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?