Probiotics, Prebiotics, and Bacteria: Perspectives and Clinical Applications in Gastroenterology - Gastroenterology Clinics of North America Vol 34 Issue 3

211 Pages·2005·3.28 MB·English

by  Gerald Friedman MD

GastroenterolClinNAm34(2005)xiii–xvi
GASTROENTEROLOGY CLINICS
OF NORTH AMERICA
PREFACE
Probiotics, Prebiotics, and Commensal
Bacteria: Perspectives and Clinical
Applications in Gastroenterology
Gerald Friedman, MD, PhD, MS, FACP, MACG
GuestEditor
This issue of the Gastroenterology Clinics of North America targets the basic
scienceandclinicalapplicationsrelatingtotheroleofcommensalbacteria
in health and disease. Unresolved controversies regarding the mecha-
nisms of action and clinical use of pre- and probiotics are being resolved by
basicscientistsandclinicalinvestigators.Theperspectivesandpotentialclinical
applicationsareofferedbyaninternationalgroupofoutstandinginvestigators.
Each investigator’s work is an expression of his analytic skills and creativity,
and this issue represents a rich diversity of investigative research. Our hope is
that this issue will provide a platform for understanding the many potential
clinical applications in this exciting area of research.
Dysregulatedimmuneresponsesingeneticallysusceptiblehostscanresultin
a group of inflammatory and allergic illnesses. The research gives particular
emphasistothemanipulationofbacterialfloraforthebettermentofhumanhost
healthbytheadditionofprobioticsandprebiotics.Theinductionofprotective
immune responses in normal hostsby commensal bacteria is fully discussed.
Dr.Tannockstatesthattheprinciplesofmicrobialecologyareessentialtoan
understanding of the relationship between the gut microbiota and the human
host. A major advance in determining culturable bacterial species involves the
revelation that the ribosomal subunit RNA (16S RNA) contained regions of
nucleotide basesequencesthatwerehighlyconserved acrossthe world.These
were interspersed with variable V regions containing the signatures of
0889-8553/05/$–seefrontmatter ª2005ElsevierInc.Allrightsreserved.
doi:10.1016/j.gtc.2005.05.013 gastro.theclinics.com
xiv PREFACE
phylogeneticgroupsandspecies.ThisallowedextractionofbacterialRNAand
DNA-PCR amplification, making it possible to enumerate the various
phylogenetic groups of bacteria inhabiting the human gut. Dr. Tannock
confirms that the bacterialcommunity ofthe large bowel iswellregulated and
resistsminorperturbations.Thecoreortruegutmicrobiotamaybeconfinedto
relativelyfewpopulationsthatprovidemajormetabolicactivities.Dr.Tannock
offers a research road map for future endeavors involving gut microbiota.
Drs. Kalliomaki and Walker provide a molecular and biochemical basis for
the protective physiologic processes of commensal bacteria. They clearly
demonstrate the interactive relationships of innate and adaptive cellular
immunitywithanemphasisonmicrobialepithelialcrosstalk.The implications
of these findings with reference to Crohn’s disease and ulcerative colitis are
examined. Possible pathways of inflammatory effects of selected probiotics
offer insight as to their mechanism of action.
Drs. MacDonald and Gordon evaluate the extent to which the products of
commensal flora regulate immune responses in the gut. A key to understand-
ing the immune response was the discovery of mammalian pattern recogni-
tion receptors, the toll-like receptors whose function is to recognize conserved
structures on bacteria and viruses. Signaling through toll-like receptors affects
dendritic cell function, which in turn will determine T cell differentiation and
antibody responses to T-dependent antigens. These authors detail the inter-
actions of microbial flora with dendritic cells and discuss the role of bacterial
flora in regulating oral tolerance.
Dr. Bengmark offers a historical perspective of the impact of dietary
evolutionary changes on the incidence and prevalence of chronic diseases
throughout theworld.Themicrobialconnectionemphasizeshowalterationof
bacterial flora may play a significant role in altering human health and in
particular inflammatory diseases. He examines the role of antioxidants and
complex carbohydrates on chronic illnesses. Finally, he offers an analysis of
commercially available pre- and probiotics and their potential clinical ap-
plication for gastrointestinal diseases.
Drs. Salminen and Isolauri introduce the topic of gut inflammation and
barrier function by describing the impact of indigenous microflora on
immunophysiologic regulation at an early age. The importance of host–
microbeinteractionismostvitalintheneonatalperiod,whentheestablishment
of a normal microbiota provides the host with the most substantial antigen
challenge, with a strong stimulatory effect on gut-associated lymphoid tissue.
Impaired gut barrier may be the explanation for an infant’s proneness to
allergic and infectious diseases. The authors discuss the promotion of gut
barrier by probiotics and the potential for clinical application.
Dr.VanderhoofandclinicalnursespecialistR.J.Youngpresentdatarelating
tobacterialcolonization atbirthandthe subsequent immunologic implications
for the infant. Mechanisms of probiotic actions are elucidated, followed by
validation of clinical studies relating to diarrheal and allergic illnesses in the
pediatric population.
PREFACE xv
Drs. Rioux, Madsen, and Fedorak discuss the role of bacteria as inciting
agents evoking inflammatory bowel disease in genetically predisposed animals
and humans. They summarize the literature examining therapeutic efficacy of
pre- and probiotics in human inflammatory bowel disease and experimental
colitis in animal studies. Evidence classifying methodologic quality of clinical
trialsinCrohn’sdisease,ulcerativecolitis,andpouchitisisoffered.Theauthors
make recommendations for future basic science and clinical research.
Drs. Doron, Snydman, and Gorbach detail historical events leading to the
discovery of the most studied lactobacillus: Lactobacillus GG. Bacteriologic
characterization and clinical applications are offered.
Strong evidence exists supporting the use of Lactobacillus GG for the
treatmentandpreventionofacutediarrheainchildrenandantibiotic-associated
diarrhea. Potential applications for the treatment and prevention of allergies,
childhood respiratory infections, prevention of dental caries, irritable bowel
syndrome, and a wide variety of other illnesses are discussed.
Drs.Gionchetti,Lammers,Rizzelo,andCampieriprovideananalysisofthe
basicandclinicalcontributionsoftheprobioticVSL#3.Theinitialstudiesofthis
agentonpouchitisprovidedanimpetusforclinicalandlaboratoryinvestigations
ofprobioticsworldwide.Bacteriologicalbackgroundofpatientswithinflamma-
toryboweldisease,possiblemechanismsofactionofprobiotics,andtherationale
for employing multiple strains of probiotics as therapy are discussed. Clinical
studies of VSL#3 in ulcerative colitis, pouchitis, and Crohn’s disease are
analyzed.Currentandfutureresearchagendasarediscussed.
An in-depth evaluation of the only yeast-derived probiotic, Saccharomyces
boulardii,isprovidedbyDrs.ButsandBernasconi.Thisagenthasbeenstudied
inFranceforover50years.Thepharmacodynamicproperties,mechanismsof
action, basic laboratory studies, and clinical applications are detailed. This
agent does not materially affect existing microflora, yet it has properties that
inactivate bacterial toxins, inhibit toxin binding, stimulate the host immune
system, and provide trophic effects on the intestinal mucosa. Treatment of
antibiotic-associated diarrhea and Clostridium difficile colitis have demonstrated
efficicacy in randomized controlled trials. Further application in traveler’s
diarrhea, AIDS-related diarrhea, and inflammatory bowel disease are being
pursued.
Dr. Quigley explains the theoretic basis for the possible use of probiotics
in functional bowel disease. Particular attention is accorded the potential
modification of mucosal immune processes following postinfective irritable
bowel syndrome. Further evidence of low-grade inflammation and immune
activation in irritable bowel syndrome suggests a role for a bacterial dysbiosis
possibly correctable with probiotics. This is clearly an area of potential study
for functional bowel disease.
Dr. Floch and Mr. Montrose provide an analysis of the literature pertain-
ingtotheuseofprobioticsinhumans.Theyhavecatalogedtheclinicalentities
and the multiple trials on adults and children. Evidence suggests efficacy
in shortening childhood and adult diarrhea, preventing and treating
xvi PREFACE
antibiotic-associated diarrhea, and treatment of pouchitis. Further clinical
applications await randomized controlled trials.
Gerald Friedman, MD, PhD, MS, FACP, MACG
Division of Gastroenterology
Department of Medicine
The Mount Sinai School of Medicine
1751 York Avenue
New York, NY 10128, USA
E-mail address: [email protected]
GastroenterolClinNAm34(2005)361–382
GASTROENTEROLOGY CLINICS
OF NORTH AMERICA
New Perceptions of the Gut Microbiota:
Implications for Future Research
Gerald W. Tannock, PhD
DepartmentofMicrobiologyandImmunology,UniversityofOtago,720CumberlandStreet,
POBox56,Dunedin,NewZealand
Ithasbeenknown,fromearlyinthehistoryofmicrobiology,thatthegutof
peopleandotheranimalsisinhabitedbymicrobialspecies, mostlybacteria.
Louis Pasteur expressed his views to the French Academy of Sciences in
1885, on the importance of bacteria in the digestion of food, believing that life
in the absence of microbes would be impossible [1]. Until the 1960s,
descriptions of the collection of normal gut inhabitants were relatively simple,
and Clostridium perfringens, lactobacilli, enterococci, and Escherichia coli were
considered to be the predominant bacteria in the feces of adult humans [2,3].
Hungate developed innovative techniques during the 1960s for cultivating
extremely oxygen-sensitive bacteria that inhabited the proximal gut of
ruminants, and demonstrated the importance of these anaerobic bacteria in
the rumen fermentation, and hence the reliance of the ruminant host on
microbial metabolic products for nutritional well-being [4]. Holdeman and
Moore [5] modified the roll-tube cultivation methods of Hungate for use in
investigations of anaerobic infections of people. Certain obligate anaerobes, it
becameclear,wereessentialtothepathogenesisoftheseinfectionsashadbeen
indicatedbythepioneeringworkofVeillonandPrevot[6].Theformulationof
selective media for the cultivation of obligate anaerobes commonly implicated
in anaerobic infections, together with the use of anaerobic glove boxes,
accelerated the acquisition of knowledge concerning the reservoir of these
anaerobes: the normal microbiota (flora or microflora) of the human body
[7,8]. Now the stage was set for two major studies of the composition of the
collection of microbes present in the feces (the fecal microbiota) in relation to
diet and colo–rectal cancer [9,10]. These studies gathered a wealth of
information concerning the composition of the fecal microbiota of people.
Usingthenewculture techniques,somebacterialpopulationsweredetected at
levelsof1010/g(wetweight)offeces,anditwasestimatedthatasmanyas400
speciesmightbecapableoflifeinthelargebowelofhumans[11].Foranygiven
E-mail address: [email protected]
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doi:10.1016/j.gtc.2005.05.006 gastro.theclinics.com
362 TANNOCK
individual,however,30to40bacterialspeciesseemedtoconstitute99%ofthe
fecal microbiota [12].
CULTURE DEPENDENCY
Investigations of the fecal microbiota, until the 1990s, relied on the use of
bacteriological culture methods and microscopic observations of Gram’s-
stained smears [13]. Culture-dependent analysis of the fecal microbiota was
flawed somewhat, however, because not all members of the microbiota could
be cultivated under laboratory conditions. Even in the 1970s, researchers had
observed that the total microscopic count of bacterial cells in fecal smears was
always higher than the total viable count (CFUs, colony-forming units)
obtained by culture on a nonselective agar medium. It was claimed, however,
thatgoodbacteriologicalmethodswouldpermitthecultureof88%ofthetotal
microscopic count. This comparison was obtained by using total microscopic
clump counts (aggregates of bacterial cells) rather than by counting individual
bacterialcellsinsmears[11].AlthoughvalidfromthepointofviewthatCFUs
on agar plates have not necessarily arisen from a single bacterial cell, the
comparisonsgaveafalsesenseofconfidencewithregardtoanalyticalresultsat
that time. Total bacteria microscopic counts, using the 4’, 6-diamidino-2-
phenylindole (DAPI) stain and epifluorescence microscopy, since have
revealed average total bacterial cell counts in human feces approaching 1 (cid:1)
1011 per gram (wet weight) [14]. State-of the-art bacteriological methodologies
still only permit about 40% of the fecal microbiota to be cultivated on
nonselective agar medium in the laboratory [14]. Thus, many bacterial cells
seen in microscope smears had not been investigated. Although some of these
cells are probably nonviable, it seemed that many could be viable but
noncultivable because of their fastidious requirements for anaerobiosis,
complex nutritional interactions that were not reproduced in vitro, nutritional
overload in complex media, or slow growth rates [15]. Clearly, alternative
analytical methods were required.
GIVE US THE TOOLS, AND WE WILL FINISH THE JOB
Woese[16]revealedthatsmallribosomalsubunitRNA(16SrRNAinthecase
of bacteria) contained regions of nucleotide base sequence that were highly
conserved across the bacterial world and that these were interspersed with
variable to hypervariable regions (V regions). These V regions contained the
signatures of phylogenetic groups and even species. With this knowledge in
hand,newmethodsfortheanalysisofbacterialcommunities,suchasthefecal
microbiota, became available. Bacterial DNA or RNA could be extracted (in
theorynucleicacidfromallofthebacterialtypesinthesamplewasrepresented
in the extracts) and polymerase chain reaction (PCR) amplification (reverse
transcription-PCRin the caseof RNA extracts) ofthe 16SrRNAgene, in part
or complete, could be accomplished. Clone libraries of the 16S rRNA genes
FUTURE RESEARCH 363
could be made, and the clones sequenced, thus producing a catalog of the
bacterialconstituentsoftheecosystem[17].Fromthissequenceinformation,it
waspossibletoderiveDNAprobesthatspecificallytargetedvariableregionsof
the 16S rRNA gene, makingit possible to enumerate the various phylogenetic
groups of bacteria inhabiting the human gut regardless of whether they could
be cultured [18,19]. Making a library of hundreds of clones for every sample
that needed to be investigated was logistically impossible. Even microscope
counts using DNA probes is a serious undertaking and really requires an
automated system for unbiased results to be obtained. Therefore, a screening
method to compare the bacterial composition of samples was needed, and
PCR, combined with temperature or denaturing gradient gel electrophoresis
(TGGE, DGGE), filled the bill [14,20]. These and other nucleic acid-based
methodsusedintheanalysisofthefecalmicrobiotaaresummarizedinTable1
[21–23]. They are critical to successful investigations of the fecal microbiota,
because they detect all bacterial species regardless of whether they have been
cultivated.
UNIQUELY STABLE AND METABOLICALLY PREDICTABLE
Clone libraries of 16S rRNA gene sequences prepared from human feces
revealed a considerable complexity in the composition of the fecal microbiota.
Moreover,anewperspectiveconcerningtheprevalenceofbacterialspecieswas
obtained: Clostridia and related gram-positive genera were among the
predominant bacteria (Table 2, Boxes 1–3), a feature not easily recognized
from the results of culture-based studies. The Bacteroides–Prevotella group, the
Eubacterium rectale–C coccoides group, and the C leptum group (containing the
Faecalibacterium prausnitzii cluster) were the predominant phylogenetic divisions
of bacteria encountered in all human fecal microbiota. Monitoring the
composition of the fecal microbiota of individual people by the use of PCR/
TGGE orPCR/DGGE,however,showedthatthedetailedcompositionofthe
fecal microbiota of each person, with respect to the numerically dominant
species, was unique and extremely stable over time [14,20]. Variation in the
composition of the human fecal microbiota between human subjects also was
detected by fluorescence in situ hybridization (FISH) [24]. Collectively, these
observations indicate that the bacterial community of the large bowel is well-
regulated and resists minor perturbations that could be induced by a varied
dailydietorminorfluctuationsinhostphysiologicalparameters.Uniquenessin
composition of bacterial communities likely reflects consistent physiological
and immunological idiosyncrasies of people that are controlled by the genetic
constitution of the host. Monozygotic twins, for example, have PCR/DGGE
profilesthataremoresimilartooneanothercomparedwiththoseofunrelated
subjects [26].
Althoughthephylogeneticcompositionsofindividualfecalmicrobiotaseem
unique among people, the overall metabolic profile of the microbiota in terms
of fermentative ability is similar. The same short-chain fatty acids in similar
364 TANNOCK
Table1
Nucleicacid-basedanalyticalmethodsusefulingutmicrobiotaresearch
Example
Technique Description reference
RNAdotblots Purpose:quantificationofspecific 19
bacterialpopulation
Summary:hybridizationofRNA
extractedfrommicrobiotaand
blottedonmembranewithspecific
labeled(oftenradioactive)
oligonucleotideprobeanduniversal
bacterialprobe.Resultgivenas
specificpopulationaspercentof
totalmicrobiota.
Fluorescenceinsituhybridization Purpose:detectionandquantification 18
(FISH)andepifluorescence ofbacterialcells
microscopy Summary:fluorescentdye-labeled
oligonucleotideprobehybridizesto
ribosomalRNAsequenceincells
fixedonslideswithwells.
Enumerationbyepifluorescence
microscopy.
Terminalrestrictionfragmentlength Purpose:profilingandquantifyingthe 21
polymorphism(T-RFLP) compositionofthebacterial
community
Summary:fluorescentdye-labeled16S
rRNAterminalgenefragmentsare
generatedbyPCRusingalabeled
primerandsubsequentrestriction
digestion.Detectionand
measurementoffluorescenceusing
sequencinggelsproducegenetic
fingerprintsthatcanberelatedto
communitycomposition.
Polymerasechainreactioncombined Purpose:profilingthecompositionof 24
withdenaturinggradientgel bacterialcommunitiesfor
electrophoresis(PCR/DGGE) comparativeanalysis
Summary:avariablesequenceregion
ofthe16SrRNAgeneisamplified
byPCRfromgenomicsequencesin
thesample.Separationof16S
rDNAfragmentsfromdifferent
bacterialtypesisbasedon
differencesinchemicalstability,
throughalinearlyincreasing
gradientofchemicaldenaturants.
TheprofileofDNAfragments
representsthegeneticfingerprintof
thecommunity.
Polymerasechainreactioncombined LikePCR/DGGEbutusingtemperature 20
withtemperaturegradientgel gradientinseparationofDNA
electrophoresis(PCR/TGGE) fragments
FUTURE RESEARCH 365
Table1
(continued)
Example
Technique Description reference
16SrRNAgenelibrary Purpose:preparingaphylogenetic 22
catalogueofthebacterial
community
Summary:PCRamplificationof16S
rRNAgenesfromasample,
subsequentcloninginaplasmid
vectorandcloninghosttoderive
alibraryofindividualrRNAgene
clonesforsequencingand
phylogeneticanalysis
Realtimequantitativepolymerase Purpose:quantificationofbacteria 23
chainreaction Summary:PCRprimersandalabeled
probe(oftenincorporating
areporter
dyeandaquenchermolecule)are
usedtomeasurethereal-time
accumulationofaspecific
targetsequence
proportions are detected in human feces regardless of the microbiota profile
obtained by PCR/DGGE [27]. This argues for considerable redundancy
amongthebacterialspeciesthat caninhabitthe gut;probablyseveralbacterial
speciescanfillagivenecologicalniche,andeachnicheisfilleddifferentlyfrom
persontoperson.Thus,nomatterwhatthecompositionofthemicrobiota,the
bowel ecosystem functions in the same, predictable manner. It may be
speculated, therefore, that amassing catalogs of bacterial species will not
promote a much clearer understanding of the gut ecosystem than that which
alreadyexists.Meredetectionofabacterialspeciesinagutsamplerevealslittle
of the role of this organism in the ecosystem, or even if it is metabolically
active. There is a striking difference in PCR/DGGE profiles of bacterial
communities in human feces generated using bacterial RNA as the PCR
template compared with DNA. Profiles generated from bacterial RNA in
a recent study showed intensely stained fragments clustered in the middle of
the denaturing gradient. The profiles were markedly different than those
generated from DNA [28]. RNA extracted from bacterial cells is mostly
ribosomal RNAand can beused asanindicatorof metabolicactivity, because
the ribosome per cell ratio is roughly proportional to growth rate of the
bacteria. Although DNA-based analytical procedures provide a phylogenetic
picture of the community, they do not reflect metabolic activity, because the
DNA could originate from living active cells, living dormant cells, lysed cells,
or dead cells [29]. On the basis of these observations, it can be proposed that
thehumangutprovidesahabitatforadiversityofbacterialspecies, asseenin
DNA-DGGE profiles, the composite of which varies from one person to