum.

[0118] This disclosure also provides in vitro use of the compositions disclosed herein for preventing or reducing the formation or growth of biofilms on surfaces, and / or for degrading or reducing biofilms on surfaces. Preferably, the method is for reducing biofilms or biofilm formation on surfaces. In some embodiments, the method includes bringing a biofilm adhering to a surface into contact with one of the compositions disclosed herein. The composition includes, for example, a cleaning composition or a disinfecting composition.

[0119] Any surface may be treated with the compositions disclosed herein for coating such surface. The surface may, for example, be sprayed, dipped, or soaked in the composition. Surfaces include glass, metal, porous and non-porous surfaces. Surfaces also relate to the exterior and interior and surfaces of equipment that may be contaminated, such as medical equipment found in the food industry or hospitals and medical facilities, as well as plumbing systems (e.g., sink drains), countertops, building materials, pipes, and surfaces found in clean rooms. Surfaces also refer to the interior or exterior of pipes, such as drains, as well as swimming pools, tanks (e.g., for aquaculture), septic filters, toilets, sinks, and surfaces in greenhouses. Surfaces also include water, such as water from drinking fountains.

[0120] In some embodiments, the surface is a medical device, such as a prosthesis (hip implant, dental implant, artificial joint, voice prosthesis, penile prosthesis), mechanical heart valve, cardiac pacemaker, arteriovenous shunt, scleral buckle, catheter (such as a central venous catheter, intravascular catheter, urethral catheter, Hickman catheter, peritoneal dialysis catheter, endotracheal catheter), tympanotomy tube, tracheostomy tube, surgical suture, bone anchor, bone screw, intraocular lens, contact lens, intrauterine device, aortofemoral graft, or vascular graft. Other infections from medical devices include those from abdominal drains, biliary stents, breast implants, cardiac pacemakers, cerebrospinal fluid shunts, contact lenses, defibrillators, dentures, electrodialysis machines, endotracheal tubes, indwelling urethral catheters, intrauterine devices, intravenous catheters, prostheses, artificial joints, artificial heart valves, nephrostomy tubes, orthopedic implants, peritoneal dialysis catheters, artificial heart valves, alloplastic orthopedic devices, tissue fillers, urethral stents, vascular prostheses, ventricular-associated pneumonia, ventricular assist devices, ventricular derivatives, ventricular shunts, and voice prostheses.

[0121] In some embodiments, the surface is a surgical instrument, such as a clamp, forceps, scissors, skin hook, tube, needle, retractor, scaler, drill, chisel, file, or saw.

[0122] Where used herein, “contains” and its conjugations are used in their non-restrictive sense, meaning that the item following the word is included, but not that items not specifically mentioned are excluded. In addition, the verb “consistes of” may be replaced with “essentially consists of,” meaning that a compound or auxiliary compound as defined herein may contain one or more additional components other than the specifically identified compound, where the one or more additional components do not alter the distinctive features of the present invention.

[0123] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “one element” means one or more elements.

[0124] When the words "almost" or "about" are used in relation to a number (almost 10, about 10), it preferably means that the value can be a given value of 10, which is about 1% of the value.

[0125] The compounds and compositions disclosed herein are useful as therapies and in therapeutic procedures, and therefore may be useful as pharmaceuticals and may be used in methods for preparing pharmaceuticals. In some embodiments, the disclosure provides methods that do not involve treatment of the human or animal body and / or methods for altering the genetic identity of the human germline, wherein the cells are not human germ cell lines.

[0126] All patents and references cited herein are incorporated in their entirety by reference.

[0127] The present invention is further illustrated in the following embodiments. These embodiments are not intended to limit the scope of the present invention, but rather to clarify it.

[0128] Examples

[0129] Several compounds isolated from Allium have been studied in vitro to determine their effects on bacteria, with the aim of developing alternatives to antibiotics. For example, Sorlozano-Puerto et al. (Biomed Research International 2018 Article ID 7861207) describe the antimicrobial activity of PTS and PTSO against numerous human pathogens (212 Gram-negative bacilli and 267 Gram-positive cocci isolated from clinical samples obtained from 479 different patients) using the Recommendation Reference Initiative (CLSI) to determine MIC50 and MIC90 values, as well as MBC50 and MBC90 values ​​(the latter used to demonstrate direct bactericidal effect), as measures of antimicrobial activity. The data for PTSO and PTS were compared with various antibiotics used in clinical practice against such pathogens. The results show that for all bacterial strains, several antibiotics from the test panel were more effective than PTSO or PTS, as indicated by the lower MIC values ​​of the antibiotics. Based on these in vitro findings, those skilled in the art would not consider PTSO or PTS as useful alternatives to common antibiotics in the treatment of bacterial infections.

[0130] In vitro assays, as described by Sorlozano-Puerto et al., measure the inhibition of growth of planktonic microorganisms. As will be understood by those skilled in the art, the effect of a compound on planktonic microorganisms does not indicate the effect of such a compound on biofilms. This has been demonstrated for many antimicrobial agents, as previously discussed, and is also well known in the literature. See, for example, Roy et al., who showed that bacteria in biofilms increased their resistance to conventional antibiotics by approximately 1000 times (2018 Virulence 9:522~554).

[0131] In contrast to studies on planktonic microorganisms, this experiment is the first to demonstrate the effects of PTSO and PTS on biofilms. In such assays, the culture medium and culture conditions (continuous agitation of the plate to induce shear stress comparable to that occurring in blood flow) stimulate microorganisms, such as bacteria, to rapidly form biofilms. Such biofilm assays can be performed in two ways: by adding the test compound (e.g., PTSO) from the beginning of the experiment to assess its potential to prevent biofilm formation, or by adding the test compound to a mature biofilm to assess its soluble effect (e.g., corresponding to Example 2, where an existing biofilm was treated with PTSO).

[0132] The primary mechanisms of action of compounds in biofilm assays are related to either the inhibition of quorum sensing and subsequent population gene switching, resulting in bacterial adhesion and synthesis, or the assembly of the extracellular matrix, thereby forming the three-dimensional structure of the biofilm. These mechanisms are distinct from the pure antibacterial / antimicrobial "killing" effect. If a compound is effectively tested in a biofilm assay but lacks antibacterial activity (e.g., an antibody developed against a specific biofilm protein to destabilize the biofilm structure), then the compound is combined with an antibiotic to ensure that bacteria released from the biofilm are killed. This is especially true when the animal's immune system is impaired. If a substance, such as PTSO, has any antibacterial activity (albeit through other mechanisms), this is considered a desirable additional effect in clinical treatment, supporting the eradication of bacterial populations by the innate immune system.

[0133] Examples 2-4 below describe the effects of the compounds of the present invention using in vitro biofilm assays. Such assays are quite different from those described by Sorlozano-Puerto et al., and measure the inhibition of growth of planktonic microorganisms. Surprisingly, these examples demonstrate that the compounds of the present invention are effective in eradicating biofilms.

[0134] Example 1: Isolation of PTSO and PTS Scope: PTSO and PTS were isolated from plant extracts containing 56% PTSO and 14% PTS (hereinafter referred to as "PTSO / PTS-rich plant extracts").

[0135] Study Design: Based on the polarity of the compound, the elution order was estimated from HPLC, and the separation conditions were developed in TLC using heptane and ethyl acetate as mobile phases. A flash column with a length of 110 cm and a diameter of 25 cm was packed with 15 kg of silica (ACROS Organics®, particle size 40-60 μm) in heptane, and the column was stabilized overnight. Purification was performed by gradually increasing the polarity from 0% to 40%. The fractions of interest, PTS (=peak 6) and PTSO (=peak 7), were both 1 Identified by 1H-NMR, elution was initiated after using 20 liters of mobile phase, and the fractions were collected in 2-liter fraction sizes. The fractions were separated according to the TLC identification of each compound, and the corresponding fractions of each compound (PTS and PTSO) were combined. The solvent was evaporated by a rotary evaporator to obtain a purified fraction of PTS (32435-2-A) and two purified fractions of PTSO (MHA32435-2-B, MHA32435-2-C), as summarized in Table 1.

[0136] The required purity of over 98% was achieved only in fraction MHA32435-2-B (=PTSO), while fractions MHA32435-2-A (PTS) and MHA32435-2-C (=the second fraction of PTSO) showed a purity of less than 98%, requiring further individual purification by a second flash chromatography to obtain the desired purity.

[0137] The PTS (MHA32435-2-A) was re-purified by packing a flash column (50 cm long and 20 cm in diameter) with 2 kg of silica (ACROS Organics®, particle size 40-60 μm), using ethyl acetate and heptane as mobile phases, and increasing the polarity from 0% to 20%. The fraction of interest was collected in 250 ml fraction sizes, identified by TLC, and combined. The solvent was evaporated by rotary evaporator to obtain PTS (EWR32514-01-1) with a purity of over 98%.

[0138] The repurification of the impure fraction of PTSO (MHA32435-2-B) was performed using the same mobile phase and flash column as MHA32435-2-A, except that 3 kg of silica (for column packing) was used, the polarity was increased from 0% to 30%, and the fraction size was 100 ml. As summarized in Table 1, the repurification resulted in two fractions of PTSO with a purity of over 98% (EWR32514-02-1 and EWR32514-02-02).

[0139] [Table 1]

[0140] This example demonstrates that PTSO and PTS were purified to a high degree of purity. Samples with a purity of over 99% are used as shown in the examples herein and are referred to as "pure PTSO" and "pure PTS".

[0141] Example 2. Biofilm in a PBR (Photo Bio-Reactor) To determine the antibiofilm effects of PTSO, PTS, and DPD (dipropyl disulfide), biofilms were grown under controlled conditions in a glass tube reactor, PBR. A photobioreactor was used as a model system because biofilm formation was clearly observed in the glass tube. After mature biofilms were formed, increased amounts of pure PTSO, PTS, or DPD were added, and the biofilms were removed by titration with pure PTSO, PTS, or DPD, and the dose at which the biofilms were eradicated was determined. The microbial composition of the biofilms was then determined by deep sequencing.

[0142] research design Heterotrophic microbial biofilms: Biofilms were cultured in a 26.5-liter glass photobioreactor (PBR) (LGEM's GemTube RD-25 Glass) with a tube length of 37 meters including bends. The PBR was filled with 22 liters of non-sterile culture medium (Table 1).

[0143] [Table 2]

[0144] The culture medium also contained 1 g / l of glucose. Inoculation was performed by adding 500 ml of trench water from Zuidpolder near Delfgauw. Air was pumped in at a rate of 1.0 liter / min. In this setup, the circulation of the medium through the PBR was kept as low as possible, but there was a stable hold-up to ensure circulation within the PBR. The PBR was covered with a dark plastic screen to prevent exposure to light. Incubation was carried out at 12–18°C, and the pH was controlled to 5.2–5.7 once every 1.5 days, five times a week. After a total dose of 5 g / l of glucose (after one week), a slimy layer (biofilm) formed covering the entire inner surface of the glass tube. When the biofilm formed, the medium was replaced with fresh medium that did not contain glucose.

[0145] As shown in the table, PTSO, PTS, or DPD were added to the PBR. Pure PTSO and PTS were obtained as shown in Example 1, and stable solutions were prepared in water by sonication of a 1000-fold diluted mixture. DPD (dipropyl disulfide, purity over 98%) was supplied by Sigma. After each addition step, the biofilm culture was incubated for 24 hours to observe the stability of the biofilm.

[0146] The number of CFUs (colony-forming units) for aerobic, anaerobic / microaerobic, and fungal organisms in the biofilm and supernatant was determined according to the following standard operating procedures: for aerobic colony-forming units (CFU / ml) per ml, see https: / / www.iso.org / standard / 53728.html; for anaerobic CFU / ml measurement, see https: / / www.thermofisher.com / order / catalog / product / AN0025A# / AN0025A (plates were cultured in an oxygen-free vessel); and for the number of colony-forming units for fungi, see http: / / edgeanalytical.com / wp-content / uploads / Food_AOAC-997.02.pdf (see Table 2).

[0147] The composition of the microbial community in the biofilm was determined using metagenomic analysis (Illumina deep sequencing).

[0148] Another sample of the biofilm was taken, and a portion was stored at -80°C as an inoculum for further study. The remaining portion of the biofilm was dissolved by gentle sonication and used, as described above, to determine the number of aerobic, anaerobic, and fungal colony-forming units. Subsequently, it was calculated whether the number of colony-forming microorganisms in the culture medium matched the number of microorganisms in the biofilm before treatment. In this way, it was calculated whether the treatment killed the microorganisms or solubilized only the biofilm while leaving the bacteria alive.

[0149] Biofilm eradication by PTSO Microscopic analysis revealed a mixture of fungi, protozoa, and bacteria.

[0150] Metagenomic analysis demonstrated that the biofilm consisted of a highly mixed population. At least 611 different genera of bacteria were identified. The five most abundant genera and their relative amounts are as follows: Pseudomonas 46%, Acidovorax 13%, Burkholderia 4%, Achromobacter 4%, and Lelliotia 3%. At least five different genera of fungi were identified. The five most abundant genera and their relative amounts are as follows: Cladobotryum 5%, Emiricellopsis 31%, Fusicolla 17%, Mucor 37%, and Rozella 10%.

[0151] The addition of PTSO resulted in the breakdown of the biofilm. This can be visualized by the release of flocs from the biofilm after the initial administration of 0.05 μl / l of PTSO, while at a total dose of 0.65 μl / l the biofilm became significantly thinner, and at the highest dose it appeared to be almost completely degraded.

[0152] To determine whether PTSO has a biofilm destabilizing effect or simply kills microorganisms, samples were taken from the biofilm before treatment, and samples of liquid culture medium were taken before and after treatment. These were inoculated as described above, and the number of colony-forming units was determined. The number of colony-forming units per ml of biofilm and in the supernatant was estimated by plating techniques and reported in Table 3. Subsequently, it was calculated whether the number of colony-forming microorganisms in the culture medium matched the number of microorganisms in the biofilm before treatment. In this way, it was calculated whether the microorganisms were killed by the treatment, or whether only the biofilm was solubilized while the bacteria remained alive (Table 6).

[0153] [Table 3]

[0154] Even at the lowest dose of PTSO, CFU increases for aerobic, anaerobic / microaerophilic, and fungal organisms. This indicates that even at low doses, PTSO initiates the breakdown of biofilm layers, subsequently releasing bacteria. At the highest concentration of PTSO, CFU decreases for aerobic, anaerobic / microaerophilic, and fungal organisms, indicating that high doses of PTSO affect planktonic microorganisms.

[0155] Biofilm eradication using PTS The biofilm was grown as described above. Metagenomic analysis demonstrated that the biofilm consisted of a highly mixed population. At least 362 different genera of bacteria were identified. The five most abundant genera and their relative proportions are as follows: Pseudomonas 72%, Raoultella 6%, Flavobacterium 3%, Enterobacter 1.5%, and Acidvorax 1%.

[0156] After the addition of a total dose of 11.65 μl / l of pure PTS, visual breakdown of the biofilm was detectable. Solubilization of the biofilm resulted in an increase in CFU of aerobic bacteria, anaerobic bacteria, and fungi in the culture (Table 3).

[0157] [Table 4]

[0158] Prior to the initial administration of PTS, there had already been some dose-independent microbial release from the biofilm, but the biofilm was visibly stable. The initial measurement at point 0 may be an artifact. Similar to PTSO, PTS results in an increase in live microorganisms in the supernatant, indicating biofilm degradation. High doses decrease CFU, indicating that high doses of PTS affect planktonic microorganisms.

[0159] Biofilm eradication by DPD The biofilm was grown as described above. Microscopic analysis showed that the biofilm consisted of both fungi and bacteria. Microbial population analysis was not performed because no significant difference was expected regarding the microbial population of the biofilm.

[0160] DPD was added to the culture medium as shown in Table 5. During the first seven doses, a biofilm was present in the reactor that did not visually grow or shrink in response to additional DPD doses. After dose 8, following a total addition of 167 μl of pure DPD / liter culture (= 160 mg of pure DPD / liter), the biofilm began to detach from the glass and dissolve into the medium. This process continued up to a total dose of 317 μl of pure DPD / liter of pure culture. At higher doses, only biofilm spots remained on the glass where previously thick biofilms had been present. Further addition of DPD did not remove the remaining thin biofilm.

[0161] [Table 5]

[0162] Based on Table 5, DPD (an exemplary compound with formula III below) appears to begin affecting biofilms after a total dose of approximately 167 μl / l. At high concentrations, CFU decreases for aerobic, anaerobic / microaerophilic, and fungal organisms, indicating that DPD affects planktonic microorganisms at high doses.

[0163] [Table 6]

[0164] From the data in Table 6, it was surprisingly revealed that despite the large fluctuations in the number of microorganisms detected in the biofilm, the eradication effect of the indicated molecules on the biofilm did not occur by solubilizing the biofilm after killing the microorganisms, likely due to the high heterogeneity of the biofilm. Rather, it was by altering the physiological function of the microorganisms that only the biofilm was destabilized and solubilized.

[0165] Example 3. In vitro biofilm assay Study Design: Biofilm formation was studied using Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa). These specimens were potential pathogens, and the biofilm eradication effect of two PTSO-containing samples was investigated in vitro.

[0166] The tested composition has the following composition: QS1: 236 mg of PTSO / PTS-rich plant extract containing 530 mg / liter of PTSO and 133 mg / liter of PTS was mixed with 250 ml of desalted water. QS2: 135 mg of pure PTSO was obtained as shown in Example 1 and diluted in 250 ml of deionized water by stirring overnight. This corresponds to 540 mg / liter of PTSO in water.

[0167] Biofilm formation was studied using Gram-positive Staphylococcus aureus ATCC 6538 (S. aureus) and Gram-negative Pseudomonas aeruginosa ATCC 9027 (P. aeruginosa). The experimental method was adopted from ASTM-E2799-17 (Standard test method for testing disinfectant efficacy against Pseudomonas aeruginosa biofilm using the MBEC assay ASTM International, West Conshohocken, PA, 2017). In this method, P. aeruginosa was used, and in this example, the same test was also applied to test the effect against S. aureus. The MBEC (Minimum Biofilm Eradication Concentration) assay is a high-throughput screening tool for determining the efficacy of drugs against biofilms of various microorganisms. The following two methods were used to test the effects of QS1 and QS2 on biofilms grown in 96-well plates. Figure 1A shows a method used to measure biofilm in a microtiter plate, and Figure 1B shows a method for measuring biofilm formation on a peg.

[0168] In short, overnight cultures of P. aeruginosa and S. aureus were diluted to 0.1 (OD600). The bacterial suspensions were then pipetted into 96-well plates in either minimal or rich medium in a 1:9 ratio. The minimal medium had the following composition: casein, 0.5%; glucose, 2 g / l; MgSO4, 1 mM; FeSO4*7H2O, 0.5 mg / L. The pH was set to 7.3 and then sterilized. The rich medium contained the following nutrients: casein peptone, 17 g / L; soy peptone, 3 g / L; dipotassium hydrogen phosphate, 2.5 g / L; sodium chloride, 5 g / L; glucose monohydrate, 2.5 g / L. The pH was set to 7.3 and then sterilized. The 96-well plates were incubated at 37°C for 24 hours, and then a biofilm was formed. Subsequently, the biofilm is exposed to the test solution as shown in Figure 1.

[0169] Initial biofilm formation occurs during the 0–24 hour period, and later biofilm formation occurs during the 24–48 hour period. The suspension is discarded, and the wells / pegs are washed with deionized H2O. Subsequently, crystal violet (0.1%) is added, and staining is performed for 15 minutes. Washing is then performed by desalting with H2O. Thus, the biofilm is destained with acetic acid and transferred to a microtiter plate as performed in Example 4. Absorbance was measured at 550 nm.

[0170] Results and discussion: The MBEC assay was used to determine the effect of QS1 and QS2 on biofilm formation, as shown in Figure 2. Results are shown as percentage growth (normalized to bacteria without 100% effector); the addition of the word "peg" (in the diagram) indicates growth in a polystyrene cone; without "peg", it indicates growth in a well on a microtiter plate. As shown in Figure 2A (late biofilm formation), growth inhibition was observed in the well when QS1 and QS2 were added to rich medium of both bacterial specimens. Furthermore, the addition of QS1 and QS2 resulted in inhibition of growth of P. aeruginosa and S. aureus in the well and on the peg under minimal medium conditions (Figure 2B).

[0171] Based on a biofilm assay (compliant with ASTM-E2799), clear biofilm inhibitory growth effects by QS1 and QS2 were observed in P. aeruginosa and S. aureus. Biofilms of both bacteria were strongly influenced by QS1 and QS2 during biofilm formation in the current assay.

[0172] Example 4. In vitro biofilm assay Scope: As described herein, the effects of compounds on planktonic microorganisms do not indicate the effects of such compounds on biofilms. However, in vitro models have been developed to study biofilm infections, and results from such models may better predict in vivo effects. For example, Bahamondez-Canas et al. Biomedicines. 2019 Jun;7(2):34 describes a number of in vitro models for studying biofilm infections of wounds. In this example, in vitro experiments were performed using various strains of Staphylococcus aureus and Streptococcus uberus, which are potentially pathogenic bacteria. These are microbial specimens involved in bovine mastitis, and similarly for Staphylococcus aureus in humans.

[0173] Research design: In this embodiment, the following strains were used: Staphylococcus aureus ATCC 25923 was obtained from the ATCC collection; Staphylococcus aureus 074 was kindly provided by Professor Fink of the Faculty of Veterinary Medicine, Utrecht University, Netherlands, and is described as a clinical isolate from a cattle with mastitis. Further information on the strain is provided below: Melchior, MB, Fink-Gremmels, J., & Gaastra, W. (2006). Comparative Assessment of the Antimicrobial Susceptibility of Staphylococcus aureus Isolates from Bovine Mastitis in Biofilm Versus Planktonic Culture. Journal of Veterinary Medicine Series B, 53(7), 326~332; Staphylococcus aureus ATCC 15564 was obtained from the ATCC collection, and Streptococcus uberis was clinically isolated from a cattle with mastitis and is part of the collection of the Department of Veterinary Microbiology, Infectious Diseases and Parasitology at the University of Thrace in the Republic of Bulgaria.

[0174] The minimum biofilm inhibition concentration (MBIC) and the microtiter biofilm eradication concentration (MBEC) are defined and measured as shown in Table 7 below.

[0175] [Table 7]

[0176] The bacteria described above were tested using the methods summarized in Table 7, with the PTSO / PTS-rich plant extract, pure PTSO, and pure PTS described in Example 1, as well as the DPD described in Example 2. The results are shown in the table below.

[0177] [Table 8]

[0178] [Table 9]

[0179] The lowest MBEC values ​​were observed in the case of PTSO / PTS-rich plant extracts, followed by PTSO alone and PTS alone. Data were obtained in a log dilution series, and differences in values ​​represent only one dilution step. Taken together, these in vitro experiments demonstrate the antibiofilm activity of the compounds of the present invention, which is supported by the clinical findings described in the examples below.

[0180] Examples 5-8: Treatment of biofilm-related disorders in vivo To demonstrate the in vivo effects of the compounds disclosed herein, PTSO and PTS were tested in vivo for numerous biofilm-related disorders.

[0181] Bovine mastitis is a disease that affects millions of cows worldwide every year. This disease is caused by a variety of very different bacteria, and in some cases, yeast that invades the mammary glands of lactating cows causes a persistent infection and inflammatory response. Bovine mastitis can be caused by a variety of Gram-positive and Gram-negative pathogens, and the prevalence of individual pathogens can vary to some extent between countries and continents. The most prominent clinical sign of all forms of mastitis is an undesirable increase in the number of somatic cells in milk intended for human consumption, caused by a (chronic) inflammatory response in the mammary tissue. This secondary inflammation reduces milk production and therefore results in a serious economic loss for farmers. Over the past several decades, numerous pharmaceuticals, including various classes of antibiotics, have been marketed to treat the major infectious agents diagnosed as causes of mastitis, administered alone or in combination. These pharmaceuticals are administered systemically (by injection) or topically via the teat channels to combat bacterial infections. However, despite these considerable efforts, antibiotic therapy is generally only temporarily effective, and since somatic cell counts (SCCs) remain high as a clinical marker of mastitis in many cases and negatively impact milk yield, bovine mastitis remains the most common disease in dairy cows.

[0182] Mastitis is a known biofilm-related disorder. Bacterial biofilm formation has been demonstrated in in vitro experiments culturing mastitis pathogens, as well as in situ by staining the biofilm matrix in the mammary tissue of infected cattle, under conditions favorable to bacterial biofilm formation (quantifiable by measuring genes that drive biofilm formation after staining). For example, Figure 1 of Schonborn S and Kromker V (2016 Journal Veterinary Microbiology, 30;96:126~128) shows the biofilm matrix from the mammary tissue of a cattle with mastitis.

[0183] Biomedical research over the past decade has revealed that biofilm formation is not only a characteristic of breast infections and mastitis, but also a common feature of microorganisms that invade human and animal tissues and cause persistent infections and inflammation. To date, microbial biofilms remain one of the major unresolved challenges in modern treatment of infections in humans and animals.

[0184] The clinical evidence provided in the following examples reveals the unexpected therapeutic effects of PTSO (and, to a lesser extent, its derivative, PTS) in the treatment of important biofilm diseases, such as bovine mastitis and infected chronic wounds (e.g., digitorum dermatitis and UCD). Several field trails have been described showing that the application of PTSO is directly related to a long-term, stable reduction in somatic cell count in treated cattle, suggesting bacterial healing and tissue regeneration. Further examples describe the effects on biofilm-related chronic wounds (i.e., digitorum dermatitis and uder cleft dermatitis (UCD)).

[0185] These findings are noteworthy because it is generally recognized that common antibiotics are ineffective against biofilm infections. These observations have been documented in numerous scientific papers in human and veterinary medicine, all suggesting that biofilm formation is a major cause of recurrent and persistent infections, as well as tissue inflammation and damage. The presented experimental (in vitro) and in vivo (clinically observed) efficacy of PTSO and PTS against prototype biofilm infections indicates that these compounds can be effectively used in clinical practice to prevent and resolve biofilm infections. Biofilm formation and degradation occur as direct interactions between molecules and microorganisms and are therefore independent of the host (animal or human). Because the mechanisms involved in biofilm formation are highly conserved among bacterial species, these compounds are also considered effective against a wide range of biofilm infections in humans and animals. This example also demonstrates that PTSO and PTS can be administered orally and exert their effects at different sites (e.g., mammary glands and nails).

[0186] Example 5: Mastitis range Experiments were conducted to investigate the effect of tablets containing 3.84 g of PTSO and 0.96 g of PTS on the cell count or somatic cell count (SCC) in milk.

[0187] SCC is related to the amount of pathogens present in the quarter of the immune system. The cell count for each cow was measured using milk production registration (MPR). This MPR is performed regularly every 30-40 days at all farms.

[0188] Somatic cell count (SCC) is a primary indicator of milk quality. The majority of somatic cells are leukocytes (i.e., white blood cells), which normally become increasingly abundant in milk as an immune response to pathogens causing mastitis, and a small number of epithelial cells, which are milk-producing cells secreted from the inside of the udder when infection occurs. Inflammation often occurs in the udder quarters, resulting in an increase in the cell count in these quarters. However, the total increase in cell count is smaller due to dilution in other quarters of the udder. SCC is quantified as the cell count per ml of milk. SCC is a primary indicator of milk quality, indicating the presence of (asymptomatic) mammary infections in animals, for example, caused by pathogens that cause mastitis. The relationship between SCC and mastitis is outlined in Sharma et al., 2011 Asian-Aust J Anim Sci 24:429~438.

[0189] SCC and MPR SCC data was extracted from the Milk Production Registry (MPR). The MPR is a database showing all the details (e.g., SCC) of the milk produced for each individual cow. The MPR for selected farms was available regularly every 30-40 days. SCC is quantified as the number of cells per ml of milk. Generally speaking, individual cows with an SCC value of 100,000 or less were "uninfected" and did not experience significant loss of yield due to asymptomatic mastitis. A threshold SCC of 150,000 determines whether a cow is infected with mastitis. Cows with a result above 150,000 have a very high probability of infection, at least one-quarter. Cows infected with serious pathogens have an SCC of 300,000 or more.

[0190] Example 5.1 Cell count survey in 18 cows using the tablets described above. Cell count analysis in 9 cows treated with the aforementioned tablets and comparison with 11 untreated cows.

[0191] Three farms were selected for the study. Table 10 below shows information about the size of the farms and the milk they produce.

[0192] [Table 10]

[0193] Cattle with a pre-treatment SCC count of 250,000 cells / ml or higher were selected for tablet administration. The following number of cattle were selected from each farm: Farm 1: 6 cows; Farm 2: 7 cows; Farm 3: 5 cows

[0194] This brought back a total of 18 cows.

[0195] SCC data was analyzed using three MPRs (Multiple Probability Ratings) before and after treatment.

[0196] Results and Discussion The numbers of the 18 cows and their average somatic cell counts are shown in Table 11 below. After treatment, the cell count of all cows was less than 150,000 cells / ml at MPR3. MPR3 is approximately 80 to 100 days after treatment. A total of 60% had cell counts even lower than 100,000 cells / ml.

[0197] [Table 11]

[0198] conclusion SCC in milk of selected cows was shown to decrease after treatment with the tablets. This was quite surprising, as it was clearly shown that PTSO / PTS was absorbed by the intestines and subsequently entered the bloodstream. PTSO / PTS was administered orally in the clinical trials for mastitis described herein. Because a clinically significant effect was observed in mammary tissue, this indicates that PTSO / PTS not only passes through the rumen (indicating stability against rumen microorganisms) but also reaches the post-hepatic bloodstream (indicating minor or insignificant hepatic in vivo changes and inactivation), and ultimately reaches the mammary gland at therapeutically effective concentrations. This example shows that the tablets effectively reduce SCC and therefore have a good therapeutic effect against mammary-related infections. Moreover, all cows showed SCC lower than 150,000 cells / ml after MPR3, which demonstrates efficacy in all individual cows in this study.

[0199] Example 5.2 Farms 1 and 2 were used for the analysis, as shown below. Cattle were selected based on cell count from MRP0 (pre-treatment). Cattle with cell counts ranging from 200,000 cells / ml to 500,000 cells / ml were either treated or untreated with tablets containing 3.84 g of PTSO and 0.96 g of PTS during the same period.

[0200] The following number of cattle were selected as the group to be treated: Farm 1: 5 cows Farm 2: 4 cows

[0201] The following number of cattle were selected as the group that was not treated: Farm 1: 5 cows Farm 2: 6 cows

[0202] For both the treated and untreated groups, a SCC level of 150,000 cells / ml was selected based on the maximum healthy cell level in the milk of young cows (heifers). The percentage of cows below this level is shown in Table 12 below. The results showed that the percentage of cows with less than 150,000 cells increased in the treated group, while it did not increase in the untreated group. The treated group eventually reached 100% after MPR4. The untreated cows showed a wide spread of SCC and no significant decrease in cell count.

[0203] [Table 12]

[0204] Tablet administration to cattle with cell counts of 200,000–500,000 cells / ml was clearly demonstrated to be effective in reducing somatic cell counts compared to untreated cattle.

[0205] Example 5.3 This study was conducted using milking robots at nine farms with a minimum herd size of 80 cows (Holstein). Only cows with increased SCC were included. Cows were considered to have increased SCC if more than 300,000 cells were measured per milliliter. Cows were in the early stages of lactation up to 5 months before the non-lactation period. In this way, cows could be monitored for a longer period.

[0206] SCC data were collected based on MPR. Cattle treated with tablets containing 3.84 g of PTSO and 0.96 g of PTS were analyzed for the effect on SCC. Subsequently, the data were evaluated for the number of cattle showing a reduction in SCC and a total reduction in cell count. Furthermore, comparisons were made between groups of cattle at different lactation stages, given the presence of age effects.

[0207] Decrease in SCC after tablet administration Results regarding the number of cows showing a decrease in SCC for each MPR dataset are shown in Table 13 below. The first MPR after tablet administration was available for all 63 cows. For 44 cows, administration of the tablets effectively reduced SCC. For 42 cows, SCC was less than 300,000 cells. The second MPR measurement was available for 48 cows. For 31 cows, SCC was lower than before tablet administration. Among them, 31 cows showed SCC of less than 300,000 cells. The third MPR measurement was available for only 12 cows. Among these 12 cows, 9 cows had a lower cell count than before the tablets were required.

[0208] <00,00882>In addition, the total decrease in cell count was also evaluated. The results of the first first first MPR are shown in Table 14 below, and the results of the second MPR are shown in Table 15 below. The results indicate that most cows showing a decrease in SCC are classified into the group of 100,000 - 50,000 cells. When the average decrease in SCC before administration of the tablets is compared with the average SCC after administration of the tablets, the average decrease is 250,000 cells / ml.

[0209] [Table 13] [[ID=1}}

[0210] [Table 14]

[0211] [Table 15]

[0212] Lactation period Younger cows (first or second lactation stage) may show a more pronounced effect on SCC reduction after tablet administration than older cows. Of the 63 cows included in the study, 32 were in the first or second lactation stage (younger cows), while the other 31 cows varied from the third to the sixth lactation stage. Among the cows in the first or second lactation stage, 23 out of a total of 32 cows showed a decrease in cell count levels (72%). Of the 31 cows that had already lactated several times, 18 showed a decrease in cell count levels (58%). Surprisingly, this indicated that the treatment was more effective in cows in the first or second lactation stage. In younger cows, cell counts remained low even after the second MPR measurement. This clearly demonstrates the effectiveness of PTSO / PTS-containing tablets in reducing SCC, which can be considered an indicator of the presence of (asymptomatic) mastitis.

[0213] Example 5.4 Milk from cows with elevated SCC values ​​was investigated in biofilms that could be selected from the milk as flocs. Flocs were isolated from fresh milk from cows with elevated SCC. Samples for microscopic observation (stained with standard Gram stain and crystal violet) were prepared and observed using a Nikon fluorescence microscope. Bacteria were detected. The biofilm was seeded onto agar plates and the bacteria were isolated.

[0214] For Staphylococcus aureus, 100 μl of milk sample was seeded onto Baird-Parker agar containing 5% egg yolk emulsion with 3.5% L-tellurite. The plate was incubated at 37°C for 24 hours. Subsequently, pure cultures of the identified Staphylococcus aureus colonies were prepared on sheep blood agar. For the isolation of Streptococcus uberis, 100 μl of milk sample was seeded onto sheep blood agar containing 10 mg / ml polymyxin and neomycin, and incubated at 37°C for 24 hours. Colonies were picked and pure cultures were performed by seeding various strains on 5% sheep blood agar. The isolated strains were frozen in 1.5 ml of TSB (tryptone soy broth) medium supplemented with 20% glycerol. The strain was identified by Vitek2 (Biomerieux Benelux BV, Amersfoort, Kingdom of the Netherlands).

[0215] For biofilm preparation, cells are placed in TSB medium for 10 minutes. 8 The solution was diluted to a concentration of CVE / ml. 20 μl was transferred to 180 μl of TSB medium containing 2 g / l glucose in a 96-well plate. The plate was incubated at 37°C for 5 days without staking. Excess medium was then removed, and the biofilm was rinsed with excess PBS phosphate-buffered saline. The wells were air-dried and prepared for microstaining and analysis as described above.

[0216] The effects on milk were monitored for several days after administering PTSO / PTS tablets (3.84 g of PTSO and 0.96 g of PTS) to cattle.

[0217] Results and discussion: Microscopic observation of biofilms isolated from milk clearly demonstrated the presence of biofilm-associated cells. These cells were used to prepare pure cultures and were identified as Staphylococcus aureus and Streptococcus uberis. The biofilms were then cultured in vitro, which clearly demonstrated the biofilm's involvement in mastitis infection.

[0218] Immediately after administering PTSO / PTS tablets to cows, the milk from the treated cows contained lumps as it flowed from the udder (Figures 3A and 3B). Crumbly milk continued to flow from the udder for several days, after which the cows began producing normal milk again. Subsequently, the red infection color of the udder disappeared, and SCC significantly decreased over the following weeks. The observations described above represent the normal healing process of mastitis after administration of PTSO / PTS tablets.

[0219] Example 5.5 Cell count is an important indicator of udder health and will be measured to study the effect of tablets containing 3.84 g of PTSO and 0.96 g of PTS on udder health. Two groups of 20 cows from two different farms will be tested. Cows will be selected if their cell count exceeds the limit of 250,000 cells / ml in the last MPR or in the second to last MPR. Each cow in the study group will be sampled before administering the PTSO / PTS tablets. After administration of the tablets, cows will be sampled once a week, and these samples will be analyzed for somatic cell count for three months, and analysis for cell count will be performed weekly for three months.

[0220] The average cell counts for both farms, measured weekly, are shown in Table 16 below.

[0221] [Table 16]

[0222] The results of both farms showed a rapid decrease in cell count in the first approximately 6 weeks and then a gradual decrease after 7 weeks. This is consistent with the expected effect of the dosage. The conclusion is that after treatment with the first administration of PTSO / PTS tablets, cows with a cell count of over 250,000 cells / ml showed a significant decrease in cell count to approximately 250,000 cells / ml. From this example, it was demonstrated that the effect on the reduction of SCC is very long-lasting.

[0223] Example 5.6 Four individual farms and a total of 47 cows were examined for the effect of tablets containing 3.84 g of PTSO and 0.96 g of PTS on SCC.

[0224] Study design: Farm 1: 11 cows, after the first treatment, at a total of 3 MPR. Farm 2: 8 cows, after the first treatment, at a total of 9 MPR. Farm 3: 16 cows, after the first treatment, at a total of 13 MPR. Farm 4: 12 cows, after the first treatment, at a total of 2 MPR.

[0225] Other data regarding the farms are shown in Table 17 below.

[0226] [Table 17]

[0227] Results and discussion: The average cell count of the milk from individual farms is shown in Table 18 below.

[0228] [Table 18]

[0229] In all cases, the number of SCCs decreased after treatment. Multiple MPRs showing consistently low SCC levels were available for Farms 2 and 3. This demonstrates the effectiveness of the tablets in reducing SCCs over the long term.

[0230] Example 5.7 At a farm in Hanford, California, with 5,500 cows, somatic cell count and milk production statistics were measured monthly to study the effects of tablets containing 3.84g PTSO and 0.96g PTS on udder health. Cows were selected based on various criteria outlined below and treated as described below. The efficacy of PTSO / PTS tablets versus a control group in reducing saccharin cornea (SCC) was tested.

[0231] The objective was to investigate whether there were significant differences when the following groups were compared after treatment. • The PTSO / PTS-treated group had a lower total mean SCC compared to the control group. • In the PTSO / PTS-treated group compared to the control group, there were more cattle with a weight of less than 200 kg. • In the PTSO / PTS-treated group compared to the control group, there were more cattle with a weight of less than 100 kg.

[0232] Initially, cattle were classified into the following groups: Selection criteria for farm trials using PTSO / PTS to support asymptomatic cattle with high SCC • Current SCC > 200,000 (SCC > 200) • Previously, SCC > 200,000 (PSCC > 200) • First and second lactating animals (LACT<3) • Number of times treated for mastitis (XMAST) < 3 • Do not ban the breeding of cattle. • Within 60 days of dry-off. DCC < 145 (This refers to the calf's gestation period (the current gestation period of an animal that is currently pregnant). SCC data for at least two additional test days is required, and therefore the cow was not dry-off before the inventors obtained such data. Divide it into two equal parts. • Sort by lactation period, sort by SCC within lactation period, and assign group AABB heads, etc. • Equalization groups for lactation period, SCC, PSCC, XMAST, and ECM (= energy-corrected milk; formulas that standardize production based on milk fat and protein content. Used to compare cows / herds of cows with different production volumes and milk components. For example, it allows comparing very high-productivity cows with low fat and protein to low-productivity cows with high fat and protein).

[0233] In total, 100 cows were selected; 50 cows were selected to be treated with one PTSO / PTO tablet, and 50 cows were left untreated for comparison (control).

[0234] After 6 weeks, the mean SCC in the PTSO / PTS-treated group (323K) was 38% lower than in the control group (523K), with significantly more cattle having SCC below 200K (24 treated cattle vs. 15 control cattle) and significantly more cattle having SCC below 100K (13 treated cattle vs. 7 control cattle).

[0235] Example 5.8 In three farms in the Netherlands, 18 cows with elevated somatic cell counts (>200,000 cells / ml) indicating mastitis were selected and given tablets containing 3.84 g of PTSO and 0.96 g of PTS. Somatic cell counts were measured three times at regular intervals of 30 days. Before treatment, the 18 cows had an average somatic cell count of over 600,000 cells / ml. The first measurement showed an average somatic cell count of less than 300,000 cells / ml, an improvement of more than 50%. On the second measurement day, the average for the group was below the 200,000 cells / ml threshold at which the cows are considered infected. The third measurement showed a decrease in the average to less than 100,000 cells / ml. These officially labeled cows with high somatic cell counts are now producing milk for which a premium can be requested.

[0236] Example 5.9 In a second trial conducted at another customer's dairy farm, the results of treatment with tablets containing 3.84 g of PTSO and 0.96 g of PTS were compared to cows that received standard antibiotic dry cow treatment (control cows). As in Example 5.8, both groups consisted of cows with high initial somatic cell counts. Nine cows were selected to receive PTSO / PTS tablets, and eleven cows were selected to be studied as controls (see Table 19). Somatic cell counts of participating cows were measured periodically for a total of three data collections. On the first measurement day after treatment (MPR1), 89 percent of PTSO / PTS-treated cows showed somatic cell counts below 150,000 cells / ml, compared to 36 percent of cows in the control group. At the fourth measurement, all cows treated with PTSO / PTS had reduced somatic cell counts to below 150,000 cells / ml, compared to only 29 percent of conventionally treated cows.

[0237] [Table 19]

[0238] Example 5.10 Changes in somatic cell counts in cattle with enhanced test results were measured over time at four farms. Before treatment, selected cattle from Farm 1 (n=11) had an average somatic cell count of over 3,500,000 cells / ml, selected cattle from Farm 2 (n=8) had an average somatic cell count of approximately 600,000 cells / ml, selected cattle from Farm 3 (n=16) had an average somatic cell count of approximately 893,000 cells / ml, and selected cattle from Farm 4 (n=12) had an average somatic cell count of 2,288,000 cells / ml. Cell counts were measured during monthly dairy product registration. Treatment with tablets containing 3.84 g of PTSO and 0.96 g of PTS (one tablet per cattle) was performed between MPR0 and MPR1. The average somatic cell count of selected cattle was significantly reduced at MPR1 in all farms. By MPR3, 78% of all selected cattle had a somatic cell count of less than 250,000 cells / ml. The average SCC for all cattle was less than 200,000 cells / ml at 4 months post-treatment and remained below 200,000 cells / ml throughout the study period (i.e., at least 6 months post-treatment).

[0239] Example 5.11 An effective alternative to traditional dry cow therapy (DCT). In an independent study conducted at Waterford Institute of Technology, PTSO / PTS was tested as an alternative dry-off treatment. Seventeen cattle were treated with PTSO / PTS tablets, while 16 cattle received traditional dry-off treatment. The PTSO / PTS tablets contained 3.84 g of PTSO and 0.96 g of PTS and were administered three weeks prior to dry-off. The control group received conventional treatment, including the usual antibiotics used for dry-off therapy, on the same day. Comparing these two dry-off methods, 23.5% of the cattle treated with PTSO / PTS became infected, compared to 60% of the cattle treated with the traditional method. The cattle were divided into three groups: low somatic cell count (<200,000), medium somatic cell count (200,000–400,000), and high somatic cell count (>400,000). When these cows were classified based on their somatic cell count before dry-off, PTSO / PTS treatment was successful in 92.9% (13 out of 14) of low SCC cows, while blanket DCT was successful in only 50% (6 out of 12) of this population. The results for the medium somatic cell count group were similar to those for the low somatic cell count cows. For high cell count cows, PTSO / PTS tablets were an effective dry-off therapy in 50% (1 / 2) of cows, and similarly, blanket DCT was effective in 50% (1 out of 2) of cows. This included an outlier PTSO / PTS-treated cow with a somatic cell count of 9,993,400 cells / ml. This cow showed a 146% reduction, having a somatic cell count of 1,560,000 cells / ml at calving, and continued to decrease in number over the following 12 weeks without further treatment.

[0240] Example 5.12 To evaluate PTSO / PTS tablets, a study was conducted by an external third-party clinic. A total of 16 animals from 5 farms were monitored for changes in high cell count after administration of tablets containing 3.84g of PTSO and 0.96g of PTS. 14 animals completed the study. An average reduction of 45% (685,000 to 378,000) in cell count was demonstrated, with an average of 44 animals.

[0241] In addition, the detection and persistence of pathogens in individual quarters were evaluated. Twenty quarters with elevated cell counts were sampled and cultured from 19 cattle from six farms. On day 1, various pathogens (Streptococcus uberis, Staphylococcus aureus, Escherichia coli, etc.) were detected in 12 quarters, and no pathogens were detected in 8 quarters. Of the 12 quarters with detected pathogens, pathogens were detected in only 4 quarters on day 42, and no pathogens were detected in 8 quarters. This represents a 67% reduction in pathogens.

[0242] Example 5.12 In a field study conducted in Upper Bavaria on asymptomatic mastitis, changes in cell count development were presented for a total of 14 cows with mastitis before and after administration of tablets containing 3.84 g of PTSO and 0.96 g of PTS. Furthermore, the presence and number of pathogens were examined at various time intervals after treatment in selected udder quarters.

[0243] research design Cell count is an important indicator of udder health and was measured to study the effect of tablets containing 3.84 g PTSO and 0.96 g PTS on udder health in 14 animals from 5 farms. Cows were selected if the cell count exceeded the cell / ml limit in the last MPR or in the second to last MPR. Milk from each cow in the study group was sampled before administering the tablets. After treatment, cows were sampled at appropriate time intervals, and the samples were analyzed for somatic cell count and infected quarters in the udder. Furthermore, pathogenic specimens of microorganisms were identified, and their numbers were determined after appropriate time intervals.

[0244] Results and Discussion Table 20 presents the results of measurements at various time intervals after treatment, with respect to somatic cell count and detected pathogens. Before treatment, the cell count was over 250,000 / ml, strongly indicating the presence of infection, and the count was increased in most cattle. Treatment was performed between MPR-0 and MRP+1. After treatment, a decrease in pathogens was observed from infection at t0, from 10 quarters where the pathogen was detected on day 1, to 7 quarters on days 14 / 15. Pathogens were still observed (a decrease of 3 quarters, equivalent to 30%), and the original pathogen was still detected in 3 quarters on days 42 / 43 / 44, which corresponds to a decrease of 7 quarters (70%) from the initially infected quarter. However, 7 cattle were infected with other pathogenic bacteria during the experimental time interval, and these cattle had to be retreated (results are not shown). In all cattle, the somatic cell count decreased on day 7 after treatment. Since SCCs are a generally accepted measure of inflammation levels, this treatment showed a clear inhibitory effect on inflammation. Interestingly, the number of SCCs was already significantly reduced by day 7 after treatment, and it was demonstrated that the cell count decreased by an average of 45% (from 685,000 to 378,000 cells / ml) between 36 and 55 days.

[0245] However, in most cases, the pathogen remained detectable. Moreover, while clumps of biofilm were detached from the mammary gland at a high rate from day two onward, they were completely removed. This strongly indicated that the biofilm was the cause of inflammation, not the floating cells still present in the mammary gland. The number of floating cells decreased over time and eventually became undetectable, a decrease that can only be caused by the action of the immune system. This demonstrates that the immune system plays a role in controlling the infection, as it is sufficient to remove the biofilm and control inflammation without killing cells.

[0246] [Table 20]

[0247] Furthermore, the detection or persistence of pathogens was assessed in each quarter. Twenty quarters with elevated cell counts from 19 cows across six farms were examined (milk samples, culture approach). On day 1, various pathogens (Streptococcus uberis, Staphylococcus aureus, Escherichia coli, etc.) were detected in 12 quarters, and no pathogens were detected in 8 quarters. Of the 12 quarters in which pathogens were detected on day 1, only 4 quarters still had pathogens detected on day 42, and none were detected in 8 quarters. This represents a 67% reduction in pathogens.

[0248] This experiment reveals that PTSO / PTS is absorbed by the intestines and then transported to the mammary glands. Subsequently, PTSO / PTS enters the biofilm (which can be considered a kind of "fortress" where bacteria hide) at a concentration high enough to cause biofilm release. Although the exact mechanism by which the dissolution and detachment process of the biofilm occurs is unknown, it is surprising that the compound can produce such an effect.

[0249] Example 6. Prototheca A highly fatal variant of mastitis is caused by the biofilm-forming microalgae of the genus Prototheca (Concalves et al., 2015, Dairy Sci 98 (6):3613~3621. Valessa et al, Cienc. Rural vol.49 no.2 Santa Maria Feb 28, 2019). Mastitis caused by Prototheca zopfii has been reported in various countries and is increasing worldwide, and is a serious problem due to the inherent resistance of these microalgae to routine treatment (Pieper et al., 2012, J. Dairy Sci. 95:5635~5644). This resistance is associated with the ability to infect and survive macrophages and invade mammary tissue, leading to persistent infections due to the regular occurrence of Prototheca zophi in milk (Marques et al., 2006, J. Dairy Sci. 89:4202~4204).

[0250] Prototheca infections occur between infected and healthy cows during milking, during routine and intensive antibiotic treatment (Pieper et al., 2012), and during poor sanitary conditions in the preparation of cows before milking. Furthermore, Prototheca zophi survives in feces and contaminates all environments. In dairy farming environments, surfaces such as stainless steel, glass, rubber, and polypropylene become contaminated by the microorganisms. Subsequently, the microorganisms can proliferate on these surfaces and form biofilms (Davies, 2003. Nat. Rev. Drug Discov. 2:114~122).

[0251] Antibiotic treatment for mastitis caused by Prototheca species only provides temporary improvement of the infection in vivo, without removing the causative agent (Costa et al., 1996. Mycopathologia 133:85~88). Therefore, culling cattle infected with Prototheca zophi is one of the recommended measures, if not the only control measure, to reduce the disease.

[0252] Scope: In a field study of (sub)clinical mastitis conducted in Upper Bavaria, changes in cell count development in a total of 14 cows with mastitis were reported before and after administration of tablets containing 4.8 g PTSO and 1.2 g PTS. In addition, the presence and number of Prototheca were examined at various time intervals after treatment in selected udder quarters.

[0253] research design Cell count is an important indicator of udder health and was measured to study the effects of PTSO / PTS tablet products on udder health in 44 animals. Cattle were selected if the cell count in the final MPR in milk exceeded the limit of 250,000 cells / ml, and the microalga Prototheca was shown in the milk. Subsequently, PTSO / PTS tablets were administered. After treatment, samples were taken from the cattle once a month, and the cell count and the presence of the microalga Prototheca in the udder were analyzed at the end of the experiment.

[0254] Results and Discussion Table 21 below shows the measurement results regarding the number of somatic cells during the time period and the presence of prototheca at the end of the experiment.

[0255] [Table 21] JPEG0007835682000044.jpg80170

[0256] Before treatment, the cell count was over 250,000 cells / ml, strongly indicating the presence of infection and an increase in the number of cells in most cattle. In all cattle, the somatic cell count decreased on day 7 after treatment. Since SCC is a measure of the level of inflammation resulting from infection, the treatment showed a clear inhibitory effect on inflammation. Interestingly, the number of SCCs had already decreased significantly on day 7 after treatment, and it was demonstrated that the cell count decreased by an average of 45% (from 685,000 to 378,000 cells / ml) after 36-55 days.

[0257] Furthermore, the detection or persistence of pathogens was assessed in each quarter. Twenty quarters with elevated cell counts from 19 cows across six farms were examined (milk samples, culture approach). On day 1, various pathogens (Streptococcus uberis, Staphylococcus aureus, Escherichia coli, etc.) were detected in 12 quarters, and no pathogens were detected in 8 quarters. Of the 12 quarters in which pathogens were detected on day 1, only 4 quarters still had pathogens detected on day 42, and none were detected in 8 quarters. This represents a 67% reduction in pathogens.

[0258] Mammary gland infections by the microalga Prototheca generally result in the culling of cattle because the algae are insensitive to most antibiotics. Unexpectedly, PTSO / PTS demonstrated complete resolution of the infection, and culling was not necessary.

[0259] Example 7. Dermatitis of the toes Another category of chronic infections is wounds infected by biofilm-forming microorganisms. Once a wound becomes infected, the microorganisms begin to form a biofilm that adheres to the wound. The production of extracellular polymers (EPS) by the microorganisms helps the biofilm form a complex three-dimensional structure within a few hours. These complex structures are resistant to the wound's defense mechanisms. When antibiotics are administered to attack the bacteria, they only partially eradicate the biofilm, leaving the wound and the tissue beneath it infected. These biofilms are known to lead to chronic infections and unhealed wounds. In the United States, approximately 16 million new biofilm-based infections are diagnosed each year. Therefore, biofilms constitute a major impediment to wound healing. Examples of wounds infected with pathogenic microorganisms include bacteria, such as Gram-positive bacteria, e.g., Staphylococcus aureus; Streptococcus; Gram-negative bacteria, e.g., Treponema species, Escherichia coli, Yersiania pestis, Pseudomonas aeruginosa; and yeasts / fungi, such as Candida species (albicans), Cladosporidium herbarum, Trichosporum, Rhodosporidium, and Malassezia.

[0260] One example of a wound infection that negatively impacts cow's milk production is digitoritis (Schlafer et al, 2008 Veterinary Microbiology 128, Issues 1~2:118~125). Diaphragmitis (synonyms include hairy heel warts, strawberry foot rot, mortellaro disease, Italian foot rot, and papillomatous digital dermatitis) is an infection that causes lameness in cattle. The lesions of digitoritis are ulcerative or proliferative masses between the bulbs (Beninger, 2018 et al, Vet Res 49:111). The cost of digitorrhea is $75 per cow per year on a farm with 65 cows (Bruijnis et al., 2010, J. of Dairy Science Volume 93, Issue 6, 2419-2432). The cost is based on premature culling, milk loss, and reduced fertility.

[0261] Finger dermatitis is caused by aerobic or anaerobic bacteria (Beninger, 2018 et al, Vet Res 49:111), particularly the genus Treponema (Demirkan et al. 2018 J. of Dairy Science vol 101 (11) p.10317~10326). Generally, infection is initiated by Treponema bacteria invading the skin around the nails. Treponema are anaerobic, Gram-negative bacteria belonging to the spirochete genus. These microorganisms can invade the skin near the nails of cattle, especially between the nails in interdigital clefts. The biofilms that develop from these dermatitis lesions constitute a heterogeneous microbial community.

[0262] Bacteria such as Treponema are present in the feces of cattle or ruminants and are thought to be a cause of toe dermatitis. Cattle with a properly functioning immune system are less susceptible to toe dermatitis. Housing, hygiene, ventilation, and nutrition are also important.

[0263] Formalin foot baths are effective in preventing and are often used against toe dermatitis. However, formalin is known to be harmful to the health of cattle and dairy farmers. If toe dermatitis is present in the nails, the nails should be completely dried and washed after trimming, and disinfected with a foot bath every two weeks. Furthermore, the infected skin is usually then treated with an antibiotic spray, and positive results can be achieved in this manner. However, often a thin layer or patch of biofilm forms within the wound where microbial cells reside in a dormant state. Only a portion of the bacterial population is killed by the (antibiotic) treatment, while the remaining cells begin to proliferate after the treatment is completed. Moreover, the infection usually penetrates deep into the dermis where local biofilms are formed, and large molecules of antibiotics have little effect. Furthermore, selective pressure is applied to other forms of antibiotic resistance other than cellular dormancy in the tissue and biological layers. For these reasons, most treatments and strategies have little to no effect on improving digitoritis.

[0264] research design The experiment was conducted at three individual farms to examine the effectiveness of two sprays (Spray #1 and Spray #2-PTSO) developed for treating wounds and injuries. Both sprays had the same composition, except that Spray #2-PTSO also contained PTSO (3.2% PTSO w / w). 0.35 ml was sprayed onto the wound for each treatment.

[0265] Cattle from 13 farms were selected based on the presence of digitoritis. These cattle with digitoritis were selected, and their gait and M score were scored. Six classes were described to classify digitoritis infections (Dopfer et al, 1997) (Berry et al., 2012). The M score is described as follows: M0, healthy; M1 (initial stage), small redness and sensitive but active inflammation, minimal injury 0-2 cm; M2, very active inflammation greater than 2 cm, strawberry red and very sensitive injury, white edges of epithelium and erect hairs; M3 (healing stage), ulcerative lesion covered with scabs; M4, change to chronic ulcer; M4.1, a combination of M4 and M1, chronic stage with swollen cleft nails. The locomotion scores are defined as follows: 1. Walks with an upright back and no limp; 2. Walks with a slight limp and a slight bend at the waist; 3. Severely limps and bows when standing and walking; 4. Severely limps and stumbles backward when standing or walking; 5. Severely limps and bows when standing, no longer stands on its own two feet, and prefers to lie down.

[0266] When an M2 lesion is treated, healing occurs as follows: The intermediate stage (M3) progresses to the healed stage (M0), or the intermediate stage (M3) changes to a chronic stage with a swollen internail fissure (M4.1). After effective treatment, M4.1 heals via M4 and M3. See, for example, Figure 4.

[0267] After scoring the wounds, the selected cattle were randomly assigned to five groups as follows: Group 1: No treatment (control); Group 2: Treatment with spray #1; Group 3: Treatment with spray #2-PTSO; Group 4: Treatment with spray #1 and administration of tablets containing 4.8g PTSO and 1.2g PTS; Group 5: Treatment with spray #2-PTSO, administration of tablets containing 4.8g PTSO and 1.2g PTS, and treatment with either spray #1 or spray #2-PTSO. The claws were scored with an M score and exercise after two weeks. Where possible, each claw was lifted again and photographed again.

[0268] Data processing The scores of the cattle were compared between Group 1 and Group 3, and between Group 2 and Group 3. Fisher's Exact Test was applied to calculate statistical significance. M1, M2, M4, and M4.1 were considered "not cured." M3 is shown as "healing," and M0 as "cured." In this study, "healing" and "cured" were grouped together.

[0269] result The M scores for each treatment group are shown in Table 22 below. Most treated cows with an M4 score had an exercise score of 1 or 2 before being treated with any of the sprays. All of these cows were scored 1 for exercise after treatment, and the skin was treated with the supplier. Cows in group 5 were scored 3 for exercise, but were scored 2 for exercise immediately after treatment, and thus rapid improvement was observed. The outcome in digitoritis was determined by the M score. All cows with active (painful) lesions of M1, M2, and M4.1 were moved to M3.

[0270] Cattle treated with both tablets and spray #1 containing 4.8g PTSO and 1.2g PTS showed that 93% of M4 scores transitioned to M3 and the remainder to M0 after treatment. When spray #2-PTSO was used, M2 scores at t=0 transitioned to M3. Cattle with an M4 score transitioned to M0 (23%), M3 (56%), or remained at M4 (21%) after treatment. Significant improvement was observed with the use of spray #2-PTSO, while additional treatment with PTSO / PTS tablets showed an additional effect.

[0271] The control group showed higher M scores because they were untreated and their condition worsened as expected. Most of the cattle remained in the chronic group (M4).

[0272] [Table 22]

[0273] Group 1: Control; Group 2: Spray #1; Group 3: Spray #2-PTSO; Group 4: Spray #1 in addition to PTSO / PTS tablets; Group 5: Spray #2-PTSO in addition to PTSO / PTS tablets. A indicates the number of cattle with a specific M score before treatment, and B indicates the number of cattle with a specific score after treatment.

[0274] Statistical analysis was performed, and Fisher's exact test showed that the p-value was 0.037 when comparing group 2 and group 3 (i.e., spray with PTSO versus spray without PTSO), and the p-value was 0.010 when comparing group 1 and group 3 (i.e., control versus spray with PTSO).

[0275] Nail health improved with both sprays, but the statistical p-value demonstrates that the spray containing PTSO showed a more improved effect than a similar spray without PTSO (p=0.032). Cattle treated with spray #1 showed better results when the treatment was combined with PTSO / PTS tablets. This embodiment demonstrates that topical administration of PTSO is useful for the treatment of biofilm-related chronic wounds such as digitoritis.

[0276] In cattle treated with spray #2-PTSO, a greater impact was observed in infection stages M1 and M2 than in chronic lesions (M4, M4.1). Further improvement is highly likely to be achieved by extending this treatment. One cow with an exercise score of 4 received the treatment; before treatment, it walked on three legs, and the remaining hooves were unusable due to severe dermatitis. After treatment, the cow walked away on four hooves, its exercise score was 2, clearly demonstrating the analgesic effect of the treatment.

[0277] Example 8. Mastitis Another example of a biofilm-related chronic wound is udder cleft dermatitis (UCD) (Sorge et al, 2019. J. Dairy Sci. 102:11470~11475; Waller et al, 2014. J. Dairy Sci. 97:310-318). UCD is a skin lesion located at the anterior junction between the mammary gland and the abdominal wall, or between the anterior quarter of the mammary gland. The lesion varies in appearance and size, with thickened skin, crusts, pus, and easily bleeding sores being common findings. Udder cleft dermatitis can be difficult to detect due to its anatomical location and the fact that affected cows rarely show common signs of the disease. Few studies have been published on the prevalence of UCD, and most studies include only one or a few herds that are primarily classified as problem herds. The intragroup prevalence in these studies varied from 0 to 22%. However, a recent Dutch study included a group of 20 cows, three of which did not have UCD, while the intragroup infection rate in the other groups varied from 2.5 to 13% (Amersfort et al., 2012). The etiology of UCD is unknown, but several factors, such as mammary gland structure and mammary edema, have been suggested to play a role. Bovine factors, such as the number of births and DIM (days in milk), have also been associated with UCD (Beattie and Taylor, 2000, J. Brit. Cattle Vet. Assoc. 8, 377-380).

[0278] The lesions are most commonly identified on the plantar lateral aspect of the interdigital clefts of the hind limbs. Treponema species are routinely present in numerous active lesions. The lesions are painful to the touch and can result in clinical lameness. Its infectivity generally leads to endemic infections of cattle herds (Plummer et al 2017, Vet Clin North Am Food Anim Pract 33(2):165-181), causing significant economic losses.

[0279] Research design: Two cows exhibiting clear characteristics of chronic UCD were selected and treated with the spray described in Example 7. Cow 1 (3076) was treated with spray #2-PTSO on day 0 and day 7. Cow 2 (2934) was treated with spray #1 at t=0 and with spray #2-PTSO on day 7. Tables 23 and 24 below show the results of the treatment.

[0280] [Table 23]

[0281] [Table 24]

[0282] Cattle 1 was treated with spray #2-PTSO on day 0 and day 7, and a reduction in inflammation was immediately observed. When cattle 2934 was treated with spray #1 on day 0, no progression was observed. After treatment with spray #2 on day 7, a clear improvement in infection reduction and subsequent wound healing was observed. These experiments were repeated in hundreds of cattle, and improvement and healing of chronic, slimy wounds of UCD were observed with spray #2-PTSO. Furthermore, the present invention may be configured as follows. [Section 1] A compound according to the following formula I, or a composition comprising a compound according to the said formula I, for use in the treatment of biofilm-related disorders, wherein the composition substantially does not contain diallylthiosulfinate. [ka] Here, R1 and R2 are independently selected from optionally substituted linear or branched alkyl groups, optionally substituted linear or branched alkenyl groups, optionally substituted linear or branched alkynyl groups, optionally substituted aryl groups, optionally substituted cycloalkyl groups, and optionally substituted heterocycloalkyl groups. [Section 2] The compound or composition for use as described in item 1, wherein the compound is propylpropanethiosulfonate (PTSO). [Section 3] For use as described in item 1, the compound or the composition, wherein the composition further comprises a compound according to the following formula II, preferably the compound according to formula II is propylpropanechosulfinate (PTS). [ka] Here, R3 and R4 are independently selected from optionally substituted linear or branched alkyl, optionally substituted linear or branched alkenyl, optionally substituted linear or branched alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl, provided that formula II is not as shown below. [ka] [Section 4] A compound according to the following formula II, preferably the compound being propylpropanethosulfinate (PTS), or a composition comprising a compound according to the formula II, wherein the composition substantially does not contain diallylthiosulfinate, for use in the treatment of biofilm-related disorders.

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Claims

1. An agent for use in the treatment of biofilm infections, comprising propylpropanethiosulfonate (PTSO).

2. The agent according to claim 1, further comprising propylpropanechosulfinate (PTS).

3. An agent for use in the treatment of biofilm infections, comprising propylpropanechosulfinate (PTS).

4. The agent according to any one of claims 1 to 3, wherein the treatment further comprises the administration of an antibacterial agent.

5. The agent according to any one of claims 1 to 4, wherein the treatment further comprises the administration of an anti-inflammatory agent.

6. The agent according to any one of claims 1 to 5, wherein the treatment is for the purpose of reducing the formation or growth of a biofilm and / or for the decomposition or reduction of a biofilm.

7. The agent according to any one of claims 1 to 6, wherein the biofilm infection is a chronic and / or persistent infection.

8. The agent according to any one of claims 1 to 7, wherein the biofilm infection is a respiratory infection, intestinal infection, breast infection, udder infection in animals, skin infection, bladder infection, ear infection, systemic infection, joint infection, cerebral infection, bacterial prostatitis, bacterial vaginosis, biliary tract infection, chronic sinusitis, chronic lung disease, dental caries, endocarditis, kidney stones, laryngitis, lung infection in cystic fibrosis, gingivitis, mastitis, middle ear infection, hospital-acquired (bloodstream) infection, obstructive pulmonary disease, osteomyelitis, otitis media, periodontitis, pneumonia, prostatitis, rhinosinusitis, sinusitis, tonsillitis, tuberculosis, urinary tract infection, and / or wound infection.

9. The agent according to any one of claims 1 to 7, wherein the biofilm infection is digitoritis or chronic wound infection.

10. The agent according to any one of claims 1 to 9, wherein the biofilm infection is a mammary gland infection.

11. The agent according to any one of claims 1 to 10, wherein the biofilm infection is mastitis.

12. The agent according to any one of claims 1 to 11, wherein the treatment is for mammals.

13. The agent according to any one of claims 1 to 12, wherein the treatment is for ruminants.

14. The agent according to any one of claims 1 to 13, wherein the biofilm comprises bacteria, yeast, fungi, microalgae, or a combination thereof.

15. A method for treating a biofilm infection in a non-human animal, the method comprising administering a composition comprising propylpropanethiosulfonate (PTSO) to a non-human animal in need thereof.

16. An in vitro method for preventing or reducing the formation or growth of a biofilm on a surface, or for degrading or reducing a biofilm on a surface, wherein the method comprises applying a composition to the surface to prevent or reduce the formation or growth of a biofilm on the surface, or degrading or reducing a biofilm on the surface, the composition comprising propylpropanethiosulfonate (PTSO).

17. The method according to claim 15, wherein the composition further comprises an antimicrobial agent.

18. The method according to claim 15, wherein the composition further comprises an anti-inflammatory agent.

19. The method according to claim 15, wherein the composition further comprises propylpropanechosulfinate (PTS).

20. The method according to any one of claims 15 and 17 to 19, wherein the biofilm infection is a chronic and / or persistent infection.

21. The method according to any one of claims 15 and 17 to 20, wherein the biofilm infection is a respiratory infection, intestinal infection, breast infection, udder infection in animals, skin infection, bladder infection, ear infection, systemic infection, joint infection, cerebral infection, bacterial prostatitis, bacterial vaginosis, biliary tract infection, chronic sinusitis, chronic lung disease, dental caries, endocarditis, kidney stones, laryngitis, lung infection in cystic fibrosis, gingivitis, mastitis, middle ear infection, hospital-acquired (bloodstream) infection, obstructive pulmonary disease, osteomyelitis, otitis media, periodontitis, pneumonia, prostatitis, rhinosinusitis, sinusitis, tonsillitis, tuberculosis, urinary tract infection, and / or wound infection.

22. The method according to any one of claims 15 and 17 to 20, wherein the biofilm infection is digitoritis or chronic wound infection.

23. The method according to any one of claims 15 and 17 to 21, wherein the biofilm infection is a mammary gland infection.

24. The method according to any one of claims 15, 17-21, and 23, wherein the biofilm infection is mastitis.

25. The method according to any one of claims 15 and 17 to 24, wherein the treatment is for ruminants.

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