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Speaker: Ladies and gentleman there is a fungus amongst us and he is on his way up here right now. A fungi, Dr. Warren Joseph is on his way to the podium to speak about drugs for bugs to save the limb.

Warren Joseph: Good morning. Yeah. Here, we got some response. Thanks all for being here this early in the morning again and I am going to be talking about something that's totally new as I mentioned a little bit yesterday. I have only given this lecture maybe three times before. First time was about a month ago. Dave Armstrong had a meeting down at USC and he gives me this topic about microbiome and treatment of diabetic foot infection. It's a great interest to me but I don't really have any slides on it. I have been reading the literature. So after about 100 hours of researching, papers and writing up slides and doing all, he gives me a 15-minute lecture. So okay, that's fine because at least it now gives me a lecture I can give meaning like this where I have a whole half-an-hour where I can expand on it a little bit and talk about some of these things and not be quite as rushed. So we are going to talk about diabetic foot. We are going to talk about microbiome and treatment. I have no financial disclosures to give for this lecture. I am not working with any of these labs or doing any work with any of these companies. We are going to talk about the ideas of a diabetic foot guidelines and what we say about diabetic foot infections, culturing and antibiotics. We are going to then talk about some of the newer technologies and give the differentiation between those newer technologies and see exactly where they fit. So let's go way back. Let's go back in history a little bit and we are going to go back in the old days.

[02:01]

What did we say back in the old days about culturing and antibiotics? Well, these are the old days 2012. Hard to believe six years is now old. These are the still the most up-to-date guidelines we have. The IDSA has not commissioned new guidelines yet for the diabetic foot section because they rank them by critical nature of the update and they just don't feel it's critical enough or there have been enough changes to warrant a new diabetic food guideline. But what did we say back in 2012 about culturing and antibiotic usage. Well, before I go into that, I just love this slide. And it really has nothing to do with anything in the lecture other than diabetic foot infection and the rapidity with which diabetic foot infection progresses. And you know it's beauty and brevity. This is a one-page paper that was published in New England Journal of Medicine. As editor of JAPMA, I get case reports in of like minor little conditions where usually written by a resident, they go on for like 10 pages with a background and it's important, it's critical that you know this background material. No, the shorter the better. And this one page article kind of proves that. This was a patient, a 50-something-year-old male who developed a diabetic foot infection and he thought it would be a good idea to sit home and just take pictures of his foot everyday as his foot started to rot. I love this guy. Thank god, he did it because we have been saying for years how rapidly diabetic foot infections progress. In the medical legal arena, we have been saying for a long time. These things can go bad overnight or in two days. Well, this actually proves it. So he sat at home and you can see day by day how this foot progressed until by day 10, he figured maybe it would be a good idea to seek treatment. So he was treated and they did a really nice job and they save the foot. It looks really good. But that just gives you an idea how rapidly these can progress.

[04:02]

I mean from day 1 to day 3 just, a major progression and you can just pick any two day in a row and you see that. So one page, one picture, really can tell you a lot and you will compare that to some of the studies I am going to show you about some of these new imaging techniques and see if you can understand those as easily. So what did we say in 2012 about culturing? We said for clinically non-infected wounds, you don't collect a specimen. We say that you diagnose infection based on clinical signs and symptoms of infection. If you don't have clinical signs and symptoms of infection, the wound isn't infected and if it's not infected you don't take a culture. If it's not infected, you don't give an antibiotic. But for infected wounds, we recommend a clinician send appropriately obtained specimens for culture prior to starting empirical antibiotic therapy except for those that are mild where we can assume they are primarily gram-positive infections, alright or the patients have been antibiotic naïve. And there is maybe nothing to culture. Cellulitis for example. Cellulitis by definition is caused by staph and strep. Group A strep and maybe MSSA. The IDSA classifies as a non-purulent cellulitis. Purulent cellulitis when you actually have pus coming out of a wound according to the IDSA skin and skin structure guidelines is MRSA until proven otherwise. So differentiation of purulent and non-purulent cellulitis. We also recommend sending a specimen for culture that's from deep tissue obtained by biopsy or curettage and after the wound has been cleansed and debrided. Swab should be avoided because of course if you swab a superficial wound, you're just going to grow a lot of junk out of it. So you don't want to do that. You do want to take some deep tissue. But the point is we are still doing this to send for a culture. The whole technique of culturing, taking a specimen, sending it to a laboratory.

[06:00]

The laboratory plating it on some agar, letting it grow for 24 hours, taking then subcultures with different looking colonies, doing a subculture on some more agar, sticking that subculture now into a machine like a Vitek or a MicroScan. This stuff goes back literally 100 years. 150 years from the days of like Pastor. They were doing it other than the automated susceptibility testing that they do in a machine nowadays, the basic techniques of culturing have not changed. And this is where I think we need to look at some of the newer technologies on microbiological diagnosis. So that's what we said about culturing. What do we say about antibiotic selection? We recommend that clinically, non-infected wounds not be treated with antibiotic therapy and that was actually a strong recommendation you know is based on a low level of evidence using the grade system for grading the evidence. We recommend prescribing antibiotic therapy for virtually all infected wounds but caution that it's often insufficient and less combined with appropriate wound care. In other words, antibiotics alone aren't going to do if you don't debride the wound and you don't use the proper dressings and you don't in particular the big thing the sine qua non of all diabetic wound treatment is offloading. So you need to do all of this along with just antibiotics. The antibiotics alone are not going to be good enough. Then for empirical selection of antibiotic, we recommend clinician selecting empirical antibiotic based on the severity of infection and the likely etiologic agents and for mild-to-moderate infections, that is going to be primarily gram positives, aerobic gram-positives. In particular, two organisms cause almost every diabetic foot infection. Staph aureus and group B streptococcus, strep agalactiae. Sure, you will grow all those organisms also but are they pathogens, are they important?

[08:01]

Well, some of us -- Ben Lipsky, Tony Barnes, some of us in the diabetic foot world, antibiotic world have talked about the concept of the two headed snake. You take a snake with two heads and those two heads are staph and strep and the body of the snake are all those gram-negatives and anaerobic organisms. What happens if you give an antibiotic what works against the staph and strep? You cut off the head of the snake. The rest of the organisms die. So for at least these infections what we assume are going to be primarily gram-positive, primarily gram-positive coverage is probably all you need. Now, we do say that for more severe infections and I direct you to the IDSA guidelines the 2012 guidelines for our definition of mild-to-moderate and severe infection but for a severe infections, which essentially mean the patients are septic, they have systemic inflammatory response syndrome, in those cases you can't afford to be wrong. So you might want to use broader spectrum therapy initially. What about MRSA? There are three situations where we say you should empirically cover for MRSA. Number one in a patient with prior history of MRSA. There is a saying in the MRSA community, once MRSA always MRSA. So if the patient had MRSA a year ago, year and half ago, two years ago and they're coming with a new wound, until proven otherwise, you need to treat that patient as if they have MRSA. Now, they may not because MRSA rates in the US are actually declining. They peaked up in early 2000 because this current MRSA epidemic started in around 1997, 1988. They peaked in the early, you know, 2005, 2006 and they have been in the decline since. So it's not uncommon to have patient who had MRSA a year or so ago coming now with methicillin susceptible staph. So until proven otherwise though, you do want to start them as if they have MRSA. So that's time number one to start MRSA therapy.

[10:00]

Number two is that when the local prevalence of MRSA colonization or infection is high, could we please be a little bit more vague? No, I mean we did that on purpose when we wrote these guidelines. What is high? Well, you have to make that determination. You know all politics are local, all microbiology is local. So what is it in your office? How often are you seeing MRSA versus non-MRSA? I will tell you from a medical legal standpoint. We used to term medical probability. Non-medically possible. You aren't held to medical possibility, you are held to medical probability. Medical probability by definition is more likely than not. I guess legal definition of probability. So more likely than not, let's say 50.1%. If you got about 50.1% MRSA in your office or in your hospital versus MSSA, then I think that's enough of a prevalence to start MRSA therapy empirically. And the third thing we say about MRSA and starting MRSA therapy is if the infection is clinically severe. Now, what do we mean by that? Well, like I said severe infection, mild-to-moderate severe are definition or classification of infection which has been validated. So severe infections just like I said earlier with a gram-negative anerobe, you can't be sure and you don't want to be wrong. So you might want to start MRSA therapy empirically if the patient has a severe infection because you don't want to have to wait three or four days to get the culture back and it says it's MRSA when you haven't used MRSA therapy. So what antibiotics can we use for diabetic foot infection? You can use any antibiotic, but only three of them carry an actual FDA indication that's for complicated skin and skin structure infections including diabetic foot infection.

[12:03]

And that's because until 2013 the FDA allowed antibiotics to be tested for diabetic foot. Today, there are only three antibiotics that hold the diabetic foot indication from the FDA. They are ertapenem, piperacillin tazobactam, and linezolid. That's it. There are only three antibiotics officially "FDA approved" for diabetic foot infections. Now, I will tell you that no new antibiotic is FDA approved for diabetic foot. Is that because they don't know work? No, it has nothing to do with that. They aren't approved for diabetic foot because in 2013 the agency, the FDA changed their guidance for industry from what they used to call complicated skin and skin structure infection guidance to what they now call the acute bacterial skin structure infection guidance. We call it ABSSI to keep it short. The ABSSI guidance specifically excludes diabetic foot infection. So the way the FDA is currently set up for new antibiotic approvals, no new antibiotic can get an indication for diabetic foot, which is a disgrace. And I have been in front of the FDA discussing this and they do understand that antibiotics are necessary for diabetic foot but they just don't believe that there is a way for them to properly set their deltas, their difference between the effect of an antibiotic versus a vehicle because there haven't been vehicle control trials. Long story but we will see what happens with that. Now, this was probably the most important slide and it's difficult to read. And I understand that but if you put a patient in the hospital, let's say today, you are admitting the patient with a diabetic foot infection, what antibiotics are they going to be started on? Usually, vanco or Zosyn. Alright, piperacillin tazobactam plus vancomycin.

[14:06]

That is the one worst possible thing you can do. Warren, what do you mean? How can you say that? That's what our ID people want. Then your ID people don't know the literature and they haven't been following it. Because I would ask you to go to pubmed.gov. Type in the word vancomycin, then use the boolean search term "AND", you will not even finish typing the A in AND before the search box will auto fill with vancomycin and piperacillin tazobactam nephrotoxicity. Now, this says 37 papers. I put this slide together about six weeks ago. I did this just about two weeks ago. I looked again. There are now 40 papers in the world literature in the last five years about synergistic toxicity, nephrotoxicity of vancomycin plus piperacillin tazobactam. Please, please do not use this combination. That's why I stopped using it a longtime ago. When I was actively at the hospital in Phili, I would start everyone on vanco ertapenem. I think it's just a much better way to go and it's a safer way to go. Then cefepime has been used a lot of other combinations, just avoid the vanc-pip-tazo. So are these recommendations, these old recommendations, still valid? I mean again even though there are only six years old, we really given the new technologies that I know still valid. Well, I thank Dr. Mark Kosinski, another ID trained podiatrist of New York College of Podiatric Medicine, very close friend of mine, we were residents together for this slide, where he starts talking about cultural evolution. And the fact is that old swab culture followed by all those techniques I mentioned before date back 100 plus years. Maybe, there is some newer technologies. Maybe there are some new things to look at.

[16:00]

And in fact if you start reading the literature, you will start hearing terms, this is the big one biofilm. Bob asked me a question about that yesterday with fungus. I will show you a couple of slides about it today. Microbiome is just a general term meaning like both either globally or in the patient just all the bacteria and all the different bacterial flora that are found. There is genomics, culturomics, metagenomics. There is different ways to look at the microbiome and genomics. They were things like 16S rRNA pyrosequencing and the latest whole genome shotgun sequencing. And then there is this whole concept that well, maybe, it's not just one organism but it's two or three or four organisms working together. And that's called sociomicrobiology. So let's just look in the last 10 to 15 minutes, just look at what's out there and I will tell you it's not easy to weigh through. Because when you start reading the papers that are out there on these topics and I love going through the scientific literature and I love reading these papers and to me the best thing are graphs. I love looking at graphs because graphs really give you visualization of what you are trying to understand and what you are reading. And let me just show you an example of some of the graphs I found in this topic. What am I supposed to do with this? What's a clinician to do with all these information? We have got scattergram, we got bar graphs and we got -- I mean every color known to man. Really what's any of this mean? So let's take a look in a little more detail at some of these topics. First, what is a biofilm? You have probably heard a lot about biofilm over the last two or three days, because it is the hottest term in the world of wound healing. Biofilm by definition - -Constantine [phonetic] [18:00] defined this back in the late 1990s, so the whole concept of biofilm was not that old.

[18:05]

A structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. I think biofilms are real. I mentioned yesterday being a biofilm agnostic and again I apologize. I think that's the wrong term. Maybe more of a biofilm nihilist. I don't know. But the point is that we aren't really -- we know biofilms are there. They have been shown. They are shown microscopically, by the way. That little slime you see over the wound is not a biofilm. You actually need to look microscopically and see this covering all the different colonies. What is the role of a biofilm? Some bacterial species produce these glycocalyx that inhibit antibiotic penetration. So biofilms are basically self-protection devices by the organism to keep them safe from antibiotics or other invaders. The most common organisms that cause biofilm are staph aureus and pseudomonas even though lots of other organisms can also cause them and within the biofilm these organisms feel safe and secure. So they basically become quiescent. They just are not continually reproducing and they just go into a nice quiet state. This makes them antibiotic resistant because not only can't the antibiotic penetrate the biofilm but when the organism is not actively reproducing in an active metabolic phase, the most antibiotics aren't going to work against them. So they become antibiotic resistant, but to me in all of the biofilm research that has been done, the most fascinating area of research is right here. It's called quorum sensing. It is this whole concept that bacterial organisms and bacterial colonies have an ability to communicate with each other.

[20:00]

They can send out chemical signals that say okay, it's time to become active again. It's time to start fighting. Or it's okay. We can go quiet. So you have these organisms actually have the ability to start communicating with each other about whether or not to become more pathogenic or less pathogenic. And according to Randy Wolcott and Lubick [phonetic] who is in the wound world is kind of Mr. Biofilm, they say that -- he says that biofilms may prevent wounds from healing by causing chronic low grade infection. I refer to that a little bit earlier or yesterday's lecture. But I have always said, yes biofilms are there, but I am not really sure whether or not they make a difference. And I have said that for years. So I was really very happy when the European Wound Management Association came out with their clinical practice guidelines discussing the use of topical antimicrobials and wounds. And this is what they said about biofilms. Biofilms may be present to non-healing wounds but their influence on wound healing in the clinical setting is uncertain. The presence of a biofilm in a wound does not always lead to treatment failure and/or delayed healing. I think that's really deep because again we have been hearing for a last couple of years from the wound gurus that we need to use biofilm based wound therapies and we need to use these different ultrasound, contact and non-contact ultrasound to breakup biofilms. We need surfactants to breakup biofilms and we need these different dressing materials of antimicrobials to breakup biofilms or maybe not. And again, I go way back to the earliest studies on vacuum assisted closure where they looked at using the VAC foam versus standard dry gauze or wet-to-dry gauze. And what did they find? They found that there was a much higher bio-burden under the sponge as opposed to the dry gauze that these wet-to-dry gauze was much cleaner.

[22:07]

It didn't have as many bacteria. Yet which healed faster? The foam with the VAC device. So I'm not sold that biofilms in of themselves really inhibit wound healing, so it was nice that you might agree with me on this. So now we look at these different genomic testing, the molecular testing and you see labs out here and you are going to be called on by labs that are doing molecular testing and they start it with PCRs where they would amplify maybe the genetic material from one organism then multiplexing PCRs where they could amplify 5 or 10 organisms in one test. Now they are doing the deep sequencing like 16S ribosomal sequencing, and I will tell you that there is lots of literature on this, lots of literature. I just choose one to give you an example. They took parallel samples from 51 chronic wounds, they found 14 genre in aerobic cultures -- standard aerobic culture. But when they used molecular techniques they increased at 10 fold, 145 different genre were found. The organisms with higher relative abundance were more likely to be detected by culture and that makes sense. Because if there are more bacteria, you are going to find more of a chance of getting them on swab or in a piece of tissue, more of a chance that laboratory is going to be able to grow them out. This is some of the work that's been done by Randy Wolcott's group looking at 16S pyrosequencing. Now this is probably the biggest study out there on wound microbiota. They looked at almost 3000 patients and they have a lab -- actually, their lab is out here advertising at this meeting and they have done a lot of great work in this area of wound microbiota and sequencing. But this is a cool paper because we have always thought that wounds are unique.

[24:02]

A decubitus ulceration is going to have a different microbiota from a diabetic foot ulceration, from a surgical wound, from a venous leg ulcer. Because they are all on different parts of the body, but actually what they found and this is what these pretty colored graph show that the composition of chronic wound microbiome is not wound dependent type. The composition is unaffected by patient demographics. Staph and pseudomonas compromise the most prevalent genre in the microbiota and chronic wounds are frequently colonized by commensal and anaerobic bacteria including coagulase negative staph, corynebacterium and finegoldia. Finegoldia magna is an anaerobic organism named after Syd Finegold who actually just passed away about two to three weeks ago, one of the top anaeorbic people on the world. Now, this is amazingly consistent throughout every study that has been done. That you find coagulase negative staph corynebacterium and finegoldia. Yet what have we been told about coagulase negative staph and corynebacterium? You probably never even heard of finegoldia. We have been told don't worry about it. It's not a pathogen. Well, is it? We don't know. But they are really, really common. This is some worked by Malone, who actually looked at frankly infected wounds, work by Wolcott's group just looked at mircobiota. They weren't particularly infected. This is actually diabetic foot infections. They stated the diabetic foot ulcers had complex microbial communities with great heterogeneity between patients. The duration of the diabetic foot ulcer prior to infection was a major driver. If a wound was relatively new with view within six weeks' duration, then staph and group B strep as I said before the most common organism were most common but more chronic wound were polymicrobial broader in diversity and higher frequency of obligate anaerobes. Wound observations really made no difference and infection severity was associated with altered communities.

[26:04]

The more severe infection, the more communities, the more organisms you are going to find. You know this is a cool study by Larry Lavery and [indecipherable] [26:11]. They just presented this at the ADA this year. Looking at osteomyelitis, I am kind of short on time, but basically what they showed is genetic sequencing using 16 RNA did not identify significantly more cases of osteomyelitis in traditional culture, which would tend to go against everything we are talking about with high sensitivity of these testing, but if you actually look at the numbers, 82.9 versus 68.6. Maybe it wasn't statistically significant with a small group, but there was at least a trend showing that the sequencing may even be better at diagnosing osteomyelitis. Again, this is just a kind of list of organisms they came up with. This is cool. This is a new test called culturomics. Culturomics takes the best of the old culturing techniques and includes with that identification through genetic sequencing. And what they found is that molecular tools are hampered. The problem with PCR or 16S ribosome or any of these is if the organism is dead, it doesn’t matter. You are still going to find the genetic material. So there are some problems. What they did here is they grew the organisms first and then after they grew the organisms they then did molecular testing to identify them. And you can see all these numbers. I find this fascinating. The only factor associated with wound improvement was presence of enterococcus faecalis. Say what? Wound improvement. We have always been taught enterococcus is a bad player. It's a nasty wound. It can be multi-drug resistant. It's a nasty bug. It can be multi-drug resistant. Yet they proposed that maybe in some wounds enterococcus is probiotic.

[28:05]

It actually improves wound healing. Wow, that's like -- that sort of moment. When you realize it, all the things we have looked at and been taught in the past about the role of these organisms may be wrong. Then in fact enterococcus may be okay, maybe other organisms are okay. Well, I will tell you that this is a very usable technique. It's very easy to understand and I will prove that by showing you the diagram they used to explain this in their paper. Yeah, this is the problem with some of these testing. I mean it's just nuts trying to read these papers and this data. This is a group that's using what's now called whole genome shotgun. Whole genome shotgun has advantages over 16S ribosome just the old PCR and that not only is it much more sensitive, you can identify more organisms but not only can you identify more organisms, you can identify virulence factors whether this organism is going to cause infection or not and you can identify resistance factors. What antibiotics can you use and can't you use. Here is a bit of a problem with this. These reports are five pages long. We are used to half-a-page SSRR SSSR, right? No, five pages long and what you see here is they were 22 bacteria, 7 antibiotic resistance factors, two fungi, a couple of viruses. This is the list. This is what one of the reports looks like. And again, it raises this questions what's the clinician to do with this material?

[30:01]

One of the companies out here likes to tell you that the organism, the most common organisms are the most pathogenic. That's absolutely nonsense. There is no proof of that whatsoever. There is no proof of that. Alright, I just love this. This little culture and way too much information. So what are we supposed to do with this information we have? We now know we have all of these organisms. What do all of these organisms mean? Well, maybe it's not just a single organism that we are looking for. Maybe it's this whole concept that there is aspect of bacteria pathogenesis that maybe you need to look at more holistic approach and there is social interaction. Wolcott and Dowd came up with a concept of functionally equivalent pathogroups. So you go back to that list of 50 different organisms or 22 organisms in this case. Maybe it's not organism 1 because it's the most common organism, 2 because it's second most common. Maybe it's organism 1, 3, 5 and 6. They together are pathogenic. And maybe all we have to is take care of organism 3 or maybe we have to take care of organism 1 and 6. We don't know this but this is the whole aspect of sociomicrobiology and functionally equivalent pathogroups. The impact of microbial communities wound healing, although the current research has made substantial progress towards our knowledge of the wound microbiome, the exact role of wound microbiome and wound healing has not yet been fully elucidated. Studies are heterogeneous and better controls are necessary to allow for analysis and comparison across different research groups. Another words, we don't quite know. So I will leave you with this slide. Those of us of a certain age remember this crew. This is the original cast of Saturday Night Live. What did they used to call themselves? Thank you. They were not ready for prime time players. And I think that's kind of where we are sitting now with some of these molecular techniques.

[32:03]

I think they are fascinating. They are rapid. You can get results within hours instead of waiting days for a traditional culture to come back. They are incredibly sensitive. You can see all of those lists of organisms we find, but what do we do with that information. What does this mean? And I think that's the question that is yet to be answered. We don't know yet. We don't know how clinically useful they are and that's what we have to work on. Finding those organisms, determining which one or ones are pathogenic either alone or together and then making it clinically available to you, the clinician so that you can determine what antimicrobial to use because the traditional SSRRSR is not going to work in this cases. Thank you very much.

[Applause]

TAPE ENDS - [33:03]