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We, as a developed
society, are told to wash our hands frequently and are taught to bathe daily at
an early age. Additionally, we stop our children when they are about to put
dirt or any other non-food substances in our mouth. We use bactericides and other
disinfectants on all surfaces in our homes to try and rid our homes of microorganisms.
We drink clean, purified water as well as pasteurized and highly processed
foods. A question that arises from this being, are we being too clean? There is
a phenomenon in science known as the hygiene hypothesis. This hypothesis
hypothesizes that a lack of early childhood exposure to infections agents,
normal flora, and parasites have increased our susceptibility to allergies and
has suppressed the development of our immune system (Hurst), (Yazdanbakhsh).
Microorganisms existed long before humans populated this earth and more
numerous than any other species. Like humans, microorganisms have evolved to
better adapt to their changing environments to increase their survivorship.

Although
these microorganisms have inhabited the planet longer than humans have, we
still try and get rid of them as best as we can. But there is evidence that
indicates the harmful effects this has on human health. A current trend in
developed nations is that there’s an increased number of cases of people reporting
allergies and asthma in the past two to three decades. (Yazdanbakhsh) Compared to developing
nations, there are less allergic and asthmatic cases. This trend is also found
when comparing urban and rural regions. The idea being proposed here is that the
“cleanliness” of developed nations is correlated with the increased prevalence
of autoimmune diseases as well as allergies and asthma as well as lack of early
exposure to microorganisms.

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Introduction

            There have been several studies that have examined the
link between cleanliness and lack of microbiota diversity to allergies and
autoimmune diseases. As mentioned previously, most studies demonstrate that
countries that are developing and have a higher exposure to microorganisms have
less numbers of people with auto immunities and asthma. The opposite can be
said for developed regions. In addition to this it can also be said that the
children in developing nations have more exposure to microorganisms and have a
higher diversity of microflora than children in developed nations exposed to
fewer microorganisms. One main difference between developed and developing
regions is the sanitation and subsequent exposure to microorganisms. Some of
these microorganisms such as parasites and bacteria are more prominent in a
developing nations environment. Most of the studies referenced in this paper
look at the exposure to microorganisms, specifically in childhood and infancy, and
some correlates them to later development of allergies and asthma.

The
history of hygiene and sanitation is not a novel concept. Hygiene practice
dates all the way back to 4000 BCE in Egypt. More recently, the Industrial
revolution of the mid-18th century. In addition, the discovery of
germ theory developed by Louis Pasteur in the late 19th century (Duncan).
Advances in hygiene have been a forefront for protection against disease. Additionally,
the implementation and widespread education on frequent and proper hand washing
has also decreased disease occurrences especially in medical practice. Hand
washing is also known to be one of the most preventative measures a person can
take to prevent the spread of disease

Sanitation
is another forefront for protection against disease. Sanitation methods have
roots in around 3200 BCE, where sewage was utilized to redirect waste and
sewage out of human contact by sending waste away from cities and homes or
underground (Abusiam). Another hallmark of
sanitation is having access to clean water free of parasites and viral and
bacterial pathogens. This has reduced the amount of water-borne illnesses such
as cholera and typhoid fever. These are instances where increased cleanliness
has improved overall global health of the population and others where harmless,
beneficial microflora is being diminished from our hygiene and sanitation
practices.

A
common theme throughout these papers is the effect that lack of exposure to
microorganisms and their infections have underdeveloped our immune systems.
This has caused our immune systems to be over reactive. A consequence of this
is that our immune cells will mount immune responses to harmless foreign
particles such as pollen and pet dander particles, and other common allergens. This
as a result causes a person to develop “allergies” to, in this instance, pollen
and animal dander. Because of this, whenever a person comes in contact with
pollen and pet dander or their specific allergens, their immune system is
activated and responds to the harmless allergen. There have been several
studies whose research questions include how the immune system becomes
sensitive to these substances, but are not relevant to this paper. There have
also been other studies that study different techniques of improving the immune
system so that there is no response mounted for harmless foreign substances by
introducing more microbiota into the gut, such as hookworms, but, these select
papers will focus on the microbiological effects of a lack of needed microflora
rather than the effects introducing more microflora into the body.

Support
for the Essentiality of Microflora

There
are some papers that demonstrated the essentiality for microflora in the gut,
currently it’s thought that our gut is considered impoverished of microflora
due to a lack of exposure to microorganisms as well as the lack of
microorganism diversity. This microorganism population develops quickly until
around toddler age, when an adult-like gut microbiome composition is established
(Yatsunenko). It is thought that if the establishment
of stable adult gut microbiota is introduced in infancy, it may serve as a
lifelong predictor of health effects (Hill). Proper establishment could suggest
a lesser likelihood of developing disorders associated with immunity. The
opposite can be said if proper gut biome establishment is not established in
early childhood. There is a study conducted by Azad et al.by the Allergy,
Asthma & Clinical Immunology, studying the hygiene hypothesis. In this
study, researchers looked at children living with siblings and pets and then
studied the established microbiota found in their guts. The notion behind this
study was that infants/children that were exposed to siblings and pets in their
homes had a higher diversity of microbiota located in their gut. The reason
behind this study was to test to identify if there was a link between infants
with low microbiota gut diversity and allergies (Azad et. al).

The
methods of this study were simple. A population comprising of twenty-four
healthy, infants found from Canadian Healthy Infant Longitudinal Development
(CHILD) birth cohort. The mothers of these infants were given a questionnaire
that included the question of whether pets or siblings were present in their
households, as well as questions unrelated to this study. Fecal samples were
then collected from infants during home visits from researchers. Next, Whole
genome DNA was extracted from 40 mg of stool using the FastPrep DNA for Soil
Kit (Azad et al.). PCR was then used before gene sequencing. 50 nanograms of
cleaned/extracted product from each sample was combined and concentrated for
sequencing. Taxonomic classifications were then made using the microbiota found
in the fecal sample. Finally, fecal microbiome biodiversity and relative
abundance of bacterial taxa according to household pets and siblings were
statistically analyzed for comparison amongst the population to yield results.

Results
from this study indicate that there was an increase in diversity of microbiota
in infants that co-habited with pets. Also, microbiota diversity was decreased
in infants living with older siblings and without peers. Other results
indicated that infants co-habiting with pets display and under-representation
of Infants living with pets exhibited under-representation of Bifidobacteriaceae and
over-representation of Peptostreptococcaceae;
infants with older siblings exhibited under-representation of Peptostreptococcaceae. In infants that
were exposed to both older siblings and pets had a low relative abundance of Bifidobacteriaceae.

This
study went a step further and used the provided fecal samples for detection of
colonized C. difficile and this was
because of its role in allergy outcomes. It was found that C. difficile prevalence tended to be higher among infants with
pets, and lower among infants with siblings: C. difficile was detected in 67% of infants with pets, compared
with 22% of infants without pets and 31% of infants with older siblings,
compared with 73% of first-born infants (Azad et al.). The conclusions drawn
from this study suggest that infants with pets had an increased microbiota
diversity than infants with older siblings. This would make sense because pets
carry a different type of microflora than a human infant would. These infants
that encounter these pets were also found to have some of the microflora
associated with household pets.

In
relation to the hygiene hypothesis, this study supports the notion that
children that have exposure to more microorganisms have more developed immune
systems. This is evidenced by the prevalence of C. difficile to be higher in fecal samples of infants with pets
because they are exposed to more microorganism than infants without pets and
only exposure to others of the species such as older siblings.

Unfortunately,
there are many shortcomings that correspond with this paper. The biggest being
that the paper could not draw concrete conclusions and could only suggest their
conclusions. This was due to their inability to exude results that were
statistically significant. One way that the paper overcame this shortcoming was
including other studies that had also suggested and/or concluded that there are
inverse associations between early-life pet exposure and allergic disease. These
studies, however were both from data in developed nations, one from Sweden and
one from America. Another shortcoming was that this study was done only using
Canadian infants which makes this study’s conclusions less applicable to other regions.
I think the study could have been strengthened by having comparable data to an
undeveloped country and then made comparisons to a developing nations data.

This
study demonstrates that the diversity of the infants’ gut microbiota is
important and gives researchers some insight about the applicability of the
hygiene hypothesis and serves as an example of the benefits of having a larger
diversity of gut microbiota. This also emphasizes that a person’s environment
and exposure of a variety of microorganisms different from one’s own
microbiota, especially at a young age, plays a role in the development of our
immune systems (Maynard).

It’s hard to know when a person’s the first
exposure to microbes is. It’s thought that it’s during birth and later skin to
skin contact, but other scientists have speculated this occurs even earlier,
which would challenge the dogma that the fetuses’ uterine environment is sterile (Aagaard). In the womb, the fetal environment is tolerant to the mother’s
alloantigens and after birth, the baby is immersed in environmental antigens (Alice).
Recently, trace amounts of
bacteria have been found throughout the amniotic sac and the placenta. It has
even been found in the fetus’ intestines (Perez-Muñoz). If this is true,
then it confirms the idea that a child’s microbiome is established earlier than
birth. This idea is still new to research and not many studies have looked
into this, so it is difficult to conclude as to when a person’s first exposure
to microorganisms happens.

There
are other studies that examined the earliest of exposure to microorganisms in
babies that were born Caesarian section. A cesarean delivery is a surgical procedure in which a fetus is
delivered through an incision in the mother’s abdomen and uterus. (American College of Obstetricians and Gynecologists) Babies that are delivered naturally,
vaginally, are introduced to microorganisms from vaginal fluid they encounter
while exiting the mother’s birth canal. Caesarian sections do not offer the
same introduction to microorganisms so the doctors coat the baby in fluids from
the birth canal of the mother. This to an extent has the same effect, but may
not be as effective. This is another example that demonstrates the essentiality
of exposure to microorganisms at a young age.

A
paper, published in the Journal of Allergy and Clinical Immunology, focused on
the concept that C-section deliveries have been associated with increased risk
of childhood immune-system related to allergies and asthma are suggestive that early
microbe colonization plays a crucial role for proper immune maturation. This
paper also focuses largely on the microbiological importance of microbe
colonization in the gut and airways that occurs at the time of birth. This
study describes that their objective was to describe the influence of delivery
method on gut and airway colonization patterns in the first year of life in the
Copenhagen Prospective Studies on
Asthma in Childhood2010 (COPSAC2010) birth cohort (Bisgaard).

This
study had several components to the methods they utilized and made it a point
to include that all protocols and ethics were considered and carried out in
their methods. The data were obtained from an ongoing Danish cohort study where
738 unselected pregnant women along with their 700 children were followed from
pregnancy in the third trimester. (Bisgaard) The category of interest was mode
of child birth delivery; natural, emergency cesarean and elective cesarean. The
delivery methods were dichotomized to be natural birth vs cesarean. In order to
test the microflora innate to the infants, fecal samples and hypopharyngeal
samples were collected at various times. Fecal samples were collected one week,
one month, and one year after birth (Bisgaard) Hypopharyngeal aspirates were
collected at the research clinic one week, one month, and three months after
birth. 

These
samples were then cultured within 24 hours of being collected and cultured on
to selective and non-selective media and aerobically incubated. Blood agar and
chocolate were used to culture the bacteria in the fecal and hypopharyngeal
aspirate samples. These cultures were incubated under microaerophilic
conditions. (Bisgaard)
Microbial identification was then performed according to growth on selective
media (blood agar and chocolate agar), characteristics of colonies, and
cellular morphology. Statistical analysis using the ?2 test was
used to yield results of this study.

Results from fecal colonization patterns
of cesarean sections revealed that at one week Cesarean section and fecal
colonization patterns there were associations found with Citrobacter freundii , Clostridium species, Enterobacter cloacae, Enterococcus faecalis, Klebsiella oxytoca, Klebsiella pneumoniae and Staphylococcus aureus whereas
colonization by Escherichia coli was negatively
associated with birth by means of cesarean section. (Bisgaard) The most significant
difference in microbial colonization between natural and cesarean births was
that E. coli has a positive
association for natural birth and a negative association for both forms of
cesarean. At one month after birth, cesarean section was significantly
associated with colonization by Clostridium species, K. oxytoca, and K. pneumoniae, whereas
colonization by E coli was negatively associated with birth
by means of cesarean section.

There were some differences between
delivery methods that were more apparent at certain ages of the neonates. At
one year of age, the study found minor differences between delivery methods.
Cesarean births were only significantly associated with increased Citrobacter
braakii. It was also found that the differences were most significant
at one month after birth and least significant at one year.

Results from the hypopharyngeal
colonization patters and cesarean sections indicate that there were no
observable effects, between different birth methods, in the colonization patterns
of the neonates airways. It was not until one week after birth where neonates
born by cesarean have a positive association by S. pneumoniae. In the airways, there was also a negative
association with E.coli. And at three
months after birth, the only difference was that there was a positive
association with the colonization of E. faecalis. Much like the fecal
colonization patter, there was a positive association with E.coli colonization in babies born by natural birth, but less diversity
of species found. This is interesting given that there is speculation about
children born cesarean section and a link to the development of allergies.

This study overall covers all its bases.
There are sections included that describe in length all the protocols followed for
each procedure. The study was also very focused and effectively presented the
data. The figures were also easy to follow, and the flow was not complicated.
An aspect I admired about this paper was it included a section about the paper’s
strengths and limitations. A great strength of this paper was how meticulous
they were in data collection to obtain fecal and hypopharyngeal samples. Along with
the strengths, there were also limitations of this study.

The largest limitation being that it is difficult
and unlikely to culture anaerobic bacteria species in the lab on selective and
non-selective media. The study also used this logic as to why natural births
were found to have a less diverse microflora. It’s thought that the natural
births of this study could have had more anaerobic species present that did not
present themselves in the lab. This however could be true for plates that had
microflora cultured on them from cesarean births.  Something that may have been a beneficial addition
to this study is the inclusion of culturing the bacteria on other types of
plates. This may help eliminate the confusion with results and the lack of
anaerobic microbes represented in the data. Also, this study’s results about
hypopharyngeal colonization patterns are slightly unclear. More research is
needed specifically on the microbe colonization patterns in the airways.

An additional study found similar
findings. Their results indicated that vaginally delivered infants’ microbiota
remained stable for both phylum and genus level during the 24-week period that
they were examined. Full term Caesarean birthed infants were found to have an
increased amount of Firmicutes when
their fecal samples were tested. These infants were also found to have
decreased abundance of actinobacteria one week after birth when compared with
full term infants born vaginally (Azad, MB). This study also demonstrated the
importance of breastfeeding after the infant is born. Breastfeeding serves as
an additional means of introducing microorganisms into the infants’ microbiome
to help establish a healthy gut biome.

Early exposure to microorganisms is
crucial to the development of the immune system, just as the stated by the “hygiene
hypothesis” and this second study’s data does represent what is being said in
the hygiene hypothesis, but there are not many studies that have examined how
important exposure to different microbes is, especially for children. It’s
important that advances in education dealing with this topic are made by
creating new and similar experiments to the three studies mentioned here. It’s
crucial that studies in this field continue to expand to try and fill in the
gaps and resolve the limitations found in the study focused on cesarean and
natural births.

Conclusion

            From these studies, it is important
for health throughout human lifespan to establish microorganism colonization in
our guts. These studies present methods of introducing microbes into the gut
and respiratory tract as well as presenting methods that serve to increase the
diversity of microorganisms found in our gut. I see further expansion of this
paper if the immune system and diseases/disorders related to immunity were examined
closer. This is a paper grounded in the benefits and importance of establishing
a healthy microbiome in the gut. Another way to strengthen this paper would be
to find studies that demonstrate the differences between gut microbiota between
developed and developing nations. I found it difficult to find studies that had
this focus and didn’t just broadly mention this in their introductory sections
of their papers. One of the most important methods that influences gut biome is
the birth mode; caesarean or vaginal. It’s during this time that initial bacterial
populations establish themselves (Azad), (Adlerberth), (Fanaro). Other
factors that influence the gut microbiota are living with siblings, or having
pets. One study lists suggest that breastfeeding also promotes benefits for
microorganism colonization.

            This all
relates to the hygiene hypothesis and emphasizes the significance of the “hygiene
hypothesis.” This hypothesis stresses that early exposure to microbes are
essential for proper maturing of our immune system. Without the presence of
these microbes, autoimmune disorders can develop as well as other health implications.
As nations are developing, there is a higher abundance of microorganisms. This
in part is due to their lack of sanitation and infrastructure in comparison to
developed nations. In addition, these underdeveloped nations have less instances
of allergies and other immune disorders which is thought to be related to the
lack of exposure to microorganisms in developed nations. This lack of exposure
is consequently thought to cause a lack of microbial diversity and abundance
colonized in the gut. 

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