Microbiology before the microbiome

The following is a slightly simplified except taken from a 2009 book chapter I wrote for the J.Craig Venter Institute. It describes research on microbes and inflammatory disease in a time before the advent of metagenomic tools:

In 1922, Ernst Almquist – a colleague of Louis Pasteur – commented, “Nobody can pretend to know the complete life cycle and all the varieties of even a single bacterial species. It would be an assumption to think so.” While Almquist’s work on idiopathic bacteria in chronic disease never received the plaudits accorded Pasteur’s work, Almquist foresaw the complexity that would later be inherent to the field of metagenomics – a field that today forces us to examine how countless microbial genomes interact with the human genome across disease states.

Yet in the decades before novel genomic technology made a metagenomic understanding of disease possible, bacteria could only be cultured in vitro on a limited range of growth media. As most major diseases of the time – tuberculosis, pneumonia, leprosy, and others – were linked to the presence of a handful of acute pathogens able to grow under these constraints, a “game over” attitude toward infectious agents dominated the thinking of much of the medical community. Little consideration was given to the possible role of these pathogens in autoimmune and inflammatory disease states. Instead, for most of the twentieth century, the predominant feeling about the treatment, control and prevention of diseases with a possible infectious etiology was optimism.

Between 1940 and 1960, the development and successes of antibiotics and immunizations added to this optimism and, in 1969, Surgeon General William H. Stewart told the United States Congress that it was time to “close the book on infectious diseases.” With “victory” declared, increasing emphasis was directed at the “non-infectious” diseases such as cancer and heart disease. In many cases, research on infectious disease or activities on their prevention and control were de-emphasized and resources were reduced or eliminated. As recently as the 1980s, pharmaceutical companies, believing that there were already enough antibiotics, began reducing the development of new drugs or redirecting it away from antibiotics.

Despite this rosy narrative, some microbiologists were never convinced that drugs like penicillin had ended the war between man and microbe. In 1932, Razumov noted a large discrepancy between the viable plate count and total direct microscopic count of bacteria taken from aquatic habitats.4 He found higher numbers (by several orders of magnitude) by direct microscopic counting than by the plating procedure. In 1949, Winogradsky confirmed Razumov’s assessment and noted that many microbes are not satisfied with laboratory cultivation conditions. He remarked that readily cultivated bacteria in natural microbial communities “draw importance to themselves, whereas the other forms, being less docile, or even resistant, escape attention.” In 1985, Staley and Konopka pointed to Razumov’s discrepancy and called it the “Great Plate Count Anomaly.” Their review describes work in which they compared the efficacy of a fluorescent dye versus standard plating procedures in detecting bacterial species in samples of water collected from Lake Washington. They found that only approximately 0.1-1.0% of the total bacteria present in any given sample could be enumerated by the plating procedure – causing them to conclude that, unless new methods for detecting bacteria were employed, “No breakthrough in determining species diversity seems likely in the near future.”

Meanwhile, some microbiologists continued their best efforts to alter the pH and growth medium of their samples in an effort to look for previously undetected bacteria in chronic disease states. Over the course of a career spanning almost 50 years, Lida Mattman of Wayne State University cultured wall-less forms of bacteria from the blood samples of patients with over twenty inflammatory diagnoses including multiple sclerosis and sarcoidosis. She authored an entire textbook on novel approaches for in vitro cultivation of bacteria.

Over his thirty-nine year career at Tulane University, Gerald Domingue published dozens of papers and book chapters devoted to the role of chronic forms of bacteria in inflammatory disease. “It is unwise to dismiss the pathogenic capacities of any microbe in a patient with a mysterious disease,” he wrote. “Clearly, any patient with a history of recurrent infection and persistent disability is sending the signal that the phenomenon [infection with chronic bacteria or viruses] could be occurring. The so-called autoimmune diseases in which no organism can be identified by routine testing techniques are particularly suspect.”

Yet scientists like Mattman and Domingue faced serious challenges in trying to convince the medical community their work was valid. Other research teams using less rigorous techniques often failed to duplicate their findings. Many of their observations were dismissed on the premise that their samples could have been contaminated. However, the greatest impediment towards the acceptance of this work was a set of rules set in motion by 19th century German physician Robert Koch. These rules, known as “Koch’s Postulates,” stipulate that in order for a microbe to be deemed a causative agent of a disease, certain criteria must be met. The same microbe must be identified in every person with a given disease; the specific microbe must be able to be grown on pure culture medium in the lab; and, when reintroduced into a healthy animal or person, must produce the disease again.

While Koch’s Postulates may have offered a certain clarity during the formative stages of the field of microbiology, the rules distracted scientists from considering the possibility that multiple species could be responsible for the onset of a single disease state. Even today, Koch’s notions about disease are regularly invoked despite the emergence of a number of counterexamples. Neither Mycobacterium leprae, which is implicated in leprosy, nor Treponema pallidum, which causes syphilis, fulfill Koch’s Postulates, because these microbes cannot be grown in conventional culture media. Viruses further invalidate Koch’s postulates because most require another living cell in order to replicate.

In the absence of clear connections between a single microbe and a single disease, most microbiologists necessarily assumed that the body was a sterile compartment and that inflammation, which might well suggest the presence of microbes, was attributed to an idiopathic causation. Unable to grow all but a fraction of bacteria found in the human body in the confines of a Petri dish, and constrained by a lack of technology with which to detect new microbes, the theory of autoimmune disease, in which the immune system loses tolerance and generates antibodies that target self gained momentum in the 1960s.