John Aitken is a free-lance microbiologist based out of Christchurch, New Zealand and the senior director of Otakaro Pathways, Ltd. Prior to his current position, he worked in medical microbiology for public and private providers for more than 40 years. His particular areas of interest are antimicrobial resistance and emerging bacterial infectious diseases. John is presently involved in research surrounding the relationship between immune diseases and the Mycobacterium species.

Here, John discusses his latest research into mycobacterium species found in Crohn’s disease patients.

Transcript

Dr. John Aitken is going to talk to us about moving into the light. Dr. Aitken is a freelance microbiologist based out of Christchurch New Zealand, and the Senior Director of the Otakaro Pathways, Ltd. Prior to starting his current position he worked in medical microbiology in the private and public sectors, for over 40 years. His particular areas of interest are antimicrobial resistance and emerging bacterial infectious diseases. John is presently involved in research surrounding the relationship between immune diseases and the Mycobacterium species.

Thank you Bob. I’m a bacteriologist, which is a depressing thing like alcoholism in that you get seduced by a very addictive hobby, which is looking at bacteria. Otakaro Pathways does research on microbiology. We do bioprospecting. We look for novel antibiotics and novel probiotics. Very interested in autoimmune diseases, particularly in Crohn’s disease.

We also develop culture media for various for various purposes. We examine the bacteria, look at what they metabolize and design culture media to suit. And we also pretty much have microscope, will travel. So if somebody comes in and asks us about a particular thing, we look at investigating it.

For example, there’s the Strep. salivaius we found recently. Strep. salivarius is a known probiotic that’s present in most humans. This strain was sent to us by…It produces an antibiotic directly into the agar that moderates biofilm production. So if the organism had biofilm, this organism swept away the biofilm quite remarkably. It doesn’t have any activity against Staph aureous or E. coli, but there’s a strong activity against rapid growing mycobacteria.

And you can see on this section of plate here that the organism has been inoculated on there, and you can see that there’s a clear zone around it. Which is pretty interesting. This is not as popular as Dietzia, which some of you may be familiar with.

This is the guy who caused all the troubles: Robert Koch. And he discovered in 1882 the causative organism of tuberculosis. And that was Mycobacterium tuberculosis. He devised a stain to see it which took 24 hours to perform. Now, the thing that you need to know what I’m going to talk about, is bacterial morphology. When Koch discovered tuberculosis, he discovered it in the bacillary form. So the long, sausagy organism. The coccus is quite different. It’s a round, spherical bacteria. And two round cocci joined together are diplococci.

That’s the theory, but if you look at the pictures, there organisms are all over the place. If you look at the Gram stain, it’s just a hodgepodge of cocci, bacilli, streptococci, staphlococci, diplococci, the whole none yards. And this is an example of a mycobacteria, which is showing an example of pleomorphism. And that is able to produce all of those shape. So these organisms are not limited to one definition. They’re depending on the atmosphere, they’re depending on the culture medium, and they’re able to take different shapes.

OK, my talk today is about seeking the primary dividends. Because with science, you can go off into all these detailed sidetracks, which lead you into doing a lot of detailed work. If you go back to where it all originated, then you can get a sense of perspective of where you are. Ellen Pierce, the researcher on Crohn’s disease and anatomical pathologist, asked a very simple question: Where are all the MAP in patients with Crohn’s disease? If you look at a patient with tuberculosis, the organism is everywhere. If you look at a patient with Crohn’s disease, you can’t see the organism.

So are they actually coccoid forms? Are we looking for bacilliary forms when we are supposed to be looking for coccoid forms? Are the inside the macrophages so they’re difficult to see? Are they hosted within the leukocytes as Dr. Pierce suggested? Or are they not there at all?

1882 Koch discovered tuberculosis. By 1883 Ehrlich, Ziehl and Neelsen had discovered the ZN stain, the Zielh-Neelsen stain, which takes about 15 minutes to do. So they went down from about 24 hours to about 15 minutes in a year. And then a chap called Henry Gabbitt, a Brit, came along and developed the Gabbitt modification decolorizer, which moved the ZN stain down to about 8-10 minutes. And you didn’t have to heat the stain any longer, because with Koch’s modifications you don’t heat the stain. You can just use cold solutions and you can do it in 10 minutes.

In 1892 it was in beautiful use, everybody using Gabbitt’s modification. Gabbitt’s modification sees not only the baciliary forms of tuberculosis, but also the coccoid forms. One of the mythologies about the Ziehl-Neelsen stain is that mycolic acid is essential for the stain to work, and therefore mycolic acid must be there before the ZN stain will work.  Coccoid forms, ie: cell walled deficient mycobacteria, CWDM, do not have cell walls. They have a semi-permeable membrane. So everybody says, How come the ZN stain works when these organisms don’t have cell walls. And the answer is, because of the semi-permeable membrane. Mycolic acid’s got very little to do with it. Although 90% of microbiologists think that mycolic acid is essential and there are papers there that demonstrate that mycolic acid is not essential but a semi-permeable membrane is. So the bag around the outside of the DNA is all that is needed for the ZN stain to work.

OK, so Gabbitt comes along and uses the ZN stain in 1884, thinks I can do better than that. So he invents his modification. The modification is very simple: 1 g. methylene blue, 20 mL sulfuric acid, 30 mL ethanol, 50 mL H2O. If you put them all in the same solution, and you just wash the it with Gabbitt’s modification, and you get cell wall deficient forms, coccoid forms, and bacillary forms.

In 1892, the Gabbitt modification was adopted by Moabit Hospital in Berlin, which was Koch’s hospital. So it went straight back to Germany.  It upset Gabbitt so much that he published in a British Journal in 1892 complaining the German’s had stolen his method.

So these are the papers that relate to the Gabbitt modification if you want to look at it. It’s a very popular modification in India because you don’t have to heat the stain and because it can be used in conditions where you do not have access to laboratory equipment.

So if you take the idea that coccoid forms were visible through the Gabbitt modification, or in other words, cell wall deficient mycobacteria or coccoid forms, you go back through the literature and everybody was seeing them. They were present as well as the bacillary form and the coccoid form. And it became a real paper chase. If you saw the coccoid form, you would publish a paper on them

When you get to the 20th century, people were still reporting the coccoid forms and them a chap called Much came along and said there’s actually a smaller form than the coccoid form. There’s a sand like granule, and if I grow it for long enough, it will produce a coccoid form and produce the baciliary form. And that granule, which was called Much’s granule, was filterable, so you could put it through very, very thin porous filters, and you would get these granular forms out, and then you would be able to grow TB again.

So this cause quite a fight. Somebody said he’s right, he’s wrong. And this went on for about 20 years or so, and during that time, more work was done on the coccoid forms. People demonstrated coccoid forms, and most of them were using Gabbitt’s modification and not the present ZN stain we use today.

Krassilnikov came along in 1935 and discovered a form of mycobacteria that was a coccoid form. And he described it. And he was a soil microbiologist, and he made a complete new genre of mycobacteria called the mycococcus. When he found these coccoid forms of mycobacteria, he would assign them to the mycococcus group.

And then Anna Csillag, a Hungarian microbiologist, Csillag means “star” in Hungarian, came to England and worked at the Health Research Council in London and also at Porton Down. Her subject was tuberculosis resistance, and she discovered a Form 2 mycobacteria. She discovered that in tuberculosis, there were 2 forms of mycobacteria. There was a coccoid form, and there was a baciliary form. And with much effort, we could isolate the coccoid form from a growing colony of tuberculosis. Very interesting publication. She made two mistakes. One was that she said that the form 2’s were not ZN positive. Because by this time she was using the modern version of the ZN stain that was used. And she also assigned it to the great mycococcus species, which was probably a mistake because the form 1 is the baciliary form, the form 2 is the coccoid form.

Because what happened then was she published a number of papers on mycococcus, and really went to town on them. And they’re tested papers. When you sit down and read them, they are examples of really good bacteriology done in the 1960’s. And then her colleagues came along. And the first paper that was written by a detractor about Csillag was called The Taxonomic Characteristics of “So-Called” form 2 mycobacteria. And the author argued that what Csillag was seeing was definitely something called mycococcus, and that mycococcus didn’t exist.

And so she published retaliatory papers, which are also very interesting to read, very personal in her views, and by 1993 DeWitt, Mitchison had published a paper based on Csillag’s original isolates which suggested that they were not mycobacteria and that Csillag was completely wrong. And so mycococcus and Anna Csillag were packed off to the side somewhere, and we all forgot about them. And particularly we forgot about form 2 mycobacteria.

This is a picture of Csillag’s form 2 mycobacteria on the right hand side. You’ll see that the coccoid, and diplococcoid, two cocci together. Now, moving on to 1971, Alan Cantwell, the confidential dermatologist, described the isolation of spheroplastic forms in scleroderma. And you can see on the right, the spheroplastic forms. And then Nadya Markova, who’s probably the one person worldwide who’s done a significant amount of work on cell wall deficient forms and form 2 mycobacteria, and L forms, started her work on looking at cell wall deficient mycobacteria. And she’s still working in the present day on it. And her work is very, very intricate, very intricate, very skilled. And she is able to isolate the cell wall deficient forms of mycobacteria in  L form cultures.

You move on to Blaine Beaman in the 1980’s. Beaman described Nocardia asteroides in allied organisms as adopting the cell wall deficient form. And on the left you’ll see one of Beaman’s photographs, the big round things there are the L forms present in mouse lungs. And on the right is a disciple of Beaman’s whose photographs are electron microscopy of the cell wall deficient forms. And the white spots are all lipid bodies deposited on the inside of the cell.

Then Edward Cunningham published a very interesting paper on what happened if you subjected a mycobaacterium to an environment that is very low in oxygen. What happened is that the cell wall thickens. You get a spore like quality to the organism.

Rod Chiodini, who probably needs no introduction to most of the people in here, Rod published a series of absolutely fascinating, ground-breaking publications on the cell wall deficient form of Mycobacterium avium paratuberculosis. And this is one of his early electron microscopy photographs of the cell wall deficient form. Big, fat and no decent cell wall. Just a semi-permeable membrane.

Ghosh in 2009 started a big argument, because he said, Oh Look, we have found that mycobacterium forms produce spores. And everybody got stuck in the Csillag like gang fest of abuse and said this is impossible, you can’t do this. But he had photographs showing the spores, what he described as the spores, forming. It’s highly probable that what Ghosh was looking at was cell wall deficient forms with a semi-permeable membrane that had become encrusted, and therefore resembled spores.

The Aitken came on the scene and produced this photograph of cell wall deficient forms present in human blood. And that was using Gabbitt’s modification taken a stage further. Because the problem with the ZN stain that we use today is that it has a lot of alcohol in it. There’s 97% alcohol, 3% HCl and a decolorizer. Alcohol is a reducing agent. Reducing agents are not conducive to ZN stains working well unless it’s a Mycobacterium tuberculosis. So the ZN stain we use today, which has alcohol and HCl in it, was optimized for Mycobacterium tuberculosis. Not for atypical mycobacteria, not for cell wall deficient strains. So from 1965 or so to now, we’ve been off track. The only reason I knew about this, was because I worked with people who had worked in the pre-streptomycin days of TB. Everybody knew about this then. We forgot.

This is a paper by Sreevatson, and he said that he had found endospore like mutation of Mycobacterium avium paratuberculosis. So this reconstituted the spore debate. And this on the right hand side you will see an electron microscopy of what he described as endospore like. And what he was looking at was an external encrustation which was not actually a spore, but it resembled a spore.

OK, so we go back to always seek the primary evidence. Where are all the Mycobacterium avium spp. paratuberculosis? My answer is that they’re probably present as form 2 mycobacteria and similar variant strains and coccoid, that are not seen on the normal ZN stain we use. Why don’t medical laboratory scientists see them? These are the people who sit in the laboratory and examine all the samples. 1) They’re not aware so they don’t look for them. Very much like Helicobacter pylori where all the anatomical pathologists remember that they’d seen these Campylobacter like forms in the stomach lesions but they all said they were probably artifacts. And then when Marshall came along they re-looked at them and said Oh, they’re not – they’re actually bacteria.

I think that in this case unless people are programmed  to look for these things they automatically discount them as artifacts. And there’s also this thing called Resource Utilization Control that laboratories are no longer encouraged to do research. So unless it fits in the narrow spectrum of diagnosis, they don’t look outside it. They use the wrong stains and the wrong media.

Now, 2018, when everything old is new again. This was on the television and it’s the new Samsung Galaxy which is a Back to the Future production. So what does the laboratory want these days? We want an efficient protocol. We want something that you can put in a book and be told how to do it. It has to be easy to interpret. It can’t be something that is complicated either in the methodology, or in the interpretation. It has to be integrated within a medical laboratory. Because you can’t have molecular pathology running around saying Oh, it’s IS900 positive there’s MAP there, and the microbiologist saying we can’t see any mycobacteria, and the anatomical pathologist looking at the sections saying I can’t see any mycobacteria there, and you get a discordant note coming out of the medical laboratory when we’re all supposed to be singing form the same sheet.

It has to be cost effective since everyone’s worried about money. It has to be skill based because everyone’s worried about losing their job if it becomes too easy to do. It has to be validated, and validation I’ll talk about soon. But most importantly, it has to be reproducible and reliable. You have to be able to sit down and see the same thing in Patient A if it doesn’t clear up, that you see in Patient B if it doesn’t clear up.

The clinicians needs are complimentary. The clinician wants to help the patient. They want a Yes/No answer. Well is it or isn’t it? They don’t want, well it depends, we see it in normal patients as well. They need a biomarker to tell objectively if the patient is getting better or not. Not a subjective observation. They want companion therapies,