Download intestinal colonization, microbiota, and probiotics

INTESTINAL COLONIZATION, MICROBIOTA, AND PROBIOTICS
SEPPO SALMINEN, PHD,
AND
ERIKA ISOLAURI, MD, PHD
The human intestine is colonized by a large number of microorganisms that inhabit the intestinal tract and support a
variety of physiological functions. The stepwise microbial colonization of the intestine begins at birth and continues during the
early phases of life to form an intestinal microbiota that is different for each individual subject. This process facilitates the
formation of a physical and immunologic barrier between the host and the environment, helping the gastrointestinal tract
maintain a disease-free state. Probiotics are viable microbial food supplements that have a beneficial impact on human health.
Health-promoting properties have been demonstrated for specific probiotic products. Scientific data are accumulating on these
properties, especially in infants; the most significant effects include prevention and treatment of antibiotic-associated diarrhea
and rotavirus diarrhea and allergy prevention. Bifidobacteria appear to be the most promising probiotic candidates, followed
by defined lactic acid bacteria, which favor specific healthy bifidobacterial growth and species composition. Because viability
appears to be important, probiotic properties also should be emphasized to meet this criterion. For future probiotics, the
most important requirements include a demonstrated clinical benefit supported by mechanistic understanding of the effect on
target population microbiota and immune functions. Genomic information and improved knowledge of microbiotic composition and its aberrancies should serve as a basis for selecting new probiotics for use in specific infant populations.
(J Pediatr 2006;149:S115-S120)
evelopment of the microbiota in the newborn gastrointestinal (GI) tract depends on the original inoculum, the
immediate living environment, and early feeding practices. The resulting mature intestinal microbiota harbors 10 times
more cells than the host. This microbiota provides not only an important barrier between the human host and the
environment, but also contact sites between microbes and the developing immune system. The barrier prevents the settlement
of unwanted or pathogenic microorganisms and facilitates predigestion of many nutrients.
Thus, the barrier function appears to support both the microbiota and the host by creating
a protective microecologic environment.
The sequencing of the human genome1,2 and specific microbial genomes has
demonstrated that human subjects harbor numerous genes in the intestinal microbiota
From the Functional Foods Forum and Department of Pediatrics, University of Turku,
that influence health by interacting with the host at the mucosal level. The contact
Turku, Finland.
between the human genome and the commensal or permanent intestinal microorganisms
Seppo Salminen is a recipient of a Bristol
(microbiome) directly influences the morphology of the gut, providing a basis for the
Myers Squibb–Mead Johnson Unrestricted
Nutrition Research grant. This is a Nutridisease-free development of the gut.3,4 The human microbiota has been suggested to play
tion, Allergy, Mucosal Immunology, and Ina significant nutritional role. By facilitating rapid salvage of energy from many nutrients
testinal Microbiota (NAMI) research group
report. Mead Johnson sponsored the symand providing diverse metabolic functions, the microbiota enables the host to survive in
posium and provided an honorarium for
different nutritional environments and to operate without having to adapt or develop all
conference attendance, presentation of the
5
article, and submission of a manuscript. The
digestive processes.
authors are entirely and exclusively responThe early and mature intestinal microbiota are unique to each human being. From
sible for its content.
birth on, during breast feeding and weaning, the microbiota diversifies and becomes stable
Presented as part of a symposium recog6-9
nizing the 25th anniversary of the Bristoland complex, to function as a barrier in the specific conditions of the host.
Myers Squibb “Freedom to Discover” NuProbiotics are live microbial food supplements with demonstrated positive effects on
trition Grants Program, University of
10,11
Cincinnati, Cincinnati, OH, June 7-8, 2005.
host health.
Probiotics often act by modifying the process of intestinal microbiota
Submitted for publication Apr 14, 2006;
development or the composition and activity of developed microbiota. Probiotics also can
accepted June 1, 2006.
act by direct contact with the mucosal cells, facilitating cross-talk with the immune system
Reprint requests: Seppo Salminen, PhD,
and microbes. Current probiotics have several demonstrated beneficial effects on infant
Functional Foods Forum, University of
Turku, 20014 Turku, Finland. E-mail:
health, including maintaining healthy intestinal microbiota development and [email protected].
ing deviations observed in gut inflammatory diseases or preceding them.
0022-3476/$ - see front matter
D
GI
Gastrointestinal
TLR
Toll-like receptor
Copyright © 2006 Mosby Inc. All rights
reserved.
10.1016/j.jpeds.2006.06.062
S115
SUCCESSION OF MICROBIAL COMMUNITIES
IN HEALTH
Source of Original Microbiota
The basis of the healthy gut microbiota is derived from
the mother. The mother’s microbiotic composition and the
original inoculum provided at birth depend on poorly understood genetic factors. Diet, environment, and stress factors
during pregnancy and birth influence the mother’s fecal microbiota composition and, consequently, the inoculum transferred to the infant at birth.12,13 The microbiota of a newborn
develops rapidly after birth and is initially strongly dependent
on feeding practices and the newborn infant’s hygienic environment.6,12
Succession of Microbial Communities
Establishment of the gut microbiota is characterized by
the following steps: early colonization at birth with facultative
anaerobes, such as the enterobacteria, coliforms, lactobacilli,
and streptococci, first colonizing the intestine, followed in
rapid succession by anaerobic genera, such as Bifidobacterium,
Bacteroides, Clostridium, and Eubacterium.12,14,15
Molecular studies have indicated that bifidobacteria
species in the intestinal tract can range from 60% to 90% of
the total fecal microbiota in breast-fed infants and lactic
acid-producing bacteria may account for ⬍ 1% of the total
microbiota, indicating the significant dominance of bifidobacteria.15 In formula-fed infants, the microbiota often is
more complex, but the composition effects depend on the
type of formula. The important differences in the microbiota
of breast-fed and formula-fed infants do not lie only in the
bifidobacterial numbers, but also in the species composition.
Bifidobacterium breve, B. infantis, and B. longum species and
strains are often found in fecal samples of breast-fed infants,
whereas formula-fed infants often also harbor other types of
bifidobacteria.6,14 The lactic acid bacteria species composition
appears to be rather similar; the most common lactobacilli in
breast-fed and formula-fed infant feces constitute Lactobacillus acidophilus group microorganisms, such as L. acidophilus, L.
gasseri, and L. johnsonii. The differences in fecal microbiota
between breast–fed and formula-fed infants may have decreased due to the development of improved infant formulas.14,16
Impact of Breast-Feeding and Weaning and Gut
Microbiota
Breast-feeding promotes a strong bifidobacterial presence in the infant gut by providing oligosaccharides that act as
favorable substrates for bifidobacteria. Breast-feeding also enhances the bifidogenic effect by providing contact and exposure to the normal microbiota of the mother’s skin and breast
milk.
Bifidobacterium longum and other intestinal bifidobacteria are adapted to the availability of scanning nutrients in the
lower GI tract in infants.17 Such strains rapidly utilize the
S116
Salminen and Isolauri
Figure. Succession of intestinal microbiota in infants and the influence of
probiotics on development.
oligosaccharides from human milk along with intestinal mucins, which are available in the colon of breast-fed infants.
Thus, they appear to be designed by nature to reside in the
healthy newborn GI tract to assist microbiotic development.
Introduction of solid foods, as well as antimicrobial
treatment periods, interrupt the constant supply of oligosaccharides and microbes from the mother, introducing new
microbial genera and species into the GI tract. This process
facilitates the development of adult-type mature microbiota.
A schematic view of microbiotic development and factors
influencing it is given in Figure 1.
HOST–MICROBE INTERACTION: FROM
COMMENSALISM TO MUTUALISM
Normal intestinal microbiota are characterized as a
complex collection and balance of microorganisms that normally inhabit the healthy GI tract. The indigenous bacteria
sometimes have been classified as potentially harmful or
health-promoting. Most of them, however, are part of the
normal commensal flora. This term indicates a relationship
between organisms of 2 or more different species in which 1
species derives benefits from the association while the other(s)
remain(s) unharmed or unaffected. Currently, the relationship
between intestinal bacteria and the host is referred to as
host–microbe cross-talk, implying peaceful coexistence and
mutual benefit. Such cross-talk has been recently characterized. Bacteroides thetaiotaomicron, a common gut commensal,
contains a large number of genes related to uptake and metabolism of carbohydrates reported for a sequenced bacterium.18 This microorganism has been shown to modulate glycosylation of the intestinal mucus and to induce the
production of antimicrobials by the mucosa.19 In this way, the
Bacteroides and other intestinal microbes may influence the
gut microecology composition and shape the immune system.
Bacteroides has been reported to evade detection by the immune system by changing the capsular polysaccharide composition and also surface antigenicity, a cellular modification,
The Journal of Pediatrics • November 2006
which may help original colonizers permanently settle in the
intestine.18,20
The intestinal mucosa and its immune system are
adapted to the proximity and to the constant microbial challenge. The GI epithelium is equipped with pattern recognition receptors, including toll-like receptors (TLRs), which
recognize specific conserved pathogen-associated molecular
patterns. Nonpathogenic microbes share these structures;
consequently, TLRs cannot distinguish between pathogens
and commensals.21 Intracellular signalling pathways of TLRs
result in production of proinflammatory cytokines through
activation of the transcription nuclear factor ␬B. These gut
microbiota impact healthy immunophysiologic regulation,
contributing to the anti-inflammatory tone in the gut. Indeed,
several characteristics of the gut epithelium have been thought
to prevent inappropriate immune responses toward indigenous gut microbiota. These include a relatively sparse expression of both certain TLRs and their essential co-receptors on
the intestinal epithelium, as well as intracellular location of a
few TLRs. Abundant immunoglobulin A antibody production at mucosal surfaces contributes to the intestinal barrier
function by binding to and excluding antigens. Maturation of
dendritic cells carrying commensals and subsequent secretion
of cytokines and chemokines then influence the polarization
of T-helper cells and thereby the adaptive immune responses.
This type of immune response has been suggested to prevent
commensals from breaching the gut mucosal barrier, whereas
pathogenic bacteria preferably destroy it.22 In experimental
studies in mice deficient in MyD88, an adaptor molecule
essential for the TLR-mediated induction of inflammatory
cytokines, demonstrated that TLR signalling pathways control the homeostasis of the epithelium and appear to be
critical for protecting the host against gut injury by controlling cytoprotective factors and epithelial cell proliferation.23
Important functions of resident bacteria on the host’s
physiology include metabolic activities, trophic effects on the
intestinal epithelium, and protection against the overgrowth
of potential pathogens in the GI tract. Along with these
effects, specific strains of the gut microbiota elicit anti-inflammatory responses in the intestinal epithelial cells, thereby
strengthening intestinal homeostasis. The importance of
host–microbe interaction is most vital in the neonatal period,
when the establishment of a normal microbiota provides the
host with its most substantial antigen challenge, a strong
stimulatory effect for the maturation of the gut-associated
lymphoid tissue.24 On this basis, the aims of probiotic therapy
are to avert deviant microbiota development, impaired gut
barrier function, abnormal immune responsiveness, and immunoinflammatory disease. Indeed, probiotics in the diet may
exert clinical effects beyond the nutritional impact of food.
Future research should provide the necessary tools, such
as DNA microarrays, to unravel the in vivo functions of gut
microbes25 or to monitor the effect of diet on microbiota and
the host’s genes and their expression.26 The goals are to
correctly interpret the complex messages provided by the
Intestinal Colonization, Microbiota, and Probiotics
multitude of microarray data and to further develop a bioinformatic approach.
ROLE OF PROBIOTICS
Probiotics have been defined by the International Life
Sciences Institute Europe as “viable microbial food supplement[s] which when taken in the right dose beneficially
influence the health of the host.”10 Practically the same definition of probiotics is used by the Food and Agriculture
Organization of the United Nations and the World Health
Organization.11 These definitions require that safety and efficacy be demonstrated scientifically for each strain and each
product.
Probiotics and Intestinal Colonization
Specific probiotics have been assessed for their ability to
attach to human intestinal mucosa, but such attachment is of
a temporary nature. For example, administering Lactobacillus
GG to infants may result in a 2- to 12-week fecal recovery of
the administered strain in feces, indicating the potential to
multiply and survive in the normal intestinal tract as well as
during rotavirus diarrhea.27-29 Fecal recovery is not necessary
for demonstrating probiotic health effects, which depend on
the interaction of the strain at the target site.
Importance of Viability
Few studies have assessed nonviable probiotics in humans. However, viability appears to be an important factor in
probiotics to facilitate health effects. Kirjavainen et al30 reported that only viable probiotics in extremely sensitive infants were able to alleviate symptoms of atopic dermatitis, as
reported by Isolauri et al.31 It is clearly of high interest to
continue such studies not only for probiotics, but also for
characterizing the viability of the residing intestinal microbiota.
Genomic Information on Probiotics
Many members of the Lactobacillus and Bifidobacterium
genera are commonly used as probiotics. In 1995, the genome
of the first free-living organism, the bacterium Haemophilus
influenzae, was sequenced.32 Since then, more than 200 bacterial genomes, mainly pathogenic microorganisms, have been
sequenced. The first genome of a lactic acid bacterium was
completed in 1999.33 Recently, the genomes of many other
lactic acid bacteria,34 bifidobacteria,17 and other intestinal
microorganisms18,20,35 have been sequenced.36 However,
genomic data on 2 of the most widely studied probiotics
(Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb-12)
are not publicly available.
Genomic information on B. longum,17 L. plantarum,34
L. johnsonii,37 or L. acidophilus,38,39 all established probiotics,
provide insight into the adhesive mechanisms present in these
microorganisms, which provide the basis for both settlement
into the gut during different age phases and communication
of regulatory signals to specific areas and sites of the gut
S117
mucosa. A eukaryotic-type serine protease inhibitor has been
identified in the genome of B. longum that may contribute to
the immunomodulatory activity of Bifidobacterium. Operons
coding for bacteriocins have been identified in L. johnsonii
and L. acidophilus that may modify the succession of microbiota in humans over time.
ABERRANT GUT MICROBIOTA: CLINICAL
CONSEQUENCES
Specific imbalances or deviations in the intestinal microbiota may make humans more vulnerable to intestinal
inflammatory diseases and systemic diseases beyond the intestinal environment. Specific Bifidobacterium species in the
healthy infant gut are the most predominant and metabolically active organisms; specific clostridia also are often
present. Changes in their quantitative and qualitative composition appear to serve as useful indicators of deviations from
the balanced microbiota. Other specific microbial biomarkers
for health remain to be defined among the developing intestinal microbiota.
Specific deviations in intestinal microbiota, including
decreased numbers, an atypical composition of bifidobacteria,
and aberrancies in clostridia content and composition, may
predispose infants to allergic disease.40,41 Similarly, deviations
from the normal microbiota are associated with antibiotic side
effects. Aberrant microbiota during childhood may predispose
a person to both inflammatory gut disease and diarrhea.42,43
Consequently, understanding the quantitative and qualitative
microbiota composition will help provide targets for probiotic
intervention.
Numerous human intervention studies have assessed
the efficacy of specific probiotics in the treatment and risk
reduction of infectious diarrhea.44,45 The effect has been
explained by a reduction in the duration of rotavirus shedding,
normalization of the gut, increased permeability caused by
rotavirus infection, and increased numbers of IgA-secreting
cells against rotavirus. Moreover, the ability of specific probiotics to increase the expression of mucins may contribute to
the barrier effect and the inhibition of rotavirus replication.
Particularly, the immunomodulatory potential of specific probiotics has introduced new potential therapeutic strategies for
combating allergic and inflammatory conditions.24 Probiotic
bacteria may counteract the inflammatory process by stabilizing the gut’s microbial environment and the intestine’s permeability barrier, as well as by enhancing the degradation of
enteral antigens and altering their immunogenicity. Another
explanation for the gut-stabilizing effect could be improvement of the intestine’s immunologic barrier, particularly the
intestinal IgA responses. Probiotic effects also may be mediated by controlling the balance between proinflammatory and
anti-inflammatory cytokines. These effects, which are exemplified in the reduced disease activity and increased intestinal
permeability, have been achieved in pediatric patients with
Crohn’s disease and atopic eczema.31,46-49 In clinical trials in
adults, results from preparations containing 4 strains of lactobacilli (L. casei, L. plantarum, L. acidophilus, and L. delS118
Salminen and Isolauri
brückii subsp. bulgaricus) and 3 bifidobacteria strains (B. longum, B. breve, and B. infantis), together with Streptococcus
salivarius subsp. thermophilus, are encouraging for preventing
relapses of chronic pouchitis.50 The preventive potential of
probiotics in atopic diseases has been demonstrated in a
double-blind, placebo-controlled study.50-52 Probiotics administered prenatally and postnatally for 6 months to children
at high risk for atopic disease reduced the prevalence of atopic
eczema to half that in infants receiving placebos, and the
effect appears to extend beyond infancy.
PERSPECTIVES
Advancing genomic research will provide data on host–
microbe interactions to identify key processes of microbiotic
development and maintenance. These include nutrient–microbe interactions and a detailed knowledge of the transfer of
microbial communities from parent to infant. Such data
should provide the basis for the development of new probiotics.53
Probiotic genome analysis will predict their properties
and interactions in human use, allowing their application to
human studies of specific target populations. Development of
genetic tools aids analysis of the functionality of these
strains,54,55 thereby facilitating the development of a new
generation of probiotics that are more site-specific and target
disorder-specific and safe.
CONCLUSION
The healthy infant microbiota acts as an organ to utilize
nutrients and also as a defense mechanism against harmful
environmental exposures. Deviations in composition can be
related to multiple disease states within the intestine but also
beyond it. Similarly, components of the human intestinal
microbiota or organisms entering the intestine may have both
harmful and beneficial effects on human health.
The current available information focuses mostly on the
role of infant microbiota and the first colonization steps in
later health. Bifidobacteria play a key role in this process;
clostridia also may serve as biomarkers of changing environmental conditions. Maternal–infant contact, breast milk oligosaccharides, and breast milk microbes exert significant effects on initial microbiotic development. The initial inoculum
at birth is promoted through prebiotic galacto-oligosaccharides in breast milk and introduces environmental bacteria
through maternal skin and other contact with the infant, thus
providing a means to guide the development of individually
optimized microbiota under existing environmental conditions.
A future goal is to apply the knowledge of microbiotic
composition and aberrancies to selecting the right probiotics
for a particular target population. Another goal is to ensure
that probiotics and prebiotics administered to infants are safe
in terms of both long-term use and effects beyond infancy.
REFERENCES
1.
International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 2001;409:860-921.
The Journal of Pediatrics • November 2006
2.
Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, et
al. The sequence of the human genome. Science 2001;291:1304-51.
3.
Bäckhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, et al.
The gut microbiota as an environmental factor that regulates fat storage. Proc
Natl Acad Sci USA 2004;101:15718-23.
4.
Falk PG, Hooper LV, Midvedt T, Gordon JI. Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from
gnotobiology. Microbiol Mol Biol Rev 1998;62:1157-70.
5.
Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon J. Hostbacterial mutualism in the human intestine. Science 2005;307:1915-20.
6.
Benno Y, Mitsuoka T. 1986. Development of intestinal microflora in
humans and animals. Bifidobacteria microflora 1986;5:13-25.
7.
Grönlund MM, Arvilommi H, Kero P, Lehtonen OP, Isolauri E.
Importance of intestinal colonisation in the maturation of humoral immunity
in early infancy: a prospective follow-up study of healthy infants aged 0-6
months. Arch Dis Child 2000;83:F186-92.
8.
Kirjavainen PV, Apostolou E, Arvola T, Salminen SJ, Gibson GR,
Isolauri E. Characterizing the composition of intestinal microflora as a
prospective treatment target in infant allergic diseases. FEMS Immunol Med
Microbiol 2001;32:1-7.
9.
Guarner F, Malagelada JR. Gut flora in health and disease. Lancet
2003;381:512-19.
10. Salminen S, Bouley C, Bouton-Ruault MC, Cummings JH, Franck A,
Gibson GR, et al. Functional food science and gastrointestinal physiology
and function. Br J Nutr 1998;80:S147–71.
11. Food and Agriculture Organization of the United Nations and World
Health Organization. Guidelines for the evaluation of probiotics in food,
2002. Available at http://www.who.int/foodsafety/fs_management/en/
probiotic_guidelines.pdf. Accessed August 2, 2006.
12. Mikelsaar M, Mändar R, Sepp E, Annuk H. Human lactic acid
bacteria microflora and its role in the welfare of the host. In: Salminen S, von
Wright A, Ouwehand A, editors. Lactic acid bacteria: microbiological and
functional aspects. New York: Marcel Dekker; 2004. p. 453-506.
13. Bailey MT, Lubach GR, Coe CL. Prenatal stress alters bacterial
colonization of the gut in infant monkeys. J Pediatr Gastroenterol Nutr
2004;38:414-21.
14. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendorp A, Klijn
N, Bindels J, et al. Analysis of intestinal flora development in breast-fed and
formula-fed infants by using molecular identification and detection methods.
J Pediatr Gastroenterol Nutr 2000;30:61-7.
15. Favier CF, Vaughan EE, De Vos WM, Akkermans AD. Molecular
monitoring of succession of bacterial communities in human neonates. Appl
Environ Microbiol 2002;68:219-26.
16. Rinne MM, Gueimonde M, Kalliomäki M, Hoppu U, Salminen SJ,
Isolauri E. Similar bifidogenic effects of prebiotic-supplemented partially
hydrolyzed infant formula and breastfeeding on infant gut microbiota. FEMS
Immunol Med Microbiol 2005;43:59-65.
17. Schell MA, Karmirantzou M, Snel B, Vilanova D, Berger B, Pessi G,
et al. 2002. The genome sequence of Bifidobacterium longum reflects its
adaptation to the human gastrointestinal tract. Proc Natl Acad Sci U S A
2002;99:14422-7.
18. Xu J, Bjursell MK, Himrod J, Deng S, Carmichael LK, Chiang HC, et
al. A genomic view of the human-Bacteroides thetaiotaomicron symbiosis.
Science 2003;299:2074-6.
19. Hooper LV, Stappenbeck TS, Hong CV, Gordon JI. Angiogenins: a
new class of microbicidal proteins involved in innate immunity. Nat Immunol
2003;4:269-73.
20. Kuwahara T, Yamashita A, Hirakawa H, Nakayama H, Toh H, Okada
N, et al. Genomic analysis of Bacteroides fragilis reveals extensive DNA
inversions regulating cell surface adaptation. Proc Natl Acad Sci U S A
2004;101:14919-24.
21. Zhang G, Ghosh S. Toll-like receptor-mediated NF-kappa activation:
a phylogenetically conserved paradigm in innate immunity. J Clin Invest
2001;107:13-9.
22. Macpherson AJ, Uhr T. Induction of protective IgA by intestinal
dendritic cells carrying commensal bacteria. Science 2004;303:1662-5.
23. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhi-
Intestinal Colonization, Microbiota, and Probiotics
tov R. Recognition of commensal microflora by toll-like receptors is required
for intestinal homeostasis. Cell 2004;118:229-41.
24. Rautava S, Kalliomäki M, Isolauri E. New therapeutic strategy for
combating the increasing burden of allergic disease: probiotics. J Allergy Clin
Immunol 2005;116:31-7.
25. De Vos WM, Bron PA, Kleerebezem M. Post-genomics of lactic acid
bacteria and other food-grade bacteria to discover gut functionality. Curr
Opin Biotechnol 2004;15:86-93.
26. Di Caro S, Tao H, Grillo A, Elia C, Gasbarrini G, Sepulveda AR, et
al. Effects of Lactobacillus GG on genes expression pattern in small bowel
mucosa. Dig Liver Dis 2005;37:320-9.
27. Schultz M, Gottl C, Young RJ, Iwen P, Vanderhoof JA. Administration of oral probiotic bacteria to pregnant women causes temporary infantile
colonization. J Pediatr Gastroenterol Nutr 2004;38:293-7.
28. Sepp E, Mikelsaar M, Salminen S. Effect of administration of Lactobacillus casei strain GG on the gastrointestinal microbiota of newborns.
Microb Ecol Health Dis 1993;6:309-14.
29. Juntunen M, Kirjavainen PV, Ouwehand AC, Salminen SJ, Isolauri E.
Gut microflora changes and probiotics in children in day-care centers. Biosci
Microflor 2003;22:99-107.
30. Kirjavainen PV, Salminen SJ, Isolauri E. Probiotic bacteria in the
management of atopic disease: underscoring the importance of viability.
J Pediatr Gastroenterol Nutr 2003;36:223-7.
31. Isolauri E, Arvola T, Sütas Y, Moilanen E, Salminen S. Probiotics in
the management of atopic eczema. Clin Exp Allergy 2000;30:1604-10.
32. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF,
Kerlavage AR, et al. Whole-genome random sequencing and assembly of
Haemophilus influenzae Rd. Science 1995;269:496-512.
33. Bolotin A, Mauger S, Malarme K, Ehrlich SD, Sorokin A. Lowredundancy sequencing of the entire Lactococcus lactis IL1403 genome. Antonie Van Leeuwenhoek 1999;76:27-76.
34. Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers
OP, Leer R, et al. Complete genome sequence of Lactobacillus plantarum
WCFS1. Proc Natl Acad Sci U S A 2003;100:1990-5.
35. Paulsen IT, Banerjei L, Myers GS, Nelson KE, Seshadri R, Read TD,
et al. Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 2003;299:1999-2002.
36. Xu J, Gordon JI. Honor thy symbionts. Proc Natl Acad Sci U S A
2003;100:10452-9.
37. Pridmore RD, Berger B, Desiere F, Vilanova D, Barretto C, Pittet A,
et al. The genome sequence of the probiotic intestinal bacterium Lactobacillus
johnsonii NCC 533. Proc Natl Acad Sci U S A 2004;101:2512-7.
38. Altermann E, Russell WM, Azcarate-Peril MA, Barrangou R, Buck
BL, McAuliffe O, et al. Complete genome sequence of the probiotic lactic
acid bacterium Lactobacillus acidophilus NCFM. Proc Natl Acad Sci U S A
2005;102:3906-12.
39. Barrangou R, Altermann E, Hutkins R, Cano R, Klaenhammer TR.
Functional and comparative genomic analyses of an operon involved in
fructooligosaccharide utilization by Lactobacillus acidophilus. Proc Natl Acad
Sci U S A 2003;100:8957-62.
40. Kalliomaki M, Kirjavainen P, Eerola E, Kero P, Salminen S, Isolauri E.
Distinct patterns of neonatal gut microflora in infants in whom atopy was and
was not developing. J Allergy Clin Immunol 2001;107:129-34.
41. Bjorksten B, Sepp E, Julge K, Voor T, Mikelsaar M. Allergy development and the intestinal microflora during the first year of life. J Allergy Clin
Immunol 2001;108:516-20.
42. Juntunen, M., Kirjavainen, PV Ouwehand, AC, Salminen, SJ, Isolauri,
E. Adherence of probiotic bacteria to human intestinal mucus in healthy
infants and during rotavirus infection. Clin Diagn Lab Immunol
2001;8:293-6.
43. Arvola T, Laiho K, Torkkeli S, Mykkanen H, Salminen S, Maunula L,
et al. Prophylactic Lactobacillus GG reduces antibiotic associated diarrhea in
children with respiratory infections: a randomized study. Pediatrics
1999;104:e64.
44. D’Souza AL, Rajkumar C, Cooke J, Bulpitt CJ. Probiotics in prevention of antibiotic associated diarrhoea: meta-analysis. BMJ 2002;324:1361.
S119
45. Szajewska H, Mrukowicz JZ. Probiotics in the treatment and prevention of acute infectious diarrhea in infants and children: a systematic review
of published randomized, double-blind, placebo-controlled trials. J Pediatr
Gastroenterol Nutr 2001;33(Suppl 2):S17-25.
46. Majamaa H, Isolauri E, Saxelin M, Vesikari T. Lactic acid bacteria in
the treatment of acute rotavirus gastroenteritis. J Pediatr Gastroenterol Nutr
1995;20:333-8.
47. Rosenfeldt V, Benfeldt E, Nielsen SD, Michaelsen KF, Jeppesen DL,
Valerius NH, et al. Effect of probiotic Lactobacillus strains in children with
atopic dermatitis. J Allergy Clin Immunol 2003;111:389-95.
48. Rosenfeldt V, Benfeldt E, Valerius NH, Paerregaard A, Michaelsen
KF. Effect of probiotics on gastrointestinal symptoms and small intestine
permeability in children with atopic dermatitis. J Pediatr 2004;145:612-6.
49. Gupta P, Andrew H, Kirschner BS, Guandalini S. Is Lactobacillus GG
helpful in children with Crohn’s disease? Results of a preliminary, open-label
study. J Pediatr Gastroenterol Nutr 2000;31:453-7.
50. Gionchetti P, Rizzello F, Venturi A, Ugolini F, Rossi M, Bridigi P, et
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Salminen and Isolauri
al. Oral bacteriotherapy as maintenance treatment in patients with chronic
pouchitis: a double-blind, placebo-controlled trial. Gastroenterology
2000;119:305-9.
51. Kalliomäki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri
E. Probiotics in primary prevention of atopic disease: a randomised placebocontrolled trial. Lancet 2001;357:1076-9.
52. Kalliomäki M, Salminen S, Poussa T, Arvilommi H, Isolauri E. Probiotics and prevention of atopic disease: 4-year follow-up of a randomised
placebo-controlled trial. Lancet 2003;361:1869-71.
53. Salminen S, Nurmi J, Gueimonde M. The genomics of probiotic
intestinal microorganisms. Genome Biol 2005;6:225-7.
54. Boekhorst J, Siezen RJ, Zwahlen MC, Vilanova D, Pridmore RD,
Mercenier A, et al. The complete genomes of Lactobacillus plantarum and
Lactobacillus johnsonii reveal extensive differences in chromosome organization and gene content. Microbiology 2004;150:3601-11.
55. Callanan M. Mining the probiotic genome: advances strategies, enhanced benefits, perceived obstacles. Curr Pharm Des 2005;11:25-36.
The Journal of Pediatrics • November 2006