Archives of Medical Research - (2018) -
REVIEW ARTICLE
Koch Postulate: Why do we Should Grow Bacteria?
Jean-Christophe Lagier, Grégory Dubourg, Sophie Amrane, and Didier Raoult
Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, Institut Hospitalo-Universitaire Méditerranée-Infection,
Faculté de médecine, Marseille, France
Received for publication October 18, 2017; accepted February 2, 2018 (ARCMED-D-17-00593).
Understanding infectious diseases has long relied on the
Koch postulate, which consists of the pure culture of
microorgan- isms. The advent of molecular methods in
clinical microbi- ology has led the phasing out of culture
as a diagnostic tool and metagenomics has become the
technique most commonly used to assess the impact of
commensal microbes on human health. However,
culturing microbes has led to substantial ad- vances, even
recently, in infectious diseases involving fastid- ious
microorganisms, as evidenced by the Tropheryma
whipplei or Bartonella species. This allows their
genomes to be sequenced and, consequently, new
diagnostic tools to be acquired, experimental model to be
created and antibiotic susceptibility testing to be
performed. In addition, extensive culture focused on
isolation of human commensals, know as the culturomics
approach, has increased the number of bacte- ria isolated
from humans by more than 35% over the past five years.
As strains belonging to the same species can have
different impacts on human health, it would appear
necessary to pursue efforts to constitute an exhaustive
bank of isolates in order to establish further proof of
concepts. This review dis- cusses recent examples,
including the influence of Akkerman- sia muciniphila and
Faecalibacterium prausnitzii, as well as the secretion of
lugdunin by Staphylococcus lugdunensis which is
efficient against the nasal carriage of Staphylococcus
aureus. Finally, responses to some anti-cancer therapies
and the treatment of Clostridium difficile infections
through the use of fecal microbiota transplantation
clearly suggest that culturing marks the beginning of
bacteriotherapy.
Introduction
The pure culture of microorganisms has long been
consid- ered as a fundamental step in microbiological
research (1). From Pasteur’s work on fermentation in
1861 through to the
Address reprint requests to: Didier Raoult, Prof Aix Marseille
Université, URMITE, UM63, CNRS 7278, IRD 198, INSERM
1095,
Marseille, France; Phone:þ 33 4 13 73 24 01; FAX: þ 33 4 13 73
24
02; E-mail: didier.raoult@gmail.com
0188-4409/$ - see front matter. Copyright © 2018 IMSS. Published by Elsevier Inc.
https://doi.org/10.1016/j.arcmed.2018.02.003
introduction of molecular methods at the end of
twentieth century, culture has been the only direct
technique used to establish a link between
microorganisms and human dis- eases (1). In the past,
clinical microbiologists and scientists focused their
research exclusively on microorganisms considered as
pathogenic for humans. With the advent of molecular
tools over the past 25 years, some authors have proposed
that using 16S rRNA amplification and sequencing (2)
and, more recently, using metagenomics (3), could
replace the culture of microorganisms.
Understanding infectious diseases has long relied on
Koch postulates which consists of the pure culture of
mi- croorganisms (4). Unfortunately, culturing has been
pro- gressively abandoned by most clinical microbiology
laboratories and is currently confined to some
microbiolog- ical examinations, such as bacteremia or
UTI etiological diagnosis, while fastidious
microorganisms are preferen- tially detected using
molecular methods. Crucially, environ- mental
microbiologists re-introduced culturing techniques
(5e8). Over the past five years, microbial culturomics, a
high throughput culture method multiplying the
number of culture conditions and using a rapid
identification method by MALDI-TOF mass
spectrometry (9e11), has been developed to study the
human gut microbiota. Conse- quently, culturomics has
doubled the number of known bacteria cultured from
human gut (11). Simultaneously, the relationship
between human microbiota and various dis- eases has
become a hot topic. This explains why interest in
commensal microorganisms has dramatically increased
in recent years.
In this paper, we propose a mini-review highlighting
the
need to go beyond Koch’s postulate through recent
changes in clinical microbiology including the study of
human microbiota and culturomics.
Koch Postulate
In 1890, Robert Koch formulated postulates defining the
criteria required to incriminate a bug (firstly a parasite)
as the causative agent of infectious diseases (4), for
which
2 Lagier et al./ Archives of Medical Research
he was awarded the Nobel Prize for Medicine in 1905.
His theory was sustained by the formulation: 1 pathogenþ
1 host 5 1 disease. First, the microorganism should be
encountered in all the cases of the diseases. Secondly,
this microorganism should not occur as commensal in
healthy individuals. Finally, the causative agent can be
isolated from one patient and, after several propagations
in pure cul- ture, can further cause this disease when re-
inoculated (12,13).
One of the most famous examples of the Koch postulate
was provided by Marshall who, after five days’ incubation,
identified small colonies from the stomach of patients
suffering from gastritis (14). Because the causative
relation- ship between this bacterium and gastritis was
challenged by the international scientific community,
Marshall demon- strated the Koch postulate., The bacteria
was orally admin- istrated to a volunteer (himself) with
histologically normal gastric mucosa who went on to
develop a histologically proven mild illness, acting as a
proof of concept of what was later known as
Helicobacter pylori infection.
This postulate generated serious criticism at that time
and Koch was himself aware that his rules were only
preliminary. Indeed, he was never able to demonstrate
his postulates for cholera because of the lack of a
reproducible animal model, despite the fact that he had
isolated vibrios as a potential cause of cholera (12). In
addition, von Pettenkofer even in- gested Koch’s cholera
vibrios orally in front of his students, without falling ill
(12). As summarized recently by Brussow et al.,
Pettenkofer rejected Koch’s hypothesis, instead formu-
lating it as ‘X (pathogen) Y (local milieu) Z (individual
host susceptibility) 5 disease’
þ þ
(13). Despite countless
criti- cisms frequently linked with the incorporation of
epidemi- ology, Koch’s postulates continued to be held
through the twentieth century.
Can we go Beyond Culture to Demonstrate Koch’s
Postulate?
The Place of 16SrRNA Amplification and Sequencing.
The advent of 16S rRNA amplification and sequencing
gave rise to the hope that molecular tools could replace
culture. Whipple’s disease is an infectious disease first
described in 1907 by George Whipple (15). First
considered as a metabolic disease, direct observation by
microscopy in 1961 of its form resem- bling a
microorganism and the efficiency of empirical anti-
biotic treatment led it to be considered as an infectious
disease (16). It was only in 1991 that Wilson directly
amplified the 16S rRNA gene from a small-bowel
biopsy of a patient suffering from Whipple’s disease. In
1992, Relman et al. then detected sequences of T.
whipplei from the tissues of five different patients (17).
For Whipple’s disease, the establish- ment of sequences
specific to the genome of the bacterium, as a result of its
culture, first performed in 2000 (18), made it possible to
identify the role of Tropheryma whipplei in numerous
pathologies, including acute infections (19), outside
- (2018) -
classic Whipple’s disease (20). This work could not have
been accomplished based on 16S rDNA alone, given the
complexity of the digestive or respiratory microbiota,
where this could be demonstrated (19). Quite clearly,
moreover, a very high num- ber of false positives were
found, initially, in the saliva ana- lyses, due to the lack of
specificity of 16S rDNA (21).
Bartonella quintana was first described in 1917 as
Rick- ettsia quintana, the agent of trench fever. It was
reclassified in 1993 as B. quintana following unification
of the genera Rochalimaea and Bartonella (22). The
proof of concept of the complementarity of techniques
and the need for bac- terial cultures, was dramatically
brought to light by an issue of the New England Journal
of Medicine, published in 1990, in which -(i) the culture
of a fastidious bacterium in the blood of a febrile
patient later turned out to be Bartonella henselae (23)
(ii) the observation, using the Warthin-Starry technique,
of bacteria during hepatic pelio- sis (24), also known to
be related to Bartonella henselae, and (iii) the
amplification and sequencing of 16S RNA of a
bacillary angiomatosis lesion also revealed Bartonella
henselae (25). It should be noted that, since then, all the
discoveries related to Bartonella henselae have been
largely due to culture (26,27), which made it possible to
find a serology that showed that Bartonella henselae
was the cause of cat scratch disease. This in turn led to
the development of a therapeutic strategy and ultimately
to the development of pathophysiological models (22).
The replacement of culture by molecular tools to expand
the spectrum of microbial pathogens, as currently
proposed, was totally irrelevant at that time (2).
The Place of Metagenomics. Metagenomics had
promised to render bacterial culture obsolete, by
detecting uncultiva- ble microorganisms. The first
studies dedicated to the description of the human gut
microbiota composition thus revealed that 80% of the
sequences were attributed to bacterial species which had
yet to be cultured (28). Never- theless, by comparing
different methods to study the same stool samples,
including electron microscopy and metage- nomics
targeting the 16S rRNA gene, Hugon P, et al.
demonstrated that metagenomics overlooked a large pro-
portion of Gram negative bacteria (29). Several biases
can explain these discrepancies, including extraction
bias, primer biases, and depth bias that were
subsequently described (30). Indeed, several studies
demonstrated that metagenomics could not be used as a
diagnostic tool for infectious diseases. First, in an
investigation into a Shiga- toxigenic Escherichia coli
(STEC) O104:H4 outbreak, the use of metagenomics by
Loman et al. did not detect the causative bacteria in
more than 30% of cases (3). As another example, in an
investigation of the causative agent of diarrhea using
V5-V3 16S RNA amplification, Singh et al. did not
detect Campylobacter and Salmonella in 42% and 66%,
respectively, of the cases diagnosed by cul- ture, while
Salmonella Shigella species were never detected
 The Need to Culture Bacteria 3

in the documented cases (31). The example of
Clostridium butyricum in necrotizing enterocolitis in
preterm neonates can also be cited, for which the role of
toxigenic C. butyr- icum strains was demonstrated by
culturomics in a case- controlled study (32).
Metagenomic analysis of the same samples had initially
ignored this bacterium and only a re-analysis of the
sequences in view of the culturomics re- sults enabled it
to be detected. In addition, as the result of culture, the
authors were able to test the toxin secretion of
C. butyricum (32).
Metagenomics does not appear to be an alternative to
culture methods, but a complementary tool that makes it
possible to mass screen a large variety of specimens.
Meta- genomics only makes it possible to find
associations be- tween bacteria and human diseases, but
by no means causal links between them.
Recent Evolution of the Koch Postulate
The Rebirth of Culture Through Microbial Culturomics.
Microbial culturomics was introduced in 2012 to
increase
knowledge of the gut microbiota repertoire. Prior to this,
environmental microbiologists mimicked the bacteria’s
nat- ural environment in order to facilitate its growth (5).
They developed some culture process, including
diffusion cham- bers and ichip systems, to dramatically
exceed the number of growing bacteria achieved through
standard cultivation (1). Based on these previous
observations, culturomics is a high throughput method
which diversifies culture condi- tions by modifying
parameters such as temperature, atmo- sphere,
incubation time, or by adding inhibitory factors such as
antibiotics, and promoters such as blood and rumen fluid
for the cultivation of minority and/or fastidious micro-
organisms (9). The use of fresh stools and incubation in
an anaerobic chamber are also essential for the
identification of anaerobes (10,11).
The rapid identification method by MALDI-TOF, a
cost and time effective technique (33) made it possible
to test more than 1,000,000 different colonies and to
identify more than 1,100 different bacteria, including
247 entirely new bacteria and 269 isolated for the first
from humans (11) (Figure 1). At the same time, other
work focused on efforts

Figure 1. Classification of cultured bacteria from human beings. Each node represents a bacterial genera for which at least one species was isolated
from human beings. Green nodes and red nodes represent the genera and the families, respectively, created as a part of culturomics studies. Green
edges represent the genera for which culturomics enabled the creation of at least one new species. (A color figure can be found in the online version
of this article.)
4 Lagier et al./ Archives of Medical Research - (2018) -

to culture sporulated bacteria using ethanol before
inocu- lating stool samples in culture media, and 66 new
bacterial genus and species were isolated (34). Overall,
the number of the bacteria isolated at least once in pure
culture from humans can be considered to have
increased by more than 35% over the past five years
(35,36).
Commensal but Critical Microorganisms. Several exam-
ples have demonstrated that commensal microorganisms
could be implicated in human diseases and could even
be used in the future as bacteriotherapy. Clostridium
difficile infections (CDI) are the first cause of
healthcare- associated diarrhea. Beyond the high-
associated mortality, CDI are also characterized by a
significant rate of recur- rence (37). Fecal microbiota
transplantation (FMT) has become the gold standard
treatment of Clostridium difficile infection. Importantly,
FMT was used for recurrent infec- tions and was
recently extended to the severe infections caused by this
bacterium (38). FMT has been demonstrated to be better
than standard antibiotics prescribed for CDI (39).
Various studies using mainly metagenomic tools have
shown that the gut microbiota composition of patients
suffering from CDI is modified. In particular, bacterial
diversity is considerably reduced, while members of the
Proteobacteria phylum are overrepresented (40). The
resto- ration of a healthy microbiota is key to
overcoming the pathogenic bacteria Clostridium difficile
(38). While most studies used whole stool samples from
healthy individuals,
some authors have proposed specific bacteriotherapy
consisting of a cocktail of bacteria (41e43).
While Koch’s postulate relies on the presence of a
microbe, recent research has highlighted that the
absence or depletion of specific microorganisms could
be strongly associated with diseases. The most striking
example is that of Faecalibacterium prausnitzii,
depletion of which is mostly associated with
inflammatory bowel diseases (IBD), including colitis
and Crohn’s disease (44). These findings were
confirmed in in vitro models (45,46). On the topic of
the relationship between cancer and microbiota, Vetizou
et al used a mouse model to demonstrate that the
absence of Bacteroidales and Burkholderiales
significantly decreased the efficiency of anti-cytotoxic T-
lymphocyte- associated protein 4 antibodies (anti-
CTLA4), which was restored through the oral
administration of some bacterial strains belonging to
these taxa (47). In addition, a recent study performed in
patients suffering from ovarian and lung cancers has
demonstrated that E. hirae and B. intestinihomi- nis
enhanced the efficacy of cyclophosphamide (48). These
findings suggest that the modulation of commensal
bacteria in the human gut could be used as adjuvant
anticancer treat- ments (49). Finally, because pathogenic
microorganisms can cause a wide spectrum of diseases in
humans (e.g., Staphylo- coccus aureus), depletion of
fundamental commensals are now associated with an
array of pathologies, perfectly reflecting the need for the
repertoire of commensals associ- ated with humans to be
comprehensively described through

Figure 2. Evolution of the Koch postulates over time: The seminal concept relies on the principle ‘ 1 bug 5 1 disease’’ (1A). Recent studies however
show that there was competition between pathogens associated with human beings, in particular through the secretion of antimicrobial molecules (1B).
In addition, it was clearly shown that some microorganisms are able to disturb an entire ecosystem as shown by infections involving toxigenic C.
difficile strains (1C). (A color figure can be found in the online version of this article.)
 The Need to Culture Bacteria
culture. Thus, after being isolated for the first time in
2004, depletion of Akkermansia muciniphila was
strongly associ- ated with several metabolic diseases
including weight gain and diabetes (50,51). These
findings led to an in vitro model of the administration of
cultivated strains to animals that established clear proofs
of concept in the field of metabolic syndrome (52) and
in inflammation (53).
Is the Koch Postulate is Still Valid on the Strain Level?
When Koch defined his postulate, the definition of
bacterial species was not yet clearly established and, in
any event, was far away from today’s definition. In his
view, in the absence of morphological differences,
species could be differentiated by their reaction to
specific antisera, which ultimately made the definition
unclear. The fact is that the current notion of the strain
of a clone enables us to understand that a single species
can have different impacts on human health. Indeed,
only some strains of Enterococcus hirae were
demonstrated to mediate the effect of cyclophosphamide
on lung and ovarian cancers, while others had no impact.
Finally, Koch’s postulate is challenged by the textbook
case of Clostridium butyricum, for which some
toxigenic strains are considered as being responsible for
NEC (32) while others are beneficial for human health,
to the point that it is allowed as a novel food adjuvant
(54). In addition, certain strains of Lactoba- cillus spp.
have an influence on weight-gain in humans and animals
while other strains of the same species do not have the
same effect (55). Currently, only some specific or type
strains are usually deposited in culture collections, but
their differential impact on human health should prompt
extensive culture along with conservation of a wide
variety of isolates within the same species.
Finally, a recent discovery has fundamentally
challenged the Koch postulate. Lugdunin is a
bactericidal agent secreted by Staphylococcus
lugdunensis that prohibits colo- nization by
Staphylococcus aureus. Zipperer A, et al. demonstrated
that human nasal colonization by S. lugdu- nensis was
associated with a significant reduction in the carriage of
S. aureus (56). The proof of concept of the bactericidal
component produced by commensal bacteria could
inhibit pathogenic bacteria (Figure 2).
Conclusion
Culturing a microorganism continues to be a fundamental
step leading to genome sequencing, the advent of new spe-
cific tools, and antibiotic susceptibility testing (36). In
addi- tion, recent advances relating to the influence of
human microbiota on human health and diseases have
created a sudden shift in interest in commensal bacteria. At
the same time, the rebirth of culture in clinical
microbiology through culturomics has led to the Koch
postulate evolving from ‘ 1 pathogen 1 host 5 1
disease’’ towards a perpetual balance between good and
þ Micro- biol Rev 2017;30:709e746.
bad microorganisms that
5
can be summarized by ‘ 1 microorganism þ other
microorganisms þ 1 host 5 1 disease’’.
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