Persisters

Persisters
Andre Willers
30 Apr 2012
“Teach an old dog new tricks”
Synopsis:
Some organisms , notably bacteria and fungi , duck into the hibernation bunker when things get tough . They use some very old evolutionary machinery and quorum systems . These can be manipulated .
Discussion:
1.What are persisters ?
http://en.wikipedia.org/wiki/Multidrug_tolerance
“Multidrug tolerance or antibiotic tolerance is the ability of a disease-causing microorganism to resist killing by antibiotics or other antimicrobials. It is mechanistically distinct from multidrug resistance:[1] It is not caused by mutant microbes, but rather by microbial cells that exist in a transient, dormant, non-dividing state. Microorganisms that display multidrug tolerance can be bacteria, fungi or parasites.”
“Relevance to chronic infections
Multidrug tolerance is caused by a small subpopulation of microbial cells termed persisters.[2] Persisters are not mutants, but rather are dormant cells that can survive the antimicrobial treatments that kill the majority of their genetically identical siblings. Persister cells have entered a non- or extremely slow-growing physiological state which makes them insensitive (refractory or tolerant) to the action of antimicrobial drugs. When such persisting microbial cells cannot be eliminated by the immune system, they become a reservoir from which recurrence of infection will develop.[3] Indeed, it appears that persister cells are the main cause for relapsing and chronic infections.[1] Chronic infections can affect people of any age, health, or immune status.”

2.Old mechanisms :
See Appendix II below . A survival mechanism for hostile environments .

3.Quorum Ratcheting :
A gene-group evolved not to risk it’s whole population on a single hibernation strategy .
See http://andreswhy.blogspot.com “Infinite probes in optimization Jun 2008 and others .
It would ratchet up or down : RatioHibernating = (1/3)^n , where n = 1,2,3,… depending on the environment . Epigenetics and prions will play a role here .
There are two potential intervention points here :
3.1The quorum sensors , but they will probably be heavily protected and buffered .
3.2The period when n changes . The metabolism is then active and things like antibiotics can work . So , timing is then critical . An interesting question would be if each n’th state is a separate gene , or an input into a more general gene . This is very old , so I would guess both , with a single gene being in the majority .
Optimal n :The immune system has co-evolved with this mechanism , and relies on it as a memory of what to guard against . Rather neat , don’t you think ?
So an optimal n for a persistent population of potential pathogens would be 2
4.Interventions that will always work :
4.1Garlic (Allicin) . See Appendix III .
Allicin (freshly crushed garlic oil) will kill bacterial films . Try it on your teeth .
Melatonin as a powerful terminal anti-feedback loop agent might be useful as an adjuvant , but extreme caution is advised . Melatonin will bring Orexins into play . We don’t know enough to predict what will happen .
See http://andreswhy.blogspot.com “Fatigue and old age” 25 Apr 2012

4.2A compound called HT61 also punches holes in persistent cells (PLoS One , vol 5 ,e11818) or NewScientist 31 Mar 2012 p31 . Probably a variant of allicin .
4.3 Temperature:
Extensively used commercially . Pasteurization . The bugs are just inactivated , not killed . They go into a persistent state of hibernation .
http://en.wikipedia.org/wiki/Pasteurization
“Unlike sterilization, pasteurization is not intended to kill all micro-organisms in the food. Instead, it aims to reduce the number of viable pathogens so they are unlikely to cause disease (assuming the pasteurized product is stored as indicated and is consumed before its expiration date). “
In the terms used above , n is dramatically increased . The bugs will reactivate later in increments of n , but the body’s defence systems can handle them easily (unless impaired) .
But note that this is an easy training method for the immune system . Pasteurised food together with a cell-burster like allicin (garlic) or HT61 makes molecular fragments available for dendritic immune cell training .

Intriguing .
(This is not medical advice)
Autoimmune diseases like asthma (known to benefit from raw milk) might be retrained by pasteurised milk with crushed garlic juice , rinsed in the mouth and drunk immediately .
Really gives that old immune system a pair of training wheels ! . At any age .
Happy pedalling !
Andre

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Appendix I
Metabolite-enabled eradication of bacterial persisters by aminoglycosides
• Kyle R. Allison,
• Mark P. Brynildsen
• & James J. Collins
• Affiliations
• Contributions
• Corresponding author
Nature 473, 216–220 (12 May 2011) doi:10.1038/nature10069
Received
22 July 2010
Accepted
24 March 2011
Published online
11 May 2011
Article tools
Bacterial persistence is a state in which a sub-population of dormant cells, or ‘persisters’, tolerates antibiotic treatment1, 2, 3, 4. Bacterial persisters have been implicated in biofilms and in chronic and recurrent infections5, 6, 7. Despite this clinical relevance, there are currently no viable means for eradicating persisters. Here we show that specific metabolic stimuli enable the killing of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) persisters with aminoglycosides. This potentiation is aminoglycoside-specific, it does not rely on growth resumption and it is effective in both aerobic and anaerobic conditions. It proceeds by the generation of a proton-motive force which facilitates aminoglycoside uptake. Our results demonstrate that persisters, although dormant, are primed for metabolite uptake, central metabolism and respiration. We show that aminoglycosides can be used in combination with specific metabolites to treat E. coli and S. aureus biofilms. Furthermore, we demonstrate that this approach can improve the treatment of chronic infections in a mouse urinary tract infection model. This work establishes a strategy for eradicating bacterial persisters that is based on metabolism, and highlights the importance of the metabolic environment to antibiotic treatment.
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Appendix II
See http://andreswhy.blogspot.com
Hibernation and Cryogenics
Andre Willers
19 Nov 2008

Discussion :
Jessica Palmer pointed out on the 16th Nov 2008 that the primary problem with cryogenics seems to be in the re-establishment of metabolic processes after un-freezing .

But we have an already existing template for doing exactly that .

Hibernation .

See Appendix A

The evolutionary explanation is that during the transition-phase from methane/sulfur to oxygen atmosphere (circa 1.5 bn years ago) , there was a major advantage in suspending oxygen-driven systems if the organism found itself in a non-oxygen environment . It went into hibernation .

At the same time , alcohol was being excreted as a poison (like oxygen) . The ancestors of mitochondria (who could use low concentrations of oxygen or alcohol ) sought refuge in cells whose cell-walls were resistant (but not impervious) to alcohol transition .

Remember , alcohol is completely soluble in water , but oxygen is not . This difference drove the process . Alcohol concentrations in water could grow large , but oxygen-concentrations could not .

Mitochondria earned their keep by mopping up alcohol first and later converting oxygen to ATP . (The glucose and ketone metabolism came after this) . The ketone metabolism has never been very popular , because of the high loss-rate in excretory products , but has been kept as a third string on the bow . (Utilization of fat and protein-muscle reserves during low-glucose periods. )

Mitochondria thus has an exclusive preference of usage : alcohol , glucose , ketones in that order .

From experimental evidence (see Appendix A) , there is a genetic switch sensitive to the concentration of H2S to bring both the host cell and the mitochondrium to a state where all programmed molecular activity is suspended . (The power is switched off) .
But , of course , random beth(0) molecular activity due to temperature does not cease . Uncontrolled and anaerobic reactions still occur .

Freezing:
We get rid of most anaerobic organisms first .
Lots of sulfur , VitC and alcohol (fermented berries or carbohydrates in the stomach . A low acidity is required in the run-up to hibernation)
Then freeze .


Starting the contraption up again is a bit of a problem .

1. The power-plant :
The mitochondria needs to be primed with their preferred fuel (alcohol)
Oxygen needs to be infused .(Hyperbaric chamber)

2. Garbage disposal
The cellular garbage-disposal systems need to be activated . ATP from the powerplant needs to be allocated to breakdown-product disposal before the ATP is allocated to DNA/RNA production processes .

The garbage-disposal uses mechanisms that use sulfur to create the various vacuoles and ropes (cf mitosis) . Enough sulfur is vital .

Once again , oxygen and alcohol is used . Both are recognized by all systems as poisons to be removed as a first priority . They activate a quite sophisticated garbage-disposal system as H2S concentrations decrease .

3 . Flushing
All that garbage has to flushed away , preferably not through the kidneys or liver .
Use machines .
As H2S concentrations decrease , damage might occur due to PH fluctuations . Acidity (H2SO4 , etc) Buffering would be advisable .

4. Temperature:
Lots of water at 105 to 107 Fahrenheit for mammals , pulsing at pulserate(about 90 cycles per minute .)
This is to activate the chaperone systems and discourage opportunistic viruses .

5. Music
See http://andreswhy.blogspot.com “Music”
Play harmonious music so the vibrations can be felt throughout organism being thawed . This enhances timing-procedures by orders of magnitudes . Emergent order .
(A Beth(0.x) effect . )

The de-hibernization process must have an exact program at molecular level to reboot the cellular metabolism . Precisely what you need after a cryogenic procedure .
But its efficiency (ie your chance of survival) can be boosted by orders of magnitude by using the steps above .

Interesting notes:
1. Do hibernating animals like bears use alcohol-producing cells in their bloodstream to time hibernation? This can be tested .
2. Are there cold-chaperone molecules ? There should be .
3. Hibernation is easy . Nature has done all the hard work . Keeping the mechanism ticking over at a very slow rate enables cellular-garbage clearing for a relatively short period (6-8 months)
4. De-cryogenics is a bit harder , Beth(1) intervention is needed .
5. Alcohol-concentrations : we are talking about 1% to 2% imbibing . About 0.06% inside the cell . Ie , the cell-wall protects by a factor of about 30
6. Pulse-Cryogenics : alternate freezing and hibernation to get a better survival factor . For those who are too stupid to design a zero-entropy system .
Try : Life=negative entropy . Non-life = positive entropy . Design it so the sum is zero .

Andre

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Appendix A
From http://andreswhy.blogspot.com “ Birdflu Update-4” dated 29 Oct 2005
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Suspended Animation (the real thing!)
In 2005, Mark Roth and other scientists from the University of Washington and the Fred Hutchinson Cancer Research Center in Seattle demonstrated that mice can be put into a state of suspended animation by applying a low dosage of hydrogen sulfide (80 ppm H2S) in the air. The breathing rate of the animals sank from 120 to 10 breaths per minute and their temperature fell from 37 °C to 2 °C above ambient temperature (in effect, they had become cold-blooded). The mice survived this procedure for 6 hours and afterwards showed no negative health consequences.
Such a hibernation occurs naturally in many mammals and also in toads, but not in mice. (Mice can fall into a state called clinical torpor when food shortage occurs). If the H2S-induced hibernation can be made to work in humans, it could be useful in the emergency management of severely injured patients, and in the conservation of donated organs.
As mentioned above, hydrogen sulfide binds to cytochrome oxidase and thereby prevents oxygen from binding, which apparently leads to the dramatic slowdown of metabolism. Animals and humans naturally produce some hydrogen sulfide in their body; researchers have proposed that the gas is used to regulate metabolic activity and body temperature, which would explain the above findings
Dosages of H2S:
Treatment involves immediate inhalation of amyl nitrite, injections of sodium nitrite, inhalation of pure oxygen, administration of bronchodilators to overcome eventual bronchospasm, and in some cases hyperbaric oxygen therapy.
Exposure to lower concentrations can result in eye irritation (because of the high alkality of the SH- anion), a sore throat and cough, shortness of breath, and fluid in the lungs. These symptoms usually go away in a few weeks. Long-term, low-level exposure may result in fatigue, loss of appetite, headaches, irritability, poor memory, and dizziness. Higher concentrations of 700-800 ppm tend to be fatal.
• 0.0047 ppm is the recognition threshold, the concentration at which 50% of humans can detect the characteristic rotten egg odor of hydrogen sulfide [2]
• 10-20 ppm is the borderline concentration for eye irritation.
• 50-100 ppm leads to eye damage.
• At 150-250 ppm the olfactory nerve is paralyzed after a few inhalations, and the sense of smell disappears, often together with awareness of danger,
• 320-530 ppm leads to pulmonary edema with the possibility of death.
• 530-1000 ppm causes strong stimulation of the central nervous system and rapid breathing, leading to loss of breathing;
o 800 ppm is the lethal concentration for 50% of humans for 5 minutes exposition (LC50).
Concentrations over 1000 ppm cause immediate collapse with loss of breathing, even after inhalation of a single breath.

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Appendix III
See http://andreswhy.blogspot.com

Garlic and the Plant-Herbivore Wars .
Andre Willers
10 Apr 2011

Synopsis:
Garlic (Allicin) is a toxin produced by binary components alliin and enzyme alliinase These are kept in separate pockets , but mix when the plant is damaged (eaten) , producing allicin .

Dicussion :
This is a very old and beautiful mechanism , verging on symbiosis .

Note the presence of Sulfur : it evolved pre-oxygen , and evolved to keep pace .
It originally evolved to zap viruses and other bacteria to give the originators a relative advantage .
In multicellular organisms , it does the same .
Notice that the chemical (Allicin) evolved first , then reversed into the genomes of various organisms because it was so useful .
Allicin remains invariant , and genomes adapt to it .
A sort of "Inverse Prion"

But it is kept in check by various mechanisms :

1.Bad taste and smell and a burning sensation

This limits eating . The burning is sensation is caused by the activation of a separate neural network that evolved to simulate a burning sensation . This evolved into capsaicum receptors in mammals . Birds , reptiles and dinosaurs had equivalents .
Note sensitivity of marine mollusks to anti-fouling paints based on capsaicum-like chemicals .

2.Deactivation of enzyme alliinase if ph<3
This led to gastric acids .
Symbiotic bacteria in the intestines must be kept protected .
Swallowing garlic plants or powders will not work
If you drink coke ,pepsi etc , with your garlic , there will be little health benefit (see Appendix A for acidity of popular drinks)
But too much of a good thing can harm . A swig of acid drink(ph<3) will reset oral bacterial systems after eating garlic . But it must be pulsed at 2 minute intervals .

3.Quick breakdown of allicin at body temperatures :

16 hours at 23 C (from Wiki)

64 hours at 3 C (the fridge) . Throw away supermarket or even frozen minced garlic as far as health effects are concerned .

5.7 hours at 38 C (doubling of chemical activity every 10 C)
Very little allicin makes it through to the small intestines to upset the gut flora .

Cooking :
5 minutes at 100 C . (doubling of chemical activity every 10 C)
There is very little allicin left after 5 minutes of cooking .

The effects of Allicin is then concentrated in the mouth and throat : where the rubber hits the tar . Where any bacteria is an enemy . Kill them all , and let the immune system sort out the remainder .
This works well in pulsed dosages , hence another reason for the rapid breakdown of allicin .

4.Breakdown accellarators :
There is some evidence that there are some other chemicals in garlic that speeds up breakdown of allicin . Presumably binary , as well .


Preservation of allicin :
See http://en.wikipedia.org/allicin
Cutler and Wilson found that an aqueous cream of allicin (0.5 g/L) compared well with 20 g/L of muporicin .
Aqueous cream is essentially an emulsion .

Make your own Allicin Cream :
Spray-on cream from an aerosol can is an emulsion .
Spray it into your bowl , then crush your garlic clove inside the cream emulsion . Hopefully , large enough percentages of the binary components alliin and enzyme alliinase are encapsulated by the lipid foams .
Smear it on , and as the foams evaporate and break down , the components interact .
Voila , allicin .
Dosages and timing will have to be found by experiment .
Be careful and err on the side of caution . 0.5 g/L allocin is kinda small .

An added advantage is that the burning sensation will be much ameliorated (cream is a milk product , which has been found to decrease the burning sensation .)

Smear it on the gums and swirl it (there will be greatly stimulated spit production) . Swallow if necessary . After a prederminate time (start at 2 minutes , then progress in sequence 4 , 8 ,16 , etc) . If discomfort is experienced , swill mouth with coke . Then milk if necessary .

It can be used as a topical cream for anti-fungal and bacteriocidal purposes on the skin.
Do not use it in capsules designed to bypass the stomach , or in suppositories without medical supervision by a really qualified doctor .

This would be like the Terminator loose in LA .

Making pure allicin , alliin and enzyme alliinase
Put clove of garlic in centrifuge test-tube , together with at least 3 balls whose diameter is slightly more than the radius of the test-tube .
Spin .
The relative motions of the balls will crush the garlic , and the centripetal forces will separate the various layers . These can then be tapped individually . But don't loiter too long , as the enzyme at the interface will eventually convert all the aliin into allicin .


Garlic Perles , oils , etc .
These are worthless as far as allicin health benefits are concerned .
These all involve macerating garlic cloves , then dissolving it into an oil .
Reaction between alliin and enzyme alliinase creates allicin . This decays within a matter of hours as set out above . What remains has very little(if any) bio-reactivity

Temperature release :
There might be a temperature release .
An old evolutionary structure would have added the extra "Oomph" of explosive micro-injection of the allicin . Ie an exothermic reaction between alliin and enzyme alliinase .
This gives opportunity of introducing retrovirals , bleach , etc into the teeth and jaw-bones by smearing a minute layer on the tooth , then biting down on the garlic clove (or cream) .

Really, really potent Garlic will blow your socks off !

Bon appetit.

Andre

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Appendix A
Acidity (PH) of some popular drinks .

Diet Mountain Dew 2.95 0.0
Dr. Pepper 2.92 9.64 tsp.
Sprite 2.90 9.29 tsp.
Gatorade (Lemon-Lime) 2.83 5 tsp.
Mountain Dew 2.80 11.07 tsp.
Minute Maid Orange Soda 2.80 11.2 tsp.
Diet Pepsi 2.77 0.0
Diet Coke 2.70 0.0
Powerade 2.63 5.36 tsp.
Pepsi 2.43 9.64 tsp.
Coca-Cola 2.30 9.64 tsp.
Battery Acid (Yikes!) 1.00 (Acidic) 0.0"

"*Acid amounts from the study “Enamel and root surface erosion due to popular U.S. beverages,” 2006. Authors: L. Ehlen, T.A. Marshall, F. Qian, J.J. Warren, J. Wefel, M.M. Hogan, and J.D. Harless. College of Dentistry, University of Iowa, Iowa City and from University of Minnesota School of Dentistry, 2000, Northwest Dentistry Vol 80, No. 2. **4.2 grams = 1 teaspoon."

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