U.S. patent application number 17/602708 was filed with the patent office on 2022-05-26 for compositions and methods for improving skin health and for the treatment and prevention of diseases, disorders and conditions associated with pathogenic microbes.
The applicant listed for this patent is DermBiont, Inc.. Invention is credited to Robert M. Brucker, Sanjay Jain, Ida Lister, Xuecheng Zhang.
Application Number | 20220160617 17/602708 |
Document ID | / |
Family ID | 1000006194478 |
Filed Date | 2022-05-26 |
United States Patent
Application |
20220160617 |
Kind Code |
A1 |
Brucker; Robert M. ; et
al. |
May 26, 2022 |
COMPOSITIONS AND METHODS FOR IMPROVING SKIN HEALTH AND FOR THE
TREATMENT AND PREVENTION OF DISEASES, DISORDERS AND CONDITIONS
ASSOCIATED WITH PATHOGENIC MICROBES
Abstract
Disclosed herein are compositions and methods for using
human-derived Janthinobacterium lividum. Compositions improve skin
health. Methods may include applying human-derived
Janthinobacterium lividum over a host or host area, such as skin or
mucosa, to minimize the presence of one or more microbes, maximize
therapeutic effects, and/or improve health. A method to minimize a
pathogenic microbe may include applying to a surface a composition
including human-derived Janthinobacterium lividum and an acceptable
carrier. Compositions and methods may include a prebiotic to
maximize growth and/or metabolites. Compositions and methods may
include human-derived Janthinobacterium lividum metabolites, such
as violacein, prodigiosin, indole-3-carboxaldehyde, and
lantibiotics, and/or other Postbiotics.
Inventors: |
Brucker; Robert M.;
(Melrose, MA) ; Zhang; Xuecheng; (Newton, MA)
; Lister; Ida; (Boston, MA) ; Jain; Sanjay;
(Shrewsbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DermBiont, Inc. |
Boston |
MA |
US |
|
|
Family ID: |
1000006194478 |
Appl. No.: |
17/602708 |
Filed: |
April 9, 2020 |
PCT Filed: |
April 9, 2020 |
PCT NO: |
PCT/US2020/027556 |
371 Date: |
October 8, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62920010 |
Apr 9, 2019 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 8/492 20130101;
A61Q 17/04 20130101; A61Q 19/08 20130101; A61K 35/747 20130101;
A61K 8/99 20130101; A61P 17/00 20180101 |
International
Class: |
A61K 8/99 20060101
A61K008/99; A61K 35/747 20060101 A61K035/747; A61K 8/49 20060101
A61K008/49; A61P 17/00 20060101 A61P017/00; A61Q 17/04 20060101
A61Q017/04; A61Q 19/08 20060101 A61Q019/08 |
Claims
1. A pharmaceutical composition comprising human-derived
Janthinobacterium lividum in an amount effective to treat, inhibit
or prevent a disease, disorder, or condition associated with a
pathogenic microorganism.
2. The composition of claim 1, formulated for topical application
to prevent, treat, or reduce a symptom of a disease of a mammalian
subject arising from an infection with a pathogenic
microorganism.
3. The composition of claim 1, wherein the Janthinobacterium
lividum comprises a nucleic acid sequence at least 95% identical to
SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
4. The composition of claim 1, wherein the Janthinobacterium
lividum comprises a nucleic acid sequence at least 99% identical to
SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
5. The composition of claim 1, further comprising an additional
isolated microbe.
6. The composition of claim 1, further comprising a first
additional isolated microbe and a second additional isolated
microbe, wherein the first and second additional isolated microbes
are independently selected from bacteria, virus, yeast, or
fungus.
7. The composition of claim 1, wherein the disease, disorder, or
condition is associated with, but not limited to dermatophytes, and
is selected from tinea barbae, tinea capitis, tinea corporis, tinea
curis, tinea pedis, or onychomycosis; or the disease, disorder, or
condition is associated with, but not limited to gram positive
bacteria and Staphylococcus, and is selected from atopic
dermatitis, impetigo, skin infections and soft tissue
infections.
8. The composition of claim 5, wherein the additional isolated
microbe comprises Lactobacillus, Lactococcus or
Propionibacterium.
9. The composition of claim 1, further comprising an anti-fungal
compound.
10. The composition of claim 9, wherein the anti-fungal compound is
present in the composition in a therapeutic amount.
11. The composition of claim 9, wherein the anti-fungal compound is
present in the composition in a sub-therapeutic amount.
12. The composition of claim 1, further comprising an
anti-bacterial compound.
13. The composition of claim 12, wherein the anti-bacterial
compound is present in the composition in a therapeutic amount.
14. The composition of claim 12, wherein the anti-bacterial
compound is present in the composition in a sub-therapeutic
amount.
15. The composition of claim 1, further comprising a prebiotic.
16. The composition of claim 15, wherein the prebiotic is selected
from one or more of an amino acid, biotin, glycerol,
fructooligosaccharide, galactooligosaccharide, inulin, lactulose,
mannan oligosaccharide, oligofructose-enriched inulin,
oligofructose, oligodextrose, tagatose,
trans-galactooligosaccharide, and xylooligosaccharide.
17. The composition of claim 1, further comprising at least one
postbiotic.
18. The pharmaceutical composition of claim 1, wherein the
diseases, disorder, or condition comprises a skin disease,
disorder, or condition in a mammalian subject.
19. The pharmaceutical composition of claim 18, wherein the
mammalian subject is a human subject.
20. The pharmaceutical composition of claim 19, wherein the
pharmaceutical composition is administered in an amount effective
to treat, inhibit, reduce a symptom of, or prevent the human skin
disease, disorder, or condition.
21. The pharmaceutical composition of claim 19, wherein the
pharmaceutical composition is administered in an amount effective
to inhibit the growth of a topical pathogenic microorganism present
on or in a tissue of the human subject.
22. The pharmaceutical composition of claim 21, wherein the topical
pathogenic microorganism is a Dermatophyte.
23. The pharmaceutical composition of claim 22, wherein the topical
pathogenic microorganism is Trichophyton mentagrophytes.
24. The pharmaceutical composition of claim 22, wherein the topical
pathogenic microorganism is Trichophyton rubrum.
25. The pharmaceutical composition of claim 21, wherein the topical
pathogenic microorganism is a bacterium.
26. The pharmaceutical composition of claim 25, wherein the
bacterium is Staphylococcus aureus.
27. The pharmaceutical composition of claim 1, wherein the
Janthinobacterium lividum are desiccated.
28. The pharmaceutical composition of claim 27, wherein the
Janthinobacterium lividum are at least 20% viable upon rehydration
from desiccated conditions.
29. The pharmaceutical composition of claim 1, wherein the
composition is at least 30% viable at room temperature for at least
thirty days.
30. The pharmaceutical composition of claim 1, wherein the
composition comprises a dried formulation of human-derived
Janthinobacterium lividum.
31. The pharmaceutical composition of claim 1, wherein the
pharmaceutical composition is formulated for topical administration
to a human or non-human subject.
32. The pharmaceutical composition of claim 31, wherein the
pharmaceutical composition is formulated as a cream, gel, foam,
ointment, powder or lotion.
33. The pharmaceutical composition of claim 31, wherein the
pharmaceutical composition is formulated as a liquid, tincture,
spray, mister, or inhaler.
34. The pharmaceutical composition of claim 31, wherein the
composition is formulated for topical administration to human
skin.
35. The pharmaceutical composition of claim 31, wherein the
composition is formulated for topical administration to human
mucosa.
36. A synthetic composition comprising human-derived
Janthinobacterium lividum comprising a nucleic acid sequence 95%
identical to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, wherein
the composition is formulated for topical application.
37. The synthetic composition of claim 36, wherein the
human-derived Janthinobacterium lividum comprises a nucleic acid
sequence 99% identical to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:
7.
38. The synthetic composition of claim 36, wherein the composition
is formulated for application to a material that will be in contact
with human skin, nails, hair or mucosa.
39. The synthetic composition of claim 36, wherein the composition
is formulated for application to the human skin and/or mucosa.
40. The synthetic composition of claim 36, wherein the composition
is formulated in an aqueous formulation.
41. The synthetic composition of claim 36, wherein the composition
is formulated for topical application to a surface that is
contacted by a human.
42. The synthetic composition of claim 39, wherein the composition
comprises a cosmetic composition.
43. The cosmetic composition of claim 42, wherein the cosmetic
composition comprises a toothpaste, mouthwash, shampoo, soap, or
dental floss.
44. The cosmetic composition of claim 42, wherein the cosmetic
composition comprises a sunscreen, moisturizer, anti-aging,
probiotic or health promoting composition.
45. A pharmaceutical composition comprising Janthinobacterium
lividum, wherein the Janthinobacterium lividum comprises a nucleic
acid sequence at least 98% identical to SEQ ID NO: 5, SEQ ID NO: 6,
or SEQ ID NO: 7 at the 16s rRNA gene sequence in an amount
effective to treat, inhibit or prevent a disease, disorder, or
condition associated with a pathogenic microorganism.
46. A pharmaceutical composition comprising Janthinobacterium
comprising a nucleic acid sequence comprising SEQ ID NO: 5, SEQ ID
NO: 6, or SEQ ID NO: 7 in an amount effective to treat, inhibit or
prevent a disease, disorder, or condition associated with a
pathogenic microorganism.
47. A pharmaceutical composition comprising an excipient and at
least one human-derived Janthinobacterium lividum in an amount
effective to treat, inhibit or prevent a disease, disorder, or
condition associated with a pathogenic microorganism.
48. A method of inhibiting or preventing a pathogenic
microorganism, the method comprising administering an effective
amount of the human-derived Janthinobacterium lividum to a subject
in need thereof.
49. A composition, comprising a population of non-pathogenic
bacteria obtained from a non-pathogenic human skin microbiome that
has a 2-fold increase in at least one metabolite in the presence of
a pathogenic microorganism, wherein the metabolite comprises
violacein, indole-3-carboxaldehyde, prodigiosin, or a lantibiotic,
wherein the composition is formulated for topical application to a
subject.
50. A method of treating a skin condition in a subject in need
thereof, comprising administering topically to the subject a
therapeutically effective amount of a pharmaceutical composition
that comprises a bacterium of the genus Janthinobacterium, wherein
the bacterium comprise a nucleic acid sequence at least 95%
identical to the 16s rRNA gene sequence of SEQ ID NO: 5, SEQ ID NO:
6, or SEQ ID NO: 7.
51. An isolated Janthinobacterium comprising a 16s rRNA nucleic
acid sequence at least 98% identical to SEQ ID NO: 5, SEQ ID NO: 6,
or SEQ ID NO: 7.
52. A pharmaceutical composition comprising the Janthinobacterium
of claim 51.
53. An isolated Janthinobacterium comprising a 16s rRNA nucleic
acid sequence at least 99% identical to SEQ ID NO: 5, SEQ ID NO: 6,
or SEQ ID NO 7.
54. A pharmaceutical composition comprising the Janthinobacterium
lividum of claim 53
55. A pharmaceutical composition, comprising at least one species
of human-derived Janthinobacterium lividum present in an amount
effective to treat, inhibit or prevent a disease, disorder, or
condition associated with a pathogenic microorganism in a subject
in need thereof, wherein the pharmaceutical composition is in a
topical dosage form.
56. A pharmaceutical composition, comprising a metabolite from at
least one species of human-derived Janthinobacterium lividum,
wherein the metabolite is present in an amount sufficient for
treatment, inhibition or prevention of a disease, disorder or
condition associated with a pathogenic microorganism in a subject
in need thereof, wherein the pharmaceutical composition is in a
topical dosage form.
57. A pharmaceutical composition, comprising a cell lysate of at
least one species of human-derived Janthinobacterium lividum
present in an amount effective to treat, inhibit or prevent a
disease, disorder, or condition associated with a pathogenic
microorganism in a subject in need thereof, wherein the
pharmaceutical composition is in a topical dosage form.
58. A pharmaceutical composition, comprising a Postbiotic of at
least one species of human-derived Janthinobacterium lividum
present in an amount effective to treat, inhibit or prevent a
disease, disorder, or condition associated with a pathogenic
microorganism in a subject in need thereof, wherein the
pharmaceutical composition is in a topical dosage form.
59. An isolated human-derived Janthinobacterium lividum that over
produces violacein, indole-3-carboxaldehyde, prodigiosin,
salicylate, 2,4-diamabutyrate and one or more lantibiotics in an
amount effective to treat, inhibit or prevent a disease, disorder,
or condition associated with a pathogenic microorganism.
60. The Janthinobacterium lividum of claim 59, comprising a nucleic
acid sequence at least 95% identical to SEQ ID NO: 5, SEQ ID NO: 6,
or SEQ ID NO: 7 at the 16S rRNA sequence.
61. A composition comprising the Janthinobacterium of claim 59 and
a physiologically acceptable carrier.
62. A method for treating a skin disorder in a subject in need
thereof, wherein the method comprises topically administering a
formulation comprising an effective amount of probiotic bacteria, a
metabolite of probiotic bacteria, postbiotic of probiotic bacteria,
and/or cell lysate of probiotic bacteria; wherein the probiotic
bacteria are human-derived Janthinobacterium lividum; and the
disorder is associated with the presence of a topical pathogenic
microorganism.
63. The method according to claim 62, wherein the formulation is
formulated for administration to skin or hair.
64. The method according to claim 62, wherein the formulation is
formulated for administration to mucosa.
65. The method according to claim 64, wherein the mucosa is
selected from the group consisting of mucosa of a vagina, penis,
urethra, bladder, anus, mouth, nose, throat, bronchi, lungs, eye,
and ear.
66. A pharmaceutical composition comprising a metabolite produced
from at least one human-derived Janthinobacterium lividum in an
amount effective to treat, inhibit or prevent a disease, disorder,
or condition associated with a pathogenic microorganism.
67. A pharmaceutical composition comprising cell lysate from at
least one human-derived Janthinobacterium lividum in an amount
effective to treat, inhibit or prevent a disease, disorder, or
condition associated with a pathogenic microorganism.
68. A pharmaceutical composition comprising postbiotic from at
least one human-derived Janthinobacterium lividum in an amount
effective to treat, inhibit or prevent a disease, disorder, or
condition associated with a pathogenic microorganism.
69. The pharmaceutical composition of any one of claims 66, 67, and
68, wherein the Janthinobacterium lividum comprises a gene sequence
that is at least 95% identical to SEQ ID NO: 5, SEQ ID NO: 6, or
SEQ ID NO: 7.
70. The pharmaceutical composition of claim 66, wherein the
composition comprises one or more compounds selected from
violacein, indole-3-carboxaldehyde, prodigiosin, salicylate,
2,4-diamabutyrate and one or more lantibiotics.
71. A synthetic composition comprising an effective amount of at
least one human-derived Janthinobacterium lividum metabolite,
formulated for topical application.
72. The synthetic composition of claim 71, wherein the
Janthinobacterium lividum comprises a 16S rRNA gene sequence that
is at least 95% identical to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID
NO: 7.
73. The synthetic composition of claim 71, wherein the synthetic
composition is a cosmetic composition.
74. The synthetic composition of claim 71, wherein the composition
comprises one of more compounds selected from violacein,
indole-3-carboxaldehyde, prodigiosin, salicylate, 2,4-diamabutyrate
and one or more lantibiotics.
75. A synthetic composition comprising an effective amount of
human-derived Janthinobacterium lividum cell lysate, formulated for
topical application.
76. The synthetic composition of claim 75, wherein the
Janthinobacterium lividum comprises a 16S rRNA gene sequence that
is at least 95% identical to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID
NO: 7.
77. The synthetic composition of claim 75, wherein the synthetic
composition is a cosmetic composition.
78. A synthetic composition comprising an effective amount of
human-derived Janthinobacterium lividum postbiotic, formulated for
topical application.
79. The synthetic composition of claim 78, wherein the
Janthinobacterium lividum comprises a 16S rRNA gene sequence that
is at least 95% identical to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID
NO: 7.
80. The synthetic composition of claim 78, wherein the synthetic
composition is a cosmetic composition.
81. The cosmetic composition of claims 73, 77, and 80 wherein the
cosmetic composition comprises a toothpaste, mouthwash, shampoo,
soap, moisturizer, or dental floss.
82. The cosmetic composition of claims 73, 77, and 80 wherein the
cosmetic composition comprises a sunscreen, moisturizer,
anti-aging, probiotic or health-promoting composition.
83. An isolated human-derived Janthinobacterium lividum that
over-produces 2-(alpha-D-mannosyl)-D-glyceric acid,
2-ketogluconate, 2-O-ethyl ascorbic acid, anthramycin,
Aprobarbital, bendiocarb, Bis(2-ethylhexyl) phthalate,
cis-5-Tetradecenoylcarnitine, Dibutyl phthalate, imidazole
propionate, indole-3-carboxaldehyde, indolin-2-one,
N-Acetyl-L-aspartic acid, Phosphoric acid, Phthalic acid,
Pimilprost, trimethadione, or Vemolate, wherein the
Janthinobacterium lividum is present in a composition in an amount
effective to treat, inhibit or prevent a disease, disorder, or
condition associated with a pathogenic microorganism.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
Provisional Patent Application No. 62/920,010, filed on Apr. 9,
2019, the entire contents of which are incorporated by reference
herein.
2. FIELD
[0002] This invention provides beneficial compositions and methods
for the improvement of skin health and the inhibition, treatment
and prevention of diseases, disorders and conditions associated
with pathogenic microbes or microorganisms using human-derived
Janthinobacterium lividum, the metabolites, the cell lysate or
postbiotic of human-derived Janthinobacterium lividum, and
Janthinobacterium lividum containing compositions, formulations and
products, for cosmetic and consumer uses.
3. BACKGROUND
[0003] Topical infections of the skin, nails, mucosa and mucous
cavities by pathogenic microorganisms is a health problem for a
large number of humans and subjects. These microorganisms cause a
variety of bacterial, viral, yeast and fungal infections. These
conditions may arise out of a dysbiosis of the skin, nails, mucosa
and mucous cavities, allowing for a pathogenic microorganism and/or
a community of pathogenic microorganisms to establish.
[0004] There are a variety of infections, such as bacterial, viral,
yeast and/or fungal infections, that affect a relatively large
number of the human population. Potentially pathogenic fungi
include yeasts (e.g., Candida albicans) and dermatophytes.
Dermatophytes are molds that require keratin for nutrition and must
live on stratum corneum, hair, or nails to survive. Human
infections are caused by Trichophyton, Microsporum and
Epidermophyton species. Trichophyton rubrum is responsible for
approximately 46% to 72% of cutaneous and nail mycoses worldwide.
Research studies have established that Trichophyton rubrum, the
most common dermatophyte, and Staphylococcus aureus are the causal
microbes for Tinea pedis and atopic dermatitis, respectively.
Onychomycosis, a common and persistent fungal infection, is
diagnosed in two to eight percent of the global population. The
disease causes disfigurement of nails and/or pain. Treatments for
dermatophytoses includes antifungal topical products (e.g.,
terbinafine, itraconazole, miconazole, etc.) and/or systemic
therapy. The ineffectiveness and toxicity of some long-term
treatments as well as anti-fungal drug resistance and recurrence of
infection has resulted in a need for an alternative treatment.
[0005] Described herein are compositions and methods for using
human-derived Janthinobacterium lividum for treating, inhibiting or
preventing pathogenic microorganisms. The compositions and methods
are useful in modulating the microbiome to effectively inhibit,
treat or prevent microbial infections. These compositions and
methods comprise products of the object of this invention,
human-derived Janthinobacterium lividum.
[0006] Also described herein are topical and cosmetic compositions
and methods for improving skin health, reducing the effects of
exposure to sun, and aging, using human-derived Janthinobacterium
lividum.
4. SUMMARY
[0007] Disclosed herein are pharmaceutical compositions comprising
at least one human-isolated Janthinobacterium lividum in an amount
effective for use in the inhibition, treatment or prevention of
topical pathogenic microorganisms. Also disclosed herein are
pharmaceutical compositions comprising one or more metabolite of
human-derived Janthinobacterium lividum in an amount effective for
use in the inhibition, treatment or prevention of a topical
pathogenic microorganism. Also disclosed herein are pharmaceutical
compositions comprising cell lysate of human-derived
Janthinobacterium lividum in an amount effective for use in the
inhibition, treatment or prevention of a topical pathogenic
microorganism. Disclosed herein are pharmaceutical compositions
comprising an excipient and human-derived Janthinobacterium
lividum, and/or materials originating from human-derived
Janthinobacterium lividum, in an amount effective for use in the
inhibition, treatment or prevention of a topical pathogenic
microorganism; and methods for using these pharmaceutical
compositions to inhibit, treat or prevent pathogenic
microorganisms. These pharmaceutical compositions can be formulated
for application to the skin, mucosa, hair, and/or nails.
[0008] Disclosed herein are synthetic compositions comprising the
probiotic human-derived Janthinobacterium lividum, metabolites from
the probiotic human-derived Janthinobacterium lividum, cell lysate
of the probiotic human-derived Janthinobacterium lividum, and/or
postbiotics from human-derived Janthinobacterium lividum formulated
for topical application. These synthetic compositions can be
formulated for application to the subject (e.g. skin, mucosa, hair,
nails) or to objects that come in contact with the subject (e.g.
cloth, floors, etc.). In some embodiments these synthetic
compositions are cosmetic compositions.
[0009] In some embodiments the compositions of this invention
further comprise a prebiotic. In preferred embodiments, the
prebiotic, is selected from one or more of an amino acid, biotin,
glycerol, fructooligosaccharide, galactooligosaccharides, inulin,
lactulose, mannan oligosaccharide, oligofructose-enriched inulin,
oligofructose, oligodextrose, tagatose,
trans-galactooligosaccharide, and xylooligosaccharide. In some
embodiments, the pharmaceutical composition further comprises an
isolated non-pathogenic additional microbe. In preferred
embodiments, the additional isolated microbe is selected from a
Lactobacillus species, a Lactococcus species, a benign fungal
species typically found on human skin, or a Propionibacterium
species. In some embodiments, the composition is formulated for
administration with additional antifungal or antibacterial
compounds. In some embodiments, the pharmaceutical composition is
formulated for topical administration to the skin or mucosa. In
some embodiments, the compositions are part of a delivery device
for mucosa cavities.
[0010] In some embodiments the human-derived Janthinobacterium
lividum nucleic acid sequence is identified by SEQ ID NO: 5, SEQ ID
NO: 6, or SEQ ID NO: 7. In some embodiments the human-derived
Janthinobacterium lividum comprises a nucleic acid sequence at
least 90% identical to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7;
or at least 93% identical to, or at least 95%, or at least 97%, or
at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 5,
SEQ ID NO: 6, or SEQ ID NO: 7 at the 16s rRNA gene sequence. In
some embodiments the human-derived Janthinobacterium lividum
comprises a nucleic acid sequence at least 90% identical to SEQ ID
NO: 5, SEQ ID NO: 6, or SEQ ID No: 7, at least 92% identical to SEQ
ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; or at least 93% identical,
or at least 95%, or at least 97%, or at least 98%, or at least 99%,
or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
In some embodiments SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 is
at least 90% identical to the human-derived Janthinobacterium
lividum nucleic acid sequence at the 16s rRNA gene sequence; is at
least 90% identical; or at least 93% identical, or at least 95%, or
at least 97%, or at least 98%, or at least 99%, or 100% identical
to human-derived Janthinobacterium lividum nucleic acid sequence at
the 16s rRNA gene sequence.
[0011] In some embodiments, the pharmaceutical composition is used
to treat a yeast pathogenic microorganism, such as Candida
albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei or
C. kefyr. In some embodiments, the pharmaceutical composition is
used to treat a fungal pathogenic microorganism, such as a
Trichophyton or a Malassezia species. In some embodiments the
pharmaceutical composition is used to treat a fungal pathogenic
microorganism such as T. rubrum, T. verrucosum, T. tonsurans, T.
terrestre, T. interdigitale. In some embodiments, the
pharmaceutical composition is used to treat a bacterium pathogenic
microorganism, such as Staphylococcus, Pseudomonas, Enterococcus
and S. aureus. In some embodiments, the pharmaceutical composition
is used to treat a virus pathogenic microorganism, such as
poliovirus, herpes simplex virus, hepatitis A virus, rotavirus,
adenovirus, SARS-CoV-2 and influenza type A virus. In some
embodiments, the pharmaceutical composition is used to treat the
pathogenic microorganism selected from the group consisting of
Gardnerella vaginalis, Candida albicans, Atopobium vaginae,
Staphylococcus aureus, Escherichia coli, Pseudomonas, and
Salmonella.
[0012] In some embodiments, the metabolite of human-derived
Janthinobacterium lividum is soluble in the formulation for topical
administration. In some embodiments, the pharmaceutical composition
comprises a metabolite selected from violacein,
indole-3-carboxaldehyde, prodigiosin, salicylate, 2,4-diamabutyrate
and one or more lantibiotics. In some embodiments, the
human-derived Janthinobacterium lividum produces an antimicrobial
metabolite, selected from violacein, indole-3-carboxaldehyde,
prodigiosin, salicylate, 2,4-diamabutyrate and one or more
lantibiotics, at a level higher than an other non-human
Janthinobacterium lividum reference strain.
[0013] In some embodiments, the pharmaceutical composition
comprising the human-derived Janthinobacterium lividum is
anhydrous, frozen at -20.degree. C., or frozen at -80.degree. C.,
before reconstitution with a separately-stored sterile liquid. In
some embodiments, the liquid for reconstitution is selected from
eye lubricant, glycerol, sucrose, mannitol, 2-Hydroxyethylstarch
(HES), Noveon AA-1 polycarbophil, Methocel F4M (HPMC),
carboxymethyl cellulose, and/or including .lamda.-Carrageenan.
[0014] In some embodiments, the human-derived Janthinobacterium
lividum compositions described herein are used in a method to
inhibit, treat or prevent a pathogenic microorganism, method
comprising administering an effective amount of a human-derived
Janthinobacterium lividum, metabolite and/or cell lysate of
human-derived Janthinobacterium lividum to a subject in need
thereof, wherein human-derived Janthinobacterium lividum,
metabolite and/or cell lysate of human-derived Janthinobacterium
lividum is present in an amount effective for inhibiting, treating
or preventing at least one pathogenic microorganism. In some
embodiments, the human-derived Janthinobacterium lividum is applied
in conjunction with an additional antifungal or antibacterial
agent. In some embodiments the compositions have an additional
probiotic or non-pathogenic microorganism.
[0015] In some embodiments, the pharmaceutical, synthetic, cosmetic
and probiotic compositions of this invention contain at least 10,
10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.10, 10.sup.20
colonizing forming units (CFUs) per a milliliter or milligram of
human-derived Janthinobacterium lividum.
[0016] Disclosed herein are methods of manufacturing a
pharmaceutical composition comprising an effective amount of
probiotic human-derived Janthinobacterium lividum, an optional
metabolite, cell lysate, and/or prebiotic, and a pharmaceutically
acceptable excipient, the method comprising preserving a
human-derived Janthinobacterium lividum by spray drying or
lyophilization in the presence of a drug substance formulation
containing an excipient which enhances preservation and packaging
the preserved human-derived Janthinobacterium lividum for
reconstitution with a second excipient formulation to generate the
formulation immediately prior to administration. In some
embodiments, the excipient is chosen from the group consisting of
amino acid, complex carbohydrate, simple carbohydrate, DMSO,
mannitol, natural tears, eye lubricant, trehalose, and
glycerol.
[0017] Described herein is a kit comprising at least one vial of
stabilized human-derived Janthinobacterium lividum and at least one
optional vial of liquid for reconstitution of stabilized
human-derived Janthinobacterium lividum, instructions for mixing
and application, and optionally one or more implements of mixing
and application. In some embodiments, implements of mixing and
application are included and comprise one or more elements selected
from a syringe, an empty sterile container, and an atomizer or
mister. In some embodiments, the kit contains multiple vials of
stabilized human-derived Janthinobacterium lividum and at least one
vial of liquid for reconstitution of stabilized human-derived
Janthinobacterium lividum, for multiple applications to one or more
subjects in need thereof. In some embodiments, the kit is prepared
for application by a medical professional. In some embodiments, the
kit is prepared for application by a patient.
5. BRIEF DESCRIPTION OF FIGURES
[0018] FIG. 1 shows representative examples of Janthinobacterium
lividum isolated from a human heel, a human forehead, and a radish,
all grown on petri dishes.
[0019] FIG. 2 shows Janthinobacterium lividum prevalence and
abundance on healthy skins, estimated using next-generation
sequencing reads as part of the 16s rRNA gene.
[0020] FIG. 3 shows a phylogenetic tree visualizing genetic
comparisons between multiple isolates of Janthinobacterium lividum
done using genome-wide ANI comparison demonstrating multiple
distinct groups of Janthinobacterium lividum were isolated.
[0021] FIG. 4 shows Janthinobacterium lividum DB02473 significantly
inhibited growth of T. rubrum growth on agar plates.
[0022] FIG. 5 shows Janthinobacterium lividum strains are sensitive
to 10 antibiotics using BD BBL Sensi-Discs.
[0023] FIG. 6 shows images from a purity assay, demonstrating lack
of contamination during manufacturing. A--F107 shake flask, B--F107
Harvest, C--F108 shake flask, D--F108 Harvest.
[0024] FIG. 7 shows Janthinobacterium lividum viability counts in
CFU/ml from F107 (A) and F108 (B) Fermenter samples plated directly
at harvest (WPI) and directly after centrifugation and resuspension
in the cryoprotectant vehicle (Tufts).
[0025] FIG. 8 shows representative images of the Ramsey assay
(Example 4). Images show 4 T. rubrum 18754 colonies alone (A, C) or
with a central cross of human-derived Janthinobacterium lividum
DB02473 from F107 at 10.sup.7 CFU/ml (B, D). A and B were imaged at
12 days; C and D were imaged at 34 days. The bars on C and D show
the position of the radial distance measured for each T. rubrum
colony.
[0026] FIG. 9 shows effectiveness of manufactured Janthinobacterium
lividum strains, as evidenced by the differences shown between mock
control T. rubrum growth radius and dilutions of F107 and F108
harvest samples after 34 days of growth.
[0027] FIG. 10 shows representative results from a S. aureus
antibiosis assay. Detailed methods are described in Example 4.
6. DETAILED DESCRIPTION
[0028] While various embodiments of the invention have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions may occur to those
skilled in the art without departing from the invention. It should
be understood that various alternatives to the embodiments of the
invention described herein may be employed.
[0029] Described herein is the probiotic bacterium human-derived
Janthinobacterium lividum. Described herein are compositions
comprising the probiotic bacterium human-derived Janthinobacterium
lividum which have antimicrobial and other beneficial properties.
The human-derived Janthinobacterium lividum is adapted to the human
host, ensuring that it is safe for human application and is
equipped to survive on a human host at least long enough to be
therapeutically effective. A preferred composition is a
pharmaceutical composition comprising at least one human-derived
Janthinobacterium lividum in an amount effective to treat, inhibit
or prevent a topical pathogenic microorganism. Another preferred
composition is a synthetic composition comprising the probiotic
human-derived Janthinobacterium lividum formulated for topical
application to modulated the microbiome of the object of
application.
[0030] In a preferred embodiment of the present invention, the
composition of human-derived Janthinobacterium lividum, metabolite,
postbiotic and/or cell lysate is formulated for administration to
the skin. In another preferred embodiment of the present invention,
the composition of human-derived Janthinobacterium lividum,
metabolite, postbiotic and/or cell lysate is formulated for
administration to the mucosa.
[0031] It will be further understood that the formulation for use
in the present invention may comprise one or more of at least one
probiotic bacteria, at least one metabolite of a probiotic
bacterium, at least one cell lysate of a probiotic bacterium or a
postbiotic or a probiotic bacterium.
[0032] It will be further understood that the formulation may
comprise more than one bacterium, soluble metabolite, cell lysate
or postbiotic. For example, the formulation may comprise at least
2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20 bacterium, a culture,
their metabolites, cell lysates or postbiotics.
[0033] It will be understood by the skilled person that as used
herein the term "probiotic" refers to a live microorganism, microbe
or living culture (including bacterium or yeasts for example)
which, provided in sufficient numbers, beneficially affects the
host organism, i.e. by conferring one or more demonstrable health
benefits on the host organism. A "probiotic bacterium" or
"probiotic microorganism" or "probiotic microbe" or "probiotic
culture" or "probiotic bacteria" is a bacterium, microorganism,
microbe, culture or bacteria which, provided in sufficient numbers,
beneficially affects the host organism, i.e. by conferring one or
more demonstrable health benefits on the host organism.
[0034] As used herein, the term "cell lysate" or "lysate" refers to
probiotic cells which have been lysed by any suitable means. In
preferred embodiments, the cell debris is removed prior to use. In
more preferred embodiments the cell lysates are filtered prior to
use. In exemplary embodiments, the cells are lysed by, for example
sonication, homogenization, shearing or chemical lysis.
[0035] As used herein the term "postbiotic" refers to functional
bioactive compounds, generated by a probiotic, which may be used to
promote health. The term postbiotics can be regarded as an umbrella
term for all synonyms and related terms of these microbial
components. Therefore, postbiotics can include many different
constituents including metabolites, short-chain fatty acids (SCFAs,
e.g. acetic, propionic and butyric acid), microbial cell fractions,
functional proteins, extracellular polysaccharides (EPS), cell
lysates, teichoic acid, phenyllactic acid, volatile organic
compounds (VOCs), B-vitamin synthesis (biotin, cobalamin, folates,
nicotinic acid, pantothenic acid, pyridoxine, riboflavin, and
thiamine), peptidoglycan-derived muropeptides, antimicrobial
peptides (AMP) and pili-type structures.
[0036] Probiotic bacterium suitable for use in the present
invention include, but are not limited to, human-derived
Janthinobacterium and any additional non-pathogenic microbe such
as, Bifidobacterium, Brevibacterium, Propionibacterium,
Lactococcus, Streptococcus, Lactobacillus (e.g., L. acidophilus),
Enterococcus, Pediococcus, Leuconostoc, and/or Oenococcus.
[0037] Soluble metabolites for use in the present invention
include, but are not limited to, soluble metabolites from
human-derived Janthinobacterium lividum and any additional
non-pathogenic microbe such as, Bifidobacterium, Brevibacterium,
Propionibacterium, Lactococcus, Streptococcus, Lactobacillus (e.g.,
L. acidophilus), Enterococcus, Pediococcus, Leuconostoc, and/or
Oenococcus.
[0038] Cell lysates for use in the present invention include, but
are not limited to, cell lysates from human-derived
Janthinobacterium and any additional non-pathogenic microbe such
as, Bifidobacterium, Brevibacterium, Propionibacterium,
Lactococcus, Streptococcus, Lactobacillus (e.g., L. acidophilus),
Enterococcus, Pediococcus, Leuconostoc, and/or Oenococcus.
[0039] As used herein, the term "soluble metabolite" refers to a
metabolite or metabolites present in the supernatant of a cell
culture from which the cells have been removed. In preferred
embodiments the culture is grown to a cell density of at least
about OD.sub.600 0.5. In a further preferred embodiment, the cells
are removed by centrifugation. In a more preferred embodiment, the
supernatant is filtered. It will be apparent that the supernatant
may be used directly in the formulations of the present invention,
or that one or more of the metabolites may be isolated form the
supernatant by any suitable means prior to use.
[0040] When used herein, the term topical includes references to
formulations that are adapted for application to body surfaces
(e.g. the skin, mucosa or mucous membranes). The skin includes the
exterior surfaces such as finger and toenails. Mucous membranes, or
mucosa, that may be mentioned in this respect include the mucosa of
the vagina, the penis, the urethra, the bladder, the anus, the
colon, the mouth (including the mucosa of the cheek, the soft
palate, the under surface of tongue and the floor of the mouth),
the nose, the throat (including the mucosa of the pharynx, the
larynx, the trachea and the esophagus), the bronchi, the lungs, the
eye and the ear.
[0041] The term "ameliorating" refers to any therapeutically
beneficial result in the treatment of a disease state, e.g., a
metabolic disease state, including prophylaxis, lessening in the
severity or progression, remission, or cure thereof.
[0042] The term "in situ" refers to processes that occur in a
living cell growing separate from a living organism, e.g., growing
in tissue culture.
[0043] The term "in vivo" refers to processes that occur in a
living organism.
[0044] The term "mammal" as used herein includes both humans and
non-humans and includes but is not limited to humans, non-human
primates, canines, felines, murines, bovines, equines, and
porcines.
[0045] As used herein, the term "derived from" includes microbes,
microorganisms or other living culture immediately taken from an
environmental sample and also microbes, microorganisms or other
living culture isolated from an environmental source and
subsequently grown in a pure culture or isolate.
[0046] As used herein, the term "strain" is defined as any nucleic
acid sequence that is 97% or greater identical to a defined 16s
rRNA nucleic acid sequence. More preferred embodiments of strain is
a nucleic acid sequence that is greater than 98%, greater than 99%
identical to a defined 16s rRNA nucleic acid sequence.
[0047] The term "percent identical," in the context of two or more
nucleic acid or polypeptide sequences, refers to two or more
sequences or subsequences that have a specified percentage of
nucleotides or amino acid residues that are the same, when compared
and aligned for maximum correspondence, as measured using one of
the sequence comparison algorithms described below (e.g., BLASTP
and BLASTN or other algorithms available to persons of skill) or by
visual inspection. Depending on the application, the percent
"identical" can exist over a region of the sequence being compared,
e.g., over a functional domain, or, alternatively, exist over the
full length of the two sequences to be compared. In some aspects,
percent identical is defined with respect to a region useful for
characterizing phylogenetic similarity of two or more organisms,
including two or more microorganisms. Percent identical in these
circumstances can be determined by identifying such sequences
within the context of a larger sequence, that can include sequences
introduced by cloning or sequencing manipulations such as, e.g.,
primers, adapters, etc., and analyzing the percent identical in the
regions of interest, without including in those analyses introduced
sequences that do not inform phylogenetic similarity.
[0048] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequent coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identical for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0049] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Ausubel et al., infra).
[0050] One example of an algorithm that is suitable for determining
percent sequence identical and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information.
[0051] The term "sufficient amount" means an amount sufficient to
produce a desired effect, e.g., an amount sufficient to alter the
microbial content of a subject's microbiota.
[0052] The term "therapeutic amount" is an amount of an
anti-microbial, for example an anti-fungal or anti-bacterial,
compound that is prescribed. Concentrations below those typically
prescribed are termed "sub-therapeutic" amounts.
[0053] The term "therapeutically effective amount" is an amount
that is effective to ameliorate a symptom of a disease. A
therapeutically effective amount can be a "prophylactically
effective amount" as prophylaxis can be considered therapy.
[0054] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0055] As used herein, the term "inhibit", "inhibiting" or
"inhibition" includes stopping the progression of a condition or
growth, substantially preventing a condition or growth or
substantially treating a condition or undesired growth.
[0056] As used herein, the term "treat", "treating" or "treatment"
includes abrogating, inhibiting substantially, slowing, or
reversing the progression of a condition, substantially
ameliorating clinical or aesthetical symptoms of a condition.
[0057] As used herein, the term "preventing" or "prevention"
includes completely or substantially reducing the likelihood or
occurrence or the severity of initial clinical or aesthetical
symptoms of a condition.
[0058] As used herein, the term "pathogen" refers to the disease,
disorder or condition and to the microorganism associated with the
disease or infection. For example: Tinea barbae is a dermatophyte
infection of the beard area most often caused by Trichophyton
mentagrophytes or T. verrucosum. verrucosum. Tinea capitis is a
dermatophytosis caused by Trichophyton tonsurans, Microsporum canis
and M. audouinii; other Trichophyton species (e.g., T.
schoenleinii, T. violaceum). Tinea corporis is a dermatophyte
infection of the face, trunk, and extremities commonly caused by
causes are Trichophyton mentagrophytes, T. rubrum, and Microsporum
canis. Tinea cruris is a dermatophytosis that is commonly caused by
Trichophyton rubrum or T. mentagrophytes. Tinea pedis is a
dermatophyte infection of the feet commonly caused by T. rubrum.
Dermatophytid (identity or id) reactions are protean; they are not
related to localized growth of the fungus but rather are an
inflammatory reaction to a dermatophytosis elsewhere on the body.
Other disease, disorder, or conditions related to, but not limited,
atopic dermatitis, impetigo, skin and soft tissue infections, are
often caused gram positive bacterium and Staphylococcus.
[0059] As used herein, the term "about" includes variation of up to
approximately +/-10% and that allows for functional equivalence in
the product.
[0060] As used herein, the term "colony-forming unit" or "CFU" is
an individual cell that is able to clone itself into an entire
colony of identical cells.
[0061] As used herein all percentages are weight percent unless
otherwise indicated.
[0062] As used herein, "viable organisms" are organisms that are
capable of growth and multiplication. In some embodiments,
viability can be assessed by numbers of colony-forming units that
can be cultured. In some embodiments, viability can be assessed by
other means, such as quantitative polymerase chain reaction.
[0063] The term "derived from" includes material isolated from the
recited source, and materials obtained using the isolated materials
(e.g., cultures of microorganisms made from microorganisms isolated
from the recited source).
[0064] "Microbiota" refers to the community of microorganisms or
microbes that occur (sustainably or transiently) in and on an
animal subject, typically a mammal such as a human, including
eukaryotes, archaea, bacteria, and viruses (including bacterial
viruses i.e., phage).
[0065] "Microbiome" refers to the genetic content of the
communities of microbes, microorganisms or living cultures that
live in and on the human body, both sustainably and transiently,
including eukaryotes, archaea, bacteria, and viruses (including
bacterial viruses (i.e., phage)), wherein "genetic content"
includes genomic DNA, RNA such as ribosomal RNA, the epigenome,
plasmids, and all other types of genetic information.
[0066] The term "subject" refers to any animal subject including
humans, laboratory animals (e.g., primates, rats, mice), livestock
(e.g., cows, sheep, goats, pigs, turkeys, and chickens), and
household pets (e.g., dogs, cats, and rodents). The subject may be
suffering from a dysbiosis, including, but not limited to, an
infection due to a pathogenic microorganism or may be at risk of
developing or transmitting to others an infection due to a
pathogenic microorganism.
[0067] The "colonization" of a host organism includes the
non-transitory residence of a bacterium or other microscopic
organism. As used herein, "reducing colonization" of a host
subject's skin (or any other microbial niche) by a pathogenic
bacterium includes a reduction in the residence time of the
pathogen on the skin as well as a reduction in the number (or
concentration) of the pathogen on the skin or adhered to the skin.
Measuring reductions of adherent pathogens may be demonstrated,
e.g., by a biopsy sample, by swabbing the skin, or reductions may
be measured indirectly.
[0068] A "combination" of two or more bacterium includes the
physical co-existence of the two bacteria, either in the same
material or product or in physically connected products, as well as
the temporal co-administration or co-localization of the two
bacteria.
[0069] As used herein, "desiccate," refers to dehydration or to
dehydrate, typically by being lyophilized, freeze dried, or spray
dried.
[0070] Throughout this application, various embodiments of this
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range.
[0071] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, can also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
can also be provided separately or in any suitable sub-combination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0072] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
[0073] Janthinobacterium
[0074] Janthinobacterium is genus of Gram negative,
betaproteobacteria that are commonly found in many environmental
niches, including the human body. Janthinobacterium lividum was
identified for its ability to protect amphibians from fungal
infection. In some embodiments, the strain of Janthinobacterium
lividum is isolated from an environmental source. In a preferred
embodiment, the strain of Janthinobacterium lividum is originally
derived from a human source. In an embodiment, a human-derived
Janthinobacterium lividum strain demonstrates superior persistence
on human skin compared to a reference strain. In an embodiment, a
human-derived Janthinobacterium lividum strain demonstrates
superior or overproduction of a metabolite or postbiotics compared
to a reference strain. In such embodiments, a reference
Janthinobacterium lividum strain may be a strain isolated from a
different environmental niche, such as salamander skin, produce, or
the like. Janthinobacterium lividum produces several metabolites
with antimicrobial effects: violacein, indole-3-carboxaldehyde,
prodigiosin, salicylate, 2,4-diamabutyrate and one or more
lantibiotics. Violacein is a bisindole compound known for its
purple color and antimicrobial properties. Indole-3-carboxaldehye
has a role as a plant metabolite, a human xenobiotic metabolite, a
bacterial metabolite and a marine metabolite. It is a heteroarene
carbaldehyde, an indole alkaloid and a member of indoles.
Prodigiosin is an alkaloid, red-pigmented, secondary metabolite,
often associated with Serratia species. Prodigiosin molecules are
identified by their common pyrrolyl pyrromethene skeleton, and have
been shown to have a variety of biological activities, including
antimicrobial activity.
[0075] Lantibiotics, a subset of bacteriocins, are
genetically-encoded peptides containing intramolecular ring
structures, many of which have been shown to have antimicrobial
properties. Lantibiotic peptides are modified post-translationally
to create their characteristic ring structures. One of the most
well-known lantibiotic is nisin.
[0076] In some embodiments, the Janthinobacterium strain is
compared to a reference strain. In some embodiments, the reference
strain is another Janthinobacterium strain isolated from another
environmental niche. In some embodiments, the human-derived
Janthinobacterium lividum of this application is compared to a
Janthinobacterium lividum strain isolated from an amphibian species
and found to provide a benefit over the amphibian-derived species
and plant-derived species. In some embodiments, the benefit is
increased longevity on human skin. In some embodiments, the benefit
is increased production of violacein, indole-3-carboxaldehyde,
prodigiosin, salicylate, 2,4-diamabutyrate and one or more
lantibiotics. In some embodiments, the benefit is increased
production of 2-(alpha-D-mannosyl)-D-glyceric acid,
2-ketogluconate, 2-O-ethyl ascorbic acid, anthramycin,
Aprobarbital, bendiocarb, Bis(2-ethylhexyl) phthalate,
cis-5-Tetradecenoylcarnitine, Dibutyl phthalate, imidazole
propionate, indole-3-carboxylate, indolin-2-one,
N-Acetyl-L-aspartic acid, Phosphoric acid, Phthalic acid,
Pimilprost, trimethadione, and Vemolate.
[0077] In some embodiments, a human-derived Janthinobacterium
lividum or an isolated human-derived Janthinobacterium lividum over
produces or over expresses its compounds (e.g., metabolites, e.g.,
violacein, indole-3-carboxaldehyde, prodigiosin, salicylate,
2,4-diamabutyrate and one or more lantibiotics) relative to other
strains (e.g., a reference strain). As used, the terms "over
produce" and "over express" refer to at least 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 100%, 110%, 120%, 130%, 140%, 1.5-fold, 2-fold, 2.5-fold, or
3-fold more production or more expression (respectively) relative
to other strains (e.g., a reference strain). In such embodiments, a
reference Janthinobacterium lividum strain may be a strain isolated
from a different environmental niche, such as salamander skin,
produce, or the like. As used, the term "over produce" refers to
the production of the compounds (e.g., metabolites) by the
organism, and the term "over expresses" refers to the expression of
a gene that produces the compounds (e.g., metabolites).
[0078] Prebiotics
[0079] Prebiotics, in accordance with the teachings of this
invention, comprise compositions that promote the growth of
beneficial bacteria. Prebiotic substances can be consumed by a
relevant probiotic, or otherwise assist in keeping the relevant
probiotic alive or stimulate its growth. When applied or consumed
in an effective amount, prebiotics also beneficially affect a
subject's naturally-occurring microbiome and thereby impart health
benefits. Prebiotic foods enter the colon and serve as substrate
for the endogenous bacteria, thereby indirectly providing the host
with energy, metabolic substrates, and essential micronutrients.
Prebiotics can also be added to any probiotic composition to
enhance effectiveness or longevity of the probiotic strains.
[0080] Prebiotics help probiotics flourish in their environment,
and accordingly, their health benefits are largely indirect. For
example, metabolites generated by colonic fermentation by
intestinal microflora, such as short-chain fatty acids, can play
important functional roles in the health of the host. Prebiotics
can be useful agents for enhancing the ability of human microflora
to provide benefits to their host.
[0081] Prebiotics, in accordance with the embodiments of this
invention, include, without limitation, amino acids, glycerol,
mucopolysaccharides, oligosaccharides, polysaccharides, amino
acids, vitamins, nutrient precursors, proteins, and combinations
thereof.
[0082] According to particular embodiments, compositions comprise a
prebiotic comprising, without limitation, amino acids, glycerol,
polysaccharides and oligosaccharides. These compounds have the
ability to increase the number of probiotics and augment their
associated benefits. For example, an increase of beneficial
Bifidobacteria likely changes the intestinal pH to support the
increase of Bifidobacteria, thereby decreasing pathogenic
organisms.
[0083] Non-limiting examples of oligosaccharides that are
categorized as prebiotics in accordance with particular embodiments
include galactooligosaccharides, fructooligosaccharides, inulins,
isomalto-oligosaccharides, lactilol, lactosucrose, lactulose,
pyrodextrins, soy oligosaccharides, transgalacto-oligosaccharides,
and xylo-oligosaccharides.
[0084] According to other particular embodiments, compositions
comprise a prebiotic comprising an amino acid.
[0085] In an embodiment, a prebiotic is included to increase
production of one or more beneficial metabolites. In an embodiment,
tryptophan and/or glycerol are included to increase production of
violacein.
[0086] In an embodiment, a prebiotic is added that additionally
serves as a cryoprotectant.
[0087] Dosage for the compositions described herein are deemed to
be "effective doses," indicating that the probiotic or prebiotic
composition is administered in a sufficient quantity to alter the
physiology of a subject in a desired manner.
[0088] Preferred embodiments are prebiotics that improve the growth
of human-derived Janthinobacterium lividum such as those selected
from D-Mannitol, Tween 20, Tween 40 and Cytidine
[0089] Preferred embodiments are prebiotics that enhance the
function of human-derived Janthinobacterium lividum such as those
selected from the Table 1.
[0090] More preferred embodiments are prebiotics that enhance the
function of human-derived Janthinobacterium lividum such as those
selected from the Table below identified by the "+".
TABLE-US-00001 Components for enhancing function of human- derived
Janthinobacterium lividum Most Concentration Preferred Range
Preferred Prebiotic Additives* Prebiotic of Efficacy
N-Acetyl-D-Glucosamine 2-20 mM L-Proline 2-20 mM D-Mannose 2-20 mM
Dulcitol 2-20 mM Glycerol 2-20 mM L-Fucose 2-20 mM D-Glucoronic
Acid 2-20 mM D-Gluconic Acid + 2-20 mM D,L-.alpha.-Glycerol
Phosphate 2-20 mM D-Mannitol + 2-20 mM L-Glutamic acid 2-20 mM
Tween 20 + 2-20 mM L-Rhamnose + 2-20 mM D-Fructose + 2-20 mM Acetic
Acid + 2-20 mM .alpha.-D-Glucose + 2-20 mM Maltose + 2-20 mM
D-Melibiose + 2-20 mM Thymidine + 2-20 mM L-Asparagine + 0.1-20 mM
D-Aspartic Acid 0.1-20 mM D-Glucosaminic Acid + 2-20 mM
1,2-Propanediol + 2-20 mM Tween 40 2-20 mM .alpha.-Keto-Glutaric
Acid 2-20 mM .alpha.-Keto-Butyric Acid 2-20 mM
.alpha.-Methyl-D-Galactoside + 2-20 mM .alpha.-D-Lactose + 2-20 mM
Sucrose 2-20 mM Uridine 2-20 mM L-Glutamine 2-20 mM
D-Glucose-1-Phosphate 2-20 mM .alpha.-Hydroxy Glutaric
Acid-g-Lactone 2-20 mM .beta.-Methyl-D-Glucoside 2-20 mM Adonitol
2-20 mM Maltotriose 2-20 mM 2-Deoxy Adenosine 2-20 mM Adenosine +
2-20 mM Mucic Acid 2-20 mM Glyoxylic Acid 2-20 mM D-Cellobiose +
2-20 mM Inosine 2-20 mM Glycyl-L-Glutamic Acid 2-20 mM
Tricarballylic Acid 2-20 mM L-Serine 2-20 mM L-Threonine 2-20 mM
L-Alanine 2-20 mM L-Alanyl-Glycine 2-20 mM
N-Acetyl-.beta.-D-Mannosamine + 2-20 mM Mono Methyl Succinate 2-20
mM Tyramine 0.1-20 mM Chondroitin Sulfate C 1-5 mM
.alpha.-Cyclodextrin 1-5 mM .beta.-Cyclodextrin 1-5 mM
g-Cyclodextrin + 1-5 mM Glycogen + 1-5 mM Inulin 1-5 mM Laminarin
1-5 mM Mannan 1-5 mM Pectin 1-5 mM N-Acetyl-D-Galactosamine 1-5 mM
N-Acetyl-Neuraminic Acid + 1-5 mM .beta.-D-Allose + 1-5 mM
Amygdalin + 1-5 mM D-Arabitol + 1-5 mM L-Arabitol + 1-5 mM Arbutin
+ 1-5 mM 2-Deoxy-DRibose + 1-5 mM i-Erythritol + 1-5 mM D-Fucose +
1-5 mM 3-0-.beta.-D-Galactopyranosyl-D Arabinose 1-5 mM Gentiobiose
+ 1-5 mM L-Glucose + 1-5 mM D-Melezitose + 1-5 mM Maltitol + 1-5 mM
.alpha.-Methyl-D-Glucoside + 1-5 mM .beta.-Methyl-D-Galactoside +
1-5 mM 3-Methyl Glucose + 1-5 mM .beta.-Methyl-D-Glucuronic Acid +
1-5 mM .alpha.-Methyl-D-Mannoside + 1-5 mM .beta.-Methyl-D-Xyloside
+ 1-5 mM Palatinose + 1-5 mM Sedoheptulosan + 1-5 mM L-Sorbose +
1-5 mM Stachyose + 1-5 mM D-Tagatose + 1-5 mM Turanose + 1-5 mM
Xylitol + 1-5 mM D-Ribono-1,4-Lactone 1-5 mM Sebacic Acid 1-5 mM
Acetamide + 1-5 mM L-Alaninamide + 1-5 mM N-Acetyl-L-Glutamic Acid
+ 1-5 mM L-Arginine + 1-5 mM Glycine + 1-5 mM L-Histidine + 1-5 mM
L-Homoserine + 0.1-5 mM Hydroxy-L-Proline + 1-5 mM L-Isoleucine +
1-5 mM dextrin 1-5 mM lactitol 1-5 mM D-Tartaric acid 1-5 mM
L-Tartaric acid 1-5 mM L-arginine 1-5 mM L-phenylalanine 1-5 mM
Ammonia + 0.1-1 mM Nitrite + 0.1-1 mM Nitrate + 0.1-1 mM Urea +
0.1-1 mM Biuret + 0.1-1 mM L-Alanine + 0.1-1 mM L-Arginine + 0.1-1
mM L-Aspartic Acid + 0.1-1 mM L-Cysteine + 0.1-1 mM L-Glutamic Acid
+ 0.1-1 mM L-Glutamine + 0.1-1 mM Glycine + 0.1-1 mM L-Histidine +
0.1-1 mM L-Isoleucine + 0.1-1 mM L-Leucine + 0.1-1 mM L-Lysine +
0.1-1 mM L-Methionine + 0.1-1 mM L-Phenylalanine + 0.1-1 mM
L-Proline + 0.1-1 mM L-Serine + 0.1-1 mM L-Threonine + 0.1-1 mM
L-Tryptophan + 0.1-1 mM L-Tyrosine 0.1-1 mM L-Valine 0.1-1 mM
D-Alanine 0.1-1 mM D-Asparagine 0.1-1 mM D-Glutamic Acid 0.1-1 mM
D-Lysine 0.1-1 mM D-Serine 0.1-1 mM D-Valine 0.1-1 mM L-Citrulline
0.1-1 mM L-Ornithine 0.1-1 mM Ethylenediamine + 0.1-1 mM Putrescine
+ 0.1-1 mM Agmatine + 0.1-1 mM Histamine + 0.1-1 mM
.beta.-Phenylethylamine 0.1-1 mM Acetamide + 0.1-1 mM Formamide +
0.1-1 mM Glucuronamide + 0.1-1 mM D,L-Lactamide + 0.1-1 mM
D-Glucosamine + 0.1-1 mM D-Galactosamine + 0.1-1 mM D-Mannosamine +
0.1-1 mM N-Acetyl-D-Glucosamine + 0.1-1 mM N-Acetyl-D-Galactosamine
+ 0.1-1 mM N-Acetyl-D-Mannosamine + 0.1-1 mM Adenine + 0.1-1 mM
Adenosine + 0.1-1 mM Allantoin 0.1-1 mM Ala-Glu 0.1-1 mM *0.1 OD
unit or more higher than neg control at 18 hour time
[0091] Formulations
[0092] Provided herein, in some aspects, are compositions that
comprise at least one strain of human-derived Janthinobacterium
lividum disclosed herein, wherein the compositions are formulated
for administration to a subject in need thereof. Generally, the
subject is a human afflicted with a topical pathogenic
microorganism infection of the skin and/or mucosa. In some
embodiments, the compositions are formulated for topical
administration to a subject in need thereof. In some embodiments,
the compositions are formulated for topical administration to the
skin of the subject. In some embodiments, the compositions are
formulated for topical administration to the scalp of the subject.
In some embodiments, the compositions are formulated for
application to mucosa surfaces. In some embodiments, a composition
is formulated for oral administration. In some embodiments, a
composition is formulated for transdermal administration. In some
embodiments, a composition is formulated for injectable
administration. In certain embodiments, the composition is a
formulation selected from a gel, ointment, lotion, emulsion, paste,
cream, foam, mousse, liquid, douche, garage, spray, suspension,
dispersion, nasal spray and aerosol. In certain embodiments, the
formulation comprises one or more excipients to provide a desired
form and a desired viscosity, flow or other physical or chemical
characteristic for effective application, coverage and adhesion to
skin.
[0093] In certain embodiments the human-derived Janthinobacterium
lividum of this invention is desiccated or dehydrated. Desiccation
may be accomplished by standard methods of practice and can be
select from such methods as lyophilization, spray drying, or freeze
drying. Rehydration of the desiccated human-derived
Janthinobacterium lividum produces at least 20% viable bacterium,
at least 30%, at least 50% viable bacterium.
[0094] Compositions disclosed herein are compositions, wherein the
composition is at least 30% viable at room temperature for at least
thirty days. Also disclosed are compositions that are at least 1%,
at least 5%, at least 10%, at least 20%, at least 30%, at least 50%
viable for at least 30, 60, 90, 120, 150, 180 or at least 360
days.
[0095] In some embodiments the compositions are pharmaceutical
compositions. In some embodiments the compositions are synthetic
compositions. In some embodiments the compositions are cosmetic
compositions. In some embodiments the compositions are probiotic
compositions.
[0096] Compositions disclosed herein may be presented in a
formulation that includes one or more excipients to improve any one
or more of shelf-life, application, skin penetration, and
therapeutic effect. In some embodiments, the excipient is necessary
to improve any one or more of shelf-life, application, skin
penetration, and therapeutic effect.
[0097] Compositions disclosed herein may be in a topical dosage
form, wherein the topical dosage form provides easy application to
a surface, such as skin, nails, hair, and/or mucosa. The surface
may be a surface that comes in contact with a subject.
[0098] In certain embodiments, the Janthinobacterium probiotic
compositions described herein are formulated for oral ingestion.
The oral ingestion form may be a pill, tablet, capsule, paste,
liquid suspension, colloid, or mixed with various foods such as
candies, chews, yogurt, milk, cottage cheese or non-dairy based or
lactose reduced substitutes. The formulation may contain additional
non-active ingredients that improve flavor, smell, or texture of
the edible composition. The formulation may also include binding
agents, encapsulating films, or excipients that preserve shelf-life
and bioavailability.
[0099] An emulsion may be described as a preparation of one liquid
distributed in small globules throughout the body of a second
liquid. In some embodiments, the dispersed liquid is the
discontinuous phase, and the dispersion medium is the continuous
phase. When oil is the dispersed liquid and an aqueous solution is
the continuous phase, it is known as an oil-in-water emulsion,
whereas when water or aqueous solution is the dispersed phase and
oil or oleaginous substance is the continuous phase, it is known as
a water-in-oil emulsion. The oil phase may consist at least in part
of a propellant, such as an HFA propellant. Either or both of the
oil phase and the aqueous phase may contain one or more
surfactants, emulsifiers, emulsion stabilizers, buffers, and other
excipients. Preferred excipients include surfactants, especially
non-ionic surfactants; emulsifying agents, especially emulsifying
waxes; and liquid non-volatile non-aqueous materials, particularly
glycols such as propylene glycol. The oil phase may contain other
oily pharmaceutically approved excipients. For example, materials
such as hydroxylated castor oil or sesame oil may be used in the
oil phase as surfactants or emulsifiers.
[0100] A lotion may be described as a low- to medium-viscosity
liquid formulation. A lotion can contain finely powdered substances
that are in soluble in the dispersion medium through the use of
suspending agents and dispersing agents. Alternatively, lotions can
have as the dispersed phase liquid substances that are immiscible
with the vehicle and are usually dispersed by means of emulsifying
agents or other suitable stabilizers. In one embodiment, the lotion
is in the form of an emulsion having a viscosity of between 100 and
1000 centistokes. The fluidity of lotions permits rapid and uniform
application over a wide surface area. Lotions are typically
intended to dry on the skin leaving a thin coat of their medicinal
components on the skin's surface.
[0101] A cream may be described as a viscous liquid or semi-solid
emulsion of either the "oil-in-water" or "water-in-oil type".
Creams may contain emulsifying agents and/or other stabilizing
agents. In one embodiment, the formulation is in the form of a
cream having a viscosity of greater than 1000 centistokes,
typically in the range of 20,000-50,000 centistokes. Creams are
often time preferred over ointments as they are generally easier to
spread and easier to remove.
[0102] The basic difference between a cream and a lotion is the
viscosity, which is dependent on the amount/use of various oils and
the percentage of water used to prepare the formulations. Creams
are typically thicker than lotions, may have various uses and often
one uses more varied oils/butters, depending upon the desired
effect upon the skin. In a cream formulation, the water-base
percentage is about 60-75% and the oil-base is about 20-30% of the
total, with the other percentages being the emulsifier agent,
preservatives and additives for a total of 100%.
[0103] An ointment may be described as a semisolid preparation
containing an ointment base and optionally one or more active
agents of this disclosure. Examples of suitable ointment bases
include hydrocarbon bases (e.g., petrolatum, white petrolatum,
yellow ointment, and mineral oil); absorption bases (hydrophilic c,
anhydrous lanolin, lanolin, and cold cream); water-removable bases
(e.g., hydrophilic ointment), and water-soluble bases (e.g.,
polyethylene glycol ointments). Pastes typically differ from
ointments in that they contain a larger percentage of solids.
Pastes are typically more absorptive and less greasy that ointments
prepared with the same components.
[0104] A gel may be described as a semisolid system containing
dispersions of small or large molecules in a liquid vehicle that is
rendered semisolid by the action of a thickening agent or polymeric
material dissolved or suspended in the liquid vehicle. The liquid
may include a lipophilic component, an aqueous component or both.
Some emulsions may be gels or otherwise include a gel component.
Some gels, however, are not emulsions because they do not contain a
homogenized blend of immiscible components. Suitable gelling agents
include, but are not limited to, modified celluloses, such as
hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol
homopolymers and copolymers; and combinations thereof. Suitable
solvents in the liquid vehicle include, but are not limited to,
diglycol monoethyl ether; alkene glycols, such as propylene glycol;
dimethyl isosorbide; alcohols, such as isopropyl alcohol and
ethanol. The solvents are typically selected for their ability to
dissolve the drug. Other additives, which improve the skin feel
and/or emolliency of the formulation, may also be incorporated.
Examples of such additives include, but are not limited, isopropyl
myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil,
squalane, cyclomethicone, capric/caprylic triglycerides, and
combinations thereof.
[0105] Foams may be described as an emulsion in combination with a
gaseous propellant. The gaseous propellant consists primarily of
hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such
as 1,1,1,2-tetrafluoroethane (HFA 134a) and
1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and
admixtures of these and other HFAs that are currently approved or
may become approved for medical use are suitable. The propellants
preferably are not hydrocarbon propellant gases which can produce
flammable or explosive vapors during spraying. Furthermore, the
compositions preferably contain no volatile alcohols, which can
produce flammable or explosive vapors during use.
[0106] Emollients may be described as externally applied agents
that soften or soothe skin and are generally known in the art and
listed in compendia, such as the "Handbook of Pharmaceutical
Excipients", 4th Ed., Pharmaceutical Press, 2003. In certain
embodiments, the emollients are almond oil, castor oil, ceratonia
extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax,
cholesterol, cottonseed oil, cyclomethicone, ethylene glycol
palmitostearate, glycerin, glycerin monostearate, glyceryl
monooleate, isopropyl myristate, isopropyl palmitate, lanolin,
lecithin, light mineral oil, medium-chain triglycerides, mineral
oil and lanolin alcohols, petrolatum, petrolatum and lanolin
alcohols, soybean oil, starch, stearyl alcohol, sunflower oil,
xylitol and combinations thereof. In one embodiment, the emollients
are ethylhexyl stearate and ethylhexyl palmitate.
[0107] Surfactants are surface-active agents that lower surface
tension and thereby increase the emulsifying, foaming, dispersing,
spreading and wetting properties of a product. In certain
embodiments, suitable non-ionic surfactants include emulsifying
wax, glyceryl monooleate, polyoxyethylene alkyl ethers,
polyoxyethylene castor oil derivatives, polysorbate, sorbitan
esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin
monostearate, poloxamer, povidone and combinations thereof. In one
embodiment, the non-ionic surfactant is stearyl alcohol.
[0108] Emulsifiers are surface active substances which promote the
suspension of one liquid in another and promote the formation of a
stable mixture, or emulsion, of oil and water. In certain
embodiments, the emulsifiers are metallic soaps, certain animal and
vegetable oils, and various polar compounds. Suitable emulsifiers
include acacia, anionic emulsifying wax, calcium stearate,
carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol,
diethanolamine, ethylene glycol palmitostearate, glycerin
monostearate, glyceryl monooleate, hydroxpropyl cellulose,
hypromellose, lanolin, hydrous, lanolin alcohols, lecithin,
medium-chain triglycerides, methylcellulose, mineral oil and
lanolin alcohols, monobasic sodium phosphate, monoethanolamine,
nonionic emulsifying wax, oleic acid, poloxamer, poloxamers,
polyoxyethylene alkyl ethers, polyoxyethylene castor oil
derivatives, polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene stearates, propylene glycol alginate,
self-emulsifying glyceryl monostearate, sodium citrate dehydrate,
sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower
oil, tragacanth, triethanolamine, xanthan gum and combinations
thereof. In one embodiment, the emulsifier is glycerol stearate. In
one embodiment, the emulsifier is glycerol. In one embodiment, the
emulsifier is glycerin.
[0109] In some embodiments, compositions disclosed herein are
formulated to be applied to a subject's scalp. In some embodiments,
the composition is formulated to be used as a product selected from
a shampoo, a conditioner, a mousse, a gel, and a spray. Such
compositions would be useful for the treatment of seborrheic
dermatitis. Treatment of seborrheic dermatitis with such
compositions may result in the reduction of a symptom selected from
dandruff and cradle cap. However, compositions disclosed herein may
be used to treat seborrheic dermatitis at other areas of the body
besides the scalp. Non-limiting examples of other areas include the
chest, stomach, skin folds, arms, legs, groin area and under
breasts.
[0110] In some embodiments, compositions disclosed herein comprise
a buffer, wherein the buffer controls a pH of the composition.
Preferably, the buffers maintain the composition from a pH of about
4 to a pH of about 7.5, from a pH of about 4 to a pH of about 7,
and from a pH of about 5 to a pH of about 7.
[0111] In some embodiments, compositions disclosed herein are
formulated to provide or maintain a desirable skin pH. In some
embodiments, the desirable skin pH is between about 4.5 and about
6.5. In some embodiments, the desirable skin pH is between about 5
and about 6. In some embodiments, the desirable skin pH is about
5.5. In some embodiments, compositions disclosed herein are
formulated with a skin pH modulating agent. Non-limiting examples
of pH modulating agents include salicylic acid, glycolic acid,
trichloroacetic acid, azeilic acid, lactic acid, aspartic acid,
hydrochloride, stearic acid, glyceryl stearate, cetyl palmitate,
urea phosphate, and tocopheryl acetate.
[0112] In some embodiments, compositions disclosed herein are
formulated to provide more oxygen to the skin. In some embodiments,
compositions disclosed herein are formulated to provide more oxygen
exposure to the skin. In some embodiments, compositions disclosed
herein are formulated to provide more oxygen diffusion into the
skin. In some embodiments, compositions disclosed herein are
formulated to provide more oxygen diffusion through the skin. In
some embodiments, compositions disclosed herein are formulated with
an agent that provides more oxygen to the skin. In some
embodiments, compositions disclosed herein are used with an agent
that provides more oxygen to the skin. In some embodiments,
compositions disclosed herein are used before use of an agent that
provides more oxygen to the skin. In some embodiments, compositions
disclosed herein are used after use of an agent that provides more
oxygen to the skin. A non-limiting example of an agent that
provides oxygen to the skin is chlorophyll.
[0113] Preservatives can be used to prevent the growth of fungi and
microorganisms. Suitable antifungal and antimicrobial agents
include, but are not limited to, benzoic acid, butylparaben, ethyl
paraben, methyl paraben, propylparaben, sodium benzoate, sodium
propionate, benzalkonium chloride, benzethonium chloride, benzyl
alcohol, cetylpyridinium chloride, chlorobutanol, phenol,
phenylethyl alcohol, and thimerosal. In one embodiment, a
concentration of a preservative that is effective to prevent fungal
growth is selected, without affecting the effectiveness of the
composition for its intended purposed upon topical application.
[0114] Excipients in the formulation are selected based on the type
of formulation intended. In certain embodiments, the excipients
include gelatin, casein, lecithin, gum acacia, cholesterol,
tragacanth, stearic acid, benzalkonium chloride, calcium stearate,
glyceryl monostearate, cetostearyl alcohol, cetomacrogol
emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers,
polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan
fatty acid esters, polyethylene glycols, polyoxyethylene stearates,
colloidol silicon dioxide, phosphates, sodium dodecyl sulfate,
carboxymethylcellulose calcium, carboxymethylcellulose sodium,
methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropylmethycellulose phthalate, noncrystalline cellulose,
magnesium aluminum silicate, triethanolamine, polyvinyl alcohol,
polyvinylpyrrolidone, sugars, and starches.
[0115] In some embodiments, compositions disclosed herein are
formulated with glycerol. In some instances, a strain of bacterium
in the composition ferments the glycerol, thereby producing short
chain fatty acids. Non-limiting examples of short-chain fatty acids
include acetic acid, lactic acid, and propionic acid. In some
instances, human-derived Janthinobacterium lividum grown in the
presence of glycerol enhances the production of violacein or one or
more antimicrobial metabolites.
[0116] Penetration enhancers are frequently used to promote
transdermal delivery of drugs across the skin, in particular across
the stratum corneum. Some penetration enhancers cause dermal
irritation, dermal toxicity and dermal allergies. However, the more
commonly used ones include urea, (carbonyldiamide), imidurea,
N,N-diethylformamide, N-methyl-2-pyrrolidine,
1-dodecal-azacyclopheptane-2-one, calcium thioglycate,
2-pyyrolidine, N,N-diethyl-m-toluamide, oleic acid and its ester
derivatives, such as methyl, ethyl, propyl, isopropyl, butyl, vinyl
and glycerylmonooleate, sorbitan esters, such as sorbitan
monolaurate and sorbitan monooleate, other fatty acid esters such
as isopropyl laurate, isopropyl myristate, isopropyl palmitate,
diisopropyl adipate, propylene glycol monolaurate, propylene glycol
monooleatea and non-ionic detergents such as BRIJ.RTM. 76 (stearyl
poly(10) oxyethylene ether), BRIJ.RTM. 78 (stearyl
poly(20)oxyethylene ether), BRIJ.RTM. 96 (oleyl poly(10)oxyethylene
ether), and BRIJ.RTM. 721 (stearyl poly (21) oxyethylene ether)
(ICI Americas Inc. Corp.).
[0117] The composition can be formulated to comprise the
Janthinobacterium probiotic composition at a particular
concentration. For example, the composition can comprise an amount
of probiotic such that the microorganisms may be delivered in
effective amounts. In certain embodiments, the amount of probiotic
delivered is at least 1.times.10{circumflex over ( )}3,
1.times.10{circumflex over ( )}4, 1.times.10{circumflex over ( )}5,
1.times.10{circumflex over ( )}6, 1.times.10{circumflex over ( )}7,
1.times.10{circumflex over ( )}8, 1.times.10{circumflex over ( )}9,
1.times.10{circumflex over ( )}10 CFU per unit dose. The
composition may be formulated with the Janthinobacterium probiotic
in a proportion of at least about 0.0001% (expressed by dry
weight), from about 0.0001% to about 99%, from about 0.001% to
about 90% by weight, from about 0.01% to about 80% by weight, and
from about 0.1% to about 70% by weight, relative to the total
weight of the composition. In general, a composition intended to be
administered topically comprises at least 1.times.10{circumflex
over ( )}3, 1.times.10{circumflex over ( )}4, 1.times.10{circumflex
over ( )}5, 1.times.10{circumflex over ( )}6, 1.times.10{circumflex
over ( )}7, 1.times.10{circumflex over ( )}8, 1.times.10{circumflex
over ( )}9, 1.times.10{circumflex over ( )}10 microorganisms per
gram of carrier, or at equivalent doses calculated for inactive or
dead microorganisms or for bacterial fractions or for metabolites
produced.
[0118] Microbes disclosed herein may be delivered in effective
amounts per unit dose, of at least about 1.times.10{circumflex over
( )}2 CFU to about 1.times.10{circumflex over ( )}20 CFU. In the
particular case of the compositions that have to be administered
topically, the concentration of each bacterial strain and/or
corresponding fraction and/or metabolite can be adjusted so as to
correspond to doses (expressed as bacterial equivalent) ranging
from about 1.times.10{circumflex over ( )}5 to about
1.times.10{circumflex over ( )}12 CFU/dose.
[0119] Compositions disclosed herein for topical application
generally comprise from about 1.times.10{circumflex over ( )}2 to
about 1.times.10{circumflex over ( )}15 CFU/g, from about
1.times.10{circumflex over ( )}5 to about 1.times.10{circumflex
over ( )}12 CFU/g, or from about 1.times.10{circumflex over ( )}6
to about 10.times.10{circumflex over ( )}12 CFU/g of bacteria.
[0120] In certain embodiments, compositions disclosed herein are
formulated in order to deliver at least 10{circumflex over ( )}6
microbes per square cm of skin. In certain embodiments, the
composition is formulated in order to deliver at least
10{circumflex over ( )}7 microbes per square cm of skin. In certain
embodiments, the composition is formulated in order to deliver at
least 10{circumflex over ( )}8 microbes per square cm of skin. In
certain embodiments, the composition is formulated in order to
deliver at least 10{circumflex over ( )}9 microbes per square cm of
skin. In certain embodiments, the composition is formulated in
order to deliver less than 10{circumflex over ( )}9 microbes per
square cm of skin. In certain embodiments, the composition is
formulated in order to deliver less than 10{circumflex over ( )}8
microbes per square cm of skin. In certain embodiments, the
composition is formulated in order to deliver less than
10{circumflex over ( )}7 microbes per square cm of skin. In certain
embodiments, the composition is formulated in order to deliver
between about 10{circumflex over ( )}7 and 10{circumflex over ( )}8
microbes per square cm of skin. In certain embodiments, the
composition is formulated in order to deliver between about
10{circumflex over ( )}6 microbes per square cm of skin and about
10{circumflex over ( )}10 microbes per square cm of skin. In
certain embodiments, the composition is formulated in order to
deliver between about 10{circumflex over ( )}6 microbes per square
cm of skin and about 10{circumflex over ( )}9 microbes per square
cm of skin. In certain embodiments, the composition is formulated
in order to deliver between about 10{circumflex over ( )}7 microbes
per square cm of skin and about 10{circumflex over ( )}10 microbes
per square cm of skin. In certain embodiments, the composition is
formulated in order to deliver between about 10{circumflex over (
)}7 microbes per square cm of skin and about 10{circumflex over (
)}9 microbes per square cm of skin.
[0121] In certain embodiments, compositions disclosed herein are
formulated at a concentration of about 10{circumflex over ( )}5
microbes per milliliter to about 10{circumflex over ( )}12 microbes
per milliliter. In certain embodiments, compositions disclosed
herein are formulated at a concentration of about 10{circumflex
over ( )}6 microbes per milliliter. In certain embodiments,
compositions disclosed herein are formulated at a concentration of
about 10{circumflex over ( )}7 microbes per milliliter. In certain
embodiments, compositions disclosed herein are formulated at a
concentration of about 10{circumflex over ( )}8 microbes per
milliliter. In certain embodiments, compositions disclosed herein
are formulated at a concentration of about 10{circumflex over ( )}9
microbes per milliliter. In certain embodiments, compositions
disclosed herein are formulated at a concentration of about
10{circumflex over ( )}10 microbes per milliliter.
[0122] In certain embodiments, compositions disclosed herein for
topical or oral use contain biologic stability compounds including
but not limited to carbohydrates such as trehalose, mannose,
fructose, glucose, sucrose, lactose, raffinose, stachyose,
melezitose, dextran, and sugar alcohols; and/or cryopreservatives
such as glycerol, bovine-free media, (e.g., tryptic soy broth),
whey protein, NaCl, phosphate buffer, MgCl, lyophilized bacteria,
or other inactive/killed bacteria.
[0123] After formulation, composition disclosed herein may be
packaged in a manner suitable for delivery and use by an end user.
In one embodiment, the composition is placed into an appropriate
dispenser and shipped to the end user. Examples of a final
container may include a pump bottle, squeeze bottle, jar, tube,
capsule or vial.
[0124] In some embodiments, compositions disclosed herein can be
added to an applicator before packaging. Non-limiting examples of
applicators include a cotton pad, a polyester pad, a Q-tip, a
sponge, and a brush. In some embodiments, the applicator is placed
in a package. Non-limiting examples of a package includes bags and
foil or wax lined paper packets. The interior of the package may be
sterile. In some embodiments, air in the package is removed with a
vacuum before sealing. In some embodiments, the package is
heat-sealed. In some embodiments, the package is sealed with
adhesive.
[0125] In another embodiment, compositions disclosed herein are
dehydrated or desiccated by being lyophilized, freeze dried, or
spray dried for reconstitution before application to the skin. In
one embodiment, lyophilization, freeze drying, or spray drying is
conducted with one or more excipients, such as glycerol or other
sugar alcohols, to improve the shelf life of the selected,
transformed, or engineered bacteria. In one embodiment, the
formulation composition does not include trehalose
(.alpha.-D-glucopyranosyl-1,1-.alpha.-D-glucopyranosyde). In some
embodiments, the composition does not have to be frozen.
[0126] Compositions disclosed herein may be packaged in one or more
containers. For example, a single bottle, tube, container, or
capsule may be divided to two equal or unequal parts wherein one
part contains the bacteria, in their packing form (desiccated,
freeze dried, etc.), and the other part contains an activation
material, which can be a liquid or a gel. The single bottle or
container can be designed so that an end user can dispense with a
single force applied to the container all or a portion of the
contents in the two container parts, to dispense onto the skin or
other surface the selected, transformed, or engineered bacterium
and the activation material. The kit may also be of the form that
comprises two or more containers, one container with the
population(s) of selected, transformed, or engineered bacterium and
the other with a formulation for admixture with the populations of
selected, transformed, or engineered bacteria. In another example,
two or more containers, one container with the population of
selected, transformed, or engineered bacteria, the other container
with natural nonpathogenic skin bacterium that are not selected,
transformed, or engineered, and a third container with a
formulation for admixture with the populations of selected,
transformed, or engineered bacteria. In another example, the two or
more containers composing the single bottle have one pump connected
to two separate tubes, each draining from a different chamber. The
kit may also include one or more complementary products, such as
soaps, body washes or moisturizing lotions with certain pH, lotions
or creams. In another embodiment, the complementary product is a
probiotic. The complementary product may include any compound
beneficial to the activity of the original product and enhance its
activity for lasting efficacy. Another contemplated packaging is
one wherein the population of selected, transformed, or engineered
bacterium is maintained as a layer on a bandage or film that is
combined with a second layer of bandage/film that will allow
activation of the bacteria, and that optionally may also limit
reproduction/growth factors. In another embodiment, the final
product is stored refrigerated, with the bacterium being in their
active state. In another embodiment, the bacterium are stored in a
small bead of water-soluble cellulose. The beads can be mixed in
any solution such as sunscreen, moisturizer, body wash or soap.
[0127] Disclosed herein are Janthinobacterium probiotic
compositions comprising human-derived Janthinobacterium lividum in
an aqueous formulation for topical application. In some embodiments
the probiotic compositions are formulated for application to the
skin. In some embodiments the probiotic compositions are formulated
for application to the mucosa.
[0128] In some embodiments the Janthinobacterium probiotic
composition is co-formulated with one or more additional active
agents. The probiotic composition can be co-formulated with one or
more additional antimicrobial agents, as detailed in the
combination section. Briefly, the additional antimicrobial agent
can be an antifungal agent, an antibacterial agent, an
anti-parasitic agent, or a combination of any of those agents.
[0129] In some embodiments, the Janthinobacterium probiotic
composition is co-formulated with one or more additional active
agents that confer additional benefits, such as an agent to relieve
itching, pain, discoloration or other undesirable effect.
[0130] In some embodiments, the Janthinobacterium probiotic
composition further contains additional microbes. In some
embodiments, the composition contains at least two, three, or four
distinct human-derived Janthinobacterium lividum, wherein at least
one is derived from a human host. In some embodiments, the
Janthinobacterium probiotic further contains a Lactobacillus
species or a Lactococcus species. In some embodiments, the
Janthinobacterium probiotic further contains a benign or beneficial
fungal strain often found on human skin or a benign or beneficial
strain of Propionibacterium.
[0131] In some embodiments, the Janthinobacterium cosmetic
composition is in the form of an emulsion composition according to
the invention is especially effective. In this case, the emulsion
may be in the form of an oil-in-water emulsion, a water-in-oil
emulsion, a water-in-oil-in-water emulsion or an
oil-in-water-in-oil emulsion. Alternatively, the cosmetic
composition of the invention may also be used in the form of a
nonaqueous composition. The form of the nonaqueous composition is
exemplified by solid, semisolid, pressed, mousse, powder and stick
forms. In this invention, "nonaqueous composition" refers to
compositions that are not formulated with water.
[0132] The human-derived Janthinobacterium lividum compositions of
this invention can be administered with other agents in a
combination therapy mode, including anti-microbial agents,
probiotics, postbiotics, and prebiotics. Administration can be
sequential, over a period of hours or days, or simultaneous.
[0133] In one embodiment, the bacterial compositions are included
in combination therapy with one or more anti-microbial agents,
which include anti-bacterial agents, anti-fungal agents, anti-viral
agents and anti-parasitic agents.
[0134] Anti-bacterial agents can include cephalosporin antibiotics
(cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin,
cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole);
fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin,
avelox, and norflox); tetracycline antibiotics (tetracycline,
minocycline, oxytetracycline, and doxycycline); penicillin
antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin,
carbenicillin, vancomycin, and methicillin); and carbapenem
antibiotics (ertapenem, doripenem, imipenem/cilastatin, and
meropenem).
[0135] Anti-viral agents can include Abacavir, Acyclovir, Adefovir,
Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine,
Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine,
Enfuvirtide, Etravirine, Famciclovir, Foscarnet, Fomivirsen,
Ganciclovir, Indinavir, Idoxuridine, Lamivudine, Lopinavir
Maraviroc, MK-2048, Nelfinavir, Nevirapine, Penciclovir,
Raltegravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine,
Tenofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine,
Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir,
Zalcitabine, Zanamivir and Zidovudine.
[0136] Examples of antifungal compounds include, but are not
limited to polyene antifungals such as natamycin, rimocidin,
filipin, nystatin, amphotericin B, candicin, and hamycin; imidazole
antifungals such as miconazole, ketoconazole, clotrimazole,
econazole, omoconazole, bifonazole, butoconazole, fenticonazole,
isoconazole, oxiconazole, sertaconazole, sulconazole, and
tioconazole; triazole antifungals such as fluconazole,
itraconazole, isavuconazole, ravuconazole, posaconazole,
voriconazole, terconazole, and albaconazole; thiazole antifungals
such as abafungin; allylamine antifungals such as terbinafine,
naftifine, and butenafine; and echinocandin antifungals such as
anidulafungin, caspofungin, and micafungin. Other compounds that
have antifungal properties include, but are not limited to
polygodial, benzoic acid, ciclopirox, tolnaftate, undecylenic acid,
flucytosine or 5-fluorocytosine, griseofulvin, and haloprogin.
[0137] In one embodiment, the bacterial compositions are included
in combination therapy with one or more corticosteroids,
mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives,
immunosuppressive drugs, cyclosporin A, mercaptopurine,
azathiopurine, prednisone, methotrexate, antihistamines,
glucocorticoids, epinephrine, theophylline, cromolyn sodium,
anti-leukotrienes, anti-cholinergic drugs for rhinitis,
anti-cholinergic decongestants, mast-cell stabilizers, monoclonal
anti-IgE antibodies, vaccines, and combinations thereof.
[0138] The methods and compositions described herein can be used to
treat and or prevent infections resulting from growth of parasitic
microorganisms susceptible to metabolites produced by human-derived
Janthinobacteria lividum. These microbes can be bacteria, viruses,
yeast or fungi.
[0139] In an embodiment, the infection is an onychomycosis. In an
embodiment, the infection is Tinea pedis. In an embodiment, the
infection is atopic dermatitis. In an embodiment, the infection is
impetigo. In an embodiment, the infection is of the skin or soft
tissue.
[0140] In an embodiment, the infection is caused by a dermatophyte.
In an embodiment, the infection is caused by a Malassezia species.
In an embodiment, the infection is caused by a Trichophyton
species. In an embodiment, the infection is caused by
Staphylococcus species. In an embodiment, the infection is caused
by Trichophyton rubrum. In an embodiment, the infection is caused
by Staphylococcus aureus. In an embodiment, the infection is caused
by a gram positive bacteria.
[0141] Prior to administration of the compositions of this
invention, the subject or patient may optionally have a
pretreatment protocol to prepare the skin to receive the bacterial
composition. In certain embodiments, the pretreatment protocol is
advisable, such as when a patient has an acute infection with a
highly resilient pathogen. In other embodiments, the pretreatment
protocol is entirely optional, such as when the pathogen causing
the infection is not resilient, or the patient has had an acute
infection that has been successfully treated but where the
physician is concerned that the infection may recur. In these
instances, the pretreatment protocol may enhance the ability of the
bacterial composition to affect the patient's microbiome.
[0142] As one way of preparing the patient for administration of
the microbial ecosystem, at least one antibiotic or antifungal may
be administered to alter the microbes on the patient. This may be
applied orally or topically.
[0143] If a patient has received an antibiotic for treatment of an
infection, or if a patient has received an antibiotic as part of a
specific pretreatment protocol, in one embodiment the antibiotic
should be stopped in sufficient time to allow the antibiotic to be
substantially reduced in concentration on the skin before the
bacterial composition is administered. In one embodiment, the
antibiotic may be discontinued 1, 2, or 3 days before the
administration of the bacterial composition. In one embodiment, the
antibiotic may be discontinued 3, 4, 5, 6, or 7 antibiotic
half-lives before administration of the bacterial composition. In
another embodiment, the antibiotic may be chosen so the
constituents in the bacterial composition have an MIC50 that is
higher than the concentration of the antibiotic on the skin.
[0144] MIC50 of a bacterial composition or the elements in the
composition may be determined by methods well known in the art
Reller et al., Antimicrobial Susceptibility Testing: A Review of
General Principles and Contemporary Practices, Clinical Infectious
Diseases 49(11):1749-1755 (2009). In such an embodiment, the
additional time between antibiotic administration and
administration of the bacterial composition is not necessary. If
the pretreatment protocol is part of treatment of an acute
infection, the antibiotic may be chosen so that the infection is
sensitive to the antibiotic, but the constituents in the bacterial
composition are not sensitive to the antibiotic.
[0145] In some embodiments, the skin of the subject is pre-treated
with a detergent substance to decrease the amount of skin pathogen
prior to application of the Janthinobacterium probiotic
composition.
[0146] In some embodiments, the Janthinobacterium probiotic is
pretreated with a substance to increase production of a beneficial
metabolite. In some embodiments, the probiotic is incubated in the
presence of a prebiotic to increase production of violacein, such
as glycerol or tryptophan.
[0147] Methods of Manufacture
[0148] Any of the compositions described herein, including the
pharmaceutical compositions, articles of manufacture, and food or
household products comprising the compositions, may contain
bacterial strains in any form, for example in an aqueous form, such
as a solution or a suspension, embedded in a semi-solid form, in a
powdered form or freeze dried form. In some embodiments, the
composition or the bacterial strains of the composition are
lyophilized. In some embodiments, a subset of the bacterial strains
in a composition is lyophilized. Methods of lyophilizing
compositions, specifically compositions comprising bacteria, are
well known in the art. See, e.g., U.S. Pat. Nos. 3,261,761;
4,205,132; PCT Publications WO 2014/029578 and WO 2012/098358,
herein incorporated by reference in their entirety. The bacterium
may be lyophilized as a combination and/or the bacterium may be
lyophilized separately and combined prior to administration. A
bacterial strain may be combined with a pharmaceutical excipient
prior to combining it with the other bacterial strain or multiple
lyophilized bacterium may be combined while in lyophilized form and
the mixture of bacteria, once combined may be subsequently be
combined with a pharmaceutical excipient. In some embodiments, the
bacterial strain is a lyophilized cake. In some embodiments, the
compositions comprising the one or more bacterial strains are a
lyophilized cake.
[0149] The bacterial strains of the composition can be manufactured
using fermentation techniques well known in the art. In some
embodiments, the active ingredients are manufactured using
anaerobic fermenters, which can support the rapid growth of
anaerobic bacterial species. The anaerobic fermenters may be, for
example, stirred tank reactors or disposable wave bioreactors.
Culture media such as BL media and EG media, or similar versions of
these media devoid of animal components, can be used to support the
growth of the bacterial species. The bacterial product can be
purified and concentrated from the fermentation broth by
traditional techniques, such as centrifugation and filtration, and
can optionally be dried and lyophilized by techniques well known in
the art.
[0150] In some embodiments, the composition of bacterial strains
may be formulated for administration as a pharmaceutical
composition. The term "pharmaceutical composition" as used herein
means a product that results from the mixing or combining of at
least one active ingredient, such as any two or more purified
bacterial strains described herein, and one or more inactive
ingredients, which may include one or more pharmaceutically
acceptable excipient.
[0151] An "acceptable" excipient refers to an excipient that must
be compatible with the active ingredient and not deleterious to the
subject to which it is administered. In some embodiments, the
pharmaceutically acceptable excipient is selected based on the
intended route of administration of the composition, for example a
composition for oral or nasal administration may comprise a
different pharmaceutically acceptable excipient than a composition
for rectal administration. Examples of excipients include sterile
water, physiological saline, solvent, a base material, an
emulsifier, a suspending agent, a surfactant, a stabilizer, a
flavoring agent, an aromatic, an excipient, a vehicle, a
preservative, a binder, a diluent, a tonicity adjusting agent, a
soothing agent, a bulking agent, a disintegrating agent, a buffer
agent, a coating agent, a lubricant, a colorant, a sweetener, a
thickening agent, and a solubilizer.
[0152] Pharmaceutical compositions of the invention can be prepared
in accordance with methods well known and routinely practiced in
the art (see e.g., Remington: The Science and Practice of Pharmacy,
Mack Publishing Co. 20th ed. 2000). The pharmaceutical compositions
described herein may further comprise any carriers or stabilizers
in the form of a lyophilized formulation or an aqueous solution.
Acceptable excipients, carriers, or stabilizers may include, for
example, buffers, antioxidants, preservatives, polymers, chelating
reagents, and/or surfactants. Pharmaceutical compositions are
preferably manufactured under GMP conditions. The pharmaceutical
compositions can be used orally, nasally or parenterally, for
instance, in the form of capsules, tablets, pills, sachets,
liquids, powders, granules, fine granules, film-coated
preparations, pellets, troches, sublingual preparations, chewables,
buccal preparations, pastes, syrups, suspensions, elixirs,
emulsions, liniments, ointments, plasters, cataplasms, transdermal
absorption systems, lotions, inhalations, aerosols, injections,
suppositories, and the like.
[0153] Articles of Manufacture
[0154] In some embodiments, the human-derived Janthinobacterium
lividum is formulated into an article of manufacture, for example a
substance impregnated with the human-derived Janthinobacterium
lividum or postbiotics, or lysates, or metabolites of the
human-derived Janthinobacterium lividum.
[0155] In some embodiments, the human-derived Janthinobacterium
lividum is associated with cloth. Cloth generally refers to a
flexible material suitable to be made into clothing, e.g., having
enough material strength to withstand everyday motion by a wearer.
Cloth can be fibrous, woven, or knit; it can be made of a naturally
occurring material or a synthetic material. Exemplary cloth
materials include cotton, flax, wool, ramie, silk, denim, leather,
nylon, polyester, and spandex, and blends thereof.
[0156] In some embodiments, the human-derived Janthinobacterium
lividum is associated with yarn. Yarn generally refers to a long,
thin spun flexible material that is suitable for knitting or
weaving. Yarn can be made of, e.g., wool, cotton, polyester, and
blends thereof.
[0157] In some embodiments, the human-derived Janthinobacterium
lividum is associated with thread. Thread generally refers to a
long, thin spun flexible material that is suitable for sewing.
Thread generally has a thinner diameter than yarn. Thread can be
made of, e.g., cotton, polyester, nylon, silk, and blends
thereof.
[0158] Articles of clothing such as, for example, shoes, shoe
inserts, pajamas, sneakers, belts, hats, shirts, underwear,
athletic garments, helmets, towels, gloves, socks, bandages, and
the like, may also be treated with the human-derived
Janthinobacterium lividum. Bedding, including sheets, pillows,
pillowcases, and blankets may also be treated with
Janthinobacterium. In some embodiments, areas of skin that cannot
be washed for a period of time may also be contacted with
Janthinobacterium. For example, skin enclosed in orthopedic casts
which immobilize injured limbs during the healing process, and
areas in proximity to injuries that must be kept dry for proper
healing such as stitched wounds may benefit from contact with
Janthinobacterium.
[0159] In some aspects, the present disclosure provides a wearable
article comprising a human-derived Janthinobacterium lividum as
described herein. A wearable article may be a light article that
can be closely associated with a user's body, in a way that does
not impede ambulation. Examples of wearable articles include a
wristwatch, wristband, headband, hair elastic, hair nets, shower
caps, hats, hairpieces, and jewelry. The wearable article
comprising a human-derived Janthinobacterium lividum described
herein may provide, e.g., at a concentration that provides one or
more of a treatment or prevention of a skin disorder, a treatment
or prevention of a disease or condition associated with low nitrite
levels, a treatment or prevention of body odor, a treatment to
supply nitric oxide to a subject, or a treatment to inhibit
microbial growth.
[0160] In some embodiments, the Human-derived Janthinobacterium
lividum is associated with a product intended to contact the hair,
for example, a brush, comb, shampoo, conditioner, headband, hair
elastic, hair nets, shower caps, hats, and hairpieces. Articles
contacting the surface of a human subject, such as a diaper, may be
associated with the Janthinobacterium of this invention.
[0161] In some embodiments, the human-derived Janthinobacterium
lividum is associated with a household item, which may otherwise
function as a reservoir for a human skin pathogen. In some
embodiments, a shower curtain, bathmat, shower mat, or drainage
tile is impregnated with Janthinobacterium or postbiotics, or
lysates, or metabolites of the human-derived Janthinobacterium
lividum.
[0162] In some embodiments, the human-derived Janthinobacterium
lividum is associated with a household or industrial cleaning
substance, such as a cleaning substance intended for cleaning a gym
shower.
[0163] In some embodiments, the product comprising the
Human-derived Janthinobacterium lividum is packaged. The packaging
may serve to compact the product or protect it from damage, dirt,
or degradation. The packaging may comprise, e.g., plastic, paper,
cardboard, or wood. In some embodiments the packaging is
impermeable to bacteria. In some embodiments the packaging is
permeable to oxygen and/or carbon dioxide.
TABLE-US-00002 SEQUENCE LISTING Seq ID No: Description Sequence 1
JL001 16S CGGTTAAGCTACCTACTTCTGGTAAAACCCGCTCCCATGGTGTGACGGGCG rRNA
GTGTGTACAAGACCCGGGAACGTATTCACCGCGACATGCTGATCCGCGATT sequence
ACTAGCGATTCCAACTTCATGCAGTCGAGTTGCAGACTACAATCCGGACTA
CGATACACTTTCTGCGATTAGCTCCCCCTCGCGGGTTGGCGGCGCTCTGTA
TGTACCATTGTATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTT
GACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTG
CCCTTTCGTAGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACC
CAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTACTG
GTTCTCTTTCGAGCACTCCCCAATCTCTCGGTGGATTCCAGCCATGTCAAG
GGTAGGTAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCACCGC
TTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCC
CCAGGCGGTCTACTTCACGCGTTAGCTGCGTTACCAAGTCAATTAAGACCC
GACAACTAGTAGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCT
GTTTGCTCCCCACGCTTTCGTGCATGAGCGTCAATCTTGACCCAGGGGGCT
GCCTTCGCCATCGGTGTTCCTCCACATATCTACGCATTTCACTGCTACACGT
GGAATTCTACCCCCCTCTGCCAGATTCTAGCCTTGCAGTCTCCAATGCAATT
CCCAGGTTGAGCCCGGGGATTTCACATCAGACTTACAAAACCGCCTGCGCA
CGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGC
GGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTTCAGGTACCGTCATTAG
CAAGAGATATTAGCTCTCACCGTTTCTTCCCTGACAAAAGAGCTTTACAAC
CCGAAGGCCTTCTTCACTCACGCGGCATTGCTGGATCAGGCTTTCGCCCAT
TGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCA
GTTCCAGTGTGGCTGGTCGTCCTCTCAGACCAGCTACTGATCGATGCCTTG
GTAGGCTTTTACCCTACCAACTAGCTAATCAGATATCGGCCGCTCCACGAG
CATGAGGTCTTGCGATCCCCCACTTTCATCCTTAGATCGTATGCGGTATTAG
CGTAACTTTCGCTACGTTATCCCCCACTCTAGGGTACGTTCCGATATATTAC
TCACCCGTTCGCCACTCGCCACCAGAGCAAGCTCCGTGCTGCCGTTCGACT
TGCATGTGTAAGGCATGCCGCCAGCGTTCAATCTGAGCCAGGATCAAACTC T 2 JL002 16S
AAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTC rRNA
ACCCCAGTCACGAATCCTACCGTGGTAAGCGCCCTCCTTGCGGTTAAGCTA sequence
CCTACTTCTGGTAAAACCCGCTCCCATGGTGTGACGGGCGGTGTGTACAAG
ACCCGGGAACGTATTCAATCTCTCGAGGATTCCAGCCATGTCAAGGGTAGG
TAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCG
GGTCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCG
GTCTACTTCACGCGTTAGCTGCGTTACCAAGTCAATTAAGACCCGACAACT
AGTAGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCT
CCCCACGCTTTCGTGCATGAGCGTCAATCTTGACCCAGGGGGCTGCCTTCG
CCATCGGTGTTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTC
TACCCCCCTCTGCCAGATTCTAGCCTTGCAGTCTCCAATGCAATTCCCAGGT
TGAGCCCGGGGATTTCACATCAGACTTACAAAACCGCCTGCGCACGCTTTA
CGCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCT
GGCACGTAGTTAGCCGGTGCTTATTCTTCAGGTACCGTCATTAGCAAGAGA
TATTAGCTCTCACCGTTTCTTCCCTGACAAAAGAGCTTTACAACCCGAAGG
CCTTCTTCACTCACGCGGCATTGCTGGATCAGGCTTTCGCCCATTGTCCAAA
ATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGT
GTGGCTGGTCGTCCTCTCAGACCAGCTACTGATCGATGCCTTGGTAGGCTT
TTACCCTACCAACTAGCTAATCAGATATCGGCCGCTCCACGAGCATGAGGT
CTTGCGATCCCCCACTTTCATCCTTAGATCGTATGCGGTATTAGCGTAACTT
TCGCTACGTTATCCCCCACTCTAGGGTACGTTCCGATATATTACTCACCCGT
TCGCCACTCGCCACCAGAGCAAGCTCCGTGCTGCCGTTCGACTTGCATGTG
TAAGGCATGCCGCCAGCGTTCAATCTGAGCCAGGATCAAACTCT 3 JL003 16S
AAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTC rRNA
ACCCCAGTCACGAATCCTACCGTGGTAAGCGCCCTCCTTGCGGTTAAGCTA sequence
CCTACTTCTGGTAAAACCCGCTCCCATGGTGTGACGGGCGGTGTGTACAAG
ACCCGGGAACGTATTCAATCTCTCGAGGATTCCAGCCATGTCAAGGGTAGG
TAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCG
GGTCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCG
GTCTACTTCACGCGTTAGCTGCGTTACCAAGTCAATTAAGACCCGACAACT
AGTAGACATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCT
CCCCACGCTTTCGTGCATGAGCGTCAATCTTGACCCAGGGGGCTGCCTTCG
CCATCGGTGTTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTC
TACCCCCCTCTGCCAGATTCTAGCCTTGCAGTCTCCAATGCAATTCCCAGGT
TGAGCCCGGGGATTTCACATCAGACTTACAAAACCGCCTGCGCACGCTTTA
CGCCCAGTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCT
GGCACGTAGTTAGCCGGTGCTTATTCTTCAGGTACCGTCATTAGCAAGAGA
TATTAGCTCTCACCGTTTCTTCCCTGACAAAAGAGCTTTACAACCCGAAGG
CCTTCTTCACTCACGCGGCATTGCTGGATCAGGCTTTCGCCCATTGTCCAAA
ATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGT
GTGGCTGGTCGTCCTCTCAGACCAGCTACTGATCGATGCCTTGGTAGGCTT
TTACCCTACCAACTAGCTAATCAGATATCGGCCGCTCCACGAGCATGAGGT
CTTGCGATCCCCCACTTTCATCCTTAGATCGTATGCGGTATTAGCGTAACTT
TCGCTACGTTATCCCCCACTCTAGGGTACGTTCCGATATATTACTCACCCGT
TCGCCACTCGCCACCAGAGCAAGCTCCGTGCTGCCGTTCGACTTGCATGTG
TAAGGCATGCCGCCAGCGTTCAATCTGAGCCAGGATCAAACTCT 4 JL004 16S
AAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTC rRNA
ACCCCAGTCACGAATCCTACCGTGGTAAGCGCCCTCCTTGCGGTTAAGCTA sequence
CCTACTTCTGGTAAAACCCGCTCCCATGGTGTGACGGGCGGTGTGTACAAG
ACCCGGGAACGTATTCACCGCGACATGCTGATCCGCGATTACTAGCGATTC
CAACTTCATGCAGTCGAGTTGCAGACTACAATCCGGACTACGATACACTTT
CTGCGATTAGCTCCCCCTCGCGGGTTGGCGGCGCTCTGTATGTACCATTGT
ATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCC
CACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCTTTCGTAGC
AACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACG
ACACGAGCTGACGACAGCCATGCAGCACCTGTGTACTGGTTCTCTTTCGAG
CACTCCTCAATCTCTCGAGGATTCCAGCCATGTCAAGGGTAGGTAAGGTTT
TTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCC
GTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCTACT
TCACGCGTTAGCTGCGTTACCAAGTCAATTAAGACCCGACAACTAGTAGAC
ATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACG
CTTTCGTGCATGAGCGTCAATCTTGACCCAGGGGGCTGCCTTCGCCATCGG
TGTTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTCTACCCCC
CTCTGCCAGATTCTAGCCTTGCAGTCTCCAATGCAATTCCCAGGTTGAGCC
CGGGGATTTCACATCAGACTTACAAAACCGCCTGCGCACGCTTTACGCCCA
GTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACG
TAGTTAGCCGGTGCTTATTCTTCAGGTACCGTCATTAGCAAGAGATATTAG
CTCTCACCGTTTCTTCCCTGACAAAAGAGCTTTACAACCCGAAGGCCTTCTT
CACTCACGCGGCATTGCTGGATCAGGCTTTCGCCCATTGTCCAAAATTCCC
CACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCT
GGTCGTCCTCTCAGACCAGCTACTGATCGATGCCTTGGTAGGCTTTTACCCT
ACCAACTAGCTAATCAGATATCGGCCGCTCCACGAGCATGAGGTCTTGCGA
TCCCCCACTTTCATCCTTAGATCGTATGCGGTATTAGCGTAACTTTCGCTAC
GTTATCCCCCACTCCAGGGTACGTTCCGATATATTACTCACCCGTTCGCCAC
TCGCCACCAGAGCAAGCTCCGTGCTGCCGTTCGACTTGCATGTGTAAGGCA
TGCCGCCAGCGTTCAATCTGAGCCAGGATCAAACTCT 5 JL005 16S
AAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTC rRNA
ACCCCAGTCACGAATCCTACCGTGGTAAGCGCCCTCCTTACGGTTAAGCTA sequence
CCTACTTCTGGTAAAACCCGCTCCCATGGTGTGACGGGCGGTGTGTACAAG
ACCCGGGAACGTATTCACCGCGACATGCTGATCCGCGATTACTAGCGATTC
CAACTTCATGCAGTCGAGTTGCAGACTACAATCCGGACTACGATACACTTT
CTGCGATTAGCTCCCCCTCGCGGGTTGGCGGCGCTCTGTATGTACCATTGT
ATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCC
CACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCTTTCGTAGC
AACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACG
ACACGAGCTGACGACAGCCATGCAGCACCTGTGTACTGGTTCTCTTTCGAG
CACTCCCCAATCTCTCGAGGATTCCAGCCATGTCAAGGGTAGGTAAGGTTT
TTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCC
GTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCTACT
TCACGCGTTAGCTGCGTTACCAAGTCAATTAAGACCCGACAACTAGTAGAC
ATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACG
CTTTCGTGCATGAGCGTCAATCTTGACCCAGGGGGCTGCCTTCGCCATCGG
TGTTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTCTACCCCC
CTCTGCCAGATTCTAGCCTTGCAGTCTCCAATGCAATTCCCAGGTTGAGCC
CGGGGATTTCACATCAGACTTACAAAACCGCCTGCGCACGCTTTACGCCCA
GTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACG
TAGTTAGCCGGTGCTTATTCTTCAGGTACCGTCATTAGCAAGAGATATTAG
CTCTCACCGTTTCTTCCCTGACAAAAGAGCTTTACAACCCGAAGGCCTTCTT
CACTCACGCGGCATTGCTGGATCAGGCTTTCGCCCATTGTCCAAAATTCCC
CACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCT
GGTCGTCCTCTCAGACCAGCTACTGATCGATGCCTTGGTAGGCTTTTACCCT
ACCAACTAGCTAATCAGATATCGGCCGCTCCACGAGCATGAGGTCTTGCGA
TCCCCCACTTTCATCCTTAGATCGTATGCGGTATTAGCGTAACTTTCGCTAC
GTTATCCCCCACTCTAGGGTACGTTCCGATATATTACTCACCCGTTCGCCAC
TCGCCACCAGAGCAAGCTCCGTGCTGCCGTTCGACTTGCATGTGTAAGGCA
TGCCGCCAGCGTTCAATCTGAGCCAGGATCAAACTCT 6 JL006 16S
AAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTC rRNA
ACCCCAGTCACGAATCCTACCGTGGTAAGCGCCCTCCTTACGGTTAAGCTA sequence
CCTACTTCTGGTAAAACCCGCTCCCATGGTGTGACGGGCGGTGTGTACAAG
ACCCGGGAACGTATTCACCGCGACATGCTGATCCGCGATTACTAGCGATTC
CAACTTCATGCAGTCGAGTTGCAGACTACAATCCGGACTACGATACACTTT
CTGCGATTAGCTCCCCCTCGCGGGTTGGCGGCGCTCTGTATGTACCATTGT
ATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCC
CACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCTTTCGTAGC
AACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACG
ACACGAGCTGACGACAGCCATGCAGCACCTGTGTACTGGTTCTCTTTCGAG
CACTCCCCAATCTCTCGAGGATTCCAGCCATGTCAAGGGTAGGTAAGGTTT
TTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCC
GTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCTACT
TCACGCGTTAGCTGCGTTACCAAGTCAATTAAGACCCGACAACTAGTAGAC
ATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACG
CTTTCGTGCATGAGCGTCAATCTTGACCCAGGGGGCTGCCTTCGCCATCGG
TGTTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTCTACCCCC
CTCTGCCAGATTCTAGCCTTGCAGTCTCCAATGCAATTCCCAGGTTGAGCC
CGGGGATTTCACATCAGACTTACAAAACCGCCTGCGCACGCTTTACGCCCA
GTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACG
TAGTTAGCCGGTGCTTATTCTTCAGGTACCGTCATTAGCAAGAGATATTAG
CTCTCACCGTTTCTTCCCTGACAAAAGAGCTTTACAACCCGAAGGCCTTCTT
CACTCACGCGGCATTGCTGGATCAGGCTTTCGCCCATTGTCCAAAATTCCC
CACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCT
GGTCGTCCTCTCAGACCAGCTACTGATCGATGCCTTGGTAGGCTTTTACCCT
ACCAACTAGCTAATCAGATATCGGCCGCTCCACGAGCATGAGGTCTTGCGA
TCCCCCACTTTCATCCTTAGATCGTATGCGGTATTAGCGTAACTTTCGCTAC
GTTATCCCCCACTCTAGGGTACGTTCCGATATATTACTCACCCGTTCGCCAC
TCGCCACCAGAGCAAGCTCCGTGCTGCCGTTCGACTTGCATGTGTAAGGCA
TGCCGCCAGCGTTCAATCTGAGCCAGGATCAAACTCT 7 JL007 16S
AAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTC rRNA
ACCCCAGTCACGAATCCTACCGTGGTAAGCGCCCTCCTTACGGTTAAGCTA sequence
CCTACTTCTGGTAAAACCCGCTCCCATGGTGTGACGGGCGGTGTGTACAAG
ACCCGGGAACGTATTCACCGCGACATGCTGATCCGCGATTACTAGCGATTC
CAACTTCATGCAGTCGAGTTGCAGACTACAATCCGGACTACGATACACTTT
CTGCGATTAGCTCCCCCTCGCGGGTTGGCGGCGCTCTGTATGTACCATTGT
ATGACGTGTGAAGCCCTACCCATAAGGGCCATGAGGACTTGACGTCATCCC
CACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCTTTCGTAGC
AACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACG
ACACGAGCTGACGACAGCCATGCAGCACCTGTGTACTGGTTCTCTTTCGAG
CACTCCCCAATCTCTCGAGGATTCCAGCCATGTCAAGGGTAGGTAAGGTTT
TTCGCGTTGCATCGAATTAATCCACATCATCCACCGCTTGTGCGGGTCCCC
GTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCTACT
TCACGCGTTAGCTGCGTTACCAAGTCAATTAAGACCCGACAACTAGTAGAC
ATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACG
CTTTCGTGCATGAGCGTCAATCTTGACCCAGGGGGCTGCCTTCGCCATCGG
TGTTCCTCCACATATCTACGCATTTCACTGCTACACGTGGAATTCTACCCCC
CTCTGCCAGATTCTAGCCTTGCAGTCTCCAATGCAATTCCCAGGTTGAGCC
CGGGGATTTCACATCAGACTTACAAAACCGCCTGCGCACGCTTTACGCCCA
GTAATTCCGATTAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACG
TAGTTAGCCGGTGCTTATTCTTCAGGTACCGTCATTAGCAAGAGATATTAG
CTCTCACCGTTTCTTCCCTGACAAAAGAGCTTTACAACCCGAAGGCCTTCTT
CACTCACGCGGCATTGCTGGATCAGGCTTTCGCCCATTGTCCAAAATTCCC
CACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCT
GGTCGTCCTCTCAGACCAGCTACTGATCGATGCCTTGGTAGGCTTTTACCCT
ACCAACTAGCTAATCAGATATCGGCCGCTCCACGAGCATGAGGTCTTGCGA
TCCCCCACTTTCATCCTTAGATCGTATGCGGTATTAGCGTAACTTTCGCTAC
GTTATCCCCCACTCTAGGGTACGTTCCGATATATTACTCACCCGTTCGCCAC
TCGCCACCAGAGCAAGCTCCGTGCTGCCGTTCGACTTGCATGTGTAAGGCA
TGCCGCCAGCGTTCAATCTGAGCCAGGATCAAACTCT
7. EXAMPLES
7.1. Example 1: Isolation of Human Janthinobacterium lividum
[0164] Materials & Methods
[0165] Samples for Bioprospecting
[0166] Samples were sourced and collected from various sources.
Skin microbiome samples were collected from 18-25 year old young
healthy adult subjects following standard scrubbing/swabbing
procedures using eSwab tubes (Fisher, Cat. No. 23-600-900). Samples
were collected from scalp, forehead, nose, antecubital fossa, palm,
heel, and toe web space for each subject.
[0167] Produce samples were obtained from farmer's market and from
farms.
[0168] Strain Isolation
[0169] Human skin samples were either processed directly or stored
at -80.degree. C. with DMSO as cryoprotectant and thawed at room
temperature for processing. Skin samples were plated onto BHI and
R2A agar plates supplied with 1% glycerol following standard
microbial practice after being diluted to the extent of 100-300
colonies per plate. Plates were incubated at ambient temperature
for 3-5 days and visually checked for bacterial colonies with
purple pigment.
[0170] Produce samples were first chopped into smaller pieces using
a pair of scissors briefly cleaned with ethanol wipes.
Approximately 50 grams of chopped pieces were mixed with 10 ml
sterile 1.times.PBS buffer in a 50 ml Falcon tube prefilled with
20-30 sterile 5-mm glass beads and vortexed at top speed for 5 min.
After vortexing, let the tubes sat at for 5-10 min to allow plant
debris settle down to the bottom. The mixture was passed through a
40 .mu.m cell strainer. 100 .mu.l of filtered liquid were plated
onto BHI and R2A plates with 1% glycerol after being diluted to the
extent of 100-300 colonies per plate. Plates were incubated at
ambient temperature for 3-5 days and visually checked for bacterial
colonies with purple pigment.
[0171] Molecular Identification of Janthinobacterium lividum
Strains
[0172] All purple colonies were purified by subculturing and
confirmed as Janthinobacterium lividum strains via 16S rRNA
sequencing. DNA was extracted from purified purple colonies and
amplified using a thermal cycler using the standard 27F/1492R
primer pair. PCR products were checked on E-gel before submission
for Sanger sequencing. Taxonomic information was determined by
search obtained sequences against NCBI database.
[0173] Results, Interpretations and Conclusions
[0174] Approximately 700 skin samples were collected from 35
subjects. Two samples, from two individual subjects, yielded one
and over 50 purple colonies (FIG. 1), respectively. One purple
colony was isolated on a R2A plate from a heel sample and confirmed
as Janthinobacterium lividum (FIG. 1, panel A). Another forehead
sample yielded over 50 purple colonies on BHI agar plates (FIG. 1,
panel B). Molecular identification indicated that 5 randomly
selected purple colonies are all Janthinobacterium lividum.
[0175] Young seedlings of several produce samples were processed,
including radish, garlic, spinach, basil, beet, asparagus, chives,
tomato, swiss chad, lettuce, and pepper. For example, three purple
colonies were isolated from a radish sample (FIG. 1, panel C).
7.2. Example 2: Genetic Studies
[0176] Summary
[0177] To survey the prevalence of Janthinobacterium lividum on
human skins, approximately 700 skin microbiome samples collected
from 42 healthy young adults were screened by quantitative PCR
(qPCR) assay. Additionally, Shotgun-sequencing was performed on
approximately 156 skin microbiome samples collected from 9 healthy
young adults. Results indicated that 19% of subjects have
Janthinobacterium lividum on their skin by qPCR and all 9 subjects
have Janthinobacterium lividum on their skin by shotgun sequencing.
Shotgun sequencing analysis also showed that Janthinobacterium
lividum was detected on multiple anatomical sites and on multiple
sample collection visits, indicating that Janthinobacterium
lividum, aside from being identified in soil, amphibians, and
plants, is ubiquitously present on human skin.
[0178] Genetic analyses and molecular evolution study of
Janthinobacterium lividum strains disclosed herein together with
strains with published genome sequences showed that
Janthinobacterium lividum strains fell into at least four distinct
subgroups and each subgroup consists of Janthinobacterium lividum
strains isolated from various sources including soil, plant,
amphibian, and human skin.
[0179] Materials & Methods
[0180] qPCR Screening of Janthinobacterium lividum from Human Skin
Microbiome Samples
[0181] Molecular screening of Janthinobacterium lividum was
performed using SensiFAST SYBR No-ROX kit (Bioline) in a 10 .mu.l
reaction in duplicates on a CFX real-time PCR detection system
(BioRad) following instructions. The screenings were carried out
sequentially using two set of primer pairs,
Janthinobacterium-specific (Janthino2F2, GCACGGAAGTGACCAAAAA and
Janthino2R2, ACATGGAGACTTGGGCTTTG) and violacein-specific (JlivF,
TACCACGAATTGCTGTGCCAGTTG and JlivR, ACACGCTCCAGGTATACGTCTTCA).
[0182] Whole Genome Shotgun Sequencing and Genome Sequence
Assembly
[0183] Shotgun sequencing library for each Janthinobacterium
lividum strain was generated using the Nextera Flex kit
manufactured by Illumina according to the instructions.
[0184] The shotgun libraries were pooled and sequenced in a HiSeq X
platform. The sequencing reads were automatically demultiplexed
into individual FASTA files, run through DermBiont's bioinformatic
analysis pipeline yielding a genome assembled from cleaned
sequencing reads.
[0185] Nucleotide-Level Genome Similarity of Janthinobacterium
lividum Strains
[0186] Genome sequences of 5 Janthinobacterium lividum strains
(JL001, JL002, JL004, JL005, and JL007), along with several
published Janthinobacterium lividum genomes, were run through
Average Nucleotide Identify (ANI,
https://img.jgi.doe.gov/docs/docs/ANI.pdf) analyses to determine
the similarity and genetic diversity.
[0187] Phylogenomic Analysis of Janthinobacterium lividum Genome
Sequences
[0188] Phylogenomic relationships of Janthinobacterium lividum
strains, including Janthinobacterium lividum strains described
herein and strains with published genomic sequences, were evaluated
using the RAxML package following the instructions.
[0189] Results, Interpretations, and Conclusions
[0190] 704 skin microbiome samples collected from 42 young healthy
adults from various anatomical sites including scalp, forehead,
alar crease, antecubital fossa, palm, heel, and toe web space were
screened. qPCR screening revealed 116 and 30 positive samples using
the Janthinobacterium-specific primer pairs and violacein-specific
primer pairs, respectively (Table 2). As expected, 8 samples from 8
individual subjects were positive for both probe sets (Table 2),
suggesting that approximately 19% of the subjects tested have
Janthinobacterium lividum on their skin.
TABLE-US-00003 TABLE 2 qPCR screening of Janthinobacterium lividum
from skin microbiome samples Violacein Janthiobacterium Probe probe
# clinical samples screened 704 (42 704 (42 subjects) subjects) #
J. lividum + Violacein- -- 116 # J. lividum - Violacein+ 30 -- # J.
lividum + Violacein+ 8 8
[0191] To further evaluate the prevalence and abundance of
Janthinobacterium lividum in skin microbiome samples, metagenomic
shotgun sequencing of a total of 127 skin microbiome samples from 9
young healthy subjects across 7 anatomical sites in 3 sample
collection visits was performed. Owing to the high sensitivity of
shotgun sequencing approach, a higher percentage of
Janthinobacterium lividum positive samples than qPCR screening were
anticipated. Indeed, and not surprisingly, all 9 subjects have
Janthinobacterium lividum on their skin samples in multiple
anatomical sites or multiple sampling visits (FIG. 2). On average,
Janthinobacterium lividum DNA accounts for 0.4% of overall skin
microbial DNA with a high abundance in antecubital fossa and palm
and very low abundance in foot area (FIG. 2).
[0192] ANI estimates the average nucleotide identity using both
best hits and reciprocal best hits between two genomes
(enve-omics.ce.gatech.edu/ani/). 8 published Janthinobacterium
lividum genomes were downloaded and compared with internally
isolated Janthinobacterium lividum genome sequences for ANI
analysis. To further evaluate evolutionary relationship of
Janthinobacterium lividum strains, phylogenomic trees of all
Janthinobacterium lividum strains were constructed using whole
genome sequences. There are at least 4 distinct subgroups of
Janthinobacterium lividum (FIG. 3). Furthermore, each subgroup
consists of Janthinobacterium lividum collected from various
sources including soil, plant, amphibian, and human skin (FIG.
3).
7.3. Example 3: Identification of Prebiotics
[0193] Using the PM1 through PM5 10 phenotype MicroArrays for
Microbial Cells form Biolog, Hayward, Calif., USA we identified
compounds that, when used in the formulation, lyophilization,
fermentation, and or vehicle of delivery for the bacterium
Janthinobacterium lividum and its constituent products to increase
growth rates (Table 3) and/or functional efficacy (Table 4) against
inhibiting microbial pathogens, such as but not limited to,
bacterial infections such as Staphylococcus, Pseudomonas,
Enterococcus; fungal pathogens including but not limited to,
Trichophyton, Malassezia, Candida; DNA and RNA viruses including
but not limited to poliovirus, herpes simplex virus, hepatitis A,
rotavirus, adenovirus, H1N1, Coronavirus and influenza type A.
TABLE-US-00004 TABLE 3 Components for enhancing growth of
Janthinobacterium in media component that enhanced DBI001 growth*
D-Mannitol Tween 20 Tween 40 Cytidine *0.1 OD unit or more higher
than standard media control at 10 and 11 hour time points at
27.degree. C.
TABLE-US-00005 TABLE 4 Components for enhancing function against
fungal or bacterial pathogens Additives to enhance efficacy of
function for DBI001* N-Acetyl-D-Glucosamine
.beta.-Methyl-D-Xyloside L-Proline Palatinose D-Mannose
Sedoheptulosan Dulcitol L-Sorbose Glycerol Stachyose L-Fucose
D-Tagatose D-Glucoronic Acid Turanose D-Gluconic Acid Xylitol
D,L-.alpha.-Glycerol Phosphate D-Ribono-1,4-Lactone D-Mannitol
Sebacic Acid L-Glutamic acid Acetamide Tween 20 L-Alaninamide
L-Rhamnose N-Acetyl-L-Glutamic Acid D-Fructose L-Arginine Acetic
Acid Glycine .alpha.-D-Glucose L-Histidine Maltose L-Homoserine
D-Melibiose Hydroxy-L-Proline Thymidine L-Isoleucine L-Asparagine
dextrin D-Aspartic Acid lactitol D-Glucosaminic Acid D-Tartaric
acid 1,2-Propanediol L-Tartaric acid Tween 40 L-arginine
.alpha.-Keto-Glutaric Acid L-phenylalanine .alpha.-Keto-Butyric
Acid Ammonia .alpha.-Methyl-D-Galactoside Nitrite .alpha.-D-Lactose
Nitrate Sucrose Urea Uridine Biuret L-Glutamine L-Alanine
D-Glucose-1-Phosphate L-Arginine .alpha.-Hydroxy Glutaric
Acid-g-Lactone L-Asparagine b-Methyl-D-Glucoside L-Aspartic Acid
Adonitol L-Cysteine Maltotriose L-Glutamic Acid 2-Deoxy Adenosine
L-Glutamine Adenosine Glycine Mucic Acid L-Histidine Glyoxylic Acid
L-Isoleucine D-Cellobiose L-Leucine Inosine L-Lysine
Glycyl-L-Glutamic Acid L-Methionine Tricarballylic Acid
L-Phenylalanine L-Serine L-Proline L-Threonine L-Serine L-Alanine
L-Threonine L-Alanyl-Glycine L-Tryptophan N-Acetyl-b-D-Mannosamine
L-Tyrosine Mono Methyl Succinate L-Valine Tyramine D-Alanine
Chondroitin Sulfate C D-Asparagine .alpha.-Cyclodextrin D-Aspartic
Acid .beta.-Cyclodextrin D-Glutamic Acid g-Cyclodextrin D-Lysine
Glycogen D-Serine Inulin D-Valine Laminarin L-Citrulline Mannan
L-Homoserine Pectin L-Ornithine N-Acetyl-D-Galactosamine
Ethylenediamine N-Acetyl Neuraminic Acid Putrescine .beta.-D-Allose
Agmatine Amygdalin Histamine D-Arabitol .beta.-Phenylethylamine
L-Arabitol Tyramine Arbutin Acetamide 2-Deoxy-DRibose Formamide
i-Erythritol Glucuronamide D-Fucose D,L-Lactamide
3-0-.beta.-D-Galactopyranosyl-D-Arabinose D-Glucosamine Gentiobiose
D-Galactosamine L-Glucose D-Mannosamine D-Melezitose
N-Acetyl-D-Glucosamine Maltitol N-Acetyl-D-Galactosamine
.alpha.-Methyl-D-Glucoside N-Acetyl-D-Mannosamine
.beta.-Methyl-D-Galactoside Adenine 3-Methyl Glucose Adenosine
b-Methyl-D-Glucuronic Acid Allantoin .beta.-Methyl-D-Mannoside
Ala-Glu *5% or more increase in diameter of zone of inhibition
compared to standard conditions outlined at 24 hours and seven
days, 27.degree. C.
[0194] Extraction and quantification of violacein and prodigiosin
are well known in the art and can be done using standard
techniques.
[0195] Violacein production can be quantified by measuring
absorbance of bacterial culture at 580 nm and 764 nm using a
spectrophotometer. By subtracting the value at 764 nm from the
value at 580 nm, the contribution to absorbance by the bacterial
cells is removed and the remaining value quantifies violacein
levels.
[0196] Levels of prodigiosin can be measured similarly, by
extracting the pigment in acidified ethanol. Prodigiosin in
acidified ethanol displays a characteristic wavelength of 535 nm.
Estimation of prodigiosin can be expressed as unit/cell after
measuring the absorbance at 600 nm at the end of incubation.
7.4. Example 4: Manufacturing
[0197] Summary
[0198] Janthinobacterium lividum DB02473 was grown in fermenters at
5 L scale. Fermenters were inoculated from 14-15 h old shake flask
culture inoculum, and growth was monitored by measuring absorbance
at 600 nm. Fermentor culture was harvested aseptically and cell
pellet was resuspended in formulation buffer containing
cryoprotectants and stabilizers, to prepare frozen formulation or
lyophilized formulation. The final formulated product was tested
for viability, purity, and functional activity against pathogens T.
rubrum and S. aureus, before and after dilution to 10.sup.8,
10.sup.7 and 10.sup.6 CFU/ml. Purity was >99.99%, there was zero
to minimal drop in viability and no change in activity between
undiluted and diluted samples.
[0199] Materials & Methods
[0200] Microbial Strains
[0201] Trichophyton rubrum strain 18754 was purchased from ATCC and
maintained according to instructions. Janthinobacterium lividum
strain JL007 cryostock 2 was stored at -80.degree. C. with either
DMSO or glycerol as cryoprotectant. S. aureus strain 25923 was
purchased from ATCC and maintained according to instructions.
[0202] Purity Assay
[0203] Shake flask cultures used to inoculate the fermenters and
harvested culture resuspended in the cryoprotectant vehicle were
diluted such that plating of 100 .mu.l would lead to an estimated
100-300 colonies per plate. This estimate was based on the
absorbance at 600 nm reading and past experience where 1 OD unit
was 5.times.10.sup.8 CFU/ml. 6-10 plates of LB50 agar or BHI
supplemented with 0.5% glycerol agar were spread plated with 100
.mu.l of culture. Plates were incubated at room temperature for up
to one week and were monitored for non-Janthinobacterium lividum
growth.
[0204] Viability Assay
[0205] At harvest, the absorbance at 600 nm was measured and the
CFU/ml calculated assuming that 1 OD unit was equal to
5.times.10{circumflex over ( )}8 CFU/ml. One sample was plated
immediately as described below and another sample was taken on ice
to the lab and analyzed for T. rubrum activity using the Ramsey
assay (see below) and the Staphylococcus antibiosis assay and
plated for counts again. Additionally, 3 samples were then made
from the harvest to equal 10.sup.8, 10.sup.7 and 10.sup.6 CFU/ml.
100 .mu.l from each dilution was added to the first well of a 96
well plate in triplicate. 30 .mu.l was removed from this well and
added to 270 .mu.l of sterile PBS in the second well of the 96 well
plate. A pipette with 100 .mu.l volume was used to mix this before
tips were changed and 30 .mu.l removed from this second well and
transferred in to a third well with 270 .mu.l sterile PBS. A 100
.mu.l volume was used to mix the culture. In this way 7 serial
10-fold dilutions were made to the original 10-fold dilution. 10
.mu.l spots of each dilution were spotted on to an LB50 agar plate
from each of the 3 replicate dilutions and allowed to dry. Plates
were incubated at room temperature for 2 days and CFU were counted.
CFU/ml was calculated using the dilution factors of the spot
counts.
[0206] Ramsey In Vitro Antibiosis Assay
[0207] In vitro assay was set up following the procedure reported
by Ramsey et al. (2015) and optimized by DermBiont. Briefly,
24-hour Janthinobacterium lividum culture grown in 50% LB-vegitone
(LB50), to roughly 2.times.10.sup.9 CFU/ml, was struck out onto 33%
Tryptone agar plates in two straight lines using a cotton swab, one
perpendicular to the other, such that a cross was formed on the
plate. Conidia spores of T. rubrum were harvested from about 2-week
old culture grown on Sabouraud Dextrose Agar, counted using a
hemocytometer under a standard microscope, and applied at a
concentration of 10.sup.6 spores/ml in four replicate spots with 5
.mu.l per spot. The positions of the Janthinobacterium lividum
cross and spore spots were replicated on each plate using a
template. Assays plates were incubated at ambient temperature or at
27.degree. C. as indicated for 2 to 3 weeks. Images were taken at
Day 14 and Day 21. These results are shown in FIG. 4.
[0208] The inhibition of T. rubrum was referred as the reduction of
colony size in mm compared to controls. Radii of T. rubrum colonies
were first measured using ImageJ in pixels (straight line) and
converted into mm based the pixel measurements of petri dishes.
[0209] S. aureus Antibiosis Assay
[0210] In vitro assay was set up following standard experimental
procedure. Briefly, S. aureus 25923 was struck out BHI agar from a
cryostock and incubated at 37.degree. C. overnight. The agar plate
was used for the assay within 1 week. 3 ml of BHI media was
inoculated with colonies from the agar plate and grown for 2-3 hr
at 37.degree. C. The absorbance at 600 nm was measured and diluted
to 1.times.10{circumflex over ( )}7 CFU/ml, assuming that 1 OD unit
is equal to 1.times.10{circumflex over ( )}9 CFU/ml. 200 .mu.l of
1.times.10{circumflex over ( )}7 CFU/ml was added to LB50 agar
plates and a lawn of bacterium dispersed over the agar using 5-10 5
mm sterile glass bead. The beads were removed and the plates
allowed to dry. 1-2 5 .mu.l spots of Janthinobacterium lividum
JL007 from frozen sample or 20-24 hr cultures were added to each
plate and allowed to dry. A mock control plate was left untouched.
Control JL007 cultures were grown as described above for the Ramsey
assay. All plates were incubated for 4-6 days at room
temperature.
[0211] Results are qualitative. A successful outcome shows a sparse
collection of S. aureus colonies on the experimental or JL007
control plated compared with a lawn on the mock control. A
qualitative score of 1-3 is given for 1 showing 50-75% growth, 2
showing 15-50% growth and 3 showing <15% growth compared with
the mock control.
[0212] Results, Interpretations, & Conclusions
[0213] Purity Assay
[0214] An estimate of the number of colonies per plate is shown in
the table 5 below. Representative images are shown in FIG. 6. No
non-Janthinobacterium lividum colonies were observed for either
F107 or F108 shake flask or harvested culture confirming that the
culture is a monoculture to >99.99% for shake flask sand harvest
cultures.
TABLE-US-00006 TABLE 5 Calculated estimate Estimated of CFU number
of Dilution per plate colonies before (# plates on all Purity
Culture plating counted) plates (%) F107 Shake 10{circumflex over (
)}3 ~2376 ~2.6 .times. >99.99 flask (2) 10{circumflex over ( )}4
(4) F107 Harvest 10{circumflex over ( )}6 23 432 >99.99 and
10{circumflex over ( )}7 (17) (17) F108 Shake flask F108 Harvest
10{circumflex over ( )}6 371 3717 >99.99 (3) (10)
[0215] Viability
[0216] CFU/ml from F107 and F108 harvest cultures were determined
before and after dilution to 10{circumflex over ( )}8,
10{circumflex over ( )}7 and 10{circumflex over ( )}6 CFU/ml. FIG.
7 shows the CFU/ml from these plated samples the comparison with
the at harvest counts. The counts show there was no significant
loss in viability after transfer of the harvest sample on ice and
that the sample was diluted to 10{circumflex over ( )}8,
10{circumflex over ( )}7 and 10{circumflex over ( )}6 CFU/ml in an
accurate manner.
[0217] Ramsey Assay
[0218] Janthinobacterium lividum-T. rubrum antibiosis assays were
mostly carried out using the lead the undiluted harvest culture and
the harvest culture diluted to 10{circumflex over ( )}8,
10{circumflex over ( )}7 and 10{circumflex over ( )}6
Representative images of the mock control, 10{circumflex over ( )}8
CFU/ml culture and the mock control are shown in FIG. 8. FIG. 10
shows the difference in mm between the size of the proximal fringe
(indicated in FIG. 8c) of the mock control and the experimental
samples. Each is an average of the four T. rubrum colonies from
each plate. It can be concluded that dilution of the sample shows
no significant effect on the inhibition of T. rubrum growth; an
approximately 5 mm decrease in growth of the T. rubrum colony is
observed at the position closest to the JL007 cross.
[0219] Staphylococcus aureus Antibiosis Assays.
[0220] Results from the S. aureus antibiosis assays are qualitative
as the method used for the assay causes JL007 from the harvested
fermenters to stop all but sporadic growth. For comparative
purposes, a representative mock and lab grown JL007 images from a
previous fermentation developmental are shown. Table 6 shows the
qualitative score for the undiluted and each dilution for F107 and
F108. It can be concluded that there is no difference between the
undiluted and dilutions of F107 and F108.
TABLE-US-00007 TABLE 6 Qualitative S. aureus antibiosis assay score
F107 Score F108 Score Undiluted 3 Undiluted 3 10{circumflex over (
)}8 dilution 3 10{circumflex over ( )}8 dilution 3 10{circumflex
over ( )}7 dilution 3 10{circumflex over ( )}7 dilution 3
10{circumflex over ( )}6 dilution 3 10{circumflex over ( )}6
dilution 3
[0221] In summary undiluted culture from F107 and F108 had just
over 10{circumflex over ( )}9 CFU/ml when analyzed directly from
the fermenter. An approximate 10-fold dilution in counts was
followed for both F107 and F108 upon dilution and plating. Purity
was >99.99% for shake and harvest samples suggesting a true
monoculture, that was confirmed for both fermenters by QPCR. The
activity assay against T. rubrum and S. aureus showed no difference
between undiluted and diluted cultures or in comparison with shake
flask grown controls confirming that fermenter grown culture was as
active in inhibiting the growth of these pathogens.
7.5. Example 5: Virulence Factors
[0222] Summary
[0223] To evaluate biosafety of Janthinobacterium lividum strains,
Janthinobacterium lividum strains were tested for virulence factors
and examined for their antibiotic resistance profiles by performing
both in silico analyses and wet lab experimental confirmation. In
silico genome mining revealed no known virulence factors and no
antibiotic resistance genes in genomes of five (5) strains of
Janthinobacterium lividum (Table 1). Antibiotic tests, showed that
the tested Janthinobacterium lividum strains are sensitive to over
24 antibiotics to various extent. Janthinobacterium lividum strains
are sensitive to cephalosporin, quinolone, and tetracycline classes
of antibiotics, less sensitive to aminoglycosides, macrolides,
aztreonam, carbapenem, meropenem, chloramphenicol, clindamycin, and
amoxicillin/clavulanate (4:1), and are resistant to bacitracin.
[0224] Materials & Methods
[0225] Whole Genome Shotgun Sequencing and Bioinformatic
Analyses
[0226] Shotgun sequencing libraries for each Janthinobacterium
lividum strain was generated using the Nextera Flex kit (Illumina).
The shotgun libraries were pooled and sequenced in a HiSeq X
platform. The sequencing reads were automatically demultiplexed
into individual FASTA files, run through bioinformatic analysis
pipeline yielding a genome assembled from cleaned sequencing
reads.
[0227] Genome sequences of each Janthinobacterium lividum strains
were run through CosmoID online service (cosmosid.com) for
searching of virulence factor genes and known antibiotic resistance
genes.
[0228] Internal Antibiotic Resistance Tests
[0229] One Janthinobacterium lividum colony of 3-day old grown in
50% BHI agar was mixed well in 1 ml of 1.times.PBS buffer, diluted
10.times. and 100.times., respectively. 200 .mu.l of diluted
Janthinobacterium lividum cells were evenly plated onto R2A/BHI
agar plates in triplicate in 150 mm petri dishes. Antibiotic discs
(BD BBL Sensi-Disc antimicrobial susceptibility test discs) were
dispensed using a Disc dispenser (BD BBL Sensi-Disc Designer
Dispenser System, 12-Place). Plates were incubated for 2-3 days at
room temperature and imaged at the end of incubation.
[0230] The sensitivity of Janthinobacterium lividum to antibiotics
is reflected by a clear zone of inhibition of Janthinobacterium
lividum cells surrounding the antibiotic discs. The zone of
inhibition was measured using ImageJ and was transformed into cm
for analysis.
[0231] Antibiotic MIC Tests
[0232] The antibiotic resistance profile and MICs tests were
performed using Janthinobacterium lividum grown from cultures
purified and grown in vegitone LB broth.
[0233] Results, Interpretations, & Conclusions
[0234] All five Janthinobacterium lividum strains were sequenced
with a coverage of at least 100.times. of the genome size. Genome
sequences were assembled into contigs and run through CosmosID
(cosmosid.com) to search for virulence factor genes and antibiotic
resistance genes. CosmosID has well-curated reference databases for
bacterial virulence factor genes and antibiotic resistance genes.
In silico searches yielded no any known genes coding for virulence
factors and antibiotic resistance.
[0235] Antibiotic sensitivities were also confirmed by laboratory
assays, using the Janthinobacterium lividum grown from
Janthinobacterium lividum culture grown in vegitone LB broth.
Antibiotic assays performed showed that all 5 Janthinobacterium
lividum strains are sensitive to the following antibiotic/doses:
penicillin 10 IU, ampicillin 10 .mu.g, streptomycin 10 .mu.g,
vancomycin 30 .mu.g, erythromycin 15 .mu.g, doxycycline 30 .mu.g,
teracycline 30 .mu.g, cephalothin 30 .mu.g, ciprofloxaxin 5 .mu.g,
and chloramphenicol 5 .mu.g (FIG. 5).
[0236] Janthinobacterium lividum strains are sensitive to
cephalosporin, quinolone, and tetracycline classes of antibiotics,
less sensitive to aminoglycosides, macrolides, aztreonam,
carbapenem, meropenem, chloramphenicol, clindamycin, and
amoxicillin/clavulanate (4:1), and are resistant to bacitracin
(Table 7 and 8).
TABLE-US-00008 TABLE 7 MIC characterization of Janthinobacterium
lividum strains MIC (.mu.g/ml)* Aminoglycosides Penicillin
Macrolide Mono- Poly- Cephalosporin Other Amika- Neo- Amoxicillin
Azithro- Erythro- bactam peptide Ceftria- Chloram- Strains cin
mycin clavulanate mycin mycin Aztreonam Bacitracin Cefepime xone
phenicol DB00118 2 2 1 8 15 32 >64 0.25 0.5 2 DB00121 1 1 0.5
0.5 8 16 >64 0.125 0.25 2 DB02378 2 2 1 2 8 16 >64 0.125 0.25
2 DB02473 2 2 1 1 15 16 >64 0.125 0.5 2 E. Coli 2 2 8 8 64 0.25
>64 <0.063 <0.063 4 ATCC25922 E. Coli 0.5-4 -- -- -- --
0.06-0.25 -- 0.016-0.12 0.03-0.12 2-8 ATCC25922 CLSI QC ranges
*MICs tests were performed by Institute for Life Science
Entrepreneurship
TABLE-US-00009 TABLE 8 MIC characterization of Janthinobacterium
lividum strains MIC (.mu.g/ml)* Quinolones Lipopetide Cabapenem
Tetracycline Strains Ciprofloxacin Levofloxacin Clindamycin
Meropenem Minocycline Tetracycline DB00118 0.5 0.25 32 2 0.125 0.5
DB00121 0.25 0.125 16 8 <0.063 0.25 DB02378 0.5 0.25 32 2 0.125
0.5 DB02473 0.5 0.25 16 2 0.25 0.5 E. Coli <0.063 0.063 >64
<0.063 0.25 1 ATCC25922 E. Coli 0.004-0.016 0.008-0.06 --
0.008-0.06 0.25-1 0.5-2 ATCC25922 CLSI QC ranges *MICs tests were
performed by Institute for Life Science Entrepreneurship
7.6. Example 6: In Vitro Efficacy
[0237] Summary
[0238] Five (5) unique strains of Janthinobacterium lividum have
been demonstrated in laboratory in vitro assays to significantly
inhibit growth of a dermatophyte, Trichophyton rubrum at ambient
temperature and at 27.degree. C. Mild growth inhibition of T.
rubrum by JL007 was observed at 30.degree. C.
[0239] Materials & Methods
[0240] Microbial Strains
[0241] Trichophyton rubrum strain 18754 was purchased from ATCC and
maintained according to instructions. Janthinobacterium lividum
strains were stored at -80.degree. C. with either DMSO or glycerol
as cryoprotectant.
[0242] Ramsey In Vitro Antibiosis Assay
[0243] In vitro assay was set up following the procedure reported
by Ramsey et al. (2015). Briefly, 24-hour Janthinobacterium lividum
culture grown in 50% vegLB (LB50), roughly 2.times.10{circumflex
over ( )}9 CFU/ml, was struck out onto 50% Tryptone agar plates in
two straight lines using a sterile Q-Tip, perpendicular to each
other. Conidia spores of T. rubrum were harvested off the
approximately 1-week old culture grown on Sabouraud Dextrose Agar,
counted using a hemocytometer under a standard microscope, applied
at a concentration of 10{circumflex over ( )}6 spores/ml in four
replicate spots with 5 .mu.l per spot. Assays plates were incubated
at ambient temperature or at 27.degree. C. as indicated for 2-3
weeks. Images were taken at Day 7, Day 14, and Day 21.
[0244] Quantification of a Zone of Inhibition
[0245] The inhibition of T. rubrum was referred as the reduction of
colony diameters in cm. Diameters of T. rubrum colonies were first
measured using ImageJ in pixels (straight line) and converted into
cm based the pixel measurements of petri dishes.
[0246] Results, Interpretations, & Conclusions
[0247] Janthinobacterium lividum-T. rubrum antibiosis assays were
carried out using the DB02473 human-isolated Janthinobacterium
lividum strain. For plates incubated at ambient temperature, T.
rubrum grows beyond initial inoculation spots starting at Day 4. At
day 7, Janthinobacterium lividum-treated T. rubrum colonies grown
in co-culture with Janthinobacterium lividum were noticeably
smaller than mock control (no Janthinobacterium lividum) colonies.
At Day 14, Janthinobacterium lividum treated colonies were
significantly smaller than control colonies (FIG. 10), indicating a
strong growth inhibition by Janthinobacterium lividum. Similar
results were observed with other unique Janthinobacterium lividum
strains.
7.7. Example 7. Metabolites
[0248] Summary
[0249] Two known metabolites of Janthinobacterium lividum,
violacein and indole-3-carbaldehyde, are active compounds involved
in Janthinobacterium lividum-exerted growth inhibition. The
metabolic profile of Janthinobacterium lividum in the presence and
absence of T. rubrum or S. aureus culture was analyzed by liquid
chromatography-mass spectrometry (LC-MS). A full spectrum of
metabolites produced by Janthinobacterium lividum isolate and those
metabolites upregulated by Janthinobacterium lividum in the
presence of T. rubrum or S. aureus culture was elucidated (Table
9). Indole-3-carbaldehyde and 17 other metabolites are uniquely
over expressed in DB02473 relative to the other Janthinobacterium
lividum isolates (Table 10). Significantly expressed (2 fold
difference compared to control) metabolites for each
Janthinobacterium lividum isolates in response to pathogenic
challenge (Staph. or T. rubrum) were compared (Table 11).
[0250] Materials & Methods
[0251] The 1.2-ml samples were sonicated using a probe sonicator
set at 40% intensity for 3.times.40 s with 20 s on ice between
sonication bursts. This procedure was based on findings from a
pilot experiment and gives 60-80% lysis of Janthinobacterium
lividum DB02473. The sonicated material was centrifuged at
14,000.times.g for 2 minutes. 700 .mu.l of the supernatant was
transferred to barcoded Micronic tubes provided by Metabolon. The
tubes were capped, flash frozen in liquid nitrogen and stored at
-80.degree. C. The remainder of the lysate supernatant was stored
in the -80.degree. C. in freezer boxes after being flash
frozen.
[0252] Snap frozen cultures were defrosted at 4.degree. C. and
centrifuged at 14,000.times.g for 10 minutes. The pellet was
resuspended in 1 ml of methanol and quantitatively transferred to
bead beating tubes. The original tubes were further washed with 1
ml of methanol and was combined into the previous 1 ml of methanol
in the bead beating tubes. 0.1 mm glass beads were added to each
bead beating tube so that the conical bottom of the tube was
filled. The tubes were placed in a Qiagene TissueLyser II and were
subject to bead beading for 10 minutes at 30 Hz. Samples were
centrifuged at 14,000.times.g for 10 minutes and the supernatant
was transferred into clean tubes. A speed vacuum was used to
evaporate the methanol at 30.degree. C. The pellet was resuspended
in 200 .mu.l of HPLC grade methanol and transferred into the insert
of autosampler vials.
[0253] Results, Interpretations, & Conclusions
[0254] Method for identification of metabolites that were
significantly enhanced or decreased by 2 fold compared to DB02473.
[0255] 1. Metabolites abundance compared with the media control or
pathogen were ranked from high to low according to DB02473. [0256]
2. The ratio of DB02473: other Janthinobacterium lividum was
calculated. If there was a 2 fold increase or decrease in at least
4 other strains, then the metabolite was considered as behaving
differently than in the DB02473 strain. [0257] 3. Metabolites where
the abundance compared with the media control or pathogen was not
significant were removed.
TABLE-US-00010 [0257] TABLE 9 Metabolic profile of all
Janthinobacterium lividum strains Biochemical Name Biochemical Name
Biochemical Name (.+/-.)-Tartaric acid Benzisothiazolone
Methohexital (+)-a(S)-butyr-amido-r-butyrolactone benzoate Methyl
Jasmonate (+)-Eudesmin beta-hydroxyisovalerate methylmalonate (MMA)
(+)-Riboflavin betaine methylphosphate (+/-)-2-Hydroxyglutaric acid
Biacetyl methylsuccinate (+/-)-Coniine Biotinl-Sulfoxide
methylxanthine (+/-)-Methoprene Bis(2-ethylhexyl) phthalate
methyjnylon (.+-.)-Malic Acid Boc-Asn-Oh mevalonolactone
(-)-trans-Methyl dihydrojasmonate Botrydial Misoprostol
(17R)-23-Amino-20-hydroxy-20-oxido- Brassicanal A MPTP
14-oxo-15,19,21-trioxa-20lambda~5~- phosphatricosan-17-yl (9Z)-9-
hexadecenoate (1R,2S)-1-(7,8-Dihydro-6-pteridinyl)- Brivaracetam
myo-inositol 1,2-propanediol (2E,4Z)-N-Isobutyl-2,4- Buflomedil
Myristamide octadecadienamide (2E)-3,7-Dimethyl-2,6-octadien-1-yl
Butabarbital Myristyl sulfate beta-D-glucopyranoside
(2R,3S,4R,5S,8R, 10R, 11R,13S,14R)-2- Butoctamide semisuccinate
Myrtillin Ethyl-3,4,10-trihydroxy-3,5,8,10,12,14-
hexamethyl-15-oxo-7-propyl-11-{[3,4,6- tride
oxy-3-(dimethylamino)-beta-D- xylo-hexopyranosyl]oxy}-1-oxa-7-
azacyclopentadecan-13- yl 2,6-dideoxy-3-C-methyl-3-O-methyl-
alpha-L-ribo-hexopyranoside (2R)-3-({[(2S)-2,3- Butyl
isothiocyanate N-(2,3,4- Dihydroxypropoxy](hydroxy)phosphoryl}
Trimethoxybenzoyl)glycine oxy)-2-[(9Z)-9- hexadecenoyloxy]propyl
(9Z)-9- hexadecenoate (2R)-3-{[(2- butyrate/isobutyrate (4:0)
N-acetyl-cadaverine Aminoethoxy)(hydroxy)phosphoryl]
oxy}-2-[(9Z)-9-hexadecenoyloxylpropyl (9Z)-9-hexadecenoate
(2R)-3-{[(2- cadaverine N-acetyl-isoputreanine
Aminoethoxy)(hydroxy)phosphoryl]oxy}- 2-hydroxypropyl (9Z)-9-
hexadecenoate (2R)-3-{[(2- Caprolactam N-acetyl-L-2-aminoadipic
acid Aminoethoxy)(hydroxy)phosphoryl]oxy}- 2-hydroxypropyl laurate
(2R)-3-{[(2- Capryloylglycine N-Acetyl-L-aspartic acid
Aminoethoxy)(hydroxy)phosphoryl]oxy}- 2-hydroxypropyl
pentadecanoate (2S)-3-(beta-D-Galactopyranosyloxy)-2- Capsi-mide
N-Acetyl-L-glutamic acid [(7Z,10Z,13Z)-7,10,13-
hexadecatrienoyloxy]propyl (9Z,12Z)- 9,12-octadecadienoate
(2S)-5-Carbamimidamido-2-(2-oxo-1- choline N-Acetyl-L-histidine
azetidinyl)pentanoic acid (2Z)-3,7-Dimethyl-2,6-octadien-1-yl 3-
choline phosphate N-Acetyl-L-leucine oxobutanoate
(3'-5')-adenylyladenosine* CILAZAPRILAT N-Acetyl-L-phenylalanine
(3'-5')-adenylyluridine Cinnamic acid N-acetylarginine
(3'-5')-cytidylyladenosine Cinnamyl alcohol N-acetylasparagine
(3'-5')-cytidylylcytidine* cis-2-Carboxycyclohexyl-acetic
N-acetylaspartate (NAA) acid (3'-5')-cytidylyluridine*
cis-5-Tetradecenoylcarnitine N-Acetylcadaverine
(3'-5')-guanylyladenosine* cis-urocanate N-Acetylcystathionine
(3'-5')-guanylylcytidine citrate N-acetylglutamine
(3'-5')-uridylyladenosine Citric acid N-acetylhistidine
(3'-5)-uridylylcytidine* citrulline N-acetylisoleucine
(3'-5')-uridylylguanosine Coprine N-acetylkynurenine (2)
(3S,8aS)-3-(4- Coumarin N-acetylleucine
Hydroxybenzyphexahydropyrrolo[1,2- a]pyrazine-1,4-dione
(3S)-3-{(Z)-[(3S)-3-{(Z)-[(3R)-3- Coumarone N-acetylmethionine
Amino-1-hydroxy-4- methylpentylidenelamino}-1-
hydroxybutylidenelamino}-5- methylhexanoic acid
(3Z)-3-[(Methylsulfanyl)methylene]-2- Cuauhtemone
N-acetylmethionine sulfoxide pyrrolidinethione
(4-Hydroxy-1H-indol-3-yl)acetonitrile cuscohygrine N-acetylmuramate
(4S)-4-{[(9Z)-3-Hydroxy-9- Cyclazocine N-acetylphenylalanine
hexadecenoyl]oxy}-4- (trimethylammonio)butanoate
(8E)-2-Amino-8-octadecene-1,3,4-triol cyclic(AMP-GMP)
N-acetylproline (9Z)-9-Octadecenamide cyclo(his-phe)
N-acetylputrescine (E)-p-coumaric acid Cyprodenate N-acetylserine
(Hydroxyethyl)methacrylate cystathionine N-acetylthreonine
1-(14-methylhexadecanoyl)pyrrolidine cysteine s-sulfate
N-acetyltryptophan 1-(4-Amino-4-carboxybutanoyl)-2- cysteine
sulfinic acid N-acetylvaline piperidinecarboxylic acid
1-(beta-D-Ribofuranosyl)-1,2- cytidine N-alpha-acetylornithine
dihydropyrimidine 1-(Propyldisulfanyl)-1- cytidine 2' or
3'-monophosphate N-butyryl-phenylalanine (propylsulfinyl)propane
(2' or 3'-CMP) 1-[(9Z)-hexadecenoyl]-sn-glycero-3- cytidine
2',3'-cyclic N-carbamoylalanine phosphocholine monophosphate
1-[1-(1-Benzothiophen-2-yl)ethyl]urea cytidine 5'-monophosphate
(5'- N-carbamoylaspartate CMP) 1-carboxyethylisoleucine cytidine
5'-monophosphate N-carbamoylputrescine 1-carboxyethylleucine
cytosine N-ethylmaleimide 1-carboxyethylphenylalanine
D-(-)-Erythrose N-formylanthranilic acid 1-carboxyethyltyrosine
D-Gluconic acid N-formylphenylalanine 1-carboxyethylvaline
D-PANTOTHENIC ACID n-Hexanamide 1-hexadecanoyl-sn-glycero-3-
D-Xylonic acid N-Hydroxy-8- phosphoethanolamine
(methylsulfanyl)octanethioamide 1-Hexadecanoylpyrrolidine
danegaptide N-methylalanine 1-Isothiocyanato-10- Deferasirox
N-Methylanhalonine (methylsulfinyl)decane 1-Isothiocyanato-7-
DEHYDROASCORBIC ACID N-methylethanolamine phosphate
(methylsulfanyl)heptane 1-methyl-5-imidazoleacetate deoxycarnitine
N-Methylpyrrolidone 1-oleoyl-sn-glycero-3- deoxythymidine
diphosphate-1- N-OLEOYL-4-AMINOBUTYRIC phosphoethanolamine rhamnose
ACID 1-palmitoleoyl-2-oleoyl-GPE deoxyviolacein
N-propionylmethionine (16:1/18:1)* 1-palmitoyl-2-oleoyl-GPE
(16:0/18:1) Desaminotyrosine N-succinyl-phenylalanine
1-palmitoyl-2-palmitoleoyl-GPC Desthiobiotin N-Tridecanoylglycine
(16:0/16:1)* 1-palmitoyl-2-palmitoleoyl-GPG Dexamisole
N-Undecanoylglycine (16:0/16:1)* 1-Pentofuranosyl-2,4(1H,3H)-
diacetylspermidine* N,N-Bis(2- pyrimidinedione
hydroxyethyl)dodecanamide 1-Piperideine Dibutyl phthalate
N('1)-acetylspermidine 1-Vinyl-2-pyrrolidone Diethylamine
N(1)-acetylspermidine 1,2-Dihydroxy-5-(methylsulfanyl)-1-
Diethylpyrocarbonate N(1)-acetylspermine penten-3-one
1,2-dipalmitoleoyl-GPE (16:1/16:1)* Diftalone
N(3)-(4-Methoxyfumaroyl)-2,3- diaminopropionic acid
1,2-dipalmitoyl-GPE (16:0/16:0)* dihydroorotate
N1,N12-diacetylspermine 1,2,3,4-tetrahydro-beta-carboline-3-
dihydroxyacetone phosphate N2-acetyl,N6-methyllysine carboxylic
acid (DHAP) 1,3-Dipropylxanthine Dimethyl sulfoxide N2-acetyllysine
1,3,7-Octanetriol Dimethylaminopropionylphenothiazine
N2,N2-dimethylguanosine 1,4-Anhydro-6-O-dodecanoyl-2,3-bis-O-
dimethylformamide N6-acetyllysine (2-hydroxyethyl)-D-glucitol
1,4-Bis(2-ethylhexyl) sulfosuccinate DL-Arginine
N6-carbamoylthreonyladenosine 1,5-DAN DL-Cerulenin
N6-carboxymethyllysine 10-undecenoate (11:1n1) DL-Glutamic acid
N6-dimethylallyladenine 11-Nitro-1-undecene DL-Histidine
N6-methyllysine 12,13-DiHOME DL-Lactic Acid N6-succinyladenosine
13-HODE + 9-HODE DL-Lysine N6,N6-dimethyladenosine
15S-hydroxyeicosatrienoic acid DL-Mevalonic acid
N6,N6,N6-trimethyllysine 16-Heptadecyne-1,2,4-triol
DL-Phenylalanine Name 1H-Imidazol-2-ol DL-Tryptophan naphazoline
2-(Hydroxy{2-[(9Z)-9- DOA Naphthalen-2-amine octadecenoyloxy]-3-
(palmitoyloxy)propoxylphosphoryl)- N,N,N-trimethylethanaminium
2-[(5Z)-5-Tetradecen-1- dopamine Niacin yl]cyclobutanone
2-aminoadipate Dulcin nicotinamide 2-Dodecylbenzenesulfonic acid
ectoine nicotinamide adenine dinucleotide (NAD+) 2-Furoic acid
Elaeokanine C nicotinamide riboside 2-hydroxy-3-methylvalerate
Embelin nicotinate ribonucleoside 2-hydroxy-4-(methylthio)butanoic
acid ENADENINE nicotinic acid mononucleotide (NaMN)
2-hydroxy-6-ketononadienedioic acid Epirizole Nitrendipine
2-Hydroxy-6-methyl-12,13-dihydro-5H- EPTAZOCINE NOP indolo
[2,3-a]pyrrolo [3,4-c]carbazole- 5,7(6H)-dione 2-hydroxyadipate
EPTC noradrenaline 2-hydroxybutyrate/2-hydroxyisobutyrate
erythronate* norhaman 2-Indolylformic acid Estramustine Phosphate
O-acetylhomoserine 2-Isocapryloyl-3R-hydroxymethyl- Ethopabate
o-Tyrosine gamma-butyrolactone 2-isopropylmalate Ethyl lactate
Octylamine 2-Mercaptobenzothiazole ethylmalonate Oleoylethanolamide
2-Methyl-4-ethyl-5-propylthiazole Fadrozole ophthalmate
2-methylcitrate/homocitrate FARNESYL ACETONE ophthalmic acid
2-methylcitric acid Fasoracetam ornithine 2-Methylthiazolidine
Fexaramine orotate 2-O-ETHYL ASCORBIC ACID flavin adenine
dinucleotide (FAD) Oryzalin metabolite 2-oxoadipate flavin
mononucleotide (FMN) Oxaceprol 2-oxoarginine* formiminoglutamate
Oxagrelate 2-pyridone Formiminoglutamic Acid oxalate (ethanedioate)
2,2-Bis(hydroxymethyl)propionic acid Frovatriptan Oxalic acid
2,3-dihydroxy-3-methylvalerate fructosyllysine Oxibendazole
2,3-dihydroxyisovalerate fumarate p-Cresol
2,4-Diacetylphloroglucinol Furaneol p-cymene 2,4-diaminobutyrate
g-Guanidinobutyrate p-hydroxybenzaldehyde 2,4-dihydroxybutyrate
Gabapentin PALGLY 2,4-dihydroxyheptadec-16-enyl acetate galactitol
(dulcitol) Palmitamide 2,4-dimethyl-4,5-dihydro-1h-imidazole
gamma-Aminobutyric acid palmitoleate (16:1n7)
2,5-Dihydroxy-3,6-di(1H-indol-3-yl)- gamma-glutamylalanine
pantetheine 1,4-benzoquinone 2'-deoxyadenosine
gamma-glutamylglycine pantethine 2'-deoxyinosine
gamma-glutamylleucine pantoate 2'-O-methyluridine
gamma-glutamylphenylalanine pantothenate 2H-Pyran
gamma-glutamylserine Paracetamol 3-(3-sulfooxyphenyl)propanoic acid
gamma-glutamylthreonine Paraldehyde 3-(4-hydroxyphenyl)lactate
Gemfibrozil PEG-4 3-aminoisobutyrate genistein pentobarbital
3-dehydroshikimate Glu-Gly Perlapine 3-deoxy-D-manno-2-octulosonic
acid gluconate PG(16:1(9Z)/18:1(9Z)) 3-deoxyoctulosonate glucose
phe-gln 3-hydroxy-2-methylpyridine glucose 6-phosphate phenacetin
3-hydroxyadipate glutamate Phenelzine 3-hydroxydodecanoylcarnitine
glutamate, gamma-methyl ester phenethylamine 3-hydroxyhexanoate
glutarate (C5-DC) phenylacetate 3-indoleglyoxylic acid Glutaric
acid phenylalanine 3-methyl-2-oxobutyrate Gly-Arg
phenylalanylalanine 3-methyl-2-oxovalerate Gly-l-pro
phenylalanylglycine 3-methylglutaconate Gly-Leu Phenylisocyanate
3-Methylsulfolene glycerate phenyllactate (PLA) 3-oxopalmitic acid
glycerol phosphate 3-phosphoglycerate glycerol 3-phosphate
phosphoethanolamine 3-phosphoserine glycerophosphoethanolamine
Phosphoric acid 3-sulfo-L-alanine glycerophosphoglycerol Phthalic
acid 3-ureidopropionate glycerophosphorylcholine (GPC) Pimilprost
3,4-Diaminopyridine glycerophosphoserine* Piracetam
3,4-dihydroxybutyrate glycine Pivagabine 3,4-Dihydroxyphenylglycol
glycolate (hydroxyacetate) porphobilinogen 4-acetamidobutanoate
glycyclamide prephenic acid 4-Aminobenzoic acid glycylisoleucine
primidone 4-Aminophenol glycylleucine Prinomide
4-hydroxy-4-(indol-3-ylmethyl)glutamic glycylvaline pro-gin acid
4-hydroxybenzoate guanine pro-hydroxy-pro 4-hydroxycinnamate
guanosine 2'-monophosphate (2'- Proflavine GMP)* 4-hydroxyglutamate
guanosine 3'-monophosphate (3'- prohydrojasmon GMP)
4-hydroxyphenylacetate guanosine 5'-monophosphate (5'-
prolylglycine GMP) 4-hydroxyphenylpyruvate Guanosine monophosphate
Propylparaben 4-Hydroxyprolylleucine guanosine-2,3'-cyclic
protoporphyrin IX monophosphate 4-imidazoleacetate harmane
pseudouridine 4-methyl-2-oxopentanoate Hept-2-ulose putrescine
4-Methylene-2-oxoglutarate heptanoate (7:0) Pyrantel
4-Morpholinylacetic acid hexahydro-2-oxo-1h-thieno(3,4- pyridoxal
d)imidazole-4-pentanoic acid 4-Nitroaniline hexobarbital pyridoxate
4-Undecylbenzenesulfonic acid Hexose pyrraline 4-Vinylcyclohexene
Hexyl 2-furoate Pyrrolidine 5-(2-Hydroxyethyl)-4-methylthiazole
Hexylresorcinol quinolinate 5-(galactosylhydroxy)-L-lysine
hippurate riboflavin (Vitamin B2) 5-aminovalerate Hippuric acid
ribonate 5-dodecenoate (12:1n7) histamine ribose
5-Methoxy-3-indoleaceate histidine betaine (hercynine)* ribose
1-phosphate 5-Methoxy-L-tryptophan histidinol
S-adenosylhomocysteine (SAH) 5-Methoxybenzimidazole histidylalanine
S-carboxymethyl-L-cysteine 5-methylthioadenosine (MTA)
homocitrulline S-methyl D-thioglycerate 5-methyluridine
(ribothymidine) homocysteine Salicylic acid 5-oxoproline
Homovanillic acid sebacate (C10-DC) 5,6-dihydrouridine
hydantoin-5-propionate Sebacic acid 5'-S-Methyl-5'-thioinosine
Hydroquinone sedoheptulose-7-phosphate
6-(1-Hydroxyethyl)-3-(hydroxymethyl)- Hydroxy(oxo)phosphoniumolate
serine 2,7-dioxabicyclo[4.1.0]hept-3-en-5-one
6-hydroxypseudooxynicotine Hydroxycitronellal diethyl acetal
shanzhiside 6-Methoxy-3-(1,3-thiazol-2-yl)-1H- Hymexazol
O-glucoside Sorbitan, monododecanoate indole
6-oxopiperidine-2-carboxylate Hypoxanthin spermidine
6-phosphogluconate hypoxanthine spermine 7-methylguanine imidazole
lactate Staurosporonine 7-methylsulfinylheptyl isothiocyanate
imidazole propionate succinate 8-[(Aminomethyl)sulfanyl]-6- Indigo
dye Succinic acid sulfanyloctanoic acid 8-Hydroxyhexadecanedioic
acid Indole Succinylacetone 9-Pentofuranosyl-3,9-dihydro-1H-
Indole-3-acetic acid sucrose purine-2,6-dione 9,10-DiHOME
indole-3-carboxylate Sulfabenzamide 9H-Fluoren-9-one indoleacetate
sulfacetamide Aceclidine Indoleacrylic acid Sulfuric acid
Aceglutamide indolelactate Suprofen Acetanilide inosine
tcmdc-125859 ACETYL ARGININE inosine 5'-monophosphate (IMP)
Tetraacetylethylenediamine ACETYL PROLINE inositol 1-phosphate
(I1P) thiamin (Vitamin B1) Acetyl-L-methionine Inspra thiamin
monophosphate acetylphosphate ionene thioproline acisoga IpA
THREO-SPHINGOSINE, (-)- adenine irdabisant THREONIC ACID, L-
adenosine isobutyrylglycine threonine adenosine 2'-monophosphate
(2'-AMP) isoleucine Threonylglutamine adenosine 3'-monophosphate
(3'-AMP) isoleucylglycine threonylphenylalanine Adenosine
monophosphate isopentenyl adenosine thymidine
adenosine-2',3'-cyclic monophosphate Isoprene thymidine
5'-monophosphate Adenylthiomethylpentose isopropylmalic acid
thymine adipate (C6-DC) isovalerate (i5:0) trans-4-hydroxyproline
agmatine Itaconic acid trans-Azobenzene ALA-PRO kynurenate
trans-urocanate alanine Kynurenic acid trans-Zeatin
alanyl-glutamyl-meso-diaminopimelate kynurenine trehalose
alanylleucine L-(+)-Leucine Tributyl citrate acetate
Alanyltyptophan L-(+)-Valine Tributyl phosphate allantoic acid
L-ACETYLTRYPTOPHAN Tridemorph allantoin L-Alanyl-L-glutamine
Trifluoroacetic acid allo-threonine
L-alpha-Aspartyl-L-phenylalanine trigonelline (N'-methylnicotinate)
Allyxycarb L-alpha- trimethadione Glycerylphosphorylcholine
alpha-hydroxyisocaproate L-gamma-Glutamyl-L-leucine tryptanthrin
alpha-hydroxyisovalerate L-Proline Tiyptoline
alpha-ketoglutaramate* L-Pyroglutamic acid tryptophan
alpha-ketoglutarate lactate Tiyptophol alpha-Ketoglutaric acid
leu-gln tryptophylglycine AMAC Leu-Gly-Pro tyrosine Amide v Leu-Leu
tyrosylglycine Aminohippuric acid Leu-pro Tyrosyltyrosine
Aminolevulinic acid Leu-Val UDP-N-acetylmuraminate (UDP- MurNAc)*
amonafide leucine uracil Ampalex leucoline urate angustine
leucylalanine urea Aniline Leucylasparagine Uric Acid anserine
leucylglutamine* uridine anthranilate leucylglycine uridine
3'-monophosphate (3'-UMP) APAZIQUONE Levetiracetam uridine
5'-monophosphate (UMP) APM Levulinic acid Uridine monophosphate
Aprobarbital Linoleamide uridine-2',3'-cyclic monophosphate
apronalide loganate val-arg Arabinosylhypoxanthine Lumichrome
valine arabitol/xylitol Lys-Pro valylglutamine Arachidonic acid
lysine valylglycine arginine lysophosphatidylcholine valylleucine
14:1(9Z)/0:0 asn-pro lysylleucine Vanillin asp-gln Lysylvaline
Vanillyl alcohol asp-leu malate Vernolate asparagine malonate
Veronal aspartate mannitol/sorbitol Vigabatrin Aspartyl-L-proline
Marimastat violacein Aspulvinone E mebutamate Vitamin C azelate
(C9-DC) mephenesin Vorinostat Bellendine meprobamate VS1150000
bendiocarb methionine xanthine Benserazide methionine sulfone
xanthosine Benzaldehyde Methionylleucine zinniol
TABLE-US-00011 TABLE 10 Metabolic compounds 2-fold expression
unique to DB02473 relative to other strains Biochemical Name
PUBCHEM ID 2-(alpha-D-mannosyl)-D-glyceric acid 5460194
2-ketogluconate 3035456 2-O-ethyl Ascorbic acid 54694369
anthramycin 5311005 Aprobarbital 6464 bendiocarb 2314
Bis(2-ethylhexyl) phthalate 8343 cis-5-Tetradecenoylcarnitine
22833575 Dibutyl phthalate 3026 imidazole propionate 70630
indole-3-carboxyaldehyde 10256 indolin-2-one 321710
N-Acetyl-L-aspartic acid 65065 Phosphoric acid 1004 Phthalic acid
1017 Pimilprost 5282140 trimethadione 5576 Vernolate 16003
TABLE-US-00012 TABLE 11 Significantly expressed (2 fold difference
compared to control) metabolites for each strain in response to
pathogenic challenge (Staph. or T. rubrum) DB00117 DB00118 DB00121
DB02378 DB02473 Biochemical Name Biochemical Name Biochemical Name
Biochemical Name Biochemical Name 1- (3'-5')- 1- (3'-5')-
1-methyladenine carboxyethylisoleucine cytidylylcytidine*
carboxyethylisoleucine guanylyluridine 1-linoleoyl-GPC (3'-5')-
1-methyladenine 1- 2-aminoadipate (18:2) guanylyluridine
carboxyethylisoleucine 1-methyladenine 1- 2-hydroxy-3- 1-methyl-4-
2-hydroxy-3- carboxyethylisoleucine methylvalerate imidazoleacetate
methylvalerate 1-oleoyl-GPC 1-palmitoyl-2- 2-hydroxy-4-
2-hydroxy-3- 2-hydroxy-4- (18:1) linoleoyl-GPC (methylthio)butanoic
methylvalerate (methylthio)butanoic (16:0/18:2) acid acid
1-palmitoyl-2- 1-palmitoyl-2- 2-hydroxyadipate 2-keto-3-deoxy-
2-hydroxyglutarate arachidonoyl-GPC palmitoleoyl-GPC gluconate
(16:0/20:4n6) (16:0/16:1)* 1-palmitoyl-2- 2-aminoadipate
2-hydroxyglutarate 2-oxoadipate 2-ketogluconate linoleoyl-GPC
(16:0/18:2) 1-palmitoyl-2- 2-hydroxy-3- 2- 3-hydroxyadipate
2-oxoadipate oleoyl-GPC methylvalerate methylcitrate/homocitrate
(16:0/18:1) 1-palmitoyl-2- 2-hydroxy-4- 2-oxoadipate 3-
2,3-dihydroxy-3- palmitoleoyl-GPC (methylthio)butanoic
hydroxyisobutyrate methylvalerate (16:0/16:1)* acid 1-palmitoyl-GPC
2- 2,3-dihydroxy-3- 3-hydroxyoctanoate 2'-O-methyluridine (16:0)
hydroxyphenylacetate methylvalerate 1-stearoyl-2- 2-oxoadipate 2,4-
3-methylglutaconate 3-hydroxyadipate arachidonoyl-GPC
dihydroxybutyrate (18:0/20:4) 1-stearoyl-2- 2,3-dihydroxy-3-
2'-deoxyuridine 4-hydroxybenzoate 3-hydroxylaurate docosahexaenoyl-
methylvalerate GPC (18:0/22:6) 1-stearoyl-2- 2,3- 3-(4-
4-hydroxyglutamate 3-indoleglyoxylic linoleoyl-GPC
dihydroxyisovalerate hydroxyphenyl)lactate acid (18:0/18:2)*
1-stearoyl-2-oleoyl- 2,4-diaminobutyrate 3-hydroxylaurate 4-
3-methylhistidine GPC (18:0/18:1) hydroxyphenylacetate
1-stearoyl-GPC 2'-deoxyadenosine 3-indoleglyoxylic 5-oxoproline
3-ureidopropionate (18:0) 5'-monophosphate acid 1,2-dilinoleoyl-GPC
2'-deoxycytidine 5'- 3-ureidopropionate allantoin
4-hydroxyglutamate (18:2/18:2) monophosphate 2-hydroxy-3-
2'-deoxyuridine 4- alpha- 5-oxoproline methylvalerate
acetamidobutanoate hydroxyisocaproate 2-hydroxy-4-
2'-O-methyluridine 4- alpha- 5,6-dihydrouridine
(methylthio)butanoic hydroxyphenylacetate hydroxyisovalerate acid
2-pyrrolidinone 3-(4- 4-methyl-2- alpha- adenine
hydroxyphenyl)lactate oxopentanoate ketoglutaramate*
2,3-dihydroxy-3- 3- 5-(2-Hydroxyethyl)- anthranilate cis-urocanate
methylvalerate deoxyoctulosonate 4-methylthiazole
3-hydroxyhexanoate 3-hydroxy-3- 5- beta-alanine
citraconate/glutaconate methylglutarate methylthioadenosine (MTA)
3-methylglutaconate 3-hydroxyadipate 5-methyluridine cis-urocanate
creatine (ribothymidine) 3-phenylpropionate 3-hydroxyhexanoate
5-oxoproline citraconate/glutaconate cyano-alanine (hydrocinnamate)
4-hydroxyglutamate 3-indoleglyoxylic 5,6-dihydrouridine creatine
cystathionine acid 5-(2-Hydroxyethyl)- 3-methylglutaconate
acetylagmatine creatinine ethyl alpha- 4-methylthiazole
glucopyranoside 5-dodecenoate 3-methylhistidine acetylphosphate
cyclo(pro- ethyl beta- (12:1n7) hydroxypro)* glucopyranoside
5,6-dihydrouridine 3-sulfo-L-alanine adenosine ethylmalonate
ethylmalonate 7-methylguanine 3-ureidopropionate adenosine 5'-
fumarate formiminoglutamate monophosphate (AMP) adenosine 3'- 4-
alpha- gluconate glycerol 3- monophosphate (3'- guanidinobutanoate
hydroxyisocaproate phosphate AMP) allantoic acid 4-hydroxybenzoate
anthranilate glutamine glycerophosphoethanolamine beta-alanine
4-hydroxycinnamate azetidine-2- glycerol 3- glycerophosphoglycerol
carboxylic acid phosphate choline phosphate 4- beta-alanine
glycerophosphoglycerol glycerophosphorylcholine
hydroxyphenylacetate (GPC) cis-urocanate 4-imidazoleacetate choline
phosphate glycerophosphorylcholine hippurate (GPC) cyano-alanine 5-
cis-urocanate hippurate hydroxymethylpyrimidine
(galactosylhydroxy)- L-lysine cystathionine 5-methyluridine
citraconate/glutaconate isobutyrylglycine imidazole (ribothymidine)
propionate cysteine s-sulfate 5-oxoproline creatine levulinate (4-
indole-3- oxovalerate) carboxylate dihydroxyacetone
5,6-dihydrouridine creatinine malate indolelactate phosphate (DHAP)
ethanolamine 7-methylguanine cyano-alanine N-acetylaspartate
indolin-2-one (NAA) ethyl alpha- aconitate [cis or cytosine
N-acetylglycine isopentenyl glucopyranoside trans] adenosine ethyl
beta- adenosine 2'- ergothioneine N-acetylisoleucine
methylsuccinate glucopyranoside monophosphate (2'- AMP)
ethylmalonate adenosine 5'- erythronate* N-acetylleucine mevalonate
monophosphate (AMP) formiminoglutamate allantoin ethyl beta- N-
mevalonolactone glucopyranoside acetylphenylalanine fumarate alpha-
ethylmalonate N-acetylserine N-acetyl-cadaverine hydroxyisocaproate
gamma- alpha- formiminoglutamate N-acetylthreonine
N-acetylhistamine glutamylglycine hydroxyisovalerate glycerol
alpha-ketobutyrate gamma- N-acetyltyrosine N-acetylhistidine
glutamylglycine glycerol 3- azetidine-2- glutamate N-acetylvaline
N- phosphate carboxylic acid acetylphenylalanine
glycerophosphoethanolamine benzoate glutarate (C5-DC) N-butyryl-
N-acetylserine phenylalanine glycerophosphoglycerol beta-alanine
glycerate N- N-acetylthreonine carbamoylaspartate
glycerophosphorylcholine beta- glycerol 3- N-formylmethionine
N-acetyltyrosine (GPC) hydroxyisovalerate phosphate homocysteine
butyrate/isobutyrate glycerophosphoethanolamine N- N-acetylvaline
(4:0) propionylmethionine hydroxymethylpyrimidine cadaverine
glycerophosphorylcholine N-succinyl- N-butyryl- (GPC) phenylalanine
phenylalanine indolelactate carnitine glycerophosphoserine*
nicotinate N-succinyl- ribonucleoside phenylalanine
isobutyrylglycine choline phosphate glycine nicotinic acid N1,N12-
mononucleotide diacetylspermine (NaMN) isopentenyl cis-urocanate
guanosine 5'- orotate N2,N2- adenosine monophosphate (5'-
dimethylguanosine GMP) mevalonolactone citraconate/glutaconate
hippurate prephenic acid N6- dimethylallyladenine
N-acetyl-cadaverine creatine histidine proline N6- methyladenosine
N-acetyl- creatinine hydroxymethylpyrimidine S-carboxymethyl-L-
nicotinamide isoputreanine cysteine riboside N-acetylhistamine
cyano-alanine indole-3-acetamide S-methylcysteine nicotinate
N-acetylhistidine cystathionine indolelactate thioproline
nicotinate ribonucleoside N-acetylkynurenine cytidine 5'- inosine
5'- trans-urocanate orotate (2) monophosphate (5'- monophosphate
CMP) (IMP) N-acetylmuramate cytosine isopentenyl trizma acetate
phosphoethanolamine adenosine N-succinyl- dihydroxyacetone
mevalonate .alpha.-Aminoadipic proline phenylalanine phosphate
(DHAP) acid N1-methylinosine ethyl beta- mevalonolactone (+/-)-2-
pseudouridine glucopyranoside Hydroxyglutaric acid N2,N2-
ethylmalonate N-acetyl-1- (+/-)-Methoprene ribitol
dimethylguanosine methylhistidine* N6- formiminoglutamate
N-acetyl-cadaverine (17R)-23-Amino- S-carboxymethyl-L-
methyladenosine 20-hydroxy-20- cysteine oxido-14-oxo-
15,19,21-trioxa- 20lambda~5~- phosphatricosan-17- yl (9Z)-9-
hexadecenoate N6-methyllysine fumarate N-acetyl-
(2E,4Z)-N-Isobutyl- sedoheptulose-7- isoputreanine 2,4- phosphate
octadecadienamide N6,N6- galactonate N-acetylarginine
(2R,3S,4R,5S,8R,10R, succinate dimethyladenosine 11R,13S,14R)-2-
Ethyl-3,4,10- trihydroxy- 3,5,8,10,12,14- hexamethyl-15-oxo-
7-propyl-11-{[3,4,6- trideoxy-3- (dimethylamino)- beta-D-xylo-
hexopyranosyl]oxy}- 1-oxa-7- azacyclopentadecan- 13- yl
2,6-dideoxy-3-C- methyl-3-O-methyl- alpha-L-ribo- hexopyranoside
O-acetylhomoserine gamma- N-acetylcitrulline (2R)-3-({[(2S)-2,3-
thymine glutamylglycine Dihydroxypropoxy] (hydroxy)phosphoryl}
oxy)-2-[(9Z)-9- hexadecenoyloxy] propyl (9Z)-9- hexadecenoate
p-hydroxybenzaldehyde glucose N-acetylglutamate (2R)-3-{[(2-
trans-urocanate Aminoethoxy)(hydroxy) phosphoryl]oxy}- 2-[(9Z)-9-
hexadecenoyloxy] propyl (9Z)-9- hexadecenoate phosphoethanolamine
glutarate (C5-DC) N-acetylhistamine (2S)-3-(beta-D- trehalose
Galactopyranosyloxy)- 2-[(7Z,10Z,13Z)- 7,10,13-
hexadecatrienoyloxy] propyl (9Z,12Z)- 9,12- octadecadienoate
proline glycerol 3- N-acetylhistidine (3Z)-3- uridine 3'- phosphate
[(Methylsulfanyl) monophosphate (3'- methylene]-2- UMP)
pyrrolidinethione pseudouridine glycerophosphoethanolamine
N-acetylleucine (8E)-2-Amino-8- .alpha.-Aminoadipic
octadecene-1,3,4- acid triol ribitol glycerophosphoglycerol N-
(9Z)-9- (+/-)-2- acetylphenylalanine Octadecenamide Hydroxyglutaric
acid S-1-pyrroline-5- glycerophosphorylcholine N-acetylputrescine
(E)-p-coumaric acid (+/-)-Coniine carboxylate (GPC)
S-methylcysteine glycerophosphoserine* N-acetylserine 1-(14-
(+/-)-Methoprene methylhexadecanoyl) pyrrolidine
thioproline guanine N-acetylthreonine 1- (.+-.)-Malic Acid
(Propyldisulfanyl)- 1- (propylsulfinyl) propane trans-urocanate
guanosine 2'- N-acetyltryptophan 1-[1-(1- (-)-trans-Methyl
monophosphate (2'- Benzothiophen-2- dihydrojasmonate GMP)*
yl)ethyl]urea trehalose guanosine 5'- N-acetyltyrosine
1-hexadecanoyl-sn- (17R)-23-Amino- monophosphate (5'- glycero-3-
20-hydroxy-20- GMP) phosphoethanolamine oxido-14-oxo-
15,19,21-trioxa- 20lambda~5~- phosphatricosan-17- yl (9Z)-9-
hexadecenoate trizma acetate guanosine-2',3'- N-acetylvaline 2-
(2E)-3,7-Dimethyl- cyclic Dodecylbenzenesulfonic 2,6-octadien-1-yl
monophosphate acid beta-D- glucopyranoside uridine histidine
N-butyryl- 2-hydroxy-6- (2R)-3-{[(2- phenylalanine
ketononadienedioic Aminoethoxy)(hydroxy) acid phosphoryl]oxy}-
2-[(9Z)-9- hexadecenoyloxy] propyl (9Z)-9- hexadecenoate
.alpha.-Aminoadipic histidylalanine N-succinyl- 2-Isocapyloyl-3R-
(2R)-3-{[(2- acid phenylalanine hydroxymethyl-
Aminoethoxy)(hydroxy) gamma- phosphoryl]oxy}- butyrolactone
2-hydroxypropyl (9Z)-9- hexadecenoate (+)-Riboflavin
hydroxymethylpyrimidine N1-methylinosine 2- (2R)-3-{[(2-
Mercaptobenzothiazole Aminoethoxy)(hydroxy) phosphoryl]oxy}-
2-hydroxypropyl laurate (+/-)-2- imidazole lactate N2,N2-
2-methylcitric acid (2R)-3-{[(2- Hydroxyglutaric dimethylguanosine
Aminoethoxy)(hydroxy) acid phosphoryl]oxy}- 2-hydroxypropyl
pentadecanoate (+/-)-Coniine indolelactate N6- 2,4- (2S)-5-
dimethylallyladenine dihydroxyheptadec- Carbamimidamido- 16-enyl
acetate 2-(2-oxo-1- azetidinyl)pentanoic acid (+/-)-Methoprene
inosine 5'- N6- 2,5-Dihydroxy-3,6- (2Z)-3,7-Dimethyl- monophosphate
methyladenosine di(1H-indol-3-yl)- 2,6-octadien-1-yl 3- (IMP)
1,4-benzoquinone oxobutanoate (.+-.)-Malic Acid isopentenyl N6,N6-
3-(3- (3S,8aS)-3-(4- adenosine dimethyladenosine sulfooxyphenyl)
Hydroxybenzyl) propanoic acid hexahydropyrrolo[1,2- a]pyrazine-1,4-
dione (-)-trans-Methyl kynurenate nicotinate 3-
(3S)-3-{(Z)-[(3S)-3- dihydrojasmonate ribonucleoside
hydroxydodecanoyl {(Z)-[(3R)-3- carnitine Amino-1-hydroxy- 4-
methylpentylidene] amino}-1- hydroxybutylidene] amino}-5-
methylhexanoic acid (1R,2S)-1-(7,8- malate O-acetylhomoserine
3-oxopalmitic acid (4-Hydroxy-1H- Dihydro-6- indol-3-
pteridinyl)-1,2- yl)acetonitrile propanediol (2E,4Z)-N-Isobutyl-
methylmalonate orotate 3,4- (E)-p-coumaric acid 2,4- (MMA)
Dihydroxyphenylglycol octadecadienamide (2R,3S,4R,5S,8R,
methylsuccinate phenyllactate (PLA) 4-hydroxy-4-(indol-
(Hydroxyethyl) 10R,11R,13S,14R)-2- 3-ylmethyl)glutamic methacrylate
Ethyl-3,4,10- acid trihydroxy- 3,5,8,10,12,14- hexamethyl-15-oxo-
7-propyl-11-{[3,4,6- trideoxy-3- (dimethylamino)- beta-D-xylo-
hexopyranosyl]oxy}- 1-oxa-7- azacyclopentadecan- 13- yl
2,6-dideoxy-3-C- methyl-3-O-methyl- alpha-L-ribo- hexopyranoside
(2R)-3-({[(2S)-2,3- mevalonate prephenic acid 4-Methylene-2- 1-
Dihydroxypropoxy] oxoglutarate (Propyldisulfanyl)-
(hydroxy)phosphoryl} 1- oxy)-2-[(9Z)-9- (propylsulfinyl)
hexadecenoyloxy] propane propyl (9Z)-9- hexadecenoate (2R)-3-{[(2-
mevalonolactone proline 4-Nitroaniline 1-[(9Z)-
Aminoethoxy)(hydroxy) hexadecenoyl]-sn- phosphoryl]oxy}- glycero-3-
2-[(9Z)-9- phosphocholine hexadecenoyloxy] propyl (9Z)-9-
hexadecenoate (2R)-3-{[(2- N-acetyl-1- pseudouridine 4- 1-[1-(1-
Aminoethoxy)(hydroxy) methylhistidine* Undecylbenzenesulfonic
Benzothiophen-2- phosphoryl]oxy}- acid yl)ethyl]urea
2-hydroxypropyl (9Z)-9- hexadecenoate (2R)-3-{[(2-
N-acetyl-cadaverine ribitol 6-Methoxy-3-(1,3- 1-Isothiocyanato-7-
Aminoethoxy)(hydroxy) thiazol-2-yl)-1H- (methylsulfanyl)heptane
phosphoryl]oxy}- indole 2-hydroxypropyl myristate (2R)-3-{[(2-
N-acetylarginine ribulose/xylulose 9-Pentofuranosyl- 1-Vinyl-2-
Aminoethoxy)(hydroxy) 3,9-dihydro-1H- pyrrolidone phosphoryl]oxy}-
purine-2,6-dione 2-hydroxypropyl pentadecanoate (2S)-3-(beta-D-
N-acetylaspartate S-1-pyrroline-5- Aceclidine 1,3,7-Octanetriol
Galactopyranosyloxy)- (NAA) carboxylate 2-[(7Z,10Z,13Z)- 7,10,13-
hexadecatrienoyloxy] propyl (9Z,12Z)- 9,12- octadecadienoate
(2S)-5- N-acetylglycine S-carboxymethyl-L- Adenylthiomethylpentose
1,4-Bis(2- Carbamimidamido- cysteine ethylhexyl) 2-(2-oxo-1-
sulfosuccinate azetidinyl)pentanoic acid (2Z)-3,7-Dimethyl-
N-acetylhistamine S-methylcysteine alpha-Ketoglutaric
16-Heptadecyne- 2,6-octadien-1-yl 3- acid 1,2,4-triol oxobutanoate
(3S,8aS)-3-(4- N-acetylhistidine sedoheptulose-7- Amide C18
2-(alpha-D- Hydroxybenzyl) phosphate mannosyl)-D-
hexahydropyrrolo[1,2- glyceric acid a]pyrazine-1,4- dione
(3S)-3-{(Z)-[(3S)-3- N-acetylleucine succinate angustine
2-(Hydroxy{2- {(Z)-[(3R)-3- [(9Z)-9- Amino-1-hydroxy-
octadecenoyloxy]-3- 4- (palmitoyloxy)propoxy} methylpentylidene]
phosphoryl)- amino}-1- N,N,N- hydroxybutylidene]
trimethylethanaminium amino}-5- methylhexanoic acid (3Z)-3- N-
thymidine apronalide 2-[(5Z)-5- [(Methylsulfanyl)
acetylphenylalanine Tetradecen-1- methylene]-2- yl]cyclobutanone
pyrrolidinethione (8E)-2-Amino-8- N-acetylputrescine
trans-urocanate asp-leu 2- octadecene-1,3,4- Dodecylbenzenesulfonic
triol acid (9Z)-9- N-acetylserine trizma acetate Benzaldehyde
2-Furoic acid Octadecenamide (E)-p-coumaric acid N-acetylthreonine
UDP-N- Biacetyl 2-hydroxy-6- acetylmuraminate ketononadienedioic
(UDP-MurNAc)* acid (Hydroxyethyl) N-acetyltyrosine uridine Biotin
1-Sulfoxide 2- methacrylate Mercaptobenzothiazole 1-(14-
N-acetylvaline .alpha.-Aminoadipic Brassicanal A 2-O-ETHYL
methylhexadecanoyl) acid ASCORBIC ACID pyrrolidine 1-(4-Amino-4-
N-butyryl- (.+/-.)-Tartaric acid Caprolactam 2,2-
carboxybutanoyl)-2- phenylalanine Bis(hydroxymethyl)
piperidinecarboxylic propionic acid acid 1-(beta-D-
N1-methylinosine (+/-)-2- Cinnamic acid 2,4- Ribofuranosyl)-1,2-
Hydroxyglutaric dihydroxyheptadec- dihydropyrimidine acid 16-enyl
acetate 1- N1,N12- (-)-trans-Methyl Cinnamyl alcohol
2,5-Dihydroxy-3,6- (Propyldisulfanyl)- diacetylspermine
dihydrojasmonate di(1H-indol-3-yl)- 1- 1,4-benzoquinone
(propylsulfinyl)propane 1-[1-(1- N2,N2- (17R)-23-Amino- cis-2-
2H-Pyran Benzothiophen-2- dimethylguanosine 20-hydroxy-20-
Carboxycyclohexyl- yl)ethyl]urea oxido-14-oxo- acetic acid
15,19,21-trioxa- 20lambda~5~- phosphatricosan-17- yl (9Z)-9-
hexadecenoate 1- N6- (2E,4Z)-N-Isobutyl- Coumarin 3-(3-
Hexadecanoylpyrrolidine methyladenosine 2,4- sulfooxyphenyl)
octadecadienamide propanoic acid 1-Isothiocyanato-7- N6,N6-
(2E)-3,7-Dimethyl- D-Xylonic acid 3-Methylsulfolene
(methylsulfanyl) dimethyllysine 2,6-octadien-1-yl heptane beta-D-
glucopyranoside 1-Pentofuranosyl- nicotinamide (2R)-3-{[(2-
DEHYDROASCORBIC 3-oxopalmitic acid 2,4(1H,3H)- riboside
Aminoethoxy)(hydroxy) ACID pyrimidinedione phosphoryl]oxy}-
2-[(9Z)-9- hexadecenoyloxy]propyl (9Z)-9- hexadecenoate
1,2-Dideoxy-3-C- nicotinate (2S)-5- deoxyviolacein 3,4-
methyl-1-(2,5,5,8a- Carbamimidamido- Diaminopyridine tetramethyl-
2-(2-oxo-1- 1,4,4a,5,6,7,8,8a- azetidinyl)pentanoic octahydro-1-
acid naphthalenyl) pentitol 1,2-Dihydroxy-5- orotate
(2Z)-3,7-Dimethyl- Desaminotyrosine 3,4- (methylsulfanyl)-1-
2,6-octadien-1-yl 3- Dihydroxyphenylglycol penten-3-one
oxobutanoate 1,4-Bis(2- phenylacetate (3Z)-3- Dexamisole
4-Aminobenzoic ethylhexyl) [(Methylsulfanyl) acid sulfosuccinate
methylene]-2- pyrrolidinethione 15S- phenyllactate (PLA)
(4-Hydroxy-1H- Dimethyl sulfoxide 4-Aminophenol
hydroxyeicosatrienoic indol-3- acid yl)acetonitrile 2-(Hydroxy{2-
prephenic acid (8E)-2-Amino-8- Diphenylamine 4-hydroxy-4-(indol-
[(9Z)-9- octadecene-1,3,4- 3-ylmethyl)glutamic octadecenoyloxy]-3-
triol acid (palmitoyloxy)prop oxy}phosphoryl)- N,N,N-
trimethylethanaminium 2-[(5Z)-5- proline (9Z)-9- DL-Glutamic acid
4-Morpholinylacetic Tetradecen-1- Octadecenamide acid
yl]cyclobutanone 2- propionylglycine (E)-p-coumaric acid
DL-Tryptophan 4-Nitroaniline Dodecylbenzenesulfonic acid
2-hydroxy-6- ribitol 1-(14- Estramustine 4- ketononadienedioic
methylhexadecanoyl) Phosphate Undecylbenzenesulfonic acid
pyrrolidine acid 2-Hydroxy-6- S-1-pyrroline-5- 1-(beta-D- Ethephon
5-Methoxy-3- methyl-12,13- carboxylate Ribofuranosyl)-1,2-
indoleaceate
dihydro-5H- dihydropyrimidine indolo [2,3- a]pyrrolo[3,4-
c]carbazole- 5,7(6H)-dione 2-Indolylformic S- 1- Ethyl lactate 5-
acid adenosylhomocysteine (Propyldisulfanyl)- Methoxybenzimidazole
(SAH) 1- (propylsulfinyl) propane 2-Isocapryloyl-3R-
S-carboxymethyl-L- 1-[1-(1- Fexaramine 6-(1-Hydroxyethyl)-
hydroxymethyl- cysteine Benzothiophen-2- 3-(hydroxymethyl)- gamma-
yl)ethyl]urea 2,7- butyrolactone dioxabicyclo[4.1.0]
hept-3-en-5-one 2- S-methylcysteine 1- Furaneol 6-Methoxy-3-(1,3-
Mercaptobenzothiazole Hexadecanoylpyrrolidine thiazol-2-yl)-1H-
indole 2-methylcitric acid succinate 1-Pentofuranosyl- hexobarbital
Aceclidine 2,4(1H,3H)- pyrimidinedione 2- tartarate 1-Piperideine
Hydroxy(oxo) Adenosine Methylthiazolidine phosphoniumolate
monophosphate 2-Pyrrolidone thioproline 1-Vinyl-2-
Hydroxycitronellal Ampalex pyrrolidone diethyl acetal 2- thymidine
1,3,7-Octanetriol hydroxypyridone angustine Quinolinecarboxylic
acid 2,4- trehalose 1,4-Anhydro-6-O- Hypoxanthin anthramycin
dihydroxyheptadec- dodecanoyl-2,3-bis- 16-enyl acetate
O-(2-hydroxyethyl)- D-glucitol 2,4- trizma acetate 10,16-
Imagabalin APAZIQUONE Dimethyloxazole Dihydroxyhexadecanoic acid
2,5-Dihydroxy-3,6- uracil 11-Nitro-1- Indigo dye Aprobarbital
di(1H-indol-3-yl)- undecene 1,4-benzoquinone 2H-Pyran urate
1H-Imidazol-2-ol Indole-3-acetic acid Arabinosylhypoxanthine 3-(3-
uridine 3'- 2-(Hydroxy{2- Inspra asp-leu sulfooxyphenyl)
monophosphate (3'- [(9Z)-9- propanoic acid UMP) octadecenoyloxy]-3-
(palmitoyloxy) propoxy}phospholyl)- N,N,N- trimethylethanaminium 3-
(+/-)-2- 2-[(5Z)-5- isopropylmalic acid Aspartyl-L-proline
hydroxydodecanoyl Hydroxyglutaric Tetradecen-l- carnitine acid
yl]cyclobutanone 3- (+/-)-Coniine 2-hydroxy-6- Kynurenic acid
bendiocarb Methylcyclohexanethiol ketononadienedioic acid
3-Methylsulfolene (.+-.)-Malic Acid 2-Indolylformic L-(+)-Valine
Benzaldehyde acid 3-oxopalmitic acid (-)-trans-Methyl 2-
L-alpha-Aspartyl-L- Benzisothiazolone dihydrojasmonate
Mercaptobenzothiazole phenylalanine 3,4- (17R)-23-Amino-
2-methylcitric acid L-Proline Biacetyl Diaminopyridine
20-hydroxy-20- oxido-14-oxo- 15,19,21-trioxa- 20lambda~5~-
phosphatricosan-17- yl (9Z)-9- hexadecenoate 3,4-
(2E)-3,7-Dimethyl- 2-Pyrrolidone L-Pyroglutamic acid Biotin
1-Sulfoxide Dihydroxyphenylglycol 2,6-octadien-1-yl beta-D-
glucopyranoside 4-Aminobenzoic (2R)-3-({[(2S)-2,3- 2,2- Leu-pro
Bis(2-ethylhexyl) acid Dihydroxypropoxy] Bis(hydroxymethyl)
phthalate (hydroxy)phosphoryl} propionic acid oxy)-2-[(9Z)-9-
hexadecenoyloxylpropyl (9Z)-9- hexadecenoate 4-Aminophenol
(2R)-3-{[(2- 2,4- Leu-Val Brassicanal A Aminoethoxy)(hydroxy)
dihydroxyheptadec- phosphoryl]oxy}- 16-enyl acetate 2-hydroxypropyl
laurate 4-hydroxy-4-(indol- (2S)-5- 2,5-Dihydroxy-3,6- leucoline
Brivaracetam 3-ylmethyl)glutamic Carbamimidamido-
di(1H-indol-3-yl)- acid 2-(2-oxo-1- 1,4-benzoquinone
azetidinyl)pentanoic acid 4-Methylene-2- (2Z)-3,7-Dimethyl- 3-(3-
lysophosphatidylcholine Butabarbital oxoglutarate 2,6-octadien-1-yl
3- sulfooxyphenyl)propanoic 14:1(9Z)/0:0 oxobutanoate acid
4-Nitroaniline (4-Hydroxy-1H- 3-Methylsulfolene meprobamate Butyl
isothiocyanate indol-3- yl)acetonitrile 4- (8E)-2-Amino-8- 3,4-
Methyl Jasmonate Caprolactam Undecylbenzenesulfonic
octadecene-1,3,4- Dihydroxyphenylglycol acid triol
4-Vinylcyclohexene (E)-p-coumaric acid 4- METHYLDOPA,
Capryloylglycine (Stearoylamino) D- butanoic acid 5-Hydroxydecanoic
1-(14- 4-Aminobenzoic methylxanthine Capsi-amide acid
methylhexadecanoyl) acid pyrrolidine 5-Methoxy-L- 1-[(9Z)-
4-hydroxy-4-(indol- Myristamide cis-5- tryptophan hexadecenoyl]-sn-
3-ylmethyl)glutamic Tetradecenoylcarnitine glycero-3- acid
phosphocholine 5'-S-Methyl-5'- 1-[1-(1- 4-Morpholinylacetic
Myrtillin Citric acid thioinosine Benzothiophen-2- acid
yl)ethyl]urea 6-(1-Hydroxyethyl)- 1- 4-Nitroaniline
N-Acetyl-L-leucine Cuauhtemone 3-(hydroxymethyl)-
Hexadecanoylpyrrolidine 2,7- dioxabicyclo[4.1.0] hept-3-en-5-one 6-
1-Isothiocyanato-7- 5-Methoxy-3- n-Hexanamide Cyclazocine
hydroxypseudooxynicotine (methylsulfanyl) indoleaceate heptane
6-Methoxy-3-(1,3- 1-Piperideine 5- N-Hydroxy-8- DEHYDROASCORBIC
thiazol-2-y1)-1H- Methoxybenzimidazole (methylsulfanyl) ACID indole
octanethioamide 7- 1-Vinyl-2- 5,6- N,N- Desaminotyrosine
methylsulfinylheptyl pyrrolidone Dihydrothymidine Dimethyladenosine
isothiocyanate 8- 1,3,7-Octanetriol 6-Methoxy-3-(1,3- N(1)-
Dexamisole [(Aminomethyl) thiazol-2-yl)-1H- acetylspermidine
sulfanyl]-6- indole sulfanyloctanoic acid 8- 1,4-Anhydro-6-O- 7-
N(3)-(4- Dibutyl phthalate Hydroxyhexadecanedioic
dodecanoyl-2,3-bis- methylsulfinylheptyl Methoxyfumaroyl)- acid
O-(2-hydroxyethyl)- isothiocyanate 2,3- D-glucitol diaminopropionic
acid 9-Pentofuranosyl- 1,4-Bis(2- Adenylthiomethylpentose
Naphthalen-2-amine Dimethyl sulfoxide 3,9-dihydro-1H- ethylhexyl)
purine-2,6-dione sulfosuccinate 9H-Fluoren-9-one 10,16- ALA-PRO
Niacin Diphenylamine Dihydroxyhexadecanoic acid Aceclidine
16-Heptadecyne- Amide C18 noradrenaline DL-Glutamic acid
1,2,4-triol Aceglutamide 1H-Imidazol-2-ol Aminohippuric acid
Octylamine DL-Histidine Acetanilide 2- amonafide Oleoylethanolamide
DL-Lactic Acid Dodecylbenzenesulfonic acid ACETYL 2-Furoic acid
Ampalex Oxagrelate DL-Mevalonic acid ARGININE Adenine
2-Indolylformic angustine p-Cresol DL-Phenylalanine acid ALA-PRO 2-
Arabinosylhypoxanthine PALGLY DL-Tryptophan Mercaptobenzothiazole
Alanyltryptophan 2-methylcitric acid Biotin 1-Sulfoxide Palmitamide
DL-TYROSINE Aminohippuric acid 2-Pyrrolidone Brassicanal A PEG-4
Elaeolcanine C amonafide 2,4- Brivaracetam Pyrantel Epirizole
Diacetylphloroglucinol Ampalex 3-(3- Butabarbital Sorbitan,
Estramustine sulfooxyphenyl) monododecanoate Phosphate propanoic
acid angustine 3-deoxy-D-manno- Caprolactam Staurosporonine
Ethopabate 2-octulosonic acid APAZIQUONE 4-Aminobenzoic
Capryloylglycine THREO- Ethyl lactate acid SPHINGOSINE, (-)-
Artemether 4-hydroxy-4-(indol- Capsi-amide Tridemorph Fadrozole
3-ylmethyl)glutamic acid asn-pro 4-Morpholinylacetic Cinnamic acid
Trifluoroacetic acid FARNESYL acid ACETONE asp-gln 4-Nitroaniline
Citric acid val-arg Fasoracetam asp-leu 5-Methoxy-3- Coprine
Vanillin Fexaramine indoleaceate Aspartyl-L-proline 5- Cuauhtemone
Vigabatrin Formiminoglutamic Methoxybenzimidazole Acid Aspulvinone
E 6-Methoxy-3-(1,3- cuscohygrine Vorinostat Furaneol
thiazol-2-yl)-1H- indole Benserazide 7- Cyclazocine zinniol
Gabapentin methylsulfinylheptyl isothiocyanate Biacetyl 8-
Cyprodenate zopiclone Gemfibrozil [(Aminomethyl) sulfanyl]-6-
sulfanyloctanoic acid Biotin 1-Sulfoxide 9H-Fluoren-9-one
Deferasirox Gly-Leu Boc-Asn-Oh Adenosine deoxyviolacein
glycyclamide monophosphate Brassicanal A ALA-PRO Desaminotyrosine
Guanine butalbital Alanyltryptophan Desthiobiotin Guanosine
monophosphate Butyl isothiocyanate alpha-Ketoglutaric Dexamisole
Hexyl 2-furoate acid Caprolactam Aminolevulinic acid Diethylamine
Hippuric acid Capryloylglycine Ampalex Dimethyl sulfoxide
Homovanillic acid Capsi-amide angustine dimethylformamide
Hydroquinone CILAZAPRILAT Artemether Diphenylamine hydroxypyridone
Cinnamic acid Benzisothiazolone DL-Arginine Hypoxanthin Cinnamyl
alcohol Biacetyl DL-Glutamic acid Indoleacrylic acid cis-2- Biotin
1-Sulfoxide DL-Histidine Isoprene Carboxycyclohexyl- acetic acid
Coumarin Botrydial DL-Lysine isopropylmalic acid Coumarone
Brivaracetam DL-Mevalonic acid Kynurenic acid Cuauhtemone
Butoctamide Eglumetad L-(-)-methionine semisuccinate Cyclazocine
Butyl isothiocyanate Embelin L-(+)-Leucine Cyprodenate Caprolactam
Epirizole L-(+)-Valine D-Gluconic acid Capryloylglycine EPTAZOCINE
L-alpha- Glycerylphospholyl choline D-PANTOTHENIC Capsi-amide
Ethopabate L-Proline ACID D-Xylonic acid Cinnamic acid Ethyl
lactate L-Pyroglutamic acid Daidzein Cinnamyl alcohol FARNESYL
Leu-Leu ACETONE danegaptide Citric acid Fexaramine Leu-Val
decanoylcarnitine Cuauhtemone Formiminoglutamic Linoleamide Acid
Deferasirox Cyclazocine Furaneol Lysylvaline DEHYDROASCORBIC
danegaptide gamma- meprobamate ACID Aminobutyric acid
deoxyviolacein DEHYDROASCORBIC Gemfibrozil Methionylleucine ACID
Desaminotyrosine deoxyviolacein Gly-Arg Methohexital Dexamisole
Desaminotyrosine Gly-l-pro Methyl Jasmonate Diethylamine Dexamisole
Gly-Leu methylxanthine Diftalone Dimethyl sulfoxide Guanine
Myrtillin Dihydroxyindole Dimethylaminoprop Guanosine
N-Acetyl-L-aspartic ionylphenothiazine monophosphate acid Dimethyl
sulfoxide Diphenylamine hexobarbital N-Acetyl-L- glutamic acid
dimethylformamide DL-Citrulline Hexyl 2-furoate N-Acetyl-L-
histidine Diphenylamine DL-Glutamic acid Hippuric acid
N-Acetyl-L-leucine DL-Arginine DL-Histidine Homovanillic acid
N-Acetylcadaverine DL-Cerulenin DL-Mevalonic acid hydroxypyridone
N- Acetylcystathionine DL-Glutamic acid Embelin Hypoxanthin
n-Hexanamide DL-Glyceric acid Epirizole Indigo dye N-Hydroxy-8-
(methylsulfanyl)oct anethioamide DL-Lactic Acid EPTAZOCINE Indole
N- methylethanolamine
phosphate DL-Phenylalanine Ethyl lactate Indoleacrylic acid
N-OLEOYL-4- AMINOBUTYRIC ACID DL-Tryptophan Fexaramine Inspra
N(3)-(4- Methoxyfumaroyl)- 2,3- diaminopropionic acid DL-TYROSINE
Formiminoglutamic ionene N1,N12- Acid Diacetylspermine Elaeokanine
C Furaneol L-(+)-Valine Naphthalen-2-amine Embelin Gemfibrozil
L-alpha- Niacin Glycerylphospholyl choline ENADENINE Gly-Arg
L-gamma- noradrenaline Glutamyl-L-leucine Epirizole glycyclamide
L-Proline Oleoylethanolamide Eslicarbazepine Guanine L-Pyroglutamic
acid Oryzalin metabolite Estramustine Guanosine Leu-Leu Oxagrelate
Phosphate monophosphate Ethyl lactate hexobarbital Leu-pro Oxalic
acid Fadrozole Hexyl 2-furoate Leu-Val Oxibendazole FARNESYL
Hippuric acid linatine p-Cresol ACETONE Fasoracetam Homovanillic
acid Linoleamide PALGLY Fexaramine hydroxypyridone Lysylvaline
Palmitamide Formiminoglutamic Hypoxanthin Marimastat Paracetamol
Acid Furaneol Imagabalin meprobamate Paraldehyde g- Indigo dye
Methyl Jasmonate pentobarbital Guanidinobutyrate Gabapentin Indole
methyprylon Phenylisocyanate Gemfibrozil Indole-3-acetic acid
Misoprostol Phosphoric acid Gly-Arg Indoleacrylic acid Myristyl
sulfate Phthalic acid Gly-l-pro Inspra N-Acetyl-L- Pimilprost
histidine Gly-Leu Isoprene N-Acetyl-L-leucine Pivagabine
glycyclamide L-(+)-Leucine N-Acetylcadaverine Pyrantel Guanine L-
N- Pyrrolidine ACETYLTRYPTOPHAN Acetylcystathionine Guanosine
L-Alanyl-L- n-Hexanamide S-methyl D- monophosphate glutamine
thioglycerate hexobarbital L-alpha- N-Hydroxy-8- Sebacic acid
Glycerylphosphoryl (methylsulfanyl)oct choline anethioamide Hexyl
2-furoate L-Proline N- shanzhiside methylethanolamine phosphate
Hippuric acid L-Pyroglutamic acid N- Staurosporonine
Methylpyrrolidone Hydroquinone laurilsulfate N-OLEOYL-4- Succinic
acid AMINOBUTYRIC ACID hydroxypyridone Leu-Leu N- Suprofen
Tridecanoylglycine Hypoxanthin Leu-pro N- trans-Zeatin
Undecanoylglycine imazamethabenz Medrogestone N,N-Bis(2- Tridemorph
hydroxyethyl) dodecanamide Indigo dye Methohexital N(1)-
Trifluoroacetic acid acetylspermidine Indole Methyl Jasmonate
N(3)-(4- trimethadione Methoxyfumaroyl)- 2,3- diaminopropionic acid
Indole-3-acetic acid methylxanthine N1,N12- Tryptoline
Diacetylspermine Indole-3-pyrubate Misoprostol Naphthalen-2-amine
Uracil Indoleacrylic acid Myristyl sulfate Niacin Uric Acid Inspra
Myrtillin noradrenaline Uridine monophosphate Isoprene N-Acetyl-L-
Octylamine val-arg histidine isopropylmalic acid N-Acetyl-L-leucine
Oleoylethanolamide Vanillin Itaconic acid N-Acetylcadaverine
Oryzalin metabolite Vernolate Kynurenic acid N- Oxagrelate Veronal
Acetylcystathionine L-(-)-methionine n-Hexanamide Oxibendazole
Vorinostat L-(+)-Leucine N-Hydroxy-8- p-Cresol Xanthine
(methylsulfanyl)oct anethioamide L-(+)-Valine N-OLEOYL-4- p-cymene
Zonisamide AMINOBUTYRIC ACID L- N- PALGLY zopiclone
ACETYLTRYPTOPHAN Tridecanoylglycine L-Alanyl-L- N(1)- Palmitamide
glutamine acetylspermidine L-alpha-Aspartyl-L- N(3)-(4- Paracetamol
phenylalanine Methoxyfumaroyl)- 2,3- diaminopropionic acid L-alpha-
N1,N12- Paraldehyde Glycerylphosphoryl Diacetylspermine choline
L-gamma- Niacin PEG-4 Glutamyl-L-leucine L-Proline noradrenaline
Phenylisocyanate L-Pyroglutamic acid Octylamine Pivagabine
L-Theanine Oryzalin metabolite porphobilinogen laurilsulfate
Oxagrelate Prinomide leu-gln Oxalic acid pro-gln Leu-Gly-Pro
Oxibendazole S-methyl D- thioglycerate Leu-Leu p-Cresol S-Propyl
hexanethioate Leu-pro PALGLY Salicylic acid Leu-Val Paracetamol
Sebacic acid leucoline Paraldehyde Sorbitan, monododecanoate
Levulinic acid pentobarbital Staurosporonine Linoleamide phe-gln
Succinic acid loganate Phenylisocyanate trans-Zeatin Marimastat
porphobilinogen Tributyl citrate acetate mebutamate Pyrantel
Tridemorph mephenesin Pyridoxal Trifluoroacetic acid meprobamate
Pyrrolidine Tryptoline Methionylleucine S-methyl D- Ulimorelin
thioglycerate Methohexital Salicylic acid Uracil Methyl Jasmonate
Sebacic acid Uric Acid methylxanthine Staurosporonine Uridine
monophosphate Misoprostol Succinic acid Vanillin Myristyl sulfate
Suprofen Veronal N-Acetyl-L- trans-Zeatin Vigabatrin glutamic acid
N-Acetyl-L-leucine Tributyl citrate violacein acetate
N-Acetylcadaverine Tridemorph Xanthine N-Acetylvaline Tryptoline
zinniol N-ethylmaleimide Tyrosyltyrosine N-Hydroxy-8- Uric Acid
(methylsulfanyl)oct anethioamide N- Uridine methylethanolamine
monophosphate phosphate N- Vanillin Methylpyrrolidone N-OLEOYL-4-
Vanillyl alcohol AMINOBUTYRIC ACID N- zinniol Tridecanoylglycine N-
Zonisamide Undecanoylglycine N,N-Bis(2- zopiclone hydroxyethyl)
dodecanamide N(1)- acetylspermidine N(3)-(4- Methoxyfumaroyl)- 2,3-
diaminopropionic acid N~6~-[(2R)-3,4- Dihydro-2H-pyrrol-
2-ylcarbonyl]-L- lysine N1,N12- Diacetylspermine naphazoline
Naphthalen-2-amine Niacin Nicotinamide Nitrendipine noradrenaline
norhaman Octylamine Oleoylethanolamide Olyzalin metabolite
Oxagrelate Oxibendazole Ozagrel p-Cresol PALGLY pentobarbital
Perlapine phenacetin Phenelzine Phenylisocyanate pro-gln Proflavine
Propylparaben Pyridine Pyrrolidine S-methyl D- thioglycerate
Salicylic acid Sebacic acid shanzhiside Staurosporonine Succinic
acid Succinylacetone Sulfabenzamide sulfacetamide
Tetraacetylethylene diamine THREO- SPHINGOSINE, (-)-
Threonylglutamine Thymine trans-Azobenzene trans-Zeatin Tributyl
phosphate Tridemorph Tiyptoline Tyrosyltyrosine Uracil Uric Acid
Uridine monophosphate val-arg Vanillin Vigabatrin violacein Vitamin
C Vorinostat Xanthine zinniol zopiclone
7.8. Example 8. Clinical Trial
[0258] Human-derived Janthinobacterium lividum, DB02473, useful to
treat and prevent skin diseases. DB02473 t showed efficacy in
treating tinea pedis. in a Phase IIa, single-dose, dose escalating
clinical trial (Reference
#[https://www.dermbiont.com/in-the-news/dermbiont-announces-positive-data-
-from-phase-2a-clinical-trial-and-start-of-phase-2b-clinical-trial-for-ath-
letes-foot-with-a-topical-live-biotherapeutic). These clinical
trials demonstrated the observations that human-derived
Janthinobacterium lividum when grown in proximity with, inhibits
the growth of T. rubrum and S. aureus.
8. INCORPORATION BY REFERENCE
[0259] All publications, patents, patent applications and other
documents cited in this application are hereby incorporated by
reference in their entireties for all purposes to the same extent
as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by
reference for all purposes.
9. EQUIVALENTS
[0260] While various specific embodiments have been illustrated and
described, the above specification is not restrictive. It will be
appreciated that various changes can be made without departing from
the spirit and scope of the present disclosure(s). Many variations
will become apparent to those skilled in the art upon review of
this specification.
Sequence CWU 1
1
1111433DNAJanthinobacterium lividum 1cggttaagct acctacttct
ggtaaaaccc gctcccatgg tgtgacgggc ggtgtgtaca 60agacccggga acgtattcac
cgcgacatgc tgatccgcga ttactagcga ttccaacttc 120atgcagtcga
gttgcagact acaatccgga ctacgataca ctttctgcga ttagctcccc
180ctcgcgggtt ggcggcgctc tgtatgtacc attgtatgac gtgtgaagcc
ctacccataa 240gggccatgag gacttgacgt catccccacc ttcctccggt
ttgtcaccgg cagtctcatt 300agagtgccct ttcgtagcaa ctaatgacaa
gggttgcgct cgttgcggga cttaacccaa 360catctcacga cacgagctga
cgacagccat gcagcacctg tgtactggtt ctctttcgag 420cactccccaa
tctctcggtg gattccagcc atgtcaaggg taggtaaggt ttttcgcgtt
480gcatcgaatt aatccacatc atccaccgct tgtgcgggtc cccgtcaatt
cctttgagtt 540ttaatcttgc gaccgtactc cccaggcggt ctacttcacg
cgttagctgc gttaccaagt 600caattaagac ccgacaacta gtagacatcg
tttagggcgt ggactaccag ggtatctaat 660cctgtttgct ccccacgctt
tcgtgcatga gcgtcaatct tgacccaggg ggctgccttc 720gccatcggtg
ttcctccaca tatctacgca tttcactgct acacgtggaa ttctaccccc
780ctctgccaga ttctagcctt gcagtctcca atgcaattcc caggttgagc
ccggggattt 840cacatcagac ttacaaaacc gcctgcgcac gctttacgcc
cagtaattcc gattaacgct 900tgcaccctac gtattaccgc ggctgctggc
acgtagttag ccggtgctta ttcttcaggt 960accgtcatta gcaagagata
ttagctctca ccgtttcttc cctgacaaaa gagctttaca 1020acccgaaggc
cttcttcact cacgcggcat tgctggatca ggctttcgcc cattgtccaa
1080aattccccac tgctgcctcc cgtaggagtc tggaccgtgt ctcagttcca
gtgtggctgg 1140tcgtcctctc agaccagcta ctgatcgatg ccttggtagg
cttttaccct accaactagc 1200taatcagata tcggccgctc cacgagcatg
aggtcttgcg atcccccact ttcatcctta 1260gatcgtatgc ggtattagcg
taactttcgc tacgttatcc cccactctag ggtacgttcc 1320gatatattac
tcacccgttc gccactcgcc accagagcaa gctccgtgct gccgttcgac
1380ttgcatgtgt aaggcatgcc gccagcgttc aatctgagcc aggatcaaac tct
143321173DNAJanthinobacterium lividum 2aaggaggtga tccagccgca
ccttccgata cggctacctt gttacgactt caccccagtc 60acgaatccta ccgtggtaag
cgccctcctt gcggttaagc tacctacttc tggtaaaacc 120cgctcccatg
gtgtgacggg cggtgtgtac aagacccggg aacgtattca atctctcgag
180gattccagcc atgtcaaggg taggtaaggt ttttcgcgtt gcatcgaatt
aatccacatc 240atccaccgct tgtgcgggtc cccgtcaatt cctttgagtt
ttaatcttgc gaccgtactc 300cccaggcggt ctacttcacg cgttagctgc
gttaccaagt caattaagac ccgacaacta 360gtagacatcg tttagggcgt
ggactaccag ggtatctaat cctgtttgct ccccacgctt 420tcgtgcatga
gcgtcaatct tgacccaggg ggctgccttc gccatcggtg ttcctccaca
480tatctacgca tttcactgct acacgtggaa ttctaccccc ctctgccaga
ttctagcctt 540gcagtctcca atgcaattcc caggttgagc ccggggattt
cacatcagac ttacaaaacc 600gcctgcgcac gctttacgcc cagtaattcc
gattaacgct tgcaccctac gtattaccgc 660ggctgctggc acgtagttag
ccggtgctta ttcttcaggt accgtcatta gcaagagata 720ttagctctca
ccgtttcttc cctgacaaaa gagctttaca acccgaaggc cttcttcact
780cacgcggcat tgctggatca ggctttcgcc cattgtccaa aattccccac
tgctgcctcc 840cgtaggagtc tggaccgtgt ctcagttcca gtgtggctgg
tcgtcctctc agaccagcta 900ctgatcgatg ccttggtagg cttttaccct
accaactagc taatcagata tcggccgctc 960cacgagcatg aggtcttgcg
atcccccact ttcatcctta gatcgtatgc ggtattagcg 1020taactttcgc
tacgttatcc cccactctag ggtacgttcc gatatattac tcacccgttc
1080gccactcgcc accagagcaa gctccgtgct gccgttcgac ttgcatgtgt
aaggcatgcc 1140gccagcgttc aatctgagcc aggatcaaac tct
117331173DNAJanthinobacterium lividum 3aaggaggtga tccagccgca
ccttccgata cggctacctt gttacgactt caccccagtc 60acgaatccta ccgtggtaag
cgccctcctt gcggttaagc tacctacttc tggtaaaacc 120cgctcccatg
gtgtgacggg cggtgtgtac aagacccggg aacgtattca atctctcgag
180gattccagcc atgtcaaggg taggtaaggt ttttcgcgtt gcatcgaatt
aatccacatc 240atccaccgct tgtgcgggtc cccgtcaatt cctttgagtt
ttaatcttgc gaccgtactc 300cccaggcggt ctacttcacg cgttagctgc
gttaccaagt caattaagac ccgacaacta 360gtagacatcg tttagggcgt
ggactaccag ggtatctaat cctgtttgct ccccacgctt 420tcgtgcatga
gcgtcaatct tgacccaggg ggctgccttc gccatcggtg ttcctccaca
480tatctacgca tttcactgct acacgtggaa ttctaccccc ctctgccaga
ttctagcctt 540gcagtctcca atgcaattcc caggttgagc ccggggattt
cacatcagac ttacaaaacc 600gcctgcgcac gctttacgcc cagtaattcc
gattaacgct tgcaccctac gtattaccgc 660ggctgctggc acgtagttag
ccggtgctta ttcttcaggt accgtcatta gcaagagata 720ttagctctca
ccgtttcttc cctgacaaaa gagctttaca acccgaaggc cttcttcact
780cacgcggcat tgctggatca ggctttcgcc cattgtccaa aattccccac
tgctgcctcc 840cgtaggagtc tggaccgtgt ctcagttcca gtgtggctgg
tcgtcctctc agaccagcta 900ctgatcgatg ccttggtagg cttttaccct
accaactagc taatcagata tcggccgctc 960cacgagcatg aggtcttgcg
atcccccact ttcatcctta gatcgtatgc ggtattagcg 1020taactttcgc
tacgttatcc cccactctag ggtacgttcc gatatattac tcacccgttc
1080gccactcgcc accagagcaa gctccgtgct gccgttcgac ttgcatgtgt
aaggcatgcc 1140gccagcgttc aatctgagcc aggatcaaac tct
117341523DNAJanthinobacterium lividum 4aaggaggtga tccagccgca
ccttccgata cggctacctt gttacgactt caccccagtc 60acgaatccta ccgtggtaag
cgccctcctt gcggttaagc tacctacttc tggtaaaacc 120cgctcccatg
gtgtgacggg cggtgtgtac aagacccggg aacgtattca ccgcgacatg
180ctgatccgcg attactagcg attccaactt catgcagtcg agttgcagac
tacaatccgg 240actacgatac actttctgcg attagctccc cctcgcgggt
tggcggcgct ctgtatgtac 300cattgtatga cgtgtgaagc cctacccata
agggccatga ggacttgacg tcatccccac 360cttcctccgg tttgtcaccg
gcagtctcat tagagtgccc tttcgtagca actaatgaca 420agggttgcgc
tcgttgcggg acttaaccca acatctcacg acacgagctg acgacagcca
480tgcagcacct gtgtactggt tctctttcga gcactcctca atctctcgag
gattccagcc 540atgtcaaggg taggtaaggt ttttcgcgtt gcatcgaatt
aatccacatc atccaccgct 600tgtgcgggtc cccgtcaatt cctttgagtt
ttaatcttgc gaccgtactc cccaggcggt 660ctacttcacg cgttagctgc
gttaccaagt caattaagac ccgacaacta gtagacatcg 720tttagggcgt
ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgtgcatga
780gcgtcaatct tgacccaggg ggctgccttc gccatcggtg ttcctccaca
tatctacgca 840tttcactgct acacgtggaa ttctaccccc ctctgccaga
ttctagcctt gcagtctcca 900atgcaattcc caggttgagc ccggggattt
cacatcagac ttacaaaacc gcctgcgcac 960gctttacgcc cagtaattcc
gattaacgct tgcaccctac gtattaccgc ggctgctggc 1020acgtagttag
ccggtgctta ttcttcaggt accgtcatta gcaagagata ttagctctca
1080ccgtttcttc cctgacaaaa gagctttaca acccgaaggc cttcttcact
cacgcggcat 1140tgctggatca ggctttcgcc cattgtccaa aattccccac
tgctgcctcc cgtaggagtc 1200tggaccgtgt ctcagttcca gtgtggctgg
tcgtcctctc agaccagcta ctgatcgatg 1260ccttggtagg cttttaccct
accaactagc taatcagata tcggccgctc cacgagcatg 1320aggtcttgcg
atcccccact ttcatcctta gatcgtatgc ggtattagcg taactttcgc
1380tacgttatcc cccactccag ggtacgttcc gatatattac tcacccgttc
gccactcgcc 1440accagagcaa gctccgtgct gccgttcgac ttgcatgtgt
aaggcatgcc gccagcgttc 1500aatctgagcc aggatcaaac tct
152351523DNAJanthinobacterium lividum 5aaggaggtga tccagccgca
ccttccgata cggctacctt gttacgactt caccccagtc 60acgaatccta ccgtggtaag
cgccctcctt acggttaagc tacctacttc tggtaaaacc 120cgctcccatg
gtgtgacggg cggtgtgtac aagacccggg aacgtattca ccgcgacatg
180ctgatccgcg attactagcg attccaactt catgcagtcg agttgcagac
tacaatccgg 240actacgatac actttctgcg attagctccc cctcgcgggt
tggcggcgct ctgtatgtac 300cattgtatga cgtgtgaagc cctacccata
agggccatga ggacttgacg tcatccccac 360cttcctccgg tttgtcaccg
gcagtctcat tagagtgccc tttcgtagca actaatgaca 420agggttgcgc
tcgttgcggg acttaaccca acatctcacg acacgagctg acgacagcca
480tgcagcacct gtgtactggt tctctttcga gcactcccca atctctcgag
gattccagcc 540atgtcaaggg taggtaaggt ttttcgcgtt gcatcgaatt
aatccacatc atccaccgct 600tgtgcgggtc cccgtcaatt cctttgagtt
ttaatcttgc gaccgtactc cccaggcggt 660ctacttcacg cgttagctgc
gttaccaagt caattaagac ccgacaacta gtagacatcg 720tttagggcgt
ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgtgcatga
780gcgtcaatct tgacccaggg ggctgccttc gccatcggtg ttcctccaca
tatctacgca 840tttcactgct acacgtggaa ttctaccccc ctctgccaga
ttctagcctt gcagtctcca 900atgcaattcc caggttgagc ccggggattt
cacatcagac ttacaaaacc gcctgcgcac 960gctttacgcc cagtaattcc
gattaacgct tgcaccctac gtattaccgc ggctgctggc 1020acgtagttag
ccggtgctta ttcttcaggt accgtcatta gcaagagata ttagctctca
1080ccgtttcttc cctgacaaaa gagctttaca acccgaaggc cttcttcact
cacgcggcat 1140tgctggatca ggctttcgcc cattgtccaa aattccccac
tgctgcctcc cgtaggagtc 1200tggaccgtgt ctcagttcca gtgtggctgg
tcgtcctctc agaccagcta ctgatcgatg 1260ccttggtagg cttttaccct
accaactagc taatcagata tcggccgctc cacgagcatg 1320aggtcttgcg
atcccccact ttcatcctta gatcgtatgc ggtattagcg taactttcgc
1380tacgttatcc cccactctag ggtacgttcc gatatattac tcacccgttc
gccactcgcc 1440accagagcaa gctccgtgct gccgttcgac ttgcatgtgt
aaggcatgcc gccagcgttc 1500aatctgagcc aggatcaaac tct
152361523DNAJanthinobacterium lividum 6aaggaggtga tccagccgca
ccttccgata cggctacctt gttacgactt caccccagtc 60acgaatccta ccgtggtaag
cgccctcctt acggttaagc tacctacttc tggtaaaacc 120cgctcccatg
gtgtgacggg cggtgtgtac aagacccggg aacgtattca ccgcgacatg
180ctgatccgcg attactagcg attccaactt catgcagtcg agttgcagac
tacaatccgg 240actacgatac actttctgcg attagctccc cctcgcgggt
tggcggcgct ctgtatgtac 300cattgtatga cgtgtgaagc cctacccata
agggccatga ggacttgacg tcatccccac 360cttcctccgg tttgtcaccg
gcagtctcat tagagtgccc tttcgtagca actaatgaca 420agggttgcgc
tcgttgcggg acttaaccca acatctcacg acacgagctg acgacagcca
480tgcagcacct gtgtactggt tctctttcga gcactcccca atctctcgag
gattccagcc 540atgtcaaggg taggtaaggt ttttcgcgtt gcatcgaatt
aatccacatc atccaccgct 600tgtgcgggtc cccgtcaatt cctttgagtt
ttaatcttgc gaccgtactc cccaggcggt 660ctacttcacg cgttagctgc
gttaccaagt caattaagac ccgacaacta gtagacatcg 720tttagggcgt
ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgtgcatga
780gcgtcaatct tgacccaggg ggctgccttc gccatcggtg ttcctccaca
tatctacgca 840tttcactgct acacgtggaa ttctaccccc ctctgccaga
ttctagcctt gcagtctcca 900atgcaattcc caggttgagc ccggggattt
cacatcagac ttacaaaacc gcctgcgcac 960gctttacgcc cagtaattcc
gattaacgct tgcaccctac gtattaccgc ggctgctggc 1020acgtagttag
ccggtgctta ttcttcaggt accgtcatta gcaagagata ttagctctca
1080ccgtttcttc cctgacaaaa gagctttaca acccgaaggc cttcttcact
cacgcggcat 1140tgctggatca ggctttcgcc cattgtccaa aattccccac
tgctgcctcc cgtaggagtc 1200tggaccgtgt ctcagttcca gtgtggctgg
tcgtcctctc agaccagcta ctgatcgatg 1260ccttggtagg cttttaccct
accaactagc taatcagata tcggccgctc cacgagcatg 1320aggtcttgcg
atcccccact ttcatcctta gatcgtatgc ggtattagcg taactttcgc
1380tacgttatcc cccactctag ggtacgttcc gatatattac tcacccgttc
gccactcgcc 1440accagagcaa gctccgtgct gccgttcgac ttgcatgtgt
aaggcatgcc gccagcgttc 1500aatctgagcc aggatcaaac tct
152371523DNAJanthinobacterium lividum 7aaggaggtga tccagccgca
ccttccgata cggctacctt gttacgactt caccccagtc 60acgaatccta ccgtggtaag
cgccctcctt acggttaagc tacctacttc tggtaaaacc 120cgctcccatg
gtgtgacggg cggtgtgtac aagacccggg aacgtattca ccgcgacatg
180ctgatccgcg attactagcg attccaactt catgcagtcg agttgcagac
tacaatccgg 240actacgatac actttctgcg attagctccc cctcgcgggt
tggcggcgct ctgtatgtac 300cattgtatga cgtgtgaagc cctacccata
agggccatga ggacttgacg tcatccccac 360cttcctccgg tttgtcaccg
gcagtctcat tagagtgccc tttcgtagca actaatgaca 420agggttgcgc
tcgttgcggg acttaaccca acatctcacg acacgagctg acgacagcca
480tgcagcacct gtgtactggt tctctttcga gcactcccca atctctcgag
gattccagcc 540atgtcaaggg taggtaaggt ttttcgcgtt gcatcgaatt
aatccacatc atccaccgct 600tgtgcgggtc cccgtcaatt cctttgagtt
ttaatcttgc gaccgtactc cccaggcggt 660ctacttcacg cgttagctgc
gttaccaagt caattaagac ccgacaacta gtagacatcg 720tttagggcgt
ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgtgcatga
780gcgtcaatct tgacccaggg ggctgccttc gccatcggtg ttcctccaca
tatctacgca 840tttcactgct acacgtggaa ttctaccccc ctctgccaga
ttctagcctt gcagtctcca 900atgcaattcc caggttgagc ccggggattt
cacatcagac ttacaaaacc gcctgcgcac 960gctttacgcc cagtaattcc
gattaacgct tgcaccctac gtattaccgc ggctgctggc 1020acgtagttag
ccggtgctta ttcttcaggt accgtcatta gcaagagata ttagctctca
1080ccgtttcttc cctgacaaaa gagctttaca acccgaaggc cttcttcact
cacgcggcat 1140tgctggatca ggctttcgcc cattgtccaa aattccccac
tgctgcctcc cgtaggagtc 1200tggaccgtgt ctcagttcca gtgtggctgg
tcgtcctctc agaccagcta ctgatcgatg 1260ccttggtagg cttttaccct
accaactagc taatcagata tcggccgctc cacgagcatg 1320aggtcttgcg
atcccccact ttcatcctta gatcgtatgc ggtattagcg taactttcgc
1380tacgttatcc cccactctag ggtacgttcc gatatattac tcacccgttc
gccactcgcc 1440accagagcaa gctccgtgct gccgttcgac ttgcatgtgt
aaggcatgcc gccagcgttc 1500aatctgagcc aggatcaaac tct
1523819DNAArtificial SequenceJanthino2F2 primer 8gcacggaagt
gaccaaaaa 19920DNAArtificial SequenceJanthino2R2 primer 9acatggagac
ttgggctttg 201024DNAArtificial SequenceJlivF primer 10taccacgaat
tgctgtgcca gttg 241124DNAArtificial SequenceJlivR primer
11acacgctcca ggtatacgtc ttca 24
* * * * *
References