Join Robert J. Snyder, DPM, MSc, CWSP, FFPM RCPS (Glasg) as he provides an overview on managing biofilm/bioburden along with his case studies.
Dr Rob Snyder. I'm professor and director of clinical research at Barry University School of Podiatric Medicine. I've been a wound, care physician and podiatrist for over 30 years. I'm involved very heavily, not only in clinical practice but clinical research as well. I published over 165 peer reviewed in journal articles and have been principal investigator for over 50 randomized controlled trial. So it is my pleasure to be here today to discuss this very important topic of biofilm within my topic today is going to be a diabetic foot ulcers, but we're going to concentrate predominantly on wound, bio burden and biofilm. This is ah, some important information that you you should look at at your leisure. Ast faras disclosures are concerned. I am a consultant and speaker for K C I, which is now part of the three m family. So why is wound healing so difficult when you think in terms of diabetes as disease in the United States alone, there are over 30 million diabetic patients. Um by 2050 will probably represent 12% off the population. 25% of those patients will develop a foot ulcer in their lifetime it's usually the reason why a diabetic patient will be hospitalized. Certainly, diabetes is the main cause of non traumatic amputations throughout the world. And there are a million amputations performed globally every year in patients with diabetes, and that is one every 30 seconds. So when you think in terms of how long it will take to go through this presentation, you could imagine how many individuals are losing a part of their body to this very devastating disease. So here's an example of a patient who had a vascular procedure done. Have a serious infection. We did a trans metatarsal amputation here, and we did not have enough skin to cover the lateral side of the foot. So, as you can see, there's a wound there that needs to breathe mint. We have been to breeding this wound on a regular basis using moist, wound healing various topical therapies. This patient, really was not converting, was not responding. Uh, take a look at this picture. This is ah, much cleaner wound wound that is much more easily managed. A Z, we go through the wounded preparation model, and and in this particular case, um, this was one week status post application of the topical anti biofilm agent. So again, something to keep in mind. We're going to go into what, those topical Asians or in just a little while, so by a film, Certainly maybe President in wounds. But I think it's a Miss Noma or some confusion, certainly at the very least as to whether or not you can actually see a biofilm. This is really, actually quite a controversial topic when you think in terms of unopened fractures, an example with hardware and plates and screws that are exposed, and you see this slimy layer across that area that is likely vile film. But the fact is that you cannot necessarily see biofilm anymore than you can see. Staph aureus or pseudomonas biofilm is, ah, microscopic. They're microscopic structures there really undetectable with the naked eye. What we're seeing, basically or the symptomology ease of the biofilm in general. And unfortunately, routine wound cultures are really not effective in in making a diagnosis of biofilm. You really need something much more sophisticated, like polymerase chain reaction testing, PCR testing again, looking at the genetic markers and genetic material off this bacteria. Eso, even if you're using a swab culture will be using, uh, the low buying technique. This certainly will not be adequate in making diagnosis of biofilm. Remember that cultures identify what we refer to as plain tonic or free floating bacteria. They're not biofilm bacteria, so there much more difficult to culture. So our objectives are pretty straight forward. We're going to discuss an overview of how Thio not only discuss but manage biofilm and bio burden. We're going to review some cases that focus on perhaps some solutions to manage this condition, And we're also going to analyze at the end some questions relating to biofilm and by a burden, particularly as it relates to the diabetic patient. So what is the problem? Well, microbiological infections certainly are there the single most important positive agent for chronic wounds. Patients with chronic wounds are stuck in the inflammatory phase wound healing, not in any small part due to high levels of bacteria. Chronic wound infections typically form biofilms. They see from platonic bacteria's deeper into the wound than they create this sticky material that will stick to substances and structures. And these are notoriously recalcitrant to conventional antibiotics. And we'll talk in just a few minutes as to why that is material biofilms on ever growing public health concern. We don't only see them in chronic wounds. We see them in other diseases as well, like cystic fibrosis. We think, as an example, that ulcerative colitis or crone's disease may in fact be a biofilm disease past, which is a normal logic condition may have its roots in Bio comes biofilms are really ubiquitous in nature. We see them as part of a bathtub ring. We see them in our toilet seats. Certainly we see them in our oral cavity dentist steps, seeing biofilm and dealing with biofilm for decades. We see this in the form of plaque. So again, a very ubiquitous problem and something which leads to recalcitrance in chronic non Henley wounds. So let's take a look at the bacterial continuum what we see in a wound. What's interesting is if through time, nature wanted wounds to be sterile when an individual had an injury, then that would exactly occur. But that's not the case. A soon as there's a breach in the epithelium, the wound becomes contaminated. Three initial entry of infectious, potentially infectious agents become a very, very prevalent there force contaminated bacteria adult replicate. But over time you will get a colonization or proliferation of these contaminants and other elements as well, and you will get a higher bacterial burden. Remember, bacterial burden means nothing more than the fact that there is bacteria in the wound. It's only when the bacterial burden gets to a critical mass that we have a problem. But there are no healing effects necessarily when you have contamination or colonization. Now, when it really gets important is when these wounds become, we commonly refer to. For years it's critically colonized, and now we refer to it as a cult infection. This is when bacteria reaches that level, where the patients immune system is overwhelmed and just cannot control thea underlying bacteria. So that's when you start to see very symptomology ease occur. You'll see increased drainage. You'll see a change in the coloration of the granule ation tissue, from a beefy red color to maybe a pale pink color or even a great color. You'll see how pocketing of the wound etcetera, and ultimately this leads to infection both locally and systemic, and this results of force and increased numbers of bacterial population So the one point to make here is that it's very important. Understand that we should not be culturing every single wound that's open, particularly the diabetic patient, because if you just have contamination of colonization, you will certainly grow something on that culture. It will be obligated to treat it with an actuality. Contaminated and colonized wounds have no effect on healing. So Dr Marty Ropes in in the nineties, had a very interesting study on acute wounds. On what he found was when you had colony forming units of bacteria greater than 10 to the fifth. Then you would either have an infection or, at the very least, have believed wound healing. Violence was really not taken into consideration to any great extent. As an example, if you have a staph infection, certainly tend to the fifth is a reasonable number two. Consider to have delayed wound healing or to have an infection. But if you have a strep infection, that could be tend to the to pretend to the three so variance plays a very important role. The dose response is also important. How many of that bacteria, how many of those pathogens are actually there so you're multiplying the virulence by the dose response. But what's really important here when you think in terms of diabetes is the host response diabetic patients or I mean a compromise. And because of that, they're not going t to to exhibit the same signs and symptoms that you would see in a patient who is not in me to compromise or did not have some type of peripheral arterial problems. So it's clearly affected by the immune system. Infection clearly complicates the treatment of wounds and impedes healing in several ways. First and foremost, that damages tissue. It reduces the wound tensile strength. It induces a very undesirable inflammatory response. Now, as we all know, chronic wounds are stuck in the inflammatory phase of wound healing. When you have high levels of bacteria, this is continual. Thus, controlling or preventing infection is certainly an essential portion off moving the wound through a normal healing cascade. Effects of bacteria are even more significant when it comes to considering things like biofilm, because biofilm will attach to host tissues. Bacteria in general produces toxins, and Proteus is the damaged tissue and delay healing on what we think of Proteus is very Often we think of Matrix, Metella, Proteus or Syrian the last days. These are the common Proteus is that we see in wound healing. What most people don't realize, however, is that bacteria not only create toxins and endo toxins but also produced their own enzymes. Thes enzymes can completely destroy the extra sailor matrix. By disconnecting that extra sailor matrix, you're disconnecting what's called dynamic reciprocity, so there's no communication between the extra sailor matrix and other cells in the wounds, like fiberglass and a filial cells. So again, wound healing is delayed or ceases entirely. Bacteria also consume nutrients. They consume oxygen. Local induced inflammatory reactions are common, and certainly these can delay wound healing. Remember, chronic wounds are stuck in the inflammatory phase of wound healing. So what is a biofilm? Biofilm is a heterogeneous mix of bacteria, pathogens, community, actually. And they're surrounded and entangled by a blank. Okay, Lex, in this, like okay, Alex is the extra cellular polemic substance. Now, what that basically is is a very, very thick coding or covering that surrounds this bacteria and actually prevent protection is entangled. Polymers are also strengthened with metallic bond. So you have this community of heterogeneous bacteria and fungi. You have entangled polymers which create this substance. And not only including this is very thick Polly Sacha ride, but also proteins, glycoprotein DNA and further. You have metallic bonds, these metallic ions buying the polymers of the MPs together so that the structure becomes very, very resilient. So if you take a closer look, what's important to understand here is that looking at this bacteria, it kind of looks like a tube. Now, within that tube, only 5 to 30% of that tube is bacteria. The rest is actually like Okay, Lex. So you have this very thick substance surrounding that heterogeneous bacterial mix, which is very real. And of course, that can lead to some serious problems because it becomes almost impossible for any topical or systemic therapies to move through it. So there's kind of an example. If you look at a and you look at this con focal laser scanning electron microscopy, you can see that in a you have a red, which means that the cells were dead and you have a green and yellow, which means the cells were alive. But these are plain tonic bacteria. You're free floating bacteria. If you look at the there's a confluence here, you don't have free floating bacteria. This is basically the formation of a biofilm. So when you look in terms of biofilm formation and you look at that continuum of bacteria and a wound, you can see the following Number one. You have plain tonic bacteria that pink platonic bacteria seeds, seeds to a deep proportion of the wound, and also the surface of the wound that initially you have this reversible attachment and that reversible attachment ultimately becomes permanent, leading to micro colonies and co aggregation. Differentiation Off sells something called quorum sensing, which we'll talk about in a moment. But you get this mature Polly microbial biofilm, which ultimately starts to disperse additional fragments of biofilm. Now you may or may not see classic signs and symptoms of infection when you see this biofilm, because again, particularly in diabetic patients, you have a scenario where there is an imminent compromised state. But clearly you will have inflammation and biofilm is truly a very formidable adversary, so most bacteria exist within biofilms. It's really amazing when you think about how significant this problem is. 10% of bacteria are plank tonic or free floating bacteria. 90% of bacteria exist in biofilm, so what's even more disconcerting is that it's 1000 times more tolerant antibiotics than any type of plank tonic bacteria that we see. This is also very interesting. 6% of acute wounds have biofilm. Up to 90% of chronic wounds have biofilm. But when we're thinking in terms of diabetes, ah, 100% of diabetic foot ulcers have some type of biofilm. Now, obviously, diabetic foot wounds will heal, and we don't really know which biofilm is bad and which by which biofilm is good. But we do know that if a wound remains recalcitrant despite what it is we're doing to treat it, then it is very, very likely that we're dealing with a biofilm issue. Imagine 100% of all wounds have biofilm patients with diet Bt's because biofilms really don't play fair. Very difficult to culture as I mentioned. Traditional cultures don't work tolerant bio sides tolerant antibiotics and they're very capable of regenerating. And very often you have what's called for sisters that hang around irrespective of what we do. Because the phenotype of the biofilm is highly adapted, and it can survive, really, in the harshest of environments, very, very important to keep that in mind. So in biofilm, bacteria will differentiate. They'll communicate, will cooperate and build the point collective defenses against antibiotics. Now, one way they do this is through a process called quorum sense. As you start to get a building up bacteria this heterogeneous, heterogeneous mixed of bacteria, you reach a critical mass. Once that critical mass is obtained, these bacteria start to speak to one another through their genetic material, and this is called quorum. Sensing is just another layer, another level of protection for the biofilm. So why is there resistant antibiotics? Why don't antibiotics work? Well, Biofilm reduces antibody penetrates. Remember, you have that thick like okay licks, and it's almost impervious to anything we throw at. The cells within the biofilm are very slow growing, and you need a quick metabolic rate in order for antibiotics actually work so antibiotics will not work with biofilm bacteria and, of course, lastly, cells within biofilm expressed many stress response genes that are resistant to antibiotic action. So most topical antimicrobial treatments are not effective in eliminating biofilm. They're unable to penetrate the like. Okay, licks. They actually had higher doses, maybe site of toxic, various strengths. So not only do they kill bacteria, but they kill other things in the Sella's well, like fibroblast and epithelial cells. Not just the pathogens, pathogens with the biofilm or metabolically inactive. They're what we call quiescent. So antibiotics, as I mentioned, or ineffective and antibiotics lose effectiveness as bacteria change genetic expression. And this will occur through that quorum sensing dynamic. And of course, clearly this could lead to bacterial resistance. So again, if you look at the microbial and antibiotic treatments and you have platonic bacteria, he's air clearly effective method. But once the biofilm form sticks to a surface, that biofilm is impervious to just about anything you will throw at it. So when you think in terms of optimal biofilm treatment requires several things. Number one you want to dismantle that like Okay, let's that E. P s structure. You need a broad spectrum antimicrobial agent without microbial resistance, you need high tissue compatibility, and you also want to prevent the biofilm from reforming. Remember that the hallmark of treatment now to treat biofilm is debridement. But even if you do an extensive debridement even in operating room setting, that biofilm will start to reform almost immediately. And within 70 you 72 hours, you will likely have a mature biofilm reforming. So you want to dismantle the GPS structure, you want to destroy it. You want to defend from re colonization, so those really are the keys. So we have fortunately developed some potential solutions Carousel a G, which is a gilling fiber dressing. Well, we know that silver has been out there very long times that is silver work. Silver works basically by the fact that it has an electron missing within the silver. And that electron is replaced with an electron from the bacteria. But Onley one electron is missing. Now we have oxy salt technology or eggy, actually, salt technology very, very interesting. And farm or sophisticated. Because not only do you have one electron missing, you have three electrons missing. And take a look at the poll here. Very substantial. Pull number three is a little slower, but if you look kind of a collaboration off all of these, you have a significant pulling power. So you have ah, very powerful substance. You have a fast kill, and it's not going to be, um, you're not going to have a situation where you have any disruption of any other normal cells in the world. Hydrogen peroxide. Another interesting dynamic, H 202 is a key stimulus to signaling tissue damage and recruits inflammatory cells that kills bacteria and, of course, in low concentrations that may actually promote healing. But in high concentrations, it does exactly opposite it inhibits healing and clearly in chronic wounds. When you see this and excessive amounts, it clearly creates a hostile wound environment. It causes tissue damage and certainly allows that wound to remain in the inflammatory phase of wound healing. But what if we could split that molecule? What if we could separate it? Well, we can with oxy salts technology. Basically, you have hydrogen peroxide and you're splitting it into oxygen and water. So when you when you take a look at what the studies look like compared to some of the competitors in the field, you can see that there is a dramatic difference in the way this product works. So carousel Aguilas jelling fiber again, it manages barriers to healing. So what are those barriers? Let's review them. Infection Excessive, exceed eight. Persistent inflammatory change. Biofilm. So you have to innovative technologies kind of built into one. You have a C M C substrate, which is going thio quickly. Absorb ah, lot of fluid in the area. Micro contours to the wounded, which will be critically important. You don't have any nooks and crannies that are exposed and, of course, reduces dramatically. This is the risk of Perry Will maceration. You couple that with Oxy salts technology with those three electrons that are missing and that now can be replaced by bacteria. This is fast and powerful silver technology. It kills bacteria within the biofilm. It quickly kills almost 100% of bacteria, while dampening inflammation to force the wound. Progress is dramatic. Just take a look at some of the benefits is the difference between using an ointment and gauze, which very, very often is used in the field. Unfortunately, and you have this exuberant amount of macerated tissue and a wound that just doesn't look very good. Clearly, this wound is not headed in the right direction, and unfortunately, a lot of what may happen here could be I atra genic due to the fact that the dressing was inappropriate. Of course, if you compare this to a jelling five, particularly with silver in it, you can see that maceration is almost completely absent. So what's the importance of micro contour? And you have to realize that when you put a dressing on and it begins to jail, it should fill all the nooks and crannies off that will. Silver will eliminate whatever's in its way. So if you have staph aureus as staph aureus is completely eliminated. If you have. Actually, if you compare it to actress L. A G extra even though it has strengthening fibers, you have these little areas here which create dead space and that dead space is a good area for bacteria to grow. And this and that and actualities what can conceivably occur. So again, reduce micro contour e may allow bacteria and biofilm growth within the dead space, and you will not see that with Carousel E g now thinking in terms of biofilm. This was very interesting study because it did not involve debridement. They looked at the control, which was moist wound healing. They looked at across L. A G extra. They looked at Carousel A G. If you take a look at what happens here, biofilm is dramatically reduced even in the absence of the agreement, which continues and prevents re formation for up to eight days. Very, very significant. Let's turn our attention to silver. Silver has been around a long time. Um, silver cell has been around for many years, but we now have a new and improved actually. Silver sell two point. Oh, if you will. It's a non adherent variety. We call it easy lift technology. It's a very, very interesting and very formidable addressing. Remember when you think in terms of infection, infection delays, healing, you have high levels of bacteria. You have high levels of extra date. You have high levels of inflammatory cytokines, and this very often leads to readiness, pain, heat and swelling. Now, if you look at this diabetic patients here, this patient may not develop primary signs and symptoms of infection. He paying register swelling the cost of the fact that they're aiming to compromise. You may have what Gardner called secondary signs and symptoms of infection pain in an otherwise painless foot and wound deterioration. You have to be looking for those. This is a more typical scenario. I know it's a little bit more difficult to see their thema here, but there is a ring of our theme around her. This is more typically noted ulceration that we see, so why silver it all? Well, Silver works at so many levels in the bacteria that inhibits the function of some bacterial enzymes. It disrupts proteins that so memory. It has an effect on targeting nucleic acids, or DNA, and it causes changes in the structure of the cell wall. So when you think in terms of how many ways it is actually attacking the bacteria, it creates very little, if any resistance at all. So here's an example of Silver Cell, not adherent. That's what the dressing looks like. It soft, sterile. It's non woven. Itt's Hydro Filic Now what you don't want and what had occurred in the past is you had residual fibers that stuck to the wound, and that's that area basically created Linton, and that's what you can see right here you see, linking very, very detrimental potentially. In fact, we've seen studies where they have actually been granulomas that have been caused by these types of dressings. They damaged graduation tissue when they're removed, and as soon as you remove them, the wound is thrown back into the inflammatory phase of wound healing, and force. Patients have pain when the dressings removed. So when you think in terms of silver sell non adherent, you're looking at a much more formidable and better product. So for selling non adherent dressing causes almost no pain and leaves no visual fibers behind. This was a study that I did several years ago. Another important fact is that this structure and this dressing is actually for back of a better word. Time released when you put it on a wound. The amount of silver that is released on Day one is the same amount of silver that's released on Day seven. Now it could be, they tend. It could be Day 20 but it's only been studied for seven days. So think about using this on a patient who who has Venus leg ulcer as an example, you want to put this under compression dressed. You have confidence that that amount of silver that's being released on Day one is going to be equivalent to the silver that's released on day set thinking in terms of biofilm, because that's really what we're talking about. What I want you to understand is that when you look at how biofilm is studying, it studied very, very differently. There are very different models, and you have to be very mindful of very careful when you're evaluating how biofilm is actually affected by various dressings. The third reason, Of course, if you start at the at the very tip of this triangle where you have biofuel, preventative strategies or preventive essays, it's very, very different from the types of more sophisticated studies that you will have in 24 hours, 72 hours and greater than 72 hours. And you can see the silver effectiveness at all of these levels based on these different types of models that are used. Of course, this was compared toa iodine and iodine, showed very effective areas and low exit dated conditions and drier wounds. Silver was effective and highly X sedated conditions and ex sedated wound, but very, very important. Understand that the older the biofilm is the fact that the antimicrobial efficacy will decrease in the force, exceed eight levels, will increase the longer than biofilm stage around. So keeping that in mind and looking at silver in general. These air some in vitro studies that are available that demonstrate that silver cell, not adherent dressing, is shown to be effective and not only preventing but disrupting biofilms first and foremost, the anti microbial action. This is published data showing stained release of silver ions for up to seven days. We talked about that. What's interesting, also to note, is that silver has an effect on 150 plus organism bacteria, yeasts and molds will put in tomorrow S A, M, r S e and B R e. It reduces wound now odor as well. We now know that it's effective in preventing and disrupting biofilm in vitro. And, of course, from a preventive standpoint, we're lowering the plan tonic bacteria. So you're not allowing that plank tonic bacteria to see to form a new biofilm. This is, of course, linked to absorb in city allows for effective management through the algae Nate. It also helps maintain a moist wound healing environment, which is of course, very beneficial for wound healing. But again, keep in mind that we feel strongly that silver does have an effect on not only preventing but disrupting by our film in vitro. So this was a monograph that I was involved with. We had some very high level individuals. Dr Mary Lavery, Um, uh, Randy Walcott, Greg Bond, etcetera. And we looked at all areas of biofilm. What was in literature and what we found most intriguing and most striking was work that was done by Greg Schultz and colleagues. And this was kind of what we referred to as the step down effect. When you first think that there's a biofilm that is going to impact, wound healing and recalcitrant way, you must start with multiple therapeutic regimes. Very aggressive debridement looking at Imperia topical therapies, some of which we've already talked about. You wanna manage host response? You want to do a glamorous chain reaction test? Of course, all of this should occur within the first 1 to 4 days, and then you start to step down within the next 5 to 7 days. You're assessing inflammatory stage of the wound you're seeing whether it you're optimizing the individual from systemic standpoint, you may want to begin systemic antibiotics at that point. Continue management host factors and then moving into the next 1 to 4 weeks, stepped down again. No, no, assess inflammation, maintain the breathing, reevaluate topical and systemic therapies and, of course, continue managing those host factors. Now you will eventually get to a point where you could move to standard care. And that's wonderful, because standard care now gives you the opportunity to create wound bed preparation, which will ultimately lead to the ability to use mawr. Advanced thirties skin substitutes, Matrix sees growth factors, skin grafts, etcetera. So really very, very important. And a very formidable and very easy way to think about how to treat this very recalcitrant problem and again, sharp debridement. Is that the center or the hallmark in treatment? But now we have a new addition. We have biofilm disruptive technology, so you can now add that biofilm disruptive technology to each step of the way. And it's very, very likely that this will not only be effective, but it will allow these days to be shortened. So perhaps 1 to 4 weeks, maybe 1 to 2 weeks. We don't know 5 to 7 days, maybe three days, etcetera, etcetera. So using that biofilm, disruptive technology can be very, very important. So here's a case study. There's a 68 year old Haitian male with insulin dependent diabetes. Yeah, his weekly palpable of Austria tibial pulses. That model physic. His ankle brachial index was 0.3. He had a necrotic great great toast. Severe pain despite severe diabetic neuropathy, which is never a good sign it prejudice. Which means that when you you put your finger on that foot, you will hear a crackling sound that's often very indicative of gas and a tissue. But his white count and the C reactive protein were normal on this very often is disconcerting to physicians who are unaware of the fact that because diabetic patients failed to mount a physiological response to infection that you're you're going to have a slower increase in that white count. That way, count may not trail up for several days. Patient was a fee brought. Now, if a non diabetic had this infection there, fever will probably be 104 and this was only five days duration. Now here is really where it gets dicey because here is an example of gas in the tissue, and this is our hot abdomen in the party actual profession. We come in at 11 12 o'clock at night to drain this out because by morning we could have liquid active. The closest bone will be mush and the tendons on muscle will be gone. So what would you do? Would you order an urgent interventional radiology council with the potential intervention prior to surgery? Would you do an urgent operating room to breathe mint, including partial first ray imputation? Would you hospitalized the patient for intravenous antibiotics and then wait until the infection begins to resolve or the patient has no constitutional symptoms. So maybe you would just culture the wound and empirically start the patient or Orel antibiotics and wound care until you get the cultural results back. So I think for a moment about what you would do, I'll tell you what I did. I took the patient immediately to got ready room for urgent debridement amputation. Hunger. First read. Now, obviously, whenever possible, we want vascular on the front end. We want them to increase vascular parity, and we want to wait a few days before we start to do any type of surgical accused. But there are situations like this where you have urgency and in that situation you have to act quickly. So the patient then had undergone successful distal and the vascular intervention and now has three vessel run off all three vessels in his floor extremity and his foot in his post. Tibial and true tibial and peroneal are open now. And of course, we've used a multidisciplinary approach. Well, now what do you do on this case? I used negative pressure. One therapy, very, very important therapeutic regime, particularly in patients that have wide open wounds, where you're not really interested in completely closing them or covering them that point. So the patient has three weeks of negative pressure. One therapy. We used adaptive touch as an interface between the phone and the wound. Why do we do that? Well, I do that for two reasons. Number one. It's easy to get the phone off when the patient comes in addressing changes, but just as importantly, that granule ation tissue could grow into the phone so that interface with something like adapted touches burglary Important. Of course, this patient also required six weeks of intravenous antibiotics, but the patients, still exhibiting copious drainage increase wound bio burden. So now what are we going to dio? Well, this is what it will look like. We're still very, very difficult to consider our options. So in this case, we use silver cell, not adherent that we use it for two weeks. We changed it every 2 to 3 days, depending on the amount of drains, the patient God and then transition to collagen or see silver for two weeks. So we didn't need that high intensity of silver after that. Two weeks. So we moved again to a lower lever silver with three parts per million. That would not be so toxic. So now we're left with this. Now what do we do? Well, very simply, in this case, we didn't need advanced therapy. Moist wound healing really was what was important. So we used the hydrogel and a full. So as far as the takeaways relative to biofilm and bio burden, biofilm clearly is a complex and synergetic relationship. You have this heterogeneous ity of bacteria. You have them in this cocoon. This like, okay, Alex, which makes them almost impervious to anything. Throw at it. The presence of biofilm and chronic wounds has been conf from, And this is clearly a deterrent wound healing. Many common modalities to address infection have been shown to be totally ineffective against biofilm for the many reasons we gave. So determining an effective way to eliminate biofilm is clearly a challenge. Drop the bereavement certainly may open up time, a time dependent windows. So you release that heterogeneous bacteria into the environment so that you can treat it. But that biofilm forms very quickly between hours to 72 hours. You have a mature biofilm, irrespective of whether you did it in your clinic. Well, you did it in the operating room. So new topical therapies certainly could help eradicate biofilm while lowering wound bio bird. So thank you very much for your time. And I hope you enjoyed this presentation as much as I enjoy giving it. So Thank you.