JMS Pearce
Hull, England

Van Leeuwenhoek (1632–1722), a Dutch botanist, using his early microscope observed single-celled bacteria, which he reported to the Royal Society as animalcules. The science of bacteriology owes its origin to two scientists of coruscating originality, Louis Pasteur and Robert Koch. Pasteur may be described as master-architect and Koch as master-builder of the science.¹

Pasteur showed in 1863 that microorganisms caused milk to sour or ferment, and meat to putrefy. He speculated that human disease was caused by the multiplication of germs or microbes in the body, but they had not then been identified.² Robert Koch in 1876 proved for the first time that a specific bacterium could cause a particular disease.³ He observed Bacillus anthracis in the tissues of anthrax victims and, crucially, showed it was transmissible. Koch extracted Bacillus anthracis from a sheep which had died of anthrax, then repeatedly injected generations of mice with it; the mice developed the disease. He then proved that bacteria could cause other infections. He was awarded the Nobel Prize in Physiology or Medicine in 1905 “for his investigations and discoveries in relation to tuberculosis.”⁴

Studying tuberculosis (1882), he formulated the Koch’s postulates with which to prove that a microbe (parasite) was the cause of the disease in question.⁵ The postulates were based on an earlier idea of Jakob Henle published in 1840 that living parasitic organisms caused infectious diseases. Koch refined and presented his postulates to the Tenth International Congress of Medicine in Berlin in 1890⁶:

Table 1. Koch’s postulates 1890

1. The parasite occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease.


2. The parasite occurs in no other disease as a fortuitous and non-pathogenic parasite.


3. After being fully isolated from the body and repeatedly grown in pure culture, the parasite can induce the disease anew.

He applied these principles to other infectious diseases: “The proof has been fulfilled in a number of diseases, anthrax, cholera, tuberculosis, tetanus, and many animal diseases.” Importantly, Koch recognized that it was not always possible to satisfy his third criterion:

In accordance with this hypothesis, we must then consider as parasitic a number of diseases in which it has not yet been possible—or only in an incomplete manner—to infect experimental animals and to prove the third part of the rules. To these diseases belong typhoid fever, diphtheria, leprosy, relapsing fever, Asiatic cholera.

He had discovered asymptomatic carriers of cholera and Salmonella typhi, but of course was not familiar with viral infections, nor had viruses or their antibodies been isolated in the laboratory. So effective were Koch’s defining rules that for many years an infectious agent was not accepted as the cause of a disease unless Koch’s postulates had been satisfied.

In his day, because infections in the absence of antibacterial drugs were usually of great severity, little attention was paid to non-pathogenic commensals, subclinical infections, or to asymptomatic carriers. These now commonplace aspects of infections constitute many of the exceptions to Koch’s postulates. But others have emerged so that Koch’s postulates have been reported as outdated or unfit for purpose.

Exceptions to Koch’s postulates

Many pathogenic organisms such as Plasmodium falciparum, herpes simplex and other viruses cannot be grown in cell-free culture, and hence cannot fulfill Koch’s third postulate.

Some infections such as human immunodeficiency virus (HIV) are restricted to humans; therefore, they cannot be transmitted to other hosts, thereby failing the third postulate.

Further, in subclinical infections and in the carrier state, a pathogenic microbe may cause disease in one person but not in another. Host resistance to infection is another factor in causing or not causing pathology depending on immunological status and genetic susceptibility. For example, many people exposed to M. tuberculosis will develop a subclinical infection detectable only by a positive tuberculin skin test, and will not develop symptomatic disease, thus violating the postulates.

Bacteria such as Clostridium tetani cause distant injury by release of a toxin, others by immune mechanisms after the causal agent has apparently disappeared. For instance, in Whipple’s disease there is a ribosomal RNA sequence but not the causal bacterium Tropheryma whipplei.

Some bacteria require co-infection with a bacteriophage or by acquiring extrachromosomal DNA to be able to cause disease (e.g., cancer pathogenesis and human papillomavirus). The hepatitis D virus relies on a second hepatitis B surface antigen for its reproduction in human tissue, termed polymicrobial causation.

Although Koch’s postulates are no longer required to prove that a pathogen such as a virus is the cause of a disease, the principles still obtain so that we have to prove that a putative causal agent is not just associated with, but actually causes the illness. Association and causation are often commonly confused, as noted by Dr Samuel Johnson:

It is incident to physicians, I am afraid, beyond all other men, to mistake subsequence for consequence.

In 1937, Koch’s postulates were modified by Rivers’ criteria to define a viral cause of a disease⁷:

Table 2. Rivers’ postulates for viruses

1.  A specific virus must be found associated with a disease with a degree of regularity.


2. The virus must be shown to occur in the sick individual not as an incidental or accidental finding but as the cause of the disease under investigation.


3. It is not obligatory to demonstrate the presence of a virus in every case of the disease produced by it, the existence of virus carriers is recognized. Finally, it is not essential that a virus be grown on lifeless media or in modified tissue culture

Falkow further modified these criteria based upon mutagenesis and noted that it was imperative when pursuing the genetic analysis of bacterial pathogenesis to apply a “molecular form of Koch’s postulates.”⁸  In 1996, Fredricks and Relman suggested criteria for the sequencing of microbial nucleic acids:⁹

Table 3. Fredricks and Relman criteria

(i) A nucleic acid sequence belonging to a putative pathogen should be present in most cases of an infectious disease. Microbial nucleic acids should be found preferentially in those organs or gross anatomic sites known to be diseased (i.e., with anatomic, histologic, chemical, or clinical evidence of pathology) and not in those organs that lack pathology.

(ii) Fewer, or no, copy numbers of pathogen-associated nucleic acid sequences should occur in hosts or tissues without disease.

(iii) With resolution of disease (for example, with clinically effective treatment), the copy number of pathogen-associated nucleic acid sequences should decrease or become undetectable. With clinical relapse, the opposite should occur.

(iv) When sequence detection predates disease, or sequence copy number correlates with severity of disease or pathology, the sequence-disease association is more likely to be a causal relationship.

(v) The nature of the microorganism inferred from the available sequence should be consistent with the known biological characteristics of that group of organisms. When phenotypes (e.g., pathology, microbial morphology, and clinical features) are predicted by sequence-based phylogenetic relationships, the meaningfulness of the sequence is enhanced.

(vi) Tissue-sequence correlates should be sought at the cellular level: efforts should be made to demonstrate specific in situ hybridization of microbial sequence to areas of tissue pathology and to visible microorganisms or to areas where microorganisms are presumed to be located.

(vii) These sequence-based forms of evidence for microbial causation should be reproducible.

This foment of new techniques expanded the realm of Koch’s postulates, but his underlying principles are plainly recognizable and still apply. New discoveries demand new criteria, even if for the clinician some of the technological minutiae are obscure. This was recognized by Fredricks and Relman who, vindicating Koch concluded: “We do not believe that every criterion for sequence-based evidence of causation needs to be fulfilled in order to incriminate a presumptive microorganism in disease, just as Koch did not believe in his day that every postulate must be fulfilled to prove causation.”

Robert Koch (Fig 4), the son of a mining engineer, was born in 1843 in Clausthal-Zellerfeld, a small mining and university town near Hanover in the Harz mountains. In 1862, he began his medical studies at Gottingen University, where the anatomist Jacob Henle was a major influence. He qualified MD maxima cum laude in 1866.

He worked in general practice in Rakwitz before joining the German Army in 1871 during the Franco-Prussian War. After his discharge in 1872, he was district physician in Wollheim, and it was here that his wife Emmy née Fraats gave him a microscope for his birthday. With this he set up a primitive laboratory and started his investigation of infectious diseases, founding the basic techniques of microscopy, bacterial staining, and culture. This led to his discoveries of anthrax, tuberculosis, cholera, and pyogenic cocci. In 1885, he was elected Professor of Bacteriology in Berlin; the Koch Institute was built for him in 1891.¹⁰

The philosopher David Hume insisted causation is not observable: only the events that suggest a conjunction between cause and effect are observable. Koch’s postulates did apply observable, valid criteria to “prove” disease causation by microbes: the inductive connection between cause and effect.

Koch’s rules can not strictly be applied to viruses nor to some other newly discovered pathogens.¹¹ But their underlying principles and his critical methods are still pertinent to provide scientific rigor when a new pathogen is suspected as the cause of an illness.

References

1. Sakula A. “Robert Koch: centenary of the discovery of the tubercle bacillus, 1882.” Thorax 1982;37:246-251.
2. Pasteur L. “Recherches sur la putrefaction,” Comptes Rendus de l’Académie des Sciences 1863;66:1189-1194.
3. Koch, R. Untersuchungen über Bakterien: V. Die Ätiologie der Milzbrand-Krankheit, begründet auf die Entwicklungsgeschichte des Bacillus anthracis. Cohn’s Beitrage zur Biologie der Pflanzen 1876;2, no. 2, 277–310.
4. Robert Koch – Facts. NobelPrize.org. Nobel Prize Outreach AB 2022. Wed. 7 Sep 2022. https://nobelprize.org/prizes/medicine/1905/koch/facts/.
5. Koch R. Die Aetiologie der Tuberculose read before the Physiological Society in Berlin on 24 March 1882, pp. 392-406. Cited in: D. H. Clark (ed.), Source book of medical history. New York: Dover Publications, 2013.
6. Koch, R. “Uber bakteriologische.” Forschung Verhandlung des X Internationalen Medichinischen Congresses, Berlin, 1890, 1, 35. August Herschwald, Berlin. 10th International Congress of Medicine.
7. Rivers TM. “Viruses and Koch’s Postulates.” Journal of Bacteriology 1937;33:1-12.
8. Falkow S. “Molecular Koch’s postulates applied to microbial pathogenicity.” Rev. Infect. Dis. 1988;10 (Suppl 2): S274–6.
9. Fredricks D and Relman D. “Sequence-Based Identification of Microbial Pathogens: a Reconsideration of Koch’s Postulates.” Clinical Microbiology Reviews 1996;9(1): 18–33. https://ncbi.nlm.nih.gov/pmc/articles/PMC172879/pdf/090018.pdf.
10. Sakula, “Robert Koch.”
11. Berman JJ. Changing how we think about infectious diseases. In: Taxonomic Guide to Infectious Diseases (Second Edition). Elsevier, 2019. pp. 321-365.