John Aitken: The Microbiology of MAP
My name’s John Aitken. I’m a medical laboratory scientist from New Zealand. New Zealand, of course, is one of the colonies of the British empire. Britain had a policy to get rid of everybody in Britain who was a nuisance to them, so the Irish were sent to America, the convicts were sent to Australia and the lunatics came to New Zealand. So don’t expect a lot of really groundbreaking stuff here. I’m going to show you some photographs.
This is the legendary Tom Borody that everybody talks about. Tom hooked me into the field of research on MAP. I’ve been playing around by myself in New Zealand on it for a number of years and I had the fortune to run into Tom at a conference and we completed a double blind on his patients very recently. And Tom has a multitude of patients in the double blind; was not the easiest thing I’ve ever done, but it went well.
If you are a Crohn’s researcher, it is like that. You are essentially alone. You are isolated from your colleagues and it’s doubly bad when you are in New Zealand, because not only are you isolated from your colleagues, but you are isolated from the rest of the world. However, you do have colleagues who will help you. Who will look at what you’re doing and discuss things with you when you present topics to them. And they’re very collaborative and they do offer a lot of very helpful contributions and also provide very helpful feedback to what you’re doing. Because nobody actually believes that MAP is causing the problem. And you can summarize the debate like this: On the one hand it is not MAP, on the other hand it is MAP. No it’s not, yes it is. It’s not MAP, it is MAP. It goes on like this in a cul-de-sac; a little street of people who are arguing with each other and do not move the argument forward any further.
One of the really good papers that was written on this was by Dr. Ellen Pierce who is a Crohn’s patient and she’s an anatomical pathologist. And it’s not often that you’ll get a great deal of common sense from anatomical pathologists but in this particular case she asked a very important question: Where is Mycobacterium avium paratuberculosis in the patient? Because everybody talks about it, but nobody can see it. Is it in the affected tissue? Is it in the blood? Is in the mesenteric lymph nodes? Why can’t we see it then? And the answer is, in my opinion, because we don’t look properly.
If you look at the literature on Unidentified Funny Objects that occur in relation to autoimmune diseases, there’s any number of them. And they’ve been described over the years. They’re in textbooks. But people don’t actually read textbooks anymore, they go to Google. So if you want to find the cool stuff on TB, you have to look prior to 1944, because in 1944 streptomycin was discovered and soon after that the other anti-tuberculosis antibiotics. So everybody actually stopped looking at TB from a close, intimate, bacteriological point of view and decided that the problem was solved.
When I started working with TB in the 1970’s, I was told that there was no point in me actually going into that area because the problem was solved and TB was going to be a disease of the past. Well, they got that wrong.
One of the places where the organism described as MAP exists is inside the white cell. So it’s well hidden. There are three schools of research. The first one, and the oldest and most trying one, is to actually isolate the causative organism and prove that is causes the disease. That’s what people do with most emerging diseases when they occur. And I’ve worked a lot with emerging disease over the years.
The second one is, if you can’t find the organism, you look for a specific DNA sequence. Now, if I break this down to a level that everybody can understand, to culture to isolate the bacteria is like me looking for my wife and talking to her face to face. And she’s just over there so I can quite effectively do that. If, however, she wasn’t here, I could ring her on the telephone because I have her telephone number. And a specific DNA sequence is like a telephone number. It finds the object or the sequence that you’re looking for. And the third one is the microbiome. And everybody talks about the microbiome at the moment. It’s a new thing; it means the organisms in the gut. If you want to look for the microbiome, in my opinion you’re picking up a telephone book and you are dialing at random, because you get all this information on every sequence that is available in the gut. And sooner or later I might strike Helen if I got really lucky. So the microbiome looks at everything. The culture looks at the organism. The specific DNA sequences look at an indication of the organism.
The other big thing is the Ziehl-Neelsen stain. The Ziehl-Neelsen stain is a stain that has a very long history, and specifically stains up mycobacteria, and nothing else really in the classic way that the ZN stain has worked. ZN/Ziehl-Neelsen are the same thing. The mythology is that the ZN stain only works if the organism has a cell wall, because inside the cell wall are the particular compounds that retain the stain, which is a red color. Everything you see in the rest of my slides that’s red is going to be the ZN or Ziehl-Neelsen stain. The problem is, that is completely incorrect. That is the mythology. The mythology was disproven in 1936. What actually happens is the Ziehl-Neelsen stain works on a semi-permeable membrane within the cell wall. So if you remove the cell wall, the Ziehl-Neelsen stain will still work. There’s nothing to do with the cell wall, although the cell wall is helpful.
What actually, what we do is we incubate our samples in a novel media that we’ve worked out, which slightly strengthens the semi-permeable membrane underneath the cell wall. So we find organisms that don’t have cell walls and check them out. The cell wall is a rigid structure that surrounds the bacterium, or the membrane of the bacterium. It keeps everything closely rigid like a corset. It can also be removed like a corset. So you can take off the cell wall and the organism will still be sitting there. But also like when you take off a corset, things may not be quite the same shape. In fact, the organism balloons into a large, sort of round shape, and it may get bigger and bigger and bigger until, helpless, it bursts. The cell membrane manages to hold the bacterial contents, including the DNA, inside it without the cell wall.
Now the cell wall, to your immune system, is a red flag. If the cell gets inside your body and travels around, the immune system gets really excited because it sees the cell wall. The cell wall is like a uniform, so a response is triggered. If the organism doesn’t have a cell wall, then it becomes a stealth pathogen and it gets away with quite a lot, because it’s very difficult for the body to detect it. What I’m talking about when organisms don’t have cell walls, is what is termed L-forms of Mycobacteria. Now, still alive, they multiply, they replicate; they just don’t have cell walls. Because they don’t have cell walls, they replicate rather quickly. They replicate a lot more quickly than an organism that contains a cell wall. So they’re living bacterial forms. They have a uniform, well they don’t have a uniform but they still actually have their underwear. And the underwear is what we are very interested in.
Now, L-forms can be made in the laboratory. They can be made in the laboratory if you strip off the outside cell wall. And you do that by using suboptimal levels of antibiotics. Which is rather interesting, if you can induce this in the environment artificially using antibiotics, the emergence of Crohn’s disease happened about the same time as the introduction of antibiotics. They’re around about the same time.
This was the first photograph I ever took on a Leica M3 I had at the time. And that is a ZN stain of the L-form in blood. And you can see the red spheres, external slightly to that blue thing, which is the white cell. So the red is the L-form, the blue is the white cell.
So do we see L-forms of mycobacteria in the environment? Because it’s quite an important question, because the question that people are going to ask is where on earth does this come from? You see it in water quite frequently, particularly in biofilm and slime that occurs in the troughs in farms. I don’t know whether you see it in milk or not. I’ve been too frightened to look, because there’s this think of plausible deniability. If you don’t know that it’s not in milk, you can answer the question honestly. Once you know it’s in milk, you have to say, yes, we saw it in milk. So, I have to say, we haven’t looked in milk. It’s in amoeba. And amoeba are those tiny little single celled organisms which are very similar to white cells, but are in the environment. And we see it in cattle and deer.
And this is a photograph taken of…This is a photograph of MAP infection in a deer. And over here on the left, you will see the cell wall version of MAP. And over here on the right you see the spheroplastic forms. So we know it exists in ruminants in combination with cell wall entire MAP.
So in amoeba, you’ll see them on the farm, containing L-forms, in the water. So a really good experiment to do, is to take a culture that we already have of a patient who has Crohn’s disease and whom we have demonstrated the L-forms in their blood. So we take the culture that’s growing, we spread it over a plate, and we put some amoeba on the plate, because one of the things that we have in the laboratory is very tiny amoeba. So we just put them on the plate and we let them have a browse. After two days you will see that the amoeba will selectively pick up the L-forms and carry them inside them. Now, this is interesting because amoeba are known to have a preference for Mycobacteria. After three days, just about all of the amoeba are gorging themselves on the L-forms and they’re starting to multiply inside of the amoeba.
Now, the great mystery of MAP. It is associated with inflammatory bowel disease, but it is seldom reported in inflammatory bowel disease cultures. It’s often detected by PCR, very variable results. It’s always dismissed by your colleagues. Whenever I start talking about MAP and spheroplasts and red spots, the reaction by my colleagues is, “Nurse, nurse! He’s out of bed again!” Targeted antibiotics work. So we know that because if we put patients on the Anti-MAP therapy, a large percentage of them improve. Steroids also work. So that’s a difficult one to figure, because in TB steroids don’t work. Steroids actually make the problem worse. We’ll come to this in a moment.
A vaccine that is used against TB is Bacille Calmette-Guérin which is a Mycobacterium bovis. It’s a tame strain of Mycobacterium bovis that you inject into patients that protects them against TB. It’s a living TB vaccine. Once you get it, it will be living for decades inside the patient. This is relatively recent news that’s been published. So it doesn’t die. The mycobacterium exists inside the patient and is passed on from macrophage to macrophage for decades. So there is a precedent there, that mycobacterium can survive inside the blood and live quite well as L-forms. If you ask people, how does BCG work, they say, Oh, it stimulates immunity against tuberculosis. Nobody knows how it actually works. We can detect it by using our tests and we can differentiate BCG from tuberculosis and MAP by DNA because it’s got a few unique sequences. So we know that we’re dealing with BCG. There’s what it looks like in a patient after 20 years. We will still be isolating those L-forms.
OK, so now we get on to the pretty pictures. What we are seeing in the blood of patients who have Crohn’s disease and what the L-forms look like, and what this has contributed to our knowledge of what we think is happening. We see a number of different forms. The important one, to my mind, is the persister form, and I’ll talk about that in a moment. The persister form is the one that lives through hell and high water. These are the large forms on the right hand side, and you can see they’re growing quite well. They’re nothing to do with macrophages at that stage, they’re just growing as a culture in the blood. The ones on the left are what we term ghost forms. They start to disappear after a short period of time in the blood. They’re probably not viable. The ones on the right will live until kingdom come.
These are rupturing forms. These are forms that are like a weather balloon. They gradually get larger and larger and larger and then they pop. And this is only in vitro. This is only outside the human body that this happens. We don’t know whether this is happening inside the human body, but if it is happening inside the human body, it is releasing DNA into the environment. It’s releasing bacterial DNA into the environment, and that may be inflammatory.
This is the thing that we think probably has a direct role in the genesis of inflammation in the bowel. It’s a single form and it has a cloud of biofilm round the outside. This organism does not multiply, it just produces biofilm. So it produces slime. And there are a lot of precedents for this phenomenon. The main one is altruism. Some bacteria sacrifice the requirement to multiply, and they swap it for something that does good for the rest of the species. And in this case, this organism is producing biofilm as a shield for other organisms to grow inside. So it’s not multiplying itself. It’s making a little nest. And you can see it happening here. There’s the biofilm being produced; the single form inside it. Over a period of time, the debris and the biofilm coalesces, and you will get a type of nest in which the L-forms can multiply. This doesn’t happen in the human body, we don’t think. This is just something the organism wishes to attain to reproduce itself.
These are the L-forms of mycobacteria on solid agar. They present as what is called poached egg colonies. They’re very small. They look like poached eggs. This colony is starting to produce biofilm. You can see it happening there. This is a later picture, maybe about two or three days later, of the L-form colonies as they form. There you can see on the lip of the colony, you can see biofilm starting to be produced. Now, if you treat the organism really nicely, and give it lots of food and secret herbs and spices, you will get it producing massive amounts of biofilm. This is something that I have never seen in any other mycobacterial species. It’s a trick I’ve never been able to do on other cultures in this genus.
We know, one of the reasons we know it’s alive is that it’s attacked by phages. And phages are viral particles that knock holes in bacterial cells. So we see these attacks going on periodically, and we know that they are living organisms that are doing it.
Interest thing is that biofilm is only produced outside the white cell. Inside the white cell everything is really cozy and the organism does not produce biofilm. There’s a suspended dormancy or condition inside the white cell. So the question is: Does the biofilm production turn the L-form into a pathogen? Because what you effectively have is an organism inside your body that is pumping out small amounts of protein, glycopeptides, herbs and spices, and they may be sufficient to sensitize your body and cause inflammation.
So, (showing images with) transmission electron microscopy and scanning electron microscopy. This is Rod Chiodini’s original scan in 1996 and in 2012 I thought this was pretty good. I looked at this picture over a number of years. It looked quite convincing, but when you look at the cell membrane there, it’s actually not a membrane so much as a torn piece of paper. It’s a good example of an L-form, but it’s not as good as some of the examples that I’m going to show you.
This is what we tried to do in 2012. There’s our L-forms. Here you can see some that resemble a bit Chiodini’s splendid photo in 1986, and this is what we are now getting on scanning electronic microscopy of the L-forms. Because people, one of the things people say is, these are not living organisms. This is some sort of artifact. These are, this is another SEM of the L-forms. These are the L-forms present, and the matrix behind is biofilm. This is our ZN stain of an organism, of the mycobacterium. There you can see the rod, which is the normal shape of mycobacterium. There is the L-form with its halo of biofilm around the outside. There are the rods here. There are the spheroplasts.
On transmission electron microscopy, which is like you slice a loaf of bread. It slices pieces of the organism so that you can see what’s inside of it, instead of the outside which is what you see on scanning EM, you can see there, over a period of time they thicken their pseudo-membranes. It’s not actually a cell wall, it’s a thickening membrane. And we can produce the same effect on our Ziehl-Neelsen stains. And you can see the same thing happening there.
One of the interesting characteristics of the organism is that it forms chains, and you can see it happening here on TEM. And here it is on the ZN stain. In fact, chains are a feature, the division of the organism on a longitudinal axis is something that is typical of mycobacteria, and we’re also seeing it happen in the L-forms.
You get the elongated forms when the organism grows very long but it doesn’t divide. And you can see it happen on the scanning electron microscopy there. These two at the top; this is a ghost form we think, and this is a normal L-form. These are TEMs of the L-form looking at it up closely. And you’ll see that it has a very thin membrane round it, and there’s the DNA inside it.
So the other question is, are these things alive? Because these are the questions that people keep beating you with; is this a living organism. So one of the, it’s important to know whether they’re living or dead to know whether a therapy works. So we designed a viability stain to see whether they were living or dead. It was a great project. We’ve now gone a little bit further and we’re now looking at PCR to detect living forms, and that seems to be working better when you combine it with antibiotics. Because we’re now looking at assays where, before the patient goes on therapy, the organism is isolated, the susceptibility tests to the different antibiotics is done, and then the patient starts on therapy.
But this is a viability study. And you can see these black dots in the top here are organisms that have taken up the marker because it’s still alive. The greenish stain around there is the biofilm, and this is just normal background material.
This is one of the ways we monitor the growth of the organism. We do counts after eight days and we do counts after thirty days, and we express them as a percentage of the total number of the clumps or clusters that we see. And you can see here in the red, 8 day column, 20, over here 32 on the 30 day. And it’s the same all the way across. We get multiplication and increases of cells over 30 days. And that is a very strong indication that we are seeing growth in our media.
The media we use is based on some of the data from the microbiome. We postulate that if the microbiome is modulating something, it is doing it by producing nutrients for the organisms. So we look at the perspective growth compounds that are coming out of the microbiome and we artificially insert them into the media to see if they have any effect on ours.
OK, so this is the preliminary data we have. The mycobacterium species probes are positive. The MAP probe is positive. We have a number of virulence factors that we have positive results for. Is it MAP? Sort of. One of the problems is that some of the virulence factors we are getting are not traditionally associated with MAP. There is some sort of hybridization going on. We actually call it Son of MAP.
What happens when you start a patient on Anti-mycobacterial therapy? You look at them beforehand. You look at them afterwards. The first thing you see is a reduction in the number of L-forms within two weeks; vast reduction. The population changes from the rupturing forms and the ghost forms, to more stable large forms that start producing quite a lot of biofilm. And then you get down to the single forms; the persister forms. And you can still see these occasional persister forms in patients after four years.
So the next step for us is animal studies. We are very fortunate in that we have viable L-forms so we can shoot them into the next rodent that comes along and see what happens. The studies for animals are very complex to set up and control. Is the mouse the best candidate to do these studies on because up to now, the mouse seems to have been the candidate. Not only a mouse, but a mouse so severely immuno-compromised, that it looks like it’s been run over by an ambulance immunosuppresively. And we would probably look at other animals, other than the mouse. I would not bother doing a virulence study on an animal that was already seriously depleted. We would try to find something that was living, hopping around well, and see what happened to it.
This is the hypothetical life cycle of MAP. Even if we ignore the idea of Crohn’s disease, it’s introduced onto the farm as Mike said. The animal gets sick. The animal excretes MAP. L-forms are present and we think that eventually on the farm they predominate in water and other animals. And the L-forms may even be transferred to humans and other animals. In humans you do not see the cell wall form. You only see the L-forms.
These are the; this is the hopeless bunch that I’m working with at the moment. We work with Dr. Nadya Markova and Georgi Slavchev at the Bulgarian Academy of Science. Absolutely brilliant people. That academy has worked on TB for 70 years. They do not see Crohn’s in Sofia. They do, however, still see TB. So us bringing in samples from outside and getting them to look at it is actually quite instructive, because they bring their TB skills to what we’re looking at. And we have molecular biologists at the University of Otago and Massey University. Dr. Will Chamberlin and Prof. Tom Borody we work with.
I’d like to acknowledge the patients, because we’re here today because of the patients. And each one of us who has come up here has played a particular tune, but you guys write the song in the first place. We’re only playing the tune. It is the patients that are presenting and are giving us so much valuable information in what we’re doing. So thank you to the patients. The Serrano family I’m deeply indebted to. They provided some seeding funding for us to do the work that I’ve just outlined. The Canterbury District Health Board in New Zealand have provided a space, and there are two organizations that encourage innovation.
This is a good slide to end up with. The quote is from Abraham Lincoln: “The hen is a very wise animal; she does not cackle until after the egg is laid.” The egg is not yet laid, so at the moment we’re all looking at the same problem from slightly different directions. As the answer emerges, we will be more confident, or we will turn around and say, we were completely wrong. Chickens, of course, are a source of a zoonosis – campylobacter. Thank you very much. Are there any questions? Anybody got any questions; good.