Biofilm

Biofilms are an assemblage of bacteria which are formed when the bacteria are living in a layer of slime which they have produced. Usually biofilms are associated with surfaces, however, they can also exist as aggregates in fluids and mucus layers. In this biofilm phenotype, the bacteria are protected from antibiotics and our host immunity, even though when they’re living as individual cells, they can be sensitive to antibiotics. When they get together in this group as a community in the slime matrix, then they become much more difficult to kill. They’re also protected from host immunity and cells such as macrophages which normally phagocytose (eat) infectious cells. They can’t do that very effectively with a biofilm because it’s physically too large. We really think biofilm formation is a protective mechanism that bacterial cells use to survive in the infection.

When pathogens exist as biofilms they grow slowly and may not necessarily produce symptoms to begin with. When patients do present with persistent fever, unwellness, pain and other symptoms following surgery and do not respond to antibiotic treatment they may have a bacterial biofilm infection. Wounds that become infected with biofilm may have drainage, delayed or incomplete healing and an unpleasant odor.

Biofilms have a complex lifecycle from initial formation to maturation. In the first phase of formation, bacteria populate the surface. Bacteria then excrete extracellular polymeric substances (EPS) that bind the colony together. The matrix of EPS protects the cells within it and facilitates communication among the bacteria through diffusible chemical signals, forming a biofilm architecture and mature into a complex three-dimensional structure. In the final dispersion phase, biofilms can propagate through seeding where individual cells are dispersed from the biofilm. These bacteria can attach to other surfaces or go on to cause sepsis.

The interesting thing is that not all biofilms are bad. We all live with biofilms. We have them on our skin. We have them in our gut. We have them on our teeth. These “commensal” biofilms actually help protect us. They might help protect us from pathogens by occupying space that such pathogens might otherwise colonize. We need biofilms in our gut to help us absorb vitamins efficiently. So certainly not all biofilms are bad. However, if we have biofilms in the wrong places, say for example in a deep tissue such as a knee then we can have issues. Biofilms are not naturally there and so Staphylococcus epidermidis, Propionibacterium acnes or even Staphylococcus aureus, which might live on our skin quite happily and causes no harm, but when they enter the body through a break in the skin may form an aggressive, invasive biofilm that’s when we have the issues, the symptoms, the difficulty in treating the biofilm.

Acute infections are infections that we’re most familiar with. These are infections from cholera, dysentery, or bacteria in our gut or bloodstream that are producing a lot of toxins. They are growing rapidly and if we don’t get immediate treatment with antibiotics to kill these rapidly growing organisms then that can be deadly. Meningococcal disease, necrotizing fasciitis and blood poisoning septicaemia are all well-known acute infections.

Now the difference when the pathogens are existing as biofilms, is that they are not as virulent. They’re growing slower. In fact, they go into an almost dormant state. What we see is rather than a rapidly expanding infection is a long lingering localized infection. Sometimes they might not even produce symptoms in the early stages of a biofilm. They might be there, and we don’t even know that they are there because they’re not growing rapidly, that is the insidious nature of biofilm infections.

Actually, what is interesting is that part of the reason that they’re resistant or, more accurately tolerant to antibiotics is because they are not growing rapidly, and antibiotics work best on rapidly growing organisms. If the organism isn’t doing anything, there is nothing for the antibiotic to attack. Biofilms are commonly associated with chronic infections.

There’s a study that was brought out in The New England Journal of Medicine (incorrectly stated as The Lancet in the video) a number of years ago, which had evidence from a patient that had osteomyelitis in her teens or early 20s. It was treated. 75 years later, she got out of her chair, felt pain in her left thigh, and the infection came back. What was really fascinating about this patient was that back when she first had the infection somebody had taken the organism, and they preserved it. They made a stock culture. Moving on 75 years, now we have the tools to do a genetic analysis. They found that it’s the same organism. From what I can tell, from talking to orthopaedic surgeons, it’s not uncommon for infections to recur when there’s been trauma. It starts to suggest that these organisms, these biofilms, can be there for extended periods of time – for years. In some of the cases that I’ve looked at, the patients haven’t presented with symptoms for 5 years. So we ask the question, “was the biofilm there all along, and we just didn’t know it? Or for some reason, did it come through the blood stream?” I think there’s certainly anecdotal evidence that biofilms can reside in patients for extended periods of time, years, and then get activated again.

Once a biofilm has become established it’s extremely difficult to treat it with conventional oral or I.V. systemic antibiotics, just because the bacteria become so tolerant. Now, one of the issues with biofilms is that because the bacteria in the biofilm stay dormant, they don’t necessarily become symptomatic until the biofilm has reached maturity. Unfortunately, with implants and deep tissue infections, one of the few treatment options is surgical. It’s actually cutting them out.

Folks are now starting to think of biofilms like cancer and we need to physically remove them. However, unlike cancer, they’re not as large as a tumor so they’re much more difficult to see. And so, there’s a number of treatments which are being used where we try our best to remove them – surgical debridement or irrigation – where we actually try to wash them out, remove them from the tissue or from the implants.

But also, what we’re finding is if you use very high levels of antibiotic locally, and this can come from bone cements, it can come from PMMA (polymethylmethacrylate) or mineral cements, which are packed right at the site of the infection, then we can actually get concentrations of antibiotics that are so high that we actually can kill biofilms. Now it seems though that it takes multiple numbers of days of exposure to kill them. We’ve got some work going on in the lab, for example, where we grew biofilms. We exposed them to high levels of antibiotics. It took us to a week before we saw eradication of those biofilms. So it seems that having very high concentrations over these extended periods can allow us to knock them down. Now there’s some question about can ever you actually eradicate biofilms? It’s an ongoing question. There are cells in biofilms called persister cells, which go into a dormant state, which are very difficult, if maybe impossible, to eradicate. However, what we’re hoping is that with these high levels of antibiotics, and also our surgical debridement, we can break up the biofilm. We can kill enough of them that our host immunity can then take care of the rest.

Prevention of biofilm is extremely difficult. And the reason is that we have bacteria on and in our skin. There’s bacteria in the environment, and even in the very highly controlled environment of the operating room there are bacteria around. During a surgery, of course, we are inflicting a wound. It’s a very controlled wound, but it’s a wound nonetheless. We maybe unintentionally letting those organisms come in.

Prevention is extremely difficult. A surface has not been found yet which totally resists bacterial adhesion. So when I say a surface, it could be a metal surface, it could be an implant surface but also just the bone itself. So basically, what I think we’re looking at here is an odds game. When a patient is undergoing surgery, as the surgeon’s knife comes through the skin, bacteria can be drawn in. If that bacteria escapes the host’s immunity for a short period of time to actually get to the surface, then they can start to form biofilm.

There’s a lot of new research being done on new surfaces, new materials, antibiotic impregnated materials, nano-textured materials, and novel antimicrobials which are being put on to surfaces; but as yet not many of these are made to be put into practical use, at least in orthopaedics. Part of that is you have to think about the constraints that an orthopaedic implant has to undergo. It has to be physically strong. There are articulating surfaces. Of course, if you have a surface which has been engineered to have an antimicrobial property, but it’s relatively weak or can be abraded, then that’s not going to work in that situation. The best we can do at the moment with prevention is to really try to stop the bacteria coming from there in the first place. Even with the extremely sophisticated drapes and infection control used in modern surgery, still 1 and 2 percent are getting infected.

From what I’ve seen in my research, I’ve taken orthopaedic implants and some of the fibrous materials, fibrous sheath associated with those and also pieces of bone, and they look quite similar. We’ve done some genetic analysis to look at the organisms involved, and again, it’s quite similar. It does not really matter what component it is, whether it’s cobalt chrome molybdenum, whether it’s titanium or polyethylene, genetically it looks like we’re getting the same sorts of organisms on all of those materials. Physically the biofilms look similar when we put them under the microscope and use confocal microscopy to see them in high resolution. As of yet, we’re just not sure and cannot fully address that question, but the biofilms look very similar wherever they are found.

An active area of research in my lab is addressing the question – are there certain areas on that implant which may be more prone to colonization than others? As we know, these implants have lots of different surfaces, some are extremely smooth, electrochemically treated, electrochemically polished to have virtually no roughness, but some of the surfaces are actually rough to help to encourage osseointegration. In addition to all these different surface’s roughness, we also have features such as screw holes, we also have modular components, we have all different sorts of geometries. So a question which nobody has fully addressed yet is, “are there certain components, certain materials and certain geometries, which might be more prone to colonization by biofilms than others in vivo?” If we start to understand that, then maybe we can start to design our implants to try to avoid those sorts of features and surfaces.

What I find when looking at waterjets, high velocity waterjets in lavage on biofilms, is that we have two things occurring. First of all, we do get some removal of the biofilm. We do see pieces of biofilm coming off and we do get quite a significant reduction of biofilm. However, biofilms can actually behave as liquids under certain circumstances such as when they are subjected to high shear stress. People don’t normally think about them as liquids. We think of them as sort of pieces of dirt almost, or solids. But biofilms can actually flow. So when biofilms are exposed to these high velocity jets, they can actually flow over the surface, and then when the jet is removed, then they can form back into biofilm. They have a viscoelastic property where they can just resume and carry on from where they left off. So I’m pretty confident that it will remove a certain amount of biofilm, but I think there’s also potential for even possibly spreading biofilm around to other places. You only start to see these effects when you use high speed imaging to look at these interactions, these fluid-biofilm interactions, to see this process occurring. Interestingly we can possibly use this time when they are liquefied to more effectively deliver more antimicrobial agents into the depths of the biofilm.

A very important consideration with antibiotic release from spacers is how is the antibiotic going to move from the spacer into the surrounding tissue. And if it is a diffusion limited situation, maybe the antibiotic will only get a certain distance at the concentrations needed to kill the biofilm. We’ve set up some experiments in my lab where we’re using agar to grow biofilms on the agar. We put in PMMA, and what we find is we get a about a 1 centimetre zone away from that surface where we can kill the bacteria – kill the biofilm. However, we have tested spacers from infected patients and found that antibiotics are still being delivered but presumably the infecting biofilm was “out of reach” of the antibiotic.

Calcium sulfate can be mixed up with antibiotics, and in my experiments, what we do is we look for the release of antibiotics over time from the calcium sulfate. And what we notice, is as the calcium sulfate dissolves we have a longer plateau of release of antibiotic from the calcium sulfate than we might from a non-absorbable material such as polymethylmethacrylate. So we see potential efficacy over much longer time periods in terms of being able to kill the biofilm. But also, when we look at elution, the concentration of antibiotics coming out of the calcium sulfate, we see that the concentration remains higher for longer periods than it does out of the PMMA. An advantage of using antibiotic beads in addition to antibiotic loaded spacers is that they increase the zone of coverage.