nutrients

Review
Therapeutic Microbiology: The Role of
Bifidobacterium breve as Food Supplement for the
Prevention/Treatment of Paediatric Diseases
Nicole Bozzi Cionci, Loredana Baffoni, Francesca Gaggìa and Diana Di Gioia *
Department of Agricultural and Food Sciences (DISTAL), Alma Mater Studiorum—Università di Bologna,
Viale Fanin 42, 40127 Bologna, Italy; nicole.bozzicionci@unibo.it (N.B.C.); loredana.baffoni@unibo.it (L.B.);
francesca.gaggia@unibo.it (F.G.)
* Correspondence: diana.digioia@unibo.it; Tel.: +39-051-209-6269




Received: 15 October 2018; Accepted: 8 November 2018; Published: 10 November 2018


Abstract: The human intestinal microbiota, establishing a symbiotic relationship with the host, plays a
significant role for human health. It is also well known that a disease status is frequently characterized
by a dysbiotic condition of the gut microbiota. A probiotic treatment can represent an alternative
therapy for enteric disorders and human pathologies not apparently linked to the gastrointestinal
tract. Among bifidobacteria, strains of the species Bifidobacterium breve are widely used in paediatrics.
B. breve is the dominant species in the gut of breast-fed infants and it has also been isolated from
human milk. It has antimicrobial activity against human pathogens, it does not possess transmissible
antibiotic resistance traits, it is not cytotoxic and it has immuno-stimulating abilities. This review
describes the applications of B. breve strains mainly for the prevention/treatment of paediatric
pathologies. The target pathologies range from widespread gut diseases, including diarrhoea and
infant colics, to celiac disease, obesity, allergic and neurological disorders. Moreover, B. breve strains
are used for the prevention of side infections in preterm newborns and during antibiotic treatments
or chemotherapy. With this documentation, we hope to increase knowledge on this species to boost
the interest in the emerging discipline known as “therapeutic microbiology”.

Keywords: Bifidobacterium breve; probiotics; paediatrics; therapeutic microbiology

1. Introduction
The use of microorganisms to treat or prevent targeted diseases was conceived at the end of the
last millennium. This concept has rapidly evolved giving rise to a new branch of applied microbiology
known as “therapeutic microbiology” [1]. Since human organisms and gut microbiota establish an
intimate symbiotic relationship that is fundamental for the maintenance of the host’s health, the
administration of beneficial microorganisms may represent a key determinant of the general health
status and diseases susceptibility. The choice for the most suitable species for a certain pathology
requires extensive studies, both in vitro and in vivo. Moreover, it is known that strains belonging
to the same species may express different functions in vivo [2]. It has also been demonstrated that
blending different microbial strains, species or even genera, may lead to a final effect that is not
predicted by results from using each single microorganism. Several Bifidobacterium species are largely
used as probiotics for their capability of reaching and colonizing the gastrointestinal tract and their
documented history of safety. Among them, Bifidobacterium breve, originally isolated from infant faeces,
represents one of the most used probiotics in infants. The multiple studies in which B. breve strains have
been successfully used in diseased humans, especially children and newborns, witness the potentiality
of strains belonging to this species for the prevention or treatment of human diseases. The aim of this

Nutrients 2018, 10, 1723; doi:10.3390/nu10111723 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 1723 2 of 27

review is to show the various applications of B. breve for preventing and treating paediatric diseases
starting from in vitro and mice model assessment of efficacy to the clinical use. To the best of our
knowledge, this work represents the first collection of works focused on the application in paediatrics
of strains belonging to the B. breve species and is aimed to shed light on the role of this Bifidobacterium
species in the scenario of “therapeutic microbiology.” Moreover, this paper explores the effectiveness
of B. breve used both as a single strain and combined with other microorganisms with a final short
outcome of its application in adulthood.

2. The Human Intestinal Microbiota

The human intestinal microbiota is a complex ecosystem that includes not only bacteria but
also fungi, Archaea, viruses and protozoans; bacteria concentration increases from the stomach and
duodenum throughout the intestinal tract and in the large intestine it rises to 1011 –1012 CFU/g of lumen
content [3]. It has been estimated that at least 1800 genera and a range of 15,000–36,000 bacterial species,
depending on whether species are conservatively (97% OTUs) or liberally (99% OTUs) classified, can
be found in the large intestine [4].
The symbiotic mutualistic relationship that the gut microbiota establishes with the host exerts
several beneficial roles, the main of which are the maintenance of the gut epithelial barrier, the inhibition
of pathogen adhesion to intestinal surfaces, the modulation and proper maturation of the immune
system, the degradation of otherwise non-digestible carbon sources such as plant polysaccharides
and the production of different metabolites including vitamins and short chain fatty acids (SCFAs) [5].
Furthermore, intestinal microorganisms seem to be responsible for a bidirectional interaction between
the gut and the Central Nervous System (CNS) via the gut-brain axis [6]. Dysfunction in this
interaction may be implicated in the development and prognosis of some neurological diseases,
including autism [7], multiple sclerosis [8] or Parkinson disease [9]. Because of this symbiotic
relationship, the human organism can be seen as a “superorganism,” which consist of not only
the microbial cells but also their genomes, that is, the microbiome and the related microproteome
and micrometabolome [10]. The microbiome represents more than 100 times the human genome
(1,000,000 genes vs. 23,000 genes) [10]. Indeed, the gut microbiome is influenced by external factors,
such as diet, health status and xeno-metabolome. These factors shape the individual intestinal
microbiota that can be considered as a “fingerprint” of the hosting organism.
Recently, the realization of global-collaborative projects has enriched the knowledge about the gut
microbiota, such as the MetaHit project [11], the Human Microbiome project [12] and the MyNewGut
project [13]. Moreover, the large amount of data from high throughput gene sequencing technology
has allowed us to gain deeper insights in the composition of the “typical” human gut microbiota. The
two principal bacterial phyla are Firmicutes and Bacteroidetes, followed by Actinobacteria, Proteobacteria
and Verrucomicrobia. Fungi and Archaea constitute approximately 1% of the species of the intestinal
microbiota [14,15]. The predominant fungal phyla are Ascomycota and Basydiomicota; some of the most
abundant genera, that is, Saccharomyces, Debaryomyces and Kluyveromyces, are found in food, confirming
the influence of diet habits also on the fungal intestinal population [16]. From the 80s some archaeal
species belonging to Methanobrevibacter genus have been identified. Methanobrevibacter is the only
genus detected in the gut probably due to the use of 16S primers not having sufficient resolution for
Archaea. Within this genus, the species’ composition depends on diet and host’s health status, as for
the entire microbiota [17,18].
Microbiologists’ attention has been also focused on bacteriophages, which, living at bacteria
expense and being vehicles of genetic transfer, could have an important role in shaping the biodiversity
of the gut ecosystem. The first metagenomic analysis of an uncultured viral community from human
faeces using partial shotgun sequencing suggested a large diversity of phages in gut microbiota [19].
The same authors investigated the viral community in the infant intestine using metagenomic
sequencing: 72% of the detected viral community resulted to be siphoviruses and prophages and over
25% resulted to be phages that infect lactic acid bacteria; faecal viral sequences were not identified
Nutrients 2018, 10, 1723 3 of 27

in breast milk, suggesting a non-dietary initial source of viruses [20]. The entire viral community
composition changed dramatically between the first and the second week of age [20], remaining then
stable during host’s life [21].
Gut colonization begins at birth, although recent evidences suggest the existence of an intrauterine
transmission of maternal bacteria to the foetus [22]. The first colonizer are facultative anaerobes
(Staphylococcus spp., Enterobacteriaceae and Streptococcus spp.), followed by strict anaerobes, such as
members of Bifidobacterium, Bacteroides and Clostridium genera [23,24]. The mode of delivery exerts a
strong influence on the first microbial colonization of newborns’ gut. Children born by natural delivery
have an intestinal microbiota profile similar to their mother’s vaginal one, characterized by Lactobacillus
and Prevotella spp., while children born by caesarean section develop a microbiota similar to that of
mother’s skin (Streptococcus, Corynebacterium and Propionibacterium spp.) [25]. In addition, the type of
feeding has a crucial role on the colonization of microbial groups in the gut. Indeed, the gut microbiota
of formula-fed infants contains a higher amount of Escherichia, Veillonella, Enterococcus and Enterobacter
members and the concentration of Lactobacillus and Bifidobacterium is lower with respect to in breast-fed
infants [26]. The abundance of these genera can be due to a more acidic pH in the colon of breast-fed
infants [27]. The prevalence of bifidobacteria in breast-fed infants is also due to their capability of
fermenting oligosaccharides (referred to as human milk oligosaccharides, HMO) [28]. Diet continues
to exert a crucial influence in the gut microbiota composition also in adulthood: De Filippis et al. [29]
showed an association between plant-based diet and a prevalence of Lachnospira and Prevotella and
a positive correlation between Ruminococcus and omnivore diet. Animal-based diets increase the
abundance of bile-tolerant microorganism (Alistipes, Bilophila and Bacteroides) and decreases the levels
of Firmicutes [30].
The use of antibiotics influences the gut microbiota composition, determining a significant
decrease of the microbial diversity in the digestive tract [31,32]. However, the microbiota is a resilient
system and tends to return to the pre-treatment state within 1 to 2 months after the end of the
administration [33]. Moreover, the use of perinatal antibiotics, such as in the intrapartum prophylaxis,
influences the establishment of a normal gut microbial composition and function, in particular reducing
the levels of bifidobacteria and increasing potential pathogens [34–36].
It is well established that a functional and balanced microbiota reflects a healthy condition of the
host; on the other hand, an unhealthy status may be associated with a compromised gut microbiota
displaying a decrease of beneficial bacteria and increase of harmful ones.

3. Probiotics with a Special Emphasis on Bifidobacterium breve

“Probiotic” means “for life” and it is currently used to name bacteria associated with beneficial
effects for humans and animals. In 2001 the Food and Agriculture Organization of the United
Nations (FAO)/World Health Organization (WHO) defined them as “live microorganism which,
when administered in adequate amounts confer a health benefit on the host” [37]. This definition has
been revised in 2014 by the International Scientific Association for Probiotics and Prebiotics, including
in the term probiotic “microorganism for which there are scientific evidence of safety and efficacy” and
excluding “live cultures associated with fermented foods for which there is no evidence of a health
benefit” [38].
Probiotics that have been largely studied in humans include species of the Lactobacillus and
Bifidobacterium genera. Probiotic administration in the first stage of life results to be more effective
in prevention and treatment of disorders, leading to a correct microbial colonization when the gut
microbiota is still in a period of establishment. Several studies have shown the beneficial effects of
Lactobacillus reuteri, one of the most used probiotics in infants, for the prevention and treatment of
infant gastrointestinal disorders, including colics, regurgitation, vomit, constipation [39–41]. This
species has been demonstrated to improve symptoms and reduce the number of anaerobic Gram
negative bacteria, Enterobacteriaceae and enterococci in colicky infants [42,43]. Furthermore, L. reuteri
ATCC 55730 was effective in children with distal active ulcerative colitis (UC) improving mucosal
Nutrients 2018, 10, 1723 4 of 27

inflammation and modulating mucosal expression levels of some cytokines involved in the bowel
inflammation [44]. Lactobacillus and Saccharomyces strains (L. casei CG, L. reuteri ATCC 55730 and a
strain of S. boulardii) exerted positive effects as supplement for rehydration therapy for infectious
diarrhoea in children by reducing the duration and stool frequency [45].
Several data are available for the use of bifidobacteria as probiotics for therapeutic purposes in
infants [46]. As an example, Bifidobacterium strains belonging to the animalis (BB-12 strain) and
longum species proved their efficacy against acute rotavirus diarrhoea in hospitalized children,
particularly by increasing the immune response and decreasing duration of disease [47–49]. In addition,
administration of Bifidobacterium bifidum and B. animalis strains in preterm and low birth weight infants
demonstrated clinical positive effects for treatment of necrotizing enterocolitis (NEC) [50–52].
Among the different species belonging to the Bifidobacterium genus, Bifidobacterium breve, is the
dominant one in breast-fed newborns [53] and one of the most used in infants. The species B. breve was
firstly described by Reuter [54], who isolated from breast-fed infant faeces and named seven species of
Bifidobacterium, including B. parvulorum and B. breve. The two species were then combined under the
name of B. breve [55]. B. breve strains are also found in the vagina of healthy women [54]. Their presence
in extra-body environments is a consequence of faecal contamination and the species is a useful
indicator of human and animal faecal pollution [56]. B. breve, like other Bifidobacterium species, possess
an array of enzymes for the utilization of different carbohydrates. These enzymes, useful to adapt
and compete in an environment with changing nutritional conditions, are inducible in the presence
of specific substrates. Amongst them, glycosidases, neuraminidases, glucosidases, galactosidase
are included as well as extracellular glycosidases that degrade intestinal mucin oligosaccharides and
glycosphingolipids [57]. B. breve also possess a glucosidase with a β-D-fucosidase activity, useful for the
utilization of fucosilated HMO [58]. B. breve is included in the list of Qualified Presumption of Safety
(QPS) biological agents [59]. Furthermore, recent studies have shown that human milk, traditionally
considered as sterile, contains commensal, mutualistic and/or potentially probiotic bacteria for the
infant gut. Among the different Bifidobacterium species found in human milk, B. breve strains have been
detected with DNA-based techniques and also isolated and characterized [60]. These bacteria from
human milk rapidly colonize the newborn’s gut, protect the infant against infections and contribute to
the maturation of the immune system [60].
Early studies by Akiyama et al. [61] showed that B. breve administration soon after birth was
effective in developing a normal intestinal microbiota and, furthermore, B. breve showed a stronger
affinity for immature bowel than other species, such as B. longum, evidencing its strong capabilities as
probiotic. These achievements stimulated the development of further studies that gave new insights to
the importance of this species as probiotic in infants.
Aloisio et al. [62] screened 46 Bifidobacterium strains for their capability of inhibiting the growth of
gut pathogens including coliforms isolated from colicky infants. The most interesting strains belonged
to the B. breve species, namely B632 strain (DSM 24706), B2274 strain (DSM 24707) and B7840 strain
(DSM 24708). In addition to the antimicrobial activity against coliforms and other pathogenic bacteria,
the strains did not possess transmissible antibiotic resistance traits and were not cytotoxic for gut
epithelium, which are important pre-requisites for their use as probiotics. B. breve B632 was also able
to stimulate the activity of mitochondrial dehydrogenases of macrophages and the production of IL-6,
linked to a considerable activation of macrophages and endothelial cells in inflammatory condition.
The potential of B. breve B632 as probiotic was also evidenced by Simone et al. [63]: it was able to inhibit
the growth of Enterobacteriaceae in an in vitro gut model system stimulating the intestinal microbiota
of a 2-month colicky infant, supporting the possibility to move to an in vivo study. Another strain
of B. breve, BR03 (DSM 16604), revealed to be effective, as well as B632, in inhibiting the growth of 4
E. coli biotypes [64]. Mogna et al. [65] also underlined the validity of these two B. breve strains (B632
and BR03) in an in vivo study. The administration of both strains for 21 consecutive days as an oily
suspension (daily dose of 100 million live cells of each strain) to healthy children was effective in
obtaining gut colonization and in decreasing total faecal coliforms.
Nutrients 2018, 10, 1723 5 of 27

A biotechnological approach could improve the gastric transit survival, gastrointestinal

persistence and therapeutic efficacy of the strain B. breve UCC2003, isolated from infant stool, via
the heterologous expression of the listerial betaine uptake system gene, BetL [66]. In addition to the
improved capability of colonizing the intestine of inoculated mice, the strain was also able to reduce
Listeria proliferation in the organs of the infected mice. Although the introduction of genes from
pathogens into probiotic cultures is unlikely to meet approval from regulatory authorities, this study
underlined that probiotic characteristics can be susceptible to improvements. Future perspectives
include the obtainment of BetL homologues from Generally Recognized as Safe (GRAS) organisms
and natural selection of probiotic cultures with elevated expression of such homologues.
B. breve strain Yakult (BBG-01) is another widely used probiotic strain. It was one of the first
B. breve strain shown to possess the ability to modulate the intestinal microbiota by reducing the
count of several pathogenic bacteria, such as Campylobacter, Candida and Enterococcus spp., after oral
administration [67,68]. This strain has also displayed an anti-infective activity against
Shiga-toxin-producing E. coli (STEC) O157:H7 in infected mice [69].
For its valid properties as probiotic, B. breve has also found a notable place in food technology in
the fermentation of milk. In this regard, the positive effects associated to B. breve-fermented soymilk
has been reported in several studies, demonstrating to improve lipid metabolism, alcohol metabolism
and mammary carcinogenesis in mice models [70–72].
Moreover, a strain of B. breve has been included in a widespread of commercial high concentrated
probiotic preparation, known as VSL#3, which contains 1011 –1012 viable lyophilized cells of different
bacterial species that are usual component of human gut microbiota. Specifically, the formulation
contains four strains of Lactobacillus (L. paracasei, L. plantarum. L. acidophilus and L. delbrueckii subsp.
bulgaricus), three strains of Bifidobacterium (B. longum, B. breve and B. infantis) and one strain of
Streptococcus salivarius subsp. thermophilus. VSL#3 exhibited an immunomodulatory capacity in in vitro
studies by increasing the production of anti-inflammatory cytokines and inhibiting the production of
pro-inflammatory cytokines [73].

4. B. breve Effectiveness in Mice Models

The strong evidence of the immune modulating capability of B. breve strains has been consolidated
and well documented in a large number of animal models studies, which are the basis for human
clinical trials.
The oral administration of B. breve YIT4064 strain, isolated from faeces of a healthy breast-fed
infant, in mice immunized orally with an influenza virus was able to increase anti-influenza virus
IgG levels in serum, thus protecting mice against infection. The authors concluded that the oral
administration of this strain may enhance antigen-specific IgG against various pathogenic antigens
taken orally and induce protection against various viral infections [74]. This conclusion was also
supported by the study of Yasui et al. [75] that proved that the same strain stimulated anti-influenza
virus hemagglutinin IgA by Peyer’s patch cells in response to addition of hemagglutinins. These
antibodies may reach the mucosal tissue and prevent influenza virus infection.
B. breve UCC2003 possessed a cell surface exopolysaccharide (EPS) able to play an important
role in immunomodulation in B cell response. Administration for 3 consecutive days of EPS+ B. breve
strains in mice infected with Citrobacter rodentium, a diarrheagenic pathogen related to human E. coli, is
effective in reducing the pathogen colonization, differently from mice fed with EPS− B. breve [76]. EPS
was involved in the production of a biofilm on the gut epithelium [77] preventing the attachment of
C. rodentium.
Natividad et al. [78] illustrated the relationship between B. breve NCC2950 and regenerating (REG)
III proteins, molecules belonging to the family of C-type lectins, which are expressed in the intestine
and involved in maintaining gut homeostasis. The group REGIII-γ was measured in the ileum and
colon of germ-free (GF) mice, mice colonized with specific pathogen free (SPF) microbiota and with a
Nutrients 2018, 10, 1723 6 of 27

low diversity microbiota (altered Schaedler flora–ASF). Monocolonization with the probiotic B. breve
NCC2950 but not with the commensal E. coli JM83, significantly induced REGIII-γ expression.
B. breve MRx0004, isolated from faeces of healthy humans, possessed a protective action in a
severe asthma condition [79]. The study remarked an important decrease of neutrophil and eosinophil
infiltration in lung bronchoalveolar lavage fluid in a mouse model of severe asthma after the probiotic
treatment. This result, together with the demonstrated reduction of pro-inflammatory cytokines and
chemokines involved in neutrophil migration, showed that B. breve MRx0004 effectiveness in reducing
the above-mentioned inflammation condition paves the way for next-generation drug for management
of severe asthma.
Many B. breve strains played an important role in prevention and treatment of various allergy
conditions. Oral administration of B. breve M-16V, isolated from faecal sample of a healthy infant, in
ovalbumin (OVA)-immunized mice significantly reduced the serum levels of total IgE, OVA-specific
IgE and OVA-specific IgG1 and ex vivo production of IL-4 by the splenocytes [80]. Schouten et al. [81]
showed that an intervention with a synbiotic formulation, comprising B. breve M-16V and a GOS/FOS
mixture, was protective against the development of symptoms in mice orally sensitized with
whey. The promising effect was confirmed by Kostadinova et al. [82] demonstrating the partially
prevention of skin reaction due to cow’s milk allergy, following the probiotic administration in
combination with specific β-lactoglobulin—derived peptides and a specific blend of short- and
long-chain fructo-oligosaccharides in mice. Particularly, the treatment, besides increasing the cecal
content of propionic and butyric acid, determined an increase of IL-22 expression, which plays an
antimicrobial role in the innate immunity response and of the anti-inflammatory cytokine IL-10 in the
Peyer’s patches. This outcome agrees with Jeon et al. [83], who demonstrated that the administration
of the B. breve Yakult strain increased the number of IL-10-producing CD4+ T cells in the large intestine
of murine models and an increased production of acetic acid [69].
B. breve was also involved in protective mechanisms against obesity; the orally administration of
B. breve B-3 in a mouse model with diet-induced obesity could suppress the increase of body weight
and epididymal fat, with improved serum levels of total cholesterol, fasting glucose and insulin and
act by regulating gene expression pathways involved in lipid metabolism and response to stress in the
liver [84,85].
Increasing evidence suggests that a brain–gut–microbiome axis exists, although its role in
cognition remains relatively unexplored [6,86]. Bifidobacteria were found to improve the behavioural
deficits and to possess a potential action on stress-related disorders in model mice [87]. B. breve strains
potential has also been investigated for the capability of conferring beneficial effects on neurological
diseases. Savignac et al. [88] showed that 6 weeks feeding of B. breve 1205 strain resulted in positive
effects on compulsive behaviour in marble burying test, anxiolytic effects in the elevated plus maze
and reduced body weight gain in model mice, contributing to a general amelioration of anxiety and
metabolism. Kobayashi et al. [89] showed that oral administration of B. breve A1, isolated from faeces of
human infants, prevented cognitive decline in Alzheimer disease (AD) model mice, with a reduction of
neural inflammation; they observed that the probiotic provided ameliorations in both working memory
and long-term memory. Furthermore, they found an increase of plasma acetate levels after the probiotic
treatment and the neural inflammation reduction can be considered as a consequence of this increase
due to B. breve administration, since SCFAs have been shown to have immune modulatory functions
in model mice [90]. This evidence suggests that B. breve A1 has therapeutic potential for preventing
cognitive impairment in Alzheimer disease and the necessity to move to a clinical intervention to
evaluate the effects on diseased humans.
B. breve supplementation can affect the metabolism of fatty acids. Among them, eicosapentaenoic
acid (EPA), which derives from α-linolenic acid metabolization, is an essential constituent of the cell
membrane, plays an important role in brain and nervous system development and in inflammatory
response [91]; docosahexanoic acid (DHA), which derives from EPA metabolization, is one of the
major n-3 polyunsaturated fatty acids (PUFA) in the brain and is essential for a correct development
Nutrients 2018, 10, 1723 7 of 27

of foetal encephalon [92]. Some studies revealed that human commensal microorganisms are able
to synthetize bioactive isomers of conjugated linoleic acids (CLA) from free linoleic acid [93]; CLA
was proven
Nutrients to xpossess
2018, 10, FOR PEER antiatherosclerotic,
REVIEW antidiabetic and immunomodulatory properties [94,95]. 7 of 27
Wall et al. [96] demonstrated that oral administration for 8 weeks to different animals (pigs and mice)
of B. breve
levels. The NCIMB
same authors702258, a CLA producer
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liver.
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strain in the
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cytokines tumour necrosis being able(TNF-α)
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of brain-derived that a 8(BDNF),
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in development of
the nervous
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acid,[97,98]. Particularly,
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resulted treatment
in an increasereduced the EPA
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exon IV, which in hasmice.
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5. B.
of breve Application
brain-derived in Clinical
neutrophic Trials inaPaediatrics
factor (BDNF), neurotrophin involved in development of the nervous
systemThe [97,98].
use of Particularly, the probiotic
B. breve strains treatmentand
for treatment reduced the expression
prevention of human of BDNF exonhave
diseases IV, which
been
has been described as being highly responsive and increased by stress [99].
increasingly expanding in the last decade. Being bifidobacteria the most abundant bacterial group in
infant gut, most of the studies are focused on paediatric subjects. Figure 1 summarizes the main
5. B. breve Application in Clinical Trials in Paediatrics
applications of B. breve in paediatric diseases.
The use of B. and
Therapeutic breveprotective
strains forrole
treatment and prevention
for human health of B.ofbreve
human diseases
strains both have beenstrain
as single increasingly
or as a
expanding in the last decade. Being bifidobacteria the most abundant bacterial group
mixture of two strains of the same species has been demonstrated. As already mentioned, several in infant gut,
most of the studies
researchers accountareforfocused on paediatric
the improved subjects.
efficacy Figure 1 summarizes
of multi-species the main
and multi-strain applicationsthat
formulations of
B. breve in paediatric diseases.
acting with a synergic effect, may enhance the effectiveness of each single strain [100,101].

Figure
Figure 1. Paediatric diseases in which an amelioration of symptoms has been obtained upon B. breve
1. Paediatric breve
strains
strains administration.
administration.

Therapeutic
5.1. Preterm Infantsand
andprotective
Necrotisingrole for human
Enterocolitis health of B. breve strains both as single strain or as
(NEC)
a mixture of two strains of the same species has been demonstrated. As already mentioned, several
A consistent
researchers account number
for the of pretermefficacy
improved infants,ofespecially thoseand
multi-species of very low birth
multi-strain weight, arethat
formulations subjected
acting
to episodes of systemic infection caused by antibiotic resistant bacteria and fungi that can lead to
with a synergic effect, may enhance the effectiveness of each single strain [100,101].
chronic diseases and brain injuries [102,103]. These episodes can result from a combination of factors,
including immature gastrointestinal tract mucosal barrier and undeveloped gastrointestinal tract
immune system, which may predispose premature infants to bacterial translocation, causing
systemic infection and necrotising enterocolitis (NEC) [104,105]. In addition, preterm infants have
revealed an altered microbiota composition, resulting in almost undetectable bifidobacteria counts
during the first and second week of life, differently for those at term [106–108]. This observation has
Nutrients 2018, 10, 1723 8 of 27

5.1. Preterm Infants and Necrotising Enterocolitis (NEC)

A consistent number of preterm infants, especially those of very low birth weight, are subjected
to episodes of systemic infection caused by antibiotic resistant bacteria and fungi that can lead to
chronic diseases and brain injuries [102,103]. These episodes can result from a combination of factors,
including immature gastrointestinal tract mucosal barrier and undeveloped gastrointestinal tract
immune system, which may predispose premature infants to bacterial translocation, causing systemic
infection and necrotising enterocolitis (NEC) [104,105]. In addition, preterm infants have revealed
an altered microbiota composition, resulting in almost undetectable bifidobacteria counts during the
first and second week of life, differently for those at term [106–108]. This observation has allowed the
formulation of the hypothesis that a bifidobacteria treatment could lead to a reintegration of beneficial
bacteria in the intestinal environment and a reduction of bacterial translocation to other districts,
stimulating researches in this sector. One of the first study that investigated the effects of a B. breve
supplementation in preterm neonates reported that the strain YIT4010, administered as a suspension
of distilled water containing 0.5 × 109 bacterial cells for 28 days, was able to colonize efficiently the
intestinal tract, to reduce abnormal abdominal symptoms and to improve the weight gain [109]. A later
study compared the effects of the administration of a B. breve strain a few hours after birth and 24 h
after birth; the supplement was prepared by dissolving 1.6 × 108 cells in 0.5 mL of 5% glucose solution
and administered twice a day for all the duration of hospitalization [110]. In newborns administered
with the probiotic soon after birth, bifidobacteria were detected significantly earlier and the number of
Enterobacteriaceae at 2 weeks after birth was significantly lower, compared to the infants treated 24 h
after birth demonstrating that a very early probiotic intervention may contribute to the establishment
of a beneficial gut microbiota and the prevention of infectious diseases [110].
A more recent work proved the suitability of B. breve M-16V administration for routine use in
preterm infants in order to control the gut microbiota colonization and shift it towards a healthy
profile [111]. Moreover, a retrospective cohort study was performed with the purpose of evaluating
whether the supplementation with the same probiotic to preterm neonates would reduce the risk of
NEC [112]. NEC represents the most life-threatening pathology of preterm neonates with incidence
and mortality of 10–12% and 40–45%, respectively. It is characterized by gastrointestinal dysfunction
progressing to pneumatosis intestinalis, systemic shock and rapid death in severe cases [113,114].
NEC is categorized into 3 different stages based on the severity of the disease, from stage I,
a suspicion for disease, to stage III, corresponding to a severe progression of the disease [115].
Although the pathogenesis of this condition remains obscure, some important prevention strategies
have been adopted, such as the use of antenatal glucocorticoids, early preferential feeding with
breast-milk, prevention and treatment of infections [116]. Since preterm infants have shown an
intestinal reduction of total bifidobacteria and a predominance of facultative anaerobes, some of
which potentially pathogens, until the 20th day of life, it has been suggested that a major etiological
factor for NEC could be an altered microbiota composition [117]. Therefore, a probiotic treatment
can be an additional strategy for NEC prevention. A 3-week B. breve M-16V supplementation
(3 × 109 CFU/day) has been associated with a lower incidence of NEC (≥stage II) in very low
birth weight infants born before 34 weeks; the incidence in those born before 28 weeks resulted lower
but not statistically significant [112]. Satoh et al. [118] had already demonstrated the efficacy of B. breve
M-16V administration in preventing NEC in extremely low and very low birth weight infants: the
probiotic was daily supplemented at a dose of 1 × 109 CFU dissolved in breast milk or breast-mixed
with formula milk several hours after birth and continued until discharge from hospital (achievement
of body weight 2300 g or gestational age of 37 weeks); the treatment led to a significant reduction of
infection and mortality rate.
Various studies suggested that an overproduction of SCFAs in the intestinal environment can
lead to mucosal injuries, which may evolve in NEC in premature infants [119,120]. Wang et al. [121]
demonstrated that a 4 weeks B. breve M-16V supplementation (1.6 × 108 cells suspended in 0.5%
glucose solution) was associated with a reduction of butyric acid levels in very and extremely low
Nutrients 2018, 10, 1723 9 of 27

birth weight newborns. Since butyric acid increases the IL-8 secretion in enterocytes, condition that
may lead to neutrophil invasion, a known hallmark of NEC, B. breve administration can be considered
protective against NEC onset.
Immediately after delivery, some physiological changes, especially in the immunologic system,
occur in newborns in order to adapt themselves to the new environment. B. breve M-16V, administered
at 109 cells in 0.5 mL of 5% glucose solution starting several hours after birth, can increase the
transforming growth factor β1 (TGF-β1) signals in preterm infants [122]. This increase has a relevant
importance as it is known to induce oral tolerance, exert anti-inflammatory effects, express mucosal
IgA and promote epithelial cell proliferation and differentiation [123]. A further study investigated the
preventive effects of the same B. breve strain against infections and sepsis in extremely and very low
birth weight newborns. The probiotic consisted on a freeze-dried preparation with a dose of 109 CFU
dissolved in breast- or formula-milk; the development of infection and sepsis resulted significantly
lower in the supplemented group compared with the non-supplemented one [124], highlighting once
more the efficacy of a B. breve treatment in the prevention of developing infections, sepsis and NEC.
According to Braga et al. [125] the combined use of B. breve Yakult and L. casei was able to reduce
the occurrence of NEC and was associated with an improvement in intestinal motility in newborns.
The intervention started at the second day of life and continued for 30 days, provided L. casei and
B. breve mixed to human milk in a daily dosage of 3.5 × 107 and 3.5 × 109 CFU, respectively. The
number of NEC confirmed cases (≥stage II) was reduced upon probiotic treatment.

5.2. Gastrointestinal Disorders

A disorder that affects up to 30% of newborns in the first months of life is infant colic. It is
characterized by paroxysmal, excessive and incontrollable crying without identifiable causes [126]
representing a serious problem for the family and, in many cases, it can cause disorders later in
life [127,128]. The aetiology remains obscure but an unbalanced intestinal microbiota has been
suggested to play a role in the disease pathogenesis. Several studies support the use of probiotics
as therapeutic or preventive agent against colics but very few clinical trials have been performed on
bifidobacteria application. A mixture of B. breve strains (BR03 and B632), whose probiotic potential,
as already highlighted in Section 3, has been extensively demonstrated in vitro, was prepared as oily
suspension and administered at a daily dosage of 5 drops containing 108 CFU of each strain to 83 infants,
involving both breast and bottle-fed subjects [129]. Preliminary results showed that administration
was effective in reducing minutes of daily crying. The clinical trial was then completed (155 infants,
130 breast- and 25 bottle-fed), as described in Aloisio et al. [130]; the B. breve mixture was able to
prevent gastrointestinal disorders in healthy breast-fed infants, principally by reducing 56% of daily
vomit frequency, decreasing 46.5% of daily evacuation over time and improving stool consistency. The
strength of this study is the interrelation among a prolonged probiotic treatment, several clinical and
anthropometric parameters (e.g., crying time, stool frequency, colour and consistency, regurgitation,
vomits, weight, length, head circumference of newborn, delivery mode, type of feeding, gestational age)
and main gut microbial groups. Epidemiological data have shown the predisposition of neonates born
by caesarean section to develop obesity later in life [131,132]. However, the B. breve supplementation
in infants born by caesarean section [130] resulted in a lower catch-up growth in weight, thus allowing
the authors to speculate a protective effect of the probiotic strains against the risk to develop metabolic
disturbance later in life.
Another common disease in childhood related to the intestinal tract is functional constipation,
a chronic condition characterized by infrequent defecation (less than three times per week) and
more than two episodes of faecal incontinence per week [133]; the pathogenesis, undoubtedly
multifactorial, has not a well-defined aetiology. It has been shown that, despite intensive medical and
behavioural therapy, 25% of patients developing constipation before the age of 5 years continue
to have constipation upsets beyond puberty [134]. A pilot study showed the beneficial effects
of 4 weeks treatment with B. breve Yakult (BBG-01) in constipated children: daily administration
Nutrients 2018, 10, 1723 10 of 27

of 108 –109 CFU led to a significantly increase in defecation frequency and amelioration of stool
consistency, frequency of episodes of faecal incontinence and abdominal pain [135]. There is a debate
of whether it is more effective the use of single strains or an association of them for constipation
treatment; however, the mentioned study demonstrated that the intake of only one B. breve strain
is even effective. Giannetti et al. [136] investigated the effects deriving from the administration of a
mixture of 3 bifidobacteria, namely B. infantis M-63, B. breve M-16V and B. longum BB536, in children
suffering from irritable bowel syndrome (IBS). IBS is a functional bowel disorder characterized
by chronic abdominal pain, discomfort, bloating and altered bowel habits including diarrhoea or
constipation [137]. The daily dose was about 109 cells for each strain administered as bacterial powder
and the treatment lasted 6 weeks. The bifidobacteria mixture intake resulted in a significant decrease
in prevalence and frequency of abdominal pain and an improvement of the quality of life, assessed by
an interview-administered validated questionnaire.
The commercial formulation VSL#3, already described in Section 3, was used in several clinical
studies targeted to different diseases in paediatrics resulting in an amelioration of the health status of
children suffering from IBS [138]. In this randomized, double-blind, placebo-controlled, multicentre
trial, patients were treated with one sachet (twice in those 12–18 years old) of probiotic mixture
containing 4.5 × 1012 bacteria for 6 weeks. The preparation was effective in improving the overall
perception of symptoms, the severity and frequency of abdominal pain, abdominal bloating and family
assessment of life disruption, leading to a general improving of quality of life in children suffering
from IBS.
Miele et al. [139] carried out the first paediatric, randomized, placebo-controlled trial using VSL#3
for the treating of ulcerative colitis (UC). This disorder belongs to the chronic inflammatory bowel
disease (IBD) category, has a prevalence of about 100 cases per 100,000 children [140] and occurs as
diffuse mucosal inflammation in the colon; it is characterized by periods of remission and relapse
episodes, not all the patients tolerate the existing treatment to induce remission for their adverse effects
and in 20–30% of paediatric patients failure of the treatment occurs [141]. Since the pathogenesis, beside
genetic susceptibility, is linked to compromised immune response and alteration in gut microbiota
composition, the idea beyond the study was that 1 year of VSL#3 administration might improve the
health status of patients. Subjects with an average age of 10 were supplemented with a weight-base
dose of probiotic (4.5 × 1011 –1.8 × 1012 bacteria per day); treated patients showed a significantly
higher rate of remission compared to placebo and a significantly lower incidence of relapse within
1 year of follow-up. According to the authors, this success may be related to the use of a mixture of
various probiotics, which might have a strong synergic action and to the high bacterial concentration
of viable cells contained in the mixture. Furthermore, the probiotic preparation showed to be safe and
well tolerable by children with a diagnosis of UC.
The efficacy of VSL#3 in paediatric diseases was also evaluated by Dubey et al. [142], who
conducted a double-blind, randomized, placebo-controlled trial treating acute rotavirus diarrhoea in
children. VSL#3, containing a total of 9 × 109 bacteria/dose and administered for 4 days, significantly
reduced, already on day 2, mean stool frequency and improved stool consistency; these results were
also reflected in the lower volume of oral rehydration salts administered in children who received
the probiotic. The functional role of VSL#3 was investigated by Sinha et al. [143], who focused on the
prevention of neonatal sepsis in low birth weight infants, one of the infections which evolves more
rapidly in this paediatric category. The mixture, containing 109 bacteria/dose, was administered for
30 days. VSL#3 intake in low birth weight was associated with a non-significant 21% reduction in the
risk of suspected sepsis; nevertheless, in the sub-group of infants weighing 1.5–1.99 kg, the reduction of
the risk of suspected sepsis was statistically significant, differently from newborns weighing 2.0–2.49 kg.
The results of the study allowed to conclude that the intervention may be useful for the most vulnerable
subjects of low birth weight.
As infant feeding has a crucial role in developing infant gut microbiota and consequently intestinal
immunity, fermented formula milk containing probiotics or prebiotics has been developed. This
Nutrients 2018, 10, 1723 11 of 27

approach is aimed at protecting infants from various gastrointestinal disorders by modulating gut
microbial composition. The first study that evaluated the effects of a fermented formula milk with
B. breve C50 and Streptococcus thermophilus 065 on the incidence of acute diarrhoea in healthy infants
was a randomized, double-blind, placebo-controlled multicentre study, which involved 971 subjects
belonging to three different areas of France [144]. The trial was planned to occur in a high risk predicted
period for diarrhoea incidence in France (from October to January) and the supplementation lasted
5 months. Although no reduction in the incidence and duration of diarrhoea episodes were observed
after the intervention, a lower number of dehydration cases, a lower number of medical consultation
cases with fewer oral rehydration solution prescriptions and changes of formula were registered. These
outcomes can be considered as indicators of probiotic positive effects on the severity of the disease.
According to the authors, these results may be related to the bifidogenic and immunomodulatory
properties of fermentation products contained in formula-milk.

5.3. Celiac Disease

The efficacy of the probiotic mixture containing B. breve B632 and B. breve BR03 was also shown
in children affected by celiac disease. In this case, the strains were administered as lyophilized
powder at a daily dosage of 109 CFU of each strain for 3 months in celiac children on a gluten free
diet (GFD). A preliminary important outcome obtained from the intervention was the reduction of
pro-inflammatory cytokine TNF-α in blood samples of celiac children on GFD [145]. The gut microbiota
composition was also studied with Next Generation Sequencing (NGS) technology. Unexpectedly, the
intervention did not cause changes at the level of the genus or phylum to which the administered
probiotics belong but the probiotic acted as a “trigger” element for the increase of Firmicutes and the
restoration of the physiological Firmicutes/Bacteroides ratio that was altered in celiacs with respect to
healthy subjects. Moreover, the intervention restored the normal amount of Lactobacillaceae members,
reaching almost the same values of healthy subjects [146]. Besides modulating inflammatory condition
and gut microbiota composition of celiac children, B. breve supplementation influenced the SCFAs
profile; acetic acid had a negative correlation with Verrucomicrobia, Euryarcheota and particularly
Synergisestes [147]. Although Synergisestes is a minor phylum in human faeces (abundance of 0.01%) of
healthy subjects, it was found to have a considerable role for human health because of its negative
correlation with TNF-α that may indicate an anti-inflammatory role [148,149]. In the study of
Primec et al. [147], the Synergisestes phylum clearly confirmed its anti-inflammatory role negatively
correlating with pro-inflammatory acetic acid after three months of probiotic treatment.

5.4. Paediatric Obesity

Another pathology in which the gut microbiota may play a notable role is obesity. Although it
is accepted that obesity results from disequilibrium between energy intake and expenditure, it is a
complex disease and not completely understood. Nowadays, obesity prevalence is spreading especially
among children and adolescents and it can be considered a worldwide epidemic. Obesity has been
associated with a chronic inflammation that may conduct to insulin resistance [150,151]. Recently,
obesity has been associated with a specific profile of the gut microbiota characterized by lower levels of
bacteria belonging to Bacteroides and Bifidobacterium genera compared to that of lean individuals [152].
In addition, bifidobacteria were shown to be higher in children maintaining normal weight at 7 years
old than in children developing overweight and their administration was able to reduce serum and
liver triglyceride levels and to decrease hepatic adiposity [153,154]. The mixture of B. breve already
mentioned (BR03 and B632) was used in a cross-over double-blind randomized controlled trial in
order to re-establish metabolic homeostasis and reduce chronic inflammation in obese children [155].
Although the study is still on-going, preliminary results related to the part previous the cross-over
demonstrated that a B. breve administration in obese children is promising: 8 weeks treatment seems
to ameliorate glucose metabolism and could help in weight management by reducing BMI, waist to
height ratio and waist circumference [155].
Nutrients 2018, 10, 1723 12 of 27

5.5. Allergies
There are increasing evidences that the intestinal microbiota plays an important role in the
development of allergic diseases, in particular, low bifidobacteria levels appear to be associated with
atopic dermatitis [156]; in the previous section, the potential of B. breve in preventing and treating
allergy conditions was reported and this impressive role has been confirmed in clinical studies. B. breve
M-16V revealed to be effective in the treatment of cow’s milk hypersensitivity infants with atopic
dermatitis [157]. B. breve, added to the casein-hydrolysed milk formula at the dosage of 5 × 109 CFU
or 15 × 109 CFU per day, increased the proportion of bifidobacteria in the gut microbial composition
and ameliorated allergic symptoms by interacting with the immune system and no remarkable dose
dependent differences were detected [157]. The synergetic combination of probiotics and prebiotics,
known as synbiotic, seems also to be promising in atopic dermatitis treatment. In this regard,
Van der Aa et al. [158] studied the effects of a synbiotic mixture on atopic dermatitis in formula-fed
infants; the formulation consisted of B. breve M-16V at a dose of 1.3 × 109 CFU/100 mL and a mixture of
90% short-chain galactooligosaccharides (scGOS) and 10% long-chain fructooligosaccharides (IcFOS),
0.8 g/100 mL added to formula milk. Although the formulation, administered for 12 weeks, had
no effect on atopic dermatitis severity, it significantly modulated the composition and the metabolic
activity of gut microbiota, leading to a decrease of pH, high lactate and low butyric levels resembling
the metabolic profile of breast-fed infants [159]. The same synbiotic mixture has demonstrated to
reduce the prevalence of asthma-like symptoms and the prevalence of asthma medications use after
the fulfilment of a 1-year follow-up [160].
The effects of a formulation containing B. breve M-16V and B. longum BB536 for the prevention
of allergies in infants enrolling both mothers and newborns was studied [161]. The formulation was
provided as powder daily doses containing 5 × 109 CFU/g of each strain. Pregnant women begun the
supplementation 4 weeks before the expected date of delivery and the newborns received the probiotic
mixed to water, breast- or formula-milk starting 1 week after birth and continuing for 6 months. The
study revealed that prenatal and postnatal supplementation with a bifidobacteria mixture reduced the
risk of developing eczema and atopic dermatitis in infants. NGS analyses of newborns’ faecal samples
showed significant differences of the major intestinal microbial phyla (Actinobacteria, Bacteroidetes,
Proteobacteria) of allergic and non-allergic infants at 4 months of age. However, these differences were
lost at 10 months of age, highlighting that the microbiota of early stages is particularly important in
regulating allergies upset in infants.

5.6. Surgical Procedures

Surgical procedures can also alter gut microbiota composition and functions and disrupt intestinal
barrier function, inducing the patient in a condition at risk for infection [162]. A probiotic therapy
may be functional for patients improving the immunological function of the intestine and competing
against harmful bacteria infection. A pilot study demonstrated that daily administration of B. breve
Yakult BBG-01 (109 freeze-dried cells per day) to children younger than 15 years 7 days before
surgery until discharge from hospital, simultaneously to intravenous antibiotics postoperatively
treatment, reduced the incidence of bacteria in blood samples. Moreover, the intestinal microbial
composition was improved by increasing Bifidobacterium spp. and reducing potential pathogens such
as Clostridium difficile, Pseudomonas and Enterobacteriaceae. Higher concentrations of faecal acetate and
lower faecal pH levels were detected in children who received the probiotic 2 weeks after surgery [163].
Improvement of intestinal environment resulting from a perioperative supplementation with the same
strain was also observed in neonates undergoing surgery for congenital heart disease [164]. Daily
dosage of 3 × 109 CFU of B. breve Yakult (BBG-01) was administered starting 1 week before surgery and
ending 1 week after the operation; infants who received the probiotic supplement showed significantly
higher bifidobacteria levels and lower Enterobacteriaceae, Staphylococcus and Pseudomonas levels in faecal
microbiota compared to infants not receiving the supplement. Moreover, probiotic treated infants
exhibited significantly higher concentration of total organic acids levels compared to non-treated ones,
Nutrients 2018, 10, 1723 13 of 27

in particular acetic acid increased immediately and 1 week after surgery; furthermore, the faecal pH
tended to decrease with the probiotic intervention.
Kanamori et al. [165] documented in a case-report the efficacy of a synbiotic therapy, consisting
in a combination of B. breve Yakult (BBG-01), L. casei Shirota and galactoolicosaccharides as prebiotic
components, in a newborn with short bowel syndrome resulting from a consistent bowel resection
performed soon after delivery. Patients affected from this pathology are subjected to an intestinal
bacteria overgrowth due to their dilated intestine [166]; this condition can lead to a bacteria
translocation in other districts inducing catheter sepsis, compromised carbohydrates fermentation
resulting in high level of lactate, with consequent acidosis [167] and a possible incontrollable growth
of intestinal pathogens. One year of synbiotic therapy, consisting in 3 g of bacteria (1 × 109 bacteria/g
per each strain) and 3 g of prebiotic per day, improved the nutritional state, prior compromised, by
increasing the intestinal motility and suppressed the intestinal pathogen overgrowth, in particular
E. coli and Candida spp.
The same synbiotic combination was used as a therapy for refractory and repetitive
enterocolitis [168]; this disorder often occurs in paediatric surgery patients and the severe type may
be fatal. The 7 recruited patients, having short bowels as a result of surgical resection and suffering
from repetitive enterocolitis, were administered with 1 g of probiotic (109 bacteria/g) 3 times daily for
36 months. All patients had an altered gut microbial composition prior to the therapy characterized by
low levels of anaerobic bacteria and high levels of resident pathogenic bacteria. In spite of the frequent
antibiotic treatments to which patients were exposed, the long synbiotic administration was effective
in highly increasing bifdobacteria and lactobacilli levels, which were almost undetectable before the
supplementation and incrementing faecal SCFAs, inducing a more normal ecosystem profile in the
intestine. Moreover, most of patients accelerated their body weight gain and showed increased serum
rapid turnover, with a general amelioration of their health status.
With the developing of therapies and surgeries in the field of perinatal and foetal cares, neonate
survival outcomes have extraordinary increased; newborns that are subjected to these interventions
need prolonged intensive care periods, which include use of antibiotics, respiratory care and restriction
of enteral feeding. All these factors may affect the normal microbial gut colonization leading to
severe infection and malnutrition [169]. A synbiotic therapy, including B. breve, as already observed,
could be effective in preventing or correcting an abnormal microbial colonization in intensive care
newborns. The same synbiotic therapy, largely and positively tested, including B. breve Yakult, L. casei
Shirota and galactooligosaccharides, was applied to newborns with diagnosis of severe congenital
anomalies [169]. The product contained 109 –1010 bacteria/g and was administered immediately after
birth via a nasogastric tube, as soon as intestinal feeding was possible, first at a dose of 0.12 g per day
in four equal dose and then, when the amount of milk increased, at 3 g per day in three equal doses.
As results of the therapy, none of patients manifested enterocolitis, they showed an improvement in
their clinical course and reached a body weight gain equivalent to that of normal infants. This last
outcome has been hypothesized to be linked to the potential metabolic activity of the administered
probiotics to promote liver lipogenesis and fat storage in the peripheral fat tissue contributing to the
growth observed in these infants despite the congenital disorders [170].

5.7. Coadjuvant in Chemotherapic Treatment

A condition in which the use of probiotics may have a reliving effect is chemotherapy. The cancer
itself and the drug-therapy inducing bone marrow suppression lead to an immunocompromising
state in which an infectious could be fatal. Since the main source of infection is endogenous intestinal
harmful bacteria [171], a probiotic treatment can certainly benefit the patient’s state by not only
competing against pathogens for nutrients and attachments sites but also by stimulating gut immunity,
producing organic acids and improving transepithelial resistance [172]. A study conducted in 2009
evaluated the effect of B. breve Yakult (BBG-01) strain in cancer paediatric subjects, administered with
109 freeze-dried cells, corn starch and hydroxipropyl cellulose in 1 g of formulation. The administration
Nutrients 2018, 10, 1723 14 of 27

was found to be effective in reducing febrile episodes, which may be the only sign of infection and the
use of intravenous antibiotics by stabilizing the intestinal microbial composition [173].
An overview in chronological order of B. breve applications as a single strain and as a component
of a multi-strain/multi-species formulation is reported in Tables 1 and 2, respectively.

Table 1. Overview of B. breve strains applications in in vitro studies, mice model and paediatric trials.

B. breve Strains Reported Effect(s) References

Strong antimicrobial activity against pathogens, stimulation of mitochondrial
B. breve B632 dehydrogenase activity of macrophages, stimulation of proinflammatory cytokines [62]
production in in vitro study
B. breve BR03 Inhibition of the growth of 4 E. coli biotypes in in vitro study [64]
Reduction of total faecal coliforms in healthy children [65]
Reduction of pro-inflammatory TNF-α in blood samples of celiac children [145]
Reduction of minutes of daily crying in healthy infants [129]
B. breve B632 + Restoration of the healthy percentage of main gut microbial components in celiac children [146]
B. breve BR03
Improvement of glucose metabolism and weight management in obese children [155]
Reduction of daily vomit frequency, daily evacuation, improved stool consistency,
[130]
protection against developing metabolic disturbance in healthy infants
Modulation of faecal SCFAs profile in celiac children [147]
Anti-infective activity against Shiga-toxin-producing E. coli in mice model [69]
Reduction of febrile episodes and use of intravenous antibiotics in cancer
[173]
paediatric subjects
Improvement of composition and metabolic activity of gut microbiota and reduction of
[163]
B. breve Yakult incidence of bacteria in blood in paediatric surgery subjects
(BBG-01) Increased defecation frequency, improvement of stool consistency, frequency episodes of
[133]
faecal incontinence and abdominal pain in constipated children
Stimulation of anti-inflammatory IL-10-producing CD4+T cells in mice model [83]
Improvement of composition and metabolic activity of gut microbiota in paediatric
[164]
surgery infants with congenital heart disease
Stimulation of anti-influenza virus hemagglutinin IgA production by Peyer’s patch cells in
[75]
mice model
B. breve YIT4064
Stimulation of antigen-specific IgG production against pathogenic antigens in mice model [74]
B. breve UCC2003 Reduction of Citrobacter rodentium gut colonization in mice model [76]
B. breve NCC2950 Induction of REGIII-γ expression in mice model and REGIII-α in in vitro study [78]
Reduction of pro-inflammatory cytokines and lung neutrophil and eosinophil infiltration
B. breve MRx0004 [79]
in severe asthma mice model
Improvement of allergic symptoms associated to cow’s milk hypersensitivity in infants [157]
Immunomodulation activity by increasing TGF-β1 in preterm infants [122]
Reduction of infections and mortality for NEC in extremely and very low birth
[118]
weight infants
Reduction of faecal butyric acid in extremely and very low birth weight infants [121]
Reduction of total IgE, OVA-specific IgE and OVA-specific IgG in mice model [80]
Protection against developing of whey allergy symptoms in model mice [81]
Reduction of infections and sepsis incidence in extremely and very low birth
[124]
B. breve M-16V weight infants
Improvement of composition and metabolic activity of gut microbiota in infants with
[158]
atopic dermatitis
Reduction of asthma-like symptoms prevalence and asthma medication use prevalence in
[160]
infants with atopic dermatitis
Shifted gut microbiota towards a healthy profile in preterm infants [111]
Low incidence of NEC (≥stage II) in very low birth weight infants [112]
Partially protection against developing skin reaction due to cow’s milk allergy, increased
cecal content of butyrate and propionate and increased antimicrobial IL-22 expression in [82]
mice model
Nutrients 2018, 10, 1723 15 of 27

Table 1. Cont.

B. breve Strains Reported Effect(s) References

Suppression of epididymal fat and body weight gain in mice model with
B. breve B-3 [84,85]
diet-induced obesity
B. breve 1205 Amelioration of anxiety condition and general metabolism in mice model [88]
Prevention of cognitive decline in Alzheimer disease and reduction of neural
B. breve A1 [89]
inflammation in mice model
B. breve NCIMB Increased CLA isomer (c9, t11), EPA and DHA in adipose tissue and reduced
[96]
702258 proinflammatory cytokines in mice model
Reduced abdominal symptoms and improved weight gain in preterm infants [109]
B. breve YIT4010
Establishment of beneficial gut microbiota and prevention of infections in preterm infant [110]

Table 2. Overview of applications of B. breve strains combined to other bacterial strains in

paediatric trials.

B. breve Strains Probiotic Mixture Reported Effect(s) References

B. breve M-16V Reduction of developing eczema and atopic
[161]
B. longum BB536 dermatitis in infants
B. breve M-16V B. breve M-16V Reduction of abdominal pain prevalence and
B. infantis M-63 frequency, improvement of quality of life in [134]
B. longum BB536 IBS children
Improvement of composition and metabolic
activity of gut microbiota and of overall health [165,168]
B. breve Yakult status in infants with short bowel syndrome
L. casei Shirota Prevention of enterocolitis, improvement of
B. breve Yakult
(BBG-01) body weight and clinical course in infants with [169]
congenital disorders
B. breve Yakult Reduction of NEC incidence and improvement
[125]
L. casei of intestinal motility in infants
Reduction of number of dehydration cases and
B. breve C50
B. breve C50 medical consultation cases in children exposed [144]
S. thermophilus 065
to risk of developing acute diarrhoea
Reduction of stool frequency and improving of
stool consistency in children with acute [142]
rotavirus diarrhoea
Manifestation of high rate of remission and low
[139]
incidence of relapse in UC children
B. breve DSM 24732 VSL#3 Improvement of symptoms, severity and
frequency of abdominal pain and bloating and
[138]
family assessment of life disruption in
IBS children
Reduction of the risk of suspected sepsis in
[144]
most vulnerable very low birth weight infants

6. B. breve Administration in Adults: A Short Outcome

The use of B. breve has been largely investigated in paediatric scenery and its therapeutic role
has been strongly supported by significant and solid outcomes; its use is not limited to paediatric
supplementation but it is also involved in improving health condition in briefly outlined.
Minami et al. [174] investigated the use of B. breve B-3 at a daily dosage of 5 × 1010 CFU/capsule
for 12 weeks in adults with a tendency for obesity. A significant decrease of the fat mass
and an amelioration of blood parameters were observed, in particular a significant reduction of
γ-glutamyltranspeptidase (γ-GTP), a marker used to evaluate liver injury and high-sensitivity protein
C-reactive (hCRP), a marker used to evaluate the inflammatory reaction, were detected. Interestingly,
a significant negative correlation between the value of fat mass and 1,5-anhydroglucitol, a marker
Nutrients 2018, 10, 1723 16 of 27

that closely reflect short-term glucose status and glycaemic variability, was recorded suggesting the
potential role of B. breve in the improvement of diabetes.
Ishikawa et al. [175] showed the effects of one year of B. breve Yakult treatment, in association
with galactooligosaccharides as prebiotic, in patients diagnosed with UC. The probiotic, containing
109 CFU/dose of freeze-dried powder, was administered immediately after every meal 3 times a
day and the prebiotic, at a dosage of 5.5 g, was administered once a day. The synbiotic intervention
improved the endoscopic score by decreasing the values of severity mucosa damage [176] and reduced
the level of myeloperoxidase, which is secreted by neutrophils and macrophages accumulated in the
inflamed lesions and positively correlated with the disease severity [177]. Regarding gut environment
results, the synbiotic treatment significantly reduced Bacteroidaceae counts and faecal pH, which may
be connected to an increment of faecal SCFAs.
An interesting relationship was evaluated by Kano et al. [178]: since a Japanese 2007 survey
evidenced that women who suffer from abnormal bowel movements also showed skin disorders,
they conducted a double-blind, placebo-controlled, randomized trial to investigate the effects of
probiotic and prebiotic fermented milk on skin of healthy adult women. The fermented milk contained
galactooligosaccharides, polydextrose, B. breve Yakult, Lactococcus lactis and S. thermophilus at a daily
dose of 6 × 1010 , 5 × 1010 , 5 × 1010 CFU/100 mL of milk, respectively. The synbiotic intake, which
lasted 4 weeks, resulted to prevent hydration level decreases in the stratum corneum. The intervention
increased cathepsin L-like protease activity, which can be considered as an indicator of keratocyte
differentiation, as proteolysis of cathepsin L activates transglutaminase 3, which plays an important
role in the stratum corneum formation [179]. Moreover, the administration reduced phenol levels in
serum and urine and since the production of phenols is inhibited at low intestinal pH, an increase of
intestinal organic acid levels might be occurred after the treatment.
The probiotic preparation VSL#3 has been extensively used for the treatment of IBD in adulthood.
Brigidi et al. [180] investigated the effects of 20 days VSL#3 administration in patients with diarrhoea
predominant-IBS or functional diarrhoea; the probiotic intake caused changes in gut microbiota
composition with a significantly increase of total lactobacilli, total bifidobacteria and S. thermophilus,
which are component of VSL#3. The treatment led also to an improvement of some enzymes
functions, whose actions are compromised in IBD, by reducing urease activity, whose products
usually allow pathogenic bacteria to survive in the gastrointestinal tract and contribute to mucosal
tissue damages [181] and by increasing β-galactosidase activity, which is involved in the metabolism
of unabsorbed carbohydrates. Pronio et al. [182] confirmed the positive role of VSL#3 upon treatment
of patients undergoing ileal pouch anal anastomosis for ulcerative colitis. The probiotic intervention
reduced signs and symptoms of inflammation inducing a significant expansion of cells associated to an
improvement of the inflammatory condition of the pouch mucosa. An interesting microbial outcome
was evidenced by Kühbacher et al. [183]: the UC remission maintained by VSL#3 administration was
accompanied by a higher bacterial diversity actually not related to the probiotic intake. However,
the increase of bacterial diversity may represent a therapeutic mechanism that supports the VSL#3
activity in maintaining UC remission. Bibiloni et al. [184] showed that 6 weeks administration with
the probiotic mixture improved UC remission and response in patients not responding to traditional
therapy. Since VSL#3 has been demonstrated to maintain remission in UC patients intolerant or allergic
to 5-aminosalicylic acid (5-ASA), known also as mesalazine [185], Tursi et al. [186] demonstrated the
efficacy on UC of another therapeutic combination: VSL#3, in association with balsalazide, 5-ASA
prodrug, was shown to be significantly superior to balsalazide alone and to mesalazide in the treatment
of active mild-to-moderate UC. One of the key points of the study is the low dosage of balsalazide used
(2.25 g/day), usually not effective in reducing UC symptoms and inducing remission. Therefore, the
low dosage appeared to be effective only in combination with VSL#3. In this regard, a more recent study,
involving a larger number of patients, highlighted the superior ability of VSL#3 to improve relapsing
mild-to-moderate UC when added to standard UC treatment with respect to patients on standard
treatment only, confirming the potential synergic action exerted by standard UC pharmacological
Nutrients 2018, 10, 1723 17 of 27

treatments and VSL#3 [187]. The reason for this synergic action may be a combined effect of the
chemotherapic on the disease and of the probiotic on the general well-being of the host. Clinical
studies proved that this probiotic mixture was particularly effective in the treatment of IBD, improving
abdominal pain duration and distention severity score in patients suffering from IBS [188]. Moreover,
it was effective in clinical condition of diarrhoea-predominant IBS subjects [189,190].

7. Conclusions
This review has outlined the large number of cases in which B. breve strains, mainly as single
strains but also in combination with other Bifidobacterium species or Lactobacillus strains, are used
for therapeutic and prevention purposes and/or to prevent further complications of the disease in
the paediatric sector. The analysis of the outlined results allows to conclude that, whereas in vitro or
animal-model study are performed with a large number of different B. breve strains, clinical studies
are performed with a restricted number of strains (mainly B. breve YIT4010, M-16V, the associations
B632/BR03 and Yakult BBG-01). Therefore, there is the opportunity of expanding the potentialities of
the strains used in clinical studies on the basis of the positive results obtained in pre-clinical studies
and, therefore, more opportunities for a further development of “therapeutic microbiology.” A second
interesting aspect outlined in this review is the frequent association of the B. breve administration
with traditional chemotherapeutic treatment. This is particularly important in the treatment of very
serious diseases in which stopping the traditional therapies may be considered risky for the patient.
The probiotic can act as a supplement to prevent complication and improve the general health status
of the patient. We are all confident that the improvement in the “therapeutic microbiology” sector will
be a great aid to medical approach in the near future.

Author Contributions: All the authors designed the outline of the review; N.B.C. wrote the main sections of the
manuscript, L.B. contributed to the general description of B. breve species, F.G. contributed to the studies on adults,
D.D.G. supervised the work and critically revised the paper.
Funding: This research received no external funding.
Acknowledgments: The authors would like to acknowledge the EU project FOODstars (Innovative Food Product
Development Cycle: Frame for Stepping Up Research Excellence of FINS, GA 692276) for the grant supplied
to N.B.C.
Conflicts of Interest: The authors declare no conflict of interest.

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