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.
