Methods and compositions for reducing malodor

Abstract

The present technology relates generally to the field of reducing, preventing, or ameliorating the generation of undesirable body odors emanating from the skin. In particular, the present technology discloses ΔpatB-Staphylococcus lugdunensis bacterial strains, which have been modified to prevent the synthesis of volatile thioalcohols from precursor molecules commonly found in sweat, compositions comprising one or more of said strain, methods of genetically engineering said strain, methods for reducing body odor using said strain, and methods for altering a microbiome using said strain.

Description

METHODS AND COMPOSITIONS FOR REDUCING MALODORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 500,784, filed May 8, 2023, the contents of which are incorporated by reference in their entirety for any and all purposes.TECHNICAL FIELD

[0002] The present technology relates generally to the field of reducing, preventing, or ameliorating the generation of undesirable body odors emanating from the skin. In particular, the present technology provides ApatB -Staphylococcus lugdunensis bacterial strains, which have been modified to prevent the synthesis of volatile thioalcohols from precursor molecules commonly found in sweat.BACKGROUND

[0003] The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the compositions and methods disclosed herein.

[0004] Body odors, particularly those originating from sweat, represent a significant irritant for many humans, as evidenced by the multi-billion-dollar deodorant, body -wash, and perfume industries. Sweat, released from glands throughout the body, is a complex solution of salts, minerals, metabolites, and, potentially, pharmaceuticals or other exogenous compounds. Once secreted, sweat provides a nutrient filled solution for the skin microbiota to use for replication. Unfortunately, this replication, and the incumbent high levels of metabolic activity, results in the production of a number of undesirable byproducts, including volatile thioalcohols, which give off a distinct and unpleasant smell commonly referred to as body odor.

[0005] Existing technologies in the anti-body odor space possess a number of limitations. Antiperspirants, so named for their sweat-blocking properties, prevent the body from engaging in natural cooling mechanisms and can be environmentally costly to produce.Deodorants, meanwhile, do not prevent the generation of body odor, but merely conceal it with a more potent odor and, in some cases, provide antimicrobial ingredients to reduce bacterial activity and abundance. Both antiperspirants and deodorants are environmentally costly to manufacture, produce significant amounts of plastic waste, and typically require multiple applications over a period of days to retain efficacy. Accordingly, there is a need for more effective deodorizing technologies that do not pose the same negative environmental and health impacts as current anti-body odor products.SUMMARY

[0006] In one aspect, the disclosure of the present technology provides a genetically engineered Staphylococcus lugdunensis strain whose genome comprises a disruption of a patB gene. In some embodiments, the strain exhibits decreased expression of the patB gene as compared to a corresponding wild-type Staphylococcus lugdunensis strain. In some embodiments, the disruption prohibits transcription of a full-length wild-type mRNA from the disrupted patB gene. In some embodiments, the disruption prohibits expression of a functional PatB enzyme, inactivates the PatB protein, or has a negative effect on the expression of the patB gene. In some embodiments, the genetically engineered strain has decreased PatB enzyme activity as compared to a corresponding wild-type Staphylococcus lugdunensis strain. In some embodiments, the disrupted patB gene comprises a deletion of all or at least a portion of the gene, optionally wherein the deletion comprises deletion of the patB promoter. In some embodiments, the disrupted patB gene comprises an insertion of a gene cassette. In some embodiments, the disrupted patB gene comprises a deletion of at least a portion of the gene. In some embodiments, the disruption comprises a knockout of the patB gene. In some embodiments, the disruption comprises an allelic replacement knockout. In some embodiments, the disruption is introduced by homologous recombination, site-directed mutagenesis, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR base editing system, a CRISPR prime editing system, a CRISPR / Cpfl system, a CRISPR / Csml system, or any combination thereof. In some embodiments, the fitness of the strain comprising the disruption is not negatively affected. In some embodiments, the strain comprising the disruption has a faster growth rate as compared to a corresponding wild-type Staphylococcus lugdunensis strain. Insome embodiments, the strain comprises Staphylococcus lugdunensis deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 250423-01, or a variant strain thereof. In some embodiments, the strain has a reduced capacity to produce a thiol when contacted with a thiol substrate as compared to a corresponding wild-type strain. In some embodiments, the strain decreases the amount of thiol production by at least 50% when contacted with a thiol substrate as compared to a corresponding wild-type strain. In some embodiments, the thiol substrate is benzyl-cysteine. In some embodiments, the strain has a reduced capacity to produce 3-methyl-3- sulfonylhexan-l-ol (3M3SH) when contacted with apocrine sweat as compared to a corresponding wild-type strain. In some embodiments, the disruption is a deletion in the DNA sequence of the patB gene encoding a PatB protein, wherein the deletion inactivates the PatB protein or has a negative effect on expression of the atB gene.

[0007] In another aspect, the present disclosure provides, a Staphylococcus lugdunensis strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 250423-01, or a variant strain derived therefrom, wherein the variant strain is obtained using the deposited strain as starting material, and wherein a reduced capacity to produce a thiol alcohol when contacted with a thiol substrate as compared to a corresponding wild-type strain is retained or improved as compared to the deposited strain. In any of the preceding embodiments, the strain decreases the amount of thiol production by at least 50% when contacted with a thiol substrate as compared to a corresponding wild-type strain.

[0008] In another aspect, the present disclosure provides a composition comprising an effective amount of one or more of the genetically engineered Staphylococcus lugdunensis strains of any one of the preceding embodiments. In some embodiments, the one or more Staphylococcus lugdunensis strains is lyophilized. In some embodiments, the composition further comprises a lyoprotectant. In some embodiments, the lyoprotectant is selected from one or more of sucrose and micellar casein. In some embodiments, the one or more Staphylococcus lugdunensis strains is viable. In some embodiments, the one or more Staphylococcus lugdunensis strains is nonproliferative. In some embodiments, the composition further comprises Staphylococcus epidermidis. In some embodiments, thecomposition further comprises an excipient. In some embodiments, the excipient is selected from one or more of oleic acid, stearic acid, almond butter, avocado butter, babassu butter, beeswax, cocoa butter, coconut butter, coconut oil, cupuacu butter, hemp seed butter, kokum butter, macadamia butter, mango butter, mowrah butter, olive butter, sal seed butter, shea butter, propylene glycol, glycerin, stearyl alcohol, myristyl alcohol, and capric triglyceride. In some embodiments, the Staphylococcus lugdunensis strain is present at a concentration of about 105to about 1014bacteria per gram. In some embodiments, the Staphylococcus lugdunensis strain is present at a concentration of about 107to about 1011bacteria per gram. In some embodiments, the composition comprises about 90 wt. % to about 99.99 wt. % excipient. In some embodiments, about 10 wt. % to about 0.01 wt. % lyophilized Staphylococcus lugdunensis bacteria. In some embodiments, the composition comprises about 95 wt. % to about 99.75 wt. % excipient. In some embodiments, the composition comprises about 5 wt. % to about 0.25 wt. % lyophilized Staphylococcus lugdunensis bacteria. In some embodiments, the composition further comprises a fragrance selected from one or more of elettaria cardamomum seed oil, eucalyptus globulus seed oil, citrus aurantium bergamia fruit oil, mentha piperita oil, salvia sclarea oil, juniperus virginiana oil, abies sibirica oil, citrus aurantium amara, citrus aurantium dulcis lower oil, citrus nobilis oil, rosmarinus officinalis oil, melaleuca alternifolia oil, citrus limon tree oil, zingiber officinale root oil, Mangifera indica oil, Lavandula angustifolia oil, and Santalum album. In some embodiments, the composition is formulated for topical administration. In some embodiments, the composition is formulated for use as a topical deodorant. In some embodiments, the composition further comprises an antiperspirant. In some embodiments, the antiperspirant comprises one or more of aluminum salt, charcoal, and witch hazel. In some embodiments, the topical deodorant is formulated as a deodorant stick, a roll-on, a cream, a lotion, a gel, a powder, a water activated cream, a water activated powder, an injectable, a patch, or a spray.

[0009] In one aspect, the present disclosure provides a method of reducing 3-methyl-3- sulfonylhexan-l-ol (3M3SH) production from a skin microbiota of a subject, the method comprising applying a composition comprising the genetically engineered Staphylococcus lugdunensis strain of any one of the preceding embodiments to the skin of the subject or the composition of any one of the preceding embodiments to the skin of the subject.

[0010] In another aspect, the present disclosure provides a method for treating, reducing, or preventing malodor on the skin of a subject, comprising treating the skin of the subject with an effective amount of the composition of any one of the preceding embodiments. In some embodiments, the composition is applied topically to the skin of the subject. In some embodiments, the composition is applied to a sweat-gland-containing area of skin of the subject. In some embodiments, the composition is administered to the subject about 1-2 times per day for about 1-7 days. In some embodiments, the treated skin of the subject comprises a reduction in the amount of malodorous bacteria as compared to a corresponding untreated portion of skin. In some embodiments, the malodorous bacteria comprise wild-type Staphylococcus lugdunensis.

[0011] In one aspect, the present disclosure provides a method of producing a genetically engineered Staphylococcus lugdunensis strain, the method comprising targeting the patB gene for disruption. In some embodiments, the disruption is introduced into the patB gene by homologous recombination, site-directed mutagenesis, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR / Cpfl system, or a CRISPR / Csml system, and any combination thereof. In some embodiments, the disruption is introduced into the patB gene by homologous recombination. In some embodiments, the homologous recombination comprises transforming the Staphylococcus lugdunensis with a vector comprising a patB gene knockout cassette. In some embodiments, the vector is methylated. In some embodiments, the methylated vector is isolated from an E. coli methylation strain. In some embodiments, the strain exhibits decreased expression of the patB gene as compared to a corresponding wild-type Staphylococcus lugdunensis strain. In some embodiments, the disruption prohibits transcription of a full-length wild-type mRNA from the disrupted patB gene. In some embodiments, the disruption prohibits expression of a functional PatB enzyme. In some embodiments, the genetically engineered strain has decreased PatB enzyme activity as compared to a corresponding wild-type Staphylococcus lugdunensis strain. In some embodiments, the disrupted patB gene comprises a deletion of all or at least a portion of the gene. In some embodiments, the strain comprises Staphylococcus lugdunensis deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 250423-01, or a variant strain thereof. In some embodiments, the geneticallyengineered Staphylococcus lugdunensis strain has a reduced capacity to produce a thiol when contacted with a thiol substrate as compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least 50% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain has a reduced capacity to produce 3-methyl-3- sulfonylhexan-l-ol (3M3SH) when contacted with apocrine sweat and / or a 3M3SH precursor (e.g., Cys-Gly-3M3SH) as compared to a corresponding wild-type strain.

[0012] In another aspect, the present disclosure provides an altered microbiota comprising a genetically engineered Staphylococcus lugdunensis strain, or a variant strain thereof, whose genome comprises a disruption of a patB gene. In some embodiments, the microbiota has a reduced capacity to produce 3-methyl-3-sulfonylhexan-l-ol (3M3SH) when contacted with apocrine sweat as compared to a corresponding wild-type microbiota.

[0013] In one aspect, the present disclosure provides a deodorant composition comprising: (i) an effective amount of a viable Staphylococcus lugdunensis strain whose genome comprises a disruption of a patB gene; (ii) a lyoprotectant; and (iii) an excipient; wherein the composition is formulated as a water activated formulation for topical administration. In some embodiments, the Staphylococcus lugdunensis strain is present at a concentration of about 105to about 1014bacteria per gram. In some embodiments, the Staphylococcus lugdunensis strain is present at a concentration of about 107to about 1011bacteria per gram. In some embodiments, the deodorant composition comprises about 90 wt. % to about 99.99 wt. % excipient. In some embodiments, the deodorant composition comprises about 10 wt. % to about 0.01 wt. % lyophilized Staphylococcus lugdunensis bacteria. In some embodiments, the deodorant composition comprises about 95 wt. % to about 99.75 wt. % excipient. In some embodiments, the deodorant composition comprises about 5 wt. % to about 0.25 wt. % lyophilized Staphylococcus lugdunensis bacteria. In some embodiments, the Staphylococcus lugdunensis strain comprises the strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 250423-01.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a sequenced verified plasmid map for pIMAY-A / / / 7>. The assembled plasmid contains regions flanking Staphylococcus lugdunensis patB, which allow for the removal of patB through controlled stepwise recombination events. The full plasmid nucleotide sequence is set forth in SEQ ID NO: 1.

[0015] FIG. 2 is a graphical overview of plasmid methylation for use in S. lugdunensis. The SL01B E. coli pass-through strain was originally developed by the Heilbronner Lab (Heilbronner S, et al, Sortase A promotes virulence in experimental Staphylococcus lugdunensis endocarditis. Microbiology (Reading). 2013 Oct; 159 (Pt 10):2141 -2152). This strain expresses the S. lugdunensis hsdM / hsdS methylation genes. Passing a plasmid through the SL01B strain provides the appropriate plasmid methylation signature to avoid destruction by the endogenous restriction modification system. The methylated plasmid can then be used directly for electroporation of S. lugdunensis.

[0016] FIG. 3 is an image of a gel showing successful transformation of pIMAY- EpatB plasmids into E. coli SL01B. Four SL01B transformants were miniprepped and digested with BstBI. BstBI cuts once in pIMAY (creating one 5700 bp fragment) and twice in pIMAY-A / / / 7> (creating 4735 bp and 2096 bp fragments). Each sample was a different transformant colony. This can be seen clearly in samples #2.1, #2.2 and #2.4, indicating that these were successful transformations with the correctly assembled pIMAY- EpatB. Sample #2.2 was subsequently used to mass-produce methylated pIMAY-A / / f.

[0017] FIG. 4 is a chart showing that the electroporation of S. lugdunensis yielded an efficiency of 34 cells per ug plasmid DNA.

[0018] FIG. 5 is a schematic showing the selection for excision of the integrated plasmid. Sensitive clones are delineated by the outlined square (1). These clones have become sensitive to chloramphenicol (Cm), evidenced by their inability to grow on TSA Cm 10. All of these clones were subsequently re-streaked on TSA Cm 10 to confirm their susceptibility (2) before being sent for amplicon sequencing (3).

[0019] FIG. 6 is a diagram showing results of amplicon sequencing to confirm that a single homologous recombination event between the plasmid and the bacterial genome had occurred, deleting a portion of the patB gene.

[0020] FIG. 7 is a graph of the growth curves for WT S. lugdunensis (WT patB) and ^patB-S. lugdunensis (KO patB) grown separately in triplicate, demonstrating that ^patB-S. lugdunensis grows faster than wild-type S. lugdunensis.

[0021] FIG. 8 is a graph of the production of thiol from benzyl-cysteine over time by wild-type S. lugdunensis (WT) and UspatB-S. lugdunensis (KO). A412 absorption, indicating benzyl-cysteine cleavage, is normalized to a no substrate control sample for each strain (AA412).

[0022] FIG. 9 is a bar graph of the production of 3M3SH by wild-type S. lugdunensis (WT), UspatB-S. lugdunensis (KO), and a cell-free control.

[0023] FIG. 10 is a line graph showing the viability (log CFU / g) of lyophilized UspatB-S. lugdunensis over time.DETAILED DESCRIPTION

[0024] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.I. Definitions

[0025] The following terms are used herein, the definitions of which are provided for guidance.

[0026] As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0027] The term “about” and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the present technology. As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0028] As used herein, “administration” of an agent, drug, bacterial strain(s) or composition of the present technology to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including topically. In some embodiments, the compositions of the present technology are formulated for topical administration. In some embodiments, the compositions are formulated as a stick, a roll-on, a cream, a lotion, a gel, a powder, a water activated formulation (e.g., a water activated cream or a water activated powder), an injectable, a patch, or a spray. In some embodiments, the compositions are formulated for use as a probiotic. As used herein, administration includes self-administration and administration by another.

[0029] As used herein, “ Staphylococcus lugdunensis TaxaOne,” or “5. lugdunensis TaxaOne” refers to a bacterial strain having been deposited under ID AC Accession No. 250423-01, or variant strain thereof, or compositions comprising the strain. S. lugdunensis TaxaOne is considered a member of Staphylococcus lugdunensis.

[0030] As used herein, “ ApatB-Staphylococcus lugdunensis" or “ApatB-S. lugdunensis" refers to a Staphylococcus lugdunensis whose genome comprises a disruption of a patB gene or compositions comprising the ApatB-Staphylococcus lugdunensis. In some embodiments, the ApatB-Staphylococcus lugdunensis exhibits a reduced PatB activity and / or patB expression. In some embodiments, the ApatB-Staphylococcus lugdunensis has a reduced capacity to produce thiol and / or 3M3SH when contacted with an appropriate substrate and / or precursor. In some embodiments, ApatB-Staphylococcus lugdunensiscomprises Staphylococcus lugdunensis TaxaOne having been deposited under IDACAccession No. 250423-01, or variant strain thereof, or compositions comprising the strain.

[0031] As used herein, “variant” refers to a strain derived from a strain of the present technology by any means, such as, but not limited to, genetic engineering, radiation and / or chemical treatment, and / or selection, adaptation, screening, etc. In some embodiments, the variant is naturally occurring. In some embodiments, the variant is selected for or engineered. In some embodiments, the variant is a functionally equivalent variant, e.g., a variant that has the same, or improved, properties with respect to its capacity to produce a thiol or 3M3SH when contacted with an appropriate substrate and / or precursor as the mother strain. Such variants are a part of the present technology. In some embodiments, the term “variant” refers to a strain obtained by subjecting a strain of the present technology to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMPS) or A-methyl-N’-nitro-N-nitroguanidine (NTG), UV light, or to a spontaneously occurring variant. In embodiments in which variants are subjected to mutagenization, one of skill in the art may refer to the variant as a mutant. A variant may have been subjected to several mutagenization treatments (a single treatment should be understood as one mutagenization step followed by a screening / selection step). In some embodiments, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of nucleotides in the bacterial genome have been changed (such as by replacement, insertion, deletion, or a combination thereof) compared to the mother strain. Such variants, which may be identified by using appropriate screening techniques, are a part of the present technology.

[0032] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention or treatment of a condition, and / or symptom(s) thereof. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the condition and on the characteristics of the subject, such as general health, age, sex, body weight, and lifestyle. It will also depend on the degree, severity, and type of condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, a singledose is administered. In some embodiments, multiple doses are administered. Additionally or alternatively, in some embodiments, multiple therapeutic compositions or compounds (e.g., pharmaceutical compositions comprising multiple bacterial strains alone or in combination with additional active agents, such as deodorizing or anti-perspiring agents). In the methods described herein, compositions comprising the bacterial strains of the present technology may be administered to a subject having, or at risk of having, body odor. In some embodiments, the compositions comprising the bacterial strains of the present technology may be administered to multiple locations on the body of a subject. For example, an “effective amount” of the compositions of the present technology, includes levels at which the presence, frequency, or severity of body odor is at a minimum reduced or ameliorated. In some embodiments, an effective amount reduces or ameliorates the presence of body odor, and / or the likelihood of developing body odor. In some embodiments, an effective amount is achieved by multiple administrations. In some embodiments, an effective amount is achieved with a single administration.

[0033] As used herein, the terms “freeze-dried” or “freeze-drying” and “lyophilized” or “lyophilization” are used interchangeably and refer to a process that removes water from a product after it is frozen and placed under a vacuum and the products produced therefrom.

[0034] As used herein, the term “lyoprotectant(s)” refers to an agent that prevents or ameliorates any damage caused to a product during the lyophilization process. In some embodiments, the lyoprotectant is a protein, a carbohydrate, or a combination thereof. In some embodiments, the lyoprotectant is a milk protein, and in particular a micellar casein. In some embodiments, the carbohydrate is sucrose. In some embodiments, the lyoprotectant prevents or ameliorates any damage caused to a bacterial strain of the present technology during the lyophilization process.

[0035] As used herein, “acceptable carrier and / or diluent” or “acceptable excipient” includes but is not limited to any combination of solvents, dispersion media, coatings, antifungal agents, isotonic and absorption delaying agents, and the like. In some embodiments, the acceptable carrier comprises oleic acid, stearic acid, almond butter, avocado butter, babassu butter, beeswax, cocoa butter, coconut butter, coconut oil, cupuacu butter, hemp seed butter, kokum butter, macadamia butter, mango butter, mowrah butter,olive butter, sal seed butter, shea butter, propylene glycol, glycerin, stearyl alcohol, myristyl alcohol, capric triglyceride, or any combination thereof. The use of such agents with biologically active substances is well known in the art. Further details of excipients are provided below. Supplementary active ingredients, such as antifungal agents, antiperspirants, fragrances, and deodorants, can also be incorporated into the compositions.

[0036] As used herein, “acceptable excipient” or “excipient” refers to substances and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or a human. As used herein, the term includes all inert, non-toxic, liquid or solid fillers, or diluents that do not react with the strains of the present technology in an inappropriate negative manner, including solvents, dispersion media, coatings, antifungal agents, isotonic and absorption delaying agents, preservatives and the like, for example liquid pharmaceutical carriers e.g., sterile water, saline, sugar solutions, Tris buffer, ethanol and / or certain oils.

[0037] As used herein, “deodorant” refers to a substance which removes and / or conceals an unpleasant smell, such as body odor. In some embodiments, the strains of the present technology are formulated as deodorant compositions. In some embodiments, the deodorant compositions further comprise a fragrance. In some embodiments, the fragrance comprises one or more of elettaria cardamomum seed oil, eucalyptus globulus seed oil, citrus aurantium bergamia fruit oil, mentha piperita oil, salvia sclarea oil, juniperus virginiana oil, abies sibirica oil, citrus aurantium amara, citrus aurantium dulcis lower oil, citrus nobilis oil, rosmarinus officinalis oil, melaleuca alternifolia oil, citrus limon tree oil, zingiber officinale root oil, Mangifera indica oil, Lavandula angustifolia oil, and Santalum album. In some embodiments, the strains and compositions of the present technology are administered with a fragrance, either simultaneously or sequentially and either as separate formulations or a single formulation.

[0038] As used herein “antiperspirant” refers to a substance that inhibits, reduces, or prevents the release of sweat from glands. In some embodiments, the strains and compositions of the present technology are administered with an antiperspirant. In some embodiments, the strains of the present technology are formulated as a deodorant that furthercomprises an antiperspirant. In some embodiments, the antiperspirant comprises one or more of aluminum salt, charcoal, and witch hazel.

[0039] The terms “disruption” and “mutation” are used interchangeably herein to refer to a detectable and heritable change in the genetic material. Disruptions or mutations may include insertions, deletions, substitutions (e.g., transitions, transversion, homologous recombination), transpositions, inversions, knockouts and combinations thereof. For example, in some embodiments, a disruption or mutation may include: (a) the introduction, substitution, or removal of one or more nucleotides in a Staphylococcus lugdunensis patB gene (or an open reading frame (ORF) thereof), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the patB gene or ORF thereof; (b) a patB gene conversion; (c) a patB gene deletion; (d) the down-regulation of the patB gene; (e) specific mutagenesis of the patB gene; and / or (f) random mutagenesis of the patB gene. Disruptions or mutations of the patB gene, as used herein, include any genetic modification that prevents or reduces the capability of the S. lugdunensis strains of the present technology from producing a functional patB gene product. Such disruptions or mutations may include complete or partial deletion of any portion of patB, including a polypeptide-coding sequence, a promoter, an enhancer, a ribosome-binding site (RBS), or other regulatory elements, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and any combinations and variations thereof that inactivate patB and prevent or reduce the production of a patB functional gene product. Mutations or disruptions may involve only a single nucleotide (e.g., a point mutation or a single nucleotide polymorphism) or multiple nucleotides. In some embodiments, the mutation or disruption causes a phenotypic change, for example, the expression level of the encoded product is altered, or the encoded product itself is altered. In some embodiments, a disruption or mutation may result in a disrupted gene with decreased levels of expression of a gene product (e.g., protein or RNA) as compared to the wild-type strain. In other embodiments, a disruption or mutation may result in an expressed protein with activity that is lower as compared to the activity of the expressed protein from the wildtype strain. In some embodiments, a gene disruption or mutation results in the removal of the gene from the genome via a substitution, such as homologous recombination.

[0040] The term “engineered” or “genetically engineered” is used herein to refer to an organism that has been manipulated to be genetically altered, modified, or changed, e.g., by disruption / editing of the genome. For example, an “engineered” or “genetically engineered” bacterial strain encompasses a bacterial strain that has been manipulated to be genetically altered, modified, or changed.

[0041] As used herein, the term “fitness” or “bacterial fitness” refers to the capacity of a bacterium, bacterial strain, or bacterial species to survive and reproduce in a given environment.

[0042] A “knocked out gene” or a “gene deletion” refers to a gene including a null mutation (e.g., the wild-type product encoded by the gene is not expressed, expressed at levels so low as to have no effect, or is non-functional). In some embodiments, the knocked out gene includes heterologous sequences or genetically engineered non-functional sequences of the gene itself, which renders the gene non-functional. In other embodiments, the knocked out gene is lacking a portion of the wild-type gene. For example, in some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 60% of the wild-type gene sequence is deleted. In other embodiments, the knocked out gene is lacking at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95% or at least about 100% of the wild-type gene sequence. In other embodiments, the knocked out gene may include up to 100% of the wild-type gene sequence (e.g., some portion of the wild-type gene sequence may be deleted) but also include one or more heterologous and / or non-functional nucleic acid sequences inserted therein.

[0043] The term “gene cassette” is used herein to refer to a DNA sequence encoding and capable of expressing one or more genes of interest (e.g., a metabolic gene, a selectable marker, or a combination thereof) that can be inserted between one or more selected restriction sites of a DNA sequence. In some embodiments, insertion of a gene cassette results in a disrupted gene. In some embodiments, disruption of the gene involves replacement of at least a portion of the gene with a gene cassette, which includes a nucleotide sequence encoding a gene of interest (e.g., a metabolic gene, a selectable marker, or a combination thereof).

[0044] As used herein, “probiotic” refers to bacteria that supplements or replaces elements of endogenous flora of a subject, which when administered to the subject confers a beneficial prophylactic and / or therapeutic effect on the subject. In some embodiments, the beneficial effect is a reduction or prevention in malodor on the skin of a subject.

[0045] As used herein, “prevention,” “prevent,” or “preventing” of a condition refers to, in a statistical sample, reduction in the occurrence or recurrence of the condition in treated subjects / samples relative to an untreated controls, or refers to delays in the onset of one or more symptoms of the condition relative to the untreated controls. In some embodiments, the condition is malodor (body odor) on the skin of the subject.

[0046] As used herein, to “reduce” or “reducing” a condition refers to results that, in a sample or specific subject, make the occurrence of the condition better or more tolerable in a sample or subject administered a therapeutic agent (e.g., a ApatB -Staphylococcus lugdunensis strain or compositions comprising the strain) relative to a control sample or subject. In some embodiments, the condition is malodor (body odor) on the skin of the subject.

[0047] As used herein “subject” and “patient” are used interchangeably. In some embodiments, the subject is an animal subject. In some embodiments, the animal subject is a mammal. In some embodiments, the mammalian subject is a human.

[0048] As used herein, the term “simultaneous” administration refers to the administration of at least two agents by the same route and at the same time or at substantially the same time.

[0049] As used herein, the term “separate” administration refers to an administration of at least two agents at the same time or at substantially the same time by different routes.

[0050] As used herein, the term “sequential” administration refers to administration of at least two agents at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one agent before administration of the other agent(s) commences. It is thus possible to administer one of the agents over several minutes, hours, or days before administering another.

[0051] A “synergistic effect” refers to a greater-than-additive effect which is produced by a combination of at least two agents, and which exceeds that which would otherwise result from the individual administration of the agents. For example, use of bacterial strain(s) of the present technology in conjunction with other agents for deodorizing or anti-perspiring may result in a greater than additive effect. In some embodiments, the synergistic effect may permit the use of lower doses of bacterial strain(s) of the present technology and / or other agents than would be required if each were used alone.

[0052] “Treating,” “treat,” “treated,” or “treatment” of a condition includes: (i) inhibiting the condition, z.e., arresting its development; (ii) relieving the condition, z.e., causing its regression; (iii) slowing progression of the condition; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the condition. In some embodiments, the condition is malodor (body odor) on the skin of the subject.

[0053] It is to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.II. Sweat and Body Odor

[0054] The armpit offers two microbial habitats: the skin surface and the skin subsurface. The skin surface is the smaller of the two skin microbial habitats, with about 2 m2of surface area. It is also a harsher environment for bacteria as it is frequently disinfected through washing and deodorant use. The skin subsurface has a much larger surface area of about 24 m2and includes the epidermis, dermis, hair follicles, and sweat glands, all of which harbor skin bacteria (Gallo RL. Human Skin Is the Largest Epithelial Surface for Interaction with Microbes. J Invest Dermatol. 2017 Jun; 137(6): 1213-1214. doi:10.1016 / j.jid.2016.11.045. Epub 2017 Apr 8. PMID: 28395897; PMCID: PMC5814118; Nakatsuji T, Chiang HI, Jiang SB, Nagarajan H, Zengler K, Gallo RL. The microbiome extends to subepidermal compartments of normal skin. Nat Commun. 2013;4: 1431. doi: 10.1038 / ncomms2441. PMID: 23385576; PMCID: PMC3655727). The hair follicle provides a stable, moist, well-perfused, and UV light-protected environment. Sweat glands interfacewith both of these habitats and produce sweat, which bacteria feed on. Sweat glands can be categorized into apocrine glands and eccrine glands, although several additional types exist.

[0055] Eccrine glands open onto the skin surface and produce eccrine sweat, a saline solution that contains trace chemicals originating from interstitial fluid, such as potassium, bicarbonate, lactate, urea, amino acids, and calcium. This sweat causes perspiration (Groscurth P. Anatomy of sweat glands. Curr Probl Dermatol. 2002;30: 1-9. Doi: 10.1159 / 000060678. PMID: 12471693). Apocrine glands open into the very upper hair follicle instead of the skin surface and produce apocrine sweat, a thick, sterile, lipid-rich, odorless fluid containing proteins, sugars, ammonia, steroids, and notably, the chemical precursors for body odor (Baker LB. Physiology of sweat gland function: The roles of sweating and sweat composition in human health. Temperature (Austin). 2019 Jul 17;6(3):211-259. doi: 10.1080 / 23328940.2019.1632145. PMID: 31608304; PMCID: PMC6773238; SanMiguel A, Grice EA. Interactions between host factors and the skin microbiome. Cell Mol Life Sci. 2015 Apr;72(8): 1499-515. doi: 10.1007 / s00018-014-1812-z. Epub 2014 Dec 30. PMID: 25548803; PMCID: PMC4376244). Under emotional stress, hormones trigger apocrine glands to release this fluid, resulting in the formation of strong body odor (Barzantny H, Brune I, Tauch A. Molecular basis of human body odour formation: insights deduced from corynebacterial genome sequences. Int J Cosmet Sci. 2012 Feb;34(l):2-l l. doi: 10.1111 / j .1468-2494.2011.00669.x. Epub 2011 Jul 25. PMID: 21790661).III. ApatB-Staphylococcus lugdunensis

[0056] The technology of the present disclosure relates to the use of ApatB- Staphylococcus lugdunensis (whose genome comprises a disruption of a patB gene), such as Staphylococcus lugdunensis TaxaOne, or variant strains thereof, to treat or prevent body odor, and methods of making the same. One of skill in the art will understand that a disruption of the patB gene that results in decreased PatB activity and / or patB expression and / or results in the strain having a reduced capacity to produce thiol and / or 3M3SH when contacted with an appropriate substrate or precursor, can be made by any means available in the art. Such methods include targeted approaches for introducing a disruption by, e.g., homologous recombination, site-directed mutagenesis, a Transcription activator-like effectornuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR base editing system, a CRISPR prime editing system, a CRISPR / Cpfl system, or a CRISPR / Csml system. Additionally or alternatively, such methods may also include random mutagenesis methods for introducing a disruption by, e.g., irradiation, chemical mutagens, passing cloned genes through mutator strains, “error-prone” PCR mutagenesis, rolling circle error-prone PCR, or saturation mutagenesis. For illustrative purposes, and for proof of concept, a patB disruption in Staphylococcus lugdunensis TaxaOne was created via homologous recombination as described in the Examples section. Staphylococcus lugdunensis TaxaOne refers to a bacterial strain, or variant thereof, having been deposited under IDAC Accession No. 250423-01, or compositions comprising the strain.

[0057] In some embodiments, bacterial strains of the present technology are used in methods and compositions for treating or preventing body odor (malodor). In some embodiments, the bacterial strains comprise a probiotic for preventing or controlling body odor. In some embodiments, compositions of the present technology comprise vegetative bacterial cells. In some embodiments, the ApatB-S. lugdunensis strains of the present technology grow faster than the corresponding wild-type S. lugdunensis strains. In some embodiments, the disruption of patB does not negatively affect the fitness of the S. lugdunensis strain whose genome comprises the disruption. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 50% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 55% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 60% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 65% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 70% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, thegenetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 75% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 80% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 85% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 90% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 95% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least about 96%, 97%, 98%, or 99% when contacted with a thiol substrate compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain has a reduced capacity to produce 3-methyl-3- sulfonylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of 3M3SH production by at least about 50% when contacted with a apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of 3M3SH production by at least about 60% when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain. In some embodiments, the genetically engineered Staphylococcus lugdunensis strain decreases the amount of 3M3SH production by at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain.

[0058] In some embodiments, bacterial strains of the present technology are used in methods and compositions for altering a microbiota. In some embodiments, the bacterial strains comprise a probiotic that is capable of engraftment to an existing microbiota, such thatthe bacterial strains persist for a period of time in the microbiota community. In some embodiments, the microbiota is a skin microbiota. In some embodiments, an agent is applied to disrupt the native microbiota, and then the bacterial strains and composition of the present technology are applied, such that the disruption of the native microbiota increases engraftment of the bacterial strains.IV. Deodorant Compositions: Methods and Modes of Administration and Application

[0059] The following discussion is presented by way of example only, and is not intended to be limiting.

[0060] One aspect of the present technology includes methods of treating or preventing body odor in a subject. In deodorizing applications, compositions comprising ApatB-Staphylococcus lugdunensis and / or the S. lugdunensis TaxaOne strain, or variant thereof, are administered to a subject suspected of, or already suffering from body odor, in an amount sufficient to reduce or eliminate the odor.

[0061] Compositions of the present technology (e.g., deodorant compositions) can be formulated for topical administration, including for administration to a subject as a topical deodorant. In some embodiments, the subject is a mammal. In some embodiments, the mammalian subject is human. Topical deodorant compositions comprising the bacterial strains of the present technology can be applied to any dermal location on a subject, including but not limited to the armpit, groin, nipples, and / or anywhere that glands are located.Additionally, topical deodorants can be formulated using any appropriate means, including as a deodorant stick, a roll-on, a cream, a lotion, a gel, a powder, a water activated cream, a water activated powder, an injectable, a patch, or a spray. In some embodiments, the compositions of the present technology comprise one or more lyophilized bacterial strains of the present technology (ApatB-Staphylococcus lugdunensis and / or S. lugdunensis TaxaOne strain, or a variant thereof), optionally in a powdered form, which can be mixed with water or a water-containing composition and subsequently applied to a subject as a topical deodorant, optionally to the armpits. In some embodiments, the topical deodorant comprises lyophilized strains of the present technology. In some embodiments, the topical deodorant composition comprises lyophilized strains of the present technology at an amount of about 0.0001% toabout 35% by weight of the total composition (wt. %). In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.001 wt. % to about 15 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.01 wt. % to about 10 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.1 wt. % to about 10 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.15 wt. % to about 10 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.2 wt. % to about 10 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 10 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 9 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 8 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 7 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 6 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 5 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 4 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 3 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 2 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 1 wt. %. In some embodiments, the topical deodorant composition comprises the lyophilized strains at an amount of about 0.25 wt. % to about 0.50 wt. %. In some embodiments, the amount of acceptable carrier or excipient in the topical deodorant composition ranges from about 65 wt. % to about 99.9999 wt. %. In some embodiments, the amount of acceptable carrier or excipient in the topical deodorant composition ranges from about 90 wt. % to about 99.9999 wt. %. In some embodiments, the topical deodorant comprises about 0.25 wt. % to about 5 wt. % lyophilized strains of the present technologyand about 95 wt. % to about 99.75 wt. % acceptable carrier or excipient. Herein, any of the upper limits of the amounts of the components of the topical deodorant compositions disclosed can be combined with any of the lower limits of the amounts of the components of the topical deodorant. In some embodiments, the topical deodorant comprises about 105to about 1014bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 107to about 1011bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 105bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 106bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 107bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 108bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 109bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about IO10bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 1011bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 1012bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 1013bacteria per gram of the deodorant composition. In some embodiments, the topical deodorant comprises about 1014bacteria per gram of the deodorant composition. In some embodiments, topical deodorant application of the strains and compositions of the present technology deposits about 0.1 to about 0.2 grams of the product at the application location.

[0062] In some embodiments, the compositions of the present technology include an acceptable carrier. In some embodiments, the acceptable carrier comprises oleic acid, stearic acid, almond butter, avocado butter, babassu butter, beeswax, cocoa butter, coconut butter, coconut oil, cupuacu butter, hemp seed butter, kokum butter, macadamia butter, mango butter, mowrah butter, olive butter, sal seed butter, shea butter, propylene glycol, glycerin, stearyl alcohol, myristyl alcohol, capric triglyceride, or any combination thereof. Additional components of the compositions of the present technology may include a preservative selected from the group consisting of sucrose, sodium ascorbate, and glutathione. In some embodiments the preservative is a cryoprotectant selected from the group consisting of a nucleotide, a disaccharide, a polyol, and a polysaccharide. In some embodiments, thecryoprotectant is selected from the group consisting of inosine-5’ -monophosphate (IMP), guanosine-5 ’-monophosphate (GMP), adenosine-5’ -monophosphate (AMP), uranosine-5’- monophosphate (UMP), cytidine-5’ -monophosphate (CMP), adenine, guanine, uracil, cytosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, orotidine, thymidine, inosine, trehalose, maltose, lactose, sucrose, sorbitol, mannitol, dextrin, inulin, sodium ascorbate, glutathione, and skim milk.

[0063] In some embodiments, the composition comprises a lyoprotectant. In some embodiments, the lyoprotectant is a protein, a carbohydrate, or a combination thereof. In some embodiments, the lyoprotectant is a milk protein, and in particular a micellar casein. In some embodiments, the carbohydrate is sucrose. In some embodiments, the lyoprotectant is present in an amount of about 0.1% by weight of the composition to about 15% by weight of the composition.

[0064] In some embodiments, the compositions of the present technology comprise an additional probiotic agent (wild-type Staphylococcus epidermidis). that may improve engraftment efficacy for the strains of the present technology.

[0065] In some embodiments, the disclosure of the present technology relates to a method of manufacturing the deodorant compositions, comprising homogeneously dispersing the ApatB-Staphylococcus lugdunensis strains of the present technology in an acceptable carrier or excipient.

[0066] The strains and compositions of the present technology can be administered to a subject one or more times per day. In some embodiments, the strains and compositions are administered more than once per day, including, for example, twice per day or three times per day. In some embodiments, the strains and compositions are administered once per day. In some embodiments, the strains and compositions are administered once every 2 days. In some embodiments, the strains and compositions are administered once every 3 days. In some embodiments, the strains and compositions are administered once every 4 days. In some embodiments, the strains and compositions are administered once every 5 days. In some embodiments, the strains and compositions are administered once every 6 days. In some embodiments, the strains and compositions are administered once every 7 days. Insome embodiments, the strains and compositions are administered once every 8 days. In some embodiments, the strains and compositions are administered once every 9 days. In some embodiments, the strains and compositions are administered once every 10 days. In some embodiments, the strains and compositions are administered once every 11 days. In some embodiments, the strains and compositions are administered once every 12 days. In some embodiments, the strains and compositions are administered once every 13 days. In some embodiments, the strains and compositions are administered once every 14 days. In some embodiments, the strains and compositions are administered once every 15 days. In some embodiments, the strains and compositions are administered once every 16 days. In some embodiments, the strains and compositions are administered once every 17 days. In some embodiments, the strains and compositions are administered once every 18 days. In some embodiments, the strains and compositions are administered once every 19 days. In some embodiments, the strains and compositions are administered once every 20 days. In some embodiments, the strains and compositions are administered once every 21 days. In some embodiments, the strains and compositions are administered once every 22 days. In some embodiments, the strains and compositions are administered once every 23 days. In some embodiments, the strains and compositions are administered once every 24 days. In some embodiments, the strains and compositions are administered once every 25 days. In some embodiments, the strains and compositions are administered once every 26 days. In some embodiments, the strains and compositions are administered once every 27 days. In some embodiments, the strains and compositions are administered once every 28 days.

[0067] In some embodiments, administering the strains and compositions of the present technology results in a reduction of malodorous bacteria, including, for example, wild-type Staphylococcus lugdunensis, at the application location.V. Combination Treatment with ApatB-Staphylococcus lugdunensis

[0068] In some embodiments, the ApatB-Staphylococcus lugdunensis strains of the present technology may be combined with one or more additional agents for the prevention or treatment of body odor. Additional agents include, but are not limited to, deodorants, antiperspirants, and / or fragrances.

[0069] In some embodiments, an additional agent is administered to a subject in combination with the ApatB-Staphylococcus lugdunensis strains of the present technology such that a synergistic effect is produced. For example, administration of ApatB Staphylococcus lugdunensis (e.g., Staphylococcus lugdunensis TaxaOne) with one or more additional agents for the prevention or treatment of body odor will have greater than additive effects in the prevention or treatment of the body odor.

[0070] In any case, the multiple agents may be administered in any order or even simultaneously. If simultaneously, the multiple agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single deodorant stick or as two deodorant sticks). One of the agents may be given in multiple doses, or both may be given as multiple doses. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.EXAMPLES

[0071] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.Materials and Methods

[0072] Bacterial Strains. A strain of Staphylococcus lugdunensis, as described in J. Freney, et al. (Staphylococcus lugdunensis sp. Nov. and Staphylococcus schleiferi sp. Nov., two species from human clinical specimens, Int J Syst Bacteriol, 38 (1988), pp. 168-172), was obtained from the cell repository ATCC (No. 49576; strain designation LRA 260.05.79). The strain was received as a freeze-dried powder, and then rehydrated and grown in TSB at 37°C for further experiments.

[0073] 3-methyl-3-sulfonylhexan-l-ol (3M3SH) Assay. To measure the capacity of the bacterial strains of the present technology to produce 3M3SH, the following assay was performed. A single inoculated colony (e.g., ApatB-Staphylococcus lugdunensis) was grown in 20mL of TSB in a 50 ml Falcon tube at 200 RPM overnight at 37 °C. The cells were pelleted at 6000 xg for 5 minutes (Thermo Fisher Sorvall X4R Pro-MD) and resuspended in 2 ml of IX M9 buffer (MP Biomedicals™ 113037022). A 200 pL reaction assay mixture wasprepared in a 96 well assay plate (Falcon, 96 well Tissue culture plate, Ref: 353072) as follows: 140uL resuspended cells normalized to an Optical Density (OD) 600nm of 5.0, 50 pL of lOmM Cys-Gly-3M3SH (Dalton Pharma services, DC-003684) dissolved in IX M9 buffer, lOul of 8mM DTNB (CAS: 68-78-3, REF: 22582, Thermo Scientific) dissolved in IM Tris-HCL, pH 8.0 (Thermo Scientific, J22638-K2). For the reagent-free negative control, the lOul of DTNB dissolved in IM Tris-HCL was replaced with lOul of IM Tris-HCL, and the 50 pL of lOmM Cys-Gly-3M3SH was replaced with IX M9 buffer. The reaction assay mixture was incubated at 37°C for 2 hours. Photometric reactions were measured using a Promega Glomax Discovery (REF: GM3000, SN: 9700100744) at A412 nm.

[0074] Thiol Production Assay. To measure the capability of the bacterial strains of the present technology to produce thiols, the following assay was performed. A single inoculated colony (e.g., ApatB -Staphylococcus lugdunensis) was grown in 20mL of TSB medium in a 50 ml Falcon tube at 200 RPM overnight at 37 °C. The cells were then pelleted at 3000 xg for 10 minutes in a microcentrifuge, and resuspended in 2ml of M9 buffer (MP Biomedicals™ 113037022). A reaction assay mixture was prepared in a 1.5 ml microcentrifuge tube as follows: resuspended cells were added to an OD600nm of 5, 50 pL of lOmM benzyl-cysteine was then added, and then M9 buffer was added to a final volume of 200 pL. The reaction assay mixture was incubated at 37°C for 5 hours, then 100 pL of the assay mixture was centrifuged for 2 min at 15000 xg. A 200 pL absorbance sample was then prepared by combining the following in a 1.5 ml microfuge tube: 50 pL reaction assay mixture, 50 mM Tris-HCL, and 0.4 mM DTNB. Photometric reactions was then measured using a BioTek Epoch 2 Microplate Spectrophotometer at A412 nm. Control samples without benzyl-cysteine were used as a baseline for normalization.

[0075] Microbiome Profiling Protocol. The following microbiome profiling protocol will be carried out to determine whether the compositions comprising the bacterial strains of the present technology alter the abundance of malodorous, wild-type bacteria present on the skin of a subject to whom the compositions are applied.

[0076] Briefly, the probiotic deodorant (deodorant composition comprising one or more bacterial strains of the present technology) will be applied to one armpit, and a control deodorant without probiotics (deodorant composition without one or more bacterial strains ofthe present technology) will be applied to a second armpit in one or more study participants. Both treatments will be applied for a specified period of days, such as study days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, with application occurring one or more times per day.

[0077] On a selected set of days, such as some combination of study days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 21, 24 and 28, both armpits for each participant will be swabbed, optionally using a zig-zag technique, for a time period sufficient to collect a sample, such as 30 seconds, with a separate eNat swab (Copan Diagnostics) or an equivalent product.

[0078] Genomic DNA will be extracted from each sample using an inBiome DNA extraction kit per the manufacturer protocol. DNA will then be analyzed for strain prevalence via amplicon sequencing analysis. First, qPCR of the samples will be performed using patB primer pairs (provided by inBiome B.V.) to determine the relative abundance of each SpatB-S. lugdunensis and wild-type S. lugdunensis species in the sample. Second, genomic DNA will be PCR amplified using inBiome IS-pro primers, and the resulting IS fragments will be sequenced using a Sanger Genetic Analyser to determine relative species and operational taxonomic unit abundance. These results will demonstrate relative bacterial abundance at a species level and will allow for differentiation between WT and SpatB-S. lugdunensis bacteria at multiple sample collection time points.Example 1 : Creation of pIMAY-A / / / 7> plasmid.

[0079] A plasmid containing two 590bp regions of homology to patB was designed and assembled into a pIMAY vector (AddGene #68939). Integrated DNA Technologies (IDT) was used to synthesizes a 1240 bp gBlock insert containing the two 590bp regions of homology to patB as well as two 30 bp Gibson cloning sites. The plasmid was then double digested with Kpnl and Notl. The linearized plasmid and the IDT insert were then assembled using Gibson Assembly and transformed into NEB chemically competent DH5a cells. The assembled plasmid was sequence verified by the sequencing provider SNPsaurus (Eugene, OR, USA). The sequence verified plasmid map is shown in FIG. 1. As shown in FIG. 1, the assembled plasmid contains regions flanking Staphylococcus lugdunensis patB, which allows for the removal of patB through controlled stepwise recombination events. Saidrecombination results in the incorporation of a chloramphenicol (Cm) resistance cassette for further selection.

[0080] Accordingly, these results demonstrate that a plasmid capable of disrupting patB in S. lugdunensis via homologous recombination, using the pIMAY backbone, was generated.Example 2: Methylation of pIMAY-A / ?atS plasmid with A. coli SL01B.

[0081] Staphylococcus lugdunensis possesses a Type III restriction modification system that controls the targeted destruction of foreign DNA. This system digests plasmids immediately upon entry to the cell. To avoid this, a pass-through E. coli SL01B methylation strain capable of methylating the plasmid as native Staphylococcus DNA, and thus avoiding destruction, was provided by Timothy J. Foster (Heilbronner S, et al.. Sortase A promotes virulence in experimental Staphylococcus lugdunensis endocarditis. Microbiology (Reading). 2013 Oct;159 (Pt 10):2141 -2152). Briefly, 5 mL of E. coli SL01B was grown up at 37°C in a 15 mL Falcon tube until an OD of 0.5 was reached. The cells were then placed on ice for 15 minutes and then spun down for 2 minutes at 12000 xg in a microcentrifuge. The supernatant was discarded and the cells were resuspended in 50 mM CaCh before being spun down again for 2 minutes at 12000 xg in a microcentrifuge. The supernatant was then discarded and the cells were resuspended in 50mM CaCh 10% glycerol. These chemically competent A. coli SL01B cells were then transformed with pIMAY-A / / / 7> using heat shock transformation at 42°C for 30 seconds. DNA was miniprepped out of successful transformants using a NEB Plasmid Miniprep Kit, according to the manufacturer protocol (FIG. 2). As shown in FIG. 3, the pIMAY-A / / / 7> plasmid was successfully transformed into the A. coli SL01B methylation strain. Briefly, four SL01B transformants were miniprepped and digested with BstBI. BstBI cuts once in pIMAY (creating one 5700 bp fragment) and twice in pIMAY-A / / / 7> (creating 4735 bp and 2096 bp fragments). Each sample shown in FIG. 3 is a different transformant colony. The dual bands present in samples #2.1, #2.2 and #2.4 indicate successful transformations with the correctly assembled pIMAY-A / / T Sample #2.2 was subsequently used to mass-produce methylated pIMAY-A / / / 7T

[0082] Accordingly, these results demonstrate that the plasmid capable of disrupting patB in S. lugdunensis was protected from restriction enzyme-based digestion via methylation in an A. coli methylation strain.Example 3 : Plasmid electroporation into S. lugdunensis.

[0083] Plasmid DNA was electroporated into S. lugdunensis obtained from the cell repository ATCC (No. 49576; strain designation LRA 260.05.79) according to the protocol established by Heilbronner et al.. 2013. Briefly, S. lugdunensis cells were incubated at 37°C in 50 ml of TSB overnight until OD578 of 0.5 (stationary phase) was achieved. Cells were then incubated with shaking at 110 rpm at 37°C for 1 hour until OD578 of 0.6 (mid-log phase) was achieved. Then the cells were transferred to ice and incubated for 15 minutes before being centrifuged for 10 minutes at 9000 xg. The supernatant was removed and the cell pellet was washed twice with 50 ml ice-cold sterile Milli-Q water and once with 50 ml ice-cold 10% glycerol. Cells were then resuspended in 1 ml 10% glycerol and aliquoted into 200 pl electrocompetent samples, which were either used immediately or frozen and stored at -80°C.

[0084] Frozen electrocompetent cells were prepared for electroporation by thawing on ice for 5 minutes and then incubating at room temperate for 30 minutes. Similarly, the frozen, methylated pIMAY-A / / / 7> plasmid was thawed on ice for 5 minutes and then incubated at room temperate for 30 minutes. Then 20 pl of 2.5M sucrose was added to 80 pl electrocompetent cells, and 5 pg plasmid DNA (suspended in 2 pl water) was added to the mixture, which was incubated at room temperature for 10 minutes. Cells were then electroporated for 2.5 milliseconds using a 1mm cuvette (Bio-Rad) according to the following parameters: Voltage: 2.1 kV, Resistance: 100 Q, Capacitance: 25 pF. Electroporated cells were incubated with shaking at 37°C for 2 hours before a 30 minute incubation on ice. Then the cells were transferred to pre-warmed 28°C TSA+CmlO plates (10 pg / ml chloramphenicol) and incubated at 28°C. As shown in FIG. 4, the procedure resulted in chloramphenicol resistance, demonstrating the successful electroporation of S. lugdunensis.

[0085] Accordingly, these results demonstrate that the plasmid capable of disrupting patB was successfully incorporated into S. lugdunensis via electroporation.Example 4: Deletion of S. lugdunensis patB.

[0086] A single transformant sample was inoculated in 5 mL liquid TSB media with lOpM chloramphenicol and incubated overnight at 37°C and 220 rpm. Overnight incubation and growth resulted in the dilution and loss of extrachromosomal plasmid due to the temperature sensitive nature of the plasmid origin (non-replicative at 37°C), resulting in a population of cells which underwent homologous recombination-insertion of the plasmid at the patB locus, resulting in patB gene disruption.

[0087] Then the plasmid was excised through non-selective growth of the patB-S. lugdunensis. Briefly, strains were grown in 5mL of TSB media without antibiotic selection for multiple days at 37°C at 220 rpm. The cells were then plated and grown on TSB and individual colonies were then patch plated onto both TSA and TSA with lOpM chloramphenicol. Successful plasmid excision was shown by normal growth on standard TSA media and a failure to grow in the presence of Cm, as shown in FIG. 5. Seven putative lpatB clonal lines with an excised Cm selection marker were identified, and the inability to grow in the presence of Cm was confirmed by subsequent plate streaking assays.

[0088] Amplicon sequencing was then performed to confirm that a single homologous recombination event between the plasmid and the bacterial genome had occurred, deleting a portion of the patB gene. Briefly, genomic DNA was extracted from each of the seven putative UspatB cell lines using an NEB Genomic DNA Extraction kit according to the manufacturer protocol. Genomic DNA was PCRed using the PBFW (GGCATGCAACATTTACCATTTGCGA; SEQ ID NO: 2) and PBRV (TGTCCAACTTTTGAGATGCACC; SEQ ID NO: 3) primers to amplify the genomic region surrounding the patB gene. The PCR reaction solution was 1 pL of genomic DNA, 1 pL each of 10 pM forward and reverse primers, 0.5 pL of 10 mM dNTPs, 0.25 pL of 5 U / pL thermostable DNA polymerase, 5 pL of 5x PCR buffer, and nuclease-free water to 25 pL. The PCR cycling conditions were as follows: an initial denaturation step at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at a 58°Cfor 30 seconds, extension at 72°C for 1 minute, and a final extension step at 72°C for 5 minutes. The resulting amplicons were sequenced using Sanger sequencing and aligned with the reference genome as shown in FIG. 6. These results confirmed that a single SpatB-S. lugdunensis strain with a deletion of the 152-610 nucleotide region of the patB gene (5. lugdunensis HKU09-01 Ref. Genome 242,878 - 243,336), which includes three amino acid active site residues Y52, Y132, and N 184, had been generated and isolated. See Rudden et al. (The molecular basis of thioalcohol production in human body odor. Scientific Reports, 2020) for a discussion of patB active site residues.

[0089] These results demonstrate that the methods of the present disclosure successfully created a SpatB-S. lugdunensis clonal cell line Staphylococcus lugdunensis TaxaOne having been deposited under ID AC Accession No. 250423-01. Accordingly, the methods and strains of the present disclosure are useful for generating, propagating, isolating, and identifying Staphylococcus lugdunensis strains wherein the patB gene is disrupted.Example 5: SpatB-S. lugdunensis Exhibits Wild-Type Growth Rates.

[0090] Triplicate biological replicates of wild-type S. lugdunensis and patB-S. lugdunensis (Staphylococcus lugdunensis TaxaOne) were grown in a 96-well plate, in 200 pL TSB liquid media for 10 hours at 37°C and 220 RPM, in a BioTek Epoch 2 Microplate Spectrophotometer with A630nm recorded at 10 minute intervals. It was anticipated that patB-S. lugdunensis would possess a growth disadvantage compared to wild-type, given that SpatB-S. lugdunensis is more metabolically restricted and disruption of metabolic pathways can have unpredictable side effects. Surprisingly, the resulting growth curve, shown in FIG. 7, demonstrated that the SpatB-S. lugdunensis cell line grew at a faster rate compared to wild-type (WT patB) bacteria, and therefore SpatB-S. lugdunensis does not possess any competitive growth disadvantage, and, in fact, possesses a competitive growth advantage compared to wild-type.

[0091] Accordingly, these results demonstrate that the SpatB-S. lugdunensis cell line of the present disclosure possesses wildtype growth capabilities. Accordingly, the patB-S. lugdunensis cell lines of the present disclosure are useful in multiple forms, including as strain isolates and compositions, and have a sufficient growth rate to persist within amicrobiota, and are useful in methods and compositions for inhibiting or reducing malodor on the skin of a subject (body odor).Example 6: patB-S. lugdunensis Reduces Body Odor.

[0092] The ability of the patB-S. lugdunensis strain of the present technology to treat, prevent, or reduce body odor (malodor) in a subject will be determined using one or more double blind odor studies.

[0093] A self-comparison odor analysis will be performed. Each study subject will receive a probiotic deodorant comprising UspatB-S. lugdunensis to one armpit, while the other armpit will receive a control deodorant without bacteria or no deodorant. Each treatment will be given for a specified number of days (e.g., study day -1, or study days -2 and -1). Then at a selection of time points post-treatment, self-reported body odor levels from each armpit will be recorded and microbiota samples will be taken. Body odor levels and microbiota composition, determined using known methods in the art, will be compared between the two treatment conditions over time and within each treatment condition over time (e.g., comparison of odor at day 0 versus day 4 for probiotic treated armpit) for each subject to determine initial impact and impact over time on reduction of body odor.

[0094] A comparative group odor analysis will be performed. Treatment group study subjects will receive a probiotic deodorant comprising ^patB-S. lugdunensis to the armpits. Control group study subjects will receive either a negative control probiotic deodorant without any bacterial strains, a standard commercial deodorant, a standard commercial antiperspirant, or some combination thereof. Multiple control groups can be tested with any of the described control treatments. Each treatment will be given for a specified number of days (e.g., study day -1, or study days -2 and -1). Then at a selection of study days taken from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 18, 20, 22, 24, and 28 days posttreatment, self-reported body odor levels from each subject will be recorded. Body odor will be compared between treatment groups, such as treatment versus control, but also within treatment groups, such as treatment group body odor at study day 0 versus study days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 18, 20, 22, 24, and / or 28.

[0095] It is anticipated that the results of these studies will demonstrate that the SpatB-S. lugdunensis strains of the present technology will successfully reduce, treat, or prevent body odor at levels that are comparable to, or greater than, the reductions observed from treatment with the control(s). Additionally, it is anticipated that the effect of the patB- S. lugdunensis strains will persist longer and at a greater level than the control deodorant effect. Accordingly, these results will demonstrate that a probiotic deodorant comprising the patB-S. lugdunensis strains of the present technology is effective for treating, reducing, or preventing body odor.Example 7: SpatB-S. lugdunensis Strain Reduces Thiol Production

[0096] The ability of the SpatB-S. lugdunensis strain of the present disclosure to produce thiols was assayed as described above in the Thiol Production Assay section, using a comparable wild-type Staphylococcus lugdunensis strain as a control.

[0097] Benzyl-cysteine is a thiol substrate with structural similarities to Cys-3M3SH, and the patB enzyme in S. lugdunensis is known to cleave benzyl-cysteine during thiol production. As shown in FIG. 8, the wild-type (WT) strain of S. lugdunensis successfully metabolized benzyl-cysteine and produced thiol, as indicated by the increased A412 absorption over time compared to the no-substrate control (AA412). In contrast, the patB-S. lugdunensis strain (Staphylococcus lugdunensis TaxaOne) was largely incapable of generating thiols, as shown by the lack of any change in A412 absorption compared to the no-substrate control. These results indicate that the patB-S. lugdunensis strain lacks a functional patB gene and cannot produce thiols. Accordingly, these results demonstrate that a probiotic deodorant comprising the patB-S. lugdunensis strain of the present technology is effective for treating, reducing or preventing body odor.Example 8: SpatB-S. lugdunensis has Reduced 3M3SH Production

[0098] The ability of the SpatB -Staphylococcus lugdunensis strains of the present disclosure to produce 3M3SH was assayed as described above in the 3-methyl-3- sulfonylhexan-l-ol (3M3SH) Assay section, using a comparable wild-type Staphylococcus lugdunensis strain as a control.

[0099] The patB enzyme in S. lugdunensis is known to cleave Cys-Gly-3M3SH during thiol production. As shown in FIG. 9, the wild-type (WT) strain of S. lugdunensis successfully metabolized Cys-Gly-3M3SH and produced 3M3SH, as indicated by the significant increase in A412 absorption over time compared to the no-substrate control (AA412). In contrast, the SpatB-S. lugdunensis strain (Staphylococcus lugdunensis TaxaOne) was largely incapable of generating 3M3SH, as shown by the lack of significant change in A412 absorption compared to the no-substrate control. These results indicate that the SpatB-S. lugdunensis strain lacks a functional patB gene and cannot produce 3M3SH. Accordingly, these results demonstrate that a probiotic deodorant comprising the patB-S. lugdunensis strain of the present technology is effective for treating, reducing, or preventing body odor.Example 9: Lyophilized SpatB-S. lugdunensis Strain Retains Viability[000100] The viability of the lyophilized SpatB-S. lugdunensis strain of the present disclosure was tested. Briefly, 50mL of SpatB-S. lugdunensis (Staphylococcus lugdunensis TaxaOne) was grown up overnight in TSB at 37°C. The overnight culture was centrifuged (4,000 g, 10 min) and resuspended (lOx dilution) in PBS with 5% (m / v) sucrose and 5% (m / v) micellar casein. ImL of this suspension was poured into 1.5mL microfuge tubes. The mixture was thoroughly vortexed and stored at -80C before freeze-drying.[000101] The samples were lyophilized in open microfuge tubes using a Labconco FreeZone Lyophilizer 7670520 per the manufacturer’s protocol. During lyophilization, samples were first kept at -40 °C w / condenser T of -55 °C and chamber pressure of 10 Pa for 18 hours, and then the condenser was turned off, and the samples remained at room temperature and 10 Pa for 5 hours. After lyophilization, samples were kept in sealed 1.5mL microfuge tubes at room temperature and pressure. Samples of lyophilized bacteria were taken directly after lyophilization and at 1 day, 1 week, 1 month, and 4 months post lyophilization, and the colony forming units per gram were measured via plating. As shown in FIG. 10, SpatB-S. lugdunensis retained high levels of viability even as late as 4 months post-lyophilization. Accordingly, these results demonstrate that the SpatB-S. lugdunensis strains of the present technology retains viability in long-term storage, and therefore aprobiotic deodorant comprising the SpatB-S. lugdunensis strains of the present technology is effective for treating, reducing or preventing body odor.Example 10: SpalB-S. lugdunensis Strain Reduces Body Odor.[000102] The ability of SpaiB-S. lugdunensis to reduce body odor was tested. Briefly, a mixture of SpatB-S. lugdunensis (Staphylococcus lugdunensis TaxaOne) in an excipient was applied to one armpit of a subject while excipient with no bacteria was applied to the other armpit of the subject. One month later the subject engaged in exercise and an odor test was performed by smelling the subject’s armpits. The armpit receiving SpatB-S. lugdunensis in excipient was identified as substantially less odorous as compared to the armpit receiving excipient alone. Accordingly, these results demonstrate that a probiotic deodorant comprising the SpatB-S. lugdunensis strains of the present disclosure is effective for treating, reducing, or preventing body odor.EQUIVALENTS[000103] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.[000104] Each and every publication and patent mentioned in the above specification is herein incorporated by reference in its entirety for all purposes. Various modifications and variations of the described methods and system of the present technology will be apparent tothose skilled in the art without departing from the scope and spirit of the present technology. Although the present technology has been described in connection with specific embodiments, the present technology as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present technology which are obvious to those skilled in the art and in fields related thereto are intended to be within the scope of the following claims.BIOLOGICAL DEPOSIT[000105] The Applicant requests that a sample of the deposited microorganisms should be made available only to an expert approved by the Applicant.[000106] Staphylococcus lugdunensis strain identified as Staphylococcus lugdunensis TaxaOne deposited with the International Depositary Authority of Canada (ID AC), National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba Canada R3E 3R2, on April 25, 2023, under ID AC Accession Number 250423-01.[000107] The deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.[000108] Name of Depositor: Xavier M. Segel. Address of Depositor: 3711 Liseter Gardens, Newtown Square, PA 19073.[000109] In respect of all designated states to which such action is possible and to the extent that it is legally permissible under the law of the designated state, it is requested that a sample of the deposited biological material be made available only by the issue thereof to an independent expert, in accordance with the relevant Patent legislation, for example EPC Rule 32, UK Patent Rules 2007 (as amended) Rule 13(1) and Schedule 1 paragraphs 6 and 7, and generally similar provisions mutatis mutandis for any other designated state.SEQUENCE LISTINGStaphylococcus lugdunensis ^XMW-SpalB plasmid nucleotide sequence (SEQ ID NO: 1):GCATGCGTTTTAGCGTTTATTTCGTTTAGTTATCGGCATAATCGTTAAAACAGGCGTTATCGTAGCGTAAAAGCCCTTGAGCGTAGCGTGGCTTTGCAGCGAAGATGTTGTCTGTTAGATTATGAAAGCCGATGACTGAATGAAATAATAAGCGCAGCGCCCTTCTATTTCGGTTGGAGGAGGCTCAAGGGAGTATGAGGGAATGAAATTCCCTCATGGGTTTGATTTTAAAAATTGCTTGCAATTTTGCCGAGCGGTAGCGCTGGAAAATTTTTGAAAAAAATTTGGAATTTGGAAAAAAATGGGGGGAAAGGAAGCGAATTTTGCTTCCGTACTACGACCCCCCATTAAGTGCCGAGTGCCAATTTTTGTGCCAAAAACGCTCTATCCCAACTGGCTCAAGGGTTTAAGGGGTTTTTCAATCGCCAACGAATCGCCAACGTTTTCGCCAACGTTTTTTATAAATCTATATTTAAGTAGCTTTATTGTTGTTTTTATGATTACAAAGTGATACACTAACTTTATAAAATTATTTGATTGGAGTTTTTTAAATGGTGATTTCAGAATCGAAAAAAAGAGTTATGATTTCTCTGACAAAAGAGCAAGATAAAAAATTAACAGATATGGCGAAACAAAAAGGTTTTTCAAAATCTGCGGTTGCGGCGTTAGCTATAGAAGAATATGCAAGAAAGGAATCAGAACAAAAAAAATAAGCGAAAGCTCGCGTTTTTAGAAGGATACGAGTTTTCGCTACTTGTTTTTGATAAGGTAATTATATCATGGCTATTAAAAATACTAAAGCTAGAAATTTTGGATTTTTATTATATCCTGACTCAATTCCTAATGATTGGAAAGAAAAATTAGAGAGTTTGGGCGTATCTATGGCTGTCAGTCCTTTACACGATATGGACGAAAAAAAAGATAAAGATACATGGAATAATAGTAATATTATACAAAATGGAAAGCACTATAAAAAACCACACTATCACGTTATATATATTGCACGAAATCCTGTAACAATAGAAAGCGTTAGGAACAAGATTAAGCGAAAATTGGGGAATAGTTCAGTTGCTCATGTTGAGATACTTGATTATATCAAAGGTTCATATGAATATTTGACTCATGAATCAAAGGACGCTATTGCTAAGAATAAACATATATACGACAAAAAAGATATTTTGAACATTAATGATTTTGATATTGACCGCTATATAACACTTGATGAAAGCCAAAAAAGAGAATTGAAGAATTTACTTTTAGATATAGTGGATGACTATAATTTGGTAAATACAAAAGATTTAATGGCTTTTATTCGCCTTAGGGGAGCGGAGTTTGGAATTTTAAATACGAATGATGTAAAAGATATTGTTTCAACAAACTCTAGCGCCTTTAGATTATGGTTTGAGGGCAATTATCAGTGTGGATATAGAGCAAGTTATGCAAAGGTTCTTGATGCTGAAACGGGGGAAATAAAATGACAAACAAAGAAAAAGAGTTATTTGCTGAAAATGAGGAATTAAAAAAAGAAATTAAGGACTTAAAAGAGCGTATTGAAAGATACAGAGAAATGGAAGTTGAATTAAGTACAACAATAGATTTATTGAGAGGAGGGATTATTGAATAAATAAAAGCCCCCTGACGAAAGTCGAAGGGGGTTTTTATTTTGGTTTGATGTTGCGATTAATAGCAATACATTCTATAATAGAAGGTATGGAGGATGTTATATAATGAGACAGAATTATGATGATCATATGTCAACTAACGGGGCAGGTTAGTGACATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCATATTATAAAAGCCAGTCATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCATGATAACCATCACAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTATTAATGAATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTAAACCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGTGTTTTGGGAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATCATAAAACTCTTTGAAGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTTTAGATACACCATCAAAAATTGTATAAAGTGGCTCTAACTTATCCCAATAACCTAACTCTCCGTCGCTATTGTAACCAGTTCTAAAAGCTGTATTTGAGTTTATCACCCTTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATCCTTCTTGTTTTATGTTTCGGTATAAAACACTAATATCAATTTCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATAATGATTAAATATCTCTTTTCTCTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATGGGTTTCACTCTCCTTCTACATTTTTTAACCTAATAATGCCAAATACCGTTTGCCACCCCTCTCTTTGATAATTATAATATTGGCGAAATTCGCTTCTAAAGATGAAACGCAATATTATATGCTTGCTTTATCGGCCGTATGTGATTATACCAGCCCCCTCACTACATGTCAAGAATAAACTGCCAAAGCATAATGGGATAATTAACCCTCACTAAAGGGAACAAAAGCTGAAACTCATTTACTGGCTTCTTTATTCATAATACGTCGTATGATTGACTCGTATGCATAGAGTGCTATCTCTTCACCCCAACTTGCTTTGTTTGGCGAATTTCTTTTCGAAATTCTCTTTGTTGGGGGGCCGGTCTTTGTCGAACACTGTATTCTCGGGTGTCTCGTTATATATATTACGTATTCATGTGTCGTTTTACGGCGAAGTTTTTTAAGAAATAAGCATGGTAACATGATGCAACATCTGCTACTAATAAAACTTCGGTTTTTATGCGTTCAAATGATATTGTGGAAACTATACTTGATAAAAAGATTTTGGCGTTGCAATTTTAATCCGTTCTATTTATGTTCCACACTGTCTTTTCACTTGTTATAGATTGTAAATGAATAAAAGAAAATAATAGGCTATAATCGGTATAAAGACTTAGCGGAGGTACTGATATGACATATAATTTTGATGAAATCATTGATAGACGTTCAACCAATGCGATGAATGTTGAAGGCTATCTGCCATATTTATTTGGTAATGCAGATGTGTCAGATTTACAACATACTGATGACTTAATTCGCTTATGGATAGCGGACATGGATTCACATGATGTGTGGTTAATTTCTGATGAAATTCACTGCGATATAATGAGAAAAGGCATGCAACATTTACCATTTGCGACCGTTCTTTCTGACTATGATAAAGTTATTACAGCCATGTCACAAAGCAAAGCGTTTAACATAGCTGGGCTTATGTTTTCAAATCTAATTATTAGGCATAAAGGACTATTAAATACATGGAAGCAACAACATTTTGGTAGTGAGAATCCATTGAGTATTGCAGCAACTCAAGCTGCTTATGAACAAGGTGAAGATTGGCTACAAGCTATGAATACTTATTTAGATGGCAACTTTGAATGGTTAAAACAATTTTTACAACAAGAGCTTCCAAATGCGAAATTTAAAATCCCAGAAGCAACATATTTGGCTTGGATAGATTTGAGTGATTATATCACGCAGCAGCATATTTCTGAACCTATGGCGAAGTATTTTATTAAGCGGGCAGGCGTGATAATCGAAGGTCAAGAACAGTTTGTACATAATGCAGATGGTCATATCAGAATTAATTTAGCTGTGCCAAGAACAGTGTTACAGCAAGGATTAACTAAAATTAAAGCAGCACTTATGTCATAGCACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACGACGTCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCCGCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCGGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGGACAGGCGAGAGGAGATCTTGATCTAATGATTCAAACCCTTGTGAACTTCTTTAGAACAAAAGAGGTTCGTAACAAGATTTTCTTCACACTAGCAATGTTAGTAATTTTTAAAATAGGGACTTATATACCAGCTCCAGGAGTAAATCCTGCAGCTTTTGATAATCCCCAAGGTTCTCAAGGTGCCACTGAGTTATTAAATACTTTTGGTGGCGGAGCCTTGAAACGATTTTCTATTTTTGCAATGGGTATTGTACCCTACATCACTGCATCAATCGTAATGCAATTATTACAAATGGATATTGTCCCTAAATTCTCAGAATGGGCAAAACAAGGTGAAGTAGGTAGAAGAAAGTTAAATAACGTTACTCGTTATTTAGCAATTTCTTTAGCATTTATCCAATCTATAGGTATGGCATTCCAATTTAATAATTATCTCAAAGGTGCGCTGATTATCAATCAGTCAATTATGAGTTATTTATTAATAGCACTAGTTTTGACAGCAGGAACTGCTTTCTTAATATGGCTTGGTGATCAAATCACTCAGTTCGGTGTTGGTAATGGTATTTCTATTATCATATTCCCATCAAGCTTATTTTAATTATACTCTATCAATGATAGAGTGTCAATATTTTTTTTAGTTTTTCATGAACTCGAGGGGATCCAAATAAAAAACTAGTTTGACAAATAACTCTATCAATGATAGAGTGTCAACAAAAAGGAGGAATTAATGATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTTAAAAGCAGCATAACCTTTTTCCGTGATGGTAACTTCAPBFW (patB forward) primer for amplicon sequencing of genome (SEQ ID NO: 2):GGCATGCAACATTTACCATTTGCGAPBRV (patB reverse) primer for amplicon sequencing of genome (SEQ ID NO: 3):TGTCCAACTTTTGAGATGCACC

Claims

CLAIMSWhat is claimed is:

1. A genetically engineered Staphylococcus lugdunensis strain whose genome comprises a disruption of a patB gene.

2. The genetically engineered Staphylococcus lugdunensis strain of claim 1, wherein the strain exhibits decreased expression of the patB gene as compared to a corresponding wildtype Staphylococcus lugdunensis strain.

3. The genetically engineered Staphylococcus lugdunensis strain of claim 1 or claim 2, wherein the disruption prohibits transcription of a full-length wild-type mRNA from the disrupted patB gene.

4. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-3, wherein the disruption prohibits expression of a functional PatB enzyme, inactivates the PatB protein, or has a negative effect on the expression of the patB gene.

5. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-4, wherein the genetically engineered strain has decreased PatB enzyme activity as compared to a corresponding wild-type Staphylococcus lugdunensis strain.

6. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-5, wherein the disrupted patB gene comprises a deletion of all or at least a portion of the gene, optionally wherein the deletion comprises deletion of the patB promoter.

7. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1- 5, wherein the disrupted patB gene comprises an insertion of a gene cassette.

8. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-5, wherein the disrupted patB gene comprises a deletion of at least a portion of the gene.

9. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1- 5, wherein the disruption comprises a knockout of the patB gene.

10. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1- 5, wherein the disruption comprises an allelic replacement knockout.

11. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-10, wherein the disruption is introduced by homologous recombination, site-directed mutagenesis, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR base editing system, a CRISPR prime editing system, a CRISPR / Cpfl system, a CRISPR / Csml system, or any combination thereof.

12. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-11, wherein the fitness of the strain comprising the disruption is not negatively affected.

13. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-12, wherein the strain has a faster growth rate as compared to a corresponding wild-type Staphylococcus lugdunensis strain.

14. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-13, wherein the strain comprises Staphylococcus lugdunensis deposited with the International Depositary Authority of Canada (IDAC) under IDAC Accession Number 250423-01, or a variant strain thereof.

15. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-14, wherein the strain has a reduced capacity to produce a thiol when contacted with a thiol substrate as compared to a corresponding wild-type strain.

16. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-15, wherein the strain decreases the amount of thiol production by at least 50% when contacted with a thiol substrate as compared to a corresponding wild-type strain.

17. The genetically engineered Staphylococcus lugdunensis strain of claim 15 or claim16, wherein the thiol substrate is benzyl-cysteine.

18. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-17, wherein the strain has a reduced capacity to produce 3-methyl-3-sulfonylhexan-l-ol(3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain.

19. The genetically engineered Staphylococcus lugdunensis strain of any one of claims 1 - 18, wherein the disruption is a deletion in the DNA sequence of the patB gene encoding a PatB protein, wherein the deletion inactivates the PatB protein or has a negative effect on expression of thegene.

20. A genetically engineered Staphylococcus lugdunensis strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 250423-01, or a variant strain derived therefrom, wherein the variant strain is obtained using the deposited strain as starting material, and wherein a reduced capacity of the deposited strain to produce a thiol alcohol when contacted with a thiol substrate as compared to a corresponding wild-type strain is retained or improved in the variant strain as compared to the deposited strain.

21. The genetically engineered Staphylococcus lugdunensis strain of claim 20, wherein the strain decreases the amount of thiol production by at least 50% when contacted with a thiol substrate as compared to a corresponding wild-type strain.

22. A composition comprising an effective amount of one or more of the genetically engineered Staphylococcus lugdunensis strains of any one of claims 1-21.

23. The composition of claim 22, wherein the one or more Staphylococcus lugdunensis strains is lyophilized.

24. The composition of claim 22 or claim 23 further comprising a lyoprotectant.

25. The composition of claim 24, wherein the lyoprotectant is selected from one or more of sucrose and micellar casein.

26. The composition of any one of claims 22-25, wherein the one or more Staphylococcus lugdunensis strains is viable.

27. The composition of any one of claims 22-26, wherein the one or more Staphylococcus lugdunensis strains is nonproliferative.

28. The composition of any one of claims 22-27 further comprising Staphylococcus epidermidis.

29. The composition of any one of claims 22-28 further comprising an excipient.

30. The composition of claim 29, wherein the excipient is selected from one or more of oleic acid, stearic acid, almond butter, avocado butter, babassu butter, beeswax, cocoa butter, coconut butter, coconut oil, cupuacu butter, hemp seed butter, kokum butter, macadamia butter, mango butter, mowrah butter, olive butter, sal seed butter, shea butter, propylene glycol, glycerin, stearyl alcohol, myristyl alcohol, and capric triglyceride.

31. The composition of any one of claims 22-30, wherein the Staphylococcus lugdunensis strain is present at a concentration of about 105to about 1014bacteria per gram.

32. The composition of claim 31, wherein the Staphylococcus lugdunensis strain is present at a concentration of about 107to about 1011bacteria per gram.

33. The composition of any one of claims 29-32, comprising about 90 wt. % to about 99.99 wt. % excipient.

34. The composition of claim 33, further comprising about 10 wt. % to about 0.01 wt. % lyophilized Staphylococcus lugdunensis bacteria.

35. The composition of claim 33, comprising about 95 wt. % to about 99.75 wt. % excipient.

36. The composition of claim 35, comprising about 5 wt. % to about 0.25 wt. % lyophilized Staphylococcus lugdunensis bacteria.

37. The composition of any one of claims 22-36, further comprising a fragrance selected from one or more of elettaria cardamomum seed oil, eucalyptus globulus seed oil, citrus aurantium bergamia fruit oil, mentha piperita oil, salvia sclarea oil, juniperus virginiana oil,abies sibirica oil, citrus aurantium amara, citrus aurantium dulcis lower oil, citrus nobilis oil, rosmarinus officinalis oil, melaleuca alternifolia oil, citrus limon tree oil, zingiber officinale root oil, Mangifera indica oil, Lavandula angustifolia oil, and Santalum album.

38. The composition of any one of claims 22-37, wherein the composition is formulated for topical administration.

39. The composition of claim 38, wherein the composition is formulated for use as a topical deodorant.

40. The composition of any one of claims 22-39, further comprising an antiperspirant.

41. The composition of claim 40, wherein the antiperspirant comprises one or more of aluminum salt, charcoal, and witch hazel.

42. The composition of any one of claims 22-41, wherein the topical deodorant is formulated as a deodorant stick, a roll-on, a cream, a lotion, a gel, a powder, a water activated cream, a water activated powder, an injectable, a patch, or a spray.

43. A method of reducing 3-methyl-3-sulfonylhexan-l-ol (3M3SH) production from a skin microbiota of a subject, the method comprising applying a composition comprising the genetically engineered Staphylococcus lugdunensis strain of any one of claims 1-21 to the skin of the subject or the composition of any one of claims 22-42 to the skin of the subject.

44. A method for treating, reducing, or preventing malodor on the skin of a subject, comprising treating the skin of the subject with an effective amount of the composition of any one of claims 22-42.

45. The method of claim 44, wherein the composition is applied topically to the skin of the subject.

46. The method of claim 44 or 45, wherein the composition is applied to a sweat-gland- containing area of skin of the subject.

47. The method of any one of claims 44-46, wherein the composition is administered to the subject about 1-2 times per day for about 1-7 days.

48. The method of any one of claims 44-47, wherein the treated skin of the subject comprises a reduction in the amount of malodorous bacteria as compared to a corresponding untreated portion of skin.

49. The method of claim 48, wherein the malodorous bacteria comprise wild-type Staphylococcus lugdunensis.

50. A method of producing a genetically engineered Staphylococcus lugdunensis strain, the method comprising targeting the patB gene for disruption.

51. The method of 50, wherein the disruption is introduced into the patB gene by homologous recombination, site-directed mutagenesis, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 system, a CRISPR / Cpfl system, or a CRISPR / Csml system, and any combination thereof.

52. The method of claim 51, wherein the disruption is introduced into the patB gene by homologous recombination.

53. The method of claim 52, wherein the homologous recombination comprises transforming the Staphylococcus lugdunensis with a vector comprising a patB gene knockout cassette.

54. The method of claim 53, wherein the vector is methylated.

55. The method of claim 54, wherein the methylated vector is isolated from an E. coli methylation strain.

56. The method of any one of claims 50-55, wherein the strain exhibits decreased expression of the patB gene as compared to a corresponding wild-type Staphylococcus lugdunensis strain.

57. The method of any one of claims 50-56, wherein the disruption prohibits transcription of a full-length wild-type mRNA from the disrupted patB gene.

58. The method of any one of claims 50-57, wherein the disruption prohibits expression of a functional PatB enzyme.

59. The method of any one of claims 50-58, wherein the genetically engineered strain has decreased PatB enzyme activity as compared to a corresponding wild-type Staphylococcus lugdunensis strain.

60. The method of any one of claims 50-59, wherein the disrupted patB gene comprises a deletion of all or at least a portion of the gene.

61. The method of any one of claims 50-60, wherein the strain comprises Staphylococcus lugdunensis deposited with the International Depositary Authority of Canada (ID AC) under IDAC Accession Number 250423-01, or a variant strain thereof.

62. The method of any one of claims 50-61, wherein the genetically engineered Staphylococcus lugdunensis strain has a reduced capacity to produce a thiol when contacted with a thiol substrate as compared to a corresponding wild-type strain.

63. The method of any one of claims 50-62, wherein the genetically engineered Staphylococcus lugdunensis strain decreases the amount of thiol production by at least 50% when contacted with a thiol substrate compared to a corresponding wild-type strain.

64. The method of any one of claims 50-63, wherein the genetically engineered Staphylococcus lugdunensis strain has a reduced capacity to produce 3-methyl-3- sulfonylhexan-l-ol (3M3SH) when contacted with apocrine sweat or a 3M3SH precursor as compared to a corresponding wild-type strain.

65. An altered microbiota comprising a genetically engineered Staphylococcus lugdunensis strain, or a variant strain thereof, whose genome comprises a disruption of a patB gene.

66. The altered microbiota of claim 65, wherein the microbiota has a reduced capacity to produce 3-methyl-3-sulfonylhexan-l-ol (3M3SH) when contacted with apocrine sweat as compared to a corresponding wild-type microbiota.

67. A deodorant composition comprising:(i) an effective amount of a viable Staphylococcus lugdunensis strain whose genome comprises a disruption of a patB gene;(ii) a lyoprotectant; and(iii) an excipient; wherein the composition is formulated as a water activated formulation for topical administration.

68. The deodorant composition of claim 67, comprising about 90 wt. % to about 99.99 wt. % excipient.

69. The deodorant composition of claim 68, wherein the Staphylococcus lugdunensis strain is lyophilized and the deodorant composition comprises about 10 wt. % to about 0.01 wt. % lyophilized Staphylococcus lugdunensis bacteria.

70. The deodorant composition of claim 68, comprising about 95 wt. % to about 99.75 wt. % excipient.

71. The deodorant composition of claim 70, wherein the Staphylococcus lugdunensis is lyophilized and the deodorant composition comprises about 5 wt. % to about 0.25 wt. % lyophilized Staphylococcus lugdunensis bacteria.

72. The deodorant composition of any one of claims 67-71, wherein the Staphylococcus lugdunensis strain is present at a concentration of about 105to about 1014bacteria per gram.

73. The deodorant composition of claim 72, wherein the Staphylococcus lugdunensis strain is present at a concentration of about 107to about 1011bacteria per gram.

74. The deodorant composition of any one of claims 67-73, wherein the Staphylococcus lugdunensis strain comprises the strain deposited with the International Depositary Authority of Canada (ID AC) under ID AC Accession Number 250423-01.

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