Intracellular microbes in opthalmic disease: a reappraisal

Abstract

In 2010, Washington University ophthalmologist Van Gelder concluded it is “highly doubtful” that all the major pathogens associated with ocular diseases have been identified.¹⁾ As we will show, the advent of metagenomic tools may pave the way for reconsidering earlier studies of difficult-to-culture microbes. Such tools have shown that, like the brain, the eyes are not nearly as sterile or “immunologically privileged” as once supposed.

Over the past decades, high throughput sequencing tools have revolutionized the way we search for, classify, and characterize microbes. Also, they have changed our understanding of microbial persistence in the human body, with unexpected or novel microbes recurrently showing up in these studies. Microbes are now known to inhabit tissues and blood, and hundreds of species are known to exist in tissues previously believed to be sterile.

Between 1970 and through 2004 and as described in dozens of papers, Columbia University-based opthalmologist Emil Wirostko and collaborators found a number of microbes in the eyes of patients with inflammatory diseases. While some of these findings were duplicated,²⁾ they were still largely ignored, if not dismissed. This publication looks at the reasons why Wirostko's work was rejected at the time, and explains why, when viewed in the context of the human microbiomeThe bacterial community in the human body. Many species in the microbiota contribute to the development of chronic disease., this body of work should be reexamined.

Wirostko used a transmissionAn incident in which an infectious disease is transmitted. electron microscope to document, within the immune cells, a range of intracellular forms, most of which lacked cell walls. The group called these forms “mycoplasma-like organisms” (MLOs). Such cells were collected from the vitreous and orbital tissues of the eyes, and occurred in a variety of chronic inflammatory conditions: uveitis, vitritis, ocular disease in juvenile rheumatoid arthritis, Crohn’s disease, and sarcoidosis.

For example, in 1989, Wirostko, Johnson, and Wirostko published a series of images of parasitized monocyctes collected from a patient with sarcoidosis associated uveitis.³⁾ In another study, cell wall deficient microbes were observed in 2 to 10% of leukocytes invading the aqueous humor.(cite?)

Many of the infected immune cells appeared to have dozens, if not hundreds, of microbe-like tubulo-spherical inclusions. MLOs varied in diameter between 0.005 and 1.0 microns and filaments between 0.005 and 0.01 microns.⁴⁾ In some monocytes, the cells were almost completely replaced by MLOs.⁵⁾ The long entities, in particular, fail to fit with anything known to be a structure in the cell.(is this true?)

The bodies found by Wirostko are structurally very similar to MLOs found in plants.⁶⁾ In both humans and plants, these MLOs replace the cytoplasm and destroy the organelles. They also produce distinct nuclear alterations, which we will discuss later. MLO may destroy the host cell, or they may continue to exist within a dysfunctional or proliferating cell.

In addition to their ultrastructural studies of MLOs, Wirostko's team also showed in several studies that these microbes can be passed to laboratory animals to induce similar symptoms and indistinguishable ultrastructural features compared to human MLOs.⁷⁾ Inoculation into mouse eyelids produced intraocular, orbital, and lethal systemic chronic progressive inflammatory disease at all disease sites. Such mice exhibited “sarcoid-like” granuloma.

Wirostko's provocative research into the presence and behavior of fastidious microbes failed to receive widespread interest perhaps because it contradicted several long-held assumptions about microbes and disease, particularly Koch's postulatesCentury-old criteria designed to establish a causal relationship between a causative microbe and a disease. Koch's belief that only one pathogen causes one disease has now been called into question as multiple postulates are increasingly considered out of date.. First, despite decades of effort, no culture system exists for these organisms. Second, Wirostko was never able to show that these microbes acted independently to cause disease as has been the case with certain other pathogens. Finally, the MLOs were found in areas of the body believed to be free of microbes, even in disease.

A cell is considered dead if it fails to give rise to a colony on any medium.

Jawetz, Melnick, & Adelberg's Medical Microbiology, 2007

The notion that microbes always grow in culture undeniably served microbiology in its formative stages as researchers like Koch (Mycobacterium tuberculosis), Fehleisen and Pasteur (Streptococcus), Schaudinn and Hoffmann (Treponema pallidum), and Hayflick (Mycoplasma pneumoniae) succeeded in culturing a range of microbes. However, the field may have been a victim of its early successes.

Wirostko's electron micrographs clearly show colonies of small cell wall-less bacteria resembling mycoplasma, but these microbes could not be cultured. At a 1972 London symposium on mycoplasma, the luminary Dr. Leonard Hayflick detailed how it took nearly a year of effort before he could isolate and cultivate Mycoplasma pneumoniae.⁸⁾ According to him, only by fulfilling Koch's postulates could he conclude that what we now know as primary atypical pneumonia is definitely caused by M. pneumoniae. For Hayflick, this experience offered an invaluable lesson as it illustrated that sufficiently diligent researchers can culture any bacteria which causes a given illness. Hayflick went on to chastise his “neophyte” colleagues who made suggestions to the contrary: “I am, therefore, earnest in my plea that the laws governing scientific proof [i.e. Koch's postulates], based on evidence under controlled conditions, be applied equally and particularly to the class Mollicutes.” Eighteen years later, an anonymous 1990 Lancet comment critical of Emil Wirostko's work echoed Hayflick's objection: “The case for uncultured organisms even existing let alone remaining uncultured has not been proven and repetitive uncontrolled observations do not serve the scientific community well and may mislead the unwary.”⁹⁾

A number of researchers studying fastidious microbes in other sites of the body including the kidneys, heart, gut, and blood also identified organisms that could not be grown easily or at all in laboratory conditions. Such discrepancies include pH, temperature, nutrient conditions as well as the presence of other microbes.^{10) 11)} Perhaps the study of microbes would have continued to be sidelined in this fashion were it not for a fundamental shift in microbiology that succeeded the sequencing of the human genome.

In Wirostko's time, there was a limited notion of the 100 trillion microbes the comprise the human microbiome, but that changed some ten years ago with the advent of high throughput genomic technologies. About a decade ago, tools originally used to sequence the human genome were first trained on the the body's microbial inhabitants, allowing them to be identified, characterized, and understood based on their DNA. As this search coalesced into the NIH-funded initiative known as the Human Microbiome Project, scientists have pointed to a growing number of microbes that defy culture-based techniques or previous assumptions about infection.

Drawing from data gathered from high throughput technologies, Relman estimated in 2007 that 90% of microbes in the human body had not yet been cultivated.¹²⁾

In 2006, the ophthalmologist Jerry Niederkorn characterized a broad number of chronic diseases including idiopathic uveitis and sympathetic ophthalmia as being caused by the “loss of immune privilege” at the brain, the pregnant uterus, and the eye.¹³⁾ This begs the question: what is responsible for the loss of immune privilege?

The assumption that the vitreous and the retina are protected from both immune cells and microbes has scarcely changed since the time of Ehrlich.^{14) 15)} The 1994 textbook Duane's Clinical opthalmology writes: “It should be noted that the intraocular contents are expected to be sterile at all times.”¹⁶⁾(There is a more recent edition. If we like this quote but not the year in which it was made, I'm sure I could find a better one.)

Now because of metagenomics, such statements have been completely undermined. Early work on the eyes is in line with this estimate. In 2011, Shestopalov et al. used deep sequencing of the 16S rRNA gene amplicon libraries generated from total conjunctival swab DNA to examine the conjunctival rDNA from four subjects.¹⁷⁾ The team found a striking degree of species variability between individuals: the conjuctiva for each individual had a different predominant genera. Even different swab pressures in at the same site in the same individual revealed significant changes in relative abundances of many microbial genera. All together, Shestopalov identified a total of five phyla and 59 distinct genera. Most striking of all though, 31% of all DNA reads belonged to unclassified or novel bacteria, and 42 out of 59 the classified bacterial genera had not been previously reported in healthy eyes. Thus far, Shestopalov and the Ocular Microbiome Project have demonstrated that as many as 300 different bacterial phylotypes are present on the human conjunctiva, which is four times more than previously identified in culture.

However, today we realize each of the body's “privileged” sites is susceptible to infection in previously unanticipated ways. For example, microbes can cross the blood-brain barrier transcellularly, paracellularly and/or in infected phagocytes.¹⁸⁾ Kim writes that a range of bacteria and bacterial toxins routinely establish intimate interactions with endothelial cells, triggering inflammatory responses and coagulation processes and modifying endothelial cell plasma membranes and junctions to adhere to their surfaces and then invade, cross and even disrupt the endothelial barrier.¹⁹⁾

A brief examination of the body's other “sterile” compartments is instructive. Alzheimer's disease has been presumed by many to be the result of a lifelong accumulation of amyloid-beta proteins.²⁰⁾ However, a 2008 Lancet paper showed that an experimental vaccine succeeded in reducing production of amyloids (and the deposition of amyloids in the brain) but without slowing neurological decline.²¹⁾ This suggested that while amyloid deposits were associated with the disease, they did not cause it. In a seminal 2010 study, a team of Harvard researchers showed that amyloid beta can act as an antimicrobial peptide, having antimicrobial activity against eight common microorganisms, including Streptococcus, Staphylococcus aureus, and Listeria.²²⁾ As a result, study author Rudolph E. Tanzi concluded that amyloid-beta is “the brain's protector.”

Several teams have found the intracellular bacterium Chlamydia pneumoniae in the brains of Alzheimer's disease patients.^{23) 24)} In humans, C. pneumoniae has been shown to reach the central nervous system via infected mononuclear cells following the breach of blood-brain barrier^{25) 26)} and to induce Alzheimer's-like amyloid deposits in mouse brain upon injection.^{27) 28)} Miklossy's 2011 review found that antibodies to several types of spirochetes including Treponema and Borrelia burgdorferi were consistently detected in patients with Alzheimer's.²⁹⁾

While Hayflick complained in 1971 of getting six to eight queries a year from “impressionable” physicians asking him to isolate microbes in cases of spontaneous abortion, we now know that microbes seem to play an important factor in whether a woman will carry a pregnancy to term. Using real-time PCR to quantify bacterial count in pregnant women's amniotic fluid, DiGiulio et al. showed in 2008 dose-response association between bacterial rDNA abundance and gestational age at delivery.³⁰⁾ The positive predictive value of PCR for preterm delivery was 100 percent.

In the last several years, opthalmologists have also generated increasing evidence that microbes play key roles in the diseases they study. Bacillus cereus produces a toxin which disrupts the function of retinal pigment epithelium (RPE) cells, the primary cells of the blood-retina barrier.^{31) 32)} Kountouras has presented evidence that Helicobacter pylori contributes to glaucoma, showing a high prevalence of H. pylori infection in Greek patients with primary open-angle glaucoma as well as exfoliative glaucoma.³³⁾ Kountaros further showed a beneficial effect of H. pylori eradication on glaucoma progression.³⁴⁾ Even so, there are also indications there is much more to learn.

Shestopalov's 2011 identification of unclassified or novel bacteria from the conjuctival microbiome³⁵⁾ raises the question: exactly how many microbes have gone undetected by PCR? Given that PCR remains the mainstay of pathogen detection in ocular diseases within the clinical setting,³⁶⁾ it is worth wondering how many microbes have gone undetected for failure to select a proper primer if such a choice is even possible.

Even the most commonly used DNA and RNA amplification techniques like PCR typically offer an incomplete picture of the human microbiome. De Groot-Mijnes et al.'s used real-time PCR to test for the presence of microbes in patients suspected of infectious uveitis.³⁷⁾ Though the team found microbes in only six of 139 patients, this work is hardly complete. The study used primers for only three bacteria and six viruses.

Hong et al. observe that “even with unlimited sampling and sequencing effort, a single combination of PCR primers/DNA extraction technique enables theoretical recovery of only half of the richness recoverable with three such combinations.”³⁸⁾ Yeung concludes that PCR's high specificity can lead to false-negative results when primers are chosen for a polymorphic region of the pathogen’s DNA where sequence variations exist between strains.³⁹⁾ Often the genetic composition of microbes change so rapidly that one does not know which target gene to use a primer. Conversely, deep sequencing tools are more precise, but they are not as sensitive, requiring hundreds of copies of DNA or RNA to make a positive match.

While Koch's postulates are no longer invoked with quite the “great solemnity” described by Hanson,⁴⁰⁾ there should be no doubt they persist. The most enduring of Koch's postulates may be the one that still remains largely fixed in the minds of researchers: a given microorganism that causes disease must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms. Even today, the failure to meet Koch's first postulate is consistently cited to partially or completely discount results. Mombaerts criticized Wirostko's conclusion that MLOs have a role in causing orbital pseudotumors stating: “Unless further studies establish the presence of MLOs in all orbital pseudotumors, including the histopathologically classical type, MLOs should not be regarded as the responsible agents for orbital pseudotumor.”⁴¹⁾ On the other hand, the eminent British ophthalmologist Wallace Foulds was able to confirm the presence of MLOs in biopsies of two patients with retinal pigment epitheliopathy, but not others.⁴²⁾

Mombaerts' criticism ignores the possibility that various species and forms interact with each other to cause disease. Recent work using culture-independent tools have revealed that the collection of microbes which comprise the human microbiome change frequently, exist even in healthy tissue, and work in concert with other microbes.

One significant liability of this postulate is the sheer scope of horizontal gene transferAny process in which a bacterium inserts genetic material into the genomes of other pathogens or into the genome of its host. (HGT), the process by which microbes exchange genetic material. According to Gorgarten, HGT occurs more frequently “than most biologists could even imagine a decade ago.” As a result, the process has turned the idea that we can classify organisms in a simple “tree of life” on its head. Some have argued convincingly that due to the frequency with which microbes exchange genetic material and change through evolutionary adaptation, there may be no such thing as a species.⁴³⁾

Another problem with this postulate is that it implicitly assumes that healthy tissue is free of pathogens. While it is accepted practice to assign one group of patients participating in a controlled trial to be the “healthy control group,” subclinical infection “showing minimal signs of infection,”⁴⁴⁾ among such patients is not the exception but the rule. Shestopalov's metagenomic study of the conjuctiva of “healthy” patients found 24 genera that include many species of common ocular pathogens. In fact, only four genera, Chrysobacterium, Enterobacter, Flavimonas and Nocardia remained undetected in this survey. Similarly, Graham et al. used PCR to measure the flora of the eye in both healthy patients and those with dry eye. The team identified a range of atypical microbes including Rhodococcus erythropolis, Erwinia sp., and Klebsiella oxytoca – the last of which is reputed to be associated an “especially poor visual prognosis.”⁴⁵⁾ These microbes were identified in cases of overt inflammationThe complex biological response of vascular tissues to harmful stimuli such as pathogens or damaged cells. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. and, most provocatively, on the normal ocular surface.⁴⁶⁾ Thus, microbes may contribute to vision conditions such as dry eye which occur in relatively healthy eyes. Even commensal ocular microbes degrade the body's antimicrobials, as Berry showed by observing the mucinolytic activity of such microbes.⁴⁷⁾

Researchers are apt to make a clear distinction between health and disease, but results like these begin to suggest the dichotomy is forced. Instead, it may be more accurate to think of the states of health and disease as poles along a spectrum. Thus, it would be overly simplistic to say there is such a thing as a truly healthy control subject.

Finally, given that, as we discussed before, about nine of ten human microbes have not yet been been proven to grow independently, it seems sensible to discount the possibility that each pathogen must cause disease independently. The best known example of how microbes work together is a collective called a biofilm A structured community of microorganisms encapsulated within a self-developed protective matrix and living together.. Biofilms A structured community of microorganisms encapsulated within a self-developed protective matrix and living together. are densely packed communities of microbial cells that grow inside or outside cells and surround themselves with secreted polymers. A given biofilm is truly an ecosystem and may have over 500 different species. The bacteria that become part of a biofilm engage in quorum sensing, a type of decision-making process in which behavior is coordinated through a “chemical vocabulary.”⁴⁸⁾ The structural and physiological complexity of biofilms has led to the idea that they are coordinated and cooperative groups, analogous to multicellular organisms.⁴⁹⁾ Biofilm have been implicated in a range of diseases: chronic wounds, atherosclerosis, chronic sinusitis, periodontal disease, osteomyelitis, urinary tract infections, and otitis media. Only several years ago researchers realized that biofilms cause most infections associated with contact lens use. In 2006, Bausch & Lomb withdrew its ReNu with MoistureLoc contact lens solution because it led to the formation of contact lens-associated fungal biofilm, causing a high proportion of corneal infections.⁵⁰⁾

We have previously defined successive infectionAn infectious cascade of pathogens in which initial infectious agents slow the immune response and make it easier for subsequent infections to proliferate. as the process by which an infectious cascade of pathogens alter expression of key antimicrobials such as those transcribed by the VDRThe Vitamin D Receptor. A nuclear receptor located throughout the body that plays a key role in the innate immune response. to slow the immune response and allow for subsequent infections to proliferate.⁵¹⁾ In successive infection, the host microbiome shifts further and further away from a natural homeostatic state. Human genes are up- or downregulated by acquired components of the microbiotaThe bacterial community which causes chronic diseases - one which almost certainly includes multiple species and bacterial forms., and infected cells progressively struggle to correctly produce human metabolites in the presence of the numerous proteins, enzymes and metabolites generated by the pathogenic genomes. As a result of this cascade, each infectious disease state becomes the sum of a person's dynamic and diverse mix of microbes.

In the arms race of host–microbe co-evolution, successful microbial pathogens have evolved a number of ingenious ways to evade or subvert the human host's immune responses.⁵²⁾ One of these is through altering transcription and translation of the nuclear receptorsIntracellular receptor proteins that bind to hydrophobic signal molecules (such as steroid and thyroid hormones) or intracellular metabolites and are thus activated to bind to specific DNA sequences which affect transcription., particularly the vitamin D receptorA nuclear receptor located throughout the body that plays a key role in the innate immune response. (VDR). When active, the VDR affects transcription of at least 913 genes and impacts processes ranging from calcium metabolism to several families of key endogenous antimicrobials including beta-defensins, cathelicidin Family of antimicrobial peptides found primarily in immune cells and transcribed by the Vitamin D Receptor., and TLR2A receptor which is expressed on the surface of certain cells and recognizes native or foreign substances and passes on appropriate signals to the cell and/or the nervous system.. In addition to the endogenous antimicrobials, a robust VDR transcribes a number of proteins responsible for vision: Epidermal retinal dehydrogenase, retinal outer segment membrane protein, retinal pigment epithelium-specific protein, retinal short chain dehydrogenase reductase; retinal outer segment membrane protein 1; retinal degeneration slow protein; arrestin 3, retinal; and retinaldehyde binding protein 1.⁵³⁾ Disruption of the VDR activity by intracellular pathogens provides a plausible mechanism for degradation of the eye whether it is through vision loss or inflammatory disease.

Perversion of VDR function would clearly ease pressure on microbes, especially intracellular communities, thus making it an ideal target. For example, Borrelia burgdorferi reduces VDR expression in monocytes (phagocytes) by 50 times, and lysates (“dead” Borrelia) reduced it by 8 times⁵⁴⁾ while another intracellular microbe Mycobacterium tuberculosis was shown to downregulate the VDR 3.3-fold ⁵⁵⁾ Equivalent actions have been documented in Epstein-Barr virus, cytomegalovirus, HIV, and Aspergillus fumigatus. Disrupting immune function through such initial insults to homeostasis could readily allow additional microbes to proliferate with greater ease.

For example, German researchers found in subjects ages three to 90 that the range of aerobic bacteria colonizing a clinically healthy conjunctiva diversifies and increases with age.⁵⁶⁾ Human immunodeficiency virus (HIV) is a pathogen associated with a variety of opthalmic manifestations including uveitis. A patient with HIV can thrive for decades, however when the infection is left untreated by antiretroviral therapy, the microbe begins to profoundly inhibit the immune response. In HIV-mediated immunosuppression, the body enters a state of immunosuppression in which otherwise easily dispatched microbes become fatal.⁵⁷⁾ Even the transmission of HIV is substantially increased by the presence of an existing infection. A 2011 study found that women with bacterial vaginosis, an infection of the vaginal canal, were three times as likely to transmit HIV to their male sexual partners.

We have discussed before how Wirostko inoculated eyelids of mice with MLO taken from inflamed ocular tissues from humans. For the first two months, no ocular inflammation was noted. But from the third month onward, these mice were significantly more likely to develop ocular diseases such as cataracts but also diseases in vital organs such as the heart,⁵⁸⁾ lungs,⁵⁹⁾ liver,⁶⁰⁾ and gastrointestinal tract.⁶¹⁾ The onset of such comorbidities in an animal model adds to the evidence both that these conditions are caused by microbes and that such microbes may be capable of spreading throughout the body in a manner consistent with successive infection. Indeed, this process may explain why in the middle Norway eye-screening study, the prevalence of exfoliative glaucoma in both members of married couples is significantly higher than expected.⁶²⁾ XX - no don't put that here.

Even while some conclude that inflammatory diseases like uveitis tend to recur “for no apparent reason,”⁶³⁾ researchers diminish the role of understudied bacteria in disease – particularly cell wall less and intracellular bacteria. The 2008 textbook Review of Medical Microbiology and Immunology writes, “Characteristic of medically important bacteria's outer surface is a rigid cell wall containing peptidoglycan.” Another recent textbook Review of Medical Microbiology and Immunology states that “the cell wall is the outermost component common to all bacteria except Mycoplasma species.” Though it is certainly true that Mycoplasma lack a cell wall, it is incorrect to claim that the balance of bacteria, or even “medically important” bacteria, always have a cell wall.

Wirostko's finding of dozens, if not hundreds, of cell wall-less intracellular microbes inside a given cell at the site of ocular disease has important implications. These cells in which inclusions were present also had profound alterations of the cell nuclei. Changes included chromatin lysis with clumping, irregular frayed nuclear contours, and/or enlarged “halo-like” paranuclear spaces. Immune cells lacking the abnormal intracytoplasmic bodies displayed a normal ultrastructural appearance.⁶⁴⁾ In 2009, Kountouras wrote that the influx of activated monocytes infected with Chlamydia pneumoniae through the BBB could have dire consequences in the brain leading to the development of degenerative diseases, including Alzherimer's disease and possibly glaucoma.⁶⁵⁾

Generally speaking, while a pathogen living in a biofilm outside a cell (for example, a biofilm on a hip joint) can elicit an immune reaction, a pathogen living inside a nucleated cell, especially an immune cell, can persist for longer periods of time,⁶⁶⁾ resulting in cell proliferation, destruction, or dysfunction⁶⁷⁾ – in essence, changing expression of our genes. In particular, an intraphagocytic pathogen can change the way that the immune system works. Kozarov et al. conclude, “the key step [towards systemic infection] is the persistence of intracellular bacteria in phagocytes.”⁶⁸⁾ Early intraphagocytic infection is a likely culprit in a cascade of events that makes the body increasingly hospitable for microbes.

We have discussed how the chronic eye diseases have long been assigned a reputation for being caused by a hyperactive immune response. Interestingly, even in the autoimmune diseases – long reputed to be driven by an overactive adaptive immune response – the innate immune responseThe body's first line of defense against intracellular and other pathogens. According to the Marshall Pathogenesis the innate immune system becomes disabled as patients develop chronic disease. is attenuated. Kanchwala showed that patients with sarcoidosis expressed the antimicrobial peptide cathelicidin less than healthy subjects, and that sicker sarcoidosis patients expressed it least of all.⁶⁹⁾ Wiken demonstrated that there was a reduced TLR2 mRNA expression in patients with Lofgren's syndrome (a type of acute sarcoidosis).⁷⁰⁾ Finally, Wang showed that Crohn's patients a decline in expression of key antimicrobial peptidesBody’s naturally produced broad-spectrum antibacterials which target pathogens. including cathelicidin and Beta-defensin An antimicrobial peptide found primarily in immune cells and transcribed by the Vitamin D Receptor.-2.⁷¹⁾

Researchers concluding that microbes play a minimal role in idiopathic ocular diseases advocate measures that “re-establish immune privilege.” For them, advances will come in fine-tuning methods for undermining an immune response that causes chronic inflammatory disease.⁷²⁾ There are currently a variety of widely used treatments that exemplify this approach including the glucocorticoids, the first-line therapy for the treatment of uveitis⁷³⁾.

However, the continued definition of certain chronic inflammatory ocular diseases as “noninfectious” seems tenuous at best. It may not be coincidental that as McCluskey writes, “Infection can closely mimic the ocular signs of endogenous inflammation”,⁷⁴⁾ In O'Connor's words, “inflammation is a clear potential link between infectious agents and chronic diseases.”⁷⁵⁾ Metagenomic sequencing tools give us the ability to characterize how infection drives inflammation.

In the 80s and 90s, the human microbiome was scarcely a concept. At the time of his work, Wirostko did not have the tools to characterize microbes absent ambiguity and consequently had to rely on direct direction via TEM. For Wirostko's critics, ultrastructural studies were not sufficient to conclude conclusively what types of microbes were observed underneath his electron microscope. It is possible that these “mycoplasma-like” organisms were another kind (or other kinds) of microbe entirely. While ophthalmology has long been “comfortable with the limits of Koch’s postulates,”⁷⁶⁾ the identification of these organisms in opthalmic disease should not be discounted just because they fail to conform to a centuries-old notion of illness. According to Van Gelder, the importance of testing intraocular fluids will increase as the next wave of technologies advancing microbiology becomes available including panmicrobial and panviral gene chips, and deep sequencing for pathogens, and characterization of short RNAs as signatures for infectious agents.⁷⁷⁾

Such tools will be especially welcome in the study of how therapies for the ocular diseases further dysregulate the ocular metagenome. Of these, it is particularly important to know how antibiotics affect microbes in ocular diseases. The immunosuppressive properties of antibiotics have been long known. Labro asserts that when it comes to treating inflammatory diseases, immunosuppression is “recognized as the basis for antibiotic action.”⁷⁸⁾ Such immunosuppression might reduce inflammation in the near term but allow microbes to spread unchecked by the immune system. One important avenue for exploration is the use of antibiotics in pulsed lower dosing to limit the effect these drugs have on immune function and host surveillance.

Look for Chapter 1 of this thesis MECHANISMS OF BLOOD RETINA BARRIER PERMEABILITY DURING BACILLUS CEREUS ENDOPHTHALMITIS

Talk about slow-growing - speed of DNA replication?

How do we explain this?

High Prevalence of Fastidious Bacteria in 1520 Cases of Uveitis of Unknown Etiology Drancourt, Michel MD, PhD; Berger, Pierre MD; Terrada, Céline MD; Bodaghi, Bahram MD, PhD; Conrath, John MD, PhD; Raoult, Didier MD, PhD; LeHoang, Phuc MD, PhD Abstract The etiologic evaluation of uveitis is frequently unsuccessful when noninvasive methods are used. We conducted a prospective study to evaluate systematic screening for pathogens of uveitis. All patients with uveitis referred to the participating tertiary ophthalmology departments from January 2001 to September 2007 underwent intraocular and serum specimen collection. The standardized protocol for laboratory investigations included universal polymerase chain reaction (PCR)-based detection of any bacteria and mycoses, specific PCR-based detection of fastidious (difficult-to-grow) bacteria and herpes viruses, and culture of vitreous fluid. Sera were tested for fastidious bacteria. Among the 1321 included patients (1520 specimens), infection was diagnosed in 147 (11.1%) patients: 78 (53%) were caused by fastidious bacteria that included spirochetes, Bartonella species, intracellular bacteria (Chlamydia species, Rickettsia species, Coxiella burnetii), and Tropheryma whipplei; 18 by herpes viruses; and 9 by fungi. Bartonella quintana, Coxiella burnetii, Paracoccus yeei, Aspergillus oryzae, and Cryptococcus albidus were found to be associated with uveitis for the first time, to our knowledge. We recommend applying a 1-step diagnostic procedure that incorporates intraocular, specific microbial PCR with serum analyses in tertiary centers to determine the etiology of uveitis. Abbreviations: ACP = anterior chamber paracentesis, EBV = Epstein-Barr virus, ELISA = enzyme-linked immunosorbent assay, HIV = human immunodeficiency virus, HSV = herpes simplex virus, PCR = polymerase chain reaction, TP-PA = Treponema pallidum particle agglutination test, VDRL = Venereal Disease Research Laboratory test.

• chlamydia harvard
• H. pylori ⁷⁹⁾ and https://bjo.bmj.com/content/93/11/1413.long
• endotoxin
• more than the usual suspects?

Even for the inarguably infectious entities corneal ulcer and postoperative endophthalmitis, culture-based methods relatively low yields.

Look at normal tension glaucoma - H. pylori; H. Pylori is nice; it defies Koch's postulates

It is of some interest that ultrastructural studies have continued to be used as evidence for a possible link between mycoplas- mas and malignancy in subsequent decades (Dvorak 1975; Johnson et al. 1996).

Talk about immune reaction uveitis?

Look at scanned PDF “Intraocular immune mechanisms in uveitis”

Over the past century, Koch's presumptions about how microbes would necessarily cause disease have sidetracked a lot of fruitful scientific inquiries.

Only 5% of the human body's microbes can be grown outside the confines of the body. Like other fields of study opthalmologyres has yet to fully come to grips with the magnitude of this false negative.

given microbiologists a distorted view of the human microbiome.

• molecular tools microbes are not where they should be
• culture-based methods offer a distorted view
• microbes are likely culprits in eye diseases
• microbiology a victim of its own success
• beyond usual suspects

The notion of microbes in the eyes would seem like new work but it is not. Scientists in the 70s and 80s identified a variety of microbes. This work faced opposition that is no longer relevant.

This paper examines how this early work is informative.

Timeline/table of important studies