NZ749273A - Method for facilitating maturation of the mammalian immune system - Google Patents
Method for facilitating maturation of the mammalian immune systemInfo
- Publication number
- NZ749273A NZ749273A NZ749273A NZ74927317A NZ749273A NZ 749273 A NZ749273 A NZ 749273A NZ 749273 A NZ749273 A NZ 749273A NZ 74927317 A NZ74927317 A NZ 74927317A NZ 749273 A NZ749273 A NZ 749273A
- Authority
- NZ
- New Zealand
- Prior art keywords
- mammal
- mmo
- feces
- composition
- dysbiotic
- Prior art date
Links
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Abstract
The inventions described herein relate generally to the use of compositions to increase output of acetate and lactate while reducing pH and the levels of pathogenic bacteria and inflammation in the gut of a nursing infant mammal including humans. These compositions generally comprise one or more bacterial strains selected for their growth on mammalian milk oligosaccharides, a source of mammalian milk oligosaccharides, and, optionally, nutritive components required for the growth of that infant mammal. terial strains selected for their growth on mammalian milk oligosaccharides, a source of mammalian milk oligosaccharides, and, optionally, nutritive components required for the growth of that infant mammal.
Description
METHOD FOR FACILITATING MATURATION OF THE MAMMALIAN IMMUNE SYSTEM
FIELD OF INVENTION
The inventions described herein relate generally to the use of
compositions to increase output of acetate and lactate while reducing pH and the levels of
pathogenic ia and ation in the gut of a nursing infant mammal including
humans. These compositions generally comprise one or more bacterial strains selected
for their growth on mammalian milk oligosaccharides, a source of mammalian milk
oligosaccharides, and, ally, nutritive components required for the growth of that
infant mammal.
BACKGROUND
The intestinal microbiome is the community of rganisms that live
within an animal’s gastrointestinal tract, the vast majority of which is found in the large
intestine or colon of mammals. In a y human, most dietary carbohydrates that are
consumed are absorbed by the body before they reach the colon. Many foods, however,
contain indigestible carbohydrates (i.e. dietary fiber) that remain intact and are not
absorbed during t through the gut to the colon. The colonic microbiome is rich in
bacterial species that may be able to fully or partially consume these fibers and utilize the
constituent sugars for energy and metabolism creating different metabolites for potential
nutritive use in the mammal. Methods for measuring dietary fiber in various foods are
well known to one of ordinary skill in the art.
The non-infant mammalian microbiome is x and contains a
diverse community of species of bacteria. This complexity begins to develop after the
cessation of human milk consumption as a sole source of nutrition. Conventional teaching
with regards to the non-infant mammalian microbiome is that xity es
stability, and maintaining a diversity of microorganisms in the microbiome while
consuming a complex diet is thought to be the key to promoting gut health. ne,
Nature, Vol. 489, pp. 220—230 (2012].
SUMMARY OF INVENTION
Creating a healthy microbiome in a mammal is necessary for the health
of the mammal. While it is difficult to understand the exact makeup of the iome at
any given time in a mammal, the inventors have found observable indicators of the health
(or, conversely, dysbiosis] of the infant microbiome in the stool composition, stool
frequency, stool consistency, and fecal pH. The presence of certain amounts of short-chain
fatty acids (SCFA) in the stool of a mammal and more specifically acetate and lactate, can
be an indication of a healthy microbiome. The inventors have ered that the se
of certain es under a controlled diet of oligosaccharides will result primarily in the
increase of lactate and acetate; the major contributors to the observed increase in SCFA in
the colon. The present ion provides for selection ques for those certain
microbes, and methods to use those microbes for the purpose of ing and
ring the achievement of healthy microbiomes.
This invention provides a method for creating, maintaining, or re-
establishing a healthy microbiome in an infant mammal by (a) administering a bacterial
composition comprising bacteria capable of and/or activated for colonization of the colon,-
and (b) administering a food composition comprising Mammalian Milk Oligosaccharides
(MMO). The MMO typically comprises ydrate polymers found in mammalian milk
which are not metabolized by any combination of mammalian digestive enzymes. The
MMO can e one or more of fucosyllactose, lacto-N-fucopentose, lactodifucotetrose,
sialyllactose, disialyllactone-N-tetrose, 2'-fucosyllactose, 3'-sialyllactoseamin, 3'-
fucosyllactose, 3'-sialylfucosyllactose, 3'-sialyllactose, 6'-sialyllactosamine, 6'-
sialyllactose, difucosyllactose, lacto-N-fucosylpentose l, lacto-N-fucosylpentose ll, lacto-N-
fucosylpentose III, lacto-N-fucosylpentose V, sialyllacto-N-tetraose, or derivatives f.
See, e.g., US. Patent Nos. 8,197,872, 8,425,930, and 9,200,091, the disclosures of which are
incorporated herein by reference in their entirety.
The MMO may be provided to the mammal in the form of a food
composition. The food composition can include mammalian milk, mammalian milk
derived t, mammalian donor milk, an infant formula, milk replacer, or enteral
nutrition t, or meal replacer for a mammal including a human. In some
embodiments, the addition of the bacterial composition and the food composition that
es MMO can occur contemporaneously, e.g., within less than 2 hours of each other.
The food composition may be sufficient to sustain the growth of the
mammal. The bacteria and the food composition can be administered in respective
amounts sufficient to maintain a level and composition of SCFA in the feces of said
mammal. The level of SCFA can be indicative of a y microbiome, and more
specifically the preferred make-up of the distribution of SCFA includes acetate and lactate.
The SCFA can include lactic, acetic, propionic, and butyric acids, and their salts. In some
embodiments, the SCFA include acetate and lactate, and these can make up at least 50% of
the SCFA. The method can include the steps of: (a) obtaining a fecal sample from the
mammal,- (b) determining the level and composition of SCFA in the sample; (c) identifying
a dysbiotic state in the mammal if the level of SCFA is too low or of skewed composition;
(d) treating the dysbiotic mammal by: (i) administering a ial composition
comprising bacteria capable of and/or activated for colonization of the colon; (ii)
administering a food composition sing MMO; or (iii) both (i) and (ii) added
contemporaneously. This embodiment can provide a method of enhancing and/or
monitoring the health of a mammal. The bacteria and/or the food composition can be
administered in tive amounts sufficient to maintain a level of SCFA in the feces of
the mammal above the threshold level in step (c).
The bacteria can be a single bacterial species of Bifidobacterium such as
B.adolescentis, B. animalis [e.g., B. animalis subsp. animalis or B. animalis subsp. Iactis), B.
bifi'dum, B. breve, B. catenulatum, B. Iongum (e.g., B. Iongum subsp. infantis or B. Iongum
subsp. Iongum), B. pseudocatanulatum, B. pseudolongum, single bacterial species of
Lactobacillus, such as L. acidophilus, L. antri, L. brevis, L. casei, L. coleohominis, L. crispatus,
L. curvatus, L. fermentum, L. gasseri, L. johnsonii, L. mucosae, L. pentosus, L. rum, L.
reuteri, L. rhamnosus, L. sakei, L. salivarius, L. paracasei, L. nsis., L. mentarius, L.
perolens, L. apis, L. ghanensis, L. dextrinicus, L. shenzenensis, L. harbinensis, or single
bacterial species of Pediococcus, such as P. parvulus, P. 1011'1', P. acidilactici, P. argentinicus, P.
claussenii, P. pentosaceus, or P. stilesii, or it can e two or more of these s. In
some embodiments, at least one of the s can be capable of consuming MMO by the
internalization of that intact MMO within the bacterial cell itself. In some embodiments, at
least one species of the bacteria composition can e bacteria activated for
colonization of the colon. The bacteria may be grown in an anaerobic culture whose sole
carbon source is wholly or partially the MMO.
In some embodiments, a method of obtaining a bacterial monoculture
suitable for this invention is described as a ial monoculture sing a bacterium
which can grow on MMO as a sole carbon source. The bacteria may grow in an bic
culture whose sole carbon source is the MMO. The method can include the steps of: (a)
ing a sample containing living microorganisms from fecal material of a nursing
infant mammal that is not dysbiotic,- (b) inoculating a culture medium whose sole carbon
source is MMO with the sample from step (a); (c) incubating the inoculated culture
anaerobically; (d) recovering a pure bacterial strain from the incubated culture of step (c),
and, optionally, exposing the sample from step (a) to mutagenesis prior to the inoculating
step [b]. The g infant mammal can be an infant human.
In some embodiments, the proportion of pathogenic bacteria in the
microbiome of the mammal is reduced by the treatment. In some embodiments, the
pathogenic bacteria are bacteriaceae (e.g., one or more of Salmonella, E. coli,
Klebsiella, or Clostridium]. In some embodiments, the pathogenic bacteria are reduced by
greater than 10%, 15%, 25%, 50%, 75%, 80%, or 85% by the ent.
In some embodiments, a method of reducing the antibiotic resistance
gene load is described. One or more genes of the antibiotic resistance gene load may be
reduced by greater than 10%, 15%, 25%, 30%, 45%, 50%, 75% or 85%. In some
embodiments, a method of reducing the levels of lipopolysaccharide (LPS) and/or
pathogenic bacteria in the gut of a mammal are described.
In some embodiments, the frequency of bowel movements in an infant
mammal can be decreased as compared to a tic mammal. In some embodiments,
the stool ition of an infant mammal can be altered as compared to a dysbiotic
mammal. The firmness/consistency of the stool composition of the infant mammal can be
increased as ed to a dysbiotic mammal. In some embodiments, the stool can be
less watery.
In the various embodiments, the mammal is a human, o, camel, cat,
cow, dog, goat, guinea pig, hamster, horse, pig, rabbit, sheep, monkey, mouse, or rat. The
mammal can be an infant. The mammal can be a nonhuman mammal, for example, a
mammal grown for human consumption. The mammal can be a companion or
performance animal.
In any embodiment according to this invention, the mammal may be an
infant mammal, and the infant mammal can be an infant human. In any of the
embodiments described herein, the infant mammal can be a pre-term infant or a term
infant, ularly an infant born by C-section, and/or a dysbiotic infant. In any of the
embodiments described herein, the infant can be a dysbiotic infant that has (a) a watery
stool, (b) Clostridium difficile levels of greater than 106 cfu/g feces, greater than 107 cfu/g
feces, or greater than 108 cfu/g feces, (c) Enterobacteriaceae at levels of greater than
greater than 106, greater than 107, or greater than 108 cfu/g feces, and/or (d) a stool pH of
.5 or above, 6.0 or above, or 6.5 or above. The infant mammal is generally receiving MMO.
In any of the embodiments described herein, the infant can be a breast-fed infant, and/or
an infant whose diet is supplemented with MMO.
The MMO can be provided at a level that is sufficient to maintain SCFA in
the stool. The MMO can be supplied chronically in amounts sufficient to maintain
colonization of the e that internalizes the MMO, and/or in SCFA in the stool.
For example, the infant mammal can be ing MMO at a dose representing over 10%,
over 15%, over 20%, over 25%, over 30%, over 35%, over 40%, over 45%, over 50%, over
55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over
95%, or up to 100% of the total dietary fiber. MMO can be administered to the infant
mammal prior to, after, or contemporaneously with the stration of the bacterial
ition.
BRIEF PTION OF THE FIGURES
Figure 1. Amount (CFU/g) of B. Iongum subsp. infantis (B. infantis] in
fecal samples as measured by qPCR during the intervention period and a follow-up period
in both vaginally- and C-section-delivered human infants. The black line and dots
represent all infants who were supplemented with B. infantis for 21 days starting at 7 days
of life. All infants receiving the standard of care (no probiotic] are depicted with the grey
line and dots. The band around each line represents a 95% confidence interval around the
line. The end of supplementation occurred at day 28 and samples were collected until day
60 of life.
Figure 2A. Abundances of different genera of intestinal bacteria in an
untreated ion baby over the study period (Day 6 to 60 of life).
Figure ZB. Abundance of different genera of intestinal ia in a C-
section baby treated from Day 7 to 28 with B. longum subsp. is.
Figure 3. ]accard stability index of unsupplemented infants delivered by
C-section (CS-UNS) or delivered vaginally (DV-UNS) compared to B. infantis supplemented
infants which included both C-section and vaginally-delivered infants together (All-SUP).
Figure 4. Predictive antibiotic (AB) resistance gene load in fecal samples
taken from unsupplemented (white bars] or supplemented (black bars) infants.
Figure 5. Mean concentration of fecal HMO (+/- SD, mg/g) in infant stools
collected at baseline (Day 6; pre-supplementation) and at the end of supplementation (Day29;
post-supplementation). Dark grey bars ent the B. infanlis supplemented group.
Figure 6. 2D density plot of all samples comparing total bacterium
measured by qPCR(Log10 CFU/g feces) with fecal pH.
Figure 7. Box plot of endotoxin levels (Log EU/ml) in fecal samples from
unsupplemented infants devoid of all bifidobacteria (Bifidobacterium-na'ive) vs. fecal
samples from s supplemented with B. infantis and replete with bifidobacteria (High
Bifidobacteria).
Figure 8. Percent change in the infant stool consistency for the
untreated (grey bars) and B. infantis-treated (black bars) groups between Intervention
and Baseline and between Post-intervention and ention. (*) P < 0.05.
Figure 9. An exemplary device to distinguish between s with a
microbiome e in bacterial from those depleted in bifidobacterial from a stool
sample.
DETAILED DESCRIPTION OF THE ION
This invention is directed to methods of monitoring, treating and
preventing sis in mammalian intestines; and to compositions and devices used in
the methods.
Definition of Dysbiosis
Generally, the phrase "dysbiosis” is described as the state of microbiome
imbalance inside the body, resulting from an insufficient level of keystone bacteria (e.g.,
bifidobacteria, such as B. Iongum subsp. infantis) or an overabundance of harmful bacteria
in the gut.
Dysbiosis in a human infant is defined herein as a microbiome that does
comprises B. Iongum subsp. infantis below the level of 108 cfu/g fecal material during the
first 12 months of life, likely below the level of detectable amount (Le, 106 cfu/g fecal
material). Dysbiosis can be further defined as inappropriate diversity or distribution of
species abundance for the age of the human or . Dysbiosis in infants is driven by
either the absence of MMO, e of B. is, or the incomplete or inappropriate
breakdown of MMO. For example, in an infant human, an insufficient level of keystone
bacteria (e.g., bifidobacteria, such as B. Iongum subsp. infantis) may be at a level below
which colonization of the bifidobacteria in the gut will not be significant [for example,
around 106 cfu/g stool or less). For non-human mammals, dysbiosis can be defined as the
presence of members of the bacteraceae family at greater than 106, or 107, or 108
cfu/g feces from the subject mammal. onally, a dysbiotic mammal (e.g., a dysbiotic
infant) can be d herein as a mammal having a fecal pH of 6.0 or higher, a watery
stool, Clostridium difficile levels of greater than 106 cfu/g feces, greater than 107 cfu/g
feces, or greater than 108 cfu/g feces, Enterobacteriaceae at levels of greater than 106,
greater than 107, or r than 108 cfu/g feces, and/or a stool pH of 5.5 or above, 6.0 or
above, or 6.5 or above. For example, a dysbiotic human infant can be a human infant
having a fecal pH of 6.0 or higher, a watery stool, Clostridium difficile levels of greater than
106 cfu/g feces, greater than 107 cfu/g feces, or greater than 108 cfu/g feces,
Enterobacteriaceae at levels of greater than greater than 106, greater than 107, or greater
than 108 cfu/g feces, a stool pH of 5.5 or above, 6.0 or above, or 6.5 or above,
lactate:acetate ratios ofless than 2:3, and/or greater than 2.5 mmol titratable acid/g feces.
Dysbiosis in a , especially an infant mammal, can be observed by
the physical symptoms of the mammal [e.g., diarrhea, digestive discomfort, inflammation,
etc.) and/or by observation of the presence of free sugar monomers in the feces of the
mammal, an absence or ion in specific bifidobacteria populations, and/or the
overall ion in measured SCFA; more specifically, acetate and e. Additionally,
the infant mammal may have an increased likelihood of becoming dysbiotic based on the
circumstances in the environment surrounding the mammal [e.g., an outbreak of disease
in the surroundings of the mammal, formula feeding, cesarean birth, etc.). sis in an
infant mammal can further be revealed by a low level of SCFA in the feces of said mammal.
The g human infant’s intestinal microbiome is quite different from
an adult microbiome in that the adult gut microbiome generally contains a large diversity
of sms, each present at a low percentage of the total microbial population. The
healthy nursing infant’s microbiome, on the other hand can be made up almost exclusively
(up to 80%] of a single species. When this species is B. infantis and the infant is a human
infant, this nt colonization unexpectedly gives rise to a very stable gut ecology.
Microbiome stability is a desirable characteristic in the first few months of life where
many developmental changes are rapidly taking place as the infant develops prior to
weaning.
The transition from the simple, non-diverse microbiome of the nursing
infant to a x, diverse adult-like microbiome (i.e., weaning) correlates with the
transition from a single nutrient source of a rather complex fiber (e.g., maternal milk
oligosaccharides) to more complex nutrient sources that have many different types of
dietary fiber.
Carbohydrates of the Infant Diet
ian milk contains a significant quantity of mammalian milk
oligosaccharides (MMO) as dietary fiber. For example, in human milk, the dietary fiber is
about 15% of total dry mass, or about 15% of the total caloric content. These
oligosaccharides comprise sugar es in a form that is not usable directly as an energy
source for the baby or an adult, or for most of the microorganisms in the gut of that baby
or adult.
The term "mammalian milk oligosaccharide" or MMO, as used herein,
refers to those indigestible glycans, mes ed to as "dietary , or the
carbohydrate polymers that are not hydrolyzed by the endogenous mammalian enzymes
in the digestive tract (e.g., the small intestine) of the mammal. Mammalian milks contain a
significant quantity of MMO that are not usable directly as an energy source for the milk-
fed mammal but may be usable by many of the microorganisms in the gut of that .
MMOs can be found as free accharides (3 sugar units or longer, e.g., 3—20 sugar
residues) or they may be conjugated to proteins or lipids. Oligosaccharides having the
chemical structure of the stible oligosaccharides found in any mammalian milk are
called "MMO" or "mammalian milk accharides" herein, whether or not they are
actually sourced from ian milk.
The major human milk oligosaccharides (“HMO”), include lacto-N-
se (LNT), lacto-N-neotetraose (LNnT) and lacto-N-hexaose, which are neutral HMOs,
in addition to fucosylated oligosaccharides such as Z-fucosyllactose (ZFL), 3-fucosyllactose
(3FL), and lacto-N-fucopentaoses I, II and Ill. Acidic HMOs include sialyllacto-N-tetraose,
3’ and 6’ sialyllactose (6SL). HMO are particularly highly enriched in fucosylated
oligosaccharides (Mills et al., US Patent No. 8,197,872). Among the enzymes that produce
HMO in the mammary gland is the enzyme encoded by the ltransferase 2 (FUTZ)
gene, which catalyzes the linking of fucose residues by an all-linkage to oligosaccharides
found in human milk. Fucosylated oligosaccharides are known to inhibit the binding of
pathogenic bacteria in the gut. HMO, and in particular the fucosylated HMO, share
common structural motifs with glycans on the infant's intestinal epithelia known to be
receptors for pathogens. (German et al., ).
Microbes ofthe Healthy Newborn Microbiome
Certain microorganisms, such as Bifi'dobacten'um Iongum subsp. infantis
(B. infantis), have the unique capability to consume ic MMO, such as those found in
human (HMO) or bovine (BMO) milk (see, e.g., US Patent No. 8,198,872 and US Patent
Application No. 13/809,556, the disclosures ofwhich are incorporated herein by reference
WO 06080
in their entirety). When B. infantis comes in contact with certain MMO, a number of genes
are specifically induced which are responsible for the uptake and internal deconstruction
of those MMO, and the individual sugar ents are then catabolized to provide
energy for the growth and reproduction of that microorganism (Sela et al, 2008). This
form of carbon source utilization is ably different from most of the other colonic
bacteria, which produce and excrete extracellular glycolytic enzymes that deconstruct the
fiber to monomeric sugars extracellularly, and only monomers are imported via hexose
and pentose transporters for catabolism and energy tion. If the appropriate gut
bacteria are not present [e.g., a consequence of the extensive use of antibiotics or cesarean
section ), or the appropriate MMO are not present (e.g., in the case of using artificial
feeds for newborns, such as infant formula or milk replacers], any free sugar rs
cleaved from the dietary fiber by extra cellular enzymes can be utilized by less desirable
microbes, which may give rise to blooms of pathogenic bacteria and symptoms such as
diarrhea resulting therefrom.
The inventors discovered that growing bacterial cultures under strong
selective pressure of MMO as the sole ional source can be used as a method to select
and/or identify certain bacterial species that were usly not known for their ability
to grow on MMO. As a result, they have developed a process with which to produce new
strains ofbacteria which can be used in the present invention.
The term "bacterial monoculture", as used herein, refers to a culture of a
single strain.
The bacteria for use in this invention may be selected and enriched from
a population ofbacteria found in a stool sample of a mammal such as, but not limited to, a
human, buffalo, camel, cat, cow, dog, goat, guinea pigs, hamster, horse, pig, rabbit, sheep,
monkey, mouse, or rat. The selection and ment can be done using a method of
providing such a population with a growth medium that comprises one or more MMO as
the sole carbon source and then cultivating said composition for a period of time required
to allow the ive enrichment of strains of ia capable of growth on said MMO. All
other growth conditions and media for the selection of bifidobacteria, pediococci, and/or
lactobacilli use standard conditions known in the art for the cultivation of these bacteria.
Following the selective enrichment of the bifidobacteria, pediococci, and/or lactobacilli
species, the mixture is plated out for the purposes of isolating individual colonies that are
then grown up as pure strains of bacteria capable of growth on the MMO. Pure colonies
isolated from a specific mammalian species can then be grown under rd conditions
for such bacteria. The population of bacteria in the stool sample or the bacteria ed
and purified from the stool sample may be treated with a chemical or physical mutagen
such as, but not limited to, ethyl methyl sulfonate (EMS), X-rays, a radioactive source
before selection on a growth medium comprising the MMO.
The bacteria may be in an activated state as defined by the expression of
genes coding for enzymes or proteins such as, but not limited to, fucosidases, sialidases,
ellular glycan binding proteins, and/or sugar permeases. Such an activated state is
produced by the cultivation of the bacteria in a medium sing a MMO prior to
harvest and the preservation and drying of said bacteria. Activation of B. infantis is
described, for example, in PCT/USZOlS/057226, the disclosure of which is incorporated
herein in its entirety.
The MMO used for cultivation, activation, selection, and/or storage of the
bacteria of this invention can e fucosyllactose (FL) or derivatives of FL including but
not limited to, N-fucopentose (LNFP) and lactodifucotetrose (LDFT), N-tetraose
(LNT) and lacto-N-neotetraose (LNnT), which can be purified from mammalian milk such
as, but not limited to, human milk, bovine milk, goat milk, or horse milk, sheep milk or
camel milk, or produced directly by chemical synthesis. The composition can r
comprise one or more bacterial strains with the ability to grow and divide using
fucosyllactose or its derivatives thereof as the sole carbon source. Such bacterial strains
may be naturally occurring or genetically modified and selected to grow on the
fucosyllactose or its derivatives if they did not naturally grow on those oligosaccharides.
The MMO can also be lactose (SL) or derivatives of SL such as, but
not limited to, 3'sialyllactose (3SL), 6’sialyllactose (6SL), and disialyllacto-N-tetrose
(DSLNT), which can be purified from mammalian milk such as, but not limited to, human
milk, bovine milk, goat milk, or mare's milk, sheep milk or camel milk, or produced
directly by al synthesis. The composition r ses one or more bacterial
strains with the ability to grow and divide using sialyllactose or derivatives thereof as the
sole carbon . Such bacterial strains may be naturally occurring or genetically
modified and selected to grow on the sialyllactose or its derivatives if they did not
naturally grow on those oligosaccharides.
The MMO can be a mixture llactose (FL) or derivatives of FL and
sialyllactose (SL) or derivatives of SL which are naturally found in mammalian milk such
as, but not limited to, human milk, bovine milk, goat milk, and horse milk. In preferred
modes, the FL and SL or tives thereof may be found in a ratio from about 1:10 to
:1.
Formulations to Treat Dysbiosis
A composition comprising (a) bacteria capable of consuming the MMO
and (b) one or more MMO can be stored in a low water activity environment for later
administration. The composition can further e a food, and the food can comprise the
complete nutritional requirements to support life of a healthy mammal, where that
mammal may be, but is not limited to, an infant. The mammal can be a human, buffalo,
camel, cat, cow, dog, goat, guinea pigs, hamster, horse, pig, rabbit, sheep, monkey, mouse,
or rat. The bacteria can include, but is not limited to, one or more of B. adolescentis, B.
animalis, (e.g., B. animalis subsp. animalis or B. animalis subsp. Iactis), B. bifi'dum, B. breve,
B. catenulatum, B. Iongum (e.g., B. longum subsp. infantis or B. longum subsp. ), B.
pseudocatanulatum, B. pseudolongum, L. acidophilus, L. antri, L. brevis, L. casei, L.
coleohominis, L. crispatus, L. curvatus, L. fermentum, L. gasseri, L. johnsonii, L. e, L.
us, L. plantarum, L. i, L. rhamnosus (e.g. LGG), L. sakei, L. salivarius, P.
acidilactici, P. argentinicus, P. claussenii, P. pentosaceus, P. stilesii L. paracasei, L. kisonensis.,
L. paralimentarius, L. perolens, L. apis, L. ghanensis, L. dextrinicus, L. shenzenensis, L.
harbinensis, P. parvulus, or P. IoIii. The composition can include at least one or more
fucosidases and/or one or more sialidases produced by at least one or more bacterial
strains of the ition that may be intracellular or extracellular. One preferred
species can be B. Iongum subsp. infantis. The B. infantis may be activated. Activation of B.
is is described in PCT/U32015/057226, the disclosure of which is incorporated
herein in its entirety.
The bacteria may be present in these itions in a dry powder
form, or as a suspension in a concentrated syrup with a water activity of less than 1.0,
preferably less than 0.9, more preferably less than 0.8, less than 0.7, less than 0.6 or less
than 0.5, or less than 0.4, or less than 0.3 or less than 0.2 or in a suspension in an oil such
as, but not limited to, medium chain triglyceride (MCT), a natural food oil, an algal oil, a
fungal oil, a fish oil, a mineral oil, a silicon oil, a phospholipid, or a ipid. The syrup
may be a concentrate of a MMO such as, but not limited to, that from human milk (HMO),
bovine milk (BMO), ovine milk (0M0), equine milk (EMO), or caprine milk (CMO). The
oligosaccharides can be obtained from a process that involves cheese or yogurt production
and can be from whey sources such as, but not limited to, the whey permeate, or a
processed whey permeate, where the processing steps may include, but are not limited to,
removal of lactose, removal of minerals, l of peptides, and removal of
monosaccharides, but which in any case, results in the concentration of the MMO to levels
that are greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater
than 60%, greater than 70%, or greater than 80% of the total dry matter of the product.
The MMO can be present in the compositions of this invention in a
powder form, in the form of a concentrated syrup with a water activity of less than 1.0,
optionally less than 0.9, less than 0.8, less than 0.7, or less than 0.6, or less than 0.5, or less
than 0.4, or less than 0.3 or less than 0.2 or in a suspension in an oil including, but not
limited to, medium chain triglyceride (MCT), a natural food oil, an algal oil, a fungal oil, a
fish oil, a l oil, a silicon oil, a phospholipid, and a glycolipid.
The ition can also include a food source that contains all the
nutritional requirements to support life of a y . That mammal may be, but
is not limited to, an infant, an adolescent, an adult, or a geriatric adult. The food source
can be a nutritional formulation ed for a human, o, camel, cat, cow, dog, goat,
guinea pigs, hamster, horse, pig, rabbit, sheep, monkey, mouse, or rat. For example, the
food source can be a food source for an infant human which further comprises a protein
such as, but not limited to, a milk protein, a cereal protein, a seed protein, or a tuber
protein. The food source can be mammalian milk including, but not limited to, milk from
human, bovine, equine, caprine, or porcine sources. The food can also be a medical food
or l food designed to meet the nutritional requirements for a mammal, for e,
a human.
Effects ofthe Compositions
The inventors have ered that providing a mammalian infant with
(a) certain isolated, purified, and activated bacteria that specifically consume milk
oligosaccharides and/or glycans, along with (b) MMO and glycans, either in the form of its
’s milk, or as ed MMO provided contemporaneously with the bacteria, results
in the production of unexpectedly high levels of SCFA, acetic and lactic acids in particular,
in the colon of that infant mammal. The inventors further found that this treatment also
significantly lowered the levels of pro-inflammatory biomarkers as well as pathogenic
bacteria and lipopolysaccharide (LPS). Similar observations found in humans, horses, and
pigs indicate that this may be a common element among many species that provide milk as
the sole source of nutrition for their infant during the first stages of life (116., all mammals).
Supplying the infant with these two components at this early stage can
further tate the nominal development of the immune system and may deflect the
appearance ofvarious disease conditions seen later in life due to a mal-development ofthe
immune . The use of food compositions with these two components can also have
an immediate impact on the reduction of pathogen blooms early in life, eliminating the
appearance of certain symptoms such as ea in certain mammals such as, but not
limited to humans and horses. One or more of the MMO ofa particular species, used as the
sole carbon source and bacteria that demonstrate the most rapid growth on that species'
MMO in culture, may be used for the purpose of colonizing the gut ofthat mammal.
The ors have discovered that the above components can be added
to foods other than milk, where such foods comprise all the nutritive ents to
sustain life of an infant mammal [e.g., artificial milks and infant formula). The inventors
have also discovered that the above compositions can be preventative and/or curative to
outbreaks of pathogens such as Clostridium difficile in in mammals such as horses if
provided immediately on delivery of the infant (foaling), and the treatment further, and
unexpectedly, completely eliminates the “foal heat diarrhea" that lly occurs on or
about day 7-10 of the life of a horse. The inventors further discovered that, although the
compositions of MMO differ from mammal to mammal, some bacteria which have the
discovered characteristics, surprisingly have r effects in mammalian species in
which they are not typically found.
The gut of a mammal can be colonized with the bacteria described
herein in combination with the oligosaccharides described herein. The mammal can be a
human, the ia can be a bifidobacteria, and the MMO can be ed from, or is
chemically identical to, a HMO or a BMO. The MMO can comprise fucosyllactose (FL) or
derivatives of FL and/or sialyllactose (SL) or as derivatives of SL. The bifidobacteria can
be provided as B. Iongum, for example, B. Iongum subsp. infantis. In some ments,
the composition is provided to the subject on a daily basis comprising from 0.1 billion to
500 billion cfu of bacteria/day. For example, the composition that is provided on a daily
basis can include from 1 n to 100 billion cfu/day or from 5 billion to 20 billion
cfu/day. The composition may be provided on a daily basis for at least 2, at least 5, at least
, at least 20, or at least 30 days. The ent of the treatment can be a human infant.
A self-sustaining, host-specific dose of SCFA can be delivered directly to
the colon by the method of this invention. Amounts of MMO to generate a ratio of about
3:2 acetate to lactate can be administered. This administration may increase the levels of
SCFA including, but not limited to lactic acid, acetic acid, propionic acid, and butyric acid
or salts thereof, in the colon of a mammal by at least , at least 5-fold, at least 10-fold,
at least 50-fold, or at least 100-fold as compared to a dysbiotic infant.
The levels of SCFA in the colon can be approximated by the levels of the
SCFA in the feces of the mammal. The SCFA will typically include acetic acid or a salt
thereof. In some embodiments, the mammal is a human, the bacteria is bifidobacteria, and
the MMO is from, or is chemically cal to, a HMO or a BMO. In some embodiments, the
mammal is a horse, the bacteria is bifidobacteria, and the MMO is from, or is chemically
identical to, an EMO, HMO or BMO. In some embodiments, the MMO comprises
fucosyllactose (FL) or derivatives of FL and/or sialyllactose (SL) or as derivatives of SL. In
some embodiments, the bifidobacteria is provided as B. Iongum, or as B. Iongum subsp.
infantis. In some embodiments, the composition is provided on a daily basis comprising
from 0.1 billion to 500 billion cfu eria/day. In some ments, the composition
is provided on a daily basis comprising from 1 billion to 100 billion cfu/day and, from
example, from 5 billion to 20 billion cfu/day. In a preferred embodiment, the composition
is provided on a daily basis for at least 2, at least 5, at least 10, at least 20, or at least 30
days. In a most preferred ment, the recipient of the treatment is a human infant.
The levels of pathogenic microorganisms in the gut of a mammal can be
reduced, as compared to a dysbiotic infant, significantly by treating that mammal with a
daily dose of a medicament comprising a MMO and bacteria that selectively grows on that
MMO. In some embodiments, the proportion of the pathogenic bacteria in the microbiome
of the mammal is reduced by the treatment. In some embodiments, the pathogenic
bacteria are reduced, as compared to a dysbiotic infant, by greater than 25%, 50%, 75%,
80%, or 85% by the treatment. The stration can occur for a period of from at least
2, at least 5, at least 10, at least 20, or at least 30 days. Pathogenic microorganisms
e, but are not limited to: Clostridium, Escherichia, Enterobacter, ella, and
Salmonella species, and their presence in the colon can be estimated by their presence in
the feces of the . The medicament composition comprising from 0.1 n to 500
billion cfu of bacteria can be provided on a daily basis. A medicament composition
comprising from 1 billion to 100 billion cfu, or from 5 billion to 20 billion cfu can also be
provided on a daily basis. The MMO can be provided in a solid or liquid form at a dose
from about 0.1—50 g/day, for example, 2—30 g/day or 3—10 g/d. The bacteria that
selectively grows on the MMO can be provided contemporaneously with the MMO, or the
bacteria can be provided separately to a g infant whose MMO are in the form of
whole milk provided by nursing or otherwise.
Optimizing colon try, reducing the ty for LPS production,
and/or reducing the levels of proinflammatory lipopolysaccharide (LPS) in the gut of a
mammal may occur by treating that mammal with a daily dose of a ment
comprising a MMO and bacteria that selectively grows on that MMO, for a period of, from
at least 2, at least 5, at least 10, at least 20, or at least 30 days. In some embodiments, the
composition is provided on a daily basis comprising from 0.1 billion to 500 billion cfu of
bacteria/day. In some embodiments, the level of LPS is d, as compared to a
dysbiotic infant, by greater than 5%, 10%, 15%, 20%, 25%, 50%, 75%, 80%, or 85% by
the treatment. In some embodiments, the level of LPS is reduced, as compared to a
dysbiotic infant, to below 0.7 endotoxin units [EU)/mL, below 0.65 EU/mL, 0.60 EU/mL,
or below 0.55 EU/mL. In some embodiments, the composition is provided on a daily basis
comprising from 1 billion to 100 billion cfu/day, for example, the composition is provided
on a daily basis comprising from 5 billion to 20 billion cfu/day. The bacteria can be
chosen from bifidobacteria, acilli, and occi, for example, the bifidobacteria
can be B. Iongum or B. longum subspecies infantis. The MMO can be ed in a solid or
liquid form at a dose from about 0.1—50 g/day, for example, 2—30 g/day or 3—10 g/d.
Levels of proinflammatory cytokines including, but not limited to, lL-Z,
lL-5, lL-6, lL-8, lL-10, lL-13, lL-ZZ and TNF-alpha, can be reduced relative to a dysbiotic
infant, particularly by greater than 50%, r than 60%, percent, greater than 70%,
greater than 80%, r than 90%, or greater than 95%. Reduction of the levels of
proinflammatory nes including, but not limited to, lL-Z, lL-5, lL-6, lL-8, lL-10, lL-13,
and TNF-alpha, and/or increasing the levels of anti-inflammatory cytokines, in the gut of a
mammal may be accomplished by treating that mammal with a daily dose of a medicament
comprising a MMO and ia that selectively grows on that MMO, for a period of from
at least 2, at least 5, at least 10, at least 20, or at least 30 days. The composition can be
provided on a daily basis, and can include from 0.1 billion to 500 n cfu of
bacteria/day. For example, the ition can be provided on a daily basis comprising
from 1 billion to 100 billion cfu/day, such as 5 billion to 20 billion cfu/day. The bacteria
can be chosen from bifidobacteria, Lactobacilli, and occi, such as B. longum or B.
Iongum subspecies infantis. The MMO can be provided in a solid or liquid form at a dose
from about 0.1—50 g/day, for example 2—30 g/day or 3—10 g/day.
Reduction of the risk of presenting certain metabolic disorders such as,
but not limited to, Juvenile Diabetes (Type I), obesity, asthma, atopy, Celiac's Disease, food
ies and autism in a human, as compared to a dysbiotic infant, may be achieved by
treating that human, beginning within the first 4 weeks of life, with a daily dose of a
medicament comprising a MMO, and bacteria that selectively grows on that MMO, for a
period of from at least 10, at least 20, at least 30, at least 60, at least 90, at least 120, at
least 150, or at least 180 days. The risk can be reduced, as compared to a dysbiotic infant,
by 20, 30, 40, 50, 60, 70, 80, or 90%. The composition that is provided can be given on a
daily basis and can include from 0.01 billion to 500 billion cfu of bacteria/day, for
example, from 1 billion to 100 billion cfu/day or from 5 billion to 20 billion cfu/day. The
bacteria can be bifidobacteria, such as B. longum or B. longum subspecies infantis. The
MMO can be provided in a solid or liquid form at a dose from about 0.1—50 g/day, for
example, 2—30 g/day or 3—10 g/d. The composition can comprise the medicament and a
food composition, and the food ition can include the complete nutritional
ements to support life of a healthy mammal n that mammal may be, but is not
d to, an infant, an adolescent, an adult, or a geriatric adult. The mammal can be a
human. The ia and the MMO can be provided contemporaneously or separately at
any time during 24 hr. The MMO could for example be provided along with an infant
formula and the bacteria provided separately within 24 hr, 12 hr, 8 hr, 6 hr, 4hr or 2hr of
consumption ofthe MMO.
A composition comprising mammalian milk of MMO and bacteria
in a concentration to provide a daily dose of from 0.1 billion to 500 billion cfu of
bacteria/day can be provided. The MMO can be provided in a solid or liquid form at a dose
from about 0.1—50 g/day, for example, 2—30 g/day or 3—10 g/d. The bifidobacteria can be
B. longum or B. longum subspecies infantis. The composition can be a medicament for a
mammal to prevent or treat a pathogenic bacterial overgrowth, which es, but is not
limited to, Enterobacteriaceae (e.g., one or more of Salmonella, E. coli, Klebsiella, or
Clostridium). For e, the pathogenic bacterial overgrowth can include bacteria of
Clostridium difficile, Escherichia coli, and/or Enterobacterium faecale.
In some ments, the mammalian milk is horse milk [mare's milk)
and the recipient of the treatment is an infant horse (a foal). The medicament can further
comprise a lactobacillus species including, but not d to, L. plantarum. In some
embodiments, the mammalian milk is human milk and the recipient of the treatment is an
infant human. The infant human can be a premature infant with a body mass of less than
2.5 kg.
A , healthy microbiome can be described as the presence of
greater than 108 cfu/g stool of a single genus of bacteria (e.g., Bifidobacterium), more
particularly, of a single subspecies or strain of bacteria (e.g., B. longum subsp. infantis).
For example, up to 80% of the microbiome can be dominated by the single bacterial
species such as Bifidobacteria sp. or, more ularly, by the single subspecies of a
bacteria such as B. longum subsp. infantis. A simple microbiome can also be described as
the presence of greater than 20%, preferably greater than 30%, more preferably greater
than 40%, greater than 50%, greater than 60%, r than 70%, greater than 75%,
greater than 80%, or greater than 90% ofa single genus eria (e.g., Bifidobacterium),
more ularly, of a single subspecies of bacteria (e.g., B. longum subsp. infantis). This
population has features of ecological competitiveness, resilience, persistence, and stability
over time, as long as MMO are present.
The level of bifidobacteria in an infant can be determined using a device
that measures pH. The inventors have determined that pH levels in a stool sample
correlate well to the levels of bifidobacteria in a microbiome (e.g., an infant microbiome).
In a healthy infant microbiome, the ors discovered that bifidobacteria will generate
at least 2.5 mmol of titratable acidity in the form of SCFA per gram of feces.
A fecal sample can be added to a mixture that includes a fixed
concentration of NaOH and an indicator. The fecal sample and NaOH can be in a ratio of
200—400 mg fecal sample per mmol of NaOH. In some embodiments, a device is designed
to match the range of titratable acid in a certain amount of fecal sample (i.e. 40-80mg) to a
fixed concentration of NaOH or other base such that the indicator changes color to
discriminate high vs low Bifidobacterium fecal samples. The device can include a on
that es 0.1M NaOH. KOH or any other appropriate base can also be used in the
invention. The solution that includes 0.1M NaOH can also include deionized water and/or
ethanol or other suitable alcohols such as but not limited to methanol, ol, and
isopropanol. The device can include a reading window and a ng devise which can
aide the user in providing a precise amount of the fecal material (e.g. 40 mg). The device
can include a filter to remove the particulate matter. The fecal sample and indicator can be
added contemporaneously into the device. In some embodiments, the tor can be in a
vessel into which the fecal sample and solution are introduced. The device can include a
reading window to view the colorimetrc reaction between the fecal sample, indicator and
NaOH. Ifthe device contains an indicator, such as phenolphthalein in dilute ethanol whose
color changes in the range of 8.2, the color of the resulting composition can indicate a
threshold level ofbifidobacteria in the sample.
If the mixture of the fecal sample plus indicator phenolphthalein and
NaOH has a pH of 8.5 or above, the fecal sample has a fecal pH of 6.0 or above and the
sample would be described as low bifidobacteria. The pH of the composition is less than
8.5, the fecal sample would have had a pH of 6.0 or less and the sample would be described
as high in bifidobacteria. Due to the discovery of the relationship n fecal pH and
bifidobacteria levels, the indication of fecal pH levels tes the bifidobacteria levels in
the sample. Thus, a fecal sample with a low level of bifidobacteria will remain pink if
phenolphthalein is the indicator. A fecal sample with a high level of bifidobacteria will
turn the indicator from pink to yellow.
Alternatively, a device that includes an indicator that indicates pH
directly can be utilized with a fecal sample that may be deproteinated and/or filtered.
Indicators such as, but not limited to, phenol red (yellow to violet), transition from
one color to another around pH 6.0 and may be used to visually discriminate a high
bifidobacteria fecal sample from a low bifidobacteria fecal sample. A pH of 6.0 or below
demonstrates that the sample has high levels of bifidobacteria. The device design may
provide a window that gives a ve (high bifidobacteria) and negative (low
bifidobacteria) sign to the user. Alternatively, users are provided a color card to match Bif
level to test result. In other ments, an optical reader may be used to ish the
colorimetric change associated with the pH differential.
EXAMPLES
Example 1: Preparation of onal HMO-selective bacteria. A
sample of feces is ed from a vaginally delivered breast-fed baby, diluted with sterile
saline and mixed to form a suspension of live bacterial cells that are representative of
those in that fecal sample. An aliquot of this suspension is then transferred to liquid
growth medium comprising deMan Rogosa Sharpe (MRS) media wherein the sole carbon
source is made up of human milk oligosaccharides (HMOs) at a concentration of from 5-20
2017/040530
g/L (the "HMO Medium"), and the cultures are grown in an anaerobic chamber for 16-72
hr allowing the selective enrichment of bacterial s that can utilize the HMOs as a
selective carbon source. The consortia from these enrichment cultures are then diluted
and erred to agar plates also ning HMO as the sole carbon source, and the
plates are incubated for an additional 24-72 hr in an anaerobic environment. Individual
pure colonies are then picked and transferred to microtiter plates with wells ning
50-200 uL of HMO Medium and these "Microcultures" are incubated for another 16-48 hr
in an anaerobic chamber. Finally, 20 uL samples from each individual Microculture are
transferred to a single well in a 96 well microplate containing 200 uL of HMO Medium (a
"Miniculture”), and the growth of each individual clone is monitored hourly by l
density of the Minicultures over a period of 72 hr. Lead candidates identified by robust
growth are then checked for ty using 163 RNA sequencing and phenotypic testing.
Example 2. Trial with Breast-fed Infants. This trial was designed
to show the effect of probiotic supplementation with bacteria in healthy term
nursing infants compared to an unsupplemented group. A dry composition of lactose and
ted Bifidobacterium Iongum subsp. is was prepared starting with the
cultivation of a ed isolate (Strain EVCOOl, Evolve Biosystems Inc., Davis, CA, ed
from a human infant fecal sample) in the presence of BMO according to
PCT/U32015/057226. The culture was harvested by centrifugation, freeze dried, and the
concentrated powder preparation had an activity of about 300 Billion CFU/g. This
concentrated powder was then diluted by blending with infant formula grade lactose to an
activity level of about 30 Billion CFU/g. This composition then was loaded into individual
sachets at about 0.625 g/sachet and provided to breast-fed infants starting on or about
day 7 of life and then provided on a daily basis for the subsequent 21 days.
This was a 60-day study starting with infants’ date of birth as Day 1.
Before postnatal day 6, women and their infants [delivered either vaginally or by
cesarean-section), were randomized into an unsupplemented lactation support group or a
B. infantis supplementation plus lactation support group. Infant birthweight, birth length,
gestational age at birth, and gender were not different between the supplemented and
unsupplemented groups. Starting with Day 7 tal, and for 21 consecutive days
fter, infants in the supplemented group were given a dose of at least 1.8 X1010 cfu of
B. infantis suspended in 5 mL of their mother’s breastmilk, once daily. Because the
provision of HMO via breastmilk was critical for supporting the colonization of B. infantis,
all participants received breast feeding t at the hospital and at home and
maintained exclusive breast feeding through the first 60 days of life.
Infant fecal samples were collected throughout the 60-day trial. Mothers
collected their own fecal and breastmilk samples as well as fecal samples from their
infants. They filled out weekly, biweekly and monthly health and diet questionnaires, as
well as daily logs about their infant feeding and gastrointestinal tolerability (GI). Safety
and tolerability was determined from maternal s of infants’ feeding, stooling
frequency, and consistency (using a ed Amsterdam infant stool scale -- watery, soft,
formed, hard; Bekkali et al. 2009), as well as GI symptoms and health outcomes. Individual
fecal samples were subjected to full microbiome analysis using lllumina sequencing based
on 16S rDNA and qPCR with primers designed specifically for B. longum subsp infantis
strain.
RLultS
B. infantis was determined to be well-tolerated. Adverse events reported
were events that would be expected in normal healthy term infants and were not different
n groups. Reports specifically monitored blood in infant stool, infant body
temperature and parental ratings of Gl-related infant outcomes such as l irritability,
upset feelings in response to ps and discomfort in passing stool or gas, and
flatulence. Furthermore, there were no differences reported in the use of antibiotics, gas-
relieving tions, or parental report of infant colic, jaundice, number of illnesses, sick
doctor visits and medical ses of eczema.
The B. infantis supplemented infants had a gut microbiome fully
dominated (on e, greater than 70%) with B. Iongum subsp infantis regardless of the
birthing mode (vaginal or C-section). This dominance continued even after
supplementation ended (Day 28) as long as the infant continued to consume breast milk
indicating that B. infantis was colonizing the infant gut to levels higher than 1010 cfu/g
feces (Figure 1). Furthermore, those s that were colonized by the B. Iongum subsp
is also had much lower levels of bacteria and enterococci (including
Clostridium and Escherichia species) (Figure 2).
Unsupplemented s (i.e., infants receiving the standard of care—
lactation support but no supplementation of B. is) did not show B. infantis levels
above 106 cfu/g (i.e., the limit of detection) in their microbiome and there were significant
differences in the microbiomes between C-section and lly delivered infants. Eighty
percent (8 of 10) unsupplemented infants delivered by C-section had no detectable
Bifidobacterium species and fifty-four percent (13 of 24) of the lly delivered infants
had no detectable Bifi'dobacterium species by day 60. Further analysis of the thirteen
unsupplemented infants that had some detectable bifidobacteria, found that the species
were primarily B. Iongum subsp Iongum, B. breve and B. catenulatum. No detectable
B. Iongum subsp. infantis was found in any ofthe unsupplemented infants in the study.
The changes in the infant gut ecology associated with the B. infantis
supplementation and its subsequent domination by B. infantis to over 80% resulted in a
icant increase in ecological stability of the microbiome. The ]accard Stability Index is
a metric of ecosystem stability in that it can be regarded as a measure of the changeability
of a complex system. See, e.g., Yassour M, et al. (2016) Natural y of the infant gut
microbiome and impact of otic treatment on bacterial strain ity and stability.
Science Translational Medicine 8(343):343ra81—343ra81. The ]accard stability index for
the microbiome of the unsupplemented, C-section delivered infants was significantly
lower than that of the unsupplemented vaginally-delivered infants [Figure 3). However,
all the B-infantis treated infants, whether delivered vaginally or by C-section, had an
exceptionally high ecological stability which reflected a very stable microbial composition.
Two different methods were used to examine the fecal samples for
antibiotic resistance gene load present in the total microbiome of unsupplemented vs. B.
infantis supplemented infants: 1) the Pfaffl method for relative nce of a gene
sequence (compared to 16S rRNA); and 2) a machine learning approach. In B. is
supplemented infants, erythromycin resistance genes [ermB) were reduced by about half
in supplemented infants ed to unsupplemented infants using the Pfaffl Method for
analyzing qPCR results (p=0.0258). To functionally classify the genes in fecal samples
from unsupplemented or B. infantis supplemented groups, the 16S rRNA amplicon
libraries generated were first zed into normalized, operational taxonomic unit
(OTUs). PlCRUSt, a publicly available bioinformatics freeware (picrust.github.io/picrust),
was used to produce a table containing predicted gene classification of all the genes
present. The genes were assigned using the Kyoto Encyclopedia of Genes and Genomes
(KEGG) database (Kanehisa et a., 2000). Differences of predicted gene content in KEGG
ries among samples were statistically analyzed using a Kruskal—Wallis one-way
ANOVA with Bonferroni correction to adjust p-values (Theodorsson-Norheim et al., 1986).
Among the KEGG Orthologies identified, mphenicol O-acetyltransferase type B, was
significantly increased in the unsupplemented samples (p: 5.50E-44; Bonferroni). Levels
of the otic ance gene annotated as 23S rRNA (adenine-N6)-dimethyltransferase
were significantly higher in the unsupplemented infants (p: 1.32E-06; Bonferroni) than
the supplemented infants. An entire group of antibiotic resistance genes were fied as
beta-Lactam resistance genes and these genes were three times higher in the
unsupplemented infants ed to the B. is supplemented infants (p: 4.94e-56;
Bonferroni) (Figure 4).
The tration of HMOs in infant feces was analyzed by liquid
tography-mass spectrometry (LC-MS). The mean fecal HMO concentration in
samples from B. infantis supplemented infants (4.75 mg/g) was 10-fold lower than in
samples from unsupplemented infants (46.08 mg/g, P < 0.001 by s le
comparison test; Figure 5).
When infant fecal samples were analyzed by LC-MS, B. infantis
supplementation significantly increased fecal organic acids—particularly lactate and
acetate. Other SCFAs (formate, propionate, butyrate, isovalerate, isobutyrate, and
hexanoate) were in low abundance in the infant stool. Supplemented infants had
significantly r fecal organic acid concentrations than unsupplemented infants
(126.55 ,u mol/g vs 52.02 ,u mol/g). The median lactate to acetate ratio of B. infantis-
supplemented infants (0.73), was near the molar ratio of the "bifid shunt" (0.67), whereas
low-bifidobacteria samples (the unsupplemented group) had a lactate to acetate ratio of
0.26 (P < 0.0001, Mann-Whitney test).
2017/040530
Monitoring pH in infant fecal samples showed a correlation between pH
and the abundance of bacteria in the sample. The mean fecal pH of the
unsupplemented group was 5.97, while the feces from B. infantis-colonized infants had a
significantly lower mean pH of 5.15 at day 21 postnatal (P < 0.0001, Mann Whitney
test) (Figure 6A). The pH of feces from that n of unsupplemented infants who had no
detectable bifidobacteria at all was 6.38, which was statistically higher than either of the
other two groups (P < 0.0001 Mann Whitney test). Overall, when compared across infants,
te bifidobacteria populations in infant stools were negatively correlated with fecal
pH (Spearman’s p = —0.62, P < 0.01) and demonstrated a bimodal distribution of fecal pH
measurements that mirrored the abundance of bifidobacteria (Fig. 6). Comparing
weighted UniFrac distance matrixes, pH was a significant discriminator of sample
community composition (Mantel Test, = 0.32, P = 0.002).
Measuring endotoxin (LPS) in the stool samples showed higher
endotoxin in the unsupplemented infants (control) than in the supplemented infants
e 7). The endotoxin load was nearly 4-fold lower in infants colonized at high levels
with B. is (>50% Bifidobacteriaceae) compared with endotoxin levels in infants with
low levels of bifidobacteria, despite a high inter-individual variation (4.64 vs 5.15 Loglo
EU/mL, P = 0.0252, Mann-Whitney U). Endotoxin was icantly correlated with
Enterobacteriaceae relative abundance (P > 0.0001, R = 0.496), but not Bacteroidaceae,
the second most abundant Gram-negative family found in the present study (P = 0.2693),
and xin concentrations were inversely correlated with Bifidobacteriaceae
abundance (P > 0.001, R = -0.431). Thus, infants that had high levels of B. is
colonization had lower endotoxin levels as compared to infants that did not have high
levels of B. infantis colonization
Fecal cytokines in stool samples were ed at day 14 of
supplementation. Such cytokines include pro-inflammatory nes like lL-8 and TNF-a.
A typical immune se to pathogens involves the rapid activation of pro-
inflammatory cytokines (e.g., IL-8 and TNF-a) that serve to initiate host defense against
microbial invasion. Since excess inflammation can give rise to systemic disturbances
harmful to the host, the immune system has evolved parallel anti-inflammatory
mechanisms that serve to curb the production of pro-inflammatory les to limit
tissue damage. Interleukin 10 ) is such a molecule that can limit host immune
se to pathogens and prevent inflammatory and mune pathologies. Elevated
levels of pro-inflammatory IL-8 and TNF-a coupled with elevated levels of lL-10, blunting
the inflammatory response, are indicative of a significant atory battle going on
within the gut of the unsupplemented s (Table 1). In contrast, in the infants
supplemented with B. infantis, the flammatory cytokines are minimized as are the
levels of IL-10, indicating that the colon of these s is in a far calmer state with
respect to inflammatory responses.
11-10 (pg/mL) 329.78 23.68 0.0398
TNF-a (pg/mL) 151.16 21.63 0.0686
Table 1: Levels of fecal cytokines in fecal samples from unsupplemented infants (-
B. infantis) and infants mented (+ B. infantis].
Infant stooling (number and consistency) was recorded in this study as a
metric of GI function (Weaver et al. 1988). The number of infant bowel movements at
Baseline was the same between the supplemented (mean, 4.0/d; range (0.80-9.6)) and
unsupplemented groups (mean, 3.9/d; range (0.80-7.6)) but was significantly (P < 0.0005)
different during the Intervention (supplemented: mean,3.2/d, range (052-72),-
unsupplemented: mean, 5.5/d; range, (2.6-10.6)) and Post-intervention (supplemented:
mean,1.7/d, range (0.30-4.8); unsupplemented: mean, 4.4/d; range, (0.97-9.9)) periods
(Figure 8). The mean number of bowel movements were not only different between
groups (P<0.01) but also different across time within each group (P<0.0005). There was a
significant time effect (P < 0.01), time X intervention interaction (P < 0.0005) and
intervention effect (P<0.0005) for the daily number of infant stools. The number ofinfant
stools significantly increased from Baseline (P< 0.0005) for s in the unsupplemented
group and decreased from Baseline (P < 0.05) for infants in the supplemented group and
significantly decreased during the Post-intervention period for both groups (P<0.0005).
Parity was unrelated to the number of reported mean number of bowel movements/d
across all three time periods.
To examine the quality of infant stool, s reported the consistency
of the first bowel movement their infants produced each day using a validated stool
consistency rating tool for infants (Bekkali et al. 2009). The proportion of each stool type
(watery, soft, formed and hard) over each time period was calculated for each infant as the
number of days each type was reported divided by the total number of days per each time
period. The majority (95%) of the mothers rated stools as watery or soft. Maternal reports
for the proportion ofwatery stools during the intervention period was lower for infants in
the supplemented vs. unsupplemented group (0.20 vs. 0.33) and higher for the number of
soft stools [0.79 vs. 0.67). The change in the percent of watery and soft stools was
significantly different between the two groups. The percent of watery stools decreased
from Baseline to the Intervention period in infants in the supplemented group by 36% but
only 7% in the unsupplemented group [P< 0.05). As expected, the percent of soft stools
increased by 36% from Baseline to the Intervention period in the mented group but
only increased by 7% in the unsupplemented group (P < 0.05) (Figure 8).
Overall, the lemented infant had average stool frequency of
4.0/day, of which 33% were watery stools, and average stool pH = 6. The mented
baby had average stool frequency of y, of which only 20% were watery , and
the stool pH was d to 4.5. xin and other inflammatory markers, including IL-
8, lL-10, IL-6, and TNFa, appeared to be reduced in the infants colonized with the B.
Iongum subsp infantis such that the gut ecology was found to be in an anti-inflammatory
condition._The supplementation of B. infantis also facilitates maturation of the gut mucosa
as supported by the data showing less frequent and more mature stool consistency in the
breast-fed infants supplemented with B. infantis.
This experiment demonstrates that sbiotic infants can be
identified as ed to dysbiotic infants by the following: (a) an increased in the
lactate:acetate ratio to around 2:3 in the feces; (b) decreased frequency of bowel
movements as compared to a dysbiotic infant; (c) more mature stool consistency (i.e.,
more firm and/or less watery); (d) decreased pro-inflammatory cytokines [e.g., lL-8 and
lL-10) by around 10X in the feces; (e) decreased inflammatory LPS by around 4X in the
feces; (f) decreased pathogenic microbe levels in the feces; (g) decreased antibiotic
resistance gene load by around 3x in the feces; (g) titratable acidity above 2 mmol/g feces,
preferably above 5 mmol/g feces; (h) bifidobacteria levels of greater than 106, preferably
greater than 108' more preferably greater than 109 or 1010 in the feces; (i) B. infantis levels
of greater than 106, preferably greater than 108' more preferably greater than 109 or 1010
in the feces; and/or (j) decreased HMO levels present in the feces of at least an order of
magnitude, compared to dysbiotic infants. These indicators may be ed to
distinguish tic infants from non-dysbiotic infants across all mammals, not just
human infants.
e 3: Trial with formula-fed infants. A dry composition of
lactose and Bifidobacterium longum subsp. infantis (Strain EVCOOl, Evolve Biosystems
lnc., Davis, CA isolated from a human infant fecal sample) produced in an activated form
by cultivation in the presence of BMO according to PCT/U32015/057226, is prepared so
that it has an activity level of about 15 Billion CFU/g. This composition is combined with a
HMO or BMO syrup prepared by defatting a sample of human milk by centrifugation,
preparing a HMO trate by iltration where milk proteins are removed, and
then concentrating the filtrate under vacuum to a water content of less than 0.5. This
HMO syrup is combined with the ted B. Iongum to provide a ition of 2.0 g
HMO with a B. infantis titer of 5 x 109 CFU/dose. The ing syrup is packaged in foil-
lined stick packs wherein one dose represents about 2 g. Alternatively, the medicament
can be prepared in a dry form and packaged in stick packs or other forms of sachet. The
contents of individual dose packs are provided to formula-fed or mixed-fed infants on or
about day 7 of life and then on a daily basis for the subsequent 180 days. Infant fecal
samples are collected hout the trial and subjected to full microbiome analysis using
lllumina sequencing based on 16S rRNA and qPCR with primers designed specifically for
B. Iongum subsp infantis. The mented infants have a significantly higher level of B.
infantis than the unsupplemented infants whether vaginally delivered or red by C-
section. When the infants terminate supplementation with HMO plus B. infantis, the levels
of B. infantis in the gut drop off precipitously. Those infants that were colonized by the B.
longum subsp infantis have much lower levels of Proteobacteria (including Clostridium and
Escherichia species]. Infant fecal samples from the supplemented infants have acetic acid
levels about 100-fold higher than the unsupplemented infants. Other pro-inflammatory
markers including lL-8, TNFa, and PPARa and PPARg are reduced in the supplemented
infants indicating that the gut ecology is in an anti-inflammatory condition.
Example 4: Eguine Trial. A major horse breeding stable with over 70
pregnant thoroughbred mares had an ak of severe hagic ea among
foals born to the mares in that stable. These animals were found to be culture- and toxin-
positive for Clostridium difi‘icile. Seventeen foals were born during the initiation of the
outbreak, of which fifteen s became ill and required intervention, according to the
standard of care (i.e., antibiotic treatment) and two died. Another eight animals were born
and initially treated with a ation comprising 6X109 CFU Bifidobacterium Iongum
subspecies infantis [Strain EVBL001, Evolve Biosystems Inc., Davis, CA) per kg
ight and 5X109 CFU of Lactobacillus plantarum (Strain EVLP001, Evolve
Biosystems Inc., Davis CA) diluted in ed bovine milk which contained BMO. All
treated animals were given doses immediately at birth and twice per day thereafter for 4
days. In total, twenty five d foals, six did not develop disease. Two foals, who were
dosed starting at 12 hours of life rather than immediately at birth, developed a mild
infection by Clostridium difficile but red within 8 hr compared to the standard
recovery time of >24 hr for sick animals given the standard of care. No adverse events
were recorded among the animals and the dosages were well tolerated. A Fisher’s exact
test of the two populations (Standard of Care and Probiotic treated) yields a significant
ence in incidences of C. difficile infection (p = 0.0036) (Table 2).
—-_——
———_
Prophalyatic B.infant1's 25 4
+ L. um at birth
Outcomes Duration ofs motoms
—Lessthan 12 hours Greater than 24 hours
No treatment at birth _—
Prophalyatic B.infantis + L. 4 0
Iantarum at 12 hours
Table 2. Summary of e Data for Foals.
gh the treatment option where the animals were dosed at 12
hours of life failed to significantly reduce incidence of diarrhea, the severity (duration)
was ically shortened to 12 hours or less (p = ,- Fisher exact test, comparing
populations of diarrheal foals segregated by duration of diarrhea). The second option,
dosing at birth, significantly reduced the incidence of diarrhea (p < 0.0001). All animals
were dosed at birth with 6.6mg/kg of ceftiofur (Excede), and this did not affect health
outcome, related to diarrhea. Furthermore, none of the 25 animals treated with the
composition of the instant ion developed foal heat diarrhea, which typically affects
>50% of animals, and requires treatment in approximately 10% of cases (Weese and
au 2005). If a >50% risk is extrapolated to a hypothetical population of 8 animals
to match the 8 observed; this yields a significant reduction in foal heat diarrhea (p =
0.0256). tative PCR of foal fecal samples obtained during the study showed 1000-
fold increase in the abundance (on average) of bifidobacteriua (all s) after
supplementation. Using the Pfaffl method for relative abundance of a gene ce
(compared to 16S rRNA), it was determined that resistance genes for gentamycin and
tetracycline (aac6-aph2 and tetQ, respectively) were both significantly reduced by about
—30% in treated foals compared to control foals. Analysis of fecal samples also revealed
at d se in SCFA after supplementation, comprised mostly of an increase in
acetate.
Example 5: Determining Bifidobacterium levels in infant stool
samples. A fresh stool sample was collected from an unsupplemented infant and from a
B. infantis supplemented infant. The stool samples were collected from soiled diapers
using a collection wand that when rolled over the stool sample collected between 40-80
mg of feces. The wand is then placed in a chamber and 800 ul phenopthalein/ethanol/O.1
M NaOH solution was added and gently shaken. The phenopthalein/NaOH fecal
ition was filtered into a second chamber to remove particulate matter. The
clarified sample was viewed though the reading window. An exemplary device to the one
used is shown in Figure 9. In the samples from unsupplemented infant, the reading
window was pink indicating that the original fecal pH was above 6 and that this infant has
a low bacteria microbiome. In contrast, the result from the B. infantis supplemented
infant was yellow indicating that the infant microbiome contains high bifidobacteria and
the fecal pH was less than 6.
Claims (25)
1. A method of monitoring the health ofa mammal, comprising: a) obtaining a fecal sample from the mammal; b) determining the level of tic indicators in the sample; c) identifying a dysbiotic state in the mammal based on the level and/or content ofthe dysbiotic indicator in the sample.
2. The method of claim 1, further comprising treating the dysbiotic mammal by: i) administering a bacterial composition comprising bacteria e of colonization of the colon; ii) stering MMO; or iii) both (i) and (ii); wherein the bacteria and/or the MMO are administered in tive amounts sufficient to change the level and/or content ofthe dysbiotic indicator in the feces of said mammal to that of a non-dysbiotic level and/or t.
3. A method of maintaining the health of the mammal, said method sing the step of: a) treating a mammal by: i. administering a bacterial composition comprising bacteria capable of colonization of the colon; ii. administering MMO; or iii. both (i) and (ii),; and further comprising the steps of: b) monitoring the mammal by: i. obtaining a fecal sample from the mammal: ii. determining the level of a dysbiotic indicator in the sample; iii. identifying a dysbiotic state in the mammal based on the level and/or content of the dysbiotic indicator in the sample; and c) administering the bacterial composition and/or the MMO in se to the identified dysbiotic state.
4. The method of any one of claims 1—3, wherein the dysbiotic indicator is stool frequency, stool consistency, amount of short-chain fatty acid (SCFA), SCFA content, pH, amount ofbifidobacteria, amount ofB. infantis, amount of pathogenic bacteria, amount of lipopolysaccharide (LPS), amount of antibiotic resistance genes, amount of human milk accharides (HMO), and/or amount of proinflammatory cytokines.
5. The method of claim 4, n the dysbiotic indicator is (a) an increase in the lactate:acetate ratio to around 2:3 in the feces; (b) decreased frequency ofbowel movements as compared to a dysbiotic infant; (c) more mature stool consistency (i.e., more firm and/or less watery); (d) decreased flammatory cytokines (e.g., lL-8 and lL-lO) by around 10X in the feces; (e) decreased inflammatory LPS by around 4X in the feces; (0 sed pathogenic microbe levels in the feces; (g) decreased antibiotic resistance gene load by around 3X in the feces; (g) titratable acidity above 2 mmol/g feces, preferably above 5 mmol/g feces; (h) bifidobacteria levels of greater than 106, preferably greater than 108' more preferably greater than 109 or 1010 in the feces; (i) B. infantis levels of greater than 106, preferably greater than 108' more preferably greater than 109 or 1010 in the feces; and/or (j) decreased HMO levels present in the feces of at least an order of magnitude, ed to dysbiotic infants.
6. The method of any one of claims 2—5, wherein the step oftreating the mammal and/or the step of administering the ial composition and/or MMO results in the proportion of one or more pathogenic bacteria in the microbiome ofthe mammal to be reduced.
7. The method of any one of claims 4—6, n the pathogenic bacteria is ella, E. coli, Enterobacteria, Clostridium, Klebsiella, or combinations thereof.
8. The method of claim 6 or 7, wherein the pathogenic bacteria is reduced by 20% by the treatment.
9. The method of any one of claims 2—8, wherein the mammal’s colon has antibiotic resistance genes, and n the step of treating the mammal and/or the step of administering the bacterial ition and/or MMO results in the antibiotic resistance gene load of at least one gene being reduced by greater than 10%, 15%, 20%, 25%, 50%, 75% 80%, or 85%.
10. The method of any one of claims 4—9, wherein the SCFA comprises one or more of acetic, propionic, and butyric acids and salts thereof, and lactic acid or salts thereof
11. The method of any one of claims 3—10, wherein the amount of the bacterial composition and/or the MMO administered in step (a) is different than the amount ofthe bacterial composition and/or the MMO administered in step (c).
12. The method of any one of claims 3—11, wherein the periodicity and/or duration of the administration of the bacterial composition and/or the MMO administered in step (a) is different than the periodicity and/or duration of the bacterial composition and/or the MMO administered in step (c).
13. A method of increasing and/or ining a level of short chain fatty acids (SCFA) or organic acids in the colon of a , comprising: a) administering a bacterial ition comprising bacteria capable of colonization of the colon,- and b) administering MMO; wherein the bacteria and the MMO are administered in respective amounts sufficient to in a level of short chain fatty acids (SCFA) or organic acids in the feces of said mammal.
14. The method of claim 13, wherein the SCFA comprises one or more of acetic, propionic, and butyric acids and salts thereof, and lactic acid or salts thereof
15. The method of claim 14, wherein acetic acid makes up at least 30% ofthe SCFA.
16. The method of any one of claims 13—15, wherein said level of SCFA or c acids is the level tive of a healthy microbiome.
17. A method of reducing the antibiotic resistance gene load of at least one gene in a mammal’s gut sing: a) stering a bacterial composition comprising at least one species capable of consuming MMO by the internalization ofthat MMO within the bacterial cell, and,- b) administering an MMO at a dose representing over 10% ofthe total dietary fiber.
18. The method of claim 17, wherein the antibiotic resistance gene load of said at least one gene is reduced by greater than 25%, 50%, 75% 80%, or 85%.
19. A method of decreasing the frequency ofbowel movements in a mammal comprising: administering to the mammal a bacterial composition comprising at least one species e of consuming MMO by the internalization of that MMO within the bacterial cell, wherein the mammal is receiving MMO at a dose representing over 10% of the total dietary fiber.
20. The method of claim 19, wherein the firmness of the stool ition ofthe mammal is sed.
21. The method of claim 13—20, wherein the bacterial composition is administered for a period and in an amount ient to in the species capable of consuming MMO by the internalization of that MMO within the bacterial cell at a level of at least 106 CFU/g feces, preferably 108 CFU/g feces.
22. A method ofaltering the stool composition in a mammal comprising: administering a bacterial ition comprising at least one species capable of consuming MMO by the internalization of that MMO within the bacterial cell to the mammal, wherein the mammal is receiving MMO at a dose representing over 10% ofthe total dietary fiber.
23. The method of claims 22, n the level of LPS in the stool composition is decreased as compared to the stool composition of a dysbiotic mammal.
24. The method of any one of claims 22 or 23, wherein the level of pathogenic bacteria in the stool composition is decreased as compared to the stool composition ofa mammal not receiving the bacterial composition.
25. The method of any one of claims 22—24, wherein the pH ofthe stool composition is decreased as compared to the stool composition of a mammal not receiving the bacterial composition.
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Application Number | Priority Date | Filing Date | Title |
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US62/397,788 | 2016-09-21 |
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