INFECTION HOT TOPIC
12032
Culturomics: a new approach to study the human microbiome
G. Greub
Institute of Microbiology, University Hospital Centre and University of Lausanne, Lausanne, Switzerland
E-mail: gilbert.greub@chuv.ch
Article published online: 12 September 2012
Humans are heavily colonized by approximately 1014 bacteria,
and the composition of our microbiota has been shown to
be associated with various diseases, such as obesity, diabetes,
atopic dermatitis, and bacterial vaginosis [1]. The medical
importance of our microbiota, which has even been consid-
ered as a ‘human organ’[2], has thus led to a growing
number of descriptive studies, mainly comparing the microbi-
omes of healthy and ill individuals. As the large majority of
microbial communities are located in the gut, an especially
high number of studies have tried to define the composition
of its core microbiota, as well as to identify specific altera-
tions that might be associated with various pathologies, such
as ulcerative colitis, colorectal cancer, and necrotizing
enterocolitis [3–5].
During the last 10 years, most of these studies have been
performed with metagenomic approaches, which allow rela-
tively fast assessment of the microbial composition by high-
throughput sequencing (Table 1). Despite completely miss-
ing the microbial species present in the studied ecosystems
at a low concentration (<104–105 mL), direct metagenomics
has progressively replaced molecular techniques based on
PCR amplification steps, such as sequencing libraries of
cloned ribosomal amplicons or pyrosequencing of 16S
rRNA amplicons, as these PCR-based approaches were lim-
ited to eubacteria, and did not provide any information on
the metabolic capacities of the studied microbiota. Micro-
arrays have also been used to define the microbial composi-
tion of the intestinal human microbiota; however, this
approach also has major limitations, including a low depth
of analysis, and the fact that it only identifies the known
bacterial species, for which corresponding sequences are
present on the chip [6].
Thus, ‘culturomics’, the new approach depicted in the arti-
cle by Lagier et al. [7], represents a completely new
approach to the study of complex microbial ecosystems,
such as the human intestinal tract, that: (i) has the potential
to detect minority populations; (ii) is not restricted to eubac-
teria; and (iii) provides strains that allow extensive character-
ization of new species and allows the study of interactions
Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases
10.1111/1469-0691.
between different bacterial strains present in a given microbi-
ota (Table 1). Another additional advantage of using culture
instead of molecular approaches is the additional information
on the viability of detected microorganisms.
Providing strains for downstream studies is not trivial.
Indeed, as metagenomics only provides sequencing data,
metagenomic-based research investigating the impact of the
microbiome on a given disease (e.g. obesity) had to be split
in two parts: first, bacterial species that are probably
involved in weight gain were identified by metagenomics;
then, animal experiments were performed with a strain of
the same species, but generally recovered from completely
different ecosystems, and thus possibly not containing the
bacterial genes that are important in triggering obesity. In
contrast, with culturomics, it is possible to directly test the
strain originating from the patient microbiota presenting
the disease of interest.
Culturomics may be defined—by analogy with meta-
genomics—as an approach allowing an extensive assessment
of the microbial composition by high-throughput culture
(Table 1). Thus, Lagier et al. have identified as many as
32 500 different colonies recovered from three human
stools [7]. Such very high-throughput identification was only
possible because of the availability of matrix-assisted laser
desorption ionization time-of-flight mass spectrometry, which
not only represents a revolution in clinical diagnostic labora-
tories [8,9], but also represents a revolution in microbial
ecology, especially when it is coupled to smart incubators
and automated colony-picking systems to constitute the next
generation of culturomic approaches. Indeed, culturomics
will further improve, thanks to automation, miniaturization,
and improved technology.
It is noteworthy that Lagier et al. not only propose a
new concept, i.e. ‘culturomics’, but also, more importantly,
they provide in this milestone article a proof-of-concept.
They applied 212 different culture conditions, and success-
fully cultured 340 different bacterial species, as well as five
fungi and the largest virus ever found in a human sample
[7]. Moreover, 32 new species have been discovered. This
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1158 Clinical Microbiology and Infection, Volume 18 Number 12, December 2012
TABLE 1. Comparison between metagenomics and culturomics
Metagenomics
Definition Method allowing the description of the microbial
composition by high-throughput sequencing
Methodology Pyrosequencing of 16S rRNA amplicons and/or
direct metagenomics without amplification step
Limitations Does not provide a strain for further studies
Misses minority population (depth bias)a
Only detects eubacteriab
Does not provide information on enzymatic abilitiesb
Advantages Detects ‘non-cultivable’ microorganisms
Rate of success Approximately 200 bacterial species/sampled
Possible future Increased depth of sequencing because of new technology
developments Coupling pyrosequencing with direct metagenomics
MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
a
Limitation of direct metagenomics (no amplification step).
b
Limitation of pyrosequencing of 16S rRNA amplicons.
c
Relevance of dead microorganisms lower than that of viable microorganisms (no metabolic activity).
d
Mean numbers of bacterial species recovered from one stool sample by Lagier et al. [7] when comparing both approaches.
e
Smart incubators, pH indicators in broth, microcalorimetry, etc.
f
Automated colony picking coupled to MALDI-TOF MS and/or full laboratory automation.
is a true breakthrough, as these 32 new species represent
approximately one-third of all new validated species recov-
ered by culture from the human intestinal tract during the
last decade.
Lagier et al. also performed a metagenomic analysis of
the same three stools by pyrosequencing of 16S rRNA
amplicons, and a total of 638 phylotypes were identified,
including 282 known bacterial species [7]. Interestingly, only
51 species identified by 16S rRNA sequencing were among
the 340 cultured species. Thus, given such a major discrep-
ancy, culturomics appears to be an ideal complementary
approach to metagenomics, as it has the ability to increase
by one-third the identified microbial repertoire. This impor-
tant discrepancy is partially attributable to the extraordi-
nary amplification power of culture, but is also related to
the restricted biodiversity observed when starting from 16S
rRNA amplicons.
It is important to stress that Raoult’s team not only pro-
vided a new approach and a proof-of-concept, but also used
highly innovative culture conditions. Thus, to circumvent
the very high rate of growth of fast-growing Enterobacteria-
ceae, and because faeces may contain a huge microbial bio-
diversity, they used various highly selective approaches,
such as: (i) the removal of Escherichia coli by specific lytic
phages; (ii) various antibiotic cocktails; (iii) heat destruction
of non-sporulated bacteria; (iv) different selective media;
and (v) amoebal co-culture.
Amoebal co-culture is a cell culture method that uses
amoebae as cells in a cell culture system [10]. This approach,
initially developed by Rowbotham [11], has the ability to
selectively grow amoeba-resisting microorganisms, such as
ª2012 The Author
Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, 1157–1159
CMI
Culturomics
Method allowing the description of the microbial
composition by high-throughput culture
Use of various selective and/or enrichment culture
conditions coupled to MALDI-TOF MS identification
Misses so-called ‘non-cultivable’ microorganisms
Does not directly provide information on enzymatic abilities
Major workload
Detect minority populations
Open approach
Detects only viable bacteriac
Approximately 100 bacterial species/sampled
Automated detection of microbial growthe
Automated identificationf
Miniaturization
Other innovative culture conditions
legionellae, mycobacteria, and chlamydiae, from heavily con-
taminated environments [12–14], and was previously applied
successfully to isolate a Legionella strain from a stool sample
[15]. Here, amoebal co-culture was successfully used to
isolate Senegalvirus [7], a giant virus related to Marseillevirus
and Lausannevirus [16,17].
In addition to these selective conditions, Lagier et al.
used various atmospheres and incubation temperatures, as
well as a large variety of enrichment broths and media.
Here again, the present study was highly innovative, as it
used rumen fluid and sterile human stools in order to have
an enrichment broth that is as similar as possible to that
encountered by the microorganisms present in our intesti-
nal microbiota. Similar studies are now needed to define
which enrichment broths and culture conditions are the
most successful, in order to define optimal culturomic pro-
tocols.
In conclusion, Raoult’s team invented culturomics, and
proved that this new approach is feasible, providing com-
pletely new insights into the microbiota. In the future, cul-
turomics will certainly be one major approach to study the
human microbiome, besides metagenomics. Besides descrip-
tive studies, culturomics may also have very specific applica-
tions, such as detailed characterization of faecal contents,
which will be used for ‘controlled faecal transplantation’.
Thus, instead of inoculating raw faeces to treat Clostridium
difficile colitis [18], we might inoculate a controlled mixture
of bacteria recovered from faeces of relatives. Moreover,
culturomics will probably be more widely used in the future,
with advances in automation and when the most effective
culture conditions have been defined.
CMI
Transparency Declaration
No conflict of interest to declare.
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