the history of microbiological 
 research has been dominated by investigations of the agents of human infectious 
 disease .
motivated by the desire to culture , characterize , and understand the 
 pathogenicity mechanisms of these organisms , several centuries of 
 microbiological research culminated in a broad range of antimicrobial 
 therapies , vaccines , and immunizations . in more recent years , similar 
 analytical methodologies have been applied to facilitate exploitation of 
 bacteria for industrial applications .
two related branches of microbiology  environmental 
 microbiology , and the study of intestinal commensals ( a branch of the first in 
 purest terms)lagged behind 
 until relatively recently . from the mid 1990s , a range of techniques allowed 
 environmental microbiologists to indentify soil microorganisms 
 in situ , without resorting to 
 culture , based upon ribosomal small subunit rna gene probes .
a natural 
 extension of this approach was to sequence large numbers of cloned ribosomal 
 rna gene amplicons , yielding catalogs of all the organisms ( the microbiota ) 
 present in complex samples .
latterly the field of metagenomics has provided 
 technical approaches to sequence large fractions of the entire microbial dna 
 present in an ecological system .
coupled with the application of molecular 
 tools for studying commensal bacteria , many of which were originally developed 
 for studying pathogens , there is now an exciting nexus between technologies and 
 research foci whereby commensal bacteria may be studied in the context of 
 intestinal ecosystems .
this review will summarize what is known about the effect 
 of introducing probiotic bacteria on the composition and activities of the microbiota , 
 with an emphasis on recent studies using culture - independent methods .
the 
 likely mechanisms whereby commensals   exert their influence are discussed , and directions for future research are outlined .
the notion that certain intestinal 
 microorganisms might benefit the host derives historically from suggestions by metchnikoff and others that putrefying bacteria that contribute to toxification and aging could be 
 deliberately replaced by fermentative organisms [ 1 , 2 ] . in this context , some of the 
 fermentative bacteria ,
probiotic bacteria are live microorganisms which when 
 administered in adequate amount confer a health benefit on the host [ 3 , 4 ] .
many microorganisms that are 
 considered probiotic have been traditionally used to preserve food products by 
 fermentation , and are present in the food in varying numbers , along with their 
 fermentation end products and other metabolites .
thus another operational definition 
 of the term probiotic requires the organism in question to be  consumed in 
 adequate amounts  to confer a benefit .
the host benefits that have 
 been attributed to consumption of probiotic microorganisms are diverse ( reviewed 
 in [ 3 , 68 ] ; some major examples are listed 
 in table 1 ) , and have been substantiated to different degrees .
probiotic 
 bacteria are now included in a wide range of consumer formulations including 
 yoghurts , drinks , capsules , and dietary supplements , and they represent a 
 significant element in the modern functional foods market .
organisms used as 
 probiotic agents are frequently members of the genera lactobacillus or bifidobacterium , 
 but escherichia coli , bacillus subtilis , saccharomyces boulardii , and enterococcus faecium are also employed , among others .
thus , an organism employed 
 as a probiotic agent may not necessarily be part of what is considered the 
  normal microbiota .
 tannock distinguishes between allochthonous and 
 autochthonous species [ 10 , 11 ] . 

autochthonous means bacteria 
 both present and replicating in situ 
 in the human gi tract , as distinct from transiently passing through 
 ( allochthonous ) .
bacteria administered as probiotic agents are not necessarily 
 autochthonous to the consuming animal , and indeed some lactobacillus species may only be autochthonous for certain human 
 individual subjects , and possibly not the majority of subjects ( see below ) . 
 rate of growth of allochthonous lactobacilli may be a critically limiting step 
 preventing their establishment .
with regard to developing 
 probiotic strains for exploitation , it may prove easier to identify beneficial 
 traits in species that are autochthonous to the human consumer , as consumer 
 acceptance is likely to be easier if the probiotic ingredient in a functional 
 food ( a food product with benefit to the consumer over and above inherent 
 nutrition ) was first cultured from humans . related to the consumption of probiotic agents
 a 
 nondigestible food ingredient that beneficially affects the host by selectively 
 stimulating the growth and/or activity of one or a limited number of bacteria 
 in the colon and thus improves host health  [ 30 , 31 ] .
it follows from this definition 
 that the bacteria capable of metabolizing prebiotics should be restricted to a 
 small number of beneficial species or strains ( reviewed in ) . in practice
, prebiotic compounds 
 must also be refractory to host digestive processes , and the combined catabolic 
 activities of bacteria higher up in the gastrointestinal tract , so that 
 prebiotic compounds are often oligosaccharides towards which probiotic bacteria 
 produce specific hydrolases .
prebiotics are commonly found in , 
 or extracted from , plant material including fruits , cereal , and vegetables , but 
 are also present in human milk and colostrum .
the best characterized prebiotics 
 include inulin , fructooligosaccharide , galactooligosaccharide , xylooligosaccharide , 
 isomaltooligosaccharide , and lactulose ( reviewed in ) .
unravelling the health benefits 
 of prebiotics is a challenging task , because these compounds have parallel 
 direct effects on the host , and potentially on multiple members of the 
 microbiota .
for example , -glucans are unbranched polysaccharides with ( 1 - 4 ) 
 and ( 1 - 3)-linked -d glucopyranosyl units , that are recognized as important 
 dietary ingredients ( reviewed in ) .
-glucans are components of plant 
 cell walls , and are abundant in the endosperm of cereals such as barley and 
 oatmeal .
consumption of -glucans has attendant health benefits that are 
 recognized by health and regulatory bodies in several jurisdictions including 
 the us 
  .
these benefits include lowering of 
 blood cholesterol and lipoprotein , lowering of postprandial glucose 
 and insulin responses , and enhancement of antitumor 
 monoclonal antibodies .
supplementation of mammalian diet 
 with -glucan , or modification by prehydrolysis of in vitro 
 bacterial growth 
 medium , leads to increased 
 numbers and proportions of lactobacilli or bifidobacteria [ 3941 ] .
barley supplementation of rat 
 diet   led to an increase in lactobacillus numbers , a decrease in bacteroides and coliforms , and an increase in the production 
 of butyrate .
butyrate is an important energy source , signalling metabolite , proliferation 
 stimulus for normal colonic epithelial cells , and anti - proliferative signal for 
 neoplastic colonocytes [ 42 , 43 ] , suggesting a potential direct 
 benefit from dietary ingredients or prebiotics that promote growth of 
 clostridia .
until recently , the composition of the microbiota was examined by 
 relatively insensitive techniques . culturing the bacteria was unrepresentative , 
 because a large proportion of the bacteria do not grow on standard laboratory 
 media .
analysis by temperature gradient 
 gel electrophoresis provided one of the earliest insights into the uncharted 
 complexity of the microbiota . using denaturing
gradient gel 
 electrophoresis ( dgge ) of 16s rrna gene amplicons , the same group later showed 
 that the colonic mucosal microbiota and faecal microbiota were different , and 
 the colonic mucosal microbiota was likely dependent on host factors . meanwhile fluorescent 
 hybridization of probes for 16s rrna genes was being applied to determine 
 species identities , numbers , and proportions of intestinal bacteria [ 47 , 48 ] , exemplified by the studies of 
 dore , blaut and colleagues [ 49 , 50 ] .
these analyses highlighted 
 extensive inter - individual variation at phylotypes level ( among northern 
 europeans ) , and some correlations of microbiota 
 with age , gender , and geographic origin but which varied between countries .
our current understanding of the intestinal 
 microbiota ( reviewed in [ 51 , 52 ] ) has been significantly shaped by 
 culture - independent methods , in particular the sequencing of 16s rrna gene amplicons , 
 either from clone libraries or direct pyrosequencing of the pcr product .
a 
 consensus appears to be emerging in the literature of somewhere between 800 and 
 1000 bacterial phylotypes being present in the healthy human intestine ; the evidence for which will be 
 selectively presented here . a relatively small - scale investigation by benno and colleagues in 2003 
 revealed an unexpectedly high number of novel phylotypes in 240 partially 
 sequenced 16s rrna gene amplicons clones derived from six elderly individuals . in a pioneering study ,
relman 
 and colleagues applied the 16s rrna molecular inventory - based approach , at a 
 much larger scale than previously published , on samples from both colonic sites 
 and faeces ; strikingly , the majority of the 
 sequences derived 
 corresponded to uncultivated species and novel microorganisms . the human 
 stomach , previously considered sterile except for infections with helicobacter pylori ,
was revealed by 16s 
 rrna gene library sequencing to be well populated by bacteria , based on 
 detection of 128 bacterial phylotypes from 23 gastric endoscopy samples .
gill and colleagues   sequenced not just 16s rrna genes , 
 but also randomly cloned bacterial dna 
gill et al . showed by this metagenomic approach that the bacteria in 
 the gut significantly expand the metabolic capabilities of the human gut . by generating two to three 454 
 pyrosequencing runs per mouse cecum , gordon and colleagues showed the existence 
 of an obesity - associated gut microbiome with increased capacity for energy 
 harvest .
significantly , this balance of the 
 microbiota was borne out when investigating obese humans , showing a seminal link between 
 human obesity and changes in the microbiota .
furthermore , the complexity of the 
 microbiota in humans and 59 other mammalian species was shown to be linked to 
 phylogeny ( of the mammal ) and the composition of the diet .
analysis of the metabolic 
 capability likely conferred by the microbial metagenome recently revealed 237 
 gene families commonly enriched in adult - type and 136 families in infant - type 
 microbiomes .
thus , any consideration of the 
 effect of probiotics on the intestinal metagenome should ultimately include 
 analysis of the downstream effects upon the host of impacting on this metabolic 
 capability .
a more fundamental consideration is that the genera whose members 
 are among the most commonly employed probiotics  bifidobacterium and lactobacillus  are not present 
 in the human gastrointestine at the high levels traditionally expected based on 
 culture - based approaches , being represented by 20 phylotypes ( ca . 2% ) and 36 
 phylotypes ( ca .
3.6% ) , respectively . thus if probiotic bacteria impart 
 health benefits to the host under
 natural conditions ,  that is , in 
 individuals who have normal nonmanipulated numbers of probiotic bacteria , they accomplish 
 this despite being at much lower numbers than are achieved by consumption of 
 probiotic products .
there have been relatively few 
 studies which have rigorously characterized the effect upon the whole microbiota 
 of administering probiotic cultures , and until recently , all such studies 
 applied targeted analysis of specific groups of bacteria . in one of the 
 earliest investigations , tannock and colleagues observed transient and modest 
 fluctuations in lactobacillus and bifidobacterium numbers following consumption 
 of a probiotic l. rhamnosus strain 
 dr20 .
lactobacilli and enterococci 
 were detected more frequently ( among 10 subjects ) and in higher numbers during 
 consumption .
interestingly , the presence of stable populations of lactobacilli 
 before the administration period precluded long - term colonization by the 
 administered probiotic strain .
most subjects ceased 
 shedding the probiotic strain in faeces soon after its consumption stopped , but 
 the l. rhamnosus strain remained 
 detectable in faeces of one subject over 2 months after the test period .
these 
 data suggest inter - host variables such as bacterium - host or bacterium - diet 
 interactions .
probiotics 
 and prebiotics are commonly applied in companion animals and production animals 
 [ 63 , 64 ] , and there have been some 
 studies of effects upon the microbiota .
administration of a cocktail containing 
 lactobacilli , bifidobacteria , enterococci , and pediococci improved weight gain 
 in broiler chickens , which was associated with an increase in numbers of bifidobacterium spp . ,
administration of a 
 probiotic enterococcus faecium strain 
 reduced e. faecalis numbers in the 
 intestines of weanling piglets , but total numbers of e. faecium remained unchanged , suggesting that the administered 
 strain had displaced part of a fixed number of niche sites occupied by the same 
 species .
many investigations have 
 been published describing the effects of probiotic bacteria on human pathogens 
 ( reviewed in ) , some of which are normal 
 components of production animal microbiota .
enterobacteriaceae numbers were reduced 
 when a cocktail of two lactobacillus strains was administered to pigs , and a five - strain probiotic 
 combination reduced salmonella enterica serovar typhimurium shedding in 
 pigs .
although data from small 
 animal models for human probiotic strains must be interpreted with caution , it was interesting to note 
 from a recent study that administration of l. 
 casei and l. plantarum affected 
 the diversity of murine intestinal lactobacilli , but not the overall bacterial 
 community structure .
there was an increase in the 
 number of lactobacilli related to the acidophilus complex in the inoculated mice .
these animal models provide an opportunity for 
 determining the effect of probiotic administration on the entire microbiota but 
 must ultimately be repeated in humans if that species is the desired host .
studies 
 in humans are currently few in number , and are often focused in nature . for 
 example , consumption of a commercial probiotic yoghurt reduced clostridium difficile - related diarrhoea 
 in hospitalized patients , but effects on the broader microbiota were not 
 studied .
alterations in the fecal 
 microbiota have been reported in irritable bowel syndrome ( ibs ) [ 72 , 73 ] .
however , administration of a 
 multispecies probiotic supplementation which alleviated ibs had negligible effect 
 upon the composition of microbiota as measured by quantitative pcr with 
 group - specific primers . however , this approach may 
 have missed changes in microbial composition within these groups .
a follow - up 
 study reported stabilization of the microbiota over time , which was related to 
 amelioration of symptomatology that was absent from the placebo control group . 

alterations of the human intestinal microbiota have also been reported in 
 inflammatory bowel disease ( ibd ) [ 7678 ] . given the clinical impetus 
 to find simple non - medicinal solutions to ibd and ibs
, one can anticipate 
 renewed vigor in studies of probiotic bacteria as agents for microbiota 
 modulation in these subjects .
probiotics also appear to be efficacious as 
 adjunct therapy for infectious diarrhea , with a recent meta - analysis revealing 
 reduction in risk and duration of diarrhea .
most of the 23 studies 
 included in this analysis were descriptive rather than investigative of the 
 microbiological aspects , and future determination of the effects on the 
 microbiota wrought by probiotic intervention will be very informative .
as 
 recently as 2006 , the effect of probiotic administration in humans was still 
 being followed by bacteriological culture , but as concluded by the authors of 
 one such study , there was a clear case for culture - independent molecular 
 methods to be applied instead .
community profiling by dgge 
 showed that lactulose increased the levels of bifidobacterium adolescentis in subjects consuming the prebiotic 
 lactulose , whereas the probiotic yeast s. 
 boulardii did not cause any significant universal changes in dgge profiles .

 figure 1 shows a 
 schematic overview of the potential mechanisms whereby probiotic 
 micro - organisms might influence the intestinal microbiota .
consumption of probiotic cultures may modulate 
 the microbiota or change its metabolic properties by competition for nutritional 
 substrates .
gordon and colleagues have used transcriptional microarrays to show 
 that introducing a probiotic into the mouse gut changes the way the endogenous microbiota 
 metabolize the diet . when germ - free mice that had been 
 monoassociated with bacteroides 
 thetaiotaomicron were challenged with bifidobacterium 
 animalis or lactobacillus 
 casei , both interventions caused shifts in the gene expression pattern of 
 the b. thetaiotaomicron genome . these differentially 
 expressed gene sets ( i.e. , in response to the two probiotics ) did not overlap , 
 emphasizing that different probiotics elicit different responses .
however , many 
 of the genes in b. thetaiotaomicron whose expression 
 was altered by presence of either probiotic strain were related to expansion of 
 the carbohydrate metabolizing capability of b. thetaiotaomicron .
thus , one of the ways in which 
 probiotics can impact upon the composition of the microbiota is apparently by 
 competing with them for substrate availability , and by altering the dynamics of 
 carbohydrate utilization by individual microbiota components .
this competition 
 is probably not restricted to the intestine , since recent evidence indicates 
 that oral bifidobacterium strains ( b. 
 adolescentis ) reduce vitamin k concentration , and may thus compete with porphyromonas gingivalis in the 
 oral cavity .
the application of 
 metabolic profiling methods to animal models has suggested another indirect way 
 in which probiotic bacteria might impact on the microbiota , namely , by 
 production of a significantly different microenvironment due to a diverse range 
 of metabolic pathway outcomes . in a recent study using germ - free mice colonized 
 by human baby microbiota and exposed to two lactobacillus strains , nicholson 
 and colleagues observed microbiome modification , measured by selected culture regimes 
 .
this was accompanied by 
 changes in cecal concentrations of short - chain fatty acids , and marked changes 
 in fecal levels of diverse metabolites including choline , acetate , ethanol , a 
 range of putative n - acetylated metabolites ( nams ) , unconjugated bile acids 
 ( bas ) , and tauro - conjugated bile acids .
while a natural focus of these studies 
 is the effect of these metabolites upon the host , it is likely that such gross 
 changes in metabolic profile also impact upon intestinal microbiota 
 composition . as noted in table 1
, some probiotic bacteria also produce vitamins 
 , enhanced availability of 
 which may modulate the microbiota .
in addition , exopolysaccharide produced by 
 probiotics including lactic acid bacteria   could act as a growth 
 substrate for selected components of the microbiota ( see figure 1 ) .
it has 
 been shown in several recent studies that they can modulate numbers of single 
 model organisms in experimental systems .
for example , probiotic l. salivarius strains inhibit the growth of h. pylori in vitro in a strain - dependent manner , by mechanisms involving 
 lactic acid secretion , and another as yet uncharacterized mechanism ( k. a. ryan 
 and p. w. o'toole , unpublished ) .
intestinal l. salivarius strains are distinguished by 
 production of a broad - spectrum bacteriocin abp118 , but this is not likely to 
 contribute to antagonism to gram - negative bacteria like h. pylori .
however , production of this bacteriocin abp118 was 
 identified as the mechanism whereby l. salivarius ucc118 eliminated listeria monocytogenes infection in a 
 murine model , providing the first definitive mechanism for anti - infective 
 activity of a probiotic bacterium in 
 vivo .
interestingly , both the 
 wild - type strain ucc118 and a bacteriocin - negative derivative were equally able 
 to suppress salmonella typhimurium 
 infection in the mouse model , suggesting that broader antimicrobial effects on 
 the gram - negative components of the microbiota may occur . from an opposite perspective , 
 production of a
bacteriocin - like substance by vaginal enterococci has been 
 linked to reduction in levels of commensal lactobacilli that is linked to 
 vaginosis .
natural competition between 
 commensals and opportunistic pathogens may therefore be mediated by mechanisms 
 such as bacteriocin production , that can be exploited for using probiotics to 
 modulate the microbiota .
competitive exclusion ( see figure 1 ) , whereby adherent 
 probiotic species occlude access of members of the microbiota to the epithelium 
 [ 89 , 90 ] , represents another way of 
 modulating the microbiota , although strong evidence for this occurring 
 in vivo is lacking .
the most subtle 
 effects wrought by probiotics on the microbiota are potentially those that 
 operate by indirect mechanisms involving the host .
improvement of the 
 intestinal epithelium barrier function   might theoretically , for 
 example , impact on efficiency of invasion of pathogens , severity of subclinical 
 tissue damage , and release rates of host - derived micronutrients ( see figure 1 ) , 
 that could translate into impacts on the microbiota . in an analogous manner
, 
 pathological changes in intestinal epithelium might also favor growth of 
 certain members of the microbiota , if inflamed or damaged epithelial cells 
 differentially affect the microbiota . it is well established that some probiotics 
 can suppress inflammation by inhibiting proinflammatory cytokine production [ 9294 ] , and although the molecular 
 basis for this is not currently understood for probiotics , mechanisms and 
 molecules have recently been identified in commensals and pathogens [ 14 , 95 ] . reduction in gut 
 inflammation by probiotics
furthermore , some probiotic bacteria 
 have been reported to stimulate the innate immune system both in animal models 
 and in elderly subjects [ 96 , 97 ] , by an unknown mechanism . 

administration of probiotic bacteria could thus bolster innate immune activity 
 against transient pathogens , or non - commensal elements in the microbiota , 
 leading to subtle changes in long - term overall composition .
however , more 
 studies are required to substantiate the mechanisms in the probiotic - host 
 interactions , and to investigate if they do in fact impact on the microbiota .
there has been a rapid recent 
 accumulation of sequence - based information on the composition of the gut 
 microbiota .
however , for pragmatic reasons of sample collection facility , this 
 is largely based on fecal analysis , and the microbiota of the colon and small 
 intestine will be different from feces .
studies of the small intestine are 
 particularly warranted because probiotics are proportionally more numerous 
 there , and may exert significant biological activity at this site .
there is adequate 
 information in the literature to support the hypothesis that administration of 
 probiotic cultures in high doses to human subjects will impact on the 
 intestinal microbiota .
a comprehensive intervention study , supporting this 
 hypothesis by deep compositional and functional metagenomics approaches , and 
 supplemented by metabolomics , is not currently available ( june 2008 ) . in this 
 hypothetical study , mechanisms whereby
changes in the microbiota that were achieved could 
 be inferred to a degree by global transcriptional analysis , but definitive linkages 
 between bacterial gene products and effects upon the microbiota could be 
 impossible to establish because of the regulatory issues surrounding human 
 trials with genetically modified organisms . as noted above , proof of principle 
 may be established in animal models , but ultimately these studies must be 
 validated in human subjects .
there remains the intriguing question of the role , 
 if any , of the relatively small numbers of potentially probiotic organisms as 
 part of the microbiota in ostensibly healthy individuals .
do these organisms 
 contribute to maintenance of health  or avoidance of 
 disease ? is the level of candidate probiotic organisms in the microbiota 
 critical , and does its importance vary with age ? as noted above ,
there is 
 reasonable evidence that changes in the microbiota accompany disease states 
 like ibd and ibs , conditions whose prevalence increases with aging .
there are attractive 
 hygiene - related hypotheses suggesting that depletion of probiotic commensal microbiota 
 in early life may be responsible for the dramatic rise in diseases involving 
 immune dysregulation .
the challenge now is to rigorously 
 tackle the interplay of diet , microbiota , and host factors in tractable 
 experiments that will elucidate the key elements in determining outcomes of 
 this interplay , and allow its manipulation .
 probiotics have a range of proposed health benefits for the consumer , which may include modulating the levels of beneficial elements in the microbiota . 
 recent investigations using molecular approaches have revealed a human intestinal microbiota comprising over 1000 phylotypes . 
 mechanisms whereby probiotics impact on the intestinal microbiota include competition for substrates , direct antagonism by inhibitory substances , competitive exclusion , and potentially host - mediated effects such as improved barrier function and altered immune response . 
 we now have the microbial inventories and genetic blueprints to begin tackling intestinal microbial ecology at an unprecedented level of detail , aided by the understanding that dietary components may be utilized differentially by individual phylotypes . 
 controlled intervention studies in humans , utilizing latest molecular technologies , are required to consolidate evidence for bacterial species that impact on the microbiota . 
 mechanistic insights should be provided by metabolomics and other analytical techniques for small molecules . 
 rigorous characterization of interactions between the diet , microbiota , and probiotic bacteria will provide new opportunities for modulating the microbiota towards improving human health . 
1. INTRODUCTORY REMARKS
2. THE CONCEPTS OF PROBIOTICS AND PREBIOTICS
3. THE NORMAL MICROBIOTA OF
HUMANS AND ANIMALS
4. PROBIOTICS AND THEIR EFFECT
UPON THE MICROBIOTA
5. MECHANISMS OF PROBIOTIC IMPACT ON
THE MICROBIOTA
6. KNOWLEDGE GAPS AND CONCLUDING REMARKS