Dr Markov CBIS Theory of ME/CFS - General Discussion

Hip

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I've now managed to isolate and identify three different bacterial species in my urine, with the help of chromogenic agar (brand: Brilliance UTI Clarity), which turns a different color depending on the species of bacteria placed on it, which aids in identification of the bacterium.

Here are some pictures of the chromogenic agar plates on which I transferred the bacteria growing on the urine dipslides, and the colors which have appeared:

On this chromogenic agar plate, the blue-green color
indicates growth Enterococcus, according to the
chromogenic agar instructions.
Brilliance UTI agar 5h after innoculation, Enterococcus.jpg



On this chromogenic agar plate, white color may indicate
Staphylococcus or Streptococcus, according to the
chromogenic agar instructions, but I am not really sure.
Brilliance UTI agar, possible Staphylococcus or Strep.jpg



On this chromogenic agar plate, the dark blue color
indicates coliform bacteria. Coliforms include Escherichia,
Klebsiella, Enterobacter, Citrobacter and Hafnia. It
cannot be E. coli, however, as the chromogenic agar
instructions show E. coli turns a pink-red color.
Brilliance UTI agar, coliform bacteria like E. coli.jpg


Interestingly, these different species of bacteria appeared on different days, captured on different dipslides.

Each day I would use a fresh new urine dipslide to expose to my morning urine, and then I would incubate the dipslide at 37°C for 24 hours. The next day, sometimes you would see growth on the dipslide, indicating that you'd caught some bacteria; but other times there would be no growth.

When there was growth, I would transfer the bacteria to the chromogenic agar with a sterile tool (I used a sterile hypodermic needle), and then incubate the chromogenic agar at 37°C.

For the first dipslide which showed growth, when I transferred some of that growth to the chromogenic agar, it turned out to be Enterococcus, as shown by the blue-green color on the first image above.

Then a few days later, I got some more growth on another dipslide. When I transferred this to the chromogenic agar, it turned out to be possibly Staphylococcus or Streptococcus, as shown by the white color in the second image.

And finally several days after that, I again got growth on a dipslide, which when transferred to a chromogenic agar plate turned out to be coliform bacteria, shown by the dark blue color in the third image above.



So for some reason, these 3 bacterial species each appeared on their own, on a separate occasion. I would have thought that if there are 3 bacterial species present in my urinary tract/kidneys, they would all appear at the same time on the same dipslide.

But these bacterial species are obviously not very sociable with each other, so they come out separately. I am not sure if this is the norm, but that's certainly what I observed in my case.


So this shows that if you have identified one bacterial species using a urine dipslide as some chromogenic agar, you probably do not want to stop there; you may want to continue urine testing to see if you can find other species.
 
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Hip

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If you want to buy some chromogenic agar plates, two brands seem to be available online:

Brilliance UTI Clarity, made by Thermo Fisher / Oxoid

Available here (ships internationally, and they say you can buy without an account).

And here (ships to UK only, and only sells to organizations, but I pretended I was one, and was able to buy).



CHROMagar Orientation, made by BioPrepare

This seems to be sold by Greek websites here, here, here and here. And a Danish site here.



Chromogenic agar plates have a shelf life of only around 6 weeks when stored in the fridge (they cannot be frozen). After that time, mold starts growing on their surface.

An alternative to buying ready-made chromogenic agar plates is chromogenic agar powder, which can be used to make your own chromogenic agar plates (you can buy empty sterile plates on Amazon).

Brilliance UTI Agar chromogenic agar powder instructions state:
Suspend 37g of Brilliance UTI Clarity Agar (CM1106) in 1 litre of distilled water. Mix well and sterilize by autoclaving at 121ºC for 15 minutes. Cool the medium to 50ºC and pour into sterile Petri dishes
You can use an pressure cooker in place of an autoclaving. If you do not have a pressure cooker, possibly just boiling at 100°C in a saucepan may suffice to sterilize (boiling kills everything except spores, which require pressure cooker temperatures of 121°C to kill).

Generally, for 1 liter of agar mixture, you can fill 50 average-sized plates (petri dishes). So 100 grams of chromogenic agar powder should make approximately 135 plates.


Chromogenic agar powder can be bought from these sellers:

HiCrome™ UTI HiVeg™ Agar neolab.de — $28 for 100 grams

HiMedia HiCrome UTI Agar labgenome — $53 for 100 grams

HiCrome™ UTI Agar Thomas Scientific — $60 for 100 grams

HiCrome UTI Selective Agar neofroxx.com — $192 for 500 grams

Buy HiCrome UTI Agar biomall.in — $142 for 500 grams

HiCrome? UTI Agar (500G) himediastore — $210 for 500 grams

Brilliance™ UTI 500g LabMal — $450 for 500 grams

Brilliance UTI Clarity Agar LabMal — $514 for 500 grams

EO Labs Primary UTI Agar analab.ie — $725 for 500 grams

HiCrome UTI Agar analab.ie— $664 for 500 grams

Brilliance UTI Agar, 400 g | analytics-shop.com — $427 for 400 grams

UTI ChromoSelect Agar, modified krackeler.com — $557 for 500 gram

UTI ChromoSelect Agar | Sigma-Aldrich — $589 for 500 grams
 
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For the first dipslide which showed growth, when I transferred some of that growth to the chromogenic agar, it turned out to be Enterococcus, as shown by the blue-green color on the first image above.
Are you able to identify what type of Enteroccus it is? Some are known to be very reistant to Antibiotic drugs.
 

Hip

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pic 3 blue cluster - could be also an Enterobacter.
Yes, dark blue means coliform bacteria, a group which includes Escherichia, Klebsiella, Enterobacter, Citrobacter and Hafnia.

But on this chromogenic agar, if there is Escherichia coli present, it appears as a pink-red (see the instructions). So I presume it cannot be E. coli, because on my agar it appears as dark blue. So I guess it must be another coliform, like Klebsiella, Enterobacter, Citrobacter or Hafnia.



Are you able to identify what type of Enteroccus it is? Some are known to be very reistant to Antibiotic drugs.
The chromogenic agar instructions say that blue-green means Enterococcus species, but you cannot tell which species, unfortunately.
 

Nuno

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It would be interesting to see what a healthy - non ME- person gets too. Might he/ she also not get some curious bacteria?
Well, I once did a test on myself and on my mother (the healthy control).
My urine dipslide test created several bacterial colonies, my mothers barely did anything.

I then showed the photos to @Hip and he can confirm to an extent
 

Hip

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Any obvious infection would surely always produce growth and be relatively consistent sample to sample I would have thought.
It's not an infection in the kidneys that Dr Markov believes causes ME/CFS, but a dysbiosis (which you might think of as a low-level infection). So this is why it's harder to catch the bacteria involved.
 

Hip

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It would be interesting to see what a healthy - non ME- person gets too. Might he/ she also not get some curious bacteria?
Dr Markov has performed urine testing on ME/CFS patients and healthy controls, and found the dysbiosis present in 95% of ME/CFS patients, but in only 7% of healthy controls.

If ME/CFS patients on this forum wanted to replicate this finding, it could be done by testing themselves, along with testing a healthy family member, as @Nuno did. But a single example will not tell you much, you would need dozens of ME/CFS patients and healthy controls to get some statistical significance.
 
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This doesn't make much sense. If there are multiple bacteria present, why would only one appear at a time? As mentioned above, it seems likely that you are looking at contaminants.
 
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This also seems like a test that could easily be done by a lab. Probably pretty much any lab could do a 24 hour culture. Why not go that route to avoid any contaminants/artifacts?
 

Hip

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This also seems like a test that could easily be done by a lab. Probably pretty much any lab could do a 24 hour culture. Why not go that route to avoid any contaminants/artifacts?
You may like to look at the Dr Markov CBIS summary thread, which should answer your question in detail.

Basically, Dr Markov says a standard lab urine culture is not sensitive enough to detect the low levels of bacteria in dysbiosis. This is why it is necessary to follow this high sensitivity urine test protocol, which is conducted at home using fresh warm urine.

In any case, I don't see how a lab urine test would be less prone to contamination than a home test. The main contamination in a urine test comes from the skin just around the exit of the urethra. Bacteria on that skin can be washed down into the urine stream, and contaminate the agar. As a precaution against this, it is advised to wash this area of skin just before urinating.



If there are multiple bacteria present, why would only one appear at a time?
It does seem strange, but then I have very little knowledge of bacteriology, and there could be reasons for this. As a guess, maybe the food you eat on the day, or the state of your immune system on the day, influences which bacterial species in your kidney or urinary tract dominate.
 
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SWAlexander

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"So I guess it must be another coliform,"

If you could test stool, maybe C-difficile will show up (is another coliform).
C-dif is complicated because there are variations. if you like listen to:
 
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Hip

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If you could test stool, maybe C-difficile will show up (is another coliform).
I did a Genova Diagnostics stool test some years ago, and the bacteria found were:

(1) Alpha hemolytic Streptococcus
(2) Gamma hemolytic Streptococcus
(3) Staphylococcus aureus
(4) Proteus mirabilis (high levels, indicating dysbiosis)
(5) Hemolytic Escherichia coli

No Clostridium difficile was found, and nothing else nasty or problematic was found.
 

SWAlexander

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I did a Genova Diagnostics stool test some years ago, and the bacteria found were:

(1) Alpha hemolytic Streptococcus
(2) Gamma hemolytic Streptococcus
(3) Staphylococcus aureus
(4) Proteus mirabilis (high levels, indicating dysbiosis)
(5) Hemolytic Escherichia coli

No Clostridium difficile was found, and nothing else nasty or problematic was found.
Yeh, no C-dif. It is a real nasty bugger.
 

BrightCandle

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I am still waiting for my dipslides. One of the ones I ordered got cancelled by amazon.de and the others aren't due until the end of the month. I do intend to run one of these tests though, its interesting at least and I do love a good diagnostic test even if it rules something out.