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The multi-room cells are called eukariots and the bacterial one-room cells are called procariotes. Prokaryotes are
divided into two large groups called Bacteria and Archaea. I will not differentiate between Bacteria and Archaea in
this lecture, although they are very different on the phylogenitic level. When I say Bacteria I mean prokaryotes.
Bacterial cells can have many shapes, a few of them is shown in this picture The spherical bacteria are called
coccus, the elongated are called bacillus and the long spiral ones are called spirochetes. Many bacteria have a flagell
- a protein tail that they can use to move around, like the tail of a tadpole.
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It is estimated that there are about a thousand billion billion billion or 1030 bacterial cells on Earth. This is a very
large number that can be difficult to imagine. It is like the number of atoms in 10.000 liters of water or about ten
million times the number of stars in the universe.
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All these bacteria weigh a million billion kilograms or 2.000 times as much as the 7 billion people on Earth. The
reason that there are so many of them is that they are extremely adaptive and very small. Bacteria are between 0.1
and 5 micrometer long. In the lab we normally see bacteria as colonies on a petri dish. A colony, so small that it is
barely visible, can contain a million bacteria, and a colony one millimeter across contains a billion. Bacteria can grow
very fast. The fastest growing organism known to man is Clostridium perfringens. Given the right conditions it has a
generation time of 6 minutes and twenty seconds. This means that one cell can become a thousand in one hour and
3 minutes, a million in two hours and 7 minutes and a billion in three hours and ten minutes. Clostridium perfringens
is an extremly fast species, but bacteria as Escherichia coli and Staphylococcus aureus can grow with a generation
time of about twenty minutes, so things move fast in the bacterial world. The Nitrogen Cycle - Bacteria makes the
world go around Nitrogen is one of the most important elements for all life. The amino acids in our proteins and the
nucleotides in our DNA and RNA contain nitrogen. There is a lot of nitrogen on Earth, 80 % of our atmosphere is
nitrogen in the form of N2. But N2 is chemically a very stable
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molecule that cannot be used by plants, fungi or animals to make amino acids or nucloetides. The nitrogen in the air
must be converted to ammonium so plants can use it as fertilizers and build it into proteins and nucleotides. Bacteria
are the only living organisms that can bind N2 from the air and convert it into ammonium, so it can be used by other
living organisms. It is called nitrogen fixation and it is a very important process for the continuation of life here on
Earth. If bacteria were not able to fixate nitrogen from the air all the nitrogen in living organisms would eventually end
as N2 in the air, and life on Earth would die out. Plants of the Legume family - such as peas, beans and lentils - can
fixate nitrogen from the air. They can do this because they have a symbiotic relationship with the nitrogen fixating
Rhizobia bacteria that live in the roots of the plant. The picture shows Rhizobia
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nodules at the roots of a bean plant. Bacteria can also convert ammonium to nitrate and nitrate to free nitrogen N2. In
that way the nitrogen cycle is completed. The process where nitrogen is extracted from solution, converted to free
nitrogen and released into the air is called denitrification. We use bacteria with this dentrification ability to remove
nitrogen from wastewater, when we process our sewage in treatment plants before leading it out in nature. Bacteria
are everywhere.Wherever there is life on this planet, there are also bacteria. They live on their own or on and within
plants and animals. The Rhizobia bacteria living in the roots of Legume plants is only a single example of their
symbiosis with multicellular organisms. In fact, all animals with a gut have bacteria living in the gut that help them
digest the food. In many ways bacteria are absolutely necessary for our utilization of the food we eat. They make
vitamins in our gut, regulate our immune system and keep pathogenic bacteria away. For cows and other grass
eating mammals the bacteria in the gut play a very important role. Grass is mostly made of cellulose - a
polysaccharide that no mammal enzyme can break down. This is also known as dietary fiber. Diatary fibers are good
for our digestion,
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but they don't contribute to our nutrition. You won't get fat by eating grass, but cows do. The bacteria living in a cow’s
gut can break down the cellulose into simple sugars that the cows can use as nutrition. In other words: it is the gut
bacteria that make it possible for grass eating animals to utilize the energy of the grass. Bacteria are highly adaptive,
and they can live in the most extreme environments. Some bacteria can survive in hot springs at 120° Celcius and
grow above 100° C. Bacteria living
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at very high temperatures are called thermophiles. In the 80° C hot water of the hot springs in Yellowstone National
Park you can find the bacteria Thermus aquaticus. The picture shows the Grand Prismatic Spring, the largest hot
spring in Yellowstone. The bright colors are due to bacteria living at different temperatures in the hot spring. The
temperature is highest in the middle of the spring and falls towards the shore. This type of bacteria has become kind
of a celebrity among molecular biologists, because it has revolutionized all work with DNA. The wonder of this
enzyme, is that it withstands the repeated heatings to 80° C, that is needed to separate the DNA strands to melt DNA
for duplication. A very well-known application of this is the DNA test on crime scenes made by the police. This would
not have been possible without the thermophilic bacteria. There are even bacteria living in the radioactive waste from
nuclear power plants. The bacteria Deinococcus radiodurans can stand extremely high amounts of radioactivity. For
a cell the greatest threat from radiation is that it can break the DNA. As a defense mechanism the D. radiodurans has
four copies of its DNA in each cell, and the ability to use the other copies to repair a broken chromosome. The water
in the Dead Sea is a saturated salt brine in which neither fish, nor shellfish nor plants can live. Some bacteria
however, have evolved to be able to live in this extremely salty environment. They are called halophiles. That means
‘salt lovers’. As these examples show, Bacteria are the forefront of life on our planet, they live in the most extreme
environments and they were the very first forms of life on this planet. In the first 2 billion years of life they were in fact
the only kind of life on the planet. Cyanobacteria is a large group of bacteria that can make photosynthesis. They do
not need organic material for life, but can make all they need from sun light, water, CO2 and inorganic salts. The
cyanobacteria make up the first step in the global food chain. The photosynthesis we know from plants is an
evolutionary descendant from cyanobacteria. Bacteria that can live using only inorganic material and sunlight are
called photoautotrophs.
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But the opposite also exists. Deep in the Earth and on the ocean floor you can find bacteria that do not even need
light to survive. These forms of bacteria extract energy from inorganic chemical reactions like oxidating Fe2+ to Fe3+,
oxidating Mangan or Sulfur. They can even build new cells from inorganic material alone. This kind of bacteria are
called chemoautotrophs. We normally consider oxygen as a prerequisite for life – but there are some bacteria to
which oxygen is toxic. These organisms are called anaerobic bacteria – or anarobes. Anarobes come in two kinds –
the obligate anarobes, to whom oxygen is toxic, and the facultative anarobes, which can tolerate oxygen, but grow
better without it. Bacteria that grow best with Oxygen are called aerobes. There are many ways to charaterise
bacteria and one of the most important methods, when
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it comes to pathogenic bacteria, was invented in 1884 by the Danish medical doctor Hans Christian Gram as a
coloring technique used to visualize bacterial cells in the microscope. The method is called Gram staining and it
divides bacteria into two large groups: the Gram-positive that become purple and the Gram-negative that becomes
pink. The picture shows Gram staining of the Gran-positive Staphylococcus aureus and the Gram-negative E. coli.
The difference between Gram-positive and Gram-negative cells is determined by the way the cell wall is arranged
relative to the cell membrane. The Gram-positive cell has the cell wall outside the membrane, while the Gram-
negative cell has two membranes and the cell wall is located between the two membranes. The Gram stain shows a
very fundamental difference between bacterial cells. A difference that determines which type of antibiotic that can be
used to combat a bacterial infection. The Gram stain is fairly easy to make and it is almost always the first step in the
identification of a bacterial infection. To summarize: Bacteria are small unicellular organisms with very simple cells,
that are extremely adaptive. They can grow in very hostile environment above 100 C in high radiation, in very dry and
in very salty environment. Bacteria are absolutely necessary for life on Earth. But they are also very dangerous ,
because they are the reason for some of our most severe diseases. Bacteria are the fastest growing organisms on
Earth, and they exist in enormous numbers everywhere on this planet. When they cause infection, they are normally
characterized by a staining technique called Gram stain, that divide them into two groups Gram-negative and Gram-
positive bacteria. This difference is very fundamental and it is determined by the way the cell wall are arranged
relative to the cell membrane. Gram-positive and Gram-positive bacteria are normally treated with different kind of
antibiotics.
BIOFILM
Today I will introduce you to: When bacteria aggregate! Bacteria can exist either as individuals or together in physical
aggregates. The individual bacteria are termed planktonic bacteria and the phenomenon of aggregation is termed
biofilms. As you see in this short movie planktonic single cells freely move around between each other. This is also
how bacteria have been studied for the last 100+ years, namely in these shaken flask where bacteria grow at their
maximum speed. Since Robert Koch scientists have grown bacteria this way, and almost all we know about bacteria
is derived from these planktonic experiments. This is in striking contrast to biofilm mode, where the bacteria
aggregate and grow in layers as you see in this movie. This is a biofilm of Pseudomonas aeruginosa growing in the
laboratory, filmed over a period of 7 days. The bacteria are visualized by inserting a Green Fluorescent Protein GFP
and the series of pictures are obtained by confocal microscopy which enables 3D visualizations. As you can see it is
bacteria on top of bacteria, layers upon layers. A normal life for bacteria are in aggregates and this is also how
bacteria live in the environment, very few bacteria live in the planktonic mode and probably only for a short while,
while they are on their way to settle in new biofilms. The term biofilm comes from this slimy green layer of BIO
material which is seen everywhere non-sterile water runs across a surface. For example in a small creek and this
was also where the Late Bill Costerton first observed bacteria attached to a surface and named the phenomenon
biofilms due to Biomaterial forming a film on a surface. Other examples is the fouling of boats, a combination of
bacteria algae which has to be removed every now and then. However bacteria in drinking water is normal and not
dangerous Also in the human body biofilms are present. We have approximately 10 times more bacteria than
human cells in our body and most if not all are situated in biofilms, on the skin, in the mouth, in the intestines, the
vagina everywhere on the human body. The exception is the inside of the body, the blood, organs, brain etc. these
areas are free of bacteria when we are healthy. A problematic event is when we have foreign bodies insert into the
human body since these are prone for attachment of bacteria forming biofilms. Every time an implant or a catheter is
inserted the risk is that they get colonies or infected by biofilms. For implants the risk is 1-10 % but urinary
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catheters will be infected by a biofilm in 100% of the cases if used for more than a week. The most obvious place for
biofilms in the human body, which we cannot and should not prevent, is the plaque on the teeth. This is the white
stuff which you can scratch of your teeth and is part of the normal flora but if not removed 1-2 times daily will turn into
carries as seen in this picture. Biofilms on the teeth is also the first place scientist observed biofilms. So what is
the problem of bacteria in biofilms? Well if we first look at the planktonic bacteria in this small animation. As you can
see these are easily phagocytosed or eaten by our white blood cells (here golf ball shaped) and antibiotics can also
easily kill the bacteria as seen in this purple haze if the bacteria are not resistant. The problem arises when the
bacteria start to aggregate forming small fortresses as seen here. Then neither the white blood cells nor antibiotics
are capable of killing the bacteria, they become tolerant, and persistent. A consequence is of course if unwanted
bacteria form a biofilm in the human body it cannot be treated and you have a chronic infection… The scenario was
pinpointed in a article published in 1956 by Stephen Elek. Here a group of volunteers were divided in two. The first
group had around 7.5 million Staphylococcus aureus bacteria injected under their skin; the other group had only
around 100 Staphylococcus aureus bacteria injected under their skin but also a small mesh. In the first group with the
high number of bacteria, around half of the volunteers experienced an infections but not severe and they all resolved
by them self. In contrast in the group with very few bacteria, but also the mesh, all the volunteers experienced an
infection and none of them could resolve or heal by themselves, they had to have the mesh removed and the
infections stopped. The reason for this was of course that the bacteria had formed a persisting biofilm on the mesh
which the immune defense could not remove, but the immune defense fairly easy removed all the planktonic bacteria
in group 1. This really pinpoints that if a few bacteria are able to settle into a biofilm we have a problem. So what is a
biofilm? Well first of all it is the physical aggregation of bacteria, layers upon layers as is seen in this electron
microscopy picture. The bacteria are immobilized in the aggregate and if we zoom in even further we can see that
they are all attached and bound together by what we call the matrix, as seen here in these strings connecting all the
bacteria. The matrix is composed of extracellular DNA, proteins polysaccharides and molecules like that. The matrix
can differ between bacterial species and even within the same species depending on the habitat like whether it is in
the human body or out in the environment. Again it is in striking contrast the planktonic single cells as seen here. So
the definition we will use in this course is that a biofilm is “A coherent cluster of bacterial cells imbedded in a matrix” –
which are more tolerant to: most antimicrobials and the host defence, than planktonic bacterial cells. So it is the
aggregation of bacteria which is important, the layer upon layers, creating oxygen and nutritional gradients going into
the biofilms slowing down the growth of the inner layers of the biofilm. How many layers of bacteria it takes to form a
biofilm we do not know and it possibly depends on where it is located. In most chronic infections we find very small
biofilms 5µm in diameter but also large biofilms cm in diameter are observed. This we will come back to. What we
know today is that all chronic infections seem to involve biofilms and in the acute infections the bacteria are more in
the planktonic state. The biofilms in the chronic infections are protected from both the immune defence and
antibiotics, whereas the planktonic bacteria in acute infections can be eradicated first of all by the immune defence
and if not by antibiotics (if the treatment is initiated early enough) So even though most of our knowledge about
bacteria comes from these planktonic culture experiments research within the last 30 years or so have also focused
on the bacteria in biofilms and the consequence of this in health and infection.
INFEC PATHOGENESIS
Hello, my name is Oana Ciofu and I am an associate professor of Medical Microbiology at the Faculty of Health and
Medical Sciences at University of Copenhagen. In this lecture I will try to answer the question “How are bacteria
causing disease?” The mechanism that causes a disease is called pathogenesis. The word comes from the Greek
pathos ("disease") and genesis ("creation") The pathogenesis of an infection is the result of complex bacterial-host
interactions that can be summarized in 4 main steps: The microorganism (bacteria and fungi) has to: 1. penetrate the
normal barriers of the body (skin or mucosa) and adhere to receptors 2. survive the several filters of the defense
system of the body: the immune system (such as antibodies, polymorphonuclear neutrophils (granulocytes),
mononuclear phagocytes from the lung, spleen and from other organs) 3. multiply and recruit immune cells at the
infection site 4. the final step is elimination of the microorganism by the recruited immune cells and antibiotics An
acute infection is by definition a short term infection, usually lasting under six months, with sudden onset and usually
the microbial pathogen replicates fast. In acute infections the virulence factors play a major role in pathogenesis,
causing tissue destruction and recruitment of the immune response. Some examples of acute bacterial infections are:
acute uncomplicated urinary tract infection, acute pneumonia, acute diarrhea, meningitis, sepsis. In spite of an
immune system that is well functioning, the elimination step is unefficient in some cases and causes chronic or
permanent infections. A common cause of an impaired elimination of the microorganism is when microorganisms
organize themselves in biofilms. Biofilms are difficult to eliminate due to their tolerance to immune system and
antibiotics, as it will be explained latter during this course and cause chronic infections. The course of the infection
(acute or chronic) is independent on the type of the microorganisms, as most, if not all, microorganisms can grow as
both free-flowing (planktonic cells) or clusters (biofilms) and are able to cause acute and chronic infections, as it will
be illustrated later in a case presentation. While the bacterial virulence and/or an impaired immune system play a
major role in the establishment of an acute infection, a variety of other host factors can predispose to biofilm-related
infections Examples of host factors that predispose to biofilm-related infections are: 1. The presence of foreign-
materials like intra- or extravascular devices. It has been shown that the risk of biofilm-infection increases >100.000
times in the presence of foreign-materials in the body 2. Necrotic tissue due to impaired blood supply
(vascularization) is also increasing the risk of microbial adherence with subsequent biofilm formation (chronic
wounds). 3. Impairment of some components of the innate defense system involved in elimination of the
microorganisms, such as: - poor functionality of the cilli on the mucosal membrane of the respiratory tract (cystic
fibrosis, primary cilli dyskinesia)
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- impairment of the function of PMNs present locally at the site of infection (implant-associated chronic osteomyelitis)
4. Lack of effective antibiotic treatment due to an undiagnosed infection or antibiotic treatment in insufficient dosages
or for short
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periods of time being insufficient to eradicate the acute infection. This is mainly the case of infections in difficult to
reach sites leading to chronicity of acute infections like the case of chronic otitis media or chronic osteomyelitis. Once
established in a biofilm, the bacteria adapt to the host, making them less invasive and less virulent and more
persistent (they down-regulates many virulence factors such as toxins, type III secretion systems, motility that are
important for the establishment of the acute infection). Biofilms have been found in pacemakers, heart valves,
vascular grafts and stents, artificial joints and pins, and breast or other kind of implants Endocarditis Chronic
osteomyelitis Chronic otitis media Chronic sinusitis Chronic wounds Chronic lung infection in cystic fibrosis Infectious
kidney stone Foreign-body associated infections: intra- and extravascular catheters Bacteria in biofilm are also
detaching and therefore a mixture of planktonic, detached cells and biofilm bacteria exist during biofilm infections.
The detached, planktonic cells
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can seed an infection in other location and cause secondary acute or chronic infections. I would like to present a
case story of a patient with acute and chronic infection caused by the same microorganism: Staphylococcus aureus
A 65 years old overweight, diabetic patient with diabetic wound on the left foot is admitted to the hospital in poor
clinical conditions with low blood pressure and fever 39.9 0C.
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The patient has undergone 5 years ago a knee replacement with a knee implant due to arthrosis of the left knee. The
patient has increased inflammatory parameters such as high C reactive protein and leukocytosis and the blood
cultures are positive for Gram-positive cocci in clusters that are identified as S. aureus. A staphylococcal sepsis is
diagnosed.
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He is treated with antibiotics for 10 days and he is dismissed from the hospital in good clinical condition. Two months
after he has been dismissed from the hospital for the staphylococcal sepsis, he feels pain in his left knee. He is
consulted by an orthopedic surgeon who finds a loose prosthesis, inflammatory markers were raised and imaging
studies were consistent with bone infection (osteomyelitis). It is decided that
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the prosthesis has to be removed and a 6 weeks antibiotic treatment started. The infected prosthesis is removed and
sent to microbiological laboratory for culturing. The prosthesis is sonicated to disrupt the biofilm and S. aureus is
cultured. This patient has been admitted with an acute S. aureus infection: S. aureus in the blood (sepsis) with
entrance port the infected diabetic wound (S. aureus grows in biofilms in the
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chronic wound infection-see later). From the wound, the bacteria enters the lymphatic vessels, pass through the local
lymph nodes and enter the blood. The virulence factors allow the bacteria to survive and toxin production lead to
inflammatory cytokine and signs of sepsis. Through the blood (hematogenous way) the bacteria reaches the patients
knee implant and it attaches to the plastic prosthesis of the knee. After adherence on the implant via fibronectin
receptors, S. aureus forms a biofilm (bacteria embedded
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in slime having a low metabolic rate). The presence of an orthopedic implant also causes
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a local polymorphonuclear cell defect, with decreased ability to kill phagocytosed bacteria (impaired host factors).
This combination of bacterial and host factors
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lead to impaired elimination of the bacteria and causes a chronic infection. During the course of infection, bacteria
induce local bone destruction (osteolysis due to surface proteins of S. aureus) and it is responsible for the septic
loosening of the knee implant.
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For his sepsis infection, the patient is treated with antibiotics administered intravenously. The antibiotics are able to
kill the free-flowing bacteria (planktonic cells) in the blood but not the biofilm on the knee implant. The bacteria persist
causing a chronic osteomyelitis manifested as pain at the prosthesis site, two months after the antibiotic treatment is
stopped. Without removal of the implant, the chronic osteomyelitis infection can be a life-long infection with minimal
or no symptoms due to low inflammatory response. Reactivation
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of osteomyelitis after 50 and 80-years has been reported, underlining the importance of implant removal, the only
efficient way to remove the biofilm. In conclusion, this case history illustrates that the bacteria can adopt both living
forms: planktonic and biofilm mode of growth. The planktonic growth allows the bacteria to spread and the pathology
of the acute infection is mainly related to toxin production and subsequent inflammatory response (sepsis), while the
biofilm mode of growth allows the bacteria to persist both in the environment, creating a reservoir for opportunistic
pathogens but also in the body, causing chronic infections (in this case chronic wound and osteomyelitis).
WEEK 2
BIOFILM PROPERTIES
Hey my name is Morten Alhede welcome to this session about biofilm properties. There are many definitions of
bacterial biofilms but the most common definition is “an aggregation of bacteria on a surface embedded in a self-
produced matrix”. In order to understand biofilms we have to study them. And there are many ways to study biofilms
in laboratory. Assays vary from very simple and cheap models to complex but also more realistic models. In the
simple end, we have the crystal violet assay. This assay is a very cheap and simple biofilm model. The assay is only
assessing the quantity of attached bacteria in wells of a plate by applying and measuring a soluble stain bound to the
bacteria. Another simple way to study biofilms is filter biofilms. Here the biofilms are grown on top of a micropore
filter, which is lying on top of an agar plate. The filter enables transfer of the bacteria onto fresh agar plates when
needed and hence the bacteria can be supplied with fresh nutrients, allowing mature biofilms to form. At third model,
is a model we developed here at the department of international health, immunology and microbiology. We call it the
semi solid model. Here the biofilms are not attached to a surface but instead kept in a semi solid matrix composed of
digested meat powder, horse Blood and serum. The idea of this model is to provide a more realistic biofilm-model
where host components are included. The importance of host components is dual. Firstly, the formed biofilms are
different to biofilms raised in simple media – but secondly, the host components effectively bind antimicrobial
molecules and hence must be considered when evaluating anti-biofilm treatments. There are as mentioned
numerous of biofilm models, however the gold standard has always been the flow-cell system. This system utilizes a
continuous flow on top of the bacteria growing in a chamber. The idea is to keep the bacteria supplied with a low but
constant supply of nutrients and flush out non-attached bacteria, and hence to form a biofilm attached to the bottom
off the chamber. Many extremely beautiful images and videos of biofilm formation have been produced using this
assay. From a distance it looks like a simple stepwise process. First the bacteria attaches to surface then they grow
into huge towers looking like mushrooms and lastly the bacteria within the mushrooms disperse into the surrounding
media to form new biofilms and colonize new surfaces. However, lets take a closer look into the mushroom-looking
towers First the stalk is formed by clonal growth – meaning that the bacteria divide and multiply to form the stalk. In
contrast, the cap is dependent on motility – see here how the motile bacteria crawl on top of the biofilm to form the
top part of the biofilm. During this stage the so-called matrix is produced. The matrix is a slime substance composed
of mainly water, but also on all available macromolecules such as i. Polysaccharides ii. DNA iii. Protein These large
structures are meant to be interspersed by channels to ensure nutrient supply. However, when measuring oxygen
concentration within a biofilm it is obvious that the supply is not sufficient. The oxygen is rapidly consumed by the
bacteria in the outer layers and subsequently, the middle part is almost depleted from oxygen. Due to this lack of
oxygen and possibly also other nutrients the growth rate within a biofilm is dramatically lower than their planktonic
counterparts The matrix components, the lack of oxygen and subsequent the slow growth, are all contributing to the
hallmark of the biofilm – namely: extreme tolerance towards antimicrobials and immune cells. Due to these factors
the biofilm can simply resist doses of antimicrobials that would otherwise be lethal to the bacteria. Recall that
antibiotics mostly target metabolic process, so if they once penetrate the matrix there are no targets to inhibit.
Interestingly, it is only physiologically dependent processes that make the biofilm tolerant – therefore it is not a
genetic or inherited trait as is antibiotic resistance. Hence the all traits making the biofilm tolerant are reversible and
can be circumvented! However, are these matrix-embedded mushrooms found outside a flow chamber? Are they
found at the site of chronic infections and thus being important? So I guess the important question is: “are flow cell
biofilms a realistic model that resembles what is going on during infections?” In a recent publication we looked into
that question. Interestingly we found that biofilms found in the body are actually very small – ranging from 5 to
100um, and none of them looked like the enormous mushrooms found in the flow cells. In addition to being very
small the biofilms are often also very heterogeneously distributed – meaning they can be extremely hard to find and
diagnose. Another interesting observation was that even though Biofilms are per definition attached to a surfaced. It
has been shown that aggregates are equally tolerant to antimicrobials and immune cells as surface attached
bacteria. This conclusion is important since many in vitro assays are evaluating the antibiofilm properties with surface
attachment as an endpoint… What if the compound is just releasing the tolerant aggregate into the blood stream?
This stresses the need for more realistic biofilm models! So to sum up: We now that the biofilms are aggregates of
bacteria embedded in a matrix of various compounds. The hallmark of the biofilm is to be extremely tolerant to
outside threads. The tolerance is reversible and thus dependent on physiological properties rather than genetic and
inherited traits. That is why biofilms is NOT a part of the resistance problem we are also facing in our environment,
but needs its own research! Biofilms can be studied in numerous of different models in the lab, but it is important to
know that all models have drawbacks and no models resembles what is found in the nature.
The non-inflammatory defence is very important for the development of biofilm infections in the lungs of patients with
cystic fibrosis. In cystic fibrosis patients thick layers of stiff dehydrated mucus compromise the mucociliary escalator
system thereby disabling the ciliary movement, which results in poor self-cleaning capacity to remove invading
microorganisms from the airways. And it is well accepted that this defective self-cleaning capacity of the mucociliary
escalator is the main reason for the higher susceptibility to biofilm infections in the lungs of cystic fibrosis patients.
The immune response is the body’s response to antigens. Antigens include pathogen associated molecular patterns
such as extra-cellular DNA, various toxins, LPS and other microbial components. Traditionally, the immune response
has been divided in the innate response and the adaptive response. The innate immune response provides the
fastest response, but it has only little specificity. Approximately 300 receptors are involved in innate immunity. In
principle the innate immune system has no memory - the innate immune system will respond the same way if
infectious microorganisms infect the host several times. The humoral components of the innate immune system
include the complement system, cytokines and bactericidal peptides. The cellular component mainly consists of
polymorphonuclear leukocytes, also called the PMNs, the macrophages, Gamma Delta T cells and epithelial cells.
The adaptive immunity provides a slower response that may take weeks to mature. Contrary to the innate immune
response the adaptive immune response has high specificity and employs 1-10 million different receptors in the body.
This allows the adaptive immune system to develop ‘memory’ of infections –This ability to remember previous
infections is an integrated mechanism of all effective vaccines. The humoral components of adaptive immune system
involve antibodies and cytokines. Lymphocytes and dendritic cells dominate the cellular components. So which part
of the immune system is activated against biofilm infections? Some information may be obtained when considering
the type of infection that predominates in patients with primary immunodeficiencies. That is patients born with
particular deficient components of the immune response. We should also take into account that the infectious biofilm
is mainly caused by bacteria and by fungi. If we take a closer look of this table, which shows the type of infection
typically associated with the major categories of primary immune deficiencies, we see that against bacterial
pathogens antibodies, phagocytes and the complement system are all important, but against mycobacteria and fungi
it appears that the phagocytes provide the most important part of the host defence. In other words we could expect a
response by the phagocytes against the biofilm formed by both bacteria, mycobacteria and as well as by fungi. The
phagocytes of the immune response are dominated by PMNs and macrophages. The third component of the host
response to infection is the inflammatory response. The inflammatory response is a reaction to tissue damage, and is
not necessarily, as with the activation of the immune system, caused by antigens. Inflammation is characterized by
pain, swelling, heat, redness and by loss of functionality. The tissue damage leading to the inflammatory response
may be caused by physical insults such as pressure, temperature and radiation and by chemical insults such as
acids, bases and oxidants. In addition antigens such as toxins may also cause the tissue damage. Within seconds of
recognizing the injury, the generation of the inflammation is mediated by chemical signaling that loosens the vascular
structure. This loosening allows migration of attracted inflammatory cells into the area of the stimulating insults. The
early inflammatory cells primarily consist of PMNs and macrophages. The phagocytes immediately start to neutralize
the initiating stimuli, which could be toxic chemicals or invading microorganisms. Successful destruction of the stimuli
is followed by removal of debris and repair and restoration of tissue as part of inflammation. Sometimes the
inflammatory response fails to destruct the stimuli. In these cases there will be a persistent inflammatory response.
This persistent inflammatory response may eventually reinforce the inflammation by causing more tissue insults
resulting in even more tissue deterioration. It should be noted that an immune response may also develop during
inflammation and this immune response may be pro- or anti-inflammatory, meaning it may accelerate or dampen the
inflammation. Before successful neutralization the inflammatory response is called an “acute-type inflammation”.
After successful neutralization it is called a “chronic-type inflammation”. The acute-type inflammation is dominated by
PMNs, while the chronic-type inflammation is dominated by macrophages, monocytes and lymphocytes. The high
tolerance of biofilm against the host response prevents removal of the stimuli resulting in prolongation of the “acute-
type inflammation”. Hence, biofilm may cause chronic infections with an acute-type inflammatory response, that leads
to collateral tissue damage. Thank you!
Pt. 2
I will give examples where the host response to biofilm infection has been most firmly demonstrated.
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The polymorphonuclear leukocytes, the PMNs, constitute the major part of the body’s first-line defence against
invading bacteria and fungi. The response of the PMNs is stereotypical and consists mainly of migration to the site of
infection, engagement of the respiratory burst, engulfment of the invading microorganisms and secretion of
antimicrobial agents. Accumulation of PMNs and activation of the respiratory burst are the most firmly documented
events of the PMN response to biofilm infections. In particular, intense PMN accumulation has been observed in the
lungs of CF patients with chronic P. aeruginosa lung infection. In this section from an explanted CF lung with chronic
P. aeruginosa infection you may see the biofilm as it is stained with red fluorescence by PNA-FISH and the PMNs
are stained with DAPI resulting in blue fluorescence.
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You can also see that the concentration of PMNs is correlated to the concentration of P. aeruginosa. This indicates
that in this chronic lung infection the host response it associated with the burden of infectious microorganisms. So
what are the PMNs doing in these infected lungs? Why are the bacteria not eradicated? The answer to the latter
question is probably that the biofilm formation protects the bacteria from the otherwise bactericidal PMNs. This
protection may rely on production of the mucoid colonies and the secretion of rhamnolipids. But the consequence of
the PMNs is far more complex than just the possible eradication of bacteria. In fact, this graph shows that in CF
patients the deterioration of the lung function is associated with the activity of the PMNs and apparently not with the
burden of P. aeruginosa. One well accepted explanation for the lung tissue deterioration in chronically infected CF
patients is the oxidative lesions caused by the activated PMNs. Here you see that biomarkers of oxidative lesions,
like protein carbonyls, in the lungs are correlated to lung tissue deterioration in chronically infected CF patients. You
may also notice that the content of protein carbonyls, in the lungs are correlated to the content of PMNs. This
indicates that the PMNs are involved in the generation of oxidative stress during the chronic P. aeruginosa lung
infection in CF. The mechanism by which the PMNs are generating oxidative stress is called the respiratory burst.
Briefly, activated PMNs consumes molecular oxygen by arming oxygen molecules with electrons resulting in
formation of very reactive oxygen species, ROS. ROS may oxidate sugars, fat and protein and the ROS are essential
parts of the PMN armory. Here you see the ongoing respiratory burst in PMNs in expectorated sputum from the lungs
of CF patients. The generation of ROS is visualized on this microphoto of PMNs from the sputum as a red
fluorescence resulting from superoxide formation. Ongoing ROS production is evidenced from this figure where the
recorded signal from the respiratory burst quickly increases and remains high for up to two hours after isolation of the
sputum sample. But if the respiratory burst is suppressed by addition of the specific inhibitor, DPI, the signal for ROS
generation decreases rapidly. On this figure you see that the respiratory burst consumes the majority of molecular
oxygen, since the inhibitor of the respiratory burst, DPI, also reduces the consumption of molecular oxygen. In
addition, aerobic respiration only accounted for 16 % of the oxygen consumption in the sputum sample, as
demonstrated by the small effect on ROS formation and oxygen consumption of the addition of cyanide. The PMNs
are also called the Janus cells due to their ambiguous activity. In CF patients with P. aeruginosa lung infection this is
evidenced by the degrading effect on the lung function and a need for PMNs since inhibition of PMN migration results
in more exacerbations.
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Accumulation of PMNs at the site of biofilm infection is also known from other diseases. In the synovial fluid from
patients with device related prosthetic knee infection the concentration of PMNs is increased. In particular, during
infection with P. aeruginosa and Staph aureus. Thus the PMNs may also be activated in biofilm infections of
indwelling devices. In addition, accumulation of PMNs has also been demonstrated in chronic wounds involving
biofilm with P. aeruginosa and S. aureus. Thus activation of PMNs is not restricted to biofilm infection of the CF lungs
or indwelling devices. These examples further support the concept of engagement of the host response during biofilm
infection. However, so far only in the chronically infected CF lungs has an ongoing respiratory burst of the PMNs
been firmly demonstrated. Activation of the adaptive immune response is important in the clinical management of CF
patients with chronic P. aeruginosa lung infection. In fact, in this infection increasing numbers of precipitating
antibodies in the sera is an important biomarker for the transition from temporary infections to chronic biofilm
infection. This engagement of humoral components of the acquired immune response is accompanied with activation
of components from the cellular adaptive immune response, the T-helper cells. During maturation of the T-helper cell
response, the naive T-helper cell may mature into a TH1 or a TH2 type depending on the type of stimuli and the
interaction with dendritic cells. The direction of the T-helper cell maturation is crucial for the CF patients with chronic
P. aeruginosa lung infection, since a TH1-dominated response is associated with a milder lung disease than a TH2-
dominated response. These presented important roles of the host response during chronic biofilm infection may
inspire further investigation of the subject. Such investigations of biomarkers of the host response and optimization of
antibiotic treatment according to oxygen depletion by the PMNs have the potential to lead to improved clinical
management of patients with biofilm infections.
ORAL BIOFILM
Hello My name is Mette Keller I am a assistent professor in Cariology at the University of Copenhagen. In this
presentation I will tell you about Oral Biofilms Earlier you have heard about biofilm formation and commensal
biofilms. One of these biofilms is the dental biofilm which we call dental plaque. This biofilm is associated with some
of the most well know oral diseases like caries and periodontal disease but it also has beneficial effects. Though all
microflora is an important part of the host defence, since the resident microbes acts as a barrier for pathogens for by
competing for nutrients, and attachment sites in the biofilm In case of depression of the resident microflora by
antibiotic treatment, we often see superinfection by a candinda spieces. The biofilm is also thought to protect the
teeth from erosion, which is de-mineralization of the teeth due to intake of fizzy acidic drinks.
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So far, almost 700 different bacterial species have been identified in the oral cavity, and about 10-20 species
constitute about 90-95% of the bacteria present in an individual. Biofilms are formed at several surfaces in the mouth
such as the tongue and the teeth. The composition of the microbiota is very varying at different locations due to the
changing ecological factors such as pH, energy supply and presence of oxygen. Biofilm formation in the oral cavity is
most widespread on the teeth because, unlike the mucosa, it’s a non-shredding surface. The formation of the dental
biofilm starts almost immediately after tooth cleaning: If the biofilm formation is left undisturbed the tooth will be
covered by bacteria after two days and visible to the eyes after 4 days. The last picture shows the bacteria stained
with erythromycin. The bacteria do not attach directly to the tooth because the enamel surface is covered by a layer
of salivary proteins called the pellicle. The first bacteria to adhere are oral streptococci such as S. sanguinis, S. oralis
and S. mitis and they are called first colonizers. Facultative Actinomyces may also occur. They act as receptors for
other bacteria which co-aggregate with the first colonizers. Gradually, more bacteria adhere and the composition of
the biofilm becomes more diverse. Fusobacterium nucleatum is thought to play a key role since it is able to co-
aggregate with all bacteria in the biofilm. If left undisturbed the biofilm develops from primarily gram positive
facultative species to an increasing number of gram negative cocci and rods. The proportion of anaerobes also
increases. When the biofilm matures the microenvironment within the biofilm changes and therefore the composition
also changes. Several of the bacterial species involved in biofilm mediated diseases are present in small numbers
even under healthy conditions but changes in the environment can favor growth of the more virulent species. The
effect of environmental factors was describe by Phillip Marsh and named the ecological plaque hypothesis. According
to this, changes in ecological factors like access to growth factors, oxygen, pH and amount of gingival crevicular fluid
determine the composition of the biofilm. The formation of cavities in the teeth is called caries. It is demineralization
of tooth substance which happens when pH drops below 5.5. The pH drop is caused by acids produced by microbial
fermentation of sugars such as sucrose, fructose and glucose. If the periods with low pH are long and/or frequent the
microbial environment will favor growth of aciduric species and the biofilm will become less diverse. The cariogenic
biofilm is often dominated by mutans streptocci, lactobacilli and Actinomyces species. Mutans streptococci are
characterized by a strong ability to adhere to the tooth surface, production of extracellular polysaccharides, ability to
metabolize sugars into mainly lactic acid and being able to survive at very low pH. Lactobacilli are most often found in
already established lesions and are even more aciduric than the streptococci. With the new sequencing methods it
has become clear that more bacteria are involved in the process although they are not all cultivable. If the caries
process is allowed to proceed the demineralization of the tooth structure will create a pathway for the oral bacteria
into the pulp where the vessels and nerves are located. At first, the tissue is not infected but the bacteria causes an
inflammatory reaction with piercing pain/ tooth ache. The initial lesion is dominated by the same bacteria that are
involved in the caries process. If the pulp tissue becomes infected, necrosis will follow. When the pulp becomes
necrotic, the available nutrients for the bacteria change from carbohydrates from the oral cavity to proteins from the
necrotic tissue; further, the environment becomes anaerobic. This favors growth of proteolytic anaerobic bacteria.
They can spread through the root canal system and can even reach the apical area of the tooth and spread into the
jawbone. This can result in either an acute or chronic infection. The bacteria are forming biofilms in the pulp cavity
which makes them very difficult to eliminate. The microflora in the root canal changes according to the stage of
infection. Other major biofilm mediated diseases are gingivitis which is characterized by swollen and bleeding gums
and periodontitis which leads to loss of supporting tissue around the teeth and loosening of the teeth. Gingivitis is
thought to be a non-specific reaction linked to the amount of bacteria in the biofilm on the tooth. Periodontitis is more
specifically linked to the concerted action of a number of certain bacterial species. Under healthy conditions, the
gums are closely in contact with the tooth and there is almost no pocket in between. When more and more bacteria
are allowed to accumulate on the tooth their metabolites can initiate an inflammatory response. This leads to an
expansion of the pocket and increased flow of gingival exudate. This changes the nutrient source for the bacteria and
favors proteolytic species. As a result of proteolysis the pH of the subgingival environment increases slightly. A
number of species have been linked to periodontal disease although they also can be found in healthy conditions in
smaller proportions. Generally we see a greater diversity in the biofilm in periodontal disease with larger proportions
of pathogenic species. The species most often associated with development of periodontitis are Fusobacterium
nucleatum, Campylobacter, Prevotella species, Porphyromonas gingivalis, Tanerella forsythia and Treponema
denticola. Aggregatibacter actinomycetemcomitans is especially associated with juvenile periodontitis. The collective
virulence of the bacteria is increased by intercellular communication. Many of the virulence factors of the periodontal
pathogens are related to either direct tissue and cell destruction or up regulation of the inflammatory response
causing an indirect tissue destruction. Besides the microbiological aspect of periodontal disease, there are several
host-related factors such as smoking and genetic aspects influencing the progression of the disease. So the bacteria
initiate the degrading of the periodontium by direct tissue destruction, but it is the host’s inflammatory response that
determines the severity of inflammation and makes the process chronic. To summarize: gingivitis is mediated by the
amount of bacteria in the biofilm, whereas both caries and periodontitis are mediated by changes in the environment
that favors growth of pathogenic species. In caries we see a decrease in pH due to fermentation of sugars while
periodontal disease is related to proteolytic activity causing a small increase in pH – partly due to the available
nutrients.
SKIN MICROBIOLOGY
I am Magnus Ågren and professor in human pathology at the University of Copenhagen. Today I will introduce you to
skin microbiology. More than 10^10 or 10 billions of microorganisms inhabit our skin. This is astonishing considering
the suboptimal growth conditions of the skin. The main skin microbes are bacteria, viruses and fungi; normally these
are friendly without causing harms. However, the skin flora is constantly challenged by our every-day life activities;
we are in contact with other people and objects but also our hygiene routines and the use soaps and other cosmetic
products will influence the normal skin flora. Many different endogenous control mechanisms maintain the delicate
balance between the host and microbes. We think most of the bacteria on the skin are aggregated in biofilms. In this
session, I will describe the variation of microbial population on our body and the many defense systems that protect
us from attacks. We distinguish between resident and transient microbes. Resident microboes live more or less
permanently on our skin while the transient do not. Resident microbes are normally commensal or harmless and can
be of benefit to us by
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out-competing the growth of pathogenic microbes. Staphylococcus aureus is a common bacterium on our skin but
can colonize and invade under certain conditions. Corynebacteria are also common and rely on lipids in the skin for
their survival; nutrients withheld from Staph. aureus preventing them to become harmful. The commensal Staph.
epidermidis can prevent overgrowth of Staph. aureus. The commensals also deliver and induce antibacterial
substances,
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activate innate immunity and prime the adaptive immune system.
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The composition of the bacterial communities varies hugely with anatomic location. Important ecological factors are
the pH, temperature, topography and moisture of the skin. For example, the groin supports growth of microorganisms
that are very different from those thriving on the buttock skin. In this case, the decisive factor is skin moisture. We
also know that sugar or glucose and sodium chloride, table salt, promote biofilm formation. Other host factors that
determine the skin microflora are age and gender. The classical example is acne that develops due to hormonal
stimulation of sebum production that nourishes an anaerobic bacterium, Propionibacterium acnes residing in the
pilosebaceous gland. Apart from the microenvironment, the surrounding macroenvironment affects the skin
microbiome. The increase of eczema has been attributed to external factors that influence the skin microbiome.
Variations in temperature and humidity will change the skin flora although this has not been studied systematically.
Sun exposure will likewise influence the skin microbiome by delivering bactericidal ultraviolet rays.
At birth the baby is initially equipped with a so called type 2 T-helper cell orientated immune system, resembling that
of individuals suffering from allergic diseases. Exposure of microbes to the Gut Associated Lymphoid Tissue in
babies is thought to mediate a shift towards a type 1 T-helper cell phenotype – common in healthy adults – thereby
providing protection against allergic reactions later in life. However, unlike originally argued by in “hygiene
hypothesis”, we now know that it is not just a lack of expositor to microbes in general, which are causing these
problems. It is also important which microbes we are exposed to and when. Almost one hundred years ago, Elie
Metchnikoff, a Russian Nobel prize laureate, first used the term dysbiosis in order to describe an imbalance in the
composition and distribution of the microbiota. It has been suggested that a dysbiotic microbiota is causing
insufficient activation of healthy immune responses early in life, which will leads to systemic immunological deviations
and possible disease. Accordingly, the gut microbiota of infants who would later develop allergy has been shown to
harbor less Enterococcus and Bacteriodes and higher amounts of Clostridia compared to healthy infants. In addition,
allergic children have been shown to harbor a microbiota that differs in composition from the microbiota in healthy
children. Studies have shown that allergic individuals are less often colonized with Bifido-bacterium and Lacto-
bacillus in the gut and more often with Staphylo-coccus than healthy subjects. A general reduction in the microbial
diversity in the gut during early childhood has also been shown to be associated with allergy development. These
findings, whether species specific, or based on diversity, support the general hypothesis that an imbalance of the
human gut microbiome in early childhood, enhances the risk of development of asthma and other aller-genic disease.
Also lifestyle illnesses such as diabetes and obesity have been shown to correlate with such dysbioses. The
establishment of a balanced gut microbiota is therefore apparently vital for many aspects of human health, and
abnormal intestinal colonization during the first weeks of life, may alter both the nutritional, the immunological, and
the protection functions of the host microbiota. Thus, the first bacteria, that the infant is exposed to, are likely to
become the ones priming the process of healthy gut microbiota maturation. Several different factors, such as mode of
delivery, diet, and environment, influence bacterial colonization of the infant gut, but their specific contribution
remains unclear. The gastro-intestinal system has traditionally been considered sterile until birth, during which it
becomes rapidly colonized with vaginal and fecal bacteria from the mother and microorganisms from the surrounding
environment. However, some data suggest that gut colonization may even start before birth through an internal
transfer of maternal bacteria to the fetal digestive tract. But the mechanism for this pre-birth bacterial transfer remains
to be substantiated. Whether or not it happens before or after birth, the transfer of bacteria from mother to child is a
type of heritance of the maternal microbiota and factors, which determine the microbiome of pregnant mothers may,
therefore, also influences whether the offspring will develop allergy or not. Facultative anaerobes, such as Entero-
bacter, Entero-coccus, Lacto-bacillus, and Strepto-coccus species, are the first bacteria to establish. As these
bacterial populations expand during the first days, they consume the oxygen in the infant gut, thus creating an
anaerobic environment, favoring the growth of strictly anaerobic bacteria, including Bacte-roides, Bifido-bacterium,
Fuso-bacterium, and Clostridium species. With time, more and more anaerobic species are able to establish and
expand in the infant gut, thus replacing some of the facultative bacteria. Within the first year of life, and typically after
the introduction of solid food, the microbial community generally stabilizes and resembles that of an adult with the
majority of the microbial species belonging to the phyla Firmicutes and Bacteriodetes. Interestingly, the intestinal
microbiota of babies delivered by caesarean section has been reported to differ from that of babies delivered vby
normal birth, both in the timing of colonization and in the species composition of the microbiota.
Children born by caesarean section show delayed and less frequent colonization with Bifido-bacterium, Lacto-bacillus
and Bacteriodes species and more frequently colonized by opportunistic pathogens typically found on human skin
and in hospitals, such as Staph. aureus, Klebsiella, and Clostridium difficile. Furthermore, the total microbial diversity
of gut microbiota in vaginal-delivered babies is significantly higher than caesarean-delivered ones, and this effect can
still be detected during the first 2 to 3 years of life. In this connection, it is very striking that it has been shown that
children delivered by caesarean section have a two-fold increased risk of developing asthma and other allergic
diseases. Other factors can also have an effect. Evidence suggests that consumption of probiotics and antibiotics by
mothers during pregnancy leads to specific changes in the gut bacteria in new borns. Since probiotics and antibiotics
have been shown to alter the composition of the intestinal and vaginal microbiota, these data demonstrate that the
specific composition and diversity of the mothers’ microbiota during pregnancy play an important role in colonization
of the infant gut. The first 1000 days of life (starting from interception) is considered as the most important period in
any individual’s life; providing the foundation for health and later development of illnesses, and the establishment of a
natural and diverse gut microbiome seems to be a very important factor here. A huge research effort has, therefore,
been put into understanding the factors that lead to a dysbiotic gut microbiota in early life. The main influence on
early gut colonization is believed to be the mothers’ microbiota but several other factors could possibly contribute.
Gut colonization patterns have also been shown to be influenced by lifestyle factors such as feeding methods, diet
and family structure, this includes number of siblings and household pets. Among these are also differential
exposures to microbes during childhood as a consequence of the use of antibiotics. The uncontrolled use of
antibiotics represents an immense interference with the natural environment, potentially influencing maintenance for
a diverse gut microbiota and elimination of important beneficial bacteria. As I have described in this presentation,
consequences of such alteration in the gut microbiome have many potential negatives effects, including providing
opportunities for colonization of pathogenic bacteria and impairing a healthy human nutrition. The recent acceleration
of prominent changes to our life-style might have significantly altered the composition of the human gut microbiota,
thereby compromising normal health and well being. In order to firmly establish causal correlation, more research is
needed, but accumulating evidence suggests that our microbial partners do play a significant role in maintaining a
healthy body and avoiding diseases such as allergy and diabetes.