Recombinant cell wall hydrolase

Abstract

This disclosure relates to novel chimeric cell wall hydrolases having anti-Staphylococcus activity. This disclosure also relates to compositions comprising these chimeric cell wall hydrolases and their use in the treatment of conditions associated with species of the genus Staphylococcus.

Description

[Technical Field]

[0001] Cross-references to related applications

[0001] This application claims the interests and priority thereto of U.S. Provisional Patent Application No. 63 / 639,416 filed April 26, 2024, and U.S. Provisional Patent Application No. 63 / 509,160 filed June 20, 2023, the respective contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation of sequence lists

[0002] The contents of the text files submitted electronically with this Specification are incorporated herein by reference in their entirety: a computer-readable copy of the sequence listing (file name: TOPB_002_02WO_SeqList_ST26.xml; size: 42,000 bytes; and creation date: June 17, 2024).

[0003]

[0003] This disclosure relates to novel recombinant cell wall hydrolases. This disclosure also relates to compositions comprising these cell wall hydrolases. [Background technology]

[0004]

[0004] Staphylococcus is a genus of Gram-positive bacteria in the family Staphylococcaceae from the order Bacillales. This genus includes at least 43 bacterial species, many of which are commensal to the skin and mucous membranes of humans and other animals without causing disease. However, some bacterial species of the genus Staphylococcus, including Staphylococcus aureus, are pathogenic and can cause infections that are particularly dangerous to immunocompromised subjects. Staphylococcus aureus is a major cause of sepsis. Overgrowth of Staphylococcus aureus in the skin microbiome is strongly associated with moderate to severe cases of atopic dermatitis and acute radiation dermatitis.

[0005]

[0005] Antibiotics can be used to treat bacterial infections, but many Staphylococcus strains are antibiotic resistant, which makes them very difficult to control. In addition, antibiotics or chemicals such as benzoyl peroxide can have a negative impact on symbiotic bacterial communities as well as pathogenic bacteria. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006]

[0006] There is an ongoing unmet need for novel therapeutic agents that are active against species of the genus Staphylococcus. [Means for solving the problem]

[0007]

[0007] This disclosure teaches a chimeric cell wall hydrolase (CWH) having desirable properties. In some embodiments, the CWH of this disclosure can selectively treat Staphylococcus aureus infections.

[0008]

[0008] In one embodiment, the present disclosure provides a chimeric cell wall hydrolase (CWH) comprising a) an enzymatically active domain (EAD) having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with sequence number 11; and b) a cell wall-binding domain (CBD) having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the sequence of sequence number 16; ii) sequence number 17; or iii) sequence number 18.

[0009]

[0009] In one embodiment, the present disclosure provides a chimeric cell wall hydrolase (CWH) comprising a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with SEQ ID NO: 22.

[0010]

[0010] In one aspect, the present disclosure provides a chimeric cell wall hydrolase (CWH) comprising a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with SEQ ID NO: 24.

[0011]

[0011] In one aspect, the present disclosure provides a topical formulation comprising the chimeric CWH of the present disclosure.

[0012]

[0012] In one aspect, the present disclosure provides a topical formulation comprising a chimeric cell wall hydrolase (CWH) comprising a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with SEQ ID NO: 21.

[0013]

[0013] In one aspect, the present disclosure provides a method of treating a condition associated with the genus Staphylococcus, comprising the step of administering a composition comprising the chimeric CWH of the present disclosure.

[0014]

[0014] The accompanying drawings, which are incorporated herein and form a part of this specification, illustrate some, but not the only or exclusive, exemplary embodiments and / or features. It is intended that the embodiments and drawings disclosed herein be considered illustrative rather than limiting.

Brief Description of the Drawings

[0015] [Figure 1]

[0015] It is a diagram showing the minimum inhibitory concentration (MIC) assay results against Staphylococcus aureus and Staphylococcus epidermidis for a chimeric cell wall hydrolase containing a LysSA12 CHAP domain fused to six different CBDs. The boxes indicate growth inhibition. [Figure 2]

[0016] Figure showing the minimum inhibitory concentration (MIC) assay results against Staphylococcus aureus and Staphylococcus epidermidis for a chimeric cell wall hydrolase comprising a Twort, LysCSA13, or LysCSA5 CHAP domain fused to a LysA72, PlySs2, or ALE-1 SH3 domain. Boxes indicate growth inhibition. [Figure 3]

[0017] Figure including two tables summarizing the MIC and selectivity results of FIGS. 1 and 2. [Figure 4A]

[0018] Figure showing the turbidity reduction assay results for exemplary chimeric cell wall hydrolases of the present disclosure compared to the Twort EAD+PlySs2 CBD chimera. Results are shown for the following chimeras: LysSA12 EAD+LysA72 CBD (FIG. 4A). [Figure 4B] Figure showing the turbidity reduction assay results for exemplary chimeric cell wall hydrolases of the present disclosure compared to the Twort EAD+PlySs2 CBD chimera. Results are shown for the following chimeras: LysSA12 EAD+PlySs2 CBD (FIG. 4B). [Figure 4C] Figure showing the turbidity reduction assay results for exemplary chimeric cell wall hydrolases of the present disclosure compared to the Twort EAD+PlySs2 CBD chimera. Results are shown for the following chimeras: LysSA12 EAD+ALE-1 CBD (FIG. 4C). [Figure 4D] Figure showing the turbidity reduction assay results for exemplary chimeric cell wall hydrolases of the present disclosure compared to the Twort EAD+PlySs2 CBD chimera. Results are shown for the following chimeras: Twort EAD+PlySs2 CBD (FIG. 4D). [Figure 5A]

[0019] Figure showing the thermal stability assay results for chimeric cell wall hydrolases of the present disclosure compared to the Twort EAD+LysA72 CBD chimera. Results are shown for the following chimeras: LysSA12 EAD+LysA7,2 CBD (FIG. 5A). [Figure 5B]This figure shows the results of a thermal stability assay for the chimeric cell wall hydrolase of the present disclosure compared to the Twort EAD+LysA72 CBD chimera. The results are shown for the following chimera: LysSA12 EAD+PlySs2 CBD (Figure 5B). [Figure 5C] This figure shows the results of a thermal stability assay for the chimeric cell wall hydrolase of the present disclosure compared to the Twort EAD+LysA72 CBD chimera. The results are shown for the following chimera: LysSA12 EAD+ALE-1 CBD (Figure 5C). [Figure 5D] This figure shows the results of a thermal stability assay for the chimeric cell wall hydrolase of the present disclosure compared to the Twort EAD+LysA72 CBD chimera. The results are shown for the following chimera: Twort EAD+LysA72 CBD (Figure 5D). [Figure 6]

[0020] This figure shows the results of LysSA12 EAD+PlySs2 CBD chimeras in turbidity reduction assays for various Staphylococcus species. [Figure 7A]

[0021] This figure shows the results of turbidity reduction assays for SA.100 and LysSA12 EAD+PlySs2 CBD chimeras against Staphylococcus aureus. Figure 7A shows the turbidity reduction assay results, while Figure 7B shows the relative activity calculated from the turbidity reduction assay. [Figure 7B] This figure shows the results of turbidity reduction assays for SA.100 and LysSA12 EAD+PlySs2 CBD chimeras against Staphylococcus aureus. Figure 7A shows the turbidity reduction assay results, while Figure 7B shows the relative activity calculated from the turbidity reduction assay. [Figure 8]

[0022] This figure shows the results of quantitative killing assays for SA.100 and LysSA12 EAD+PlySs2 CBD chimeras against both Staphylococcus aureus and Staphylococcus epidermidis. [Figure 9]

[0023] This figure shows the results of pH assays for SA.100 and LysSA12 EAD+PlySs2 CBD chimeras across a pH range of pH 7.4 to pH 4.5. [Figure 10]

[0024] This figure shows the results of an assay testing the effects of LysSA12 EAD+PlySs2 CBD chimera compared to SA.100 or PBS alone in a 3D skin model of the skin microbiome. Error bars represent the standard error of the mean of three biological replicates. [Figure 11A]

[0025] This figure shows the activity of LysSA12 EAD+PlySs2 CBD chimeras against Staphylococcus aureus and Staphylococcus epidermidis in several topical formulations: formulation #1 containing hyaluronic acid (Figure 11A). The control formulation is identical to the test formulation but does not contain the LysSA12 EAD+PlySs2 CBD protein. [Figure 11B] This figure shows the activity of LysSA12 EAD+PlySs2 CBD chimeras against Staphylococcus aureus and Staphylococcus epidermidis in several topical formulations: Formulation #2 containing hydroxymethylcellulose (Figure 11B). The control formulation is identical to the test formulation but does not contain the LysSA12 EAD+PlySs2 CBD protein. [Figure 11C] This figure shows the activity of LysSA12 EAD+PlySs2 CBD chimeras against Staphylococcus aureus and Staphylococcus epidermidis in several topical formulations: Formulation #3 (Figure 11C) is a cream-based formulation. The control formulation is identical to the test formulation but does not contain the LysSA12 EAD+PlySs2 CBD protein. [Figure 12]

[0026] This figure shows the activity of the LysSA12 EAD+PlySs2 CBD chimeric topical formulation compared to the control formulation at 1, 2, and 4 weeks. The control formulation is identical to the test formulation but does not contain the LysSA12 EAD+PlySs2 CBD protein. [Figure 13A]

[0027] This figure shows the results of applying the LysSA12 EAD + PlySs2 CBD chimera to dry, red, and itchy skin regarding pruritus (Figure 13A). The figure shows the group mean with standard deviation; individual data points are also plotted. "ns" indicates p > 0.05; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. [Figure 13B] This figure shows the results of applying the LysSA12 EAD+PlySs2 CBD chimera to dry, red, and itchy skin, regarding the number of days of skin itching (Figure 13B). The figure shows the group mean with standard deviation; individual data points are also plotted. "ns" indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001. [Figure 13C] This figure shows the results of applying a LysSA12 EAD + PlySs2 CBD chimera to skin with dryness, redness, and itchiness related to insomnia (Figure 13C). The figure shows the group mean with standard deviation; individual data points are also plotted. "ns" indicates p > 0.05; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. [Figure 13D] This figure shows the results of applying the LysSA12 EAD+PlySs2 CBD chimera to dry, red, and itchy skin regarding the frequency of sleep disturbance due to skin (Figure 13D). The figure shows the group mean with standard deviation; individual data points are also plotted. "ns" indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001. [Figure 13E]This figure shows the results of applying the LysSA12 EAD + PlySs2 CBD chimera to skin with dryness, redness, and itchiness, regarding skin redness (Figure 13E). The figure shows the group mean with standard deviation; individual data points are also plotted. "ns" indicates p > 0.05; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. [Figure 13F] This figure shows the results of applying the LysSA12 EAD+PlySs2 CBD chimera to skin exhibiting dryness, redness, and itchiness, regarding skin dryness (Figure 13F). The figure shows the group mean with standard deviation; individual data points are also plotted. "ns" indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001. [Figure 13G] This figure shows the results of applying the LysSA12 EAD+PlySs2 CBD chimera to skin exhibiting dryness, redness, and itchiness, with regard to skin peeling (Figure 13G). The figure shows the group mean with standard deviation; individual data points are also plotted. "ns" indicates p>0.05; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001. [Figure 13H] This figure shows the results of applying the LysSA12 EAD + PlySs2 CBD chimera to skin exhibiting dryness, redness, and itchiness, related to skin irritation (Figure 13H). The figure shows the group mean with standard deviation; individual data points are also plotted. "ns" indicates p > 0.05; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. [Figure 13I]This figure shows the results of applying a LysSA12 EAD + PlySs2 CBD chimera to dry, red, and itchy skin, regarding pain caused by dry, red, and itchy skin (Figure 13I). The figure shows the group mean with standard deviation; individual data points are also plotted. "ns" indicates p > 0.05; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. [Figure 14A]

[0028] This figure shows representative facial images (Figure 14A) before and 14 days after treatment using the LysSA12 EAD+PlySs2 CBD chimeric composition. [Figure 14B] This figure shows representative palm images (Figure 14B) before and 14 days after treatment using the LysSA12 EAD+PlySs2 CBD chimeric composition. [Figure 14C] This figure shows representative elbow images (Figure 14C) before and 14 days after treatment using the LysSA12 EAD+PlySs2 CBD chimeric composition. [Modes for carrying out the invention]

[0016]

[0029] All publications, patents, and patent applications, including any drawings and annexes, are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

[0017]

[0030] The following statements contain information that may be useful in understanding this disclosure. This does not constitute an endorsement that any information provided herein is prior art, or that any publication specifically or implicitly referenced is prior art.

[0018] definition

[0031] The terms "a" or "an" mean one or more of the whole, that is, they can refer to multiple objects. Therefore, the terms "a," "an," "one or more," and "at least one" are interchangeable within this specification. In addition, a reference to "an element" by the indefinite article "a" or "an" does not exclude the presence of two or more elements unless the context explicitly requires that there be only one element.

[0019]

[0032] Throughout this application, the term “about” is used to indicate that a value includes intrinsic error variability in the device or the method used to determine the value, or variability present between the samples being measured. Unless otherwise specified or it is evident from the context, the term “about” means within 10 percent above or below the reported numerical value (except where such a number exceeds 100% of the possible value or falls below 0%). When used in conjunction with a range or sequence of values, unless otherwise indicated, the term “about” applies to each of the values ​​listed at the end of the range or within the sequence. As used herein, the terms “about” and “approximately” are used interchangeably.

[0020]

[0033] As used herein, the term “cell wall hydrolase” or “CWH” means a bacterial cell wall hydrolase, which is an enzyme that degrades peptidoglycan in the bacterial cell wall by cleaving bonds in the peptidoglycan chain and side chain branching. These terms also encompass any recombinant enzyme of the present disclosure that has cell wall hydrolase activity. Cell wall hydrolases can have a variety of domain structures. A CWH includes an “enzymatically active domain” or “EAD,” which is a domain responsible for the degradation of peptidoglycan. In some embodiments, the EAD has glycosidase, amidase, and / or peptidase enzymatic activity. In some embodiments, a CWH includes a “cell wall-binding domain” or “CBD,” which is a domain that binds to the bacterial cell wall.

[0021]

[0034] The term "natural" protein is used to indicate a protein that exists naturally and has not been artificially modified or recombinant.

[0022]

[0035] The term “recombinant” is used herein to describe nucleic acids, proteins, vectors, and host cells in configurations that are not naturally occurring or, in the context of nucleic acids, not naturally found. Thus, “recombinant protein” means a protein that is not naturally occurring. In some embodiments, as used herein, recombinant protein means a chimeric protein. In some embodiments, recombinant protein means the expression product of either an EAD or CBD sequence disclosed herein, either alone or within a protein that is not naturally occurring. For example, this disclosure assumes a recombinant EAD or CBD sequence of this disclosure fused to any protein tag, such as 6×His.

[0023]

[0036] As used herein, “heterogeneous” means any genetic material that is artificially introduced into a non-natural context. For example, a heterogeneous domain means a domain such as EAD or CBD that is artificially introduced into a recombinant protein sequence, where the resulting recombinant protein sequence is non-natural.

[0024]

[0037] As used herein, “chimeric protein” refers to any recombinant protein containing two or more heterogeneous domains, for example, EAD and / or CBD.

[0025]

[0038] As used herein, a protein “domain” is a functional and / or structural subunit within a protein. In some embodiments, they are responsible for specific functions or interactions that contribute to the overall role of the protein. Protein domains are fundamental units of protein structure, folding, function, evolution, and design. See, for example, Wang et al., “Protein domain identification methods and online resources,” Comput Struct Biotechnol J 2021;19:1145–1153, incorporated herein by reference.

[0026]

[0039] As used herein, “chimeric cell wall hydrolase” or “chimeric CWH” refers to a chimeric protein that functions as a cell wall hydrolase and contains at least one heterologous domain, e.g., heterologous EAD or CBD, compared to an innate CWH sequence. Chimeric CWH also refers herein to a recombinant protein containing two heterologous CWH domains, e.g., EAD and CBD.

[0027]

[0040] As used herein, “activity” means the ability of a protein (e.g., a chimeric CWH protein) to inhibit the growth of cells from Staphylococcus species and / or lyse cells. The term “active against” (e.g., a chimeric CWH protein) as used herein with reference to a target Staphylococcus species means a protein (e.g., a chimeric CWH protein of this disclosure) that can inhibit the growth of cells belonging to a target Staphylococcus species and / or lyse cells. Activity can be calculated in various ways depending on the assay performed. In some embodiments, the level of activity is expressed based on the minimum inhibitory concentration ("MIC"), for example, the minimum concentration of protein required to prevent the growth of a target Staphylococcus species in an MIC assay. In some embodiments, the level of activity is expressed, for example, -ΔOD 600Activity is expressed based on turbidity reduction, with activity calculated as / min / (mg of enzyme). In some embodiments, activity is expressed based on the reduction of viable bacterial cells in the culture after incubation time with the protein (e.g., 2 hours).

[0028]

[0041] In the context of anti-Staphylococcus activity, the terms “selective” and “selectivity,” as used herein, mean the property of exhibiting higher activity against one target bacterial species of the genus Staphylococcus compared to a second bacterial species of the genus Staphylococcus. Selectivity can be calculated by comparing the reciprocal of the MIC of the chimeric protein for the first bacterial species to the MIC of the protein for the second bacterial species. In some embodiments, selectivity is determined based on relative activity in a turbidity reduction assay.

[0029]

[0042] As used herein, the term “sequence identity” means the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant across the entire alignment window of residues, e.g., nucleotides or amino acids. “Identity percentage” of aligned segments of the test sequence and reference sequence is the number of identical residues shared by the two aligned sequences, divided by the total number of residues in the reference sequence segment, i.e., the entire reference sequence or a predetermined smaller portion of the reference sequence. “Percent identity” is the identity percentage multiplied by 100. Sequence comparisons to determine percentage identity can be performed by a number of well-known methods, including, for example, by using mathematical algorithms, such as those in the BLAST suite of sequence analysis programs. Unless otherwise stated, the term “sequence identity” in the claims means sequence identity as calculated by MUSCLE (www.ebi.ac.uk / Tools / msa / muscle / ) using default parameters.

[0030]

[0043] As used herein, the term "w / v" means the concentration of a component in a composition as measured by the weight or mass of the component compared to the volume of the composition. The weight of the solute is often provided in grams, and the volume is often provided in milliliters; the resulting value is provided as a percentage.

[0031] overview

[0044] This disclosure provides novel chimeric cell wall hydrolases (CWHs). Compositions comprising chimeric CWHs, as well as their use in targeting species of the genus Staphylococcus and in the treatment of conditions associated with species of Staphylococcus, are also provided herein.

[0032]

[0045] This disclosure relates in part to the growing appreciation for the critical role that the skin microbiome plays in skin health and function. Among other benefits, a healthy microbiome helps prevent colonization by pathogenic microorganisms, trains the immune system and prevents inflammation, strengthens the skin barrier, and promotes wound healing. Dysbiosis of the skin microbiome can cause and / or exacerbate skin conditions such as atopic dermatitis and acne vulgaris. For example, overgrowth of Staphylococcus aureus has been strongly associated with moderate to severe cases of atopic dermatitis and acute radiation dermatitis.

[0033]

[0046] Current therapeutic tools do not allow for precise treatment of skin diseases. Common approaches such as topical / oral antibiotics or chemicals like benzoyl peroxide are used to target the causative bacteria, but they also negatively impact the overall skin microbiome. In other cases, long-term steroid use can lead to undesirable and severe side effects.

[0034]

[0047] This disclosure provides compositions and treatments for Staphylococcus infections based on the identification of novel chimeric cell wall hydrolases (CWHs). CWHs are enzymes that degrade bacterial peptidoglycans by cleaving bonds in the peptidoglycan chain and side chain branching. Degradation of the peptidoglycan cell wall by CWHs can lead to rapid lysis of bacterial cells due to their inability to withstand internal turgor pressure.

[0035]

[0048] As demonstrated in the examples herein, this disclosure provides novel and highly effective chimeric CWHs. These chimeric CWHs possess high lytic activity and / or specificity for Staphylococcus species. In some embodiments, the chimeric CWHs are composed of domains derived from parent proteins and possess superior properties compared to any of the parent proteins. In some embodiments, the CWHs herein bind to highly specific epitopes in target cell walls. In some embodiments, the CWHs herein have lytic activity against a minimum of a single bacterial species or a group of closely related bacterial species. Due to these properties, in some embodiments, the CWHs herein function as highly specific skin microbiome regulators. For example, in the case of atopic dermatitis or acute radiation dermatitis, in some embodiments, the CWHs herein can specifically kill Staphylococcus aureus while exhibiting significantly lower activity against healthy symbiotic bacteria.

[0036] Chimeric cell wall hydrolases of the present disclosure

[0049] This disclosure is based on the inventors' development of a novel and highly active chimeric CWH. As disclosed in the examples herein, the exemplary CWHs of this disclosure are remarkable in that they possess highly effective anti-Staphylococcus properties resulting from the unique combination of CBD and EAD contained in the CWH.

[0037]

[0050] In some embodiments, the disclosure provides recombinant proteins comprising sequences of EAD and / or CBD according to any one of the embodiments disclosed herein. In some embodiments, the recombinant protein is a chimeric protein. In some embodiments, the chimeric protein is a chimeric cell wall hydrolase (CWH).

[0038]

[0051] The chimeric CWHs described herein include at least one heterologous domain, such as a heterologous EAD or CBD, compared to an innate CWH sequence. In some embodiments, the chimeric CWHs described herein are chimeric proteins containing EADs and CBDs derived from different proteins.

[0039] Enzymatically active domain (EAD)

[0052] In some embodiments, the chimeric protein of this disclosure includes EAD. In some embodiments, the EAD is derived from Staphylococcus phage SA12, philPLA35, Twort, CSA13, CSA5, phiH5, or SA97. In some embodiments, the EAD is derived from a prophage in Streptococcus suis strain 89 / 1591. In some embodiments, the EAD is derived from Staphylococcus capitis. In some embodiments, the EAD is derived from LysSA12, LysA72, PlySs2, Twort, LysCSA13, LysCSA5, LysH5, ALE-1, LysSA97, or LysPALS1. In some embodiments, the EAD is derived from LysSA12, Twort, LysCSA13, or LysCSA5. In some embodiments, the EAD is derived from LysSA12. The term "derived" in the context of describing the source of EAD or CBD indicates that the domain is contained within the referenced protein. Therefore, EAD derived from LysSA12 is contained within the innate LysSA12 endolysin protein.

[0040]

[0053] In some embodiments, the EAD consists of sequence numbers 11, 12, 13, or 14. In some embodiments, the EAD includes the sequence of sequence numbers 11, 12, 13, or 14. In some embodiments, the EAD has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with sequence numbers 11, 12, 13, or 14. In some embodiments, the EAD differs from the sequence of sequence numbers 11, 12, 13, or 14 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0041]

[0054] In some embodiments, the EAD consists of sequence number 11. In some embodiments, the EAD includes the sequence of sequence number 11. In some embodiments, the EAD has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with sequence number 11. In some embodiments, the EAD differs from the sequence of sequence number 11 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0042]

[0055] The sequence for sequence number 11 is as follows:

[0043]

[0056] MQAKLTKKEFIEWLKTSEGKQYNADGWYGFQCFDYANAGWQVLFGYNLKGVGAKDIPSANDFNGLATVYQNTPDFLAQPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWTDGVQQPGSGWEKVTRRQHAYDFPMWFIRPNFKSETETAPRSVQSPTQASKKET.

[0044]

[0057] In some embodiments, the EAD consists of sequences included in Table 1. In some embodiments, the EAD includes sequences included in Table 1. In some embodiments, the EAD has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with sequences included in Table 1. In some embodiments, the EAD differs from sequences included in Table 1 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0045] [Table 1]

[0046]

[0058] In some embodiments, CWH includes an EAD derived from lysine. In some embodiments, the lysine is endolysine, tail lysine, exolysine, bacteriocin, or autolysine. In some embodiments, the EAD is derived from any one of the endolysines listed herein. In some embodiments, the EAD is a glycosidase. In some embodiments, the EAD is an amidase. In some embodiments, the EAD is a peptidase. In some embodiments, the EAD is a CHAP domain. In some embodiments, the EAD is an M23 domain.

[0047]

[0059] In some embodiments, the chimeric CWH includes an EAD according to any one of the embodiments described above.

[0048]

[0060] In some embodiments, the chimeric CWH includes one EAD. In some embodiments, the CWH includes two or more EADs. In some embodiments, the chimeric CWH includes two EADs. In some embodiments, the chimeric CWH includes three, four, five, six, seven, eight, nine, or ten EADs.

[0049] Cell wall-binding domain (CBD)

[0061] In some embodiments, the chimeric CWH includes a cell wall-binding domain (CBD). In some embodiments, the CBD is derived from a Staphylococcus phage protein. In some embodiments, the CBD is derived from a Staphylococcus phage CWH. In some embodiments, the CBD is derived from a Staphylococcus phage lysine. In some embodiments, the lysine is endolysine, taillysine, exolysine, bacteriocin, or autolysine. In some embodiments, the CBD is an SH3b domain.

[0050]

[0062] In some embodiments, CBD is derived from Staphylococcus phages SA12, philPLA35, Twort, CSA13, CSA5, phiH5, or SA97. In some embodiments, CBD is derived from prophage in Streptococcus porcine strain 89 / 1591. In some embodiments, CBD is derived from Staphylococcus capitis. In some embodiments, CBD is derived from LysSA12, LysA72, PlySs2, Twort, LysCSA13, LysCSA5, LysH5, ALE-1, LysSA97, or LysPALS1. In some embodiments, CBD is derived from LysH5, LysA72, PlySs2, ALE-1, LysSA97, or LysPALS1. In some embodiments, CBD is derived from PlySs2, LysA72, or ALE-1.

[0051]

[0063] In some embodiments, CBD comprises sequence numbers 15, 16, 17, 18, 19, or 20. In some embodiments, CBD includes the sequence of sequence numbers 15, 16, 17, 18, 19, or 20. In some embodiments, CBD has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with sequence numbers 15, 16, 17, 18, 19, or 20. In some embodiments, CBD differs from the sequence of sequence numbers 15, 16, 17, 18, 19, or 20 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0052]

[0064] In some embodiments, CBD comprises sequence numbers 16, 17, or 18. In some embodiments, CBD includes the sequence of sequence numbers 16, 17, or 18. In some embodiments, CBD has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with sequence numbers 16, 17, or 18. In some embodiments, CBD differs from the sequence of sequence numbers 16, 17, or 18 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0053]

[0065] The sequence of sequence number 16 (LysA72 CBD) is as follows:

[0054]

[0066] KNPPVPAGYTLDKNNVPYKKEAGNYTVANVKGNNVRDGYSTNSRITGVLPNNATIKYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRISSFGKFSTI.

[0055]

[0067] The sequence of sequence number 17 (PlySs2 CBD) is as follows:

[0056]

[0068] SRSYRETGTMTVTVDALNVRRAPNTSGEIVAVYKRGESFDYDTVIIDVNGYVWVSYIGGSGKRNYVATGATKDGKRFGNAWGTFK.

[0057]

[0069] The sequence of sequence number 18 (ALE-1 CBD) is as follows:

[0058]

[0070] MPFLKSAGYGSNSTSSSNNNGYKTNKYGTLYKSESASFTANTDIITRLTGPFRSMPQSGVLRKGLTIKYDEVMKQDGHVWVGYNTNSGKRVYLPVRTWNESTGELGPLWGTIK.

[0059]

[0071] In some embodiments, the CBD consists of sequences included in Table 2. In some embodiments, the CBD includes sequences included in Table 2. In some embodiments, the CBD has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with sequences included in Table 2. In some embodiments, the CBD differs from sequences included in Table 2 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0060] [Table 2]

[0061]

[0072] In some embodiments, the chimeric CWH includes CBD according to any one of the embodiments described above.

[0062]

[0073] In some embodiments, the chimeric CWH contains one CBD. In some embodiments, the CWH contains two or more CBDs. In some embodiments, the chimeric CWH contains two CBDs. In some embodiments, the chimeric CWH contains three, four, five, six, seven, eight, nine, or ten CBDs.

[0063] Linker

[0074] In some embodiments, the chimeric protein comprises two or more domains linked together by linkers. In some embodiments, the linkers are flexible linkers. In some embodiments, the linkers are amino acid sequences of 1 to 100 amino acids in length, encompassing all values ​​and partial ranges between them. In some embodiments, the linkers comprise one or more glycine and / or serine.

[0064] Protein tags

[0075] In some embodiments, the recombinant protein of the Disclosure includes a protein tag. The protein tag is typically a short sequence of amino acids or a protein domain fused to the recombinant protein to facilitate purification and / or visualization. In some embodiments, the protein tag improves protein solubility. In some embodiments, the tag is a His tag, GST tag, MBP tag, Strep tag, FLAG tag, GFP tag, HA tag, V5 tag, Avi tag, CBP tag, ZZ tag, SUMO tag, Fc tag, thioredoxin tag, protein kinase A (PKA) tag, Myc tag, or S tag, or any combination thereof. In some embodiments, the tag is a His tag and contains six histidine residues.

[0065] Nucleic acids, vectors, and host cells of this disclosure

[0076] The Disclosure also provides nucleic acids encoding chimeric proteins of the Disclosure, such as CWH. The Disclosure also provides vectors and host cells for the expression of chimeric proteins of the Disclosure. In some embodiments, the vector is a plasmid, cosmid, bacteriophage, or virus containing the nucleic acid of the Disclosure. In some embodiments, the host cell contains the nucleic acid of the Disclosure or the vector of the Disclosure. In some embodiments, the host cell is a bacterial cell, yeast cell, insect cell, mammalian cell, or plant cell.

[0066] Formulations of the Disclosure

[0077] This disclosure provides compositions comprising novel chimeric proteins (e.g., CWH), nucleic acids, vectors, or host cells disclosed herein. In some embodiments, these compositions are formulated for delivery to subjects for the treatment of conditions associated with species of the genus Staphylococcus.

[0067] Topical, parenteral, and enteral formulations

[0078] In some embodiments, the compositions of this disclosure are formulated for topical, parenteral, or enteral administration.

[0068]

[0079] In some embodiments, the compositions herein are formulated for topical administration. Formulations for topical administration include lotions, hydrogels, creams, ointments, gels, drops, transdermal patches, colloidal patches, powders, suppositories, sprays, liquids, semi-solids, single-phase compositions, multi-phase compositions (e.g., oil-in-water, water-in-oil), foams, microsponges, liposomes, nanoemulsions, aerosol foams, polymers, fullerenes, and powders. In some embodiments, carriers, bases, thickeners, penetration enhancers, buffers, diluents, emulsifiers, humectants, dispersants, binders, and / or excipients are added to the formulation. In some embodiments, the composition is formulated as a hydrogel. In some embodiments, the composition is formulated as a lotion. In some embodiments, the composition is formulated as a cream. In some embodiments, the composition is formulated as a lyophilized powder, which can be reconstituted with a liquid prior to use, for example. In some embodiments, the composition is a colloidal patch.

[0069]

[0080] In some embodiments, the compositions of this disclosure are formulated for parenteral administration. As used herein, “parenteral administration” of a composition includes administration via any route of administration characterized by physical destruction of the target tissue, resulting in direct administration into the bloodstream, intramuscularly, or into an internal organ, and administration of the composition through a site of damage in the tissue. Accordingly, parenteral administration includes, but is not limited to, administration of the composition by injection, application of the composition via surgical incision, application of the composition via a tissue-penetrating nonsurgical wound. In particular, parenteral administration may include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, intrabursal injection or infusion; and renal dialysis infusion techniques.

[0070]

[0081] In some embodiments, the compositions herein are prepared for oral administration. The terms “oral,” “enteral,” “enterally,” “oral,” “non-parenteral,” and “non-parenteral” all refer to the administration of a compound or composition to an individual via a route or manner along the gastrointestinal tract. Examples of “oral” administration routes of a composition include, but are not limited to, swallowing of a liquid or solid dosage form of the composition by mouth, administration of the composition through a nasojejunal or gastrostomy feeding tube, intraduodenal administration of the composition, and rectal administration, for example, using a suppository to the lower intestinal tract of the gastrointestinal tract. The compositions herein can be formulated into a number of possible dosage forms, including, but are not limited to, tablets, capsules, liquid syrups, softgels, suppositories, aerosols, and enemas. The compositions herein can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain certain substances that increase the viscosity of the suspension, such as, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. Suspensions may also contain stabilizers.

[0071] Pharmaceutical ingredients

[0082] In some embodiments, the composition includes an emulsifier. In some embodiments, the composition includes a mixture of emulsifiers.

[0072]

[0083] In some embodiments, the composition contains about 0.5% to about 5% w / v of an emulsifier or a mixture of emulsifiers.

[0073]

[0084] Examples of emulsifiers suitable for use in some embodiments of this disclosure include xanthan gum, polysorbate 80, oleyl polyoxyl-6 glyceride, polyoxyl 35 hydrogenated castor oil, sucrose distearate, saponins, sodium alginate, guar gum, tocopherol polyethylene glycol 1000 succinate, lauroyl polyoxyl-32 glyceride, sorbitan monooleate, glyceryl stearate, cetearyl alcohol, sodium stearoyl lactylate, their salts, their derivatives, and mixtures thereof. In some embodiments, the emulsifier is xanthan gum.

[0074]

[0085] In some embodiments, the emulsifier component is polyglycolized glycerides and polyoxyethylene glycerides of medium- to long-chain mono, di, and triglycerides, for example: almond oil PEG-6 ester, almond oil PEG-60 ester, apricot kernel oil PEG-6 ester (Labrafil® M1944CS), caprylic / capric triglyceride PEG-4 ester (Labrafac® HydroWL1219), caprylic / capric triglyceride PEG-4 complex (Labr afac(registered trademark) Hydrodrophile), Caprylic / Capric Glyceride PEG-6 Ester (Softigen(registered trademark) 767), Caprylic / Capric Glyceride PEG-8 Ester (Labrasol(registered trademark)), Castor Oil PEG-50 Ester, Hydrogenated Castor Oil PEG-5 Ester, Hydrogenated Castor Oil PEG-7 Ester, Hydrogenated Castor Oil PEG-9 Ester, Corn Oil PEG-6 Ester (Labrafil(registered trademark) M2125CS), Corn Oil PEG-8 Ester (Labrafil(registered trademark) WL2609 BS), corn oil glyceride PEG-60 ester, olive oil PEG-6 ester (Labrafil® M1980CS), hydrogenated coconut / palm kernel oil PEG-6 ester (Labrafil® M2130BS), palm kernel oil-containing hydrogenated coconut / palm kernel oil PEG-6 ester, PEG-6, coconut oil (Labrafil® M2130CS), palm kernel oil PEG-40 ester, peanut oil PEG-6 ester (Labrafil® M1969CS), saturated C8-C18 fatty acid glycerol ester (G Gelucire® 33 / 01), Glyceryl esters of saturated C12-C18 fatty acids (Gelucire® 39 / 01 and 43 / 01), Glyceryl laurate / PEG-32 laurate (Gelucire® 44 / 14), Glyceryl laurate / PEG-20 laurate, Glyceryl laurate / PEG-32 laurate, Glyceryl, Glyceryl laurate / PEG-40 laurate, Glyceryl oleate / PEG-20 glyceryl, Glyceryl oleate / PEG-30 oleate,Glyceryl palmitostearate / PEG-32 palmitostearate (Gelucire® 50 / 13), glyceryl stearate / PEG stearate, glyceryl stearate / PEG-32 stearate (Gelucire® 53 / 10), saturated polyglycolated glycerides (Gelucire® 37 / 02 and Gelucire® 50 / 02), triisostearin PEG-6 ester (i.e., Labrafil® I Selected from sostearique, triolein PEG-6 ester, trioleate PEG-25 ester, polyoxyl 35 castor oil (Cremophor® EL or Kolliphor® EL), polyoxyl 40 hydrogenated castor oil (Cremophor® RH40 or Kolliphor® RH40), polyoxyl 60 hydrogenated castor oil (Cremophor® RH60), lecithin, phospholipids, and mixtures thereof.

[0075]

[0086] In some embodiments, the emulsifier is caprylic / capric diglyceride, glyceryl monooleate, glyceryl ricinoleate, glyceryl laurate, glyceryl dilaurate, glyceryl dioleate, mono / dioleate, caprylic / capric glyceride, medium-chain (C8 / C10) mono and diglycerides (Capmul® MCM, Capmul® MCM(L)), mono and diacetylated monoglycerides, polyglyceryl oleate, polyglyceryl-2 dioleate, polyglyceryl-10 trioleate, polyglyceryl-10 laurate, polyglyceryl-10 oleate, and polyglyceryl-10 monodioleate. Polyglycolated derivatives and polyoxyethylene esters or ester derivatives of medium- to long-chain fatty acids that can be used include polyglycolated derivatives and polyoxyethylene esters or ester derivatives, various surfactants trade names Brij and Myrj, and propylene glycol esters of medium- to long-chain fatty acids, including polyglycolated derivatives of

[0076]

[0087] In some embodiments, the composition includes a humectant. In some embodiments, the composition is a topical formulation and includes a humectant, which may be referred to as a soothing agent, smoothing agent, moisturizing agent, or protective agent. The humectants of this disclosure function to stabilize the moisture content of the tissue to which they are applied in the presence of fluctuating humidity.

[0077]

[0088] In some embodiments, the humectants include polyglycols (as defined below herein), propylene glycol, sorbitol, lactic acid, sodium lactate, glycerol, glycerin, ethoxylated castor oil, calamine, dodecyl sulfate, sodium lauryl sulfate (SLS); polyoxyethylene esters of polysorbitan, such as monooleic acid, monolauric acid, monopalmitic acid, monostearate; esters of sorbitan, polyoxyethylene ethers, sodium dioctyl sulfosuccinate. Selected from thorium (DOSS), lecithin, sodium docusate, hexylene glycol, butylene glycol, aloe vera gel, aloe vera powder, hyaluronic acid, alpha hydroxy acids, such as lactic acid, egg yolk, egg white, glyceryl triacetate, honey, molasses, polymerizable polyols, such as polydextrose, quillaja, sodium hexametaphosphate e452i; sugar alcohols (sugar polyols), such as glycerol, sorbitol, xylitol, maltitol; urea, and castor oil.

[0078]

[0089] In some embodiments, the composition includes a humectant selected from the list consisting of aloe vera, betaine, butylene glycol, caprylyl glycol, dimethicone, fructose, glucomannan, glucose, glycerin, glyceryl glucoside, honey, hyaluronic acid, lactic acid, panthenol, polyethylene glycol, propylene glycol, propanediol, sodium hyaluronate, sodium lactate, sodium pyrrolidone carboxylate, sorbitol, and urea. In some embodiments, the composition includes a humectant in the range of 0.1 to 50% w / v, encompassing all values ​​and partial ranges in between. In some embodiments, the composition includes a humectant in the range of 0.5 to 10% w / v.

[0079]

[0090] In some embodiments, the composition contains hyaluronic acid. In some embodiments, the composition is a hyaluronic acid-based hydrogel for topical application. In some embodiments, the composition contains 0.1 to 10% w / v hyaluronic acid. In some embodiments, the composition contains 0.5 to 5.0% w / v hyaluronic acid. In some embodiments, the composition contains 1 to 2% w / v hyaluronic acid. In some embodiments, the composition contains a hydrogel. In some embodiments, the hydrogel contains a cellulose polymer. In some embodiments, the hydrogel contains hydroxypropyl methylcellulose.

[0080]

[0091] In some embodiments, the composition contains a cellulose polymer. In some embodiments, the cellulose polymer is hydroxyethylcellulose, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, microcrystalline cellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, or cellulose acetate. In some embodiments, the composition contains a cellulose polymer in a concentration of 0.1 to 20% w / v, encompassing all values ​​and partial ranges in between. In some embodiments, the composition contains a cellulose polymer in a concentration of 0.5 to 10% w / v. In some embodiments, the composition contains a cellulose polymer in a concentration of 1 to 5% w / v.

[0081]

[0092] In some embodiments, the composition comprises a thickener, a gelling agent, and / or a polymer. In some embodiments, the composition comprises an acrylate. In some embodiments, the composition comprises a carbomer.

[0082]

[0093] In some embodiments, the composition contains salt. In some embodiments, the composition contains salt selected from the list consisting of calcium chloride, Dead Sea salt, Epsom salt, Himalayan pink salt, magnesium chloride, sea salt, and sodium chloride. In some embodiments, the composition contains salt in concentrations of 10 to 500 mM, encompassing all values ​​and partial ranges in between. In some embodiments, the composition contains salt in concentrations of 50 to 250 mM.

[0083]

[0094] In some embodiments, the composition contains a buffer. In some embodiments, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, acetic acid, ammonium acetate, boric acid, citric acid, glycine, phosphoric acid, potassium hydroxide, potassium phosphate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium hydroxide, sodium phosphate, sodium tetraborate, tris(hydroxymethyl)aminomethane, or trisodium phosphate. In some embodiments, the composition contains a buffer in the range of 1 to 250 mM, encompassing all values ​​and partial ranges in between. In some embodiments, the composition contains a buffer in the range of 5 to 50 mM.

[0084]

[0095] In some embodiments, the composition contains a surfactant. In some embodiments, the composition contains a surfactant selected from the list consisting of ceteareth-20, cocamidopropyl betaine, cocoglucoside, decylglucoside, decylpolyglucose, disodium laureth sulfosuccinate, glycereth-26, laurylglucoside, laurylpolyglucose, sodium cocoyl glutamate, sodium cocoyl isethionate, sodium laureth sulfate, and sodium lauryl sulfate. In some embodiments, the composition contains a surfactant of 0.1 to 20% w / v, encompassing all values ​​and partial ranges in between. In some embodiments, the composition contains a surfactant of 1 to 10% w / v.

[0085]

[0096] In some embodiments, the composition contains oil. In some embodiments, the composition contains oil selected from the list consisting of argan oil, avocado oil, baobab oil, camellia oil, carrot seed oil, coconut oil, evening primrose oil, grapeseed oil, hemp seed oil, jojoba oil, macadamia nut oil, marula oil, mineral oil, olive oil, pomegranate seed oil, raspberry seed oil, rosehip seed oil, squalane oil, sunflower seed oil, sweet pea oil, and tamanu oil. In some embodiments, the composition contains oil in a w / v range of 0.1 to 20%, encompassing all values ​​and partial ranges in between.

[0086]

[0097] In some embodiments, the composition contains an alcohol. In some embodiments, the composition contains an alcohol selected from the list consisting of cetyl alcohol, ethyl alcohol, isopropyl alcohol, and stearyl alcohol. In some embodiments, the composition contains an alcohol of 0.1 to 20% w / v, encompassing all values ​​and partial ranges in between. In some embodiments, the composition contains an alcohol of 1 to 10% w / v.

[0087]

[0098] In some embodiments, the composition contains free amino acids. In some embodiments, the composition contains alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, the composition contains amino acids selected from the list consisting of alanine, arginine, cysteine, glutamine, glycine, histidine, lysine, methionine, proline, serine, and threonine. In some embodiments, the composition contains amino acids in concentrations of 10 to 250 mM, encompassing all values ​​and partial ranges in between. In some embodiments, the composition contains amino acids in concentrations of 25 to 150 mM.

[0088]

[0099] In some embodiments, the composition contains glycerol. In some embodiments, the composition contains 0.5 to 50% w / v glycerol, encompassing all values ​​and partial ranges in between. In some embodiments, the composition contains 1 to 30% w / v glycerol. In some embodiments, the composition contains 1 to 5% w / v glycerol.

[0089]

[0100] In some embodiments, the composition contains petrolatum. In some embodiments, the composition contains petrolatum in an amount of 0.1 to 20% w / v, encompassing all values ​​and partial ranges in between.

[0090]

[0101] The compositions of this disclosure may contain one or more additional agents, whether active or passive. Examples of such agents include sweeteners, flavorings, colorants, filling agent binders, lubricants, excipients, preservatives, emollients, hydrating agents, smoothing agents, or manufacturing agents. Additional excipients or additives may be added to the compositions. For example, if desired, any generally acceptable soluble or insoluble inactive filler (diluent) material may be included in the final product (e.g., solid dosage form). Such inactive fillers may include monosaccharides, disaccharides, polyhydric alcohols, inorganic phosphates, sulfates or carbonates, and combinations thereof. Examples of suitable inactive fillers include sucrose, dextrose, lactose, xylitol, fructose, sorbitol, calcium phosphate, calcium sulfate, calcium carbonate, microcrystalline cellulose, and combinations thereof. An effective amount of any generally acceptable lubricant, such as calcium or magnesium soap, may be added.

[0091]

[0102] Depending on the dosage form, optional additives and modifiers may further include one or more of the following: acids, bases, acidity modifiers, alcohols, anticoagulants, defoamers, antioxidants, bulking agents, coagulants, color retainers, emulsifiers, flavor enhancers, milling agents, gelling agents, gloss enhancers, humectants, leavening agents, tracer gases, preservatives, stabilizers, sweeteners, softeners, and thickeners.

[0092]

[0103] The compositions of this disclosure may additionally contain other conventional auxiliary components. For example, the compositions may contain additional compatible active substances such as antipruritics, astringents, topical anesthetics, or anti-inflammatory agents, or additional substances useful in the physical formulation of various dosage forms of the compositions of this disclosure, such as colorants, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, if such substances are added, they should not excessively interfere with the biological activity of the components of the compositions of this disclosure. The formulations are sterile and, if desired, may be mixed with auxiliary agents that do not adversely affect the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0093]

[0104] In some embodiments, the composition includes a skin protectant. In some embodiments, the composition includes an ingredient approved by the FDA for the treatment of atopic dermatitis. In some embodiments, the composition includes a skin protectant approved by the FDA. In some embodiments, the composition includes colloidal oatmeal. In some embodiments, the composition includes a skin protectant selected from the list consisting of allantoin, aluminum hydroxide gel, calamine, cocoa butter, cod liver oil, colloidal oatmeal, dimethicone, glycerin, hard fat, kaolin, lanolin, mineral oil, petrolatum, sodium bicarbonate, topical starch, white petrolatum, zinc acetate, zinc carbonate, and zinc oxide. In some embodiments, the composition comprises one of the following skin protectants within the following ranges: allantoin, 0.5-2%; aluminum hydroxide gel, 0.15-5%; calamine, 1-25%; cocoa butter, 50-100%; cod liver oil, 5-13.56%; colloidal oatmeal, at least 0.007%, or at least 0.003% in combination with mineral oil; dimethicone, 1-30%; glycerin, 20-45%; hard fat, 50-100%; kaolin, 4-20%; lanolin, 12.5-50%; mineral oil, 50-100%, or 30-35% in combination with colloidal oatmeal; petrolatum, 30-100%; sodium bicarbonate; topical starch, 10-98%; white petrolatum, 30-100%; zinc acetate, 0.1-2%; zinc carbonate, 0.2-2%; zinc oxide, 1-25%. See, for example, Section 347.10, Chapter 21, Volume 5, “Skin protectant active ingredients,” of the Federal Regulations, which is incorporated herein by reference in its entirety.

[0094]

[0105] In some embodiments, other components such as antibiotics; preservatives, antifungal agents; corticosteroids; sedatives; anti-aging agents; smoothing agents; moisturizers; and protective agents are also present in the composition.

[0095] Characteristics of the composition disclosed herein

[0106] This disclosure provides chimeric proteins and compositions comprising these chimeric proteins. These compositions possess characteristics beneficial for therapeutic use against target Staphylococcus species.

[0096]

[0107] In addition to selectivity issues, prior CWHs and endolysins known in the art have properties that make them unsuitable for therapeutic applications, such as topical application. For example, many previously characterized CWHs have weak activity, poor thermal stability, and / or narrow or unsuitable pH ranges.

[0097]

[0108] This disclosure provides, but is not limited to, chimeric proteins having beneficial characteristics such as high anti-Staphylococcus activity, Staphylococcus species specificity, thermal stability, and a broader or more suitable pH range. For example, the exemplary LysSA12 EAD+PlySs2 CBD chimera of this disclosure exhibits very high activity, selectivity, and thermal stability, attributes that make it well suitable for applications where it is necessary to selectively remove Staphylococcus aureus from a microbial community that also includes other Staphylococcus species (e.g., Staphylococcus epidermidis). The skin microbiome is one example of such a community, where Staphylococcus epidermidis is a symbiotic organism and an important component of a healthy microbiome, while Staphylococcus aureus is typically pathogenic and associated with skin conditions such as atopic dermatitis.

[0098] Anti-Staphylococcus activity

[0109] The compositions disclosed herein are active against Staphylococcus species, such as target Staphylococcus species.

[0099]

[0110] In some embodiments, the compositions of the present disclosure have activity against a target Staphylococcus species, the degree of which is determined based on their minimum inhibitory concentration (MIC) against the target bacterial species. In some embodiments, the MIC is less than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 μg / mL. In some embodiments, the MIC is less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μg / mL. In some embodiments, the MIC is less than 5.0, 4.5, 4.0, 3.5, 3, 0, 2.5, 2.0, 1.5, 1.0, or 0.5 μg / mL. In some embodiments, the MIC is less than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μg / mL.

[0100]

[0111] The genus Staphylococcus consists of Gram-positive facultative anaerobic bacteria found in the skin and mucosal microbiota of mammals and birds. It is a genus of non-spore-forming cocci belonging to the family Micrococcaceae. Species of Staphylococcus are often found as part of the normal human microbiota of the skin and nasal cavity. This genus includes clinically important opportunistic pathogens in both human and veterinary medicine. Bacterial species belonging to this genus can be grouped and identified according to the production of coagulase enzymes, which are enzymes capable of converting fibrinogen to fibrin, a feature readily detectable in the laboratory and enabling practical classification.

[0101]

[0112] In general, coagulase-positive staphylococci (CoPS), such as Staphylococcus aureus, S. intermedius, and S. pseudointermedius, are usually pathogenic, although in some cases they can cause asymptomatic colonization in healthy individuals. In some embodiments, the compositions of the present disclosure are active against coagulase-positive staphylococcal (CoPS) ​​bacterial species. In some embodiments, the bacterial species is Staphylococcus aureus. In some embodiments, the compositions of the present disclosure are active against the bacterial species S. intermedius. In some embodiments, the compositions of the present disclosure are active against the bacterial species S. pseudointermedius.

[0102]

[0113] Coagulase-negative staphylococci (CoNS), represented by a larger group of bacterial species, have been associated with opportunistic infections. CoNS bacterial species such as Staphylococcus epidermidis, S. haemolyticus, and S. lugdunensis have been associated with opportunistic infections in humans. Certain subspecies of Staphylococcus epidermidis and S. schleiferi can cause skin and ear infections in dogs. S. felis can cause lower urinary tract disease, eye infections, and otitis in cats. In some embodiments, the compositions of this disclosure are active against coagulase-negative staphylococcal (CoNS) bacterial species.

[0103]

[0114] In some embodiments, the compositions of the present disclosure are selective for one or more CoPS bacterial species compared to one or more CoNS bacterial species.

[0104]

[0115] In some embodiments, the compositions of this disclosure are active against Staphylococcus aureus. Staphylococcus aureus is considered the most important pathogen of this genus. In humans, Staphylococcus aureus can be found in the social environment and hospital settings and constitutes a significant source of medically associated infections. The bacteria can cause infections in humans associated with the skin and soft tissues, pneumonia, sepsis, and osteomyelitis. These conditions have also been reported in animals. Staphylococcus aureus can cause numerous superficial purulent (pus-forming) infections of the dermis and underlying tissues, as well as severe systemic infections. Staphylococcus aureus can produce a wide range of toxins, including enterotoxins (food poisoning), cytotoxins (common systemic toxins), and toxic shock superantigens.

[0105]

[0116] In some embodiments, the compositions of this disclosure are active against S. pseudointermedius, a common cause of skin and soft tissue infections in humans, as well as in dogs and cats.

[0106]

[0117] In some embodiments, the compositions of this disclosure are active against Staphylococcus epidermidis. Staphylococcus epidermidis is a common cause of infections associated with medical devices such as catheters, pacemakers, and artificial joints. Staphylococcus epidermidis is also a major cause of bloodstream infections in hospitalized patients.

[0107]

[0118] In some embodiments, the compositions of this disclosure are active against S. saprophyticus, a common cause of urinary tract infections in sexually active young women.

[0108]

[0119] In some embodiments, the compositions of this disclosure are active against S. haemolyticus, a major cause of infections associated with central venous catheters and other medical devices, particularly in immunocompromised patients.

[0109]

[0120] In some embodiments, the compositions of this disclosure are active against S. lugdunensis. S. lugdunensis is increasingly recognized as an important pathogen, particularly in skin and soft tissue infections, endocarditis, and bone and joint infections.

[0110]

[0121] In some embodiments, the compositions of the present disclosure are active against bacterial species of the genus Staphylococcus. In some embodiments, the compositions of the present disclosure are active against S. agnetis, S. argensis, S. argenteus, S. arlettae, Staphylococcus aureus, S. auricularis, S. capitis, S. caprae, S. carnosus, S. chromogenes, S. cohnii, S. condimenti, S. cornubiensis, S. delphini, S. devriesei, S. edaphicus, Staphylococcus epidermidis, S. equi, S. equiorum, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis, S. hyicus, S. intermedius, S. kloosii, S. lentus, and S. lugdunens. is, S.lutrae, S.massiliensis, S.microti, S.muscae, S.nepalensis, S.pasteuri, S.petrasii, S.pettenkoferi, S.piscifermentans, S.pseudintermedius, S.pseudoxylosus, S.rostri, S.saccharol yticus, S.saprophyticus, S.schleiferi, S.schweitzeri, S.sciuri, S.simiae, S.simulans, S.stepanovicii, S.succinus, S.vitulinus, S.warneri, and S.xylosus.

[0111]

[0122] In some embodiments, the compositions of the disclosed herein are active against Staphylococcus species in the same phylogenetic classification as Staphylococcus aureus. In some embodiments, the compositions of the disclosed herein are active against S. argenteus, Staphylococcus aureus, S. schweitzeri, or S. simiae. For example, see Madhaiyan et al., Int. J. Syst. Evol. Microbiol 2020;70:5926-5936, incorporated herein by reference, which provides a phylogenetic analysis of Staphylococcus species.

[0112]

[0123] In some embodiments, the compositions of the present disclosure are active against Staphylococcus species belonging to the same phylogenetic classification as Staphylococcus epidermidis. In some embodiments, the compositions of the present disclosure are active against S. capitis, S. caprae, Staphylococcus epidermidis, or S. saccharolyticus.

[0113] Selectivity

[0124] The inventors have discovered that exemplary novel chimeras disclosed herein, such as LysSA12 EAD+PlySs2 CBD, LysSA12 EAD+LysA72 CBD, and LysSA12 EAD+ALE-1 CBD, exhibit remarkable selective activity against Staphylococcus aureus compared to Staphylococcus epidermidis. Surprisingly, it was found that the CBD and EAD contained in these chimeras did not exhibit the same selectivity in other chimeric combinations. As is known in the art, the majority of CWHs that have lytic activity against Staphylococcus aureus also have similar lytic activity against Staphylococcus epidermidis. This means that while they can kill undesirable Staphylococcus aureus overgrowth, they will also kill beneficial Staphylococcus epidermidis populations, which is highly undesirable for topical skin microbiome applications. In contrast, the exemplary chimeric CWHs of this disclosure can distinguish between Staphylococcus aureus and Staphylococcus epidermidis by including specific combinations of CBD and EAD.

[0114]

[0125] In some embodiments, the chimeric proteins described herein have species-specific activity of Staphylococcus species. In some embodiments, the chimeric proteins described herein have selectivity for a particular species of Staphylococcus compared to another species of Staphylococcus. Selectivity in this context means that the chimeric proteins of this disclosure have higher activity for a particular species of Staphylococcus than for another species of Staphylococcus. In some embodiments, the chimeric proteins described herein are selective for a particular group of Staphylococcus species compared to another group of Staphylococcus species. For example, in some embodiments, the chimeric proteins of this disclosure are selective for a phylogenetic classification including Staphylococcus aureus compared to a phylogenetic classification including Staphylococcus epidermidis. In some embodiments, the chimeric proteins of this disclosure are selective for coagulase-positive staphylococcus (CoPS) ​​species (e.g., Staphylococcus aureus) compared to coagulase-negative staphylococcus (CoNS) species (e.g., Staphylococcus epidermidis).

[0115]

[0126] In some embodiments, the chimeric proteins described herein are selective for Staphylococcus aureus compared to other bacterial species of the genus Staphylococcus. In some embodiments, the chimeric proteins described herein are selective for Staphylococcus aureus compared to CoNS bacterial species. In some embodiments, the chimeric proteins of this disclosure are selective for Staphylococcus aureus compared to Staphylococcus epidermidis. In some embodiments, the chimeric proteins of this disclosure are selective for Staphylococcus aureus compared to S. hominis.

[0116]

[0127] The degree of selectivity exhibited by a given chimeric protein towards a given bacterial species, for example, towards bacterial species A and bacterial species B, is generally determined by comparing the activity of the chimeric protein towards bacterial species A with its activity towards bacterial species B. If the activity is defined, for example, by the minimum inhibitory concentration (MIC) via an MIC assay, the selectivity for bacterial species A compared to bacterial species B is calculated by the inverse ratio of the MICs of the chimeric protein towards each bacterial species. That is:

[0117]

number

[0118]

[0128] In some embodiments, the chimeric proteins of the Disclosure have selectivity of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the chimeric proteins of the Disclosure have selectivity of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times. In some embodiments, the chimeric proteins of the Disclosure have measurable activity against one bacterial species while being substantially inactive (or undetectable) against another bacterial species.

[0119]

[0129] In some embodiments, the compositions of this disclosure have broad-spectrum anti-Staphylococcus activity. In some embodiments, the compositions have high activity against multiple bacterial species of the genus Staphylococcus. In some embodiments, the compositions have high activity against a group of closely related Staphylococcus bacterial species. In some embodiments, the compositions have high activity against a diverse group of Staphylococcus bacterial species. In some embodiments, the compositions are suitable for use as broad-spectrum therapeutic agents.

[0120] thermal stability

[0130] The inventors of this disclosure have also surprisingly discovered that some of the novel chimeric CWHs of this disclosure, for example, SEQ ID NO: 21, exhibit higher thermal stability in the chimeric CWHs of this disclosure than in the control CWH containing Twort EAD (SEQ ID NO: 27).

[0121]

[0131] In some embodiments, the compositions of the Disclosure exhibit thermal stability at temperatures up to 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60°C, including all values ​​and ranges in between.

[0122]

[0132] As used herein, thermal stability at a given temperature means the ability of a protein to maintain its activity level at that temperature or after exposure to that temperature. In some embodiments, thermal stability at a given temperature is measured after exposure to that temperature over a period of time. In some embodiments, thermal stability is determined based on experiments testing activity at a given temperature or after exposure to that temperature over a period of time (e.g., showing a measurable reduction in target bacterial density at or after exposure to that temperature). Thus, in some embodiments, an EAD, CBD, or chimeric protein is considered thermally stable at a given temperature if it still exhibits measurable activity at or after exposure to that temperature. In some embodiments, an EAD, CBD, or recombinant chimeric protein is considered thermally stable at a critical temperature if it still exhibits measurable activity at its intended use temperature after exposure to a critical temperature (e.g., activity tested after the protein has been exposed to a critical temperature for 30 minutes). In some embodiments, thermal stability after exposure to a given temperature is determined based on assays performed at room temperature. In some embodiments, thermal stability is determined after exposure to a given temperature based on an assay that measures activity. In some embodiments, the assay is a turbidity reduction assay.

[0123]

[0133] In the context of comparing two types of proteins (e.g., two types of EAD, two types of CBD, or two types of chimeric CWH), one protein can be considered more thermally stable at a given temperature than the other if it exhibits higher absolute activity at or after exposure to that temperature (e.g., resulting in a greater reduction in microbial density over the measured time). In other embodiments, one protein can be considered more thermally stable at a given temperature than the other if it exhibits higher relative activity at or after exposure to that temperature (e.g., if the protein shows a smaller reduction in activity after exposure to a given temperature compared to a relative reduction in activity of a second protein after exposure to the same temperature).

[0124]

[0134] In some embodiments, thermal stability at a given temperature is determined based on the ability to maintain activity after exposure to that temperature for 10, 20, 30, 40, 50, or 60 minutes, including all values ​​and ranges in between. In some embodiments, thermal stability at a given temperature is determined after exposure to that temperature for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, including all values ​​and ranges in between. In some embodiments, thermal stability at a given temperature is determined after exposure to that temperature for 1, 2, 3, 4, 5, 6, or 7 days, including all values ​​and ranges in between. In some embodiments, thermal stability at a given temperature is determined after exposure to that temperature for 1, 2, 3, or 4 weeks, including all values ​​and ranges in between. In some embodiments, thermal stability at a given temperature is determined after exposure to that temperature over a period of 1, 2, 3, 4, 5, or 6 months, including all values ​​and ranges in between.

[0125]

[0135] In some embodiments, thermal stability is measured by maintaining the composition at a given temperature for a certain period, for example, several weeks or several months, and then measuring the activity. In some embodiments, thermal stability is determined based on the activity retained after two months at a given temperature.

[0126]

[0136] In some embodiments, the compositions herein are thermally stable at 45°C for at least 4 weeks. In some embodiments, the compositions herein are thermally stable at 45°C for at least 2 months. In some embodiments, the compositions herein are thermally stable at 50°C for at least 2 months.

[0127]

[0137] In some embodiments, the compositions herein are considered thermally stable or storable at room temperature if, after exposure to a temperature of 45°C for four weeks, they retain at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of their original activity, including all values ​​and ranges during that period, at room temperature.

[0128] pH range

[0138] In some embodiments, the compositions herein are stable within a range of pH values. A composition, for example, a chimeric protein, is considered stable at a given pH level if it exhibits activity at that pH level. In some embodiments, pH stability at different pH values ​​is determined based on activity assays performed at different pH values. For example, in some embodiments, pH stability is determined by incubating the composition of the disclosure (e.g., a chimeric protein) with target bacterial cells at different pH values ​​(e.g., in a turbidity reduction assay). The assay results provide activity levels for the composition at different pH values, including the maximum activity level. In some embodiments, a composition is stable at a certain pH level if it has the same activity at that pH as its maximum activity level at that pH. In some embodiments, a composition is stable at a certain pH level if it exhibits at least 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of its maximum activity at that pH. In some embodiments, the composition is stable at a certain pH level if, at that pH, it exhibits about 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of its maximum activity.

[0129]

[0139] In some embodiments, pH stability is determined based on an activity assay. In some embodiments, the assay is a turbidity reduction assay.

[0130]

[0140] In some embodiments, the compositions herein are stable at pH 3, 4, 5, 6, 7, 8, 9, or 10, or within any range therein. In some embodiments, the compositions herein are stable in the pH range of 6 to 8. In some embodiments, the compositions herein are stable in the pH range of 5 to 8. In some embodiments, the compositions herein are stable at the pH value most suitable for topical skin application. For example, in some embodiments, the compositions herein are stable at pH 4, 5, or 6.

[0131] Synergistic effect

[0141] In some embodiments, the Disclosure provides a combination composition comprising at least two compositions, for example, a chimeric protein of the Disclosure. In some embodiments, at least two compositions of the Disclosure are administered together. In some embodiments, two compositions of the Disclosure are administered one after the other or simultaneously. In some embodiments, the combination composition of the Disclosure exhibits synergistic results compared to the individual constituent compositions.

[0132]

[0142] In some embodiments, the chimeric proteins of this disclosure exhibit synergistic effects from the combination of CBD and EAD contained therein. In some embodiments, this synergistic effect is measured in comparison to a control protein. In some embodiments, the control protein is the original protein from which CBD is derived. In some embodiments, the control protein is the original protein from which EAD is derived. In some embodiments, the control protein is a control chimera containing CBD and control EAD. In some embodiments, the control protein is a control chimera containing EAD and control CBD. In some embodiments, the control CBD is Twort CBD. In some embodiments, the control EAD is Twort EAD. The discussion of synergistic effects provided herein applies to the combination compositions of this disclosure and chimeras containing synergistically interacting domains.

[0133]

[0143] As used herein, the term “synergistic” means, when referring to the compositions of this disclosure, a composition that exhibits an effect exceeding the predicted effect of the composition as calculated by the (y) reference model. In some embodiments, “synergistic” means an effect greater than a simple additive effect. In some embodiments, a synergistic combination is one in which the MIC of the combination is lower than the MIC of its components. In some embodiments, a synergistic combination is one in which the MIC of the combination is lower than the MIC of its components when calculated by the percentage composition. For example, in some embodiments, a synergistic combination of CBD and EAD is one in which the MIC of the CBD-EAD chimera is lower than the MIC of the control protein.

[0134]

[0144] In some embodiments, the presence of a synergistic effect with respect to the combination composition is determined based on the fractional inhibitory concentration (FIC) index value. To quantify the potency of the drug combination compared to the individual activity of each drug, the fractional inhibitory concentration (FIC) index value for each protein combination is calculated using the following equation:

[0135]

number

[0136]

[0145] In some embodiments, synergistic effects are calculated using the Synergyfinder web application. The following reference, which describes the Synergyfinder web application in detail, is incorporated herein by reference in its entirety: Ianevski, A.; He, L.; Aittokallio, T.; Tang, J. SynergyFinder: A Web Application for Analyzing Drug Combination Dose-Response Matrix Data Bioinformatics 2017, 33(15), pp. 2413–2415.

[0137]

[0146] In some embodiments, the reference model is a "simple deductive model." The simple deductive model determines that a composition exhibits a synergistic effect if the observed effect is greater than the effect predicted from the sum of the effects of the individual components. The synergistic effect according to the deductive model can be calculated using the following equation: y = observed effect of the composition - (sum of expected effects of the individual active ingredient components). If y is greater than zero, the composition exhibits a synergistic effect. An adjusted synergistic effect percentage model can also be calculated. The equation for the adjusted synergistic effect percentage is observed effect of the composition - additive inhibitory value. The additive inhibitory value for a composition containing two components (e.g., A and B) can be calculated according to the following equation: (expected effect of component A) + ((1 - expected effect of component A) × expected effect of component B) / 100.

[0138]

[0147] In some embodiments, the reference model is a “highest single agent” (HSA). The HSA model states that the expected combined effect is equal to the higher effect of the two individual drugs. HSA =max(y1,y2). The following references describe this model in detail and are incorporated herein by reference in their entirety: Berenbaum, MC (1989). What is synergy? Pharmacol. Rev., 41(2): pp. 93-141.

[0139]

[0148] In one embodiment, the reference model is the Loewe additive model. This model defines the expected effect y as if the drug were combined with itself LOEWE The Loewe additive model takes into account the dose-response curves of the individual drugs. The expected effect y LOEWE is

[0140]

Number

[0141]

Number

[0142]

[0149]

[0143]

Number

[0144]

[0150] In some embodiments, the reference model is the Bliss model. The Bliss model is incorporated herein by reference in its entirety and is described in detail in the following references: Bliss, CI (1939). The toxicity of poisons applied jointly. Annals of Applied Biology, 26(3): pp. 585-615. Bliss assumes a stochastic process in which two components exert their effects individually, and the expected combined effect can be calculated based on the probability of independent events as follows: BLISS = y1 + y2 - y1 × y2.

[0145]

[0151] In some embodiments, the reference model is the zero-interaction potency (ZIP) model. The ZIP model is described in detail in the following reference, which is incorporated herein by reference in its entirety: Yadav, B., Wennerberg, K., Aittokallio, T., and Tang, J. (2015). Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model. Comput Struct Biotechnol J, 13: pp. 504-513. ZIP calculates the expected effects of two components under the assumption that they do not enhance each other:

[0146]

number

[0147] Methods for treating Staphylococcus genus disease

[0152] This disclosure provides a method for treating conditions associated with the genus Staphylococcus, comprising administering a composition of this disclosure.

[0148]

[0153] The compositions disclosed herein can be used to treat subjects suffering from conditions associated with the Staphylococcus bacterial species as defined herein. In some embodiments, the subjects are animals. In some embodiments, the animals are mammals. In some embodiments, the subjects are humans.

[0149] situation

[0154] In some embodiments, the compositions of this disclosure are used in the treatment of a condition associated with the genus Staphylococcus. In some embodiments, the condition is a Staphylococcus infection.

[0150]

[0155] In some embodiments, the condition is associated with the skin. In some embodiments, the condition is a skin infection. In some embodiments, the condition is impetigo, cellulitis, folliculitis, atopic dermatitis, acute radiation dermatitis, acne, or abscess. In some embodiments, the condition is atopic dermatitis. In some embodiments, the condition is acute radiation dermatitis. In some embodiments, the condition is dry, itchy, and / or reddened skin. In some embodiments, the condition is dry skin. In some embodiments, the condition is itchy skin. In some embodiments, the condition is reddened skin.

[0151]

[0156] In some embodiments, the condition is a wound infection, pneumonia, food poisoning, toxic shock syndrome, bloodstream infection, urinary tract infection, bone or joint infection (e.g., osteomyelitis, septic arthritis), endocarditis, meningitis, sepsis, ear infection (e.g., otitis externa), eye infection (e.g., conjunctivitis, keratitis), sinus infection, gastroenteritis, mastitis, peritonitis, prosthesis infection, sternal wound infection, catheter-related infection, or tonsillitis. In some embodiments, the condition is a skin infection. In some embodiments, the condition is a soft tissue infection. In some embodiments, the condition is an opportunistic infection. In some embodiments, the condition is a wound infection. In some embodiments, the condition is a chronic wound.

[0152]

[0157] In some embodiments, the condition is the presence of Staphylococcus species in the environment, for example, on surfaces in a hospital. In some embodiments, the compositions herein are used as disinfectants to reduce the concentration or presence of Staphylococcus species in the environment.

[0153] Dosage

[0158] In some embodiments, the compositions of the Disclosure contain recombinant proteins disclosed herein, such as chimeric proteins, in a concentration of 0.1 to 100 μg / mL, encompassing all values ​​and partial ranges in between. In some embodiments, the compositions contain 0.5 to 50 μg / mL of the proteins of the Disclosure. In some embodiments, the compositions contain 1 to 25 μg / mL of the proteins of the Disclosure. In some embodiments, the compositions contain 2 to 15 μg / mL of the proteins of the Disclosure. In some embodiments, the compositions contain approximately 4 μg / mL of the proteins of the Disclosure.

[0154]

[0159] In some embodiments, the compositions of the present disclosure are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , comprising 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μg / mL of the protein of this disclosure.

[0155] Administration

[0160] For the purpose of administration, the compositions of the present invention can be formulated in various forms. The term “dosage form” refers to any form of formulation containing a sufficient amount of the chimeric protein of the present disclosure to achieve at least a partial therapeutic effect by single or repeated administration. In some embodiments, the dosage form is a topical dosage form. In some embodiments, the dosage form is a lotion, oil, hydrogel, ointment, or body balm. In some embodiments, the dosage form is a lotion. In some embodiments, the dosage form is a hydrogel.

[0156]

[0161] The compositions can be formulated in forms including, but are not limited to, liquids, gels, semi-solids, and solids. The compositions disclosed herein can be further processed into forms including, but are not limited to, solids, liquids, suspensions, gels, lotions, balms, and other forms discussed herein.

[0157]

[0162] In some embodiments, an effective dose of the composition is administered to the subject. The terms “effective dose” or “therapeutic dose” mean, but are not limited to, the amount of the composition described herein that is sufficient to achieve the intended application, including a reduction in the Staphylococcus population. The therapeutic dose may vary depending on the subject and condition being treated, e.g., the subject’s weight and age, the severity of the disease condition, the mode of administration, etc., and can be readily determined by those skilled in the art. The term also applies to the dose that will induce a specific response at the target site, e.g., a reduction in inflammation, pain, acne, fever, etc. The specific dose will vary depending on the particular formulation of the composition, whether or not it is administered in combination with other compounds, the subsequent drug regimen, the timing of administration, the tissue to which it is administered, the route of administration, and the physical delivery system through which it is carried.

[0158]

[0163] In some embodiments, compositions as disclosed herein are said to be active, functional, or therapeutically active, or capable of treating, preventing, and / or delaying conditions associated with the Staphylococcus genus, in that they reduce or improve one or more symptoms associated with the condition. In some embodiments, a composition is considered therapeutically active if it reduces the symptoms after treatment by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% compared to the severity of the symptoms before treatment. In some embodiments, a composition is considered therapeutically active if it reduces symptoms by up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% after treatment, compared to the severity of symptoms before treatment. In some embodiments, a composition is considered therapeutically active if it reduces symptoms by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% after treatment, including all values ​​and partial ranges in between, compared to the severity of symptoms before treatment. In some embodiments, symptoms include pain, fever, swelling, redness, dry skin, number of lesions, lesion size, rash, warmth, drainage, secretion, cough, shortness of breath, tachycardia, low or high heart rate, chills, nausea, vomiting, diarrhea, stomach cramps, chest pain, or organ failure.

[0159]

[0164] In some embodiments, the compositions herein are therapeutically active if they reduce the amount of a target Staphylococcus species present in a subject body or in vitro system. In some embodiments, the compositions herein are therapeutically active if about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less of the initial amount of Staphylococcus species are still detectable after treatment. In some embodiments, the compositions herein are therapeutically active if up to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% of the initial amount of Staphylococcus species are still detectable after treatment. In some embodiments, no Staphylococcus species are detectable after treatment. In this specification, the expression "amount of Staphylococcus species" means the amount of living Staphylococcus species. In some embodiments, Staphylococcus species are determined using sequencing techniques such as 16S sequencing or shotgun sequencing to quantify the amount of different Staphylococcus species present in a sample and to assess the bacterial species present throughout the microbiome in question. Staphylococcus species can also be detected using standard techniques known to those skilled in the art, such as immunohistochemistry using Staphylococcus-specific antibodies, test tube coagulase tests to detect staphylocoagulase or "free coagulase," clamping factors (slide coagulase tests), and / or detection of surface proteins such as protein A (commercial latex tests). Viable Staphylococcus species can be detected using standard techniques known to those skilled in the art, such as microbiological bacterial culture techniques and / or real-time quantitative reverse transcription polymerase chain reaction for assays with respect to bacterial mRNA. In some embodiments, the reduction is assessed in the tissue or cells of the individual or patient by comparison to the amount present in the individual or patient before treatment with the composition disclosed herein.In some embodiments, the comparison is made using the tissue or cells of the individual or patient who has not yet been treated with the compositions disclosed herein, if the treatment is topical.

[0160]

[0165] In some embodiments, application of the compositions herein improves the health, appearance, characteristics, texture, or feel of the skin. In some embodiments, application of the compositions herein reduces itching, redness, dryness, peeling, irritation, or pain of the skin. In some embodiments, application of the compositions herein reduces the severity or frequency of insomnia caused by skin problems. In some embodiments, application of the compositions herein to the skin once or twice daily over a period of 7 to 14 days reduces itching, redness, dryness, peeling, irritation, or pain of the skin in the subject who needs it. In some embodiments, application of the compositions herein to the skin once or twice daily over a period of 7 to 14 days reduces the severity or frequency of insomnia caused by skin problems.

[0161]

[0166] The compositions disclosed herein may be administered to subjects requiring them, or to cells, tissues, organs, or patients, for a period of at least one day, one week, one month, six months, one year, or longer. In some embodiments, the compositions herein are applied over a period of 1 to 30 days. In some embodiments, the compositions herein are applied over a period of 1 to 4 weeks. In some embodiments, the compositions herein are applied over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the compositions herein are applied until the condition improves. In some embodiments, the compositions herein are applied to maintain the condition.

[0162]

[0167] In some embodiments, the compositions herein are applied 1 to 10 times per day. In some embodiments, the compositions herein are applied 1 to 50 times per week. In some embodiments, the compositions herein are applied as needed. In some embodiments, the compositions herein are applied once or twice per day.

[0163]

[0168] Accordingly, compositions as disclosed herein are provided for use by subjects requiring such use. Preferably, the compositions are used pharmaceutically in the prevention, delay or treatment of a condition in a subject, where the condition is associated with an infection by a Staphylococcus species such as coagulase-positive or coagulase-negative Staphylococcus species.

[0164]

[0169] Further compositions as disclosed herein are provided for systemic or topical administration to a subject.

[0165]

[0170] Local administration can be used, for example, locally at the site of infection or implantation during surgery. The medical uses disclosed herein can be formulated as the product as disclosed herein for use as a pharmaceutically acceptable treatment of the specified conditions, but they can equally be formulated as a method of treating the specified conditions using the product as disclosed herein, the product as disclosed herein for use in the preparation of a pharmaceutically acceptable treatment of the specified conditions, and the use of the product as disclosed herein for the treatment of the specified conditions. All such medical uses are envisioned by this disclosure. Subjects requiring treatment, delay, and / or prevention of the enumerated conditions may be any animal subject, preferably a mammal, more preferably a pet such as a cattle, dog or cat, or a human subject.

[0166]

[0171] Further provided are compositions as disclosed herein, or nucleic acid constructs as disclosed herein, or expression constructs as disclosed herein, or in vitro use of host cells as disclosed herein, as antimicrobial agents or bactericidal agents.

[0167]

[0172] Further, the use of compositions as disclosed herein, or nucleic acid constructs as disclosed herein, or expression constructs as disclosed herein, or host cells as disclosed herein, or compositions as disclosed herein, for the detection of Staphylococcus species such as Staphylococcus aureus and Staphylococcus epidermidis in ex vivo diagnostic applications is provided. [Examples]

[0168] Example 1: Construction of a novel chimeric cell wall hydrolase

[0173] The inventors of this disclosure combined the cell wall hydrolases CBD and EAD to form a novel chimeric cell wall hydrolase.

[0169]

[0174] Sources of EAD and CBD. Chimeras were constructed using the following protein CHAP enzymatically active domains (EADs): LysSA12 EAD (SEQ ID NO: 11), Twort EAD (SEQ ID NO: 12), LysCSA13 EAD (SEQ ID NO: 13), and LysCSA5 EAD (SEQ ID NO: 14). Chimeras were constructed using the following endolysin / bacteriocin cell wall-binding domains (CBDs): LysH5 CBD (SEQ ID NO: 15), LysA72 CBD (SEQ ID NO: 16), PlySs2 CBD (SEQ ID NO: 17), ALE-1 CBD (SEQ ID NO: 18), LysSA97 CBD (SEQ ID NO: 19), and LysPALS1 CBD (SEQ ID NO: 20).

[0170]

[0175] Construction of chimeric cell wall hydrolases. The DNA sequences of EAD and CBD were codon-optimized and synthesized. Separate enzyme domains containing up to 50 aa upstream and downstream were synthesized using the NdeI / SpeI site. Separate cell wall-binding domains containing up to 25 aa upstream and downstream were synthesized using the SpeI / HindIII site. Chimeric CWHs were constructed by ligating both EAD and CBD to the NdeI / HindIII site of pET24a(+). The vector also included a C-terminal sequence encoding a 6×His protein tag used for protein purification. Chimeric enzymes consisting of one EAD and one CBD combination were constructed as shown in Table 3 below.

[0171] [Table 3-1]

[0172] [Table 3-2]

[0173] [Table 3-3]

[0174] [Table 3-4]

[0175]

[0176] Bacterial strains and culture conditions. Escherichia coli DH10B (Invitrogen®, Carlsbad, CA) was used for cloning and storage. Escherichia coli BL21 (DE3) (EMD Biosciences, San Diego, CA) was used for protein expression. All Escherichia coli strains were grown at 37°C in 2×YT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl) with shaking, or on plates containing LB (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract) supplemented with 2% (w / v) agar. 50 μg / mL kanamycin was used for proper selection of Escherichia coli clones.

[0176]

[0177] Protein production and purification. An expression vector containing a chimeric cell wall hydrolase was chemically transformed into an inducible BL21 E. coli expression vector for downstream protein expression and purification. BL21 cells containing the appropriate expression plasmid were grown overnight in 2×YT medium at 37°C with shaking. The following morning, the cells were re-diluted 1:1000 into a flask containing 50 mL of ZYM-5052 autoinducible medium (Fisher Scientific® catalog no. NC1093977) and incubated at 37°C for 2-3 hours with shaking. Subsequently, the flask was transferred to 22°C and incubated overnight with shaking. The culture was centrifuged, the supernatant was drained, and the pellet was stored at -80°C for at least 30 minutes. Each frozen pellet was resuspended in 5 mL of lysis buffer (NPI-10 (100 μM Tris pH 8, 300 mM NaCl, 10 mM imidazole) supplemented with 5 mg lysozyme and 100 units of DNASeI) and incubated at 30°C for 30 minutes with gentle shaking. Subsequently, the cells were centrifuged until a clear lysate was obtained and a solid pellet was formed. The clear lysate was transferred to a column containing nickel-NTA agarose resin (Gold Biotechnology®) suspended in NPI-10. The column was inverted several times to completely resuspend the resin, and then incubated at 4°C for at least 1 hour to allow protein binding. Once the resin had completely settled, the lysate was drained from the column and the column was washed with 2 column volumes of NPI-20 (100 μM Tris pH 8, 300 mM NaCl, 20 mM imidazole). Next, the protein was eluted by adding 3 mL of NPI-250 (100 μM Tris pH 8, 300 mM NaCl, 250 mM imidazole), and the fraction was collected. The protein was quantified and its purity confirmed by Bradford assay, SDS-PAGE gel, and Coomassie staining. Subsequently, the protein was concentrated using an Amicon® Ultra-15 filter unit and the buffer was changed to a protein storage buffer (50 mM Tris pH 6.8, 300 mM NaCl). For long-term storage, the protein was stored at -80°C with 30% glycerol.

[0177] Example 2: Experimental assay to test the anti-Staphylococcus activity of exemplary chimeras

[0178] Using the following materials and methods, the chimera of Example 1 was tested in various assays to determine its activity against different Staphylococcus species, as described in the Examples below.

[0178]

[0179] Bacterial strains and culture conditions. Following the purification of proteins in Example 2, chimeric enzymes were tested for lytic activity against Staphylococcus species as described in the following examples. All Staphylococcus strains were grown at 37°C with shaking in tryptic soy broth (TSB; BD Difco®, Franklin Lakes, NJ) or on TSB plates containing 2% (w / v) agar.

[0179]

[0180] Turbidity reduction assay. The lysis activity of chimeric cell wall hydrolases was assayed via a turbidity reduction assay as described in the Art. Briefly, the target Staphylococcus species was grown overnight in TBS at 37°C with shaking. In the morning, the cells were diluted again and grown in TBS at 37°C with shaking until the exponential growth phase (OD600 approximately 0.5) (approximately 2-3 hours). Subsequently, approximately 1 × 10⁶ per reaction were performed. 6Cells were mixed in a flat-bottom microtiter plate with a 2-fold dilution of purified protein (e.g., chimeric enzyme and / or control at exemplary concentrations of 12 μg / mL to 0.75 μg / mL) in a final volume of 200 μL PBS. Subsequently, the OD600 of each well was measured every 2–5 minutes using a microplate reader. The lysis activity of cell wall hydrolase resulted in a decrease in Staphylococcus cell integrity, which led to a decrease in OD600 readout. A more rapid decrease in optical density correlated with higher enzyme activity. Specific activity was calculated as described in Briers et al., "A standardized approach for accurate quantification of murein hydrolase activity in high-throughput assays," J Biochem Biophys Methods 2007;70(3):531–533, which is incorporated herein by reference.

[0180]

[0181] Thermal stability assay. To test the thermal stability of the proteins, aliquots of the proteins were incubated at temperatures ranging from 37°C to 54°C for 30 minutes. Subsequently, the proteins were immediately tested in the room-temperature turbidity reduction assay as described above. The activity results from the heat-exposed proteins were compared with each other to evaluate the ability of each enzyme to maintain activity after exposure to a certain range of temperatures, and the results at 37°C served as a positive control for enzyme activity.

[0181]

[0182] pH assay. To test the activity of enzymes over a range of pH values, cells and enzymes were resuspended in buffers at each pH value and subsequently tested in the turbidity reduction assay as described above. The buffers used were as follows: PBS pH 7.4; Tris pH 6.8 + 120 mM NaCl; Citrate buffer pH 6.5 + 120 mM NaCl; Citrate buffer pH 6.2 + 120 mM NaCl; Citrate buffer pH 5.7 + 120 mM NaCl; Citrate buffer pH 5.3 + 120 mM NaCl; Citrate buffer pH 4.9 + 120 mM NaCl; Citrate buffer pH 4.5 + 120 mM NaCl.

[0182]

[0183] Minimum Inhibitory Concentration (MIC) assay. The MIC of the protein was determined using the conventional microliquid dilution technique in TSB (CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 11th edition. CLSI standard M07. Clinical and Laboratory Standards Institute; 2018). Briefly, progressive 2-fold dilutions of the protein were used, with each well containing 1 × 10⁶ wells of the target Staphylococcus species. 6 The protein was added to microtiter plates containing cells. The minimum inhibitory concentration (MIC) was defined as the lowest protein concentration that inhibited bacterial growth visible after incubation at 37°C for 20-24 hours.

[0183]

[0184] Quantitative killing experiment. The antimicrobial activity of chimeric cell wall hydrolases was assayed via quantitative killing assays as described in the Art of this. Briefly, the target Staphylococcus species were grown overnight in TBS at 37°C with shaking. In the morning, the cells were diluted again and grown in TBS at 37°C with shaking until the exponential growth phase (OD600 approximately 0.5) was reached (approximately 2-3 hours). Subsequently, approximately 1 × 10⁶ cells were added per reaction. 6Cells were mixed with the desired amount of protein in a final volume of 200 μL of TSB medium and incubated at room temperature. At appropriate time points (e.g., time 0 and 2 hours), 20 μL of the reaction mixture was taken out, and serial dilutions were plated onto TSB agar plates and grown at 37°C for approximately 16 hours. Subsequently, the number of viable cells was calculated by counting the CFU.

[0184]

[0185] Checkerboard assay. A standard checkerboard assay was used to measure the potential synergistic activity resulting from the combination of chimeric CWH proteins. This assay measures the minimum inhibitory concentration (MIC) of the combined proteins in double serial dilutions. Briefly, in a 96-well microtiter plate, columns 1-11 contained double serial dilutions of protein A, and rows A-G contained double serial dilutions of protein B. Row H contained protein A alone, and column 12 contained protein B alone; these served as controls for measuring the MIC of protein A and B individually. Approximately 1 × 10⁻⁶ 6 Nine Staphylococcus aureus cells were added to each well in a final total volume of 200 μL of TSB. Visible growth of Staphylococcus aureus was assessed after incubation at 37°C for 20–24 hours. To quantify the efficacy of the antibiotic combinations in comparison to their individual activities, the fractional inhibitory concentration (FIC) index for each protein combination was calculated using the following equation:

[0185]

number

[0186] Example 3: LysSA12-PlySs2, LysSA12-LysA72, and LysSA12-ALE-1 chimeric CWH showed higher selectivity for Staphylococcus aureus compared to Staphylococcus epidermidis in MIC assays.

[0186] As described in Example 1, the inventors created a chimeric cell wall hydrolase by combining CWH CBD and EAD in a novel combination. MIC experiments were performed on the chimeric CWH containing LysSA12 EAD, which was fused to a set of six CBDs (PlySs2, LysA72, LysH5, PALS1, ALE-1, and LysA97) derived from CWH that have anti-Staphylococcus aureus activity. To test the activity of these enzymes against both Staphylococcus aureus and Staphylococcus epidermidis, MIC experiments were performed as described in Example 2.

[0187]

[0187] Results: The results of the minimum inhibitory concentration (MIC) assays against Staphylococcus aureus and Staphylococcus epidermidis are shown in Figure 1. All tested chimeric CWHs exhibited activity against Staphylococcus aureus. The MIC values ​​for Staphylococcus aureus ranged from 1 μg / mL to 50 μg / mL. The LysSA12 EAD+PALS1 CBD and LysSA12+LysSA97 chimeric proteins had equal MIC values ​​against both Staphylococcus species and therefore did not show selectivity for Staphylococcus aureus compared to Staphylococcus epidermidis. However, the LysSA12 EAD+LysH5 CBD chimera showed relatively lower selectivity for Staphylococcus aureus compared to Staphylococcus epidermidis (4×). In contrast, surprisingly, the LysSA12 EAD+PlySs2 CBD, LysSA12 EAD+LysA72 CBD, and LysSA12 EAD+ALE-1 CBD chimeric CWHs were strongly selective against Staphylococcus aureus compared to Staphylococcus epidermidis (32×~128×). These results demonstrate that not all active chimeric proteins containing LysSA12 EAD were selective against Staphylococcus aureus compared to Staphylococcus epidermidis, suggesting that while the LysSA12 domain lacks intrinsic selectivity for Staphylococcus aureus, some chimeric combinations containing this domain exhibit remarkable selectivity.

[0188] Example 4: Exemplary chimeric CWH selectivity arises from specific combinations of CBD and EAD.

[0188] To investigate whether the selectivity of chimeric CWHs in LysSA12 EAD+PlySs2 CBD, LysSA12 EAD+LysA72 CBD, and LysSA12 EAD+ALE-1 CBD is due to CBD, the inventors created a second set of chimeric proteins by fusing the same three types of CBD (PlySs2, LysA72, and ALE-1 CBD) to the EADs of three other CWHs with anti-Staphylococcus activity: Twort, LysCSA5, and LysCSA13. Similar to the EAD of LysSA12, the Twort, LysCSA5, and LysCSA13 EADs are classified as CHAP domains (cysteine-histidine-dependent aminohydrolase / peptidase).

[0189]

[0189] The second set of chimeric proteins was assayed in MIC experiments against both Staphylococcus aureus and Staphylococcus epidermidis, as described in Example 2.

[0190]

[0190] Results: The results of the MIC assay are shown in Figure 2. All nine chimeric proteins were active against both Staphylococcus aureus and Staphylococcus epidermidis, and showed no significant selectivity for either bacterial species compared to the other. This suggests that LysSA12 EAD, PlySs2 CBD, LysA72 CBD, and ALE-1 CBD are not intrinsically selective. Instead, selectivity arose from the specific combinations of LysSA12 EAD + PlySs2 CBD, LysSA12 EAD + LysA72 CBD, and LysSA12 EAD + ALE-1 CBD. The MIC and selectivity data are summarized in Figure 3, which illustrates that the selectivity was not an intrinsic property of any of the domains tested, but rather arose from specific, unique combinations.

[0191] Example 5: LysSA12-PlySs2, LysSA12-LysA72, and LysSA12-ALE-1 exhibit high activity and selectivity against Staphylococcus aureus in a turbidity reduction assay.

[0191] To further test the selectivity of LysSA12 EAD+PlySs2 CBD, LysSA12 EAD+LysA72 CBD, and LysSA12 EAD+ALE-1 CBD chimeric CWH, each chimera was tested for activity against both Staphylococcus aureus and Staphylococcus epidermidis in a turbidity reduction assay performed as described in Example 2. The Twort EAD+PlySs2 CBD chimera was included as a control because it showed nearly equivalent activity against both strains in the MIC experiment (Example 4). The Twort EAD+PlySs2 CBD chimera was tested at 12 μg / mL. LysSA12 EAD+PlySs2 CBD and LysSA12 EAD+ALE-1 CBD were tested at 6 μg / mL. LysSA12 EAD+LysA72 CBD was tested at 3 μg / mL.

[0192]

[0192] Results: The results of the turbidity reduction assay are shown in Figures 4A to 4D. Consistent with the results from the MIC assay, LysSA12 EAD+PlySs2 CBD, LysSA12 EAD+LysA72 CBD, and LysSA12 EAD+ALE-1 CBD chimeric CWH were found to have potent lytic activity against Staphylococcus aureus, as demonstrated by a larger and more rapid decrease in the optical density of Staphylococcus aureus in the reactant, but were far less active against Staphylococcus epidermidis. As a control, the Twort-PlySs2 chimera was found to have comparable activity against both Staphylococcus aureus and Staphylococcus epidermidis.

[0193] Example 6: Exemplary chimeras exhibit thermal stability up to 45-51°C.

[0193] The thermal stability of enzymes is a crucial factor in their potential commercial applications. In the case of enzymes targeting Staphylococcus aureus, many CWHs exhibit very limited heat resistance. To evaluate the thermal stability of the LysSA12 EAD+PlySs2 CBD, LysSA12 EAD+LysA72 CBD, and LysSA12 EAD+ALE-1 CBD chimeric CWHs, aliquots of each enzyme were tested in a thermal stability assay performed as described in Example 2, in which the chimeric CWHs were incubated at temperatures ranging from 37 to 54°C for 30 minutes, followed by testing in a standard turbidity reduction assay at room temperature. Twort EAD+LysA72 CBD was included for comparison.

[0194]

[0194] Results: The results of the thermal stability assay are shown in Figures 5A to 5D. The Twort-LysA72 chimera showed activity only after exposure to 37°C and became inactive after incubation at 45°C, thus exhibiting the lowest thermal stability. Both LysSA12-LysA72 and LysSA12-ALE-1 were more stable and retained activity at 45°C. LysSA12-LysA72 became inactive after incubation at 48°C, and LysSA12-ALE-1 became inactive after incubation at 50°C. LysSA12 EAD+PlySs2 CBD showed remarkable thermal stability, retaining full activity up to 51°C and showing limited activity even after incubation at 54°C.

[0195] Example 7: LysSA12 EAD+PlySs2 CBD shows selectivity against Staphylococcus aureus compared to several other Staphylococcus strains.

[0195] To confirm that the selectivity of LysSA12 EAD+PlySs2 CBD was consistent across a broader panel of Staphylococcus species, LysSA12 EAD+PlySs2 CBD was assayed in turbidity reduction assays against six Staphylococcus aureus strains, four Staphylococcus epidermidis strains, one S. hominis strain, and one S. simulans strain. The turbidity reduction assays were performed as described in Example 2.

[0196]

[0196] Results: The results of the turbidity reduction assay are shown in Figure 6. In all cases, the Staphylococcus aureus strains were susceptible to lysis with LysSA12 EAD + PlySs2 CBD, while the enzyme showed reduced activity against Staphylococcus epidermidis, S. hominis, and S. simulans strains.

[0197] Example 8: LysSA12 EAD + PlySs2 CBD chimera is superior to commercially available chimeric endolysins in terms of activity and selectivity.

[0197] SA.100 is a well-known, commercially available chimeric endolysin that has been shown to have selectivity for Staphylococcus aureus compared to Staphylococcus epidermidis (see Staphefekt®, www.staphefekt.com / en / ). The activity of LysSA12 EAD+PlySs2 CBD was compared with that of SA.100 in a turbidity reduction assay for Staphylococcus aureus performed as described in Example 2. A quantitative killing assay was also performed to compare the effects on the cell viability of Staphylococcus aureus and Staphylococcus epidermidis when incubated with SA.100 or LysSA12 EAD+PlySs2 CBD.

[0198]

[0198] Results: The results of the turbidity reduction assay are shown in Figures 7A and 7B. Relative activity was calculated by normalizing the activity of both enzymes relative to the SA.100 activity against Staphylococcus aureus. The activity of LysSA12 EAD+PlySs2 CBD was approximately 3.5 times higher than that of SA.100 (Figure 7B), indicating that LysSA12 EAD+PlySs2 CBD is significantly more effective than SA.100 in lysing Staphylococcus aureus.

[0199]

[0199] The results of the quantitative killing assay are shown in Figure 8. Two hours of incubation with SA.100 resulted in a two-order-order reduction in surviving Staphylococcus aureus cells. Notably, treatment with LysSA12 EAD+PlySs2 CBD for the same duration resulted in a four-order-order reduction in surviving Staphylococcus aureus, again demonstrating that LysSA12 EAD+PlySs2 CBD is significantly more potent than SA.100. Neither SA.100 nor LysSA12 EAD+PlySs2 CBD showed significant activity against Staphylococcus epidermidis in these quantitative killing assays, but LysSA12 EAD+PlySs2 CBD had a significantly larger selectivity ratio against Staphylococcus aureus compared to Staphylococcus epidermidis than SA.100.

[0200] Example 9: LysSA12 EAD+PlySs2 CBD exhibits pH tolerance down to a minimum of pH 5.

[0200] To evaluate the pH tolerance of LysSA12 EAD+PlySs2 CBD in comparison with commercially available SA.100 enzyme, each enzyme was tested in a pH assay as described in Example 2, and the enzymes were tested at the following pH values: 7.4, 6.8, 6.5, 6.2, 5.7, 5.3, 4.9, and 4.5.

[0201]

[0201] Results: The results of the pH assay are shown in Figure 9. Relative activity was calculated by normalizing all activity to the activity of SA.100 at pH 7.4. SA.100 lost all activity below pH 6.2. In contrast, LysSA12 EAD+PlySs2 CBD maintained significant activity across all tested pH values ​​and, in fact, showed higher activity than the maximum activity of SA.100 at all pH values. These results demonstrate that LysSA12 EAD+PlySs2 CBD has much higher pH tolerance than commercially available SA.100 enzyme.

[0202] Example 10: Selective activity of LysSA12 EAD+PlySs2 CBD chimera against Staphylococcus aureus in a 3D skin model of the skin microbiome.

[0202] Considering the potent Staphylococcus aureus-selective activity of the LysSA12 EAD+PlySs2 CBD chimeric protein in MIC, turbidity reduction, and quantitative killing assays, the action of this protein was tested against a 3D skin model of the skin microbiome. In this experiment, a mixture of four skin bacteria was colonized overnight on reconstituted human epidermis (RHE): Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium xerosis, and Propionibacterium acnes. Subsequently, the RHE was treated for 4 hours with a solution of LysSA12 EAD+PlySs2 CBD at 8 μg / mL, SA.100 at 32 μg / mL, or PBS alone. The RHE was gently washed three times, and then residual adherent bacterial cells were collected and quantified using quantitative PCR.

[0203]

[0203] Results: The results are shown in Figure 10. Treatment with LysSA12 EAD + PlySs2 CBD resulted in a three-fold greater reduction in adherent Staphylococcus aureus cells compared to SA.100. No significant differences were observed in the amounts of Staphylococcus epidermidis, C. xerosis, or Propionibacterium acnes among the three treatment groups, which is consistent with the results of previous examples and indicates that treatment with LysSA12 EAD + PlySs2 CBD results in a highly selective reduction of Staphylococcus aureus.

[0204] Example 11: Predictive synergistic anti-Staphylococcus aureus activity from a combination of a chimera containing M23 EAD and a chimera containing CHAP2 EAD.

[0204] The LysSA12 EAD+PlySs2 CBD chimeric protein contains a CHAP family EAD. The inventors hypothesize that LysSA12 EAD+PlySs2 CBD may function synergistically with selective enzymes containing EADs derived from different classes. To test this, a standard checkerboard assay is used to characterize the activity of combinations of the M23S1 EAD+LysH5 CBD (SEQ ID NO: 36) chimeric protein and the LysSA12 EAD+PlySs2 CBD (SEQ ID NO: 31) chimeric protein. The combinations of the two proteins are tested to identify combinations that inhibit the growth of Staphylococcus aureus and to calculate the FIC index of 0.5 or less, indicating synergistic activity between the two proteins.

[0205] Example 12: Predictive comparison of exemplary chimeras against innate cell wall hydrolases

[0205] LysSA12 EAD + PlySs2 CBD chimeric CWH is compared to natural LysSA12 and PlySs2 cell wall hydrolases to compare their species-specific activity in Staphylococcus bacteria.

[0206]

[0206] The limited or absent selectivity for either of the innate proteins suggests that the remarkable selectivity of the LysSA12 EAD+PlySs2 CBD chimeric enzyme is a characteristic arising from the combination of the two domains.

[0207] Example 13: LysSA12 EAD + PlySs2 CBD chimeric CWH showed potent selective activity against Staphylococcus aureus across multiple topical formulations. material and method

[0207] Formulation: LysSA12 EAD+PlySs2 CBD chimeric CWH from previous examples was prepared for topical formulations. Three topical formulations were prepared: a hydrogel formulation containing hyaluronic acid (Formulation #1), a hydrogel formulation containing hydroxymethylcellulose (Formulation #2), and a cream-based formulation containing cetereth-20 / ceteryl alcohol (Formulation #3). The two hydrogel formulations can be used to treat Staphylococcus infections while improving skin characteristics by combining the hydrating effect of hyaluronic acid or the texture-improving effect of hydroxymethylcellulose with the beneficial microbiome effects of CWH, while the cream-based formulation allows for the addition of lipid-soluble components to the formulation. Each formulation contained 4 μg / mL of LysSA12 EAD+PlySs2 CBD chimeric CWH. The formulations were stored at room temperature for 7 days and subsequently tested for antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis using a quantitative sterilization assay.

[0208]

[0208] Quantitative Killing Assay: The antimicrobial activity of chimeric cell wall hydrolase was assayed using a standard quantitative killing assay. Briefly, the target Staphylococcus species was grown overnight in TBS at 37°C with shaking. In the morning, the cells were rediluted and incubated in TBS at 37°C with shaking until the OD was approximately 0.5 600 The cells were allowed to grow to the exponential growth phase. Subsequently, approximately 1 × 10⁶ cells were added per reaction. 6Cells were mixed with 100 μL of the test formulation and incubated at room temperature. Upon selection, 20 μL of the reaction mixture was taken out, and serial dilutions were plated onto TSB agar plates and grown at 37°C for approximately 16 hours. Subsequently, the number of residual viable cells was calculated by counting the CFU.

[0209] result

[0209] In all three formulations, the number of viable CFUs of Staphylococcus aureus remaining after 2 hours of treatment with LysSA12-PlySs2 was reduced by 3 to 4 orders of magnitude compared to less than one order of magnitude for Staphylococcus epidermidis (Figures 11A to 11C). Therefore, all three formulations demonstrated potent selective killing of Staphylococcus aureus, showing the ability of LysSA12 EAD+PlySs2 CBD chimeric CWH to retain enzyme activity in various formulation types suitable for topical skin application.

[0210] Example 14: Long-term stability of LysSA12 EAD + PlySs2 CBD chimeric CWH in topical formulations

[0210] The long-term stability of enzyme activity in formulations at room temperature is an important characteristic for the commercial feasibility of topical skincare products. Accelerated life testing at high temperatures is a common method for determining the shelf life at room temperature. Stability at 45°C for 4 weeks is roughly equivalent to stability at room temperature for 5-6 months, based on the Arrhenius function.

[0211]

[0211] Aliquots of Formulation #2 from Example 13 were selected as exemplary formulations for stability testing. The formulations contained 4 μg / mL of LysSA12 EAD+PlySs2 CBD chimeric CWH and were stored at 45°C for 4 weeks. The formulations were tested for antimicrobial activity against Staphylococcus aureus at 1 week, 2 weeks, and 4 weeks using the quantitative killing assay from Example 13.

[0212]

[0212] The results are shown in Figure 12. The enzyme maintained significant activity at all time points tested, demonstrating the long-term stability of LysSA12 EAD+PlySs2 CBD chimeric CWH in exemplary topical skincare formulations.

[0213] Example 15: Clinical trial of topical application of LysSA12 EAD + PlySs2 CBD chimeric CWH demonstrates improvement in 9 skin parameters. material and method

[0213] 15.1. Participants

[0214]

[0214] A total of 15 male and female participants aged 18-65 years were recruited for this study. One participant dropped out of the study. Statistical analysis was performed on the intention-to-treat (ITT) population. All participants met the following inclusion and exclusion criteria.

[0215]

[0215] Inclusion criteria: • Male or female between the ages of 18 and 65. • Individuals with moderate to severe redness, dryness, and itchiness of the skin in at least one of the following areas: face, neck, front of the forearm, and inner elbow. • You are not currently using, and have not used in the past month, any oral / topical steroids, oral immunosuppressants, topical / oral antibiotics, antimicrobial baths, prescription creams, or other prescription medications to treat eczema. • You wish to avoid oral / topical steroids, oral immunosuppressants, topical / oral antibiotics, antimicrobial baths, or prescription creams during the 2-week study period. • You wish to discontinue and are able to discontinue systemic medication or therapy (e.g., oral medication, phototherapy, herbal medicine, or acupuncture) used to alleviate dry, itchy skin or eczema.

[0216]

[0216] Exclusion criteria: • Individuals in poor health. • Individuals with any chronic health condition, such as a tumor or mental disorder. • Individuals with severe allergic reactions requiring the use of an EpiPen. • Pregnant, breastfeeding, or planning to become pregnant within the next two weeks. • Individuals who are unable or unwilling to engage with the research protocol. • Individuals with a history of drug abuse. • Individuals who have undergone an invasive medical procedure within the three weeks prior to the study or who have a procedure planned during the duration of the study. Individuals with autoimmune skin diseases such as psoriasis, infectious skin diseases, ichthyosis, lupus erythematosus, or dermatomyositis.

[0217]

[0217] 15.2. Study Design and Intervention Procedures

[0218]

[0218] This practical study required participants to complete questionnaires at home and take before-and-after photographs of their skin. A consent form outlining the research process, instructions, evaluation methods, and bill of rights was provided to participants before they agreed to participate in the study. Following the consent process, participants took photographs of their affected skin and completed a baseline questionnaire that included answering research-specific questions about their skin.

[0219]

[0219] Participants were instructed to apply a LysSA12 EAD+PlySs2 CBD chimeric CWH topical formulation to the affected area in the morning and evening (before going to bed).

[0220]

[0220] Participants filled out questionnaires at the end of week 1 and week 2, and had their photos taken at the end of week 2.

[0221]

[0221] 15.3. Data Analysis and Statistics

[0222]

[0222] Data was collected from questionnaires using a written 5, 9, or 10-point Likert scale for each question. The written Likert data was converted to numerical values, and the scores from each question were analyzed independently. The data were checked for normality using the Pearson test. Based on the normality of the data, the data was analyzed using mixed-effects analysis or the Kruskal-Wallis test, with Dunnett or Dunn tests for multiple comparisons. Statistical analysis was performed using GraphPad Prism 10.0, with a significance level of 0.05. For product-specific questions not evaluated at baseline, the results were expressed as the percentage of subjects reporting each answer.

[0223] result

[0223] The effects of LysSA12 EAD + PlySs2 CBD chimeric CWH application on skin exhibiting redness, dryness, and itchiness, as well as related outcomes including pain, sleep, and mood, were measured via questionnaires completed at baseline, on day 7, and on day 14.

[0224]

[0224] Statistical analysis of the Day 7 questionnaire results revealed that participants experienced improvement in all nine tested parameters, with statistically significant improvements in six of them (Table 4 and Figures 13A-13I). This included significant reductions in the number of days participants experienced itching; the severity and frequency of insomnia caused by skin problems; skin dryness; skin roughness; and pain resulting from dry, red, and itchy skin.

[0225]

[0225] Statistical analysis of the 14-day questionnaire results revealed that after 14 days of applying the LysSA12 EAD+PlySs2 CBD chimeric CWH, participants experienced a statistically significant improvement in all 9 tested skin-related parameters (Table 4 and Figures 13A - 13I). This included a significant reduction in the severity of skin itching (from 5.80 to 1.86); the severity (from 5.27 to 1.29) and frequency (from 2.60 to 0.57) of insomnia caused by skin problems; skin dryness (from 2.13 to 1.00); skin roughness (from 2.20 to 1.14); and pain resulting from skin with dryness, redness, and itching (from 4.00 to 2.14).

[0226]

Table 4

[0227]

[0226] On the 14th day, participants answered an 18-item product recognition questionnaire, and the majority showed positive responses to all 18 items, with over 70% showing favorable responses to 14 items (Table 5). It should be noted that 85.71% of the participants agreed that the product effectively alleviated skin with dryness, redness, and itching, and that their skin had less redness and irritation since using it. Regarding their overall satisfaction with the product, 71.43% of the participants agreed that their skin was in the best condition in months, and 78.57% stated that they wanted to add the product to their normal skin care regimen. A significant 78.57% also agreed that they would recommend the product to friends and family.

[0228]

Table 5

[0229]

[0227] Photographs taken by participants at the start (day 0) and end (day 14) of the trial period also demonstrated exemplary improvements in skin conditions at various locations on the body, including the face (Figure 14A), hands (Figure 14B), and elbows (Figure 14C).

[0230] References

[0228] The following references are incorporated herein by reference in their entirety for all purposes. 1. Bonar E, Bukowski M, Chlebicka K, Madry A, Bereznicka A, Kosecka-Strojek M, Dubin G, Miedzobrodzki J, Mak P, Wladyka B. Human skin microbiota-friendly lysostaphin. International Journal of Biological Macromolecules. 2021;183:852-60. 2. Chang Y, Ryu S. Characterization of a novel cell wall binding domain-containing Staphylococcus aureus endolysin LysSA97. Applied microbiology and biotechnology. 2017;101:147-58. 3. Choi Y, Ha E, Kong M, Ryu S. A novel chimeric endolysin with enhanced lytic and binding activity against Clostridium perfringens. LWT. 2023 May 1;181:114776. 4. Duyvejonck L, Gerstmans H, Stock M, Grimon D, Lavigne R, Briers Y. Rapid and high-throughput evaluation of diverse configurations of engineered lysins using the VersaTile technique. Antibiotics. 2021;10(3):293. 5. Eichenseher F, Herpers BL, Badoux P, Leyva-Castillo JM, Geha RS, van der Zwart M, McKellar J, Janssen F, de Rooij B, Selvakumar L, Rohrig C. Linker-Improved Chimeric Endolysin Selectively Kills Staphylococcus aureus In Vitro, on Reconstituted Human Epidermis, and in a Murine Model of Skin Infection. Antimicrobial Agents and Chemotherapy. 2022;66(5):e02273-21. 6. Gutierrez D, Garrido V, Fernandez L, Portilla S, Rodriguez A, Grillo MJ, Garcia P. Phage lytic protein LysRODI prevents staphylococcal mastitis in mice. Frontiers in microbiology. 2020;11:7. 7. Gutierrez D, Rodriguez-Rubio L, Ruas-Madiedo P, Fernandez L, Campelo AB, Briers Y, Nielsen MW, Pedersen K, Lavigne R, Garcia P, Rodriguez A. Design and selection of engineered lytic proteins with Staphylococcus aureus decolonizing activity. Frontiers in Microbiology. 2021;12:723834. 8. Kost Y, Deutsch A, Mieczkowska K, Nazarian R, Muskat A, Hosgood HD, Lin J, Daily JP, Ohri N, Kabarriti R, Shinoda K. Bacterial Decolonization for Prevention of Radiation Dermatitis: A Randomized Clinical Trial. JAMA oncology. 2023 May 4. 9. Kost Y, Rzepecki AK, Deutsch A, Birnbaum MR, Ohri N, Hosgood HD, Lin J, Daily JP, Shinoda K, McLellan BN. Association of Staphylococcus aureus Colonization With Severity of Acute Radiation Dermatitis in Patients With Breast or Head and Neck Cancer. JAMA oncology. 2023 May 4. 10. Lee C, Kim J, Son B, Ryu S. Development of advanced chimeric endolysin to control multidrug-resistant Staphylococcus aureus through domain shuffling. ACS Infectious Diseases. 2021;7(8):2081-92. 11. Matsuzaki S, Rashel M, Uchiyama J, Sakurai S, Ujihara T, Kuroda M, Ikeuchi M, Tani T, Fujieda M, Wakiguchi H, Imai S. Bacteriophage therapy: a revitalized therapy against bacterial infectious diseases. Journal of infection and chemotherapy. 2005;11:211-9. 12. Obeso JM, Martinez B, Rodriguez A, Garcia P. Lytic activity of the recombinant staphylococcal bacteriophage ΦH5 endolysin active against Staphylococcus aureus in milk. International journal of food microbiology. 2008;128(2):212-8. 13. Son B, Kong M, Lee Y, Ryu S. Development of a novel chimeric endolysin, Lys109 with enhanced lytic activity against Staphylococcus aureus. Frontiers in Microbiology. 2021;11:615887. 14. Vermassen A, Leroy S, Talon R, Provot C, Popowska M, Desvaux M. Cell wall hydrolases in bacteria: insight on the diversity of cell wall amidases, glycosidases and peptidases toward peptidoglycan. Frontiers in microbiology. 2019;10:331. 15. WO 2004 / 020635 A1 16. WO 2009 / 024327 A2 17. WO 2019 / 105936 A1 WO 2007 / 130655 A2

[0231] Built-in by reference

[0229] All references, articles, publications, patents, patent gazettes, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, no reference, article, publication, patent, patent gazette, or patent application cited herein is, and should not be received as, an endorsement or suggestion, of constituting valid prior art or forming part of common general knowledge in any country of the world.

[0232] Numbered Embodiments of the Present Disclosure

[0230] Notwithstanding the attached claims, this disclosure illustrates the following numbered embodiments: 1.a) Enzymatically active domains (EADs) having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence of sequence number 11; and b)i) Sequence ID 16; ii) Sequence ID 17; or iii) Sequence ID 18 Cell wall-binding domains (CBDs) having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence of Chimeric cell wall hydrolase (CWH) containing this enzyme. 2. A chimeric CWH of Embodiment 1, wherein EAD contains the sequence of Sequence ID No. 11. 3. A chimeric CWH according to Embodiment 1 or Embodiment 2, comprising the sequence of Sequence ID No. 16. 4. A chimeric CWH according to Embodiment 1 or Embodiment 2, comprising the sequence of Sequence ID No. 17 for CBD. 5. A chimeric CWH according to Embodiment 1 or Embodiment 2, comprising the sequence of Sequence ID No. 18. 6. Chimeric cell wall hydrolase (CWH) containing a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 21. 7. A chimeric CWH of Embodiment 6, comprising the sequence of Sequence ID No. 21. 8. Chimeric cell wall hydrolase (CWH) containing a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with sequence number 22. 9. A chimeric CWH of Embodiment 8, comprising the sequence of Sequence ID No. 22. 10. Chimeric cell wall hydrolase (CWH) containing a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with sequence number 24. 11. A chimeric CWH of Embodiment 10, comprising the sequence of Sequence ID No. 24. 12. A chimeric CWH according to any one of embodiments 1 to 11, which is active against bacterial species of the genus Staphylococcus. 13. A chimeric CWH according to any one of embodiments 1 to 12, which is active against bacterial species of the genus Staphylococcus, and whose minimum inhibitory concentration (MIC) of activity is determined by an MIC assay. A chimeric CWH according to any one of Embodiments 1 to 13, which is active against bacterial species of the genus Staphylococcus with an MIC of 14.50 μg / mL or less. A chimeric CWH according to any one of Embodiments 1 to 14, which is active against bacterial species of the genus Staphylococcus with an MIC of 15.10 μg / mL or less. 16. Active against bacterial species of the genus Staphylococcus, including S. agnetis, S. argensis, S. argenteus, S. arlettae, Staphylococcus aureus, S. auricularis, S. capitis, S. caprae, S. carnosus, S. chromogenes, S. cohnii, S. condimenti, S. cornub iensis, S.delphini, S.devriesei, S.edaphicus, Staphylococcus epidermidis, S.equi, S.equorum, S.felis, S.fleurettii, S.gallinarum, S.haemolyticus, S.hominis, S.hyicus, S.intermedius, S.kloosii, S.lentus, S.lu gdunensis, S.lutrae, S.massiliensis, S.microti, S.muscae, S.nepalensis, S.pasteuri, S.petrasii, S.pettenkoferi, S.piscifermentans, S.pseudintermedius, S.pseudoxylosus, S.rostri, S.sacc The chimeric CWH of any one of embodiments 1-15, which is S. harolyticus, S. saprophyticus, S. schleiferi, S. schweitzeri, S. sciuri, S. simiae, S. simulans, S. stepanovicii, S. succinus, S. vitulinus, S. warneri, or S. xylosus. 17. A chimeric CWH according to any one of embodiments 1 to 16, which is active against Staphylococcus aureus. A chimeric CWH according to any one of Embodiments 1 to 17, which is active against Staphylococcus aureus with an MIC of 18.50 μg / mL or less. A chimeric CWH according to any one of Embodiments 1 to 18, which is active against Staphylococcus aureus with an MIC of 19.10 μg / mL or less. 20. A chimeric CWH according to any one of Embodiments 1 to 19, exhibiting selective activity against one bacterial species of the genus Staphylococcus compared to a second bacterial species of the genus Staphylococcus. 21. Any one of chimeric CWHs of Embodiments 1-20 that exhibits selective activity against coagulase-positive bacterial species (CoPS) of the genus Staphylococcus compared to coagulase-negative bacterial species (CoNS) of the genus Staphylococcus. 22. Any one of chimeric CWHs of Embodiments 1-21 that has at least 2-fold, at least 5-fold, or at least 10-fold selectivity against one bacterial species of the genus Staphylococcus compared to a second bacterial species of the genus Staphylococcus. 23. Any one of chimeric CWHs of Embodiments 1-22 that exhibits selective activity against Staphylococcus aureus compared to Staphylococcus epidermidis. 24. Any one of chimeric CWHs of Embodiments 1-23 that has at least 2-fold selectivity against Staphylococcus aureus compared to Staphylococcus epidermidis. 25. Any one of chimeric CWHs of Embodiments 1-24 that has at least 10-fold selectivity against Staphylococcus aureus compared to Staphylococcus epidermidis. 26. Any one of chimeric CWHs of Embodiments 1-25 that is active against bacterial species of the genus Staphylococcus and retains activity after exposure to a temperature of up to 45°C. 27. Any one of chimeric CWHs of Embodiments 1-26 that is active against bacterial species of the genus Staphylococcus and retains activity after exposure to a temperature of about 45°C. 28. Any one of chimeric CWHs of Embodiments 1-27 that is active against bacterial species of the genus Staphylococcus and retains activity after exposure to a temperature of about 45°C for about 4 weeks. 29. Any one of chimeric CWHs of Embodiments 1-28 that is active against bacterial species of the genus Staphylococcus and retains activity after exposure to a temperature of up to 50°C. 30. Any one of chimeric CWHs of Embodiments 1-29 that is active against bacterial species of the genus Staphylococcus and retains activity after exposure to a temperature of about 50°C. 31. Any one of the chimeric CWHs of Embodiments 26-30 that retains at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of activity. 32. A chimeric CWH according to any one of embodiments 1 to 31, which is active within the pH range of pH 6 to 8. 33. A chimeric CWH according to any one of embodiments 1 to 32, which is active within a pH range of pH 5 to 8. 34. A topical formulation containing a chimeric CWH according to any one of embodiments 1 to 33. 35. A topical formulation comprising a chimeric cell wall hydrolase (CWH) containing a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 21. 36. A topical formulation of Embodiment 35, wherein the chimeric CWH contains the sequence of Sequence ID No. 21. 37. A formulation according to any one of embodiments 34 to 36, which is a hydrogel, lotion, cream, or gel-cream. 38. A formulation according to any one of embodiments 34 to 37, which is a hydrogel. 39. A formulation according to any one of embodiments 34 to 37, which is a cream. 40. A formulation according to any one of embodiments 34 to 39, comprising a humectant. 41. Any one formulation of Embodiments 34 to 40, comprising a humectant selected from the list consisting of aloe vera, betaine, butylene glycol, caprylyl glycol, dimethicone, fructose, glucomannan, glucose, glycerin, glyceryl glucoside, honey, hyaluronic acid, lactic acid, panthenol, polyethylene glycol, propylene glycol, propanediol, sodium hyaluronate, sodium lactate, sodium pyrrolidone carboxylate, sorbitol, and urea. A formulation according to any one of Embodiments 34 to 41, comprising 42.0.1 to 50% w / v of a humectant. 43. A formulation according to any one of Embodiments 34 to 42, comprising 0.5-10% w / v of a humectant. 44. A formulation comprising any one of embodiments 34 to 43, comprising a cellulose polymer. 45. Any one of the formulations of Embodiments 34 to 44, comprising a cellulose polymer selected from the list consisting of hydroxyethylcellulose, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, microcrystalline cellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, and cellulose acetate. 46. ​​A formulation comprising a cellulose polymer, wherein the cellulose polymer is hydroxymethylcellulose, any one of embodiments 34 to 45. 47.0.1-20% w / v cellulose polymer formulation, any one of embodiments 34-46. 48. A formulation comprising any one of Embodiments 34 to 47, comprising 0.5 to 10% w / v cellulose polymer. A formulation according to any one of embodiments 34 to 48, comprising 49.1-5% w / v cellulose polymer. 50. A formulation according to any one of Embodiments 34 to 49, comprising a thickener, a gelling agent, and / or a polymer. 51. A formulation according to any one of embodiments 34 to 50, comprising a carbomer or an acrylate polymer. A formulation according to any one of Embodiments 34 to 51, comprising 52.0.05 to 5.0% w / v of a carbomer or acrylate polymer. 53. A formulation comprising a salt, one of any of embodiments 34 to 52. 54. Any one formulation of Embodiments 34 to 53, comprising a salt selected from the list consisting of calcium chloride, Dead Sea salt, Epsom salt, Himalayan pink salt, magnesium chloride, sea salt, and sodium chloride. A formulation according to any one of embodiments 34 to 54, containing a salt concentration of 55.10 to 500 mM. A formulation according to any one of embodiments 34 to 55, containing a salt concentration of 56.50 to 250 mM. 57. A formulation according to any one of embodiments 34 to 56, comprising a buffering agent. A formulation of any one of embodiments 34 to 57, comprising a buffer selected from the list consisting of 58.4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, acetic acid, ammonium acetate, boric acid, citric acid, glycine, phosphoric acid, potassium hydroxide, potassium phosphate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium hydroxide, sodium phosphate, sodium tetraborate, tris(hydroxymethyl)aminomethane, and trisodium phosphate. A formulation according to any one of embodiments 34 to 58, comprising 59.1 to 150 mM of buffering agent. A formulation according to any one of embodiments 34 to 59, comprising 60.5 to 50 mM of buffering agent. 61. A formulation comprising a surfactant, any one of embodiments 34 to 60. 62. Any one formulation of Embodiments 34 to 61, comprising a surfactant selected from the list consisting of ceteareth-20, cocamidopropyl betaine, cocoglucoside, decyl glucoside, decyl polyglucose, disodium laureth sulfosuccinate, glycereth-26, lauryl glucoside, lauryl polyglucose, sodium cocoyl glutamate, sodium cocoyl isethionate, sodium laureth sulfate, and sodium lauryl sulfate. A formulation according to any one of embodiments 34 to 62, comprising 63.0.1 to 20% w / v of a surfactant. A formulation according to any one of embodiments 34 to 63, comprising 64.1 to 10% w / v of surfactant. 65. A formulation according to any one of embodiments 34 to 64, comprising a free amino acid. 66. Any one of embodiments 34 to 65 comprising a free amino acid selected from the list consisting of alanine, arginine, cysteine, glutamine, glycine, histidine, lysine, methionine, proline, serine, and threonine. A formulation according to any one of embodiments 34 to 66, containing 67.5 to 500 mM of free amino acids. A formulation according to any one of embodiments 34 to 67, containing 68.10 to 250 mM of free amino acids. 69. A formulation comprising oil, any one of embodiments 34 to 68. 70. Any one formulation of Embodiments 34 to 69, comprising an oil selected from the list consisting of argan oil, avocado oil, baobab oil, camellia oil, carrot seed oil, coconut oil, evening primrose oil, grapeseed oil, hemp seed oil, jojoba oil, macadamia nut oil, marula oil, mineral oil, olive oil, pomegranate seed oil, raspberry seed oil, rosehip seed oil, squalane oil, sunflower seed oil, sweet pea oil, and tamanu oil. A formulation according to any one of Embodiments 34 to 70, comprising 71.0.1-20% w / v oil. A formulation according to any one of embodiments 34 to 71, containing 72.1 to 10% w / v oil. 73. A formulation comprising any one of embodiments 34 to 72, comprising alcohol. 74. Any one of the formulations of Embodiments 34 to 73, comprising an alcohol selected from the list consisting of cetyl alcohol, ethyl alcohol, isopropyl alcohol, and stearyl alcohol. A formulation according to any one of Embodiments 34 to 74, comprising 75.0.1 to 20% w / v alcohol. A formulation according to any one of embodiments 34 to 75, containing 76.1 to 10% w / v alcohol. 77. A formulation comprising glycerol, one of any one of embodiments 34 to 76. 78.0.A formulation according to any one of Embodiments 34 to 77, containing 50% w / v glycerol. A formulation according to any one of embodiments 34 to 78, containing 79.1-30% w / v glycerol. A formulation according to any one of embodiments 34 to 79, containing 80.1-5% w / v glycerol. 81. A formulation comprising petrolatum, any one of embodiments 34 to 80. A formulation according to any one of embodiments 34 to 81, comprising 82.10 to 99% w / v petrolatum. A formulation according to any one of embodiments 34 to 81, comprising 83.0.1-20% w / v petrolatum. 84. Any one of the formulations from Embodiments 34 to 83, which is thermally stable at 45°C for 4 weeks. 85. Any one formulation of Embodiments 34 to 84, which is thermally stable at 45°C for at least 4 weeks. 86. Any one of the formulations of Embodiments 34 to 85, which is thermally stable at 45°C for at least two months. 87. Any one formulation of Embodiments 34 to 86, which is thermally stable at 50°C for at least two months. A formulation according to any one of embodiments 34 to 87, which is active within a pH range of 88.6 to 8. A formulation according to any one of embodiments 34 to 88, which is active within a pH range of 89.5 to 8. 90. A method for treating a condition associated with the genus Staphylococcus, comprising the step of administering a composition comprising a chimeric CWH according to any one of Embodiments 1 to 33 or a formulation according to any one of Embodiments 34 to 89. 91. The method of Embodiment 90, wherein the composition or formulation is administered topically, enterally, or parenterally. 92. The method of Embodiment 90 or 91, wherein the composition or formulation is administered topically. 93. Any one of the embodiments 90 to 92, wherein the composition or formulation is administered 1 to 4 times every 1 to 7 days. 94. Any one of embodiments 90 to 93, wherein the composition or formulation is administered 1 to 4 times per day. 95. Any one of embodiments 90 to 94, wherein the composition or formulation is administered once or twice daily. 96. Any one of the embodiments 90 to 95, wherein the composition or formulation is administered over a period of 1 to 14 days. 97. Any one of embodiments 90 to 96, wherein the composition or formulation is administered over a period of 1 to 12 weeks. 98. Any one of the embodiments 90 to 97, wherein the composition or formulation is administered until the symptoms are resolved. 99. Conditions include S.agnetis, S.argensis, S.argenteus, S.arlettae, Staphylococcus aureus, S.auricularis, S.capitis, S.caprae, S.carnosus, S.chromogenes, S.cohnii, S.condimenti, S.cornubiensis, S.delphini, S.devr iesei, S.edaphicus, Staphylococcus epidermidis, S.equi, S.equorum, S.felis, S.fleurettii, S.gallinarum, S.haemolyticus, S.hominis, S.hyicus, S.intermedius, S.kloosii, S.lentus, S.lugdunensis, S.lutrae, S.ma Any one of the methods of Embodiments 90 to 98 relating to a bacterial species of the genus Staphylococcus selected from ssiliensis, S. microti, S. muscae, S. nepalensis, S. pasteuri, S. petrasii, S. pettenkoferi, S. piscifermentans, S. pseudinttermedius, S. pseudoxylosus, S. rostri, S. saccharolyticus, S. saprophyticus, S. schleiferi, S. schweitzeri, S. sciuri, S. simiae, S. simulans, S. stepanovicii, S. succinus, S. vitulinus, S. warneri, and S. xylosus. 100. Any one of the embodiments 90 to 99, wherein the condition is associated with Staphylococcus aureus. 101. Any one of the embodiments 90 to 100, wherein the condition is an infection by a bacterial species of the genus Staphylococcus. 102. Any one of the methods of Embodiments 90 to 101, wherein the condition is an overgrowth of a bacterial species of the genus Staphylococcus. 103. Any one of the embodiments 90 to 102, wherein the condition is dry skin, itchy skin, and / or red skin. 104. Any one of embodiments 90 to 102, wherein the condition is atopic dermatitis. 105. Any one of embodiments 90 to 102, wherein the condition is a wound infection. 106. Any one of the embodiments 90 to 102, wherein the condition is acute radiation dermatitis. 107. Any one of embodiments 90 to 102, wherein the condition is a chronic wound. 108. A method according to any one of Embodiments 90 to 107, comprising the step of applying a second topical formulation after administering a composition or formulation according to any one of Embodiments 1 to 89. 109. The method of Embodiment 108, wherein the second topical formulation is a hydrated formulation. 110. The method of Embodiment 108 or 109, wherein a second topical formulation restores the skin barrier. 111. Any one of embodiments 108 to 110, wherein the second topical formulation is a cream or a lotion. 112. Any one of the embodiments 108 to 111, wherein the second topical formulation is applied within 60 minutes of the application of the composition or formulation according to any one of embodiments 1 to 89. 113. Any one of the embodiments 108 to 112, wherein the second topical formulation is applied within 15 minutes of the application of the composition or formulation according to any one of embodiments 1 to 75.

Claims

1. a) Enzymatically active domains (EADs) having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 11; and b) i) Sequence ID 17; ii) Sequence ID 16; or iii) Sequence ID 18 Cell wall-binding domains (CBDs) having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the above. Chimeric cell wall hydrolases (CWHs) that include these.

2. The chimeric CWH according to claim 1, wherein EAD includes sequence number 11.

3. CBD, a) Sequence ID 17; b) Sequence ID 16; or c) Sequence ID 18 A chimeric CWH according to claim 1, including the above.

4. A chimeric cell wall hydrolase (CWH) comprising a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with sequence number 21.

5. The chimeric CWH according to claim 4, comprising the sequence of sequence number 21.

6. a) at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity, or 100% identity, with Sequence ID No. 22; and b) At least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with Sequence ID No. 24, or 100% identity. A chimeric cell wall hydrolase (CWH) containing a sequence having the following properties.

7. A chimeric CWH according to any one of claims 1 to 6, which is active against bacterial species of the genus Staphylococcus, and may have a minimum inhibitory concentration (MIC) of 50 μg / mL or less or 10 μg / mL or less, the MIC of which may be determined by an MIC assay.

8. Bacterial species of the genus Staphylococcus are S. agnetis, S. argensis, S. argensis. argenteus, S. arlettae, Staphylococcus aureus, S. auricularis, S. capitis, S. caprae, S. carnosus, S. chromogenes, S. cohnii, S. condimenti, S. cornubiensis, S. cornubiensis. delphini, S. devriesei, S. edaphicus, Staphylococcus epidermidis, S. equi, S. equirum, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis, S. hyicus, S. intermedius, S. kloosii, S. lentus, S. lugdunensis, S. lutrae, S. massiliensis, S. microti, S. Muscae, S. nepalensis, S. pasteuri, S. petrasii, S. Pettenkoferi, S. piscifermentans, S. pseudointermedius, S. pseudoxylosus, S. rostri, S. saccharolyticus, S. saprophyticus, S. schleiferi, S. Schweitzeri, S. sciuri, S. simiae, S. simulans, S. stepanovicii, S. succinus, S. vitulinus, S. warneri, or S. warneri. The chimeric CWH according to claim 7, which is P. xylosus.

9. The chimeric CWH according to claim 7, wherein the Staphylococcus species is Staphylococcus aureus, and the chimeric CWH may be active against Staphylococcus aureus with a MIC of 50 μg / mL or less or 10 μg / mL or less.

10. A chimeric CWH according to any one of claims 1 to 6, which exhibits selective activity against a first bacterial species of the genus Staphylococcus compared to a second bacterial species of the genus Staphylococcus, wherein the first bacterial species may be a coagulase-positive bacterial species (CoPS) ​​of the genus Staphylococcus and the second bacterial species may be a coagulase-negative bacterial species (CoNS) of the genus Staphylococcus, and which may have at least 2 times, at least 5 times, or at least 10 times the selectivity against the first bacterial species compared to the second bacterial species of the genus Staphylococcus.

11. The chimeric CWH according to claim 10, which exhibits selective activity against Staphylococcus aureus compared to Staphylococcus epidermidis, and may have at least twice or at least ten times the selectivity against Staphylococcus aureus compared to Staphylococcus epidermidis.

12. A chimeric CWH according to any one of claims 1 to 6, which is active against bacterial species of the genus Staphylococcus and retains its activity after exposure to a temperature of up to 45°C or up to 50°C.

13. A chimeric CWH according to any one of claims 1 to 6, which is active within a pH range of 6 to 8 or within a pH range of 5 to 8.

14. A topical formulation comprising the chimeric CWH described in claim 1.

15. A topical formulation comprising a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with Sequence ID No. 21, or a chimeric cell wall hydrolase (CWH) comprising the sequence of Sequence ID No.

21.

16. The formulation according to claim 14 or 15, which is a hydrogel, lotion, cream, or gel-cream, and may be a hydrogel.

17. The formulation according to claim 14 or 15, comprising a humectant, which may be selected from the list consisting of aloe vera, betaine, butylene glycol, caprylyl glycol, dimethicone, fructose, glucomannan, glucose, glycerin, glyceryl glucoside, honey, hyaluronic acid, lactic acid, panthenol, polyethylene glycol, propylene glycol, propanediol, sodium hyaluronate, sodium lactate, sodium pyrrolidone carboxylate, sorbitol, and urea.

18. The formulation according to claim 17, comprising 0.1 to 50% w / v of a humectant, and possibly comprising 0.5 to 10% w / v of a humectant.

19. The formulation according to claim 14 or 15, comprising a cellulose polymer, the cellulose polymer may be selected from the list consisting of hydroxyethylcellulose, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, microcrystalline cellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, and cellulose acetate.

20. The formulation according to claim 19, comprising 0.5 to 10% w / v of cellulose polymer, and possibly comprising 1 to 5% w / v of cellulose polymer.

21. The formulation according to claim 14 or 15, comprising a salt, the salt may be selected from the list consisting of calcium chloride, Dead Sea salt, Epsom salt, Himalayan pink salt, magnesium chloride, sea salt, and sodium chloride.

22. The formulation according to claim 21, comprising 10 to 500 mM of salt, and possibly comprising 50 to 250 mM of salt.

23. The formulation according to claim 14 or 15, comprising a buffer, the buffer may be selected from the list consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, acetic acid, ammonium acetate, boric acid, citric acid, glycine, phosphoric acid, potassium hydroxide, potassium phosphate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium hydroxide, sodium phosphate, sodium tetraborate, tris(hydroxymethyl)aminomethane, and trisodium phosphate.

24. The formulation according to claim 23, comprising 5 to 50 mM of buffering agent.

25. The formulation according to claim 14 or 15, comprising a surfactant, the surfactant may be selected from the list consisting of ceteareth-20, cocamidopropyl betaine, cocoglucoside, decyl glucoside, decyl polyglucose, disodium laureth sulfosuccinate, glycereth-26, lauryl glucoside, lauryl polyglucose, sodium cocoyl glutamate, sodium cocoyl isethionate, sodium laureth sulfate, and sodium lauryl sulfate.

26. The formulation according to claim 25, comprising 0.1 to 20% w / v of a surfactant, and possibly comprising 1 to 10% w / v of a surfactant.

27. The formulation according to claim 14 or 15, comprising a free amino acid, the free amino acid may be selected from the list consisting of alanine, arginine, cysteine, glutamine, glycine, histidine, lysine, methionine, proline, serine, and threonine.

28. The formulation according to claim 27, comprising 10 to 250 mM of free amino acids.

29. The formulation according to claim 14 or 15, comprising an oil, which may be selected from the list consisting of argan oil, avocado oil, baobab oil, camellia oil, carrot seed oil, coconut oil, evening primrose oil, grapeseed oil, hemp seed oil, jojoba oil, macadamia nut oil, marula oil, mineral oil, olive oil, pomegranate seed oil, raspberry seed oil, rosehip seed oil, squalane oil, sunflower seed oil, sweet pea oil, and tamanu oil.

30. The formulation according to claim 29, comprising 0.1 to 20% w / v oil.

31. The formulation according to claim 14 or 15, comprising an alcohol, the alcohol being selected from the list consisting of cetyl alcohol, ethyl alcohol, isopropyl alcohol, and stearyl alcohol.

32. The formulation according to claim 31, comprising 0.1 to 20% w / v alcohol, and possibly comprising 1 to 10% w / v alcohol.

33. The formulation according to claim 14 or 15, which contains glycerol, and may contain 0.5 to 50% w / v glycerol, 1 to 30% w / v glycerol, or 1 to 5% w / v glycerol.

34. The formulation according to claim 14 or 15, comprising petrolatum, which may contain 0.1 to 20% w / v petrolatum.

35. The formulation according to claim 14 or 15, which is heat-stable at 45°C or 50°C for at least four weeks or at least two months.

36. The formulation according to claim 14 or 15, which is active in a pH range of 6 to 8, and may also be active in a pH range of 5 to 8.

37. A method for treating a condition associated with the genus Staphylococcus, comprising the step of administering a first composition containing a chimeric CWH according to any one of claims 1 to 6, or a formulation according to claim 14 or 15.

38. The method according to claim 37, wherein the first composition or formulation is administered topically, enterally, or parenterally, and the composition or formulation may be administered topically.

39. The method according to claim 37, wherein the first composition or formulation is administered 1 to 4 times every 1 to 7 days, and the composition or formulation may be administered once or twice daily over a period of 1 to 12 weeks.

40. The method according to claim 37, wherein the first composition or formulation is administered until the symptoms are resolved.

41. The condition is S. agnetis, S. argensis, S. argensis. argenteus, S. arlettae, Staphylococcus aureus, S. auricularis, S. capitis, S. caprae, S. carnosus, S. chromogenes, S. cohnii, S. condition, S. cornubiensis, S. cornubiensis. delphini, S. devriesei, S. edaphicus, Staphylococcus epidermidis, S. equi, S. equirum, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis, S. hyicus, S. intermedius, S. kloosii, S. lentus, S. lugdunensis, S. lutrae, S. massiliensis, S. microti, S. Muscae, S. nepalensis, S. pasteuri, S. petrasii, S. Pettenkoferi, S. piscifermentans, S. pseudointermedius, S. pseudoxylosus, S. rostri, S. saccharolyticus, S. saprophyticus, S. The method according to claim 37, wherein the bacterial species is associated with a Staphylococcus species selected from schleiferi, S. schweitzeri, S. sciuri, S. simiae, S. simulans, S. stepanovicii, S. succinus, S. vitulinus, S. warneri, and S. xylosus, and the bacterial species may be Staphylococcus aureus.

42. The method according to claim 37, wherein the condition is infection by a bacterial species of the genus Staphylococcus and / or overgrowth of a bacterial species of the genus Staphylococcus.

43. The state is, a) Dry skin, itchy skin, and / or red skin; b) Atopic dermatitis; c) wound infection; d) Acute radiation dermatitis; and / or e) chronic wounds The method according to claim 37.

44. The method according to claim 37, comprising the step of applying a second topical formulation after administering a first composition or formulation.

45. The method according to claim 44, wherein the second topical formulation is a hydrated formulation and / or the second topical formulation restores the skin barrier.

46. The method according to claim 44, wherein the second topical preparation is a cream or lotion.

47. The method according to claim 44, wherein the second topical formulation is applied within 60 minutes of the application of the first composition or formulation, or the second topical formulation may be applied within 15 minutes of the application of the first composition or formulation.

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