The Crohnie

Antibacterial Activities of Naturally-Occurring Compounds against Mycobacterium avium subspecies paratuberculosis.
Wong SY, Grant IR, Friedman M, Elliott CT, Situ C.

http://aem.asm.org/cgi/content/abstract/AEM.00981-08v1
http://www.ncbi.nlm.nih.gov/pubmed/18676709

Antibacterial activities of 18 naturally-occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols and other plant extracts) against three strains of Mycobacterium avium subspecies paratuberculosis (Map), a bovine isolate NCTC 8578, a raw milk isolate 806R and a human isolate ATCC 43015, were evaluated using a macrobroth susceptibility testing method. Map was grown in 4 ml Middlebrook 7H9 broth containing 10% OADC, 0.05% Tween 80 (or 0.2% glycerol) and 2 microg/ml mycobactin J supplemented with five concentrations of each test compound. The changes in optical density (OD) of the cultures at 600 nm as a measure of CFUs was recorded at intervals over an incubation period of 42 days at 37 degrees C. Six of the compounds were found to inhibit the growth of Map. The most effective compound was trans-cinnamaldehyde with a minimum inhibitory concentration (MIC) of 25.9 microg/ml, then cinnamon oil (26.2 microg/ml), oregano (68.2 microg/ml), carvacrol (72.2 microg/ml), 2,5-dihydroxybenzaldehyde (74 microg/ml), and 2- hydroxy-5-methoxybenzaldehyde (90.4 microg/ml). With the exception of carvacrol, a phenolic compound, three of the four most active compounds are aldehydes, suggesting that the structure of the phenolic group or aldehyde group may be important to the antibacterial activity. No difference in compound activity was observed between the three Map strains studied. Possible mechanisms of the antimicrobial effects are discussed.

Another piece of research which establishes the anti-paratuberculosis antibiotic activity of molecules which are currently thought to have an immuno-suppressant effect in Crohn’s Disease.

http://www.paratuberculosis.org/pubs/proc9/abst185f_o4.htm

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BACKGROUND:

We have shown that the “immuno-modulators” methotrexate and 6-MP and the “anti-inflammatory” 5-ASA inhibit MAP growth (www.PLoSONE.org) and concluded that their most plausible mechanism of action in several idiopathic diseases is as antiMAP antibiotics. Thalidomide is an “immunomodulator” used in multiple “auto-immune” and “inflammatory” diseases and the mycobacterial diseases leprosy and tuberculosis. We now test the hypothesis that thalidomide inhibits MAP growth.

METHODS

Thalidomide (+-) and (+) and (-) and its two components, phthalimide and 1-hydroxy 2,6 piperidine dione (HPD) were evaluated in culture of two strains each of MAP (ATCC 19698 [bovine] & Dominic [human]) and M. avium subspecies avium (ATCC 25291 & 101.) We used a radiometric (14CO2 Bactec(R)) detection system. Inhibition is indicated by “percent decrease in cumulative Growth Index” (%-DcGI) from control.

RESULTS:

Phthalimide has no dose dependent inhibition on any strain. There was no dose dependent inhibition on either M. avium strain with thalidomide or its components. With the two MAP strains, there is dose dependent inhibition with thalidomide (+1); Dominic (31%-DcGI) and ATCC 19698 (26%-DcGI) at 64microg/ml. Thalidomide (+) is more inhibitory than (-). HPD is, on a weight for weight basis, the most inhibitory agent evaluated; Dominic (46%-DcGI) and ATCC 19698 (44%-DcGI at 64microg/ml)

CONCLUSIONS:

We show in vitro heretofore-undescribed inhibition of MAP growth by racaemic thalidomide. Thalidomide (+) is more potent than (-). Of thalidomide’s two moieties, phthalimide has no antiMAP activity and HPD is the active component in inhibiting MAP growth. We suggest that since 1942, initially with 5-ASA, the medical profession has unknowingly been treating MAP infections. These data are compatible with our concern that MAP is zoonotic. We conclude that all idiopathic “autoimmune” and “inflammatory” diseases, empirically treated with medications that we show are active against MAP, should now be evaluated for MAP as the etiological agent.

It appears that some Azathioprine and 6-MP, which are used for the treatment of Crohn’s Disease, and whose mechanism of action is currently unknown, have antibiotic activity against Mycobacterium avium subspecies paratuberculosis (MAP).

http://www.paratuberculosis.org/pubs/proc9/abst182f_o6.htm

As noted by the authors, “These data may partially explain the paradoxical response of Crohn’s disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs i.e. they do not worsen with anti-inflammatory treatment as would be expected with a microbial etiologic pathogen.”
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The in vitro susceptibility of human and bovine-origin Mycobacterium paratuberculosis to the thiopurine drugs 6-mercaptopurine (6-MP) and azathioprine (AZA) were established using conventional plate counting methods and the MGIT 960 ParaTB culture system. Both 6-MP and AZA had antibacterial activity against M. paratuberculosis; isolates from Crohn’s disease patients tended to be more susceptible than were bovine-origin isolates. Isolates of Mycobacterium avium, used as controls, were generally resistant to both AZA and 6-MP even at high concentrations (>=64.0 microg/mL). Among rapidly growing mycobacteria, M. phlei was susceptible to 6-MP and AZA whereas M. smegmatis strains were not. AZA and 6-MP limited the growth of, but did not kill, M. paratuberculosis in a dose-dependent manner. Anti-inflammatory drugs in the sulfonamide family (sulfapyridine, sulfasalazine, and 5-amino- salycilic acid (mesalamine)) had little or no antibacterial activity against M. paratuberculosis. The conventional antibiotics azithromycin and ciprofloxacin (CPX) used as control drugs were bactericidal for M. paratuberculosis, exerting their killing effects on the organism relatively quickly. Simultaneous exposure of M. paratuberculosis to 6- MP and CPX resulted in significantly higher CFUs as compared to use of CPX alone. These data may partially explain the paradoxical response of Crohn’s disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs i.e. they do not worsen with anti-inflammatory treatment as would be expected with a microbial etiologic pathogen. These findings also should influence the design of therapeutic trials to evaluate antibiotic treatments of Crohn’s disease: azathioprine drugs may confound interpretation of data on therapeutic responses both antibiotic-treated and control groups.

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Sulfasalazine (Azulfidine) is an anti-inflammatory medication that belongs to a class of drugs called sulfa drugs. The active ingredients in sulfasalazine consist of salicylate (the main ingredient in aspirin) combined with a sulfa antibiotic.
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American College of Rheumatology
http://www.rheumatology.org/public/factsheets/sulfasalazine.asp

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[Sulfasalazine] is split by the action of bacterial azoreductases in the large intestine into sulfapyridine and mesalazine (mesalamine, 5- aminosalicylic acid),
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Sulfasalazine. A review of its pharmacological properties and therapeutic efficacy in the treatment of rheumatoid arthritis.
http://www.ncbi.nlm.nih.gov/pubmed/7588084

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Sulfapyridine is a sulfonamide antibiotic. The sulfonamides are synthetic bacteriostatic antibiotics with a wide spectrum against most gram-positive and many gram-negative organisms. However, many strains of an individual species may be resistant. Sulfonamides inhibit multiplication of bacteria by acting as competitive inhibitors of p- aminobenzoic acid in the folic acid metabolism cycle. Bacterial sensitivity is the same for the various sulfonamides, and resistance to one sulfonamide indicates resistance to all.
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DrugBank: Sulfapyridine
http://redpoll.pharmacy.ualberta.ca/drugbank/cgi-bin/getCard.cgi?CARD=APRD00491.txt

Note also that the antibiotic para-aminosalicylic acid (4-ASA) is closely related to the 5-ASA portion of sulfasalazine, and is used for antibiotic treatment of mycobacterial infections, particularly tuberculosis.

http://www.answers.com/topic/para-aminosalicylic-acid?cat=health
http://www.rxlist.com/cgi/generic/paser.htm

The anti-mycobacterial agent 4-ASA has also been successfully used in the treatment of IBD, particularly Crohn’s Disease.

On the action of 5-amino-salicylic acid and sulfapyridine on M. avium including subspecies paratuberculosis.
Greenstein RJ, Su L, Shahidi A, Brown ST.
PLoS ONE. 2007 Jun 13;2(6):e516

http://www.ncbi.nlm.nih.gov/pubmed/17565369

Full Text

Abstract

BACKGROUND: Introduced in 1942, sulfasalazine (a conjugate of 5-aminosalicylic acid (5-ASA) and sulfapyridine) is the most prescribed medication used to treat “inflammatory” bowel disease (IBD.) Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We have shown that two other agents used in the therapy of IBD (methotrexate and 6-MP) profoundly inhibit MAP growth. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. We herein hypothesize that the mechanism of action of 5-ASA and/or sulfapyridine may also simply be to inhibit MAP growth. METHODOLOGY: The effect on MAP growth kinetics by sulfasalazine and its components were evaluated in bacterial culture of two strains each of MAP and M. avium, using a radiometric ((14)CO(2) BACTEC(R)) detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (%-DeltacGI). PRINCIPAL FINDINGS: There are disparate responses to 5-ASA and sulfapyridine in the two subspecies. Against MAP, 5-ASA is inhibitory in a dose-dependent manner (MAP ATCC 19698 46%-DeltacGI at 64 microg/ml), whereas sulfapyridine has virtually no effect. In contrast, against M. avium ATCC 25291, 5-ASA has no effect, whereas sulfapyridine (88%-DeltacGI at 4 microg/ml) is as effective as methotrexate, our positive control (88%-DeltacGI at 4 microg/ml). CONCLUSIONS: 5-ASA inhibits MAP growth in culture. We posit that, unknowingly, the medical profession has been treating MAP infections since sulfasalazine’s introduction in 1942. These observations may explain, in part, why MAP has not previously been identified as a human pathogen. We conclude that henceforth in clinical trials evaluating antiMAP agents in IBD, if considered ethical, the use of 5-ASA (as well as methotrexate and 6-MP) should be excluded from control groups.

On the action of methotrexate and 6-mercaptopurine on M. avium subspecies paratuberculosis.
Greenstein RJ, Su L, Haroutunian V, Shahidi A, Brown ST.
PLoS ONE. 2007 Jan 24;2(1):e161

http://www.ncbi.nlm.nih.gov/pubmed/17252054

Full Text

Abstract

BACKGROUND: Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6-MP) is associated with a decrease in pro-inflammatory cytokines. This has been presumed to indicate the mechanism of action of methotrexate and 6-MP. Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP. METHODOLOGY: The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in cell culture of two strains each of MAP and M. avium using a radiometric ((14)CO(2) BACTEC detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (% -DeltacGI). PRINCIPAL FINDINGS: The positive control antibiotic (clarithromycin) has >or=85% -DeltacGI at a concentration of 0.5 microg/ml. The negative control (ampicillin) has minimal inhibition at 64 microg/ml. MAP ATCC 19698 shows >or=80% -DeltacGI for both agents by 4 microg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate. CONCLUSIONS: We show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different % -DeltacGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in pro-inflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. We conclude that henceforth, in clinical studies that evaluate the effect of anti-MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control groups.

On the Action of Cyclosporine A, Rapamycin and Tacrolimus on M. avium Including Subspecies paratuberculosis
Greenstein RJ, Su L, Juste RA, Brown ST.
PLoS ONE. 2008 Jun 25;3(6):e2496

http://www.ncbi.nlm.nih.gov/pubmed/18575598

Full Text

Abstract

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. Recently the “immuno-modulators” methotrexate, azathioprine and 6-MP and the “anti-inflammatory” 5-ASA have been shown to inhibit MAP growth in vitro. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. The “immunosuppressants” Cyclosporine A, Rapamycin and Tacrolimus (FK 506) treat a variety of “autoimmune” and “inflammatory” diseases. Rapamycin and Tacrolimus are macrolides. We hypothesized that their mode of action may simply be to inhibit MAP growth. METHODOLOGY: The effect on radiometric MAP (14)CO(2) growth kinetics of Cyclosporine A, Rapamycin and Tacrolimus on MAP cultured from humans (Dominic & UCF 4) or ruminants (ATCC 19698 & 303) and M. avium subspecies avium (ATCC 25291 & 101) are presented as “percent decrease in cumulative GI” (%-DeltacGI.) PRINCIPAL FINDINGS: The positive control clofazimine has 99%-DeltacGI at 0.5 microg/ml (Dominic). Phthalimide, a negative control has no dose dependent inhibition on any strain. Against MAP there is dose dependent inhibition by the immunosuppressants. Cyclosporine has 97%-DeltacGI by 32 microg/ml (Dominic), Rapamycin has 74%-DeltacGI by 64 microg/ml (UCF 4) and Tacrolimus 43%-DeltacGI by 64 microg/ml (UCF 4) CONCLUSIONS: We show heretofore-undescribed inhibition of MAP growth in vitro by “immunosuppressants;” the cyclic undecapeptide Cyclosporine A, and the macrolides Rapamycin and Tacrolimus. These data are compatible with our thesis that, unknowingly, the medical profession has been treating MAP infections since 1942 when 5-ASA and subsequently azathioprine, 6-MP and methotrexate were introduced in the therapy of some “autoimmune” and “inflammatory” diseases.

Leprosy is caused a member of genus Mycobacteria, M. leprae, and some forms of leprosy are treated by “tamping down the immune system” with anti-TNF agents, such as thalidomide. Steroids are also used for the treatment of leprosy[1].

FDA approves Thalidomide for Hansen’s disease side effect, imposes unprecedented restrictions on distribution.
http://www.fda.gov/bbs/topics/ANSWERS/ANS00887.html

Redeeming thalidomide
http://pubs.acs.org/hotartcl/mdd/00/jun/mddkling.html

Remicade’s anti-TNF activity might also be useful for leprosy (a known mycobacterial disease), but there haven’t been any trials of remicade/infliximab for Leprosy (Hansen’s Disease), presumably because the manufacturers know that the average third-world leper can’t afford $30K+ a year to be maintained on remicade, and thus won’t spend the huge money necessary for the relevant clinical trials.

But Thalidomide, the poor man’s anti-TNF drug, is long out of patent, and is cheap, and so gets used for the poor lepers. But there is a fightback in the leprosy medical community, primarily because of the nasty birth defects that Thalidomide causes.

No Role for Thalidomide in Leprosy
http://www.paho.org/English/AD/DPC/CD/thalidomide.htm

Thalidomide has been trialled for Crohn’s Disease.

Thalidomide reduces tumour necrosis factor {alpha} and interleukin 12 production in patients with chronic active Crohn’s disease
http://gut.bmjjournals.com/cgi/content/abstract/50/2/196

Thalidomide: New uses for notorious drug
http://www.mayoclinic.com/health/thalidomide/HQ01507

So the relative success of treatment of Crohn’s Disease with Remicade is NOT an argument against a mycobacterial cause. In fact, if the Crohn’s Disease to Leprosy analogy that has been made by some researchers[2,3] is true, then Remicade treatment in Crohn’s could be an argument FOR a mycobacterial cause.

1. Steroid prophylaxis for prevention of nerve function impairment in leprosy: randomised placebo controlled trial.
http://bmj.bmjjournals.com/cgi/content/abstract/328/7454/1459

2. Comparisons with leprosy, tuberculosis and Johne’s disease: is Crohn’s disease caused by a mycobacterium?
http://www.paratuberculosis.org/proc7/abst6_p6.htm

3. Mycobacterium avium subspecies paratuberculosis in the causation of Crohn’s disease
http://www.wjgnet.com/1007-9327/6/630.asp

Here’s a few references about “Herbal Antibiotics”.

Antimicrobial properties of allicin from garlic.
http://www.ncbi.nlm.nih.gov/pubmed/10594976

In-vitro antimicrobial activity of four diallyl sulphides occurring naturally in garlic and Chinese leek oils.
http://www.ncbi.nlm.nih.gov/pubmed/11444776

Allylsulfide constituents of garlic volatile oil as antimicrobial agents.
http://www.ncbi.nlm.nih.gov/pubmed/11185736

There’s a lot of controversy of which forms of garlic are more effective: fresh pressed garlic vs. aged garlic vs. garlic oil vs. etc, etc. But there is little independent science out there: a lot of published results seem to be from companies that are pushing their own specific (and expensive) garlic preparation.

You can read a good introduction to the chemistry of garlic here:

http://www.herbalchem.net/GarlicIntroductory.htm

Other plants with antimicrobial properties include ginger, galanga, turmeric, lemon grass and chilli. Which is great if you like Thai or Indonesian food.

Although ginger is readily available, you’ll probably have to go to an Asian food shop to get the rest. Believe me, fresh turmeric is worth the effort: so much tastier than that nasty dried yellow powder!

Coconut oil also has significant antimicrobial properties, particularly the lauric acid which makes up ~ 50% of coconut oil. Other active ingredients of coconut oil are capric and caprylic acid.

http://www.mercola.com/fcgi/pf/2001/jul/28/coconut_health.htm

And when you get into essential oils, there are many that have antimicrobial effects. Tea tree oil and peppermint oil are purported to be effective against candida.

http://www.carenewengland.org/body.cfm?id=170&chunkiid=21475

It will be great to finally see published the results of that double-blind, placebo-controlled and multi-center clinical trial: reliable data are desperately needed in this area.
http://www.crohns.org/treatment/austrial.htm

I expect to see results that show that RMATC (the Pharmacia trial is using RMAT + Clofazimine) is equally or more effective than existing treatments for Crohn’s Disease, and in a minority subgroup of patients, induces significantly longer clinical remission periods than existing treatments.

However, even when the results are published, we will still be in the following dissatisfactory position

1. We will not know in advance who will benefit from anti-MAP treatment and who will not.

2. If the treatment achieves a successful clinical outcome in any given patient, we will not know why.

3. If the treatment fails to achieve a successful clinical outcome, we will not know why. Which means that we will be unable to tell if the same treatment can be used again in the same patient, or if an alternative antibiotic treatment is required (e.g. due to antibiotic resistance).

4. We will not know the parameters which will define a successful end of the treatment period, e.g. organism clearance, antibody reduction, etc, etc. The currently proposed 2-year treatment period is essentially a guesstimate, based on experience with the treatment of related mycobacterial infections such as M avium subsp. avium. Since these antibiotics are expensive, and can be toxic, we need to be able to much more clearly define clinical outcome parameters. If 2 years is too short, then we risk antibiotic resistance. If 2 years is too long, then we need to reduce it. But we have little data to base conclusions upon.

5. We will not know if the treatment is microbiologically working in a patient with physical stricturing symptoms. RMAT treatment can accelerate stricture formation in a (hard to identify) subgroup of patients, due to fibrotic tissue formation, just as with other mycobacterial diseases. Physicians inexperienced with the protocol can misread the consequent temporary disimprovement in the patient’s clinical status as a treatment failure and take the patient off treatment, thus risking antibiotic resistance. We will have no tools, other than physician experience and intuition, to differentiate success from failure.

Or as Chiodini puts it, we will still be “at the whim of the individual physician”.

I believe that the bottom line is that most Gastroenterologists simply don’t know enough about infectious diseases, are not taught enough about infectious diseases: it’s not a large enough part of their educational, medical, or clinical culture. For example, some of the research published by gastroenterologists about anti-MAP antibiotic treatment for Crohn’s Disease displays fundamentally flawed reasoning and lack of microbiology experience.

I don’t find it at all strange that RMAT treatment was pioneered by a surgeon, John Hermon-Taylor. Fortunately, there are many excellent, open-minded and energetic gastroenterologists who are at the forefront of a fundamental rethink of Crohn’s Disease: Ira Shafran(Florida), Tom Borody(Sydney), Will Chamberlin(US Army), etc, etc. I’ve had the great fortune to work with all three: three things stand out about all of them: 1. They concern themselves deeply with the health of their patients, 2. they are not afraid to admit that they don’t know everything and are willing to learn, and 3. they are not driven by money.