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Early Human Development (2007) 83, 165–170

Bacteria and early human developmentMark Wilks ⁎,1

Department of Microbiology, 3rd Floor, Path and Pharm Block, 80 Newark St, E1 2ES, London, United Kingdom

⁎ Tel.: +44 20 3246 0295; fax: +44 20E-mail addresses: m.wilks@qmul.ac

mark.wilks@bartsandthelondon.nhs.uk1 Lead Clinical Scientist, Department

London NHS Trust. Honorary Senior LeDiseases, Barts and the London SchooQueen Mary University of London.

0378-3782/$ - see front matter © 200doi:10.1016/j.earlhumdev.2007.01.007

Abstract Our understanding of the relationship between microbes and their animal hosts havechanged dramatically in the last decade. The development of powerful new molecular methods aswell as different animal models, particularly germ free rodents, has enabled precise characterisa-tion of the ways in which commensal bacteria in the gastrointestinal tract and other areas interactwith their hosts. It is now clear that animal development, far from being genetically determined isintimately bound up with the microbial flora of that particular animal. In germ free mice, theaddition of Bacteroides thetaiotaomicron mediates the maturation and function of thegastrointestinal tract in several different ways. Similarly Bacteroides fragilis directs the deve-lopment of the immune systemboth in and outside the gastrointestinal tract. The relevance of thesefindings and others to our understanding of human development and disease is discussed.© 2007 Published by Elsevier Ireland Ltd.

KEYWORDSMicrobial flora;Germ free models;Probiotics

The concept that bacteria might have a pivotal role in earlyhuman development is relatively new, and may seem at firstabsurd. Hitherto, it was generally assumed that all the majorsteps in the development of complex organisms weregenetically determined, yet the evidence from models ofanimal–bacterial interaction which have been developedover the last 10 or so years provide powerful evidence thatthis is not the case and that bacteria have a major role inanimal development.

Previously the microbial flora was largely ignored orunderstood in terms of a more or less continuous battlebetween humans and bacteria, with occasional areas ofapparent truce such as the gastrointestinal tract where largenumbers of bacteria appeared to coexist with the host to noapparent detriment [1]. This has been supplanted in the last

3246 0303..uk,.of Microbiology, Barts and thecturer, Centre for Infectiousl of Medicine and Dentistry,

7 Published by Elsevier Ireland Ltd

ten years or so by the gradual realization that bacteria andtheir hosts have coevolved and that coevolved bacterial–animal partnerships represent a common fundamentaltheme in the biology of animals [2].

This paradigm shift in our understanding has come aboutin the last few years for a number of reasons, both technicaland conceptual. Firstly the development of a number ofdifferent animal–microbe models which are readily amen-able to experimental manipulations and some of the keyfindings from these are discussed below. Particularlyimportant has been the development of germ free animalmodels which show striking differences from conventionallyraised animals in their development, some of which areshown in the Table 1.

Secondly, complementing the sequencing of the humangenome, has been the development of metagenomics, whereapplication of high throughput sequencing methods andother molecular techniques have been applied to character-ise the microbial flora of a particular site, regardless ofwhether the bacteria there can be cultured in the laboratory[3]. A ‘second human genome project’ aimed at characteris-ing the microbes of the gastrointestinal tract has beenproposed [4]. So far over 2000 different phylotypes have

.

Table 1 Selected differences between Germ Free (GF)and Conventionally Raised (CR) rodents

GF rats live longer than CR ratsGF mice and rats have lower cardiac weight and outputGF mice and rats have a lower metabolismGF mice have increased susceptibility to vitamindeficiencies

GF mice have reduced epithelial renewal ratesGF mice have lower gut motilityGF rats have reduced bile acid deconjugationGF rats produce more bile acidsGF mice absorb more cholesterolGF mice have reduced capillary network in their villusmesenchymal cores

GF mice have 40–100 times serum IgM/IgG and intestinalIgA

GF mice are more resistant to developing IBD and have areduced susceptibility to arthritis

GF rats are very susceptible ( approximately 50%) todeveloping kidney stones

166 M. Wilks

been described that are normally found associated with man.There have also been numerous other technical advancessuch as the development of microarrays, allowing changes inthe expression of both host and bacterial genes to be studiedunder a variety of different conditions when they areinteracting.

This recent research has shown that the microbiota of thegut consists of at least 1013–14 microorganisms [5]. Thisincludes not only 800–1000 species of bacteria, but fungi,viruses, archea and eukaryotic microorganisms such ashelminthes and protozoa. In fact although apparentlyimpossibly complex, only 8 of the possible 55 main divisionsof bacteria are present, so there is already some selection offlora by the host. More recently these have been referred toas the ‘microbiome’ [6]. This collective genome is approxi-mately 100–1000 fold larger than that of the human host(1011 cells). Blaser proposed an integration of the human 46chromosome genome and the extremely large microbiome toproduce the term ‘metabolome’ to represent the totalmetabolic activity of the host and microbial flora, an ideawhich he suggests might proof helpful in the understanding ofnormal physiology and disease [6]. In some cases the relativeimportance of the microbial flora may be overstated, arecent review described the role of the human host as ‘anadvanced fermenter, carefully designed to maximise theproductivity of the microbiome’ [7]. The burgeoning numberof terms coined by adding the suffices ‘ -omic’ or ‘-ome’ toexisting terms to indicate the emerging fields of large-scale,data-rich biology, which might appear facetious, actuallyreflects this conceptual change in thinking [8].

1. Developmentof animalmodels of host-microbeinteraction

Much of the evidence for bacteria influencing host develop-ment has been derived from invertebrate model systems ofhost microbe interaction, but whilst some specific featuresof the interaction may not be applicable, their relevance

should not be discounted for that reason, indeed one of thestriking features of cellular microbiology is how widespreadsome of the mechanisms of host–microbe interaction are.Thus it is often forgotten that the Toll receptor system whichhas been the subject of recent intensive study in humans wasfirst described in the fruit fly Drosophila, and even has ahomologue in plants — the nucleotide-binding/leucine richrepeat proteins (NB/LRR) which confer protection on theplant against some bacterial pathogens.

The first clue that bacteria might induce morphologicalchanges in their hosts came from studies of the luminousbacterium Vibrio fischeri that lives in the light organ of themarine squid Euprymna scolopes. The bacteria live in aprotected nutrient rich environment where they producelight that illuminates the lower surface of the animal, providingcamouflage to the squid and protecting it from predatorsbeneath [2]. The bacteria are specifically acquired within amatter of a few hours of the host's hatching when V. fischerialone is selectively taken up from the surrounding sea water,which contains about 106 organisms per ml. The epithelial cellsresponsible for this selective acquisition are then lost. Thebacterial cells also promote differentiation of light organepithelial cells, causing profound changes in size and shape [9].

This model has two features of particular interest — howdoes the host selectively acquire V. fischeri alone from theseawater and how are the host cells lost once the bacteriahave been acquired? The squid epithelial cells secretecopious amounts of mucus and it is here that selection ofthe bacterium occurs [10]. The precise molecular basis of theselective take up is not yet clear but the bacterial cells dohave to be viable, suggesting that active metabolism ratherthan passive adherence to the mucus is important. Interest-ingly bacterial motility is not important, suggesting that thisone species is actively taken up by the host.

Once the squid has acquired the bacteria from thesurrounding seawater, it is also clear that the presence ofthe bacteria induces morphogenesis in the developing lightorgan. The ciliated epithelial cells which are responsible forharvesting the bacteria are lost as programmed cell death isinduced by specific monomeric peptidoglycan fragmentsreleased from the bacterial cell wall.

At first sight this model system seems so esoteric that itcould not have any relevance to humans. However, theimportance of cross talk between components of the bacterialcell wall and mammalian cells first studied in the squid Vibriosystem is increasingly recognised and has direct relevance tohuman infections and disease. The same peptidoglycan frag-ments that are released by V. fischeri are released by thehuman pathogens Bordetella pertussis and Neisseria gonor-rhoeae. In whooping cough, B. pertussis releases a peptidogly-can fragment called tracheal cytotoxin (TCT), which actssynergistically with lipopolysaccharide (LPS) inducing theproduction of IL-1a and nitric oxide, causing the selectiveepithelial damage that occurs in whooping cough [11].

Determining the precise mechanisms by which V. fischerialone is able to traverse the mucus may have importantimplications for the study of human disease such as IBD andcystic fibrosis, where Pseudomonas aeruginosa appears tohave a unique interaction with the copious quantities ofmucus in the lungs and other organs.

These specific molecules which are recognised by the hostare now collectively known as pathogen-associated molecular

167Bacteria and early human development

patterns (PAMPs). They include the peptidoglycan fragmentsmentioned above but also unmethylated dinucleotidesequences (CpG) in the microbial DNA, LPS in Gram negativebacteria, lipoteichoic acid (LTA) in Gram positive bacteria anddouble stranded RNA replication intermediates in viruses. Thesame LPS structure can act as an agonist by stimulating a Tolllike receptor response in one mammalian host whilst acting asan antagonist in another [12].

The squid—V. fischeri system poses the question do host–bacterial interactions effect morphogenesis in mammals? Thisquestion has now been unequivocally answered with thefinding that the microbiota appears to direct intestinalangiogenesis in the germ free mouse. Amongst other differ-ences, the germ free mouse has a very poorly developed bloodsupply to the gut. Surprisingly it appeared that addition of thenormal flora was capable of inducing intestinal angiogenesis inthe germ free mouse, and even more surprisingly the additionof one organism, Bacteroides thetaiotaomicron, was capableof this dramatic effect [13]. Thepeptidemolecules responsiblefor this, called angiogens,which are released fromPaneth cellsof the crypt also have potent antimicrobial properties.

The developing microbial flora of the gut also has majoreffects on the host metabolism, effecting the ability tometabolise carbohydates, fats and proteins in response to achanging diet as weaning occurs. Sequencing of the genome ofB. thetaiotaomicron shows that it has a very large number ofgenes involved in acquiring and digesting large carbohydratemolecules which cannot be digested by the mammalian host.For example B. thetaiotaomicron has 64 different genesresponsible for the degradation of xylan-, pectin- andarabinose containing polysaccharides which are major compo-nents of dietary fiber, energy from these would otherwise beinaccessible to the host [14]. Also produced are short chainfatty acids such as acetate, propionate and especiallybutyrate, which stimulate intestinal blood flow, affectintestinal proliferation and differentiation and maintain tightjunctions [15].

Equally as surprising is thework of Kasper and colleagues onB. fragilis, a close relative of B. thetaiotaomicron studied byGordon's group. This work suggests that B. fragilis directs thedevelopment of the immune system of the mouse host [16].Germ free mice are characterised by a decreased number ofCD4 Tcells in the spleen, this can be corrected by colonisationwith wild type B. fragilis, but not a polysaccharide (PSA)deficient mutants of B. fragilis. Similarly the whole architec-ture and organization of the spleen can be restored by wildtype B. fragilis but not the PSA deficient mutant.

CD4 cells differentiate into cytokine secreting effectorcells of two types, Th1 cells that secrete IFNg and Th2 cellsthat secrete IL-4. CD4 cells from germ free mice (and someatopic patients) are more skewed to a Th2 phenotype,producing more IL-4 and less IFNγ than CD4 cells from wildtype mice. This imbalance can be corrected by colonisationof the germ free mouse by wild type B. fragilis but again notby the PSA deficient mutant, there is specific production ofIFNγ which inhibits the Th2 reaction and reduces IgEproduction, restoring cytokine balance. It is unclear howPSA presentation to the mesenteric lymph node can affectthe whole CD4 population outside the gut, but the implica-tions of this work for human disease are obvious. Asthma andallergies are characterized by overproduction of Th2cytokines and of IgE — another component of the Th2

response [17]. It is worth emphasising that here the effects ofthe microbial flora occur at a distance from the actual site ofhost–microbe interaction.

It would be very unlikely that these dramatic effectsproduced in the mouse model systems by B. thetaiotaomi-cron and B. fragilis are restricted to these species alone , butrather are an inevitable consequence of the limitations ofmodel systems. In vitro studies with probiotic strains ofLactococcus and Bifidobacterium suggests that these alsoproduce immunostimulatory molecules [18,19].

2. Microbial host interactions in utero

If the development of mammalian intestinal structure,immune system andmetabolism aremediated by the presenceof a bacterial flora, what other mammalian systems might bedependent in the same way? Might this process actually beginin utero? Many aquatic invertebrates have evolved a specificmonoassociation with a Gram negative bacillus Alteromonassp which can be consistently isolated from the surface of theembryos, and produces 2,3-indolinedione (isatin), an anti-fungal compound that inhibits a widespread pathogenicfungus. If exposed to the fungus, bacteria-free embryosquickly die, as do normal embryos treated with antibiotics,whereas similar embryos reinoculated with the bacteria ortreated only with 2,3-indolinedione survive well [20].

It seems highly unlikely that early human developmentcould be so intimately associated with bacteria as in theAlteromonas transovarian system, presumably their pre-sence would have been detected by scanning electronmicroscopy or other means regardless of any difficulties inculturing them. Indeed the presence of bacteria in theuterus is universally regarded as a negative event associatedwith premature birth (PTB) and possibly still birth [21]. Forexample a number of studies have linked the presence ofbacteria and Fusobacterium necrophorum in particular withchorioamnionitis and preterm birth, but it now appears thatthe relationship between the presence of bacteria in theuterus and PTB is far more complex than was first thought.Recent work showed that organisms could be detected deepwithin human fetal membranes by in situ hybridization at allstages of pregnancy and suggested that the presence ofbacteria within the uterus during pregnancy is common.Most preterm tissues were found to contain bacteria,whether preterm tissues were delivered by cesarean sectionwithout labor or were collected after preterm labor orPPROM suggesting that the presence of bacteria is commonand insufficient to cause preterm labor or PPROM [22]. Thisdata if confirmed, clearly suggests the need for a reassess-ment of the relationship between bacteria and the hostbefore birth.

Might the presence of bacteria in the uterus actually bebeneficial in the same way as the intimate association ofbacteria and egg in the model systems given above? Using asheep model of chorioamnionitis based on intra-amnioticadministration of E. coli LPS, it has been shown that foetallung inflammation causes striking improvements in pretermlung function, caused primarily by increases in pulmonarysurfactant [23]. Pulmonary primitive epithelium expresses IL-6 constitutively throughout all gestational ages, displayingthe highest levels during the earliest stages. Previous studieshave shown that chorioamnionitis, with increased IL-6,

168 M. Wilks

promotes fetal lung maturation and decreases the incidenceof respiratory distress syndrome in premature neonates [24].If there is a physiological role for IL-6 on pulmonary branchingmechanisms, possibly involving p38-MAPK intracellular sig-nalling pathway, might this be regulated by Gram negativebacteria such as Fusobacteria?

3. Modulations of the normal flora by antibioticsand probiotics

If bacteria are important in early humandevelopment then thishas obvious implications for the use of antibiotics andprobiotics. The use of peripartum antibiotics has becomeprevalent in modern obstetric and neonatal practice, mainlyaimed at reducing the incidence of gp B streptococcalinfection. Whilst this aim has been achieved, there was adoubling in the rate of E. coli early onset sepsis in VLBW infantsin a decade [25]. Similarly, in a recent case control study,intrapartum antibiotic use was associated with an increase inlate onset serious bacterial infections [26]. In the US, revisedguidelines now recommend universal screening for GBS andintrapartum chemopropylaxis for all GBS colonized women, sothis trend is likely to be accentuated in the future [27]. In arecent large study of over 1000 infants at one month of age,antibiotic usage was associated with decreased numbers ofbifidobacteria and Bacteroides [28]. Antibiotic use is known tocause changes in the faecal flora which may persist long aftercessation of antibiotic treatment [29,30].

As well as leading to changes in the organisms causingsepsis and the spread of antibiotic resistance, the wide-spread use of antibiotics particularly in pre term infants mayhave other unforeseen effects beyond these, particularlywith respect to the later development of allergies [31].Clearly, the decision to treat an individual infant for asuspected or proven infection cannot at the moment takeinto account possible effects on the normal flora or specificcomponents of it which cannot yet be precisely defined.However, the widespread practice of treating prematureinfants prophylactically should be reconsidered.

In the distant future, as the importance of the gutmicrobiota in influencing the disposition, fate and toxicity ofdrugs in the host becomes more defined appropriate con-sideration of individual human gut microbial activities maybecomes a necessary part of future personalised health-care.Recently Nicholson and co workers introduced the concept ofglobal systems biology to describe the investigation of theinteractions of themammalian transcriptomes, proteomes andmetabolomes, the gut microbiota and other environmentalfactors on host metabolic regulation. A model was proposedthat might explain variations, especially unexpected toxico-logical reactions and drug interactions based on the likelihoodof interactions between the host genome and the indigenousflora. Although presently largely a theoretical framework, inthe longer term it is hoped that drug treatment could bemodified to take account of the individual [7].

Arguably the same strictures regarding the use of anti-biotics should apply to the use of probiotics, where the gutflora is directly rather than indirectly modified. During 2005alone 300 peer reviewed articles and 60 literature reviews onprobioics were published. Very few of these articles wereprimarily concerned with mechanism of actions, yet if the use

of probiotics is to become fully credible and gain equal rankingwith other therapeutic approaches a clear understanding ofmechanisms of action is essential. Previous criteria which ledto the designation of and use of certain strains of bacteriaparticularly lactobacilli and bifidobacteria as probiotics, suchas acid and bile resistance and the ability to survive GI tractpassage may not be as relevant as was previously thought andshould be supplanted by more precise molecular descriptionsof their effects.

For example there is a growing body of evidence thatoverproduction of Il8 is a critical factor in components of thesystemic inflammatory response in neonates leading to NEC,CLD and neuro developmental delays [32]. Some probioticshave been shown to specifically inhibit TNFα induced Il8secretion in HT29 cells. It should also be pointed out thatimmunostimulatory effects have been described with deadprobiotic bacteria and even unmethylated dinucleotidecomponents (CpG) of the microbial DNA [33,34]. Whilstsuch studies of individual properties are of course valuable,the availability of microarrays now makes it possible to studymultiple effects of a probiotic on introduction into an animalmodel. Thus work by Kleerebezem and co-workers on theprobiotic strain L. plantarum WCFS1 using a microarrayshowed that 72 genes were induced upon introduction into amouse GI tract model [35]. These predominant in vivoresponses were in genes which were associated with nutrientacquisition, intermediate and/or cofactor biosynthesis,stress response and cell surface proteins. Conversely analysisof the host responses in vivo and in vitromodels showed thatthere was increased transcriptional activity in genes encod-ing fatty acid metabolism and lipid transfer factors as well ascytoskeletal factors that suggested an increased turnover ofepithelial cells [35].

4. Necrotising enterocolitis (NEC)

Whilst inflammatory bowel diseases may well have theirorigins in early host–microbe interactions when the host islearning to react appropriately to it's developing microbialflora, clinical manifestations may not be apparent for manyyears. This makes applying the new approaches and findingsdescribed above extremely difficult. However, they may givenew insights when applied into NEC. The pathogenesis of NECis poorly understood, but is often said to bemultifactorial andis highly associated with immaturity of the gastrointestinaltract, where in premature infants the intestinal mucosa maybe overlayed by a single layer of epithelial cells, colonisationby the intestinal microbiota, and the immaturity of theimmune system. Important pathological findings includeischemia, changes in the intestinal flora and inflammation.

At this early stage of development the flora is undergoingrapid change and the host must respond in a matter of daysfrom germ free to the presence of high numbers of bacteria[36]. These bacteria include bifidobacteria and lactobacillifrom breast milk, which arguably the intestine has evolved torespond to, albeit at a later stage in the case of the prematureinfant, but this is not always the case. The neonatemight alsoreceive sterile feeds by different routes. In these circum-stances, relatively low numbers of different bacteria whichare inevitably acquired from staff and surroundings maybecome established. The situation is further complicated by

169Bacteria and early human development

the use of antibiotics which will not only effect the pattern ofcolonisation but may release potent microbial cell wallproducts. Analysis of changes in the intestinal flora precedingthe onset of NEC using molecular methods might provide aninsight into this process. Several recent studies have reportedbeneficial effects of probiotics, but the molecular basis ofthese effects have not been established. Testing the effectsof these organisms which have given benefits in trials inanimalmodels of NEC and analysing the host response to themshould also be a research priority.

In a pragmatic response to the complexity of NEC, a multi-modal approach to NEC prophylaxis was reported, consisting ofearly trophic feeding with human breast milk, and enteral ad-ministration of an antibiotic, an antifungal agent, and probioticshas been used [37]. In fact, given the extraordinary range ofeffects that even individual species of bacteria show in themodel systems described above, the pathogenesis may not bemultifactoral at all. If as we have seen bacteria can stimulateangiogenesis, and control inflammation and the development ofthe immune system, then it is entirely possible that the cascadecould be initiated by a change in the intestinal flora or aninappropriate host response to the intestinal flora, which couldpave the way for novel prophylactic and therapeuticapproaches.

5. Conclusion

The aim of this article has been to draw attention to new andrapidly changing fields of researchwhichwill have increasinglyimportant implications for clinical practice. There are there-fore no formal conclusions as such to this article, nor can therebe at this stage any recommendations for changes in clinicalpractice. Nevertheless, clinicians should be alert to possiblenew solutions to old clinical problems which will arise from abetter understanding of host–microbe interactions, which it isnow possible to study both inmodel systems and in vivo inwayswhich have hitherto not been possible.

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