REVIEW

Clostridium butyricum: from beneficial to a new emerging pathogen

N. Cassir, S. Benamar and B. La Scola

Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63 CNRS 7278 IRD 198 INSERM U1095, Facultés de Médecine
et de Pharmacie, Aix-Marseille Université, Marseille, France

Abstract

Clostridium butyricum, a strictly anaerobic spore-forming bacillus, is a common human and animal gut commensal bacterium, and is also
frequently found in the environment. Whereas non-toxigenic strains are currently used as probiotics in Asia, other strains have been
implicated in pathological conditions, such as botulism in infants or necrotizing enterocolitis in preterm neonates. In terms of the latter,
within the same species, different strains have antagonist effects on the intestinal mucosa. In particular, short-chain fatty acids, which are
products of carbohydrate fermentation, have a dose-dependent paradoxical effect. Moreover, toxin genes have been identified by genome
sequencing in pathological strains. Asymptomatic carriage of these strains has also been reported. Herein, we provide an overview of the
implications of C. butyricum for human health, from the beneficial to the pathogenic. We focus on pathogenic strains associated with the
occurrence of necrotizing enterocolitis. We also discuss the need to use complementary microbiological methods, including culture, in
order to better assess gut bacterial diversity and identify new emergent enteropathogens at the strain level.
Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All
rights reserved.

Keywords: Clostridium butyricum, culturomics, metagenomics, necrotizing enterocolitis, toxins

Article published online: 20 October 2015

including C. butyricum [3]. Ferraris et al. found that 44% of faecal

Corresponding author: B. La Scola, Unité de Recherche sur les
samples collected from asymptomatic preterm neonates
Maladies Infectieuses et Tropicales Emergentes, URMITE, UM63,
CNRS 7278, IRD 198, INSERM 1095, Faculté de médecine, (n = 76), whose median gestational age was 32.8 weeks, tested
Aix-Marseille Université, 27 Boulevard Jean Moulin, 13385 Marseille positive for C. butyricum [4]. Surveys have identified C. butyricum
cedex 05, France
in 10–20% of human faecal samples by microbial culture [5].
E-mail: bernard.la-scola@univ-amu.fr
Moreover, this bacterium has been widely used as a probiotic in
Asia (particularly in Japan, Korea, and China) [1]. However,
some C. butyricum strains detected in stool samples have also
Introduction been implicated in pathological conditions such as botulism in
infants and necrotizing enterocolitis (NEC) in preterm neonates
[6,7].
Clostridium butyricum is a strictly anaerobic, Gram-positive,
Several Clostridium species involved in infectious diseases are
spore-forming bacillus named for its capacity to produce high
known to be pathogenic, essentially by producing potent toxins
amounts of butyric acid. It was first isolated from pig intestines
that are responsible for life-threatening diseases in humans and
by Prazmowski in 1880 [1]. Since then, several strains of
other animals [8]. Interestingly, these species produce the
C. butyricum have been described in a variety of environments,
highest number of toxins of any type of bacteria, and the genetic
and are common human and animal gut commensal bacteria. A
characteristics of toxigenic clostridia support horizontal toxin
recent study found C. butyricum strains in 302 of 978 environ-
gene transfer [8,9]. Notably, it has been shown that toxigenic
mental samples tested (31%) [2]. The highest percentage of
and non-toxigenic Clostridium difficile strains form part of the
isolates was found in soil, vegetables, soured milk, and cheeses.
normal human gut microbiota [10]. Moreover, within the same
In humans, following birth, the neonate’s intestine is progres-
species, antagonist effects have been described. A recent pilot
sively colonized by facultative and strictly anaerobic bacteria,

Clin Microbiol Infect 2016; 22: 37–45

Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved
http://dx.doi.org/10.1016/j.cmi.2015.10.014
38 Clinical Microbiology and Infection, Volume 22 Number 1, January 2016
study showed that the administration of spores of a non-
toxigenic C. difficile strain was effective for the prevention of
recurrent C. difficile infection caused by toxigenic strains [11].
As fermentative bacteria, clostridia produce short-chain fatty
acids (SCFAs), primarily butyrate and acetate, through
fermentation of mainly undigested dietary carbohydrates. This
has the primary effect of stimulating the proliferation of
enterocytes [12]. It has also been shown that butyrate may
downregulate the expression of several virulence genes [13].
Although previous reports have suggested that, besides
C. difficile, other Clostridium species may also cause antibiotic-
associated diarrhoea, anaerobic research in clinical practice is
frequently limited to C. difficile and its toxins [14]. Detection of
clostridia in stool samples remains challenging. Although the
advent of modern molecular microbiological methods has
expanded the degree of detection, it is also hampered by
several biases [15]. Consequently, there has been renewed in-
terest in culture methods [16,17]. In a recent comparative
analysis of stool samples using culture and 16S rRNA gene
sequencing data, only 15% overlap was found [16]. Comple-
mentary approaches are therefore necessary to explore the gut
microbiota. Through the use of both techniques, we recently
ascertained a specific association between NEC and C. butyricum
[17]. These data were confirmed by the use of a specific
quantitative real-time PCR (qPCR) assay to screen a larger set
of samples. Genome sequencing allowed the detection of genes
encoding toxin-like proteins shared by this C. butyricum strain.
In this article, we will provide an overview of the various
implications of C. butyricum for human health, from beneficial to
pathogenic. We will focus on its role in the pathogenesis of
NEC occurring in preterm neonates as compared with that of
C. difficile in pseudomembranous colitis occurring in the elderly.
We will also discuss the need to use complementary microbi-
ological methods, including culture, in order to better assess
gut bacterial diversity and identify new emergent enter-
opathogens at the strain level.
Probiotic
The WHO has defined probiotic bacteria as ‘live microorgan-
isms which when administrated in adequate amounts confer a
health benefit on the host’ [18]. Several studies have shown
probiotic properties of specific C. butyricum strains, mainly
performed in Japan, Korea, and China, where those probiotic
strains are commercialized [19,20]. A recent study found that
the C. butyricum strain MIYAIRI 588 (CBM588) promoted reg-
ulatory T-cell generation in the intestine through induction of
transforming growth factor-β1 from lamina propria dendritic
cells. This pathway was mainly Toll-like receptor 2 dependent,
Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, 22, 37–45
CMI
with a suggested role of fermentation metabolites such as
butyrate [21]. However, the same beneficial mechanism has
been observed with 17 other Clostridium species belonging to
clusters IV, XIVa, and XVIII [22]. This C. butyricum strain also
induced interleukin-10-producing macrophages in inflamed
mucosa to prevent acute experimental colitis in mice [19].
Other experimental studies have shown the capacity of this
strain to improve non-alcoholic fatty liver disease [23] and
prevent C. difficile infection [24], enterohaemorrhagic Escher-
ichia coli O157:H7 infection [25], and gastric ulcers [26]. In
clinical practice, the administration of CBM588 was effective for
both the treatment and the prophylaxis of antibiotic-associated
diarrhoea in children [27]. It increased the level of anaerobes
and prevented the decrease in the level of Bifidobacterium
species in subjects who received antibiotics.
Moreover, it has been shown that CBM588 harbours a
plasmidic bacteriocin that is very similar to the previously
described butyricin 7423 [28], and is related to its probiotic
effect [29]. The supernatant of cultured CBM588 inhibited the
growth of Clostridium beijerinkii ATCC25752 and Clostridium
pasteurianum, but not of C. beijerinkii ATCC6015, Clostridium
acetobutylicum, and C. butyricum ATCC19398. In addition, the
purified recombinant bacteriocin showed bactericidal activity
against C. difficile and Clostridium perfringens, but had no effect on
Gram-negative bacteria. These results suggest a narrow, strain-
specific spectrum of activity of this bacteriocin that cannot fully
explain the beneficial effects previously cited. Similarly, Gebhart
et al. have shown that some strains of C. difficile produce R-type
bacteriocins, termed diffocins, which have specific bactericidal
effects against other strains of the same species [30].
When orally administered, C. butyricum spores germinate and
grow in intestinal tracts [5], and produce large amounts of
SCFAs, such as butyrate and acetate [31]. SCFAs constitute an
important energy source for intestinal cells, and have prolifer-
ative effects on enterocytes [12]. Additionally, SCFAs have been
noted to have immune-modulatory effects on colonic inflam-
mation. They suppress inflammatory cytokine secretion in
cultured epithelial cells, facilitating tolerance of the intestinal
mucosa to the presence of vast quantities of living microor-
ganisms, and controlling the overgrowth of pathogens [12]. It is
therefore hypothesized, but not proven, that the main mech-
anism by which probiotic C. butyricum strains exert their
beneficial effect is through equilibrated SCFA production.
Pathogenic
Botulism
Botulism is an acute paralytic disease caused by botulinum
neurotoxin (BoNT), which is most frequently secreted by
CMI
Clostridium botulinum [6]. Botulism can occur when BoNT-
producing clostridia colonize wounds or the intestine and
subsequently secrete the toxin, or when contaminated food is
ingested (foodborne) [32]. In 1986, the first infant botulism case
caused by a BoNT/E-producing C. butyricum strain was reported
in Italy [6]. Since then, C. butyricum has been associated with
botulism in other countries (i.e. China, India, Japan, Ireland, and
the USA) [32]. Notably, the operon encoding the BoNT/E toxin
harboured by the neurotoxigenic C. butyricum strains is very
similar to that carried by group II type E toxin-producing
C. botulinum strains [33]. The presence of the BoNT/E toxin
gene within either plasmids or the chromosome in different
Clostridium species is consistent with horizontal transfer events
mediated by plasmids or phage, and recombination events
mediated by mobile genetic elements such as transposons [33].
NEC
NEC is a devastating gastrointestinal disease causing high
morbidity and mortality, affecting predominantly preterm neo-
nates during outbreaks [34]. Its clinical presentation is charac-
terized by abdominal distension, gastrointestinal bleeding, mucosal
ulcerations and necrosis, portal venous gas, and pneumatosis
intestinalis, with different degrees of severity [34]. Despite de-
cades of research, the pathogenesis of NEC remains elusive.
Although no aetiological microorganism has been definitively
established, the most often implicated bacteria have been Clos-
tridium species [35]. Howard et al. first described an association
between C. butyricum and NEC in 1977 [7]. They identified
C. butyricum in blood and stool cultures in nine of ten preterm
neonates during an outbreak of NEC. In another study, Gorham
et al. reported the presence of C. butyricum on the hands of
members of the medical and nursing staff during an NEC outbreak
[36]. This was consistent with the effectiveness of preventive
measures in controlling such outbreaks [37]. Additionally, Sturm
et al. demonstrated a cytotoxic effect of the supernatant of a
C. butyricum strain isolated from a preterm neonate with NEC [38].
Recently, Smith et al. detected the presence of C. butyricum with
high density in two surgical samples from preterm neonates with
NEC. Both specimens were characterized by histological pneu-
matosis intestinalis [39]. In a previous study, we analysed 30 stool
samples from preterm neonates with and without NEC by using
16S rRNA pyrosequencing and culture-based methods, and 163
samples by using C. butyricum qPCR [17]; C. butyricum was specif-
ically associated with NEC, and culture supernatants of
C. butyricum strains from preterm neonates with NEC showed
significant cytotoxic activity.
Genome sequencing allowed the identification of various
toxin genes present in these pathogenic C. butyricum strains
[17,40]. We previously identified, in NEC-associated
C. butyricum strains, four genes encoding polypeptides that are
Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, 22, 37–45
Cassir et al. Clostridium butyricum antagonist effects 39
very similar to haemolysins shared by Brachyspira hyodysenteriae,
the aetiological agent of swine dysentery [41]. Among those
haemolysins, the pore-forming β-haemolysin has been
described as the main virulence factor capable of inducing
enterocyte necrotic lesions via the culture supernatant [42,43].
Interestingly, the polypeptide encoded by the β-haemolysin
homologue gene was identified in the secretome of an NEC-
associated C. butyricum strain, confirming expression of this
potential toxin [17]. However, the involvement of this toxin in
the pathophysiology of NEC needs further confirmation.
Experimental studies showed the capacity of C. butyricum to
reproduce NEC-like lesions in animal models. Popoff et al.
developed an experimental chicken model for C. butyricum
caecitis with lesions mimicking neonatal NEC by using a human
NEC strain [44]. In germ-free quails, C. butyricum was shown to
be the Clostridium species inducing the increased occurrence of
NEC-like caecal wall lesions and the only Clostridium species
inducing pneumatosis intestinalis, preceded by high levels of
butyric acid production [31]. In this study, when quails were fed
a diet without lactose, no lesions were observed. Interestingly,
like preterm neonates, birds show low endogenous lactase
activity, and their caeca have a physiological stasis favouring
overgrowth with lactose-fermenting bacteria [45]. Among the
other Clostridium species inducing NEC-like lesions,
C. perfringens and Clostridium paraputrificum were those that
were also involved in human NEC cases [35,39,46]. Other
experimental findings showed that C. butyricum could play a
primary role in the pathogenesis of NEC via the fermentation of
carbohydrate products, depending on the lactase deficiency of
preterm neonates [47,48]. By using a Caco-2 cell monolayer
model of the intestinal barrier, Peng et al. showed that butyrate
induced apoptosis and reduced the number of viable Caco-2
cells in a dose-dependent manner [49]. Additionally, Bousse-
boua et al. showed that lactose fermentation was a prerequisite
for the expression of C. butyricum enteropathogenicity.
Numerous gas cysts were always observed in the caecal wall of
quails mono-associated with C. butyricum [50]. Neuraminidase
production has also been described as a major virulence factor
in C. butyricum strains causing NEC in preterm neonates [51].
Azcarate-Peril et al. demonstrated, in an experimental piglet
model of NEC, that the occurrence of disease was associated
with significantly higher loads of C. butyricum, as ascertained by
qPCR in ileal mucosa samples, and overall bacterial dysbiosis
[52]. These results suggest that lactose fermentation could be a
prerequisite for the pathological changes observed in NEC
patients, and stress the roles played by both the strain and the
host in the expression of C. butyricum enteropathogenicity.
Further studies are needed to confirm the toxigenic mechanism
by which C. butyricum is involved in the pathogenesis of NEC.
Given that C. perfringens and the new species Clostridium
40 Clinical Microbiology and Infection, Volume 22 Number 1, January 2016
neonatale have also been associated with hospital outbreaks of
NEC [46,53], the potential involvement of other Clostridium
species represents an exciting area of future research.
Other conditions
A new pathogenic C. butyricum strain (NOR33234) has recently
been isolated from an elderly patient with antibiotic-associated
diarrhoea; tests for C. difficile toxins gave negative results [40].
Among the proteins annotated from genome sequencing of this
strain, there was an enterotoxin (OA81_00270). Moreover, two
annotated proteins had sequence similarity to phage holins in
C. botulinum. A previous report has shown that holin-like tcdE is
required for exporting enterotoxins tcdA and tcdB in C. difficile
[54]. Whether the enterotoxin or holin plays a pathogenic role
in C. butyricum infection remains to be examined.
To the best of our knowledge, only one case has been re-
ported of C. butyricum bacteraemia and sepsis, which occurred
in an injecting drug user [55].
Complementary tools for identification
Detection of bacteria from stool samples is challenging, given
that the human gut contains approximately 1014 bacteria [56],
and that, for a single person, the number of different phylotypes
has been estimated to be at least 130–200 by molecular
methods [57]. Clostridia belong to a complex phylogenetically
heterogeneous group, with some clusters having high genetic
strain diversity [58]. Depending on the microbiological method
used, the species may be incorrectly identified. For example, a
strain was recently isolated and identified by biochemical
analysis (VITEK2) as Clostridium clostridioforme, whereas 16S
analysis identified the same sample as C. butyricum with 99.0%
nucleotide identity, within the species definition threshold [40].
The following two sections discuss the available culture-
independent and culture-based methods.
Culture-independent analysis
Although 16S rRNA gene pyrosequencing is now widely used
in microbial ecological analysis, expanding the depth of
detection within gut microbiota, it is also hampered by several
biases [15]. First, automated taxonomic assignment and the
short length of the sequences amplified allow only limited
identification from phylum to genus [59]. However, in a pre-
vious study, we showed that it was possible to optimize the
assignment at species level for each separate read, by using
BLAST similarity searches against the National Center for
Biotechnology Information database. Species level was defined
with a minimum sequence identity of 98.7% as the best BLAST
hit [17]. Second, it has recently been shown that molecular
Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, 22, 37–45
CMI
studies overlooked Gram-negative populations as well as mi-
nority species [60]. Enteropathogens that can be present with
a subdominant status (<105 bacteria/mL) may therefore be
neglected, adding to the difficulty in achieving clear, repro-
ducible detection [15]. Third, microbial diversities measured
in the same sample and relative taxon abundances may differ
with the DNA extraction methods, targeted 16S rRNA re-
gion(s), and sequencing technology [15]. Although the V6
region is only 58 bp in length, it shows considerable sequence
variability, and appears to be the best region for distinguishing
Clostridium species [61]. We have recently analysed faecal
samples from preterm neonates by using 16S rRNA (V6) gene
sequencing, and identified, for the first time, C. butyricum in 12
of 15 samples from patients with NEC [17]. The high het-
erogeneity of results obtained by previous studies character-
izing the bacterial profile associated with NEC may, in part, be
explained by the use of different molecular methods for bac-
terial identification, including the target 16S rRNA region
(Table 1). Finally, 16S rRNA gene sequencing results are only
semiquantitative, and cannot distinguish between living and
dead bacteria [62]. This is a major limitation when the clinical
relevance of the results obtained with molecular methods is
considered.
Culture-based analysis
At the strain level, clones with high virulence and conta-
giousness could emerge among populations of pathogenic and
non-pathogenic species [63]. So far, despite all of the recent
technological advances in molecular biology, pure culture is
the only way to characterize the physiological properties of
living bacteria and to assess their potential for virulence at the
strain level [64]. Despite this, culture methods have rarely
been used in studies analysing the bacterial profile associated
with NEC (Table 2).
In the 1970s, the application of anaerobic conditions to the
culture of gut microorganisms enabled the recovery of a large
variety of anaerobes, including clostridia, from clinical samples
[65]. With the advent of metagenomics, molecular tools
replaced culture methods in microbial ecological analysis; those
were considered to be time-consuming, to be fastidious, and to
lack sensibility [15]. Regarding anaerobes, Hayashi et al. stressed
the need to reintroduce culture methods [66]. They compared
gut bacterial diversity assessed by 16S rRNA gene sequencing
with that assessed by anaerobic culture, and identified compa-
rable numbers of phylotypes or species with both methods, with
imperfect overlap. Goodman et al. later demonstrated that the
use of straightforward anaerobic culture conditions made it
possible to enlarge the repertoire of cultivable bacteria from the
gut [67]. In 2012, the new approach known as ‘microbial cul-
turomics’ (large-scale culture conditions with identification of
 CMI
TABLE 1. Gut microbiota profile associated with necrotizing enterocolitis (NEC) (case–control studies), analysed with culture-independent methods

Culture- Number of
independent Region patients Sample
Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, 22, 37–45

analysis (16S rRNA) (NEC/controls) collection Location Phylum level Genus/family level Species level Diversity Reference

454-pyrosequencing V6 15/15 Stool; NEC onset 3 NICUs, South-eastern No difference No difference Clostridium butyricum Lower Cassir, 2015 [17]
France (12 vs. 2)
V3–V5 12/26 Stool; daily until 1 NICU, Boston, USA NA Clostridium associated with NA Lower Zhou, 2015 [70]
discharge NEC in early-onset cases
and with
Gammaproteobacteria in
late-onset cases
V1–V3 21/74 Stool; 0–5 days 1 NICU, Louisiana, USA Lower prevalence and abundance Lower abundance in NA Lower McMurtry, 2015 [71]
before NEC onset in Actinobacteria and clostridia Veillonella and
Streptococcus
V3–V5 12/44 Stool; weekly until 1 NICU, London, UK No difference Clostridium associated with Clostridium OTU in 4 No Sim, 2014 [46]
NEC onset NEC NEC patients; difference
Klebsiella OTU in 7
NEC patients
V2–V4 16/10 Intestinal samples 1 NICU, Pittsburgh, USA No difference Clostridium and Bacteroides Clostridium perfringens Lower Brower-Sinning,
enrichment in 3 NEC samples; 2014 [72]
Clostridium
paraputrificum in 2
NEC samples
V3–V4 5/5 Stool; weekly until 1 NICU, Chicago, USA Proteobacteria increase; Firmicutes Enterobacteriaceae increase; NA Lower Claud, 2013 [73]
NEC onset decrease Veillonellaceae decrease
V3–V5 11/21 Stool; NEC onset 2 NICUs, Cincinatti, USA Increased Firmicutes in 4 patients Propionibacerium lack; NA Lower Morrow, 2013 [74]
with NEC increased
Enterobacteriaceae in 7
patients with NEC
V1–V3 9/9 Stool; 1 week and 3 NICUs, Florida, USA Bloom of Proteobacteria and Enterobacteriaceae NA NA Mai, 2011 [75]
72 h before NEC Firmicutes decrease in NEC cases sequences present more
onset between the 1-week and 72-h frequently
samples
V1–V2 4/6 Stool; 1 week before 1 NICU, Florida, USA No difference Increased abundance of Citrobacter-like species NA Mshvildadze,

Cassir et al.
NEC onset Enterococcus in 3 NEC samples 2010 [76]
Genome-resolved — 3/4 Stool; NEC onset 1 NICU, Pittsburgh, USA No difference No difference No difference NA Raveh-Sadka,
analysis 2015 [77]
454-pyrosequencing; V1–V3 18/35 Stool; weekly until 3 NICUs, Florida, USA Proteobacteria and Actinobacteria No difference Klebsiella granulomatis, No Torrazza, 2013 [78]
Bifidobacterium NEC onset increase; Bacteroidetes decrease Klebsiella difference
qPCR pneumoniae,
Clostridium
perfringens and

Clostridium butyricum antagonist effects

Staphylococcus
epidermidis more
frequent in NEC
samples
PCR-DGGE V3 8/17 Stool samples before 1 NICU, Newcastle, UK No difference Enterobacter, Flavobacterium, NA NA Stewart, 2012 [79]
and after NEC onset Propionibacterium and
Staphylococcus more
frequent
PSQMA- V1 24/0 Intestinal samples 1 NICU, Copenhagen, Proteobacteria (49.0%), Firmicutes Ralstonia most frequent; Clostridium butyricum NA Smith, 2011 [39]
pyrosequencing; Denmark (30.4%), Actinobacteria (17.1%) Clostridium in 4 samples and Clostridium
FISH and Bacteroidetes (3.6%) paraputrificum with
high density in 2
NEC samples with
Pneumatosis
intestinalis
PCR and T-RFLP V1–V6 10/10 <1 day after NEC 1 NICU, Chicago, USA Proteobacteria increase; Firmicutes Increased abundance of NA Lower Wang, 2009 [80]
analysis onset decrease Gammaproteobacteria
PCR-TTGE — 3/9 Stool samples at 7 1 NICU, Nantes, France NA Clostridium only in patients NA Lower De la Cochetière,
and 14 days of life with NEC 2004 [81]

DGEE, denaturing gradient gel electrophoresis; FISH, fluorescence in situ hybridization; NA, not available; NICU, neonatal intensive-care unit; OTU, operational taxonomic unit; qPCR, real-time PCR; T-RFLP, terminal restriction fragment length
polymorphism; TTGE, temporal temperature gradient electrophoresis.

41
42 Clinical Microbiology and Infection, Volume 22 Number 1, January 2016 CMI

TABLE 2. Gut microbiota profile associated with necrotizing enterocolitis (NEC) (case–control studies), analysed with
culture-dependent methods

Methods for Anaerobic Number of

bacterial culture patients Sample Genus/family Species
identification conditions (NEC/controls) collection Location level level Reference

MALDI-TOF MS; Anaerobic chamber; 15/15 <1 day after 4 NICUs, No difference Clostridium butyricum Cassir, 2015 [17]
16S rRNA PCR anaerobic blood NEC onset South-eastern (14 vs. 2)
culture bottles; France
anaerobic agar
MALDI-TOF MS Anaerobic conditions 12/44 1 stool sample per 1 NICU, London, — Clostridium perfringens Sim, 2014 [46]
(unspecified); alcohol week until NEC UK type A (β2 toxin gene)
shock; anaerobic agar onset in 4 NEC patients
API kit Anaerobic conditions 8/17 Stool samples 1 NICU, Newcastle, — CoNS more frequent Stewart,
(unspecified); before and after UK 2012 [79]
anaerobic agar NEC onset
API kit, gas Anaerobic conditions 24/41 <1 day after NEC 1 NICU, Rotterdam, Increased No difference Westra-Meijer,
chromatography (unspecified); onset The Netherlands abundance 1983 [82]
anaerobic agar of Klebsiella

CoNS, coagulase-negative staphylococci; NICU, neonatal intensive-care unit; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

FIG. 1. The ambiguous roles of Clostridium butyricum in its host. Some C. butyricum strains have been reported to be pathogenic, expressing virulence
factors (i.e. toxins such as enterotoxins or botulinum neurotoxin; enzymes such as neuraminidase; adhesion molecules; and secretion of high levels of
butyric acid). Other C. butyricum strains have been reported to be beneficial, expressing host protective factors (i.e. bacteriocins such as butyricin,
pathogen inhibition, and secretion of butyric acid).
Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, 22, 37–45
CMI
colonies by the use of matrix-assisted laser desorption ionization
time-of-flight mass spectrometry or 16S rRNA PCR) provided
results comparable to those obtained with molecular methods,
in terms of diversity [16]. However, the 15% overlap found in
the inaugural study demonstrates that, like molecular techniques,
culture is far from identifying the totality of gut bacteria, and that
complementary approaches are therefore required.
Cultivation of strictly anaerobic species is challenging.
Although they are thought to be the most common microor-
ganisms in the human microbiota, many anaerobic bacteria
detected in stool samples with metagenomic methods remain
uncultured [68]. In particular, only a few studies analysing the gut
bacterial profile associated with NEC have used strategies to
optimize the growth of strict anaerobic bacteria (i.e. heat shock
for sporulating anaerobes, direct inoculation in anaerobic culture
bottles, selective culture media, and the use of an anaerobic
chamber) (Table 2). Promising strategies, such as a reduction in
the sample transport time before inoculation and the use of
antioxidant agents such as ascorbic acid and glutathione, will
dramatically improve the culture of anaerobic bacteria, including
potential new emerging enteropathogens [69].
Conclusions
C. butyricum has various implications for human health, from
beneficial to pathogenic (Fig. 1). Whereas a non-toxigenic strain
has been validated in clinical practice for its probiotic properties,
other strains have been linked with pathological conditions such
as botulism in infants or NEC in preterm neonates. As toxigenic
and non-toxigenic clostridia form part of the normal gut
microbiota, it remains challenging to understand the triggers of
the expression of their beneficial or virulence factors. The fact
that different strains within the same species may have antagonist
effects on human health reinforces the need to accurately
identify gut bacterial species at the strain level. To date, bacterial
culture is the only method that fulfils this criterion, which is also
essential to discover new emerging enteropathogenic strains.
Transparency declaration
The authors declare that they have no conflicts of interest.
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