supplemental references for dr ...€¦ · i find the maypa remedies are very well tolerated and...

www.KlinghardtInstitute.com

Supplemental references for Dr.Kl inghardt Lyme seminar London 2/2016

A. Immunemodulation

How to make an auto-nosode from stool, sal iva o urine

Auto-NosodePreparation

Materialsneeded:

Sterileurinecontainers

10mLsterileglassemptyvials

10mLsyringes

18Gx1”needle

Sterilewater

0.3ccinsulinsyringes

Filterneedle0.2micron

Fillsterileemptyvialswith10mLofwaterandlabeleachasC1,C2,C3,etcuptoC12.

Fill2sterileurinecupswith10mLwithsterilewater.

Mixstoolwithtapwaterinasterileurinecontainer.Allowtoincubateatroomtemperaturefor48hours.After48hours,mixthoroughlyanddrawup1mLintoasyringe.


Putthe1mLmixtureintoanewsterileurinecontainerwith10mLsterilewater.Mixthoroughly.

Withanewsyringeandfilterneedle,drawup1mLofmixture.Takethefilterneedleoffandsquirtintoanewsterileurinecupwith10ccsterilewater.Mixthoroughly.

Drawup0.01mLwiththe0.3mLinsulinsyringeandaddtosterileviallabeledC1(pre-filledwith10mLsterilewater).Succuss(hitbottomofbottleagainstpalmofhand)50times.

Drawup0.01mLfromC1andaddtovialC2,succuss50times.

Drawup0.01mLfromC2andinjectintoC3andsuccuss50times.

ContinuedilutinguntilC12.

B . Important Reading: Lyme Treatment Guidel ines by Dr Burrascano - Lyme disease www.lymenet.org/BurrGuide200810.pdf Artesunate source:


C . Other herbal remedies that are useful (observation


by Scot Forsgren) [email protected]

NutraMedix - https://www.nutramedix.com/ Burbur - excellent for detoxification; can be used every 10 minutes to help pull someone out of a Herxheimer reaction Samento / Banderol - this is a good antimicrobial combination and was studied by Eva Sapi's team with positive results. Note have to be separated by 15-20 minutes. Cumanda - another broad spectrum antimicrobial Enula - seems to be helpful for Babesia and protozoa Houttuynia - seems to be helpful for Bartonella Stevia - this is the stevia used in Eva Sapi's research on Stevia for Lyme Beyond Balance - http://www.beyondbalanceinc.com/ BB-1, BAR-1, and BAB-2 - these may be helpful for the "Big 3" TOX-EASE GL and TOX-EASE - detoxification support BFM-1 - biofilm (generally later in treatment) CYFLACALM II - I really like this one for nervous system and brain related inflammation There are others in this line I really like too like MYCOREGEN for fungal issues, MC-CH for CPn, ENL-MC for Mycoplasmas, etc. Byron White Formulas - http://www.byronwhiteformulas.com/ A-L Complex, A-BART, A-BAB - these may be helpful for "the Big 3" A-FNG - may be helpful for fungal issues A-P - may be helpful for parasites A-MYCO - may be helpful for Mycoplasmas A-CPN - may be helpful for Chlamydias


From Dr. Wayne Anderson - http://www.gordonmedical.com/unravelling-complex-chronic-illness/services-offered-at-gordon-medical-associates/guidebook-for-the-byron-white-formulas-table-of-contents/ Supreme Nutrit ion - http://www.supremenutrit ionproducts.com/ Takesumi Supreme - my favorite binder Morinda Supreme (noni) - a good broad spectrum antimicrobial Melia Supreme (neem) - a good broad spectrum antimicrobial Gloden Thread Supreme (coptis) - a good broad spectrum antimicrobial I use these antimicrobials generally when people are through the more specific, targeted treatments (Beyond Balance, etc.) and need something broad-spectrum to maintain an antimicrobial focus. Maypa Herbals - http://maypaherbalremedies.com/ Formula L or L Plus - Borrelia Formula Bart Formula Bab Plus I find the Maypa remedies are very well tolerated and people find they work well. Other things I like: Biobotanical Research Biocidin, Olivirex, and GI Detox (good binder) ACZ Nano spray - liquid zeolite that tests very well for many (Daniela is sending me the new zeolite you like to test on people). Another one I have been using is CytoDetox but I still think I like the ACZ Nano better so far. RESTORE - seems to be excellent for GI/leaky gut support. restore4life.com Devices I like: MAS PEMF


Biomat - you have a site for this where you get commissions - http://sophialife.biomat.com/

C . Astragalus 1. Zhao KW, Kong HY. Effect of Astragalan on secretion of tumor

necrosis factor in human peripheral mononuclear cells. Chung Kuo HsiI Chieh Ho Tsa Chih 1993;13:263-265.

2. Huang ZQ, Qin NP, Ye W. Effect of Astragalus membranaceus on T-lymphocyte subsets in patients with viral myocarditis. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 1995;15:328-330.

3. Peng T, Riesemann H, Kandolf R. The inhibitory effect of Astragalus membranaceus on coxsackie B3 virus RNA replication. Chin Med Sci J 1995;10:146-150.

4. Guo Q, Peng TQ, Yang YZ. Effect of Astragalus membranaceus on Ca 2+ influx and coxsackie virus B3 replication in cultured neonatal rat heart cells. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 1995;15:483-485.

5. Zhao XZ. Effects of Astragalus membranaceus andTripterygium hypoglancum on natural killer cell activity of peripheral blood mononuclear cells in systemic lupus erythematous. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 1992;12:679-671.

6. Luo HM, Dai RH, Li Y. Nuclear cardiology study on effective ingredients of Astragalus membranaceus in treating heart failure. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 1995;15:707-709.

7. Chu DT, Lin JR, Wong W. The in vitro potentiation of LAK cell cytotoxicity in cancer and AIDS patients induced by F3-a fractionated extract of Astragalus membranaceus. Chung Hua Chung Liu Tsa Chih 1994;16:167-171.

8. He J, Li Y, Wei S, et al. Effect of mixture of Astragalus membranaceus, Fructus Ligusti lucidum and Eclipta prostrata on


immune function in mice. Hua Hsa I Ko Ta Hseuh Hseuh Pao 1992;23:408- 411.

9. Li SQ, Yuan RX, Gao H. Clinical observation on the treatment of ischemic heart disease with Astragalus membranaceus. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 1995;15:77-80.

10. Lei ZY, Qin H, Liao JZ. Action of Astragalus membranaceus on left ventricular function of angina pectoris. Chung Kuo Chung Hsi I Ho Chieh Ho Tsa Chih 1994;14:199-202.

11. Astragalus membranaceus stimulates human sperm motility in vitro. Am J Chin Med 1992;20:289-294.

D. AstraSmile Buhner SH. Healing Lyme - Naturai Healing and Prevention of Lyme Borreiiosis and Its Coinfections. New York: Raven Press; 2005: 272. Duke JA. The Green Pharmacy. Rodale Press, Emaus, PA: Rodale Press; 1997: 507. Koch HP, Lawson LD. Garlic: The Science and Therapeutic Application of Allium sativum L. and Related Species. Baltimore: Williams andWiikins; 1996. Winston D. Tick-borne diseases: Their effective treatment, including the use of botanicai & complimentary therapies. Available at: http://www.botanicalmedicine.org/proceedings/me06book.htm. Accessed February 9. 2006.

“Healing Lyme” (Buhner SH. Healing Lyme - Naturai Healing and Prevention of Lyme Borreiiosis and Its Coinfections. New York: Raven Press; 2005)

Herbs with Anti-Lyme Potential Townsend letter, James Duke PhD, Apri l 2007, pg 114-117


Clinical Roundup: Selected Treatment Options for Lyme Disease

Suvarna Reshma

Alternative and Complementary Therapies.August 2012, Vol. 18, No. 4: 220-225

E. Coriandolo

Cilantrofordetoxofleadandaluminum1. Y.Omura et al: Preventative Effects of Chinese Parsley on

Aluminum Deposits in ICR MiceAcupuncture&Electro-TherapeuticsResearch28(1/2)1-44(2003)

SHIGEHARU Fukuda Ph.D., MIHO Aga, KANSO Iwaki, SHINPEI Ushio Ph.D., NAOYA Masaki, MASAO Ikeda, MASASHI Kurimoto Ph.D.YASUHIRO Shimotsuura MD., F.I.C.A.E.and YOSHIAKI Omura MD., Sc. D., F.I.C.A.E., F.A.A.I.M.,

F.R.S.M. The preventive effect of Chinese parsley on aluminum (Al) deposition was investigated in male ICR mice exposed to Al. Seven weeks old ICR male mice were exposed to 1000 ppm Al as Al chloride in drinking water for 39 days. Administration of Chinese parsley to mice by gastric intubation was performed for 25 days from 14 days after beginning of Al exposure to the end of experiment. After 39 days, the mice were sacrificed for the comparison of Al distribution. The localized Al in various tissues was analyzed by kinetic differentiation mode of HPLC. After Al exposure, Al was found to accumulate in the brain, kidney and femur. Localized Al deposition in brain was significantly decreased by the administration of2.4mg/body of Chinese parsley as shown in Fig.1. The similar results were obtained in thefemur (Fig.2). Surprisingly, Al levels in femur on Chinese parsley administered group were lower than that on control. Orally administered Chinese parsley is effective at reducing the deposition of Al in the tissues. These findings suggest the possibility that Chinese parsley may be useful as a natural antidote for Al intoxication. Fig.1 Effect of Chinese parsley on Al concentration in the brain Fig.2 Effect of Chinese parsley on Al concentration in the femus


concentration in the brain SHIGEHARU Fukuda, PhD. Amase Institute, Hayashibara Biochemical Labs. Inc. 77, Amase MinamiMachi, Okayama, 7000834, Japan Tel:+81862316731; Fax:+81862316738; email:[email protected]

2. Preventive effect of Coriandrum sativum (Chinese parsley) on localized lead deposition in ICR mice. J Ethnopharmacol 2001 Oct;77(2-3):203-8 (ISSN: 0378-8741) Aga M; Iwaki K; Ueda Y; Ushio S; Masaki N; Fukuda S; Kimoto T; Ikeda M; Kurimoto M

The preventive effect of Coriandrum sativum, Fam. UMBELLIFERAE (Chinese parsley) on lead deposition was investigated in male ICR mice given lead (1000 ppm) as lead acetate trihydrate in drinking water for 32 days. Administration of Chinese parsley to mice by gastric intubation was performed for 25 days from day 7 after the start of lead exposure up to the end of the experiment. The mice were then sacrificed for comparison of lead distribution. The lead reached its highest concentration in the femur but localized lead deposition in the femur was significantly decreased by meso-2,3-dimercaptosuccinic acid (DMSA), a chelating agent used as a positive control to validate this experimental model. Administration of Chinese parsley also significantly decreased lead deposition in the femur and severe lead- induced injury in the kidneys. In addition, urinary excretion of delta- aminolevulinic acid (ALA) which is known to increase with lead intake was significantly decreased after administration of Chinese parsley. The MeOH extract of Chinese parsley also reduced lead-induced inhibition of delta-aminolevulinic acid dehydratase (ALAD) activity in vitro. These results suggest that Chinese parsley has suppressive activity on lead deposition, probably resulting from the chelation of lead by some substances contained in Chinese parsley.

3. D. Karunasagar*, M.V. Balarama Krishna, S.V. Rao, J. Arunachalam Removal and preconcentration of inorganic and methyl mercury from aqueous media using a sorbent prepared from the plant Coriandrum sativum (National Center for Compositional Characterization of Materials (CCCM), Bhabha


Atomic Research Centre) Journal of Hazardous Materials B118 (2005) 133–139

4. Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils Int J Food Microbiol 2002 Mar 25;74(1-2):101-9 (ISSN: 0168-1605) Delaquis PJ; Stanich K; Girard B; Mazza G

Essential oils from dill (Anethum graveolens L.), coriander (seeds of Coriandrum sativum L.), cilantro (leaves of immature C. sativum L.) and eucalyptus (Eucalyptus dives) were separated into heterogeneous mixtures of components by fractional distillation and were analyzed by gas chromatography-mass spectroscopy. Minimum inhibitory concentrations against gram-positive bacteria, gram-negative bacteria and Saccharomyces cerevisiae were determined for the crude oils and their fractions. Essential oil of cilantro was particularly effective against Listeria monocytogenes, likely due to the presence of long chain (C6- C10) alcohols and aldehydes. The strength and spectrum of inhibition for the fractions often exceeded those determined in the crude oils. Mixing of fractions resulted in additive, synergistic or antagonistic effects against individual test microorganisms.

5. Role of mercury (Hg) in resistant infections & effective treatment of Chlamydia trachomatis and Herpes family viral infections (and potential treatment for cancer) by removing localized Hg deposits with Chinese parsley and delivering effective antibiotics using various drug uptake enhancement methods. Acupunct Electrother Res. 1995 AugDec;20(34):195-229. Omura Y, Beckman SL. Heart Disease Research Foundation, New York, USA.

Abstract The authors found that antibiotics used to treat various infections often were ineffective in the presence of abnormal localized deposits of heavy metals like Hg and Pb, which were often observed to co-exist with Chlamydia trachomatis, Herpes Simplex Types I & II, Cytomegalovirus(CMV), and other microorganisms. Our earlier research revealed that despite rigorous treatment with antibiotics together with various drug uptake enhanceme


nt techniques, subjects who had been treated for Chlamydia trachomatis infections, seemingly successfully with disappearance of their symptoms, were often experiencing recurrences within several months after completion of their treatment despite taking precautions against reinfection. Careful examination of the entire body of these symptomfree patients with the Bi-Digital ORing Test revealed that the Chlamydia trachomatis had retreated to 3 approximately 5 hiding places with localized increase in uric acid levels: 1) sublingual caruncle, 2) a small round area in the right and/or left axillae, 3) the genitals (Corona Glandis area of the Glans Penis at the Fossa Navicularis of the urethra in the male, and near the orifice of the urethra in the female), 4) Insulinlike Growth Factor positive horizontal lines, particularly above and below the knees, 5) the maxillary, ethmoid and frontal sinuses and the horizontal lines at the base of the nostrils (particularly small areas where Insulin-like Growth Factors exist). We found that all these areas contain Insulinlike Growth Factors I & II which are reduced in the presence of infection. Even when drug uptake of antibiotics was selectively increased in these 3 approximately 5 areas by various drug uptake enhancement methods developed by the 1st author, still the infection persisted. In the spring of 1995, use of Chinese parsley for successful elimination of Hg deposits existing in various organs of the first author as the result of the decay of radioactive Thallium 201 injected for cardiac SPECT, was accidentally discovered after eating Vietnamese soup, which happened to contain Chinese parsley, also called ci lantro. We also found Chinese parsley accelerates the excretion of Hg, Pb, and A1 from the body through the urine. Our subjects were given a course of antibiotics (Doxycycline for Chlamydia trachomatis infection) or antiviral agents (EPA with DHA for Herpes Family Viruses) together with Chinese parsley. Since these vegetable/herbs were eaten, the amount of effective substance absorbed varied and some people did not like the taste of these relatively large amounts of either cooked or raw parsley or its juice, but together with effective antibiotics delivered by drug uptake enhancement methods to the infected areas, the substances worked synergistically, rapidly reducing the generalized symptoms and infection. The micro-organisms retreated to the 3 approximately 5 areas listed above where, with continued treatment, they were significantly reduced, but not completely eliminated. Because of these problems, a pharmaceutical company was asked to produce a Chinese parsley tablet containing a controlled amount in a highly absorbable form. When 11 subjects were treated with Doxycycline for Chlamydia trachomatis infection, or antiviral agents (EPA with DHA) for Herpes Family Viruses, drug uptake enhancement methods to selectively increase delivery of the drugs to the affected areas, and Chinese parsley tablets to remove


the heavy metal deposits, the last traces of the infections and clinical symptomsdisappeared completely. Therefore we hypothesized that the infectious micro organisms mentioned above, somehow utilize the Hg or Pb to protect themselves from what would otherwise be effective antibiotics, and/or that heavy metal deposits in some way make antibiotics ineffective. Since the micro-organisms retreat to areas in which Insulinlike Growth Factors I & II normally exist, they may be utilizing them for their own growth and multiplication. PMID: 8686573 [PubMed indexed for MEDLINE]

6. Signif icant mercury deposits in internal organs fol lowing the removal of dental amalgam, & development of pre-cancer on the gingiva and the sides of the tongue and their represented organs as a result of inadvertent exposure to strong curing l ight (used to sol idify synthetic dental f i l l ing material) & effective treatment: acl inical case report, a long with organ representation areas for each tooth. Acupunct Electrother Res. 1996 Apr-Jun;21(2):13360. Omura Y, Shimotsuura Y, Fukuoka A, Fukuoka H, Nomoto T.

Heart Disease Research Foundation, New York, USA. Abstract Because of the reduced effectiveness of antibiotics against bacteria (e.g. Chlamydia trachomatis, alphaStreptococcus, Borrelia burgdorferi, etc.) and viruses (e.g.Herpes Family Viruses) in the presence of mercury, as well as the fact that the 1st author has found that mercury exists in cancer and precancer cell nuclei, the presence of dental amalgam (which contains about 50% mercury) in the human mouth is considered to be a potential hazard for the individual's health. In orderto solve this problem, 3 amalgam fillings were removed from the teeth of the subject of this case study. In order to fill the newly created empty spaces in the teeth where the amalgams had formerly existed, a synthetic dental-filling substance was introduced and to solidify the synthetic substance, curing light (wavelength range reportedly between 400 and 520 nm) was radiated onto the substance in order to accelerate the solidifying process by photopolymerization. In spite of considerable care not to inhale mercury vapor or swallow minute particles of dental amalgam during the process of removing it by drilling, mercury entered the body of the subject. Prec


autions such as the use of a rubber dam and strong air suction, as well as frequent water suctioning and washing of the mouth were insufficient. Significant deposits of mercury, previously nonexistent, were found in the lungs, kidneys, endocrine organs, liver, and heart with abnormal lowvoltage ECGs (similar to those recorded 13 weeks after i.v. injection of radioisotope Thallium-201 for Cardiac SPECT) in all the limb leads and V1 (but almost normal ECGs in the precordial leads V2toV6) the day after theprocedures were performed. Enhanced mercury evaporation by increased temperature and microscopic amalgam particles created by drilling may have contributed to mercury enteringthe lungs and G.I. system and then the blood circulation, creating abnormal deposits of mercury in the organs named above. Such mercury contamination may then contribute to intractable infections or precancer. However, these mercury deposits, which commonly occur in such cases, were successfully eliminated by the oral intake of 100 mg tablet of Chinese parsley (Ci lantro) 4 times a day (for average weight adults) with a number of drug-uptake enhancement methods developed by the 1st author, including different stimulation methods on the accurate organ representation areas of the hands (which have been mapped using the BiDigital ORing Test), without injections of chelating agents. Ingestion of Chinese parsley, accompanied by druguptake enhancement methods, was initiated before the amalgam removal procedure and continued for about 2 to 3 weeks afterwards, and ECGs became almost normal. During the use of strong bluish curing light to create a photo-polymerization reaction to solidify the synthetic filling material, the adjacent gingiva and the side of the tongue were inadvertently exposed. This exposure to the strong bluish light was found to produce precancerous conditions in the gingiva, the exposed areas of the tongue, as well as in the corresponding organsrepresented on those areas of the tongue, and abnormally increased enzyme levels in the liver. These abnormalities were also successfully reversed by the oral intake of a mixture of EPA with DHA and Chinese parsley, augmented by one of the noninvasive druguptake enhancement methods previously describedby the 1st author, repeated 4 times each day for 2 weeks. PMID: 8914687 [PubMed indexed for MEDLINE 6.“Ci lantro—Culinary Herb or Miracle Medicinal Plant” Abascal Kathy and Yarnell Eric. Alternative and Complementary Therapies. October 2012, 18(5): 259-264. doi:10.1089/act.2012.18507.


F. Chlorella BioPure uses a patented process by which stiff structural elements of the cell wall are cracked using sound waves. This keeps the extra cellular chlorella barrier intact, thus protecting all of the cell's organelles. It has a better absorption of toxins, but is harder to digest than some other forms of Chlorella. Super nutrient: with 50-60% amino acid content. Methylcobolamin - the most easily absorbed and utilized form of B12, B6, minerals, chlorophyll, beta-carotene etc. Directions for use: 4 Tablets 2 to 3 times per day with meals, or as directed by your practitioner. For mold/biotoxin binding/elimination and active phases of mercury/lead detox, use higher dosages ( 20-30 tbl 3-4 times per day, typically 30 min before meals or at bedtime)

Both C.pyreneidosa (better absorption of toxins, but harder to digest) and C.vulgaris (higher CGF content – see below, easier to digest, less metal absorbing capability) are available. A scientific literature list is available on www.KlinghardtAcademy.com. Be aware that there are huge differences in quality. We only recommend BioPure chlorella.

Chlorella has multiple published health inducing effects:

• Toxin binding (mucopolysaccharide membrane) all known toxic metals, environmental toxins such as dioxin and others

• Antiviral (especially effective against the cytomegaly virus from the herpes family)

• Repairs and activates the body’s detoxif ication functions: • Dramatically increases intra-cellular reduced glutathion, • Sporopollein is as effective as cholestyramin in binding

neurotoxins and more effective in binding toxic metals then any other natural substance found.


• Various peptides restore coeruloplasmin and metallothioneine, • Lipids (12.4 %) alpha-and gamma-linoleic acid help to balance the

increased intake of fish oil during our detox program and are necessary for a multitude of functions, including formation of there peroxisomes.

• Methyl-coblolamine is food for the nervous system, restores damaged neurons and has its own detoxifying effect.

• Chlorella growth factor helps the body detoxify itself in a yet not understood profound way. It appears that over millions of years chlorella has developed specific detoxifying proteins and peptides for every existing toxic metal.

• The porphyrins in chlorophyll have their own strong metal binding effect. Chlorophyll also activates the PPAR-receptor on the nucleus of the cell which is responsible for the transcription of DNA and coding the formation of the peroxisomes (see fish oil), opening of the cell wall (unknown mechanism) which is necessary for all detox procedures, normalizes insulin resistance and much more. Medical drugs that activate the PPAR receptor (such as pioglitazone) have been effective in the treatment of breast and prostate cancer.

• Super nutrient: 50-60% amino acid content, ideal nutrient for vegetarians, methylcobolamin - the most easily absorbed and utilized form of B12, B6, minerals, chlorophyll, beta carotene etc.

• Immune system strengthening • Restores bowel f lora • Digestive aid (bulking agent) • Alkal iniz ing agent ( important for patients with

malignancies) Both C.pyreneidosa (better absorption of toxins, but harder to digest) and C.vulgaris (higher CGF content – see below, easier to digest, less metal absorbing capability) are available. A scientific literature list is available on www.KlinghardtAcademy.com. Be aware that there are huge differences in quality. We only recommend BioPure chlorella.

Chlorella has multiple published health inducing effects:

• Toxin binding (mucopolysaccharide membrane)


all known toxic metals, environmental toxins such as dioxin and others

• Antiviral (especially effective against the cytomegaly virus from the herpes family)

• Repairs and activates the body’s detoxif ication functions: • Dramatically increases intra-cellular reduced glutathion, • Sporopollein is as effective as cholestyramin in binding

neurotoxins and more effective in binding toxic metals then any other natural substance found.

• Various peptides restore coeruloplasmin and metallothioneine, • Lipids (12.4 %) alpha-and gamma-linoleic acid help to balance the

increased intake of fish oil during our detox program and are necessary for a multitude of functions, including formation of there peroxisomes.

• Methyl-coblolamine is food for the nervous system, restores damaged neurons and has its own detoxifying effect.

• Chlorella growth factor helps the body detoxify itself in a yet not understood profound way. It appears that over millions of years chlorella has developed specific detoxifying proteins and peptides for every existing toxic metal.

• The porphyrins in chlorophyll have their own strong metal binding effect. Chlorophyll also activates the PPAR-receptor on the nucleus of the cell which is responsible for the transcription of DNA and coding the formation of the peroxisomes (see fish oil), opening of the cell wall (unknown mechanism) which is necessary for all detox procedures, normalizes insulin resistance and much more. Medical drugs that activate the PPAR receptor (such as pioglitazone) have been effective in the treatment of breast and prostate cancer.

• Super nutrient: 50-60% amino acid content, ideal nutrient for vegetarians, methylcobolamin - the most easily absorbed and utilized form of B12, B6, minerals, chlorophyll, beta carotene etc.

• Immune system strengthening • Restores bowel f lora


• Digestive aid (bulking agent) • Alkal iniz ing agent ( important for patients with

malignancies) Literature on Heavy metal detoxification and Chlorella (selected by Dietr ich Kl inghardt MD, PhD t i l l 2008)

We only use and recommend the pristinely grown sound cracked chlorella from BioPure. There has been trouble with other products.

Chlorel la safety

500 Gramm Chlorella per day in experiment without serious side effects except bloatedness (Algae Feeding in Humans R.Powell et al, J of Nutrition 75: 61, pg 7-12). Exempt in Japan from necessity of further safety studies

NIN report: no LD 50 in rats

South Korea: 4000 tons of chlorella used annually by humans without reports of worrysome side effects

Over 10 Million people take chlorella as daily supplement (ref 335) – Mason R: Altern Compl Therap, 2001;7 (3):161-165

Chlorel la and metal detox Uranium

Horikoshi, T./ Nakajima, A., et al.: Uptake of uranium by various cell fractions of chlorella vulgaris. Radioisotopes 28: 485-488, 1979

Nakajima, A; Horikoshi, T; Sakagushi, T.: Recovery of uranium by immobilised micro-organisms. Evr. J. Appl. Microbiol. Biotech, 16: 88-91, 1982.

Lead


Protective effects of chlorella vulgaris in lead exposed mice infected with Listeria monocytogenes M.Queiroz et al International Immunopharmacology 3 (2003) 889-900

Cadmium

Hagino et al.: Effect of chlorella on fecal and urinary cadmium excretion in Itai-itai. Jap. J. Hyg. 30: 77, 4/1975

Nagano, T./Suketa, Y., et al.: Absorption and excretion of chlorella ellipsoidea cadmium-binding protein and inorganic cadmium in rats. Jpn. J. Hyg., 38: 741-747, 1983

Carr, H.P., Carino, F.A., et al.: Characterization of the cadmium-binding capacity of chlorella vulgaris. Bull. Environ. Contam. Toxicol., 60: 433-440, 1998

Mercury

Shieh, Y.J.; Barger, J: Uptake of mercury by chlorella and its effect on potassium regulation. Planta, 109: 49-60, 1973

Klinghardt,D. :Algenpraeparat hilfreich bei der Amalgamausleitung

Erfahrungsheilkunde Band 48, Heft 7, Juli 1999

D.Klinghardt and J. Mercola: Mercury toxicity and systemic elimination agents D.Klinghardt and J. Mercola, J of Nutritional and environmental Medicine (2001) 11, 53-62

Parachlorella beyerinckii CK-5 is found to accelerate excretion of methyl-mercury both into feces and urine: “Japan Society for Bioscience, Biotechnology and Agro-chemistry”(JSBBA: http://www.jsbba.or.jp) Meeting in Nagoya City, Japan, March 29~30, 2008 .


Ben-Basset,D.; Mayer, A.M.: Reduction of mercury chloride by chlorella: Evidence for a reducing factor. Physiol. Pl., 40, 157-162, 1977).

Krisenon J: Inauguraldissertation (=PhD thesis) Fachbereich Chemie Universitaet GH Essen, 2002 (pp. 230, 231,232, 236, 247, Ref. 922, 923, 924 u. 926, 1000-1003): Chlorella and CVE remove mercury, tin, antimonum, bismuth and arsenic (toxins) from the bloodstream and from the oral cavity Chlorel la used tradit ional ly to save l ives after environmental catastrophes Nishijo et al: Toxicol Letters 1999; 108 (2-3): 321-7 Iwata K et al, Tohoku J Exp Med 1991; 164(2):93-102 Prof Lin, Ichiumura S: Rept Chlorella Res 1973, p 195 (only in Japanese) Hagino N et al: Nippon Eiseigaku Zasshi, 1975 ;30 (1):77

CVE: treatment of intestinal infections (L isteria, pathogenic e.col i and CMV) and lead toxicity:

Hasegawa, T./ Okuda, M./ Nomoto, K., et al.: Augmentation of the resistance against Listeria monocytogenes by oral administration of hot water extract of chlorella vulgaris in mice. Immnuopharmacology and Immunotoxicology, 16(2): 191-202, 1994

Queiroz, M. et al: Protective effects of chlorella vulgaris extract CVE) in lead-exposed mice infected with Listeria monocytogenes . Int Immunopharmacol 2003, Jun 3(6): 889-900

Chlorel la in cancer therapy


Komiyama, K.: Hirokawa, Y.; Mocota, T., et al: An acidic polysaccharide chlon A, from chlorella pyrenoidosa. Anti-tumour activity and immunological response, Chemotherapy, 34: 302-307, 1986.

Konishi, F.; Tanaka, K. ; Himeno, K., et al: Anti-tumour effect induced by a hot water extract of chlorella vulgaris: Resistance to meth-A tumour growth mediated by CE-induced polymorphonuclear leucocytes. Cancer Immunology and Immunotheraphy, 19 : 73-78, 1985.

Kuniaki, T.; Yoshifumi, T.; Tsuruta, M. et al: Oral administration of chlorella vulgaris augments concomitant anti-tumour immunity. Immuno-pharmacology and Immunotoxicology, 12 (2): 277-291, 1990.

Miyazawa, Y.; Murayama, T.; Ooya, N. et al: Immunomodulation by unicellular green algae (chlorella pyrenoidosa) in tumour-bearing mice. Journal of Ethnopharmacology, 24, 135-146, 1988.

Tanaka, K.; Konishi, F.; Himeno, K: Augmentation of anti-tumour resistance by a strain of unicellular green algae, chlorella vulgaris. Cancer Immunology and Immunotheraphy, 17: 90-94, 1984. 83

Merchant, R.E.; Rice, C.C.; Young, H.F.: Dietary chlorella pyrenoidosa for patients with malignant glioma: Effects on immunocompetence, quality of life, and survival. Phytotherapy Research, Vol. 4, No. 6, 220-230, 1990.)

Chlorel la and chemical detox

Effect of chlorella pyreneidosa on fecal excretion and liver accumulation of polychlorinated dibenzo-p-dioxin in mice Chemosphere 2005;59 297-304

Pore, R.S.; Detoxification of chlordecone poisoned rats with chlorella and chlorella-derived sporopollenin. Drug. Chem. Toxicol, 7: 57-71, 1984


Urey, J.C., et al.: Bioconcentration of four pure PCB Isomers by chlorella pyrenoidosa. Bull. Envir. Contam. Toxicol 16: 81-85, 1976

Morita, K., Matsueda T., Iida, T., Hasegawa, T.: Chlorella accelerates dioxin excretion in rats. Journal of nutrition 129 (9): 1731-6, 1999

Kunimasa M., Masahiro O., Hasegawa, T.: Chlorophyll derived from chlorella inhibits dioxin absorption from the gastrointestinal tract and accelerates dioxin excretion in rats. Environmental Health Perspectives 109: 289, 2001

Nutr J 2006 Jun 8;5:16Nick, G. Addressing human exposure to environmental toxins with chlorella Pyrenoidosa. Townsend Letter for Doctors and Patients. Apr 2003. (237), 28-32.

Chlorel la as general supplement

Over 10 Million people take chlorella as daily supplement (ref 335) – Mason R: Altern Compl Therap, 2001;7 (3):161-165

Tamiya, N., et al.: Preliminary experiments in the use of chlorella as human food. Food Technology Vol. VIII, 4: 179-182, 1954),

CGF - optimal facial development, optimal skeletal growth and development of intelligence:

Yamagishi, Y., et al.: School children’s growth and the value of chlorophyll. Nihon Iji Shimpo, S. 2196, 1961 (in Japanese)

R.Pratt et al :Production of thiamine, riboflavin, folic acid and biotin by chlorella vulgaris und chlorella pyreneidosa J of Pharmaceutical Sciences Vol 54, No.6, June 1965: chlorella contains significant amounts of: Vit B2, B3, methyl B12, D-3, Vit K, Vit C, Vit E, beta carotine and other carotinoids, all essentiell aminoacids, magnesium, iron, potassium, chlorophyll

Tokuyasu, M.: Examples of diets for infant’s and children’s nutritional guidance, and their effects of adding chlorella and C.G.F. to food schedule. Totori City, Japan: Comference proceedings at the


nutritional Illness Counseling Clinic 1983, siehe auch: Jpn. J. Nutr. 41(5): 275-283, (1980 u.) 1983)

Merchant, R., Andre, C.: A review of recent clinical trials of the nutritional supplement chlorella pyrenoidosa in the treatment of fibromyalgia, hypertension, and ulcerative colitis. Alternative Therapies 7: 79, 2001)

The chlorel la membrane: contents and properties

Bohumil Voelsky: Biosorption of Heavy Metals. CRC Press, 1990: several papers on the cell wall of chlorella. It contains: Hemizellulose A and B, C. P membrane contains Sporopollenin, not C.V, carotenoids, polyphenols and more

Ben-Basset,D.; Mayer, A.M.: Reduction of mercury chloride by chlorella: Evidence for a reducing factor. Physiol. Pl., 40, 157-162, 1977).

Pregnancy and Breastfeeding: the protect ive effect of chlorel la

S.Nakano et al: Maternal-fetal distribution and transfer of dioxins in pregnant women in Japan, and attempts to reduce maternal transfer with Chlorella (Chlorella pyrenoidosa) supplements Chemosphere, April 2005

Shiro Nakano et al: Chlorella Pyreneidosa supplementation decreases Dioxin and increases Immunoglobulin A concentrations in breast milk J Med Food 10 (1) 2007, 134-142).

Chlorel la lowers l ipids

A hot water extract of chlorella pyreneidosa reduces body weight and serum lipids in ovarectomized rats S.Hidaka et al Phytotherapy Research 18 (2004) 164-168

Effect of Chlorella on the level of serum cholesterol in rats C-J Wang et al, J Formosan Med Assoc 80 (1981) 929-933)


Chlorel la and the intestinal tract (col it is , IBS, metal binding) Merchant, Altern Ther Health Med. 2001 May-Jun;7(3):79-91. Merchant, R. & Andre, C. Dietary supplementation with chlorella pyrenoidosa produces positive results in patients with cancer or suffering from certain common chronic illnesses. Townsend Letter for Doctors and Patients. Feb 2001. (211), 74-80. Nucleotide (CGF/CVE chlorella extracts) und IBS Nucleotide supplementation: a randomised double-blind placebo controlled trial of IntestAidIB in people with Irritable Bowel Syndrome [ISRCTN67764449]. (Significant improvement over placebo) Full text:http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1513247&blobtype=pdf) Dancey CP, Attree EA, Brown KF Chlorophyll from chlorella binds toxic metals in the gut: Yun CH et al: Micros Res Tech 1997; 36(4): 313-23), Kensler TW et al: ibidem

Sporopollein from chlorella attaches itself to toxins in the intestinal mucosa and is excreted in this form together with the toxin (ref 233 pg 96): Pore, RS – Drug Chem Toxicol, 1984;7(1):57-71

Use of chlorel la for toxin el imination

Guzelian PS, Z Gastroenterologie, 1984; 22:16-20, symptoms of toxicity reduced (ref 28): Biesalski HK et al: Ernaehrungsmedizin 1999, Thieme Verlag, Stuttgart, Germany)

Mercola, J. & Klinghardt, D. Mercury toxicity and systemic elimination agents. Journal of Nutritional and Environmental Medicine. 2001. V. 11, 53-62.

Ray, T. The mitigation of methyl mercury vapor inhalation and exhalation in people with dental amalgam fillings. Townsend Letter for Doctors and Patients. Nov 2002. (232), 86-88. In this study a


mercury vapor analyzer is used to show that “nanonized” chlorella (Matrix Metals) and regular chlorella bind mercury

Papers in English by D. Klinghardt, MD,PhD, containing definitive references about the use of chlorella in a detoxification programme:

Klinghardt, D. Amalgam/mercury detox as a treatment for chronic viral, bacterial, and fungal illnesses. J Explore! 1997. V. 8, (3).

Klinghardt, D. The five levels of healing. J Explore! 2005. V. 14, (4).

Klinghardt, D. Metal toxicity. J Explore! 2000. V. 10 (1).

Klinghardt, D Chlorella – Erfahrungsheilkunde 1999;7:435-438

Klinghardt, D Lecture at the ETH in Zurich: Heavy Metal Toxicity Update (with full discussion on chlorella), Oct 31, 2001 –available on video

Reference about the gold and mercury detoxifying property of chlorella in the book

“The Biosorption of Heavy Metals”; Volesky B, CRC press 1990

Chlorel la References, unsorted 1 Aksu, Z.; T. ; The usage of chlorella vulgaris in waste water treatment

containing heavy metal irons. Proc. 4th

Eur. Cong. Biotech, 2, 80-83, 1987


2 Ben-Basset,D.; Mayer, A.M.: Reduction of mercury chloride by chlorella: Evidence

for a reducing factor. Physiol. Pl., 40, 157-162, 1977.

3 De Filippis, L.F.; The effect of sub-lethal concentrations of mercury and zinc on chlorella IV characteristics of a general reducing enzyme system for metallic irons. Z. Pflanzenphysiol. 86S: 339-352, 1978.

4 De Fillips, L.F.; Pallaghy, C.K.: The effect of sub-lethal concentrations of mercury and zinc on chlorella III. Development and possible mechanisms of resistance to metals. Pflanzenphysiol. 79S: 332-335, 1976.

5 Jensen, S.; Jenelov, A.: Biological methylation of mercury in aquatic organisms. Nature, 223: 753, 1969.

6 Nakajima, A; Horikoshi, T; Sakagushi, T.: Recovery of uranium by immobilised micro-organisms. Evr. J. Appl. Microbiol. Biotech, 16: 88-91, 1982.

7 Shieh, Y.J.; Barger, J: Uptake of mercury by chlorella and its effect on potassium regulation. Planta, 109: 49-60, 1973

8 Sneddon. J.; Pappas, C.P: Binding and removal of metal irons in solution by an algae biomass. Am. Environ. Lab, 10: 9-13, 1991.

9 Wilkinson, S.C.; Goulding, K.H.; Robinson, P.K. Mercury removal by immobilised Algae (Chlorella) in batch culture systems. Journal of Applied Phycology, 2:

223-230, 1990.


10 Pore, R.S.; Detoxification of chlordecore poisoned rats with chlorella and chlorella-derived sporopollenin. Drug. Chem. Toxicol, 7: 57-71, 1984

11 Kojima, M.; Kasajima, t.; Imai, Y., et al: A new chlorella polysaccharide and its

accelerating effect on the phagocytic activity of the reticuloendothelial system.

Recent Adv. Res., 13, 11, 1973

12 Merchant, R.E.; Rice, C.C.; Young, H.F.: Dietary chlorella pyrenoidosa for

patients with malignant glioma: Effects on immunocompetence, quality of life, and

survival. Phytotherapy Research, Vol. 4, No. 6, 220-230, 1990.

13 Tanaka, K.; Koga, T.; Konishi, F., et al: Augmentation of host defence by a

unicellular alga, chlorella vulgaris, to escherichia coli infection. Infect. Immun., 53:

267-271, 1986.

14 Ibusuki, K.; Minamishima, Y.: Effect of chlorella vulgaris extracts on murine cytomegalovirus infections. Nat. Immun. Cell Growth Regul., 9 : 121-128, 1990.

15 Kanazawa Medical College Dept. of Serology: Effects of various

preparation


made from chlorella pyrenoidosa cells on the defence mechanism (immune Resistance), 66-70, 1980. 16 Komiyama, K.: Hirokawa, Y.; Mocota, T., et al: An acidic

polysaccharide chlon A, from chlorella pyrenoidosa. Anti-tumour activity and immunological response, Chemotherapy, 34: 302-307, 1986.

17 Konishi, F.; Tanaka, K. ; Himeno, K., et al: Anti-tumour effect

induced by a hot water extract of chlorella vulgaris: Resistance to meth-A tumour growth mediated by CE-induced polymorphonuclear leucocytes. Cancer Immunology and Immunotheraphy, 19 : 73-78, 1985.

18 Kuniaki, T.; Yoshifumi, T.; Tsuruta, M. et al: Oral administration of chlorella vulgaris augments concomitant anti-tumour immunity. Immuno-pharmacology and Immunotoxicology, 12 (2): 277-291, 1990.

19 Miyazawa, Y.; Murayama, T.; Ooya, N. et al: Immunomodulation by unicellular green algae (chlorella pyrenoidosa) in tumour-bearing mice. Journal of Ethnopharmacology, 24, 135-146, 1988. 20 Tanaka, K.; Konishi, F.; Himeno, K: Augmentation of anti-tumour resistance by a strain of unicellular green algae, chlorella vulgaris. Cancer Immunology and Immunotheraphy, 17: 90-94, 1984. 83


21 Morita, K., Matsueda T., Iida, T., Hasegawa, T.: Chlorella accelerates dioxin excretion in rats. Journal of nutrition 129 (9): 1731-6, 1999

22 Kunimasa M., Masahiro O., Hasegawa, T.: Chlorophyll derived from

chlorella inhibits dioxin absorption from the gastrointestinal tract and accelerates dioxin excretion in rats. Environmental Health Perspectives 109: 289, 2001

23 Merchant, R., Andre, C.: A review of recent clinical trials of the

nutritional supplement chlorella pyrenoidosa in the treatment of fibromyalgia, hypertension, and ulcerative colitis. Alternative Therapies 7: 79, 2001

24 Japan Food Research Laboratories, Analysis Certificate No.

100021448-010, 2000 25 Wong, S.L., et al..: Detection of toxic organometallic complexes in

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26 Travesio, L./ Canizares, R.O.: Heavy metal removal by microalgae.

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Hier&Jetzt 3: 10-13, 2000

28 Urey, J.C., et al.: Bioconcentration of four pure PCB Isomers by chlorella pyrenoidosa. Bull. Envir. Contam. Toxicol 16: 81-85, 1976

29 Palacios, Mayorga S., et al.: Algunas modificationes al aislamiento y

cultivo de chlorella vulgaris Beijerinck. Rev. Lat-amer. Microbiol 19: 95-103, 1977


30 Chang, J.: Wild swans – three daughters of China. New York, Simon Schuster, 1991

31 Spoehr, H.A./ Milner, H.W.: Plant. Physiol. 24: 120f., 1949 32 Tamiya, N., et al.: Preliminary experiments in the use of chlorella as

human food. Food Technology Vol. VIII, 4: 179-182, 1954 33 Hagino et al.: Effect of chlorella on fecal and urinary cadmium

excretion in Itai-itai. Jap. J. Hyg. 30: 77, 4/1975 34 Mündliche Mitteilung während der 3rd International Exhibition &

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35 Isidori, A., et al.: A study of human growth hormone (HGH) release

in man after oral administration of amino acids. Current-Medical Research and Opinion, 7, 1981

36 Fried, R./ Merrel, W.C.: The arginine solution, New York: Warner

Books, 1999 37 Lombard, J./ Germano, C.: The brain wellness plan, New York:

Kensington Publishing, 1997 38 Kojima, M./ Shishido, K., et al.: A clorella polysaccharide as a factor

stimulating RES activity. J. reticuloendothel Soc., 14(2): 192-208, 1973

39 Kojima, M./ Kasajima, Y.: A new chlorella polysaccharide and its

accelerating effect on the phagozytose activity of the reticuloendothelial system. Recent Adv. R.E.S., 13: 11, 1973


40 Matusiak, K./ Krzywicka, A.: Influence of the extract of chlorella vulgaris on growth of fungi. Acta Microbiologica Polonica Ser. B, 7(24), No.1: 51-54, 1975

41 Forest, W.: Ullmanns Enzyklopädie der technischen Chemie,

München/Berlin Verlag Urban & Schwarzenberg, 136-141, 1956 42 Popp, F.A.: Das Licht in unseren Zellen, 6.Auflage, Verlag

Zweitausendeins, 1995 43 Koch, H.P./ Hühnler, G.:Chlorophyll – Blattgrün als Arzneimittel,

Deutscher Apotheker Verlag, 1995 44 Economics Club: Latest report on functional study of chlorella.

Nihonbashi, Japan (unveröffentlicht), 25.06.1997

45 Yasukawa, K./ Akihisa, T., et al.: Inhibitory effects of sterols isolated

from chlorella vulgaris on 12-0-Tetradecanoylphorbol-13-acetate-induced inflammation and tumor promotion in mouse skin. Biol. Pharm. Bull. 19(4): 573-576, 1996

46 Vortrag von Dr. Tahao Iida (Health Ecology Research Lab., Fukoka,

Japan [July 9-10th, 1998]: Chlorella has remarkable effects of excreting dioxin out of the body. Sonderdruck aus Health Life Business Magazine 179, 1998

47 Jensen, B.: Chlorella, Jewel of the Far East. Escondido/CA, USA:

Bernard Jensen Publisher, 1992 48 Koerber/Männle/Leitzmann: Vollwerternährung – Konzeption einer

zeitgemäßen Ernährung. Heidelberg: Haug Verlag, 1994 49 Epstein, S.S.: The politics of cancer revisited, New York: East Ridge

Press, 1998


50 Singh, A./ Singh, S.P., et al.: Inhibitory potential of chlorella vulgaris

(E-2S) on mouse skin papillomagenesis and xenobiotic detoxicartion system. Anticancer Research, 19: 1887-1892, 1999

51 Nagano, T./Suketa, Y., et al.: Absorption and excretion of chlorella

ellipsoidea cadmium-binding protein and inorganic cadmium in rats. Jpn. J. Hyg., 38: 741-747, 1983

52 Carr, H.P., Carino, F.A., et al.: Characterization of the cadmium-

binding capacity of chlorella vulgaris. Bull. Environ. Contam. Toxicol., 60: 433-440, 1998

53 Horikoshi, T./ Nakajima, A., et al.: Uptake of uranium by various cell

fractions of chlorella vulgaris. Radioisotopes 28: 485-488, 1979 54 Nach Pinnang Soong in Shelef/Soeder (Hrsg.): Algae Biomass,

North-Holland: Biomedical Press, 1980 55 Tanaka, K./ Yamada, A./ Noda, K., et al.: A novel glycoprotein

obtained from chlorella vulgaris strain CK22 shows antimetastatic immunopotentiation. Cancer Immunol. Immunother. 45: 313-320, 1998

56 Konishi, F./ Mitsuyama, M./ Okuda, M., et al.: Protective effect of

an acidic glycoprotein obtained from culture of chlorella vulgaris against myelosuppression by 5-fluorouracil. Cancer Immunol. Immunother., 42: 268-274, 1996

57 Noda, K./ Tanaka, K., et al.: A water-soluble antitumor glycoprotein

from chlorella vulgaris. Planta medica, 62: 423-426, 1996 58 Fujimaki, M., et al.: About C.G.F., Effects on actions of chlorella cell

itself, Tokyo, Japan: People’s scientific Research Center at Koganei (unveröffentlichter Bericht)


59 Hasegewa, T./ Okuda, M./ Makino, M., et al.: Hot water extracts of

chlorella vulgaris reduce opportunistic infection with listeria monocytogenes in C57BL/6 mice infected with LP-BM5 murine leukaemia viruses. Inter. J. of Immunopharmacology, 17(6): 505-512, 1995

60 Watzl, B./ Leitzmann, C.: Bioaktive Substanzen in Lebensmitteln,

Stuttgart: Hippokrates-Verlag, 1999 61 Lee, W.H./ Rosenbaum, M.: Chlorella – The sun-powered

supernutrient an dits benefiial properties. A good Health Guide, New Canaan, Connecticut: Keats Publishing, 1987

62 Huang, S.-W./ Kondo, N.: Studies on the effect of feeding edible

microalgae to honey bee colonies. Res. Inst. Evolut. Biol. Sci. Rep., (8): 57-83, 1996

63 Yamagishi, Y., et al.: School children’s growth and the value of

chlorophyll. Nihon Iji Shimpo, S. 2196, 1961 (in japanischer Sprache)

64 Tokuyasu, M.: Examples of diets for infant’s and children’s

nutritional guidance, and their effects of adding chlorella and C.G.F. to food schedule. Totori City, Japan: Comference proceedings at the nutritional Illness Counseling Clinic 1983, siehe auch: Jpn. J. Nutr. 41(5): 275-283, (1980 u.) 1983

65 Statistics and Information Departement: Abridged life table for

Japan, average expectancy 1945-1992. Minister’s Secretariat, Ministry Health Welfare: Japanese Government, 1992

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prevention: Investigating the roles of micronutritient, New York: Marcel Dekker, 1989

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resistance against Listeria monocytogenes by oral administration of hot water extract of chlorella vulagris in mice. Immnuopharmacology and Immunotoxicology, 16(2): 191-202, 1994

72 Hasegawa, T./ Yoshikai, Y./ Okuda, M. et al.: Accelerated

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74 Hasegawa, T./ Tanaka, K., et al.: Augmentation of the resistance

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75 Konishi, F./ Tanaka, K. et al.: Antitumor effect induced by a hot

water extract of chlorella vulgaris (CE): Resistance to Meth-A tumor growth mediated by CE-induced polymorphonuclear leukocytes. Cancer Immunology Immunotherapy, 19(2): 73-78, 1985

76 Tanaka, K./ Konishi, F./ Himeno, K., et al.: Augmentation of

antitumor resistance by a strain of unicellular green algae, chlorella vulgaris. 17(2): 90-94, 1984

77 Hasegawa, T./ ito, K., et al.: Oral administration of hot water of

chlorella vulgaris reduces IgE production against milk casein in mice. International Journal of Immunopharmacology, 21(5): 311-323, 1999

78 Tanaka, K./ Yamada, A./ Noda, K., et al.: Oral administration of a

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Golden Pamphlet, Heidelberg: Haug-Verlag, 1993

G. Bartonella


Click album below for photos of Bartonella

rashes http://www.lymediseaseassociation.org/index.php/resources/

medical-photos/category/27-bartonella

2002 Paper by Martin D Fried MD and Aswine Bal MD; MD Fried, J

Schairer, G Madigan, A Bal - J Pediatr Gastroenterol Nutr, 2002

Bartonella henselae is associated with heartburn, abdominal pain, skin

rash, mesenteric adenitis, gastritis and duodentis in children and

adolescents.

H. “Bell ’s Palsy of the Gut” and Other GI Manifestations of Lyme and Associated Diseases

A SPECIAL ARTICLE Virginia T. Sherr, M.D., DLFAPA, Distinguished Life Fellow of the American Psychiatric Association, Solo Private Practice of Medicine, Holland, PA. Virginia T. Sherr 74 PRACTICAL GASTROENTEROLOGY • APRIL 2006 Bell ’s palsy signif ies paralysis of facial muscles related to inf lammation of the associated seventh Cranial Nerve. Physic ians may not real ize that this syndrome is caused by the spirochetal agent of Lyme disease unti l proven otherwise . Whether it is a ful l or hemifacial paralysis, Bel l ’s palsy is cosmetical ly disf iguring when ful ly expressed. Sudden loss of normal facial expression terrif ies patients who natural ly fear they are having a stroke. When a smile is asked for, normal countenances warp into bizarre


grimaces. The amount of tooth area exposed in this attempt to smile helps doctors evaluate the degree of paralysis and its change over t ime (Figure 1). In every case of Bel l ’s , doctors need to careful ly investigate by history, physical , and laboratory work every shred of evidence that might suggest the presence of cryptic tert iary Lyme, a serious mult isystem, gut a patients have no evidence whatsoever of having had a t ick-bite. Gastrointestinal Lyme disease may cause gut paralysis and a wide range of diverse GI symptoms with the underlying etiology l ikewise missed by physicians. Borrel ia burgdorferi , the microbial agent often behind unexplained GI symptoms—along with numerous other pathogens also contained in t ick sal iva—influences health and vital ity of the gastrointestinal tract from oral cavity to anus. Disruptions caused by GI borrel iosis (Lyme) may include, amongst many others, distortions of taste, fai lure of other neural functions that supply the entire GI tract—paralysis or partial paralysis of the tongue, gag ref lex, esophagus, stomach and nearby organs, small and/or large intestines INTRODUCTION Until proven otherwise, a patient’s unexplained facial paralysis is caused by the tick-borne spirochetes of Lyme disease (LYD) (1). The widely endemic bacteria are easily capable of inducing distal inflammation of the Seventh Cranial (Facial) Nerve (2). “Considering the incidence of Bell’s palsy in Lyme, it is improper to treat it as viral in origin without a work-up for Lyme disease” (3). In an early study with nearly 1000 LYD cases studied, Bell’s palsy occurred in at least 10% of validated cases (4). The frequency of Lyme’s Bell’s palsy etiology is unfamiliar to many physicians. Likewise many physicians are unfamiliar with the spirochetal cause of paralyses of muscles that facilitate normal gastrointestinal transit. Yet, these vital muscles also may be greatly compromised by the same offending neurotropic spirochete, Borrelia burgdorferi (Bb) in patients who are totally unaware of having Lyme disease. Their physicians are often


surprised to learn that persistent Lyme disease is outstandingly a disease of the brain as well as involving one or all components and sub-systems of the entire (“ i leus”), bowel pseudo-obstruction, intestinal spasms, excitabi l ity of gut muscles, inf lammation of lumen l ining t issues, spirochetal hepatit is , possibly cholecystit is , dysbiosis, jejunal or i leal incompetence with resultant small intestine bacterial overgrowth (SIBO), megacolon, encopresis and rectal muscle cramping (proctalgia fugax). In cerebral hypothalamic and pituitary centers, usual s ites of borrel ial disruptions of the brain’s normal hormonal cascades, there are strong inf luences on human att itudes, ideation, and behavior relating to gastronomic issues. Newly discovered Lymeendangered cerebral hormones and renegade cytokines regulate brain-gut interactions thus init iat ing behavioral tendencies such as anorexia or a fai lure of satiety with resultant obesity. Ticks and other vectors of Lyme disease attract their own infections from many microbes, some known and some unknown (viruses, amoebas, bacteria, and possibly parasit ic f i lar ia), which they then also can pass on to humans. The GI tract is especial ly vulnerable to machinations of such co-infections as bartonel losis, mycoplasmosis, human anaplasmosis (HA), and human monocytic ehrl ichiosis (HME). Syndromes exactly s imilar to Irr itable Bowel Syndrome (IBS), Crohn’s Disease, and cholecystit is , for example, may not have readi ly suggested a borrel ial et iology to the diagnostic ian but Lyme increasingly is known to be a potential contributor to each. Al l known Lyme-gut syndromes are treated by combining several effective antimicrobials ( including use of azole medications with specif ic antibiotics) with agents that boost gut l ining repairs and overal l immunity enhancement. Azole medications are borrel iacidal


(against the anti-Bb spirochetal cyst form) medications such as metronidazole (Flagyl) . Needed GI heal ing agents may include gut st imulants or relaxants, Ph agents, bi le salts, nutriceuticals, immunity-enhancers, neurotoxin absorbents, and steri l izers of gut-specif ic microbes. Paral lel ism between Lyme borrel iosis-caused paresis of facial muscles supplied by Cranial Nerve VII and Lyme-caused gastrointestinal paralyses suggested a pseudonym to the author—Bell ’s palsy of the Gut—despite the fact that these syndromes are related to different types of neural f ibers and only occasional ly occur together. S ince s imilar injury to al l s ites may be etiological ly related, however, otherwise unexplained gastrointestinal symptoms should be considered as possibly related to Lyme borrel iosis and/or its co-infections unti l proven otherwise.

References

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29. Coulter P, Lema C, Flayhart D, et al. Two-year evaluation of Borrelia burgdorferi culture and supplemental tests for definitive diagnosis of Lyme disease. J Clin Microbiol, 2005; 43(10):5080-5084. 30. Picha D, Moravcova L, Zdarsky E, et al. PCR in Lyme neuroborreliosis: a prospective study. Acta Neurol Scand, 2005; 112(5): 287-92. [Czech Republic]. 31. Medical Diagnostic Laboratories, L.L.C, 2439 Kuser Road, Hamilton, NJ 08690 USA. http://www.mdlab.com/html/testing/available_ tests.html#tick ) 32. Guarner J, Shieh WJ, Morgan J, et al. Leptospirosis mimicking acute cholecystitis among athletes participating in a triathlon. Hum Pathol, 2001; 32(7):750-752. 33. Chen W, Li D, Paulus B, et al. High prevalence of Mycoplasma pneumoniae in intestinal mucosal biopsies from patients with inflammatory bowel disease and controls. Dig Dis Sci, 2001; 46(11):2529-2535. 34. IGeneX, Inc. 795 San Antonio Rd., Palo Alto, CA 94303 http://www.igenex.com/about.htm 35. Boltri JM, Hash RB, Vogel RL. Patterns of Lyme disease diagnosis and treatment by family physicians in a southeastern state. J Community Health, 2002; 27:395-402. 36. Association of State and Territorial Public Health Lab Directors (ASTPHLD). Proceedings of the second national conf. on the serological diagnosis of Lyme disease. Dearborn, Michigan USA. 1994; 27-29. 37. Aguero-Rosenfeld ME, Nowakowski J, McKenna DF, et al. “Evolution


of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans.” J Clin Microbiol, 1996; 34:1-9. 38. Cameron D, Gaito A, Harris N, et al. Evidence-based Guidelines for the management of Lyme disease. Expert Rev Anti-infect Ther, 2004; 2(1):1-13. 39. Summary: Evidence-based Guidelines for the management of Lyme disease. Expert Rev Anti-infect Ther, 2004; 2(1):1-13. http://www.ilads.org/guidelines_ilads.html 40. Honegr K, Hulínská D, Dostál V. Persistence of Borrelia burgdorferi sensu lato in patients with Lyme borreliosis. Epidemiol Mikrobiol Imunol, 2001; 50(1):10-6. 41. Lin HC. Small intestinal bacterial overgrowth: a framework for understanding irritable bowel syndrome. JAMA, 2004; 292(7):852- 858. 42. Singh V, Toskes P. Small bowel bacterial overgrowth: presentation, diagnosis, and treatment. Curr Gastroenterol Rep, 2003; 5(5):365-372. 43. Genova (Great Smokies) Laboratory: http://www.gsdl.com/ home/ 44. Doctors’ Data Laboratory http://www.doctorsdata.com/home.asp 45. Mormont E, Esselinckx W, De Ronde T, et al. Abdominal wall weakness and lumboabdominal pain revealing neuroborreliosis: a report of three cases) Clin Rheumatol, 2001; 20(6):447-450.


46. Krishnamurthy KB, Liu GT, Logigian EL. Acute Lyme neuropathy presenting with polyradicular pain, abdominal protrusion, and cranial neuropathy. Muscle Nerve, 1993; 16(11):1261-1264. 47. Daffner KR, Saver JL, Biber MP. Lyme polyradiculoneuropathy presenting as increasing abdominal girth. Neurology, 1990; 40:373-375. 48. Pokorny P. Incidence of the spirochete Borrelia burgdorferi in arthropods (Arthropoda) and antibodies in vertebrates (Vertebrata). Cesk Epidemiol Mikrobiol Imunol, 1989; 38(1):52-60. 49. Savely GR. The Belly Acher: My Most Unusual Patient. Beyond the Textbook, in Clinician News, 2005; 9(9):14-15. 50. Fried MD, Abel M, Pietrucha D, et al. The spectrum of gastrointestinal manifestations in children and adolescents with Lyme disease. JSTBD, 1999 Fall/Winter; 6. 51. Rodríguez LA, Gonzales PA, Johansson S. Centro Español de Investigación Farmacoepidemiológica (CEIFE) Aliment Pharmacol Ther, 2005; 22(4):309-315. ©2005 Blackwell Publishing, Mölndal, Sweden. 52. Molecular self-loathing. The Economist, 2005. 53. Fetissov SO, Harro J, Jannisk M, et al. Autoantibodies against neuropeptides are associated with psychological traits in eating disorders. PNAS, 2005; 102:14865-14870. 54. Cone RD. Anatomy and regulation of the central melanocortin system). Nat Neurosci, 2005; 8(5):571-578. 55. Fallon BA, Nields JA. Lyme disease: a neuropsychiatric illness,


Am J Psychiatry, 1994; 151(ll):1571-1583. 56. Dattwyler RJ, Halperin JJ, Volkman DJ, et al. Treatment of late Lyme borreliosis—randomised comparison of ceftriaxone and penicillin. Lancet, 1988; 1(8596):1191-1194. 57. Craft JE, Fischer DK, Shimamoto GT, Steere AC. Antigens of Borrelia burgdorferi recognized during Lyme disease. Appearance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J Clin Invest, 1986; 78(4):934-939. 58. Kalish RA, McHugh G, Granquist J, et al. Persistence of immunoglobulin M or immunoglobulin G antibody responses to Borrelia burgdorferi 10–20 years after active Lyme disease. Clin Infect Dis, 2001; 33(6):780-85. [email protected] 59. Burrascano JJ. Diagnostic hints and treatment guidelines for Lyme and other tick borne illnesses. 2005. www.ilads.org [See “Articles and presentations”]. 60. Lauritano EC, Gabrielli M, Lupascu A, et al. Rifaximin dose-finding study for the treatment of small intestinal bacterial overgrowth. Aliment Pharmacol Ther, 2005; 22(1): 31-35. 61. Sherr VT. Human babesiosis—an unrecorded reality. Absence of formal registry undermines its detection, diagnosis and treatment, suggesting need for immediate mandatory reporting. Med Hypotheses, 2004; 63(4):609-615. 62. Zaidel O, Lin HC. Uninvited guests: the impact of small intestinal


bacterial overgrowth on nutritional status. Practical Gastroenterology, 2003; 27(7):27. 63. Parrish CR (Ed), Yoshida CM. Nutrition intervention for the patient with gastroparesis: an update. Pract Gastroenterol—Nutrition issues in gastroenterology. 2005; 29(8):29. 64. Nichols TW, Faass N. Optimal Digestive Health: A Complete Guide. 2005. Healing Arts Press, Rochester, VT. (continuedfrompage88)

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1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373078/?page=2 2. Hall-Stoodley L et all, Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media, JAMA. 2006 Jul 12;296(2):202-11.http://www.ncbi.nlm.nih.gov/pubmed/16835426 3. "Research on microbial biofilms (PA-03-047)". NIH, National Heart, Lung, and Blood Institute. 2002-12-20. 4. Ana-Monica Pais-Correia et al., Biofilm-like extracellular viral assemblies mediate HTLV-1 cell-to-cell transmission at virological synapses, Nature Medicine, 16, 83-89, 2010,http://www.nature.com/nm/journal/v16/n1/abs/nm.2065.html 5. The Lyme-Autism Connection: Unveiling the Shocking Link Between Lyme Disease and Childhood Developmental Disorders Paperback, Tami Duncan, Bryan Rosner 6. R.F.Itzhaki, Herpes Simplex Virus Type 1 in Alzheimer’s Disease: The Enemy Within, Journal of Alzheimer’s Disease, Vol 13, N.4/2008


7. Dementia Caused by Borellia infection of the Central Nervous System’, Alan B. McDonald, M.D.http://alzheimerborreliosis.net/wp-content/uploads/2012/10/Final_version_Philadelphia_Presentation.pdf 8. Alzheimer’s disease – a neurosporochetosis. Analysis of the evidence following Koch’s and Hill’s criteria. Judith Miklossy,MD, Journal of Neuroinflammation 2011, 8:90http://www.jneuroinflammation.com/content/8/1/90 9. Direct Visualization of Fungal Infection in Brains from Patients with Alzheimer's Disease. Pisa D. et al.; J.Alzheimers Dis. Aug.13,2014. http://www.ncbi.nlm.nih.gov/pubmed/25125470 10. Alzheimer’s disease and disseminated mycoses. R. Alonso et.al.; European Journal of Clinical Microbiology & Infectious Diseases, July 2014, Volume 33, Issue 7, pp 1125-1132http://link.springer.com/article/10.1007/s10096-013-2045-z 11. http://www.cdc.gov/vaccines/schedules/downloads/child/0-18yrs-combined-schedule-bw.pdf 12. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1393226/ 13. Suppression of bacterial cell–cell signalling, biofilm formation and type III secretion system by citrus flavonoids, A.Vikram et.al.; Journal of Microbiology, Vol.109,Iss.2, August, 2010.http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2010.04677.x/full 14. Resveratrol Inhibits Periodontal Pathogens In Vitro, D.J.O’Connor et. al., Phytotherapy Research, Vol 25.11.November 2011.http://onlinelibrary.wiley.com/doi/10.1002/ptr.3501/abstract?deniedAccessCustomisedMessage=&userIsAuthenticated=false


15. Apple Flavonoid Phloretin Inhibits Escherichia coli O157:H7 Biofilm Formation and Ameliorates Colon Inflammation in Rats. Jin Hyung-Lee et.al.; Infection and Immunity, Vol.79,no.12,December 2011. http://iai.asm.org/content/79/12/4819.short 16. Adam M. Brickman et.al.; Enhancing dentate gyrus function with dietary flavanols improves cognition in older adults. Nature Neuroscience, October 2014.http://www.nature.com/neuro/journal/vaop/ncurrent/full/nn.3850.html 17. Effect of enterosorption on animal lifespan. Frolkis VV, et al. Biomater Artif Cells Artif Organs, 1989; 17(3):341-51. Institute of Gerontology, AMS USSR. 18. Antibiofilm activity of sodium bicarbonate, sodium metaperiodate and SDS combination against dental unit waterline-associated bacteria and yeast. Gawande PV. Et al., J.Appl. Microbiol. 2008 Oct., http://www.ncbi.nlm.nih.gov/pubmed/18422552 19.

I. Depression and Infection

Biol Mood Anxiety Disord. 2014; 4: 10. Published online 2014 Oct 21. doi: 10.1186/2045-5380-4-10

PMCID: PMC4215336

Reconceptualizing major depressive disorder as an infectious disease Turhan Canli 1,2,3,4

Background

Despite decades of substantial research efforts, major depressive disorder (MDD) remains among the most common mental disorders, with a 16.6%


lifetime prevalence rate [1]. Pharmacological treatment approaches have not changed during this period, targeting primarily receptor-ligand interactions [2]. These types of antidepressants may bring relief to patients with severe symptoms but are not clinically more effective than placebos in mild to moderate cases [3]. Indeed, recurrence rates of 50% for first-episode patients and of 80% for second-episode patients [4] suggest that the core of the illness goes untreated.

Given this track record, I argue that it is time for an entirely different approach. Instead of conceptualizing MDD as an emotional disorder, I suggest to reconceptualize it as some form of an infectious disease. I propose that future research should conduct a concerted search for parasites, bacteria, or viruses that may play a causal role in the etiology of MDD. I present three arguments why this may be a fruitful endeavor. I have outlined the idea in much greater detail elsewhere [5], but will highlight some key points here.

Main text

My first argument is that patients with MDD exhibit sickness behavior. Patients experience loss of energy; they commonly have difficulty getting out of bed and lose interest in the world around them. Although our Western conceptualization puts affective symptoms front-and-center, non-Western patients who meet DSM criteria for major depression report primarily somatic symptoms [6-11], reflecting in part cultural differences in the stigmatization of mental illness.

Yet, studies of inflammatory biomarkers in major depression strongly suggest an illness-related origin. For example, a meta-analysis of 24 studies confirmed prior reports of elevated TNFα and IL-6 in patients with major depression [12]. A second meta-analysis of 29 studies further extended the list of significantly elevated inflammatory markers to also include the soluble interleukin-2 receptor [13].

Several postmortem studies report the presence of inflammatory markers in the brains of depressed or mood-disordered patients. For example, compared to controls, female suicide victims showed elevated levels of IL-4 and male suicide victims showed elevated levels of IL-13 in Brodmann Area (BA) 11 [14], a brain region previously associated with suicidal ideation [15,16]. Compared to age-matched controls, patients diagnosed with major depression showed elevated levels of transmembrane TNFα (tmTNFα) in BA46 [17], a region associated with emotion regulation [18-


20]. Patients with major depression, relative to controls, showed differential expression of a large set of both anti- and pro-inflammatory markers (including IL1α, 2, 3, 5, 8, 9, 10, 12A, 13, 15, 18, and IFNγ) in BA10 [21], a region associated with reward processing [22].

These inflammatory markers may represent activation of the immune system in response to some kind of pathogen, which could be a parasite, bacterium, or virus, and which could play a causal role in the etiology of depression. There is currently no direct evidence that major depression is caused by such microorganisms, but nature has offered some examples to illustrate that such a process is conceivable.

Thus, my second argument is that nature has already provided examples by which parasites, bacteria, or viruses can affect emotional behavior. The best-known example of a parasite that affects emotional behavior and that is relevant to human health is Toxoplasma gondii. T. gondii lives in the feline intestinal tract, where it lays its eggs, which are dispersed into the environment upon excretion. When a rat comes in contact with these eggs and becomes infected, it becomes attracted to the scent of cat urine [23,24]. This manipulation of the rat’s behavior involves the deposit of parasitic cysts across the rodent brain including the amygdala [25]. The mechanism for loss of fear to the scent of cat urine appears to involve a reduction in circulating corticosterone and dendritic retraction in the basolateral amygdala [26]. The mechanism for the rat’s attraction to the odor may involve activation of sexual arousal pathways [27].

The specificity of the behavioral change in the rat’s behavior appears to reflect functional changes that are limited to catecholaminergic neurons [28]. Infected animals have elevated levels of dopamine [29], but T. gondii can only synthesize tyrosine hydroxylase (which converts tyrosine to L-DOPA), and would therefore need to rely on catecholaminergic neurons to provide the needed DOPA decarboxylase to convert the L-DOPA to dopamine.

Human exposure to T. gondii is pervasive, with one-third of the world’s population [30] and one-fifth of the U.S. population [31] believed to be infected. Infection is associated with elevated inflammatory cytokines IL-6, IL-12, TNF, and IFN-γ [32,33], similarly as observed in depressed patients. A study of 20 European countries reported a positive correlation between T. gondii prevalence rates and national suicide rates [34]. Among patients with diagnosed major depression or bipolar disorder, those with a history of suicide attempt had higher T. gondii antibody titers [35]. Yet,


large-scale studies of major depression and T. gondii or systematic searches to discover other potential parasitic infections have not yet been conducted.

Bacteria could be another causal factor for major depression. Studies of bacterial colonies residing in the gastrointestinal tract have begun to examine links to emotional behavior. In the first study of this kind, germ-free (GF), specific pathogen-free (SPF), and gnotobiotic mice were compared in their response to restraint stress [36]. GF mice exhibited higher levels of plasma ACTH and corticosterone and had lower levels of brain-derived neurotrophic factor in the cortex and hippocampus, compared to SPF mice. The elevated stress response of GF mice was normalized with administration of the bacterium Bifidobacterium infantis. Another rodent study showed that administration of B. infantis in rats reduced the levels of IFN-γ, TNF-α, and IL-6 following mitogen stimulation and altered tryptophan, 5-HIAA, and DOPAC levels in the frontal cortex and amygdala [37]. Administration of the Lactobacillus rhamnosus strain in mice was shown to alter GABAergic expression in the brain: elevating GABAB1b mRNA in the cingulate and prelimbic cortices, while reducing it in hippocampus and amygdala, among other regions [38].

The ‘leaky gut’ hypothesis proposes a mechanism by which gastrointestinal bacteria may contribute to major depression [39,40]. According to this hypothesis, cytokines or other stressors may render the intestinal tract permeable to lipopolysaccharides (LPS) from gram-negative bacteria to activate the immune system. Indeed, the model is supported by data showing elevated serum concentrations of IgM and IgA against LPS of the gram-negative enterobacteria in depressed patients [39,40]. These studies were conducted with relatively small numbers of patients and suggested that this mechanism may apply to some subgroups of patients but not others. It would be useful to expand the search using large patient cohorts and a wide range of different antibodies. Future work should then examine potential neural mechanisms.

Viruses represent the third pathogenic route in the etiology of major depression. A meta-analysis of 28 studies explicitly examined the link between infectious agents and depression [41]. Among viruses that had significant associations with the illness were the Borna disease virus (BDV), herpes simplex virus-1, varicella zoster virus, and Epstein-Barr virus. Among these, BDV has been studied most extensively and was 3.25 times more likely to be found in depressed patients than in normal controls [41]. One postmortem study reported BDV infection in 2 out of 30


depressed patients in the frontal and temporal cortex, olfactory bulb, and hippocampus [42], although a larger study failed to detect any infection [43]. A small open-label study of BDV-infected depressed patients reported a reduction in both depressive symptoms and BDV infection upon treatment with the antiviral drug amantadine [44].

The mechanism between BDV infection and depression could involve glutaminergic transmission, because amantadine is an antagonist of the N-methyl-D-aspartate (NMDA) receptor, one of the receptors targeted by glutamate. The related NMDA antagonist memantine has been evaluated in a randomized, double-blind study of patients diagnosed with bipolar depression, where it was applied to augment treatment with the presynaptic glutamate release inhibitor lamotrigine, and found to accelerate treatment response [45]. Another NMDA receptor antagonist, ketamine, also has antidepressant effects [46], which appear to be mediated by changes in mTOR signaling [47]. However, the literature on BDV infection and depression remains controversial, with several studies failing to replicate any association between the two [48-51].

My third argument is that reconceptualizing major depression as being causally linked to parasites, bacteria, or viruses is useful when thinking about the genetics of this illness. Evidence from twin studies notwithstanding, the search for specific genes linked to major depression has come up empty [52,53]. Perhaps, we have been looking at the wrong organism. Genetic studies to date have focused the search on human genes within our genome. Yet, 8% of the human genome is based on exogenous sequences from retroviruses [54]. These retroviral insertions may sometimes benefit the human host and therefore be protected from mutational degeneration [55]. Indeed, the BDV discussed earlier inserted some of its sequences into vertebrate genomes approximately 40 million years ago [56], and presence of these sequences correlates with disease resistance to BDV. Parasites could also add exogenous sequences to the human genome through the process of horizontal gene transfer [57]. It is possible that polymorphisms within such exogenous sequences, or interactions between these exogenous sequences and other variables such as human gene polymorphisms or stressful life experiences, could render some individuals vulnerable to major depression.

Furthermore, if we view the human body as an ecosystem that is a host to numerous microorganisms which may be passed across generations, the opportunity for genetic discoveries is vastly amplified. For example, an estimated thousand species of bacteria reside in the human gastrointestinal


tract [58], and these could be passed during birth or through common environmental exposure between parents and offspring [59]. Humans also carry vast numbers of viruses, which can be unknown and go undetected until subjected to a concerted search using new approaches such as deep sequencing [60].

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Conclusions In light of the above considerations, an important point of reflection concerns the relation between the immune response and MDD and the specificity of any putative mechanism. The literature implicating the immune system in MDD [61] can be read as suggesting that the immune response itself is the causal mechanism in depression. Indeed, conditions such as hypoxia known to produce sterile inflammation ([62], i.e., activation of the immune system sans a pathogen) may increase the risk of depression [61] in conditions such as obstructive sleep apnea [63] or chronic obstructive pulmonary disease [64]. Yet, most cases of MDD are not attributable to sterile inflammation. Thus, I suggest that some unknown pathogen(s) could play a causal role, and that the immune response is secondary to the infection; interventions that only target the immune response may bring symptom relief but would not address the root cause of the illness.

If a pathogen played a causal role in MDD, the next question would concern the specificity of the mechanism. One perspective would favor a very general, non-specific mechanism. For example, chronic fatigue syndrome (CFS)—which is characterized by sickness behavior that may include depressive symptoms—has been hypothesized to be caused by vagus nerve infection, regardless of the type of pathogen [65]. My view is that, for MDD, the type of pathogen may matter a great deal, and that it plays a very specific causal role: the examples I presented above suggest plausible mechanisms by which pathogens may alter neurotransmission. However, there may not be a single pathogen that causes all cases of MDD. Instead, there may be a class of pathogens, similar to those discussed above, which share common modes of action. This class of pathogens would specifically target the nervous system in a manner that causally contributes to MDD. I use the term ‘contribute’ to indicate that these pathogens may act in concert with other variables. For example, an individual may carry a latent infection and be asymptomatic for depressive symptoms. This individual would be characterized by susceptibility to


MDD which may only emerge after the pathogen was activated by other factors such as stressful life events; this activation could then also trigger a concomitant immune response. It is possible that such a pathogen-driven mechanism is not limited to MDD but may contribute to other psychopathologies. For example, posttraumatic stress disorder could be one such extension of the same mechanism: not every individual develops the disorder in response to a traumatic experience (suggesting individual differences in susceptibility), and the illness is accompanied by immune system activation [66,67].

In closing, I think it would be worthwhile to conduct large-scale studies of carefully characterized depressed patients and healthy controls, using gold-standard clinical and infectious disease-related study protocols, as have already been developed for bacteria [68,69] and viruses [70-76]. Such efforts, if successful, would represent the ‘end of the beginning’ , as any such discovery would represent the first step toward developing a vaccination for major depression.

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Abbreviations BA: Brodmann Area; BDV: Borna disease virus; GABA: gamma-aminobutyric acid; IFN-γ: interferon gamma; IgA: immunoglobulin A; IgM: immunoglobulin M; IL: interleukin; L-DOPA: L -3,4-dihydroxyphenylalanine; LPS: lipopolysaccharides; MDD: major depressive disorder; NMDA: N-methyl-D-aspartate; TNFα: tumor necrosis factor alpha; tmTNFα: transmembrane tumor necrosis factor alpha.

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Competing interests

The author declares that he has no competing interests.

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Healing Lyme with Antibiotics 2016 ORIGINAL RESEARCH ARTICLE

Front. Microbiol., 10 February 2016 | http://dx.doi.org/10.3389/fmicb.2016.00062

Eradication of Biofilm-Like Microcolony Structures of Borrelia burgdorferi by Daunomycin and Daptomycin but not Mitomycin C in Combination with Doxycycline and Cefuroxime

Jie Feng, Megan Weitner, Wanliang Shi, Shuo Zhang and Ying Zhang*

• Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA

Lyme disease, caused by Borrelia burgdorferi, is the most common vector-borne disease in the United States and Europe. While the majority of Lyme disease patients can resolve their symptoms if


treated promptly, 10–20% of patients suffer from prolonged symptoms called post-treatment Lyme disease syndrome (PTLDS). Although the cause for PTLDS is unclear, one possibility is the presence of bacterial persisters not effectively cleared by the current Lyme antibiotics. Recent studies identified several drug candidates including daptomycin, daunomycin, doxorubicin, and mitomycin C that had good activity against B. burgdorferi persisters. However, their relative activities against B. burgdorferi persisters have not been evaluated under the same conditions. In this study, we tested the anti-persister activities of these drugs against both 7-day and 15-day old stationary phase cultures of B. burgdorferi individually as well as in combination with Lyme antibiotics doxycycline and cefuroxime (Ceftin). Our findings demonstrate daunomycin and daptomycin were more active than mitomycin C in single drug comparison at 10 and 20 µM, as well as in drug combinations with doxycycline and cefuroxime. In addition, daunomycin was more active than doxorubicin which correlated with their ability to stain and accumulate in B. burgdorferi. The two drug combination of doxycycline and cefuroxime was unable to eradicate biofilm-like microcolonies of B. burgdorferi persisters. However, the addition of either daunomycin or daptomycin to the doxycycline + cefuroxime combination completely eradicated the biofilm-like structures and produced no visible bacterial regrowth after 7 and 21 days, while the addition of doxorubicin was unable to prevent regrowth at either 7 or 21 day subculture. Mitomycin C in combination with doxycycline and cefuroxime caused no regrowth at 7 days but visible spirochetal regrowth occurred after 21 day subculture. Furthermore, we found that cefuroxime (Ceftin), the third commonly used and most active antibiotic to treat Lyme disease, could replace cefoperazone (a drug no longer available in the US) in the daptomycin + doxycycline combination with complete eradication of the biofilm-like structures as shown by lack of any regrowth in subcultures. Our findings may have implications for improved treatment of Lyme disease.

Introduction Borrelia burgdorferi is the causative agent of Lyme disease, which is the most common vector-borne disease in the United States with an estimated 300,000 cases in 2013(CDC, 2015a). The infection is transmitted to


humans by tick vectors that feed upon rodents, reptiles, birds and deer (Radolf et al., 2012). In the early stage of Lyme disease, approximately 50% of patients have localized erythema migrans, a target-shaped rash that expands as the bacteria disseminate from the cutaneous infection site (CDC, 2015a). Late stage Lyme disease is a multi-system disorder with symptoms including arthritis, carditis, and neurologic impairment (CDC, 2015a). The majority of Lyme disease patients can resolve their symptoms if treated promptly with doxycycline, amoxicillin, or cefuroxime (Wormser et al., 2006). However, at least 10–20% of Lyme disease patients experience prolonged symptoms such as neurologic impairment, muscular pain, and fatigue 6 months after antibiotic treatment, a collection of symptoms called Post-Treatment Lyme Disease Syndrome (PTLDS;CDC, 2015b).

The cause of PTLDS is unknown, though there are several theories including co-infections (Swanson et al., 2006), autoimmune response (Steere et al., 2001), immune response to continued presence of antigenic debris (Bockenstedt et al., 2012), as well as B. burgdorferi persisters that are not killed by the current antibiotics (Hodzic et al., 2008, 2014; Embers et al., 2012). Using a combination of diagnostic techniques including xenodiagnosis and PCR, studies have found evidence of B. burgdorferi persistence in dogs (Straubinger et al., 1997), mice (Hodzic et al., 2008, 2014), monkeys (Embers et al., 2012), and humans (Marques et al., 2014) after antibiotic treatment, though no viable bacteria could be cultured.

Borrelia burgdorferi develops persisters stochastically in stationary phase which are tolerant to the antibiotics used to treat Lyme disease (Feng et al., 2014a,2015a; Caskey and Embers, 2015; Sharma et al., 2015). These persister bacteria have been found to have an altered RNA profile, making them phenotypically drug tolerant (Feng et al., 2015c). In log phase cultures (3–5 days old), B. burgdorferi is primarily in motile spirochetal form which is highly susceptible to current Lyme antibiotics doxycycline and amoxicillin, however, in stationary phase cultures (7–15 days old), increased numbers of atypical forms such as round bodies and aggregated


biofilm-like microcolonies develop (Feng et al., 2014a, 2015a). These atypical forms have been shown to have increased tolerance to doxycycline and amoxicillin when compared to the growing spirochetal forms (Feng et al., 2014a, 2015a; Caskey and Embers, 2015; Sharma et al., 2015). Therefore, stationary phase cultures (7–15 days old) which are enriched in persisters were used as a model for high-throughput drug screens against persister populations (Feng et al., 2014a, 2015a,b,d).

Drugs with high activity against the B. burgdorferi stationary phase persisters were identified through screens of FDA approved drug library and NCI compound libraries (Feng et al., 2014a, 2015b,d). Among them, daptomycin, a lipopeptide antibiotic targeting bacterial cell membranes, was found from the FDA drug library to have the highest anti-persister activity against B. burgdorferi (Feng et al., 2014a). In addition, anticancer anthracycline antibiotics, such as daunomycin and doxorubicin, and also mitomycin C were found from the NCI compound library screen as having excellent or good activity against B. burgdorferi persisters (Feng et al., 2015b). Daunomycin, doxorubicin and mitomycin C were all isolated from Streptomyces and are used in the treatment of a wide range of cancers. Daunomycin and doxorubicin belong to anthracycline anti-cancer antibiotic and kill the bacteria by inhibiting DNA and RNA synthesis, causing DNA damage and producing reactive oxygen species. Mitomycin C blocks DNA replication and causes cell death by DNA crosslinking. Although the anti-persister drugs such as daptomycin are more active than the current Lyme antibiotics such as doxycycline or amoxicillin against B. burgdorferi persisters (Feng et al., 2014a), they alone could not completely eradicate the more resistant biofilm-like microcolonies and a drug combination approach is required to do so (Feng et al., 2015a). The more effective drug combination approach to eradicate biofilm-like microcolonies is consistent with the drug combination principle for treatment of persistent infections like tuberculosis (Zhang et al., 2012; Zhang, 2014). In a recent study using a relatively young 5 days old culture, mitomycin C was found to have higher activity than daptomycin against B.


burgdorferi persisters (Sharma et al., 2015). However, their relative activity against B. burgdorferi persisters has not been compared or evaluated in the same study under the same conditions. In this study, four of the identified drugs with the highest activity against stationary phase B. burgdorferi persisters were tested to determine their anti-persister activity at more clinically achievable levels. In addition, we assessed these persister active drugs in combination with the commonly prescribed Lyme antibiotics doxycycline and cefuroxime, which have high activity against growing log phase cultures, with the aim to increase the activity of these drugs for more effective treatment of Lyme disease.

Materials and Methods Strain, Media, and Culture Techniques Borrelia burgdorferi strain B31 (ATCC 35210) was obtained from American Type Tissue Collections (Manassas, VA, USA). B. burgdorferi was grown in BSK-H medium (HiMedia Laboratories, Mumbai, India) and supplemented with 6% rabbit serum (Sigma Aldrich, St. Louis, MO, USA). The medium was filter-sterilized via passage through a 0.22 µM filter. The inoculated medium was incubated in sterile 50 mL conical tubes (BD Biosciences, San Jose, CA, USA) in a 33°C incubator without shaking. The culture was maintained in these conditions for 7 or 15 days until the culture reached stationary phase, when it was transferred to a 96 well plate for evaluation with the drugs or their combinations.

Drugs The following drugs were obtained from Sigma–Aldrich, St. Louis, MO, USA and dissolved in the solvents suggested by the Clinical and Laboratory Standards Institute to make a stock solution: doxycycline (Dox), cefuroxime (CefU), cefoperazone (CefP), daptomycin (Dap), mitomycin C (MitC), doxorubicin (DoxR), daunomycin (Dau), (Clinical and Laboratory Standards Institute, 2007). The drug stock solutions were filter-sterilized using a 0.22 µM filter and stored at –20°C.

Microscopy


The B. burgdorferi cultures were examined using a Zeiss AxioImager M2 microscope with differential interference contrast and epifluorescent illumination. Pictures were taken using a SPOT slider camera. A SYBR Green I/PI assay was performed as previously described to assess cell viability using the ratio of green:red fluorescence to determine the live:dead cell ratio, respectively, as measured by a plate reader (Feng et al., 2014b). This residual cell viability reading was confirmed by analyzing three representative images of the bacterial culture using epifluorescence microscopy. Image Pro-Plus software was used to quantitatively determine the fluorescence intensity.

Evaluation of Drugs Against Biofilm-Like Structures inB. burgdorferi Stationary Phase Cultures For single drug evaluation, an aliquot of the drug stock solution was added to each 96 well plate containing 100 µL of 7-day old stationary phase B. burgdorferi culture to obtain the desired drug concentration. The plate was then sealed and was incubated at 33°C without shaking for 7 days. After incubation, the viability of the residual viable cells was assessed using the SYBR Green I/PI viability assay and confirmed using epifluorescence microscopy (Feng et al., 2014b). Each sample was analyzed in triplicate and the mean residual viable cells remaining were calculated.

For assessing the activity of anthracycline compounds and daptomycin and mitomycin C in combination with current Lyme antibiotics against biofilm-like structures, a 15-day old B. burgdorferi stationary phase culture was used. Aliquots of the drugs were added to 96 well plate containing 100 µL of the 15-day old stationary phase B. burgdorferi culture which was enriched in aggregated biofilm-like structures to create a final drug concentration of 10 µg/mL for each drug. This drug concentration was chosen as most drugs evaluated in this study fell within or close to their Cmax values (maximum serum concentration; Table 1). The plate was then sealed and was incubated at 33°C without shaking for 7 days, when the residual viable cells remaining were measured using the SYBR Green I/PI viability assay and confirmed using epifluorescence microscopy as described (Feng et al., 2014b).


TABLE 1

TABLE 1. Relative activity of daunomycin, daptomycin, doxorubicin, and mitomycin C on a 7-day old B. burgdorferistationary phase culturea.

Subculture Study to Assess the Effect of Drug Combination on the Biofilm-Like Structures in B. burgdorferi Stationary Phase Cultures A 15-day old B. burgdorferi culture (500 µL of 1 × 107 spirochetes/mL) was exposed to the indicated drug combinations in Eppendorf tubes, and incubated at 33°C for 7 days without shaking. After incubation, the bacteria were spun down and washed with 1 mL fresh BSK-H medium. The cultures were resuspended in 500 µL BSK-H medium, and a 50 µL aliquot was used to inoculate a new tube of 1 mL fresh BSK-H medium for subculture. The cultures were allowed to grow for either 7 or 21 days, when they were evaluated for regrowth with viable cells using the previously described SYBR Green I/PI assay and epifluorescence microscopy (Feng et al., 2015a).

Results and Discussion Comparison of the Relative Anti-Persister Activity of Daunomycin, Doxorubicin, Daptomycin, and Mitomycin C in Single Drug Exposure Against Stationary Phase B. burgdorferi Culture Although daptomycin (Feng et al., 2014a), daunomycin (Feng et al., 2015b), doxorubicin (Feng et al., 2015b), and mitomycin C (Feng et al., 2015b; Sharma et al., 2015) were identified to have high activity against B. burgdorferipersisters, their relative activities have not been compared under the same conditions. To do so, we compared them for their activity against the same 7-day old B. burgdorferi stationary phase culture at the same concentrations (5, 10, and 20 µM), using SYBR Green I/PI viability


stain followed by epifluorescence microscopy. The anthracycline drug daunomycin was shown to have the highest activity against the stationary phase cultures even at the lowest concentration (5 µM) with a dose-dependent increase in killing activity resulting in a near total clearance of bacteria at the highest concentration (20 µM) as shown by mostly red (dead) cells and dispersed, smaller aggregated microcolony size, revealed by the SYBR Green I/PI viability assay (Figure 1, Table 1). Daptomycin was the second most active drug against the B. burgdorferi stationary phase culture, followed by doxorubicin (Figure 1, Table 1). Mitomycin C was the least active drug among the four persister-active drugs, and even at 20 µM had poor activity against the aggregated biofilm-like microcolony form of B. burgdorferipersisters, as shown by mostly green (live) microcolonies remaining after the drug treatment for 7 days (Figure 1).

FIGURE 1

FIGURE 1. Comparison of anti-persister activity of daunomycin, doxorubicin, daptomycin, and mitomycin C. A 7-day old B. burgdorferistationary phase culture containing aggregated microcolonies was incubated for 7 days with daptomycin (Dap), daunomycin (Dau), doxorubicin (DoxR), or mitomycin C (MitC) at the same drug concentrations of 5, 10, or 20 µM, respectively, followed by viability assessment using the SYBR Green I/PI assay. Representative images were taken using epifluorescence microscopy at 400× magnification. Green cells indicate live cells while red cells indicate dead cells.


Doxorubicin was less active than daunomycin as shown by higher percentage of viable cells remaining after drug exposure (Table 1) despite their both belonging to the same anthracycline class. These results could be explained by structural differences between those compounds (Figure 2A). Doxorubicin possesses a hydroxyl group as opposed to a methyl group in the corresponding position of daunomycin, with the remainder of the anthracycline structure being identical. Interestingly, although doxorubicin and daunomycin both have orange-red color in solution (Figure 2B), we found daunomycin visibly stained the B. burgdorferi cells red as seen in the red cell pellet while doxorubicin only stained the cells rather faintly (Figure 2C). This finding suggests that daunomycin may cross the B. burgdorferi cell membrane more efficiently to accumulate in the cell while doxorubicin may have poor ability to enter or accumulate in B. burgdorfericells.

FIGURE 2

FIGURE 2. Differences in structures of daunomycin and doxorubicin and their ability to accumulate in B. burgdorferi. (A)Chemical structures of daunomycin and doxorubicin. Red box shows the difference between the structures of daunomycin (methyl group) and doxorubicin (hydroxyl group). (B) Daunomycin and doxorubicin show the same orange–red color at 10 mM solution. (C) Cell pellets of 7-day old B. burgdorferi treated with 10 µM daunomycin (left-side tube) 10 µM doxorubicin (right-side tube) for 7 days, where daunomycin stained B. burgdorferi red while doxorubicin hardly stained the organism.

Comparison of the Relative Anti-Persister Activity of Daunomycin, Daptomycin, Doxorubicin, and


Mitomycin C in Drug Combinations Using SYBR Green I/PI Viability Assay Both two-drug combinations doxycycline + cefuroxime and doxycycline + cefoperazone showed poor activity against the 15-day old stationary phase culture, with 67% residual viable (green) cells remaining in comparison to 79% viable cells in the drug-free control (Figure 3, Table 2). Consistent with the single drug exposure experiment (Figure 1), daunomycin, doxorubicin and daptomycin when added to the drug combination doxycycline + cefuroxime had a high anti-persister activity as seen by 12, 18, and 30% viable cells remaining (Table 2) as well as mostly red (dead) cells after treatment (Figure 3). In contrast, when mitomycin C was added to the drug combination doxycycline + cefuroxime, the anti-persister activity of these compounds was moderately increased as shown by 45% residual viable cells remaining (Table 2), but more green (live) cells were seen with the mitomycin C drug combination than with the daunomycin or daptomycin drug combination (Figure 3).

FIGURE 3

FIGURE 3. Comparison of the activity of daunomycin, daptomycin, and mitomycin C in combination with currently used Lyme antibiotics. A 15-day old B. burgdorferi stationary phase culture was incubated with the indicated drug combinations at a final concentration of 10 µg/mL for each antibiotic for 7 days followed by SYBR Green I/PI stain and epifluorescence microscopy. Abbreviations: Dox, doxycycline; CefU, cefuroxime; CefP, cefoperazone; Dap, daptomycin; MitC, mitomycin C; DoxR, doxorubicin; Dau, daunomycin.

TABLE 2


TABLE 2. Viability of stationary phase B. burgdorferi after antibiotic treatmenta assessed by direct SYBR Green I/PI viability assay and subculture.

Subculture Study to Assess the Relative Anti-Persister Activity of Daunomycin, Daptomycin, and Mitomycin C in Drug Combinations To validate the activity of these drug combinations, samples of the above drug-treated cultures were subjected to subculture after removal of the drugs by washing followed by incubation in fresh BSK medium for 7 or 21 days. A lack of visible regrowth when measured by microscopy suggests that few to no viable cells remain after drug treatment, while visible regrowth of the culture indicates the presence of viable cells after drug treatment. The addition of daunomycin or daptomycin to the doxycycline + cefuroxime drug combination showed no visible regrowth after 7 and 21 days, suggesting no viable B. burgdorferiorganisms were left after the treatment (Figure 4). Despite the high anti-persister activity of doxorubicin + doxycycline + cefuroxime in the microscopic analysis (Figure 3), with only 18% residual viable cells after treatment (Table 2), this triple drug combination was unable to prevent bacterial regrowth at either 7 or 21 days subculture, indicating it is not as active as daunomycin or daptomycin (Table 2, Figure 4). However, doxorubicin + doxycycline + cefuroxime was more active than mitomycin C + doxycycline + cefuroxime as shown by less regrowth than the latter combination (Figure 4). This is consistent with the single drug data where doxorubicin was more active than mitomycin C (Figure 1).

FIGURE 4


FIGURE 4. Subculture (21 days) of 15-day old B. burgdorferi stationary phase culture treated with different drug combinations.The 15-day old B. burgdorferi culture was incubated with the indicated drug combinations at a final concentration of 10 g/mL for each antibiotic for 7 days followed by washing and resuspension of cells in fresh BSK medium and subcultured for 21 days. The viability of the subculture was examined by SYBR Green I/PI viability assay and epifluorescence microscopy (400 magnification). NG, no growth.

The discrepancy in the activity of doxorubicin in epifluorescence microscopy based viability analysis and subculture study was noted in our previous studies (Feng et al., 2015a,b). This is due to the red orange color of the anthracycline drug doxorubicin, which stains the cells red and could give false impression of a high killing activity. However, subculture studies were able to show the inability of doxorubicin + doxycycline + cefuroxime to eradicate the microcolony form ofB. burgdorferi persisters as shown by regrowth after subculture. Thus, the subculture study is crucial in validating the results of other forms of viability assays such as SYBR Green I/PI assay in persister drug evaluations.

When mitomycin C was added to doxycycline + cefuroxime combination treatedB. burgdorferi stationary phase culture, there was no regrowth at 7 days, but visible spirochetal regrowth occurred after 21 days subculture (Figure 4). This finding suggests that the addition of mitomycin C to the commonly used Lyme antibiotics doxycycline + cefuroxime is not as active as the addition of daunomycin or daptomycin, but is more active than doxorubicin. Furthermore, this result indicates that 7 days subculture is not sufficient to reveal the small number of residual bacteria remaining after drug treatment and that a prolonged incubation to 21 days is needed to demonstrate the small numbers of viable bacteria for more reliable evaluation of drug combinations against B. burgdorferi persisters.


In a recent study, mitomycin C was shown to be more active than daptomycin and to eradicate all B. burgdorferi persisters (Sharma et al., 2015). This is in contrast to the results of this study which found daptomycin to have higher anti-persister activity than mitomycin C in both single drug exposure (Table 1, Figure1) and drug combination studies (Figures 3 and 4). Several possibilities exist to explain the discrepancy. First, we used older 7 and 15 days stationary phase cultures containing an increased number of persisters and biofilm-like microcolonies previously shown to have increased tolerance to antibiotics (Feng et al., 2015a), while the other study used a younger culture of 5 days (Sharma et al., 2015), which would have more growing cells and fewer persister cells. The difference in persister numbers in these cultures would result in the bacteria in the younger culture of 5 days being more easily killed by mitomycin C but not by daptomycin. Indeed, daptomycin is known to have relatively high MIC (12.5–25 mg/mL) for growing spirochetes despite its high activity against B. burgdorferipersisters (Feng et al., 2014a), and this may also explain why daptomycin had limited activity in that study as a younger culture was used (Sharma et al., 2015). The use of a younger culture in the other study that contained mainly growing spirochetes is also consistent with their finding that the 5-day old culture was readily killed by even amoxicillin and ceftriaxone (Sharma et al., 2015), which are known to kill mainly growing bacteria. Second, we used different viability assays. In this study, we used SYBR Green I/PI viability stain along with microscopy and subculture in liquid medium to assess the viability of residual bacteria after drug treatment. In contrast, the other study used colony forming unit (CFU) assay on solid agar to determine the viable bacteria after drug exposure (Sharma et al., 2015). Based on studies with other bacteria like M. tuberculosis (Dhillon et al., 2004), the CFU assay favors the detection of more viable organisms and is less sensitive than culture in liquid medium which can detect small numbers of viable cells which may not grow on solid medium after drug exposure. Third, we used BSK-H medium, which is richer than the BSK-II medium used by the other study (Sharma et al.,


2015), which might have also contributed to the higher activity of mitomycin C than daptomycin in that study (Sharma et al., 2015).

Cefuroxime (Ceftin) Could Replace Cefoperazone in the Daptomycin + Doxycycline Combination to Completely Eradicate Biofilm-Like Structures In our previous drug combination study, we found that daptomycin + doxycycline + cefoperazone was able to completely eradicate the most resistant aggregated biofilm-like microcolonies (Feng et al., 2015a). However, since cefoperazone is not available in the US, we replaced it with the current Lyme antibiotic cefuroxime (Ceftin) in the daptomycin + doxycycline combination and found they had equivalent activity as shown by the same 30% residual viable cells after antibiotic treatment using SYBR Green I/PI viability stain and microscopy (Figure 3, Table 2). In subculture studies, we found replacement of cefoperazone with cefuroxime (Ceftin) in the daptomycin + doxycycline combination similarly resulted in complete eradication of the biofilm-like structures as shown by lack of any regrowth in 7 and 21 days subcultures (Figure4).

Cefuroxime and cefoperazone, which are second and third generation cephalosporins, respectively, function as a highly penetrative beta-lactam antibiotic by disrupting the bacterial cell wall biosynthesis (Barriere and Flaherty, 1984). Despite being reported as the best beta-lactam antibiotic in the 7-day stationary phase persister model (Feng et al., 2014a), cefoperazone did not give any advantage over the use of the commonly prescribed Lyme antibiotic cefuroxime (Ceftin) in the context of drug combination with daptomycin + doxycycline (Figures 3 and 4). This data suggests that replacing cefoperazone with the commonly used cefuroxime (Ceftin) will maintain comparable efficacy against the biofilm-like microcolony form of B. burgdorferi persisters in the drug combination with daptomycin + doxycycline.

In this study, we were able to confirm our previous observations of the high anti-persister activity of daptomycin (Feng et al., 2014a, 2015a) and daunomycin (Feng et al., 2015b) alone and in drug combination with


doxycycline + cefuroxime.It is worth noting that the high anti-persister activity of daptomycin and the anthracycline antibiotic daunomycin is due to the unique mechanisms of action through disruption of cell membrane and damage of DNA, respectively (Feng et al., 2014a, 2015b). These observations suggest that bacterial membranes and DNA integrity are important targets for bacterial persister drugs. The complete eradication of biofilm-like structures of B. burgdorferi by daunomycin or daptomycin in drug combination with doxycycline + cefuroxime, again supports the Yin–Yang treatment principle of combining drugs that target growing bacteria (Yang; with doxycycline + cefuroxime) and drugs like daunomycin or daptomycin that target non-growing persisters (Yin) for more effective treatment of persistent infections (Zhang, 2014). This strategy may be generally useful for treatment of persistent infections including biofilm infections, which cannot be eradicated by a single drug alone. Future studies are needed to validate this principle.

Although our findings that daunomycin or daptomycin plus doxycycline + cefuroxime could completely eradicate the biofilm-like structures are encouraging, they are in vitro studies and have limitations and cannot be equated to the clinical situation. Moreover, daunomycin and daptomycin are intravenous drugs and not convenient to administer. Future studies to develop oral regimens as effective as the above combinations are needed for more convenient administration. In addition, the toxicity associated with the anticancer drug daunomycin calls for caution with its use in clinical settings. Further in vivo animal studies are needed to validate the highly active drug combinations identified in this study before they can be used for patient treatment in the clinic.

Conclusion In summary, we found that daunomycin and daptomycin were more active against B. burgdorferi biofilm-like structures than mitomycin C and doxorubicin in single drug comparisons as well as in drug combinations. Daunomycin or daptomycin when added to doxycycline + cefuroxime completely eradicated the biofilm-like structures, while the two drug


combination doxycycline + cefuroxime alone or mitomycin C and doxorubicin when added to the above combination failed to do so. Additionally, we showed that cefuroxime (Ceftin) could replace cefoperazone in the daptomycin + doxycycline combination and caused complete eradication of the biofilm-like structures. Future studies are needed to evaluate these promising drug combinations in vivo in animal models, and if promising, in patients. Our findings may have implications for improved treatment of Lyme disease.

Author Contributions YZ conceived the experiments; JF, MW, WS, SZ, performed the experiments; JF, MW, and YZ analyzed the data; and MW, JF, YZ wrote the paper.

Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments We acknowledge the support of our work by Global Lyme Alliance, and Johns Hopkins Fisher Center for Environmental Infectious Diseases. YZ was supported in part by NIH grants AI099512 and AI108535.

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Citation: Feng J, Weitner M, Shi W, Zhang S and Zhang Y (2016) Eradication of Biofilm-Like Microcolony Structures of Borrelia burgdorferi by Daunomycin and Daptomycin but not Mitomycin C in Combination with Doxycycline and Cefuroxime. Front. Microbiol. 7:62. doi: 10.3389/fmicb.2016.00062

Received: 08 December 2015; Accepted: 14 January 2016; Published: 10 February 2016.

Edited by: Octavio Luiz Franco,UniversidadeCatólicadeBrasília,Brazil

Reviewed by: César De La Fuente-Núñez,MassachusettsInstituteofTechnology,USASuzana Meira Ribeiro,UniversidadeCatólicaDomBosco,Brazil

Copyright © 2016 Feng, Weitner, Shi, Zhang and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance


with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Ying Zhang, [email protected]

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