doi: 10.1136/gut.2010.211706 2010 59: 1476-1484Gut

 M S Malo, S Nasrin Alam, G Mostafa, et al. normal homeostasis of gut microbiotaIntestinal alkaline phosphatase preserves the

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Intestinal alkaline phosphatase preserves the normalhomeostasis of gut microbiota

M S Malo,1 S Nasrin Alam,1 G Mostafa,1 S J Zeller,2 P V Johnson,2 N Mohammad,1

K T Chen,1 A K Moss,1 S Ramasamy,1 A Faruqui,1 S Hodin,1 P S Malo,1 F Ebrahimi,1

B Biswas,1 S Narisawa,3 J L Millan,3 H S Warren,4 J B Kaplan,5 C L Kitts,6

E L Hohmann,2 R A Hodin1

ABSTRACTBackground and aims The intestinal microbiota playsa critical role in maintaining human health; however, themechanisms governing the normal homeostatic numberand composition of these microbes are largely unknown.Previously it was shown that intestinal alkalinephosphatase (IAP), a small intestinal brush borderenzyme, functions as a gut mucosal defence factorlimiting the translocation of gut bacteria to mesentericlymph nodes. In this study the role of IAP in thepreservation of the normal homeostasis of the gutmicrobiota was investigated.Methods Bacterial culture was performed in aerobic andanaerobic conditions to quantify the number of bacteriain the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment lengthpolymorphism, phylogenetic analyses and quantitativereal-time PCR of subphylum-specific bacterial 16S rRNAgenes were used to determine the compositional profilesof microbiotas. Oral supplementation of calf IAP (cIAP)was used to determine its effects on the recovery ofcommensal gut microbiota after antibiotic treatment andalso on the colonisation of pathogenic bacteria.Results IAP-KO mice had dramatically fewer and alsodifferent types of aerobic and anaerobic microbes in theirstools compared with WT mice. Oral supplementation ofIAP favoured the growth of commensal bacteria,enhanced restoration of gut microbiota lost due toantibiotic treatment and inhibited the growth ofa pathogenic bacterium (Salmonella typhimurium).Conclusions IAP is involved in the maintenance ofnormal gut microbial homeostasis and may havetherapeutic potential against dysbiosis and pathogenicinfections.

INTRODUCTIONThrough millions of years of evolution metazoanshave developed mechanisms that maintain a mutu-ally beneficial symbiotic relationship with commensalmicrobiotadfor example, intestinal microbes playa pivotal role in maintaining human health and well-being.1e4 The human gastrointestinal tract harboursapproximately 1014 bacteria composed of 300e1000different species.5 Dysbiosis, defined as dysregula-tion of the normal homeostasis of the intestinalmicrobiota, has been implicated in the pathogenesisof various disease conditions including (but notlimited to) antibiotic-associated diarrhoea (AAD),6 7

Clostridia difficile-associated disease (CDAD),8

inflammatory bowel disease (IBD),9 AIDS10 andobesity.2

The fundamental mechanisms that govern thenormal homeostatic number and composition ofthe intestinal microbiota remain poorly under-stood, although a few factors have been implicatedin influencing the gut microbiota including anti-microbial peptides, age, immune status, luminalpH, available fermentable materials and generalliving conditions.11 However, no specific endoge-nous factor has been identified that functions eitherdirectly or indirectly to preserve the normalhomeostatic number and composition of theintestinal microbiota.Over the last decade, intestinal alkaline phos-

phatase (IAP), a small intestinal brush border

< Additional data are publishedonline only. To view these filesplease visit the journal online(http://gut.bmj.com).1Department of Surgery,Massachusetts GeneralHospital, Harvard MedicalSchool, Massachusetts, USA2Infectious Disease Division,Department of Medicine,Massachusetts GeneralHospital, Harvard MedicalSchool, Massachusetts, USA3Sanford Children’s HealthResearch Center,Sanford-Burnham MedicalResearch Institute, California,USA4Infectious Disease Unit,Departments of Pediatrics andMedicine, MassachusettsGeneral Hospital, HarvardMedical School,Massachusetts, USA5Department of Oral Biology,New Jersey Dental School,New Jersey, USA6Environmental BiotechnologyInstitute, California PolytechnicState University, California, USA

Correspondence toM S Malo, Department ofSurgery, MassachusettsGeneral Hospital, Jackson 812,55 Fruit Street, Boston, MA02114, USA;mmalo@partners.org

Revised 25 June 2010Accepted 29 June 2010

Significance of this study

What is known about the normal homeostasisof intestinal microbiota?< Intestinal microbiota is involved in maintaining

human health and well-being.< Imbalances of normal intestinal microbiotal

homeostasis (dysbiosis) are associated withvarious disease conditions.

< Molecular mechanisms regulating the normalhomeostatic number and composition of intes-tinal microbes are largely unknown.

What are the new findings?< Compared with wild-type animals, mice defi-

cient in intestinal alkaline phosphatase (IAPknockout) harbour fewer and different types ofintestinal bacteria.

< Oral supplementation of IAP rapidly restorescommensal gut microbiota lost due to antibiotictreatment.

< Oral supplementation of IAP dramaticallyreduces colonisation of Salmonella typhimurium.

How this may impact future clinical work?< The endogenous brush border enzyme IAP

appears to maintain the normal homeostasis ofintestinal microbiota.

< IAP might be an effective therapeutic agentagainst dysbiosis.

< IAP might represent a novel therapy againstpathogenic bacterial infections.

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enzyme, has been recognised as a gut mucosal defence factor.IAP has the ability to detoxify lipopolysaccharides (LPS) fromGram-negative bacteria and exogenous IAP has been shown toattenuate LPS-mediated toxicity.12 13 Bates et al14 demonstratedin zebrafish that, by preventing LPS-mediated inflammatoryresponses, IAP plays a major role in promoting mucosal toler-ance to the commensal gut bacteria. Recently, we found thatmice deficient in IAP (IAP knockout, IAP-KO mice) had increasedbacterial translocation to mesenteric lymph nodes when theintestine was subjected to a local or distant ischaemic injury.15

This property of IAP as a gut mucosal defence factor led us toinvestigate its potential interaction with the intestinal micro-biota. Here we report that IAP acts to preserve the normalhomeostasis of the gut microbiota and it may have therapeuticpotential to prevent/treat dysbiosis as well as infections due topathogenic bacteria.

MATERIALS AND METHODSDetails of the experimental Materials and Methods are shown inthe online supplement

AnimalsIAP-KO mice (Mus musculus C57BL/6) construction has beendescribed elsewhere.16 Heterozygous mice were obtained fromthe Burnham Institute for Medical Research, La Jolla, California,USA. These animals were subsequently bred at the Massachu-setts General Hospital (MGH) animal facility to create homo-zygous IAP-KO, heterozygous and wild-type C57BL/6 (WT)littermates. Confirmation of genotype was performed by PCRanalysis.16 The animal experiments were reviewed and approvedby the IACUC at MGH. Animals in this study were maintainedin accordance with the guidelines of the Committee on Animalsof Harvard Medical School (Boston, Massachusetts, USA) andthose prepared by the Committee on the Care and Use ofLaboratory Animals of the Institute of Laboratory Resources,National Research Council (Department of Health, Educationand Human Services, publication no. 85e23 (National Instituteof Health), revised 1985).

Terminal restriction fragment length polymorphism analysesTerminal restriction fragment length polymorphism (TRFLP)was performed following the protocol described in Kaplan et al17

(also see online supplement).

Construction of the library of bacterial 16S small subunitribosomal RNA genesStool samples of equal weight from 10 individual WT mice werepooled and used for isolation of bacterial DNA using the DNAisolation kit from Qiagen (Valencia, California, USA). Similarly,DNA was isolated from pooled stool samples of 10 IAP-KOanimals. The forward and reverse primers17 used in PCRamplification of the 16S rRNA gene fragments for TRFLP anal-yses (see table 1 in online supplement) were also used to amplifythe same approximately 500 bp 16S rRNA gene fragments forcloning. PCR was performed using Taq DNA polymerase ina thermocycler (PTC-200, MJ Research, Waltham, Massachu-setts, USA). The PCR conditions were: initial denaturation for2 min, then 32 cycles of denaturation (948C for 30 s), annealing(458C for 30 s) and extension (728C for 60 s) followed by a finalextension step of 5 min at 728C. The PCR products were verifiedby electrophoresis through a 2% agarose gel. Using a TOPO TAcloning kit (Invitrogen, Carlsbad, California, USA), fresh PCRproducts were cloned into pCR2.1 TA cloning vector following

the manufacturer ’s protocol. The transformants were plated onLuria-Bertani (LB) agar plates containing ampicillin (100 mg/ml)and X-gal (40 mg/ml) and incubated at 378C overnight.Approximately 1000 white transformant colonies from eachgroup (WT and KO) were grown at 378C overnight in 96-wellplates, each well containing 150 ml LB broth with 100 mg/mlampicillin.

Phylogenetic analysesCloned 16S rRNA gene sequences were analysed with theClassifier program developed by Michigan State University(http://rdp.cme.msu.edu/). The program produced the nameand number of 16S rRNA gene sequences and arranged them inthe taxonomical hierarchy. The percentage of each sequence wascalculated using the Microsoft Excel program. c2 analysis wasperformed to determine statistical significance in the distribu-tion of clones in WT and KO libraries; p<0.05 was consideredsignificant.

Quantitative real-time PCRSemiquantitative limited-cycle PCR (<20 cycles) was performedon WT and KO stool DNA using Taq DNA polymerase with Ecoli LacZ and subphylum-specific 16S rRNA gene primers (seetable 1 in online supplement) in a thermocycler (MJ Research).Quantitative real-time PCR was performed in an IQ5 Thermo-cycler (Bio-Rad, Hercules, California, USA) using a SYBR GreenPCR kit (New England Biolabs, Ipswich, Massachusetts, USA).Primers were synthesised by the MGH Core DNA SynthesisFacility.For absolute quantitation of bacterial DNA, serial dilutions of

a known amount of E coli DH5a genomic DNA were subjectedto qPCR amplification with the 16S rRNA gene universalprimers (synthesising 175 bp fragment, see table 1 in onlinesupplement), and a standard curve was generated by plottingCTs against the known amounts of DNA. DNA isolated fromWT and KO stools were subjected to qPCR using universal aswell as subphylum-specific 16S rRNA gene primers. Quantita-tion of Eubacterial DNA as well as subphylum-specific bacterialDNAwas calculated by comparing the known CT values againstthe standard values. Each PCR was repeated at least three times.

Colonisation assayFor studying colonisation of E coli a commensal E coli wasisolated from the stool of a WT mouse and a spontaneousstreptomycin-resistant mutant was isolated by culturing thebacterial sample in a MacConkey plate containing streptomycin(100 mg/ml). Spontaneous streptomycin-resistant mutants arefrequently a result of mutations in the rpsL gene and arephenotypically stable.18 S typhimurium SL1344 was grown in LBbroth and the colony-forming units (CFU) were determined byplating on Hektoen plates. Doses of bacteria for oral gavagevaried from 2 3 104 to 2.5 3 106 CFU depending on theexperiments. After oral gavage, the presence and quantity ofbacteria were determined by stool culture on selective media.

Restoration of gut microbiota after antibiotic treatmentTwo groups of wild-type (C57BL/6) mice (n¼5 for each group)were allowed to drink autoclaved tap water containing 5 mg/mlstreptomycin for 3 days. One group also received 200 U/ml calfIAP (cIAP, 20 ml/ml, New England Biolabs) along with strepto-mycin (cIAP+ group), and cIAP was continued until normal gutmicrobiota was re-established (usually by day 7). The othergroup received an equal amount (20 ml/ml) of vehicle for cIAP(see Materials and Methods in online supplement) for the total

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duration of the experiment (cIAP� group). Water containingcIAP and vehicle for cIAP was replaced daily. The faecal samplefrom each animal was plated everyday on MacConkey agarplates to determine the restoration of Gram-negative bacteria,especially E coli.

The experiment was repeated six times and the duration ofthe experiments varied from 7 to 35 days. Data on the day offirst appearance of E coli in an animal’s stool were compiled for39 animals in each group. Statistical significance of the differ-ence in the number of animals with E coli at a specific point oftime (day) was determined by the two-tailed Fisher exact testand p<0.05 was considered significant.

RESULTSGram-negative aerobic bacteria are absent from the stools ofIAP-KO miceBecause IAP is a gut mucosal defence factor that detoxifies LPS,the toxic outer membrane component of Gram-negativebacteria, we investigated the effects of IAP on the intestinalmicrobiota in WT and IAP-KO mice.16 We plated stool sampleson a variety of microbiological media, including MacConkeyagar, which primarily allows the growth of Gram-negativebacteria (see Materials and Methods in online supplement).Stool samples from WT animals grew numerous colonies onMacConkey plates (figure 1A); however, faecal samples from KOanimals grew no colonies on these plates (figure 1B). We platednumerous faecal cultures from KO mice and never observedaerobic Gram-negative bacteria on MacConkey plates. Thepredominant aerobic faecal bacterium isolated from the stools ofWT animals grown on MacConkey plates was E coli; however,occasionally we observed the growth of Enterobacter, Citrobacter,Proteus, Alcaligenes, Stenotrophomonas and Acinetobacter spp.

To ascertain whether the absence of E coli in IAP-KO micestools was a cohort or cage effect, we performed mixed housingexperiments in which adult WT animals were caged with KOlittermates. Even when housed together for 60 days, KO animalsfailed to acquire an aerobic Gram-negative microbiota from theWT littermates as measured by plating of the stool samples onMacConkey plates. This observation that Gram-negativebacteria are absent in the stools of IAP-KO mice suggests thatIAP is involved in regulating the intestinal microbiota.

Total number of aerobic bacteria is greatly reduced in the stoolsof IAP-KO miceQuantitative cultures of WTand IAP-KO stools were performedunder aerobic conditions using a variety of rich solid agar media(see Materials and Methods in online supplement). Figure 1Cshows that, when plated aerobically on LB, Brain Heart Infusion(BHI) and Brucella agar plates, the stools of KO mice yieldeddramatically fewer bacterial colonies than the stools of WTanimals (13105 vs 53106 CFU/g stool). Stool culture on theMacConkey media showed approximately 106 Gram-negativebacteria/g stool of WTanimals and again, as expected, there wasno bacterial growth from the stools of KO animals (figure 1B).

Anaerobes are moderately reduced in the stools of IAP-KO miceStool samples from WT and KO mice were cultured on Brucellaagar plates in an anaerobic chamber. The number of anaerobicbacteria in the stools of KO mice was approximately half of thatpresent in WT littermates (4.431010 vs 9.731010 CFU/g stool)and the difference was statistically significant (p<0.01, figure 1D).

We next used limited-cycle PCR (<20 cycles) to compare theamount of bacterial DNA in equal amounts (weight) of stools

from WTand KO animals. Two pairs of 16S rRNA gene-specificuniversal primers were used (see table 1 in online supplement),amplifying 523 and 175 bp fragments, respectively. Figure 1E(top two gel photographs) shows that the quantity of 16S rRNAgene fragments is relatively higher in the stools of WT animals

Figure 1 Number of bacteria in the stools of wild-type (WT) andintestinal alkaline phosphatase knockout (IAP-KO) mice. Stool samplesfrom individual mice were separately collected in Brain Heart Infusion(BHI) medium on ice, weighed and homogenised followed by serialdilution of samples and plating on selective plates under aerobic andanaerobic conditions. For PCR analyses, DNA was isolated from an equalamount (weight) of individual stool samples. Bacterial counts wereexpressed as mean6SEM colony forming units (CFU)/g stool. Eachexperiment was repeated at least five times and similar results wereobtained. (A) Growth of Gram-negative bacteria from the stools of WTmice on MacConkey agar plates (0.01 mg stool plated). (B) Growth ofGram-negative bacteria from the stools of KO mice on MacConkey agarplates (10 mg stool plated). (C) Total count of bacteria from the stools ofWT and KO mice (n¼7) grown in aerobic conditions on Luria-Bertani(LB), BHI, MacConkey (Mac) and Brucella agar plates. (D) Total count ofbacteria from the stools of WT and KO mice (n¼7) grown in anaerobicconditions on Brucella agar plates. (E) Semiquantitative limited-cyclePCR (<20 cycles) amplifying bacterial DNA from equal amounts of WTand KO mice stools. **p<0.01, ***p<0.001 (two-tailed Student t testof all data points).

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compared with KO animals. These data confirmed the stoolculture data that the number of bacteria is higher in the stools ofWT animals than of KO animals.

We then amplified E coli DNA from the stool DNA samplesfrom WT and KO animals. While multiple pairs of externalprimers failed to amplify E coli LacZ-specific fragments, we wereable to amplify a target 280 bp LacZ fragment only from WTstools using internal primers (see Materials and Methods inonline supplement) as shown in figure 1E (bottom gel photo-graph). These data confirm the stool culture data that stools ofWTanimals contain E coli, whereas stools of KO animals containno detectable E coli.

Taken together, these stool culture and PCR results demon-strate a generalised decrease in bacterial microbiota in IAP-KOmice. The most dramatic finding was a complete absence ofE coli in the KO animals. These observations suggest that theendogenous brush border enzyme IAP plays a role in theregulation of the gut microbiota.

Terminal restriction fragment length polymorphism (TRFLP) of16S rRNA genes reveals differential intestinal microbiotal profilein the stools of IAP-KO miceFaecal bacterial differences between WT and KO mice werefurther refined using TRFLP analyses of the bacterial 16S rRNAgenes (see Materials and Methods in online supplement). DNAwas isolated from the stool samples of eight mice in each group(WTand KO) and from each sample we amplified approximately500 bp of the 59 end of 16S rRNA genes using fluorescent PCRprimers universal to bacteria.17 The PCR products were thendigested with specific restriction endonucleases and the profile ofthe resulting terminal restriction fragments (TRF) was analysedby electrophoresis through a sequencing gel and detected asa peak in fluorescence (see Materials and Methods in onlinesupplement). Figure 2A shows the number of TRF peaks persample after Dpn II, Hae III and Hpa II digests as well as TRFpeaks from the combination of all three restriction digests. Therewere significantly more TRF peaks in KO samples (p<0.05),indicating a difference in the types of bacteria present comparedwith WT. Figure 2B shows the ordination of Dpn II, Hae III andHpa II TRF peaks using multidimensional scaling (MDS). TheTRFLP data were similar among the animals within each groupand significantly different between the groups (p#0.05).

Phylogenetic analyses reveal an altered profile of intestinalmicrobiota in the stools of IAP-KO miceWe used the pCR2.1 vector to construct two libraries carryingthe same 16S rRNA gene fragments from the WT and IAP-KOmice as described above (also see Materials and Methods inonline supplement). Approximately 1000 clones from eachlibrary were sequenced, resulting in 805 and 877 sequences of16S rRNA genes from bacteria in WT and KO mice stools,respectively. Sequences were subjected to phylogenetic analysesand a distribution of the bacterial types to the level of family isshown in table 1. The results show that the Bacteroidetesconstitute more than 50% of bacterial populations in eithergroup followed by the Firmicutes (25%) and Proteobacteria(1%); there was no statistically significant difference at thephylum level between the WTand KO groups. Interestingly, wefound that a higher number of Clostridia species was present inthe stools of KO mice than in the stools of WT animals (4.56%vs 2.36%, p<0.05). For Unclassified Firmicutes, the WT stoolshad a greater number than the KO stools (11.8% vs 8.78%,p<0.05). About 21% of the bacteria could not be classified in thestools of WT animals, whereas the number of Unclassified

Bacteria made up to 25% of the microbiota in the stools of KOmice; this difference was statistically significant (p<0.05). Itshould be noted that, because <1000 clones were sequencedfrom each library, we could not expect to find statisticallysignificant differences in aerobic Gram-negative bacteria (eg, Ecoli) present in relatively low numbers (Proteobacteria, <1%).

Quantitative PCR reveals subphylum-specific differencesbetween intestinal microbiotas of WT and IAP-KO miceTo confirm the phylogenetic data we performed semi-quantitative limited-cycle PCR (<20 cycles) as well as quanti-tative real-time PCR (qPCR) on stool DNA samples from WTand IAP-KO mice. For semiquantitative PCR, equal amounts of

Figure 2 Terminal restriction fragment length polymorphism (TRFLP) ofthe 16S rRNA gene sequences of the bacteria obtained from the stoolsof wild-type (WT) and intestinal alkaline phosphatase knockout (IAP-KO)mice. Bacterial DNA was isolated from pooled or individual stoolsamples (n¼8 per group) and 16S rRNA gene fragments were amplifiedusing PCR with dye-labelled primers followed by restriction digestionwith a specific enzyme, electrophoresis through a sequencing gel andcounting of terminal restriction fragment (TRF) peaks. TRF peaks wereordinated using multidimentional scaling (MDS). (A) TRF peaks persample of restriction digests (Dpn II, Hae III and Hpa II and all combined).(B) Ordination using MDS showing the similarity in the TRF profiles ofindividual as well as all combined samples of Dpn II, Hae III, and Hpa IIrestriction digests of 16S rRNA gene fragments. *p<0.05 (ANOSIM).

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DNA from each sample were used and, as expected, we observedsimilar band intensity of the 175 bp 16S rRNA gene fragments(amplifiedbyuniversal primers) inbothWTandKOstools (figure3).We then used subphylum-specific 16S rRNA gene primers (seetable 1 in online supplement) to amplify Clostridiales, Lacto-bacillaceae, Enterococcus and Bacteroidetes. The results indi-cate that, when the ratio of Bacteroidetes in WT and KOstools is not changed, Clostridiales are increased and Lacto-bacillaceae and Enterococcus are decreased in KO animals(figure 3).

The absolute amounts of Eubacterial DNA and subphylum-specific bacterial DNA/g stool, as determined by qPCR, areshown in table 2. The calculated fold changes of the respectivebacterial groups are also shown in table 2. The ratio of Clos-tridiales is increased by 3.47-fold and Lactobacillaceae andEnterococcus are reduced by approximately 70% in IAP-KOanimals compared with WT animals (table 2). On the otherhand, although the ratio of Bacteroidetes increased by 1.62-foldin the IAP-KO animals, this difference was not statisticallysignificant. It should be noted that, because amplification of

E coli DNA even from WT mouse stools requires use of internalprimers (see figure 1E), we could not generate any qPCR data onE coli from mouse stool DNA. These qPCR data also confirm thesemiquantitative PCR data shown above (figure 1E and figure 3).The culture, quantitative PCR, TRFLP and phylogenetic data

together establish a specific role for IAP in the regulation of gutmicrobiota. While bacterial counts are decreased in the IAP-KOanimals, TRF peaks are increased. There are populations (Clos-tridia and Unclassified Bacteria) that appear to be present insmall numbers in WT mice but are enhanced in KO animals,whereas some organisms that predominate in WT animals(Unclassified Firmicutes) are depressed in KO animals.

Commensal E coli fails to colonise IAP-KO miceThe absence of detectable E coli in the stools of IAP-KO miceprompted us to investigate whether the luminal environmentwas unfavourable for E coli colonisation in these animals. Inorder to study E coli colonisation we introduced a streptomycin-resistant mutant of commensal E coli (6.03105 CFU) to WTandIAP-KO animals (n¼8) by oral gavage and monitored its presencein the stool over time (see Materials and Methods in onlinesupplement). The inoculated organism was initially present inonly 6/8 KO animals 1 day after inoculation, and it was subse-quently eliminated by all but one animal after 15 days when theexperiment was terminated (figure 4A), suggesting that IAP-KOanimals have an intraluminal environment that inhibits colo-nisation by exogenous E coli. As expected, WT mice carried theinoculated streptomycin-resistant E coli in their stools alongwith the pre-existing commensal E coli.

Oral supplementation of IAP favours the survival of commensalE coli in IAP-KO miceWe next tested whether the unfavourable intraluminal envi-ronment for E coli in IAP-KO mice could be reversed by oralsupplementation with IAP. We first assayed the activity and

Table 1 Phylogenetic profile of bacteria in the stools of wild-type (WT)and intestinal alkaline phosphatase knockout (KO) mice as determined byanalyses of 16S rRNA gene sequences

Number ofsequence

Percentage (%) ofsequence

Taxonomic rank WT KO WT KO

Domain Bacteria 805 877 100 100

Phylum Proteobacteria 6 6 0.75 0.68

Class Deltaproteobacteria 2 1 0.25 0.11

Unclassified Deltaproteobacteria 2 1 0.25 0.11

Class Epsilonproteobacteria 2 3 0.25 0.34

Order Campylobacterales 2 3 0.25 0.34

Family Helicobacteraceae 2 3 0.25 0.34

Class Betaproteobacteria 1 2 0.12 0.23

Unclassified Betaproteobacteria 1 2 0.12 0.23

Class Gammaproteobacteria 1 0 0.12 0

Order Enterobacteriales 1 0 0.12 0

Family Enterobacteriaceae 1 0 0.12 0

Phylum Firmicutes 187 201 23.23 22.92

Class Clostridia 19 40* 2.36 4.56

Order Clostridiales 19 40 2.36 4.56

Family Ruminococcaceae 1 5 0.12 0.57

Family Lachnospiraceae 9 18 1.12 2.05

Family Incertae Sedis XV 0 1 0 0.11

Unclassified Clostridiales 9 16 1.12 1.82

Class Bacilli 73 84 9.07 9.58

Order Lactobacillales 67 76 8.32 8.67

Family Lactobacillaceae 1 1 0.12 0.11

Family Carnobacteriaceae 0 1 0 0.11

Unclassified Lactobacillales 66 74 8.20 8.44

Unclassified Bacilli 6 8 0.75 0.91

Unclassified Firmicutes 95 77* 11.80 8.78

Phylum Bacteroidetes 445 450 55.28 51.31

Class Bacteroidetes 289 299 35.90 34.09

Order Bacteroidales 289 299 35.90 34.09

Family Prevotellaceae 7 8 0.87 0.91

Family Rikenellaceae 12 12 1.49 1.37

Family Bacteroidaceae 5 9 0.62 1.03

Family Porphyromonadaceae 0 1 0 0.11

Unclassified Bacteroidales 265 269 32.92 30.67

Unclassified Bacteroidetes 156 151 19.38 17.22

Unclassified Bacteria 167 220* 20.75 25.09

Note: Phylogenetic profiles were assigned by the Classifier program developed by theMichigan State University (http://rdp.cme.msu.edu/).*p<0.05 (c2 test).

Figure 3 Semiquantitative limited-cycle PCR (<20 cycles) amplifyingsubphylum-specific bacteria in equal amounts of stool DNA from wild-type (WT) and intestinal alkaline phosphatase knockout (IAP-KO) mice.Sequences of subphylum-specific primers are shown in table 1 in theonline supplement. The experiment was repeated three times and similarresults were obtained. M, 100 bp DNA markers; P, control PCR withprimers only (no template DNA).

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stability of cIAP in the drinking water itself as well as in thestools of five animals receiving cIAP in their water. The activityof cIAP in drinking water was determined by following theprotocol described in Baykov et al19 (see Materials and Methodsin online supplement). We found that cIAP is reasonably stablein water and approximately 80% of cIAP enzymatic activityremains after 24 h at room temperature (see figure S1 in onlinesupplement). Faecal IAP enzyme activity increased in a dose-dependent fashion in mice drinking water with the added cIAP(see figure S2 in online supplement).

IAP-KO animals were then treated or not treated with cIAP(n¼6) and each animal was gavaged twice (2 days apart) with lowdoses of streptomycin-resistant E coli (20000 CFU). The presenceof E coli in the stools was monitored each day. We observed thatonly a few of the KO animals from either group carried E coli intheir stools on any day, and on day 6 most of the animals carriedno E coli in their stools (figure 4B). We then stopped cIAPsupplementation and treated the animals with streptomycin for3 days, thereby killing most of the native microbiota but allowingproliferation of the surviving streptomycin-resistant E coli. Weobserved that all animals in the group receiving cIAP had a veryhigh number of E coli in their stools after streptomycin treatment(days 10 and 15), whereas animals receiving no cIAP had no E coliin their stools (p<0.01). The experiment was repeated twice andsimilar results were obtained. These data indicate that exogenouscIAP administration promoted the survival of E coli in IAP-KOanimals, further supporting a role for this enzyme in the main-tenance of commensal bacteria.

Temporal enhancement of colonisation of E coli in WT micereceiving oral IAP supplementationTo determine the temporal effects of IAP on the restoration ofgut microbiota, groups of WT animals (n¼6) were treated withstreptomycin and ciprofloxacin for 3 days and the eradication ofaerobic Gram-negative microbiota in their stools was docu-mented. This was followed by oral gavage of small doses(20 000 CFU) of streptomycin-resistant E coli for two alternatedays in the presence or absence of cIAP (see Materials andMethods in online supplement). The results (figure 4C) showthat animals receiving cIAP had more rapid recolonisation(p<0.015) with E coli, which suggests that IAP could be used tohelp restore the gut microbiota after antimicrobial therapy.

Oral administration of the IAP protein enhances recovery of theendogenous gut microbiota in antibiotic-treated WT miceThe experiments described above show that cIAP enhancescolonisation with re-fed enteric bacteria after antimicrobial

eradication. To further investigate a possible therapeutic use ofIAP, we examined whether treatment with exogenous cIAPmight promote the restoration of endogenous enteric microbiotaand therefore potentially be useful in disorders like C difficilecolitis and AAD. Accordingly, experiments were performed inwhich groups of WT mice were treated orally with the anti-biotic streptomycin in the presence or absence of cIAP. Allanimals had enteric Gram-negative organisms present at theoutset of the experiment and these organisms were eradicated bystreptomycin. After discontinuation of the antibiotic, stoolswere cultured daily and the day of appearance of Gram-negativebacteria (usually E coli, occasionally Proteus mirabilis) wasrecorded for each animal (see Materials and Methods in onlinesupplement). The experiment was repeated six times (totaln¼39 per group), but the duration of each experiment varied(7e35 days). The data from all animals were combined andpresented as the number of animals with faecal cultures positivefor E coli in each group by day. Faecal culture data are shown forday �4 to day +3, at which point all animals receiving cIAP hadE coli in their stools (figure 5A). On each given day the number ofanimals with Gram-negative bacteria was much higher in thecIAP+ group than in the cIAP� group, and the differences werestatistically significant for all time points (p<0.00004).

IAP inhibits the growth of Salmonella typhimurium in vivoIt is well known that enteric pathogenic bacteria compete withthe endogenous microbiota and that enteric infections are morecommon in settings where the normal intestinal microbiota islost or disrupted. Given our findings with regard to the resto-ration and/or maintenance of the normal microbiota, we spec-ulated that cIAP could work to inhibit infection by pathogenicbacteria. Groups of WT mice (n¼5) were therefore treated withstreptomycin for 3 days in the presence or absence of of cIAP.Four days after discontinuation of streptomycin, when allanimals in the cIAP+ group but none in the cIAP� group had Ecoli in their stools, oral gavage of 500 000 CFU streptomycin-resistant virulent S typhimurium SL1344 was performed and thepresence of S typhimurium was monitored by plating stools onHektoen plates (see Materials and Methods in online supple-ment). The results (figure 5B) show that the number of Styphimurium was dramatically lower in animals in the cIAP+group (4 log fewer CFU, p<0.001). It is worth noting that E colialso returned 2 days after Salmonella infection in the cIAP�group, and both the cIAP+ and cIAP� groups maintained E coliin their stools during salmonellosis (data not shown). We believethat the delayed return of E coli in the cIAP� group is the normalconsequence of antibiotic treatment and not related to

Table 2 Absolute amounts of DNA of specific groups of bacteria in the stools of wild-type (WT) and intestinal alkaline phosphatase knockout (IAP-KO) mice determined by quantitative real-time PCR

GroupTaxonomicrank Phylum

Genespecificity

Amount of DNA inWT mice (mg/gstool)

Amount of DNA inKO mice (mg/g stool)

Fold change(KO/WT) p Value

Eubacteria Kingdom 16S rRNA 660.74679.13 408.57637.50 0.62 0.035433*

Clostridiales Order Firmicutes 16S rRNA 29.1765.35 65.7166.84 3.47 0.002960*

Lactobacillaceae Family Firmicutes 16S rRNA 13.7862.24 3.6160.45 0.36 0.002984*

Enterococcus Genus Firmicutes 16S rRNA 0.3560.09 0.0860.03 0.33 0.034392*

Bacteroidetes Class Bacteroidetes 16S rRNA 220.46613.41 260.54614.27 1.62 0.093682

Amount of DNA expressed as mean6SEM.Quantitative real-time PCR was performed on WT and KO stool DNA samples using kingdom-specific as well as subphylum-specific bacterial 16S rRNA gene primers.37 The sequences ofprimers are also provided in table 1 in the online supplement.The fold change for Eubacteria was determined by dividing the total amount of IAP-KO stool DNA with the total amount of WT stool DNA, whereas the fold change of a specific bacterial groupwas calculated by dividing the percentage of the specific bacterial group in IAP-KO mice stool with the percentage of the respective bacterial group in WT mice stool.*p<0.05 (Student t test).

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Salmonella infection. In addition to the E coli species, we alsooccasionally observed a few P mirabilis colonies. The experimentwas repeated with oral gavage of 2.53106 CFU S typhimuriumand similar results were obtained. Combined data from twoexperiments showed that, after 7 days of infection, 70% ofanimals from the cIAP+ group survived compared with only20% of the animals from the cIAP� group.

DISCUSSIONBased on the present data, we believe that the endogenous gutenzyme IAP functions in regulating the gut microbiota.Furthermore, it appears that exogenous IAP could be a usefultreatment for maintaining and/or restoring the normal micro-biota under a variety of disease-related conditions including, forexample, in the setting of antibiotic therapy.Intestinal alkaline phosphatase has been known to physio-

logists for more than half a century.20 IAP is a brush borderenzyme that is exclusively expressed in villus-associated enter-ocytes and hence it has been recognised as an enterocyte differ-entiation marker.21 The human IAP gene maps to chromosome2q34-37 and produces a 528-amino acid membrane-bound glyco-protein.22 IAP hydrolyses a wide variety of monophosphate estersat high pH optima. The enzyme is thought to be involved in

Figure 4 Effects of oral intestinal alkaline phosphatase (IAP)supplementation on colonisation and survival of E coli in IAP knockout(IAP-KO) and wild-type (WT) mice. The streptomycin-resistant E coliwas a spontaneous derivative of a commensal E coli isolated from a WTmouse stool (see Materials and Methods in online supplement). Groupsof IAP-KO and WT mice (n¼5) were allowed to drink water with orwithout calf IAP (cIAP, 200 U/ml) in the presence or absence ofstreptomycin and/or ciprofloxacin. After oral gavage of streptomycin-resistant E coli, stool samples were collected every day, homogenised inphosphate-buffered saline and plated on Luria-Bertani (LB) and/orMacConkey agar plates with or without streptomycin. (A) CommensalE coli failed to colonise IAP-KO mice. (B) Oral supplementation of IAPfavoured the survival of E coli in IAP-KO mice. (C) Temporalenhancement of colonisation of E coli by IAP in WT mice treated withtwo antibiotics (streptomycin and ciprofloxacin). *p<0.05, **p<0.01(two-tailed Fisher exact test of all data points).

Figure 5 Oral intestinal alkaline phosphatase (IAP) supplementationenhances the restoration of antibiotic-associated loss of gut microbiotaand inhibits Salmonella typhimurium infection. (A) Groups of wild-type(WT) mice were treated with streptomycin with or without calf IAP(cIAP, 200 U/ml drinking water). Stool culture was performed daily andtime of appearance (day) of Gram-negative (E coli) bacteria recorded foreach animal. Data from six independent experiments (for each groupn¼39 in total) were compiled to calculate the number of animals withE coli in each group on a specific day. ***p<0.001 (two-tailed Fisherexact test) difference in number of animals with E coli between the twogroups at a specific point of time (day). (B) Oral IAP administrationinhibits growth of S typhimurium in WT mice. Groups of animals (n¼5)were treated with streptomycin with or without cIAP as describedabove. Each animal received an oral gavage of 500 000 colony-formingunits (CFU) streptomycin-resistant S typhimurium. Stool culture wasperformed on Hektoen plates containing streptomycin. Bacterial countswere expressed as mean6SEM CFU/g stool. The experiment wasrepeated twice. *p<0.05, **p<0.01, ***p<0.001 (two-tailed Student ttest of all data points).

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phosphate and fat metabolism, and is known to be a majorcomponent of the fat-containing surfactant-like particlesobserved in enterocytes after high fat feeding and are secreted intothe intestinal lumen as well as the interstitial spaces.23 Narisawaet al16 reported on the phenotype of IAP-KO mice, showing thatthese animals displayed accelerated fat absorption and becameobese compared with their pair-fed WT littermates when feda high-fat diet.

Over the past decade evidence has accumulated to suggest animportant physiological role for the endogenous IAP enzyme ingut mucosal defence.14 15 Based on the interaction of IAP with theluminal microbial environment, we examined the status of thegut microbiota in IAP-KO mice and found dramatic differencescompared with their WT littermates. Given that IAP works todephosphorylate LPS, the toxic cell wall component of Gram-negative bacteria, we initially wondered whether the absence ofIAP in KO mice might result in a greater number of intestinalGram-negative bacteria. However, to our surprise we observed nogrowth of aerobic Gram-negative bacteria in the stools of KOmice, as determined by culturing the stool samples in MacConkeyagar plates (figure 1B). Although MacConkey media preferentiallyallow the growth of Gram-negative bacteria, there are someGram-negative bacteria that do not grow in this medium. We aretherefore unable to say that Gram-negative bacteria arecompletely absent in the stools of IAP-KO mice. However, it isclear that the overall bacterial count is greatly reduced in thestools of KO mice (figure 1BeE), as determined by stool cultureand semiquantitative PCR. We focused much of our work on thecommon enteric commensal E coli and observed that the luminalenvironment of IAP-KO mice is particularly unfavourable to thisbacterium. In future studies it will be interesting to determinewhether colonisation by other types of Gram-negative bacteria issimilarly affected by the absence of IAP.

The numerous beneficial roles of gut commensal bacteria arewell known and include effects on development as well asvitamin and nutrient absorption.4 It will be interesting toinvestigate whether there are differences between IAP-KO andWT mice with regard to gut development or vitamin andnutrient absorption. However, under controlled laboratoryenvironmental conditions, we and others have found no grossphenotypic differences except for KO mice being obese when feda high-fat diet.

Dysregulation of the normal homeostasis of intestinalmicrobiota is associated with a wide variety of disease statessuch as AAD,6 7 CDAD,8 IBD,9 AIDS,10 irritable bowelsyndrome,24 obesity,2 diabetes,25 colorectal carcinoma26 andrheumatoid arthritis.27 The recent use of probiotics to treatsome of these disease conditions is entirely based on the conceptof a beneficial role for commensal microbiota.7 11 28

Tuin et al29 recently reported that IAP attenuates the colonicinflammation in DSS-induced IBD in rats. Compared withcontrols, animals treated with IAP showed dramatic inhibitionof the DSS-induced inflammation. In addition, oral IAP treat-ment may be of benefit in human IBD.30 The mechanismsresponsible for these beneficial effects of IAP have not yet beendetermined. In light of the present work and the known role ofthe gut microbiota with regard to IBD, it will be of interest toexamine the status of commensal bacteria in these animals.

We have used TRFLP analyses of 16S rRNA to show evidencethat the gut microbiota in WTand KO mice is different (figure 2).However, phylogenetic analysis ultimately determines thecomposition of bacteria in a given specimen.31 Comparison ofphylogenetic data shows that IAP-KO mice have more Clostridiaclass of bacteria belonging to the Firmicutes phylum than WT

mice (table 1). Recent studies have demonstrated a relationshipbetween the gut microbiota and body weight, the Firmicutesbeing the pro-obesity bacteria whereas the opposite occurs withthe Bacteroidetes.2 Interestingly, IAP-deficient mice have beenshown to become obese when fed a high-fat diet16 but themechanism for the weight gain is not entirely clear. The obesity inthe IAP-KO mice was initially attributed to enhanced fat absorp-tion due to loss of the Akp3 gene (IAP-deficient)16 but, morerecently, to the increased expression of the Akp6 gene.32 Based onour data on the gut microbiota, it is possible that Clostridia spp.,which are greatly increased in KO mice (tables 1 and 2 figure 3),may contribute to the obesity seen in the IAP-KO mice. However,Clostridia probably contributes to obesity only in the presence ofa high-fat diet as IAP-KO mice on a normal diet and harbouringincreased Clostridia do not become obese. Also, because IAP-KOmice on a normal diet do not become obese, we believe thatdifferences in the microbiota observed in IAP-KO mice are notrelated to fat absorption, fat metabolism or body weight.Bacteria of the Clostridia class are mostly commensals but a few

of them are responsible for many critical illnesses, most notablyC difficile infection which generally occurs following antibioticexposure and has become a major health problem, especiallyamong hospitalised patients. Many of these hospitalised patientstake in little or no enteral nutrition and we have previouslyshown that starvation leads to villus atrophy and loss of IAPexpression.15 33 It is possible that the loss of IAP leads to anincrease in the number of Clostridia in the gut and increasedsusceptibility to subsequent infections. Our data (figure 5A)showing enhanced recovery of the gut microbiota in WTanimalstreated with an antibiotic in the presence of cIAP suggests thatIAP could be an effective therapeutic agent to prevent or treatAAD and CDAD. Along the same lines, inhibition of Salmonellacolonisation in the presence of cIAP (figure 5B) suggests a prom-ising possibility of a therapeutic use for IAP in the prevention ortreatment of pathogenic infection. We believe that the IAP-medi-ated increased growth of commensal bacteria limits the ‘food andshelter ’ (nutrition, anchorage sites, space, etc) for the invadingpathogens and consequently inhibits pathogenic infection.The molecular mechanisms of how IAP favours the growth of

E coli and other enteric bacteria are unclear. We initially specu-lated that IAP might exert a direct effect on the growth ofcertain bacteria. However, we have observed no direct growth-promoting effects of exogenous cIAP on several commensal andpathogenic bacteria (E coli, S typhimurium, S aureus, L mono-cytogenes) in vitro (see figure S3 in the online supplement), whichsuggests that the effects of IAP on the gut microbiota occurthrough a more indirect mechanism.Because IAP detoxifies LPS12 and IAP expression is silenced by

proinflammatory cytokines,34 we wondered whether the IAP-mediated regulation of the microbiota was related to changes inthe inflammatory state of the intestine. However, histologicalanalyses of IAP-KO intestine revealed no inflammatory changes(data not shown). Interestingly, we have found elevated MHCclass II expression in the liver of IAP-KO mice (in press),suggesting some degree of chronic inflammation within theportal system of these mice. Therefore, although the changes inthe gut microbiota in the IAP-KO mice are probably not due toan altered gut inflammatory state, it remains possible thatinflammatory factors may play some role in mediating theeffects of IAP.Another possible mechanism for the action of IAP with regard

to the gut microbiota relates to pH. It is known that pH affectsbacterial growth,35 and IAP has been shown to regulate luminalpH through dephosphorylation of ATP (the higher the ATP

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concentration, the higher is the luminal pH).36 Accordingly, wedetermined the pH of luminal content and stool. Comparedwith WT animals, the pH levels of the luminal contents andstools of IAP-KO mice were marginally higher but the differencewas not statistically significant (data not shown). It should berecognised, however, that the pH of the microenvironmentadjacent to the mucosal membrane (bacterial attachment site)may not be reflected in the assessment of the total luminalcontents. We speculate that IAP regulates the pH of the mucosalmicroenvironment by altering the luminal ATP concentration,since ATP is a known target of IAP.36 A change in pH and/orATP concentration might then regulate bacterial growth. Futurestudies will be needed to determine whether changes inpH-specific luminal microenvironments play a role in the IAPregulation of the gut microbiota.

Bates et al14 showed that IAP plays a major role in promotingmucosal tolerance to commensal microbiota. It would be inter-esting to colonise newborn WT or IAP-KO mice with E coli toinvestigate whether the absence of E coli colonisation in IAP-KOadult animals is due to abnormal maturation of the intestinalimmune system.

Taken together, it appears that IAP exerts its effects on the gutmicrobiota via some indirect mechanism, perhaps related to pH,inflammation, immunity or some other factors. In addition, it ispossible that the effects of IAP are confined to certain speciesand the changes in other species result from the competitioneffects for nutrients and other factors.

In summary, our data show that the endogenous brush borderenzyme IAP appears to provide a favourable environment for Ecoli and perhaps other enteric microbes. Furthermore, exogenousadministration of oral IAP promotes restoration of the normal gutmicrobiota following antibiotic exposure and also appears toinhibit the colonisation and infection of the gut pathogenSalmonella. Based on these data, we suggest that orally adminis-tered IAP might be an effective treatment for bacterial patho-genesis as well as a variety of disease conditions associated withdysregulated intestinal microbiota.

Acknowledgements We thank our colleagues Drs Ramnik Xavier andHans-Christian Reinecker for their critical review of the manuscript.

Funding This work was supported by NIH grants R01DK050623 and R01DK047186 toRAH, a Junior Faculty Award to MSM from the MGH Department of Surgery and a GrandChallenge Exploration Grant from the Bill and Melinda Gates Foundation to MSM.

Competing interests None.

Contributors Study concept and theory (MSM, RAH); research design (MSM, RAH,ELH, SHW, CLK); data acquisition (MSM, GM, SNA, SJZ, PVJ, CLK, NM, KTC, AKM,AF, SH, PSM, FE, BB, SN); data analyses and interpretation (MSM, RAH, ELH, SHW,CLK, JBK, JLM, SN, SR); statistical analyses (MSM, RAH, ELH, SHW, JBK, CLK);drafting of the manuscript (MSM, RAH, ELH); critical review of the manuscript forimportant intellectual content (all authors); obtained funding (RAH, MSM); approval ofthe manuscript (all authors); study supervision (RAH, MSM).

Provenance and peer review Not commissioned; externally peer reviewed.

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