This course will give you an introduction to bacteria and chronic infections. Leading experts in the field will make you familiar with the fundamental concepts of microbiology and bacteriology such as single cell bacteria, biofilm formation, and acute and chronic infections.
5.1 Diagnosis of Chronic infections, by Professor Thomas Bjarnsholt
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來自 University of Copenhagen 的課程
细菌和慢性感染
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從本節課中
Diagnosis and treatment of chronic infections
Hi hello Biofilmers welcome to module 5.During this module you will learn about the difficulties of diagnosing and treating chronic infections.
I hope you will enjoy
CheersThomas
與講師見面
Thomas BjarnsholtProfessor
Costerton Biofim Centre
Hello my name is Thomas Bjarnsholt and I am professor in Chronic infections and Biofilms
at the University of Copenhagen. Today I will introduce you to Diagnosis of chronic infections
Diagnosing chronic infections is just as complicated as treating these infections. There are 3
main issues when we talk about diagnosing biofilms in chronic infections, sampling the
bacteria, identifying the bacteria in the sample and finally proofing whether the bacteria
were in a biofilm. I will in this presentation tell you about some the experience we have
within this. In the chronic infection of cystic fibrosis
patients it is very easy to get good samples since they cough up phlegm all the time and
it is fairly easy to culture and diagnose the bacteria here. This is not the case in
other Chronic infections . Here it is difficult to sample to culture and diagnose bacteria.
This is the only exception all other chronic infections are difficult. I will start by
telling about aabout a study we did on patients with chronic wounds. We examined wounds from
22 patients and we did both standard culturing and we looked in the tissue for aggregated
bacteria using the same methods of staining and microscopy as you have already seen. In
the study we could culture bacteria from 19 of the 22 wounds and we could culture Staphylococcus
aureus in 12 of the wounds and Pseudomonas aeruginosa in 5 of the wounds. This is very
much in line with what other research teams have observed. Then we turned to investigate
the wounds by microscopy to look for biofilms. Using microscopy we could find biofilms in
13 out of the 22 wounds, again this correlates to another study which was published shortly
after ours. However when we looked for individual species of bacteria we could only find 2 out
of the 13 wounds with Staphylococcus aureus biofilms but suddenly 9 out of the 13 wounds
contained biofilms of Pseudomonas aeruginosa. This was a little strange.
This let us to the question are bacteria in biofilms/chronic infections difficult to culture?
So I have a quiz for you. Are bacteria in biofilms unculturable?
Yes and no. The question arose from an earlier publication looking at menses tampons from
women. Here the scientist could detect bacteria by molecular techniques meaning that they
could find traces of bacterial DNA but they could not culture any. They concluded that
bacteria in biofilms are unculturable. Since we culture bacteria all the time from biofilms
in our laboratory we went to look for another explanation. However we also know that most
of the bacteria on planet earth are unculturable using the techniquesKirsten Mottram
and growth media we have today. We find only whatr we are looking for.are
In our study it turned out that it was not because the bacteria are unculturable but
that they were not sampled the right way. When we examined the wounds closer we could
see that Staphylococcus aureus here in green resided on top or close to the surface of
the wounds, whereas Pseudomonas aeruginosa in red was observed deeper into the wound.
Since the samples for culturing the wounds were done by cotton sticks swiping across
the wound surface they could easily pick up Staphylococcus aureus but only in a few cases
Pseudomonas aeruginosa since they were buried in the wounds.
This was pointed out even further by another study where the distance from the wound surface
to the bacterial biofilms was measured. As can be seen from the graph Staphylococcus
aureus resided in the upperpart down to 30µm and Pseudomonas aeruginosa resides in the
area from 40-70µm. This clearly indicates that the sampling from a chronic infection
is very important since the bacteria are not evenly distributed.
This we pinpointed in another study. Here we collected wounds from another group of
patients and divided each wound into 5 pieces, a central piece, a 12 o’clock, 3, 6, and
9 o’clock piece. Hereafter we determined the number of bacteria present in each small
part of the wound by molecular techniques. As can be seen form the table the number of
bacteria vary very much depending on the position in the wound. This indicates that not even
a biopsy taken from a chronic wound or a chronic infection is a representative sample, again
because the bacteria are so unevenly distributed. So this was a little about the difficulties
of sampling representative material from a chronic infection. However one would might
ask why not just take a blood samples and look for signs of infections? This is possible
for acute infections and can in many cases reveal the presence of bacteria. BUT for chronic
infections, this is not possible since the inflammation is restricted to the site of
infection, i.e. the wound, the implant etc. As it is now we do not have any good methods
to get the right samples. However if we take a sample from a chronic
wound and we DO find bacteria, then they have to be identified to be able to treat them
with the right antibiotics. The identification itself is not really a problem since we can
identify most bacteria, at least the ones involved in human infections, either by culturing
or by molecular methods, i.e. their DNA finger print. The problem arises since we often find
several bacteria present in these infections. As can be seen from these studies most often
chronic wounds contain more than 5 different bacteria – which to treat? This is the problem,
should they all be treated, are they all in a biofilm?
This table gives an overview of the advantages and difficulties of some of the diagnostic
methods we use: If we start by culturing: the advantage here
is that if we culture bacteria we know they are present (if it is not a contamination
but that can happen for all the methods). Since we have cultured the bacteria we can
easily test which antibiotics they are susceptible to, by growing them in the presence of antibiotics
and look for those which kill the bacteria can be used for treatment. Another advantage
is that we can get an estimate of how many of each species were present in the sample.
The pitfalls are that as I just mentioned, that bacteria are very heterogeneously distributed
so the sampling might miss some of the bacteria and of course then they are not cultured.
Another problem is to find the focus to actually get some bacteria. When we have cultured say
5-6 bacteria the next question is which ones are causing the infection and are some of
them contaminations? Lastly we could experience bacteria which were unable to grow, and they
would of course not be detected by conventional culturing. An additional problem is that just
by growing the bacteria does not reveal anything about whether the bacteria were planktonic
or in a biofilm. For the molecular methods – the search for
bacterial DNA, we can find bacteria even though we cannot culture them and we can also find
low numbers of bacteria. These are the advantages. The disadvantages are as with culturing, to
get the bacteria in the sample – the heterogeneous distribution, finding the focus and whether
the identified bacteria are pathogens or contaminations. Also as or culturing were the sampled and
identified bacteria biofilm or planktonic. The last main method for diagnosing bacteria
is microscopy. The advantage of this is that if bacteria are observed it can be directly
confirmed whether or not they are in a biofilm. It is also possible to see the interaction
with the tissue and whether inflammatory cells are present, all adding to the diagnosis.
As for the molecular techniques bacteria can be observed even though they are culture negative.
The disadvantages is as with the other methods the heterogeneous distribution and finding
the focus. The contamination issue is not a problem here since the microscopy will reveal
the interaction with the tissue and whether inflammatory cells are present indicating
the bacteria are causing the infection. However also important, microscopy is really slow
and time consuming and it is often not possible to identify all the species present.
In addition to this, microscopy is also a skill one has to learn, many artefacts are
present in human tissue and samples. Just look at these pictures here… The bacteria
are really only easy to identify in the upper left picture. The 3 other pictures are likely
not bacteria even though some of it might look like it.
So another quiz question for you, what should we look for?
Should it be bacteria on the surface, should it be large biofilm or should it be bacteria and inflammation?
Based on what we've learned so far what should you look for in a microscope?
Recently we surveyed the literature to find
evidence and to understand biofilms in infections, the in vivo biofilm. As can be seen from this
list these 3 representative pictures we found the in vivo biofilm to lack these mushroom
structure, often seen in the laboratory (in vitro). Most of the biofilms in infections
were small microcolonies; they all had an additional layer of host material. This is
important since this creates additional layers for oxygen and nutritional gradients. A hallmark
was also the presence of host defence and inflammation; of course a heterogeneous distribution
and importantly many of the biofilms were not associated to surfaces most often they
were imbedded in puss or host material. Also the biofilms were often not connected to healthy
tissue. So to sum up: Both culture and molecular identification
reveal on the presence of bacteria but not on the orientation and or the distribution
of the bacteria. Microscopy does this but is slow and time consuming. However the biggest
problem so far is the sampling, to get the right samples contain the bacteria causing
the infection. We are of course working on solving this and
some of the questions we are trying to answer are:
To determine the role, if any, of the numerous bacteria identified in chronic wounds and
other chronic infections Are they all contributing to the pathogenesis?
Are they all situated as biofilms? Are they all homogenous distributed?
Do the anaerobes play a role? In addition, can we find an inflammatory marker
which can be used to diagnose the presence of bacterial biofilms? We need to know if
bacteria or fungi are present to initiate treatment, and early diagnosis is equal to
early treatment maybe before the bacteria situate themselves in biofilms and become
chronic. So are we totally lost if we cannot see or
find anything? No we can still look at the patient:
We can look at the medical history of the patient, does he or she have any predisposing
factors such implanted medical devices, cystic fibrosis, diabetes obesity act.
We can look at recurrence of infection; does the patient experience an infection over and
over again, especially if it is the same bacterial species and even more suspicious if it is
the same bacterial strain? This indicates that a biofilm reservoir is present somewhere.
Does the patient have a history of antibiotic failure or persistent infection despite adequate
choice of antibiotics? Does the patient have evidence of local or
systemic signs and symptoms of infection that resolve with antibiotic therapy only to recur
after therapy has stopped? This all adds up to the diagnosis of a biofilm infection.
