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Home  >  Medical Research Archives  >  Issue 149  > The Presence and Impact of Bacteria and Biofilms in Chronic Skin and Systemic Diseases
Published in the Medical Research Archives
Jul 2023 Issue

The Presence and Impact of Bacteria and Biofilms in Chronic Skin and Systemic Diseases

Published on Jul 29, 2023




This will be an overview of our studies on biofilms and the diseases with which they are associated. Where possible, it will include the microbes that create these biofilms, their locations in the body, and their impact on the innate and adaptive immune systems. It will include all the diseases which we have recently classified as eczema in addition to those previously considered as such. It will also include psoriasis, tinea versicolor, leprosy, tertiary Lyme disease and Alzheimer’s disease, the deposition diseases (arteriosclerosis, gout pretibial myxedema), the necrobiotic granulomas (granuloma annulare, rheumatoid nodules, and necrobiosis lipoidica), molluscum contagiosum and squamous cell carcinoma in situ in pigmented transplant patients. Consequently, many chronic skin and systemic diseases have been shown to be biofilm diseases. This implies that these disorders have a large, often determinative microbiological component. Where known, the mechanism whereby these biofilms interact with the immune system will be discussed.

Author info

Herbert Allen, Rachel Allen, Kiertana Kannan, Lucy Fransko

Stipulations re Biofilms
1.    Biofilms are common in nature; more than 90% of microbes in nature live in biofilms.
2.    All organisms, bacteria, mycobacteria, viruses, fungi and others can and do form biofilms.
3.    Biofilms are made of extracellular polysaccharides that make up the bulk of the biofilm. They also contain amyloid (ihat forms the infrastructure of the biofilm), microbial cells, DNA, and water channels.
4.    10 microbial cells in every direction are necessary to begin biofilm formation.
5.    The size of the biofilm depends on the size of the microbe making it. For example, Malassezia furfur/ovale (3.0) yeasts form a biofilm that is too large to fit into an eccrine duct (30), thus it cannot cause eczema. The ultimate size of the spirochetal biofilm needs to fit inside the neuronal cytoplasm to cause Alzheimer’s disease; also, the streptococcal biofilm needs to fit into the cytoplasm of the ectodermal cells lining the tonsils in psoriasis.
6.    In the tonsil, the extracellular polysaccharides (slime) are outside the microbial cells.
7.    Biofilms have attachment sites for other organisms, so the biofilms may be polymicrobial. The best example of this is dental plaque: streptococcus mutans initiates the biofilm and attaches to the tooth; next porphyromonas gingivalis joins in; and last; T. denticola (representing all dental spirochetes) joins. The biofilm is conceptually 3 layered.
8.    The biofilm has attachment sites for Toll-like receptor 2 molecules of the innate immune system. These ordinarily are involved with gram-positive bacteria, but they attach to gram-negative biofilms as well.
9.    Exporter cells are extruded from the biofilm and can leave and establish a new biofilm elsewhere.
10.    Biofilms are multidrug resistant; they pass drug resistant genes horizontally between cells inside the biofilm.
11.    Many topical and systemic biofilm dispersing agents are available. The best systemic agent studied has been rifampin which “pokes holes” in the biofilm. This must be taken with an antibiotic which will kill the organisms, otherwise the cells in the biofilm become exporter cells and will make new biofilms. Nicotine is an example of this.

Disease- Atopic Dermatitis/Eczema

The first disease to be discussed is eczema; we have written many papers and have given many presentations on this chronic disease.1-5 (Figs. 1-4) Sulzberger found occluded eccrine ducts (Fig 5), in 19477 and, 60 years later, we showed these occlusions were biofilms created by normal flora staphylococci. 6 (Fig. 3) Eczema results from occluded eccrine ducts by normal flora staphylococci that make biofilms because of the salt and water present.2 These occluded ducts then react with the inate immune system molecule toll- like receptor 2 (TRL2), (Fig.4) which produces proteinase-activated receptor 2 (PAR2) to create the itch that rashes which is eczema.8-10 The biofilm/TLR2 also produces TNF that causes spongiosis. Spongiosis is the hallmark pathological feature of eczema.1

We have shown how this is a double-hit phenomenon disease: one hit is the genetic component filaggrin (or another gene) and the second hit is the environmental component comprised of staphylococcal biofilms that occlude sweat ducts. The filaggrin and other genes make a faulty SC.1 We have shown how this process carries over to the other form of eczema where the genetic component is altered. Examples of this would include seborrheic dermatitis, where the stratum corneum (SC) is altered by yeasts (primarily Malassezia oleosa) and granular parakeratosis, where the alteration in the SC comes from the disruption caused by the myriad of granules.12 The following table includes all the “eczema” diseases we have studied.

Each of these disorders has shown occlusion of the sweat ducts with normal flora staphylococcal biofilms, and each disease has the characteristic pathology, microbiology, and physiology of eczema.1 Sulzberger also found ductal occlusion in seborrheic dermatitis.7 The initial itch of eczema is caused by TLR2 that activates proteinase-activated receptor 2 (PAR2) which is a pruritogen stronger that histamine. Incidentally, the pathological finding of spongiosis is created by the interaction of the TLR2 with Myeloid Differentiation Primary Response 88 (MYD88) Pathway. Thus, the physiologic and pathological responses in eczema are generated by the innate immune system TLR2.1

Medium strength corticosteroid creams and ointments can be used to treat mild to moderate eczema. For more severe signs and symptoms, high patency corticoid creams and ointments can be employed. With all groups of patients, minimal use of soap and aggressive moisturization must be employed; for example, if soap (in any form) continues to be used excessively, the eczema cannot get better. And, moisturizing by itself cannot affect the overuse of soap. Admittedly, these general measures are aimed at correcting the genetic component of the double-hit phenomenon. This may be the first time in history that clinical measures can aid in correcting the genetic component of a disease. Further, all the foregoing has been aimed at the initiation (and presentation) of the disease; it does not include that which occurs when the epidermis is breached by scratching and the adaptive immune system comes into play in the dermis. Oral antibiotics are a helpful addition when that occurs.1 If all the above is carried out, antimetabolite and biological treatments are seldom necessary. Treatments such as azithromycin, methotrexate, cyclosporine, dupilumab, crisaborole, and others often have toxicities, along with being seldom necessary.

References Atopic dermatitis/eczema
1.    Allen HB. The Etiology of Atopic Dermatitis (2015). DOI: 1-4471-6545-3
2.    Allen HB, Mueller JL. A novel finding in atopic dermatitis: biofilm-producing Staphylococcus epidermidis as an etiology. Int J Dermatol. 2011; 50(8):992- 993.
3.    Haque MS, Hailu T, Pritchett E, Cusack CA, Allen HB. The oldest new finding in atopic dermatitis: subclinical miliaria as an origin. JAMA Dermatol. 2013;149(4):436-438.
4.    Allen HB, Jones NP, Bowen SE. Lichenoid and other clinical presentations of atopic dermatitis in an inner city practice. J Am Acad Dermatol. 2008;58(3):503-504.
5.    Choi C, Hailu T, Cusack CA, Allen HB, Lodha S, Hailu T. The earliest immunologic finding in atopic dermatitis: periductal Toll-like receptor 2 expression in response to ductal occlusion by Staphylococcus epidermidis biofilm. J Am Acad Dermatol. 2012;66:AB71.
6.    Allen HB, Vaze ND, Choi C, Hailu T, Tulbert BH, et al. (2014) The presence and impact of biofilm-producing staphylococci in atopic dermatitis. JAMA Dermatol 150: 260-265.
7.    Sulzberger MB, Herrmann F, Zak FG. Studies of sweating; preliminary report with particular emphasis of a sweat retention syndrome. J Invest Dermatol.1947;9(5):221-242.
8.    Steinhoff M, Corvera CU, Thoma MS, et al. Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase. Exp Dermatol 1999:8(4) 282-94
9.    Lee SE, Jeong SK, Lee SH. Protease and protease-activated receptor-2 signaling in the pathogenesis of atopic dermatitis. Yonsei Med J. 2010 Nov;51(6):808-22. doi: 10.3349/ymj.2010.51.6.808. PMID: 20879045; PMCID: PMC2995962.
10.    Kerstan A, Bricker E-B, Trautmann A. Decisive
role of tumor necrosis factor–α for spongiosis formation in acute eczematous dermatitis. Arch Dermatol Res. 2011;303(9):651-658.

Streptococcal biofilms are present in the tonsils in psoriasis. (Fig. 1,2) They are both “intra”cellular (in the ectodermal lining cells) and extra cellular (between the lymphoid cells). (Fig.2)1 They cause upregulation of both the innate and adaptive arms of the immune system. Streptococcal-specific IgG has been found in the serum (Fig.3) of psoriasis patients;2 this is likely from the ectodermal lining cells that shed into the pharynx and have their intracellular contents exposed to oral enzymes. TLR-2 is present in the dermal capillaries (Fig.4) on immunopathology of psoriatic lesions.3 This is likely from the biofilms amongst the lymphoid cells.

Penicillin treats streptococci, but the organisms in biofilms are resistant. It appears that the serum IgG is more important than TLR2 because treatment with PCN and a biofilm disperser is minimally effective compared to penicillin given after a long course (9-12 months). After such long treatment, more than 90% of treated patients are clear (PASI 100). Saxena gave benzathine PCN every 2 weeks to achieve those results.4,5 Given for 2 years, 80% of those patients were cured! Additional proof of streptococcus was found by McFadden who discovered locations with no streptococcus in their environments had no psoriasis.6
Psoriasis is also a double-hit phenomenon like eczema with the environmental component bring the streptococcal biofilms and the genetic component being one of the PSORS genes.7 Ordinary treatment with topical corticoids plus/ minus vitamin D is moderately effective. UVB which impairs or kills Langerhans cells (antigen presenting cells) is very helpful in psoriasis. The biologics (TNFa, IL12, 23, IL17, IL23, and T Cell inhibitors) are very effective in psoriasis and some require as little as an injection four times yearly after a loading dose.7 We have treated multiple patients with biologics together with daily penicillin simultaneously and after 2 years have been able to discontinue the biologic successfully. Continuing one daily dose of penicillin leads to prolonged remission.8

References psoriasis
1.    Allen HB, Jadeja S, Allawh RM, Goyal K (2018) Psoriasis, chronic tonsillitis, and biofilms: Tonsillar pathologic findings supporting a microbial hypothesis. Ear Nose Throat J 97: 79-82.
2.    El-Rachkidy RG, Hales JM, Freestone PP, Young HS, Griffiths CE, Camp RD. Increased blood levels of IgG reactive with secreted Streptococcus pyogenes proteins in chronic plaque psoriasis. J Invest Dermatol. 2007;127(6):1337-42.
3.    Zhang J, Shaver C, Neidig L, Jones K, Cusack CA, Allen HB. Toll-Like Receptor 2 and Its Relationship with Streptococcus in Psoriasis. Skinmed. 2017;15(1):27-30.
4.    Saxena VN, Dogra J. Long-term use of penicillin for the treatment of chronic plaque psoriasis. Eur J Dermatol. 2005;15(5):359-62.
5.    Saxena VN, Dogra J. Long-term oral azithromycin in chronic plaque psoriasis: a controlled trial. Eur J Dermatol. 2010;20(3):329-33.
6.    McFadden JP, Baker BS, Powles AV, Fry L. Psoriasis and streptococci: the natural selection of psoriasis revisited. Br J Dermatol. 2009;160(5):929-37.
7.    Chang YT, Chou CT, Shiao YM, et al. Psoriasis vulgaris in Chinese individuals is associated with PSORS1C3 and CDSN genes. Br J Dermatol. 2006;155(4):663-9.
8.    Biologics for Psoriasis Treatment. https://www,>biofilms. 12/14/23
9.    Personal observation

Tinea Versicolor
Tinea versicolor (TV) is a yeast disease caused by Malassezia furfur/ovale. (Fig.1) It ordinarily is asymptomatic presenting with skin color change (white, pink, brown, red) and peeling.1,2 We have shown that biofilms made by the yeast are present amongst corneocytes in the stratum corneum where they do not interact with living cells and thus do not generate TLR2.1 (Fig.2) The biofilms are too large to fit in and occlude the eccrine sweat ducts, so they cannot generate the pathology of eczema or miliaria. (See stipulations)
The organisms generate azelaic acid which causes most of the color change. Selenium sulfide not only kills the M. furfur/ovale but it disrupts the biofilms (heavy metals topically are biofilm dispersers).1,2,3 Ketoconazole topically works similarly, and systemically (orally).4 It localizes in the SC, kills the organisms, and disrupts the biofilm. The azoles are biofilm dispersers, in addition to being antifungal.4 TV is an excellent control for the impact of TLR2 in eczema. It is activated by live cells immediately proximal to biofilms; however, in TV, the biofilms are present only in the acellular SC, so they do not generate an innate immune system response.

References Tinea versicolor
1.        Allen HB, Goyal K, Ogrich L, Joshi S (2015) Biofilm formation by Malassezia furfur/ovale as a possible mechanism of pathogenesis in Tinea versicolor. J Clin Exp Dermatol Res 6: 311.
2.    Gupta AK, Lane D, Paquet M. Systematic Review of Systemic Treatments for Tinea Versicolor and Evidence-Based Dosing Regimen Recommendations. J. Cutan. Med. Surg. 18, 79–90 (2014).
3.    Spence-Shishido A, Carr C, Bonner MY, Arbiser J. In vivo gram staining of tinea versicolor.JAMA    Dermatol. doi:10.1001/jamadermatol.2013.2699.
4.    Kaplan JB. Biofilm Dispersal: Mechanisms, Clinical Implications, and Potential Therapeutic Uses. J. Dent. Res. 89, 205–218 (2010)

The mycobacterium M. Leprae, makes biofilms and causes leprosy. The biofilms are initially in the liver, spleen, and kidneys. (Figs,1,2) Only in lepromatous (late stage) leprosy are biofilms found in the skin; they are in globi (histocytes filled with organisms and biofilm).1 (Fig.3) The addition of rifampin to dapsone has changed the incidence of 15 million worldwide 40 years ago to less than one million today. Rifampin pokes holes in the biofilm and allows the dapsone to kill the M. leprae inside.1

The immune reaction to M. leprae follows the bacterial load and disease progression; this has been summarized by Ridley and Jopling.2 Britton has also reviewed the immunologic actions in depth.3,4 Suffice it to say, the dapsone by itself would be (and was) relatively ineffective because of the biofilms made by M. leprae, but with added rifampin, the disease has been and could be controlled.1 There are 5 types ranging from tuberculoid leprosy (TT), borderline tuberculoid (BT), borderline (BB), borderline lepromatous (BL), and lepromatous leprosy (LL). The TT and LL types of leprosy have been compared based on the immune responses the elicit in the body.2 The TT type instigates a vigorous immune response which confines bacterial load to granulomas in the dermis and peripheral nerves. This type of reaction is consistent with the Type 1 reaction in leprosy, which is mostly cellular immunity. The lepromatous types have been associated with the later stage complications, including secondary amyloidosis in the kidney, liver, and spleen (Sanz- Martín, Sharma).5,6 The LL type is consistent with the type II immune reaction, which activates proinflammatory cytokines such as TNF, IL-1, IL-6, and IL-8 (Nery).7

We believe that the LL type can avoid specific cellular immunity due to the presence of biofilms, rather that with another type of evasion.. Mycobacterium leprae have been shown to form biofilms in nature (Chakraborty).8 Biofilm formation in LL type would explain the late manifestations of disease and unusually low specific cellular immunity to M. leprae (Britton and Lockwood). This is also consistent with LL type histopathology, since there are mostly microphages and foam cells present with large groups of leprosy bacilli.

The link between the biofilm formation in leprosy to the secondary amyloidosis is pertinent to understanding risk factors of other biofilm disease. Biofilms interact with the innate immune system through toll-like receptors on the surface of macrophages (mainly TLR2) to activate them.9The activation of the TLR2 receptors will elicit a proinflammatory response from the macrophage through release of cytokines, most notably TNF alpha though the MyD88 pathway.10 As more macrophages come into contact with and are activated by the biofilm, more proinflammatory cytokines are being produces. If the biofilm is constantly stimulating the release of proinflammatory cytokines are being produced. If the biofilm is constantly stimulating the release of proinflammatory cytokines, then there is an increase of TNF alpha in the body. TNF alpha stimulates the release of serum amyloid A (SAA) from the liver. SAA in large amounts can become cleaved and deposited in the body, and certain organs are more commonly affected by secondary amyloidosis like the liver, spleen, and kidney (Simmons).11
The biofilm itself can also produce amyloid, and amyloid fibers protect the integrity of the extracellular matrix and the biofilm itself (Romero).12 Therefore, biofilm and amyloid coexist with one another and diseases that are known to produce amyloid should be researched extensively to determine if there is an infectious cause.

References Leprosy
1.        Allen HB, Moschella SL. The Role of Rifampin in Leprosy: Leprosy Through a New Lens. JAMA Dermatol. 2017;153(3):261–262. doi:10.1001/jamadermatol.2016.5506
2.    Ridley DS, Jopling WH. Classification of leprosy according to immunity: a five-group system, Int J Lepr Other Mycobact Dis, 1966, Vol. 34 (pg. 255-73).
3.    Warwick J. Britton, 3. Immunology of leprosy, Transitions of the Royal Society of Tropical Medicine and Hygiene, Volume 87, Issue 5, 1993, Pages 501-514, ISSN 0035-9203, 9203(93)90066-Y.
4.    Britton WJ, Lockwood DN. Leprosy. Lancet. 20014 Apr 10;363(9416):1209-19. doi:10.1016/S0140-6736(04)15952-7. PMID: 15081655.
5.    Sanz-Martín N, Samillán-Sosa Kdel R, De Miguel J, Martínez-Miguel P. Renal amyloidosis in leprosy, an infrequent cause of nephrotic syndrome in Europe. BMJ Case Rep. 2016 Aug 3;2016:bcr2016216038. doi: 10.1136/bcr-2016-216038. PMID: 27489069; PMCID: PMC4986063.
6.    Sharma S, Sarin R C, Prakash S. Secondary Amyloidosis in Leprosy. Indian J Dermatol Venereol Leprol 1978;44;31-33.
7.    Nery JA, Bernardes Filho F, Quintanilha J, Machado AM, Oliveria Sde S, Sales AM. Understanding the type 1 reactional state for early diagnosis and treatment: a way to avoid disability in leprosy. An Bras Dermatol. 2013 Sep-Oct; 88(5):797-92. doi:10.1590/abd1806-4841.20132004/ PMID: 24173185; PMCID: PMC3718356.
8.    Chakraborty P, Kumar A. The extracellular matrix of mycobacterial biofilms: could we shorten the treatment of mycobacterial infections? Microb Cell. 2019 Jan 18;6(2);105-122 doi: 10.15698/mic2019.02.667 PMIDL 30740456; PMCID: PMC636425
9.    Tukel C, Wilson RP, Nishimori M, Pezeshki M, Chromy BA, Baumier AG. Responses to amyloids of microbial and host origin are mediated through toll-like receptor 2. Cell Host Microbe. 2009;6:45-53.
10.    Allen HB. The Etiology of Atopic Dermatitis (2015). DOI: 1-4471-6545-3
11.    Simons JP, Al-Shawi R, Ellmerich S, Speck I, Aslam S, Hutchinson WL, Mangione PP, Disterer P, Gilbertson JA, Hunt T, Millar DJ, Minogue S, Bodkin K, Pepys MB, Hawkins PN. Pathogenetic mechanisms of amyloid A amyloidosis. Proc Natl Acad Sci U S A. 2013 Oct    1;110(40):16115-20.    doi: 10.1073/pnas.1306621110. Epub 2013 Aug 19. PMID: 23959890; PMCID: PMC3791773
12.    Romero D, Aguilar C, Losick R, Kolter R. Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2230-4. doi: 10.1073/pnas.0910560107. Epub 2010 Jan 13. PMID: 20080671; PMCID: PMC2836674.

Tertiary Lyme Disease and Alzheimers Disease
We have previously written about two different forms of tertiary Lyme disease. (Figs.1,2) Lyme arthritis manifests as Montauk knee (arthritic) and tertiary spirochetosis of the brain (Alzheimer’s disease [ALZ]).1 Our first patient was a premedical student doing a rotation in our department. After hearing a talk about Lyme being a biofilm disease when is becomes chronic, she said, I have Lyme Arthritis and revealed her knee, which was swollen like a balloon. She had been taking doxycycline 200 MG daily for 1 month without any improvement, and in fact was gradually getting worse. After discontinuing the doxycycline, she began a regimen of Amoxicillin 500 MG 3 times daily and Rifampin 300 MG daily. Within one month, she was clear and wanted to know when she could begin running again. (Fig.3) Neither Amoxicillin nor Rifampin by themselves would have cured her, but the combination “busted apart” the biofilm and the amoxicillin was able the to kill the Lyme spirochetes inside the biofilm.

Spirochetes (Lyme and dental) (Fig.4) are found in the postmortem brain; an older woman who died with severe dementia was analyzed.1,2 The pathological changes in ALZ were similar in every respect to syphilitic dementia. She had biofilms in her brain intracellularly in her neurons, and extracellularly in the senile plaques.3 (Fig.5) Research has shown the presence of Lyme spirochetes in 25% of these patients with ALZ.4,5 More recently, it has been shown that the intracellular spirochetes made beta amyloid (Abeta) simultaneously with making a biofilm.6(Fig.5) It has also been shown that the Abeta induces ordinary tau to make hyperphosphorylated tau (p-tau).7 This is likely the most important development in ALZ because p-tau causes neuronal dendrite destruction.(Fig.6)This leads not only to loss of the synaptic connection from one neuron to another, but also to death of the neuron itself. This is ultimately viewed as the atrophy of the brain which is uniformly found.8 (Fig. 7)

A pilot trial with amoxicillin 500 MG TID and rifampin 300 MG QD was done for 6 ALZ disease patients. In all, the disease stopped progressing: and, in 4 patients, there were moments of lucidity. We have proposed taking amoxicillin 500 MG daily for 2 weeks once yearly to prevent the disease.9 Theoretically, this would include patients 65 or older and those younger who have a family history of the disease.

References Tertiary Lyme Disease and Alzheimers Disease
1.    Allen HB, Allawh RM, Gresham K, Donnelly K, Goyal K (2018) Tertiary Lyme Disease. Clin Microbiol 7: 309. doi:10.4172/2327- 5073.1000309
2.    Allen HB (2021) Alzheimer’s Disease: A Chronic Infection. DOI: 10.9734/bpi/mono/978-81- 9479-7-5
3.    Allen HB, Allawh R, Touati A, Katsetos C, Joshi SG. Alzheimer’s disease: The novel finding of intracellular biofilms. J Neuroinfect Dis. 2017;8:247.
4.    MacDonald AB (1986) Borrelia in the brains of patients dying with dementia. JAMA 256, 2195-2196.
5.    Riviere GR, Riviere GH, Smith KS (2002) Molecular and immunological evidence of oral treponemes in the human brain and their association with Alzheimer’s disease. Oral Microbiol Immunol 17, 113-118.
6.    Miklossy J. Bacterial amyloid and DNA are important constituents of senile plaques: Further evidence of the spirochetal and biofilm nature of senile plaques. J Alz Dis. 2016;53:1479-1473.
7.    Iqbal K, Alonso AC, Chen S, Chohan MO, El- Akkad E, Gong C Khatoon S, Liu F, Rahman A, Tanimukai H, Grundke-Iqbal I (2005) Tau pathology in Alzheimer disease and other tauopathies. Biochim Biophys Acta 1739: 198- 210.
8.    Allen HB, Allen,RA (2022) The Historical Development of the Pathological Changes in Alzheimer’s Disease Based on Microbiology, Anatomy, and Physiology.
9.    Allen HB (2021) A Novel Approach to the Treatment and Prevention of Alzheimers Disease Based on the Pathology and Microbiology. J Alz Dis 84(1): 61-67

The Deposition Diseases
Three deposition diseases have occurred in settings where biofilms have been found. First, in arteriosclerotic diseases, they have been found colocalized with cholesterol deposits in intraluminal and intramural plaques. Biofilms also create calcium deposits and attract TLR2.1-6 (Figs.1,2 It is uncertain which is first, the biofilm or the cholesterol, but the fact is they are localized together. This was shown in multiple patients with arteriosclerotic disease (endarterectomy) specimens. The presence of calcium in the plaques makes dental organisms likely inhabitants of the plaques because they consistently make calcium in dental plaque.
Gout is another deposition disease in which uric acid deposits in gouty tophi (Fig.3) Biofilms again co-localize with the uric acid; these biofilms are made by different microbes because they stain pathologically with colloidal iron stain instead of PAS staining as seen with the other microbes.7 Congo red staining is present in all. The biofilms, in gout then, are likely made by gram negative organisms because the staining pattern favor them. They also do not attract TLR2, so they are asymptomatic. Gouty tophi tend to develop clinically at points of trauma, but again it is unknown which comes first, the biofilm or the uric acid.

A third deposition disease is pretibial myxedema.8 The staining pattern of the mucin in this condition is similar to gouty tophi and shows either large pools of hyaluronic acid in the upper dermis or interlacing bacterial between the collagen bundles. (Figs.4,5)The Congo red staining is less pronounced than in arteriosclerotic plaques and gouty tophi.

References Deposition Diseases
1.    Fatourechi V. Pretibial myxedema: pathophysiology and treatment options. Am J Clin Dermatol. 2005;6(5):295-309. doi: 10.2165/00128071-200506050-00003. PMID: 16252929.
2.    Allen HB, Cusack CA, Allen RA (2019) The Presence of Biofilms in Gouty Tophi. Clin Microbial    Res    DOI: org/10.31487/j.CMR.2018.01.007
3.    Purohit MB, Purohit TM, Tandon RK. FNAC of gouty tophi-a case alreport. Indian J Pathol Microbiol. 2006;49:42–3.
4.    McCarty    DJ.    Gout    without hyperuricemia. JAMA. 1994;271:302–3
5.    Sah SP, Rani S, Mahto R. Fine needle aspiration of gouty tophi: a report of two cases. Acta Cytol. 2002;46:784–5.
6.    Allen HB, Boles J, Morales D, Ballal S, Joshi SG (2016) Arteriosclerosis: The Novel Finding of Biofilms and Innate Immune System Activity within the Plaques. J Med Surg Pathol 2016; 1:135.doi: 10.4172/jmsp.1000121
7.    Den Dekker WK, Cheng C, Pasterkamp G, Duckers HJ (2010) Toll like receptor 4 in atherosclerosis and plaque destabilization. Atherosclerosis 209: 314-320.
8.    Ishikawa Y, Satoh M, Itoh T, Minami Y, Takahashi Y, et al. (2008) Local expression of Toll-like receptor 4 at the site of ruptured plaques in patients with acute myocardial infarction. Clin Sci (Lond) 115: 133-140.
9.    Lanter BB, Sauer K, Davies DG (2014) Bacteria present in carotid arterial plaques are found as biofilm deposits which may contribute to enhanced risk of plaque rupture. MBio 5: e01206-01214.
10.    Hall-Stoodley L, Stoodley P (2009) Evolving concepts in biofilm infections. Cell Microbiol 11: 1034-1043.
11.    Darveau RP (2010) Periodontitis: a polymicrobial disruption of host homeostasis. Nat Rev Microbiol 8: 481-490.
12.    Amar S, Engelke M (2015) Periodontal innate immune mechanisms relevant to atherosclerosis. Mol Oral Microbiol 30: 171-185.
13.    Rosenfeld ME, Campbell LA (2011) Pathogens and atherosclerosis: update on the potential contribution of multiple infectious organisms to the pathogenesis of atherosclerosis. Thromb Haemost 106: 858-867.
14.    Slocum C, Kramer C, Genco CA (2016) Immune dysregulation mediated by the oral microbiome: potential link to chronic inflammation and atherosclerosis. J Intern Med 280: 114-128.

Necrobiotic Granulomas
The necrobiotic granulomas all show biofilms in the areas of the necrobiosis. These include granuloma annulare, rheumatoid nodules, and necrobiosis lipoidica.1-3 The pathology specimens of these three disorders stained for PAS weakly, but strongly for colloidal iron. The Congo red stained positively in all the specimens in the areas of necrobiosis. A different microbe, likely a gram-negative bacterium, is creating the biofilm in these three disorders.2 Granuloma annulare (GA) and necrobiosis lipoidica (NL) do not have similar serologic finding as RA nodules but are biofilms none the less. (Figs1-3)
NL is linked to diabetes in 30% of patients, but correcting the diabetes has no impact on the disease. RA nodules are associated with rheumatoid factor and treatment with biologics has represented marked improvement in joint symptoms and disappearance of nodules.2 The patients need to continue taking the biologic because it treats the symptoms and not the cause of the disease. This is like psoriasis where the biologic treats the symptoms with considerate or total clearing, but once stopped, the disease will reappear. Biofilms have been found in other cutaneous diseases such as eczema, and systemic diseases such as leprosy,4,5

References Necrobiotic GranULOMas
1.    Allen HB, Encarnacion IN, Roberts AA (2023) Biofilms in Necrobiotic    Granulomas. J Bio Innov 12(1) DOI: 10.6084/m9.figshare.22312759
2.    Allen HB, Allawh RM, Larijani M, Cusack CA (2019) Biofilms in Granuloma Annulare. Clin Dermatol Dermatitis 2: 1 DOI: 10.6084/m9.figshare.20960617.
3.    Allen HB, Yi Z, Roberts A, Allen RA (2019) Biofilms in Rheumatoid Arthritis Nodules: a Novel Clue Relating to Microbial Origin. Microbial Infect Dis 3(4): 1-2.
4.    Allen HB (2015) The Etiology of Atopic Dermatitis. Springer-Verlag, London.
5.    Allen HB, Moschella SL (2017) The Role of Rifampin in Leprosy: Leprosy Through a New Lens. JAMA Dermatol 153: 261-262.
6.    Jacovides CL, Kreft R, Adeli B, Hozack B, Ehrlich GD, et al. (2012) Successful identification of pathogens by polymerase chain reaction (PCR)-based electron spray ionization time-of-flight mass spectrometry (ESI-TOF-MS) in culture-negative periprosthetic joint infection. J Bone Joint Surg Am 94: 2247- 2254.

Viral Diseases with Biofilms
We have discovered viral biofilms in 2 diseases: one benign (molluscum contagiosum) and one malignant (squamous cell in situ in transplant patients of color).(Figs1,2) These were caused by MC virus and oncogenic human papilloma virus 16, and 18 respectively.1,5 The pathology specimen of MC showed viral effect and intracellular staining with PAS and Congo red in all layers except the basal layer. The SCC specimens were not from sun exposed skin where they are prevalent in non- pigmented patients. These were from the wart-like lesions in non-sun exposed skin in transplant patients of color.2 More than half of these patients had specimens that stained immunopathologically with anti HPV 16, 18 and these same specimens stained positively for PAS.(Fig.3) The Congo red staining was inhibited by transthyretin which does not allow production of amyloid, so the amyloid in biofilm was inhibited.6 This further documents this as a malignant lesion. (Fig.4)
Both showed these biofilms to be intracellular. Being viral, they need to overtake the DNA of the cell to make more viruses. There are likely more viruses that make biofilms; the first that comes to mind are herpes zoster, which has the earmarks of a biofilm disease, and herpes simplex, which may already be present in the biofilms of ALZ made by the Lyme and dental spirochetes. This has already been shown in spirochetal biofilms with C. pneumonia in the middle of the biofilm. Perhaps something akin to that occurs with HSV.

References Viral Diseases
1.    Allen HB, Allawh RM, Ballal S (2017) Virally- Induced, Intracellular Biofilms; Novel Findings in Molluscum Contagiosum. Clin Microbiol 6: 302.
2.    Allen HB, Chung CL, Allawh RM, Larijani M, Cusack CA (2019) Biofilms in Squamous Cell Carcinoma in Situ Clin Dermatol and Dermatitis 2(1):110.
3.    Durkin J, Fine JL, Sam H, Pugliano-Mauro M, Ho J. Imaging of Mohs micrographic surgerysections using full-field optical coherence tomography: a pilot study. Dermatol Surg. 2014 Mar;40(3):266-74. doi: 10.1111/dsu.12419. Epub 2014 Jan 16
4.    Chung CL, Nadhan KS, Shaver CM, et al. Comparison of Posttransplant Dermatologic Diseases    by    Race. JAMA    Dermatology. 2017;153(6):552-558. doi:10.1001/jamadermatol.2017.0045.
5.    Pais-Correia A-M, Sachse M, Guadagnini S, Robbiati V, Lasserre R, Gessain A, Gout O, Alcover A, Thoulouze M-I. (2010) Biofilm-like extracellular viral assemblies mediate HTLV-1 cell-to-cell transmission at virological synapses. Nature Medicine 16 (1): 83-90.
6.    Jain N, Aden J, Nagamatsu K, et al. Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin. PNAS 2017; 114(46): 12184-12189
7.    Ding H, Liu J, XUE R, et al. Transthyretin as a potential biomarker for the differential diagnosis between lung cancer and lung infection. Biomedical Reports. 2014;2(5):765- 769. doi:10.3892/br.2014.313.

Bioethical Challenges of Biofilm Diseases
We have written previously about the bioethical challenges of psoriasis, Lyme disease, and Alzheimer’s disease.1-3 Each disease has a different microbe, and each has significant challenges. Psoriasis requires a long treatment time with penicillin for treatment and cure. Further, the treatment with biologics and penicillin simultaneously (once the causative microbe is known) is very effective at treating the symptoms and signs of a particular disease.
Lyme disease, when biofilms are present, requires treatment with penicillin (or azithromycin for those allergic to penicillin) plus rifampin which pokes holes in the biofilm. It must be noted that the necessary dose of doxycycline for initial and secondary or tertiary Lyme disease is double the ordinary dose and 400 MG daily is incompatible with gastrointestinal health in nearly all patients.

Alzheimers disease has no current effective treatment; we have recently proposed preventative treatment of penicillin for a 2-week course once yearly which logically would prevent the ALZ from developing and a clinical trial in MCI to halt the disease. This preventive approach corresponds to the treatment for primary and secondary syphilis. ALZ appears identical to (tertiary) syphilitic dementia pathologically. 200 completed clinical trials have not produced any effective treatment (mostly because they have been focused on the faulty beta amyloid theory). From the pathology, this is clearly a spirochetal/biofilm disease.
Eczema is a disease in which minimizing bathing, limiting the use of soap, and aggressively moisturizing are all helpful in the treatment of the disease. Biologics seem rarely necessary in that situation; plus, they do not have the same efficiency in eczema as in psoriasis. As more is learned about the viral diseases, the deposition diseases, and the necrobiotic granuloma diseases, their treatments may be radically changed because of the microbe/ biofilm nature of the disorders.

1.    Jariwala N, Ilyas E, Allen HB (2016) Lyme Disease: A Bioethical Morass. J Clin Res Bioeth 7: 1000288. doi: 10.4172/2155- 9627.1000288
2.    Allen HB, Allawh R, Ogrich L, Jariwala N, Ilyas E (2017) Bioethics and Psoriasis. J Clin Res Bioeth 8: 304. doi: 10.4172/2155- 9627.1000304
3.    Allen HB, Allawh R, Ilyas E, Joshi SG. (2018) Bioethical Challenges Arising from the Microbiology and Pathology of Alzheimers Disease.    Current Neurobiology DOI: 10.6084/m9.figshare.12071958

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