Analysis of bacterial bowel communities of ibd patients: what has it revealed?

Analysis of Bacterial Bowel Communities of IBD Patients: Harry Sokol, MD,* Christophe Lay, PhD,† Philippe Seksik, MD, PhD,* and Gerald W. Tannock, PhD‡ the tissue, it is probably best for the present to consider it a Abstract: The bacterial community, in whole or in part,
pathogen and for its presence to be termed an infection.7 The resident in the bowel of humans is considered to fuel the terminal ileum and the large bowel are hospitable places for chronic immune inflammatory conditions characteristic of microbial proliferation because bowel motility is slower, and Crohn’s disease and ulcerative colitis. Chronic or recurrentpouchitis in ulcerative colitis patients is responsive to antibi- microbial communities (mostly bacterial species) reside in otic therapy, indicating that bacteria are the etiological these sites. The surfaces of undigested plant fragments in the agents. Microbiological investigations of the bacterial com- digesta have adherent bacterial associates that are involved in munities in stool or of biopsy-associated bacteria have so far the breakdown of complex carbohydrate molecules, just as failed to reveal conclusively the existence of pathogens or occurs in the rumen of sheep and cows.8 The microbiota of bacterial communities of consistently altered composition in feces represents the microbiology of the distal large bowel IBD patients relative to control subjects. Confounding factors and does not necessarily apply to other regions, even of the need to be eliminated from future studies by using better- colon, and certainly not of the terminal ileum.
defined patient populations of newly diagnosed and untreated The existence of a discrete bacterial community asso- individuals and by improved sampling procedures.
ciated with the mucosal surface of the human bowel has been (Inflamm Bowel Dis 2008;14:858 – 867) postulated by researchers who have drawn on knowledge ofthe distribution of bacteria in the proximal colon of mice.9 Key Words: bowel bacteria, microbiota, microbial communities,
Accurate definition of the mucosa-associated community re- commensals, inflammatory bowel diseases, Crohn’s disease, ulcer-ative colitis, pouchitis mains difficult because mucosal biopsy specimens must becollected: an invasive procedure that is ill-defined in terms ofcollection procedure from study to study. Prior to colonos- The remnants of microbial cells, particularly bacterial copy and biopsy the patient is purged to remove bowel DNA, can be detected along the entire length of the contents. The effect of this preparative treatment on the gastrointestinal tract.1–5 This does not mean, however, that composition of the bowel community has been little investi- bacterial communities colonize all regions of the gut. Not all gated.10 Further, the bowel is not completely decontaminated parts of the gut are suitable for microbial persistence: the acid by this procedure and a fecal fluid continues to be present in secreted in the stomach and the swift flow of contents in the the bowel and to bathe mucosal surfaces. Therefore, it is not duodenum and jejunum ensure that the more proximal re- clear what is being sampled: the mucosal surface contami- gions contain only transient microbial cells in the healthy nated with luminal bacteria or true mucosal inhabitants.11 human host.6 Helicobacter pylori associates with the epithe- Additionally, the extent of contamination of the colonoscope lial surface of the gastric and duodenal mucosa, but since the with bacteria from the fecal fluid has never been determined.
presence of this organism is associated with inflammation of Therefore, reports about the mucosa-associated communityof humans must be treated with caution. At the least, thesestudies have relieved the previous focus on the fecal bacterial Received for publication December 13, 2007; Accepted December 17, community: they now seek to define the microbiology of From the *Gastroenterology and Nutrition Unit, Saint-Antoine Hospital, Feces, and therefore by inference distal large bowel APHP, Paris, France, †Institute of Microelectronics, A*STAR (Agency for contents, contain about 1011 bacterial cells per gram (wet Science, Technology and Research), Singapore, ‡Department of Microbiol- weight) and bacterial cells comprise about 50% of fecal ogy and Immunology, University of Otago, Dunedin, New Zealand.
Reprints: G. W. Tannock, Department of Microbiology and Immunology, mass.12 Four bacterial phyla are represented (Firmicutes, University of Otago, PO Box 56, Dunedin, New Zealand Bacteroidetes, Actinobacteria, and Proteobacteria) and 3 phy- (e-mail: [email protected]).
logenetically broad groups, each containing many genera and Copyright 2008 Crohn’s & Colitis Foundation of America, Inc.
species, are numerically dominant in the feces of healthy DOI 10.1002/ibd.20392Published online 14 February 2008 in Wiley InterScience (www.interscience.
humans (clostridial cluster XIVa [Clostridium coccoides group], clostridial cluster IV [Clostridium leptum subgroup], Inflamm Bowel Dis ● Volume 14, Number 6, June 2008 Inflamm Bowel Dis ● Volume 14, Number 6, June 2008 Bacterial Bowel Communities of IBD Patients Bacteroides-Prevotella group).13,14 Bacteria dominate the as-yet-unidentified factors associated with the pouch con- bowel community but fungi and Archaea may also be resi- tents. Empirical success in the treatment of pouchitis with dent, comprising less than 0.05% and 1% of the total inhab- antibiotics points, however, to bacteria or bacterial products itants, respectively.15,16 Much of the information pertaining to as the likely factors with which the immune system reacts.31 the residents of the bowel has been generated through theapplication of nucleic acid-based methodologies, most of BOWEL COMMENSALS AND CD: FECAL ANALYSES
which target the nucleotide base sequence of small ribosomal A variety of molecular methods have been used to subunit RNA (16S rRNA in the case of bacteria), which analyze the composition of the fecal community of IBD provides a cornerstone of microbial taxonomy. An earlier patients relative to controls. The phylogenetic composition of estimate of the number of bacterial species that might be the fecal community has notable temporal stability in healthy resident in the human large bowel was based on bacteriolog- humans considered individually. In CD patients, however, ical culture. Four hundred species seemed a likely number by temporal instability of the fecal microbiota has been reported.
extrapolation from what had already been cultured.17,18 Nu- Seksik et al32 showed that the dominant members of the fecal cleic acid-based methods of detection suggest that about 50% microbiota varied markedly between remission and flare.
of the bacterial cells seen microscopically in feces cannot yet Scanlan et al33 showed that there was less temporal stability be cultured in the laboratory, even when accounting for the of the dominant fecal microbiota in CD patients compared fact that some of them are dead.19,20 “Operational taxonomic with controls, even in those who stayed in remission until the units” (OTU; molecular species) never encountered in cul- end of the study. A decrease in the number of OTU (reduced ture-based bacteriology are detectable by molecular analyti- biodiversity) has also been reported in relation to the fecal cal methods. Estimates of biodiversity now seem to contin- community of CD patients. The main observation was that ually inflate but pollution of databanks with chimeric and fewer types of Firmicutes (43 in controls versus 13 in CD) other inaccurate sequences mean that about 5% of sequences were detected. Clostridial cluster IV (Clostridium leptum are unreliable.21,22 Curiously, therefore, despite the applica- subgroup) formed a smaller proportion of the fecal commu- tion of state-of-the-art technology, we still do not really nity in CD patients than in controls. Bacteroides and clos- know, in any accurate detail, the composition of the bowel tridial cluster XIVa populations were not different between groups. This outcome has been replicated in other studies.
Crohn’s disease (CD) and ulcerative colitis (UC) are Hence, Scanlan et al33 reported that they failed to detect chronic immune inflammatory conditions of the alimentary members of clostridial cluster IV in 27% of CD fecal samples tract referred to collectively as inflammatory bowel diseases (polymerase chain reaction, PCR) but all control samples (IBD). CD lesions can occur even in upper regions of the tract contained these bacteria (P Ͻ 0.0001). Taken together, these but are usually located where there are microbial residents results showed a quantitative and a qualitative (biodiversity) (ileum and colon), whereas UC is limited to the large bowel.
reduction in representation of the Firmicutes phylum, and Experimental animal models of colitis do not mimic exactly particularly clostridial cluster IV members in the feces of CD CD or UC but can be used to examine the role of specific patients. This phylogenetic group contains several butyrate- bacteria in the etiology of enterocolitis in general terms. The producing bacteria, such as Faecalibacterium prausnit- results of this work provide the best evidence that bacteria zii.34–36 Butyrate and other short chain fatty acids are be- resident in the bowel of the animals have an essential role in lieved to be important sources of energy for colonic epithelial the pathogenesis of colitis because, when maintained germ- cells and may have immunomodulator and antiinflammatory free, the animals do not develop disease.23 Current interest properties.37–39 Hence, the decrease in butyrate-producing therefore focuses on the bowel community as the source of bacteria in the colon might have a detrimental effect on the antigens that fuel the chronic inflammation seen in IBD.
colonic epithelium. There seems to be little agreement as to Chronic or recurrent pouchitis is the most common differences in fecal populations of lactobacilli and bifidobac- cause of troublesome, long-term functional disturbance for teria, sometimes regarded as “beneficial” bacteria, between patients with pouches created by ileo-anal anastomosis fol- lowing removal of the large bowel to cure UC.24 Pelvic Quantitative differences in enterobacterial populations pouches show varying degrees of chronic mucosal inflamma- have been reported. Giaffer et al41 reported that patients with tion and other alterations described as “colonic phenotype active CD had significantly larger Escherichia coli fecal change.”25–27 In some patients, however, acute inflammation populations than did patients with quiescent disease or nor- and clinical pouchitis develop by a process that possibly mal controls. In a further study, they showed that enteroad- parallels UC.28 Pouchitis is less common in pouches formed herent E. coli were frequently associated with CD.42 Simi- in patients because of familial adenomatous polyposis (FAP) larly, Seksik et al32 detected enterobacteria in all samples than in patients with UC.29,30 It appears, therefore, that the from CD patients but in none of the samples from controls.
dysfunctional immune system of UC patients reacts with Conflicting data are available with respect to members Inflamm Bowel Dis ● Volume 14, Number 6, June 2008 of the Bacteroidetes phylum. In some studies a decrease in difference in the microbiology of affected and unaffected the Bacteroides group32 or in the B. fragilis subgroup33 was mucosa. One study has contradicted these results,62 but in that reported, whereas other authors have reported an increase in report the authors pooled all inflamed and noninflamed bi- the B. fragilis subgroup, particularly in B. vulgatus and re- opsy data and then made comparisons regardless of the sub- lated species.44,45 The relative biodiversity of the B. fragilis ject, gut location, or disease (CD as well as UC).
subgroup may be reduced,33,46 although the Bacteroidetes As in fecal studies reviewed above, members of the phylum biodiversity seemed to be conserved.46 Firmicutes have been reported to be less prevalent in CDpatients. Two studies using clone library analysis retrieved BOWEL COMMENSALS AND CD: MUCOSAL
fewer sequences from the Firmicutes,52,63 particularly from SURFACE ANALYSIS
the Lachnospiraceae subgroup.63 Among this subgroup, Fae- The preparation of mucosal specimens (biopsies) has calibacterium prausnitzii seems to be lacking in CD patients.
differed from one study to another: samples have sometimes Martinez-Medina et al61 compared the biopsy-associated bac- been washed (with different buffers) or not, and the fixative teria of 19 CD patients with mildly active disease and 15 used to prepare specimens has varied. These differences in controls; F. prausnitzii was found in 13 of 15 controls sample preparation, and the analytical methods used in their (86.7%), whereas the prevalence in CD patients was 52.6% analysis, doubtless influence the outcome of assays and help (P ϭ 0.035). Frank et al63 also reported that F. prausnitzii to explain the variation in results reported from laboratory to laboratory. There is a consensus, however, that larger num- Outcomes of analyses with respect to members of the bers of bacteria are associated with bowel biopsies collected phylum Bacteroidetes have differed. Some authors have from CD patients compared to controls.47–51 This increased noted an increase in the prevalence of this phylum,50–52 while concentration seems to concern anaerobes as well as faculta- others report a decrease.63 On the other hand, a pediatric tive bacteria52,53 and may be due to a disruption in antibac- study detected a decrease in the concentration of B. vulga- terial barrier function.54 The ileal expression of alpha-de- tus.47 It is difficult to reach a consensus view about this from fensins (HD-5 and HD-6) is diminished in ileal CD,5,6 while the literature. The concentration of enterobacteria may be beta-defensin (HBD-2 and HBD-3) expression is weakly increased50,61,64 and may be phenotypically different than induced in colonic CD compared to UC.7,8 This relative commensal E. coli. Darfeuille-Michaud et al65 isolated E. coli defensin deficiency could also allow intestinal microbes to from resected chronic ileal lesions and from neoterminal invade the mucosa, as reported in some studies48,51 or, more ileum (with and without CD recurrence) after surgery. Many rarely, to permit colonization of intestinal crypts.51,59 How- of the isolates from diseased ileum adhered to Caco-2 cells.
ever, other authors did not observe bacteria directly attached Those authors confirmed, in a subsequent study, that an to the epithelial cells or intracellular bacteria.49,59 It is notable adherent-invasive type of E. coli (AIEC; reference strain that the biopsy specimens were not fixed in the same manner LF82) was specifically associated with ileal mucosa in some in these studies (some samples were fixed in nonaqueous CD patients.66 Kotlowski et al64 studied rectocolonic biopsies Carnoy solution,51 whereas others were fixed in 4% buffered from IBD patients and healthy subjects by generating elec- formalin48,49,59), perhaps indicating the importance of sample trophoretic fingerprints. DNA fragments unique to patients originated from E. coli. Culture-based studies detected larger As in fecal microbiota analysis, a reduction in the numbers of enterobacteria associated with IBD patient biop- biodiversity of biopsy-associated bacteria has been reported sies compared to controls. Moreover, the abundance of the in CD patients. Ott et al60 used an electrophoretic method to “B2ϩD” E. coli groups was significantly greater in patients detect biopsy-associated bacterial collections of 26 active CD than in controls. Genotypic characteristics such as serine patients, and 46 controls (31 noninflammatory controls and protease autotransporters (a unique class of transporters 15 inflammatory controls composed of undetermined colitis, found in the Enterobacteriaceae that have been implicated in infectious colitis, and radiation colitis). They found a reduc- virulence), as well as possible adhesins have been detected in tion in biodiversity that was indicated by a decrease in the these E. coli strains.64 Other bacterial species which, although number of bands and of weighted diversity indices in elec- not bowel commensals, have been investigated in relation to CD (Listeria and Mycoplasma)67,68 but a role for these bac- The patchy distribution of the intestinal tract lesions in teria in the pathogenesis of IBD has not been confirmed.
CD in normal-looking mucosa, side-by-side with deep ulcer- Mycobacterium avium subspecies paratuberculosis (MAP) ations, encouraged investigators to search for a localized has long been suspected to be involved in CD pathogenesis.
detection of possible “pathogens.” Three studies using 16S MAP was isolated by culture from the bowel of 6 of 7 (86%) rRNA gene clone libraries to catalog the composition of the CD patients in the United States,69 detected by IS900 in situ bacterial collections and 3 other studies using electrophoretic hybridization with fixed gut tissue from 27 of 33 (82%) CD methods1,32,52,53,61 did not find any statistically significant patients in Italy,70 detected by nested PCR IS900 with a Inflamm Bowel Dis ● Volume 14, Number 6, June 2008 Bacterial Bowel Communities of IBD Patients TABLE 1. Summary of Bacteriological Observations in CD
Increased concentration of total bacteria Increased concentration of total and facultative anaerobes Bacterial invasion of the mucosa and presence of No difference between injured and healthy ileo-colonic Firmicutes (particularly C. leptum group) decreased quantitatively and in biodiversity Decreased concentration of Faecalibacterium prausnitzii Increased concentration of the pathogenic E. coli B2ϩD aReferences related to fecal microbiota.
bReferences related to mucosa-associated bacteria.
MAM, mucosa-associated microbes; AIEC, adherent, invasive E. coli; MAP, Mycobacterium avium subspecies pseudotuberculosis.
significantly higher rate in CD patients than in controls in otrexate, 6-mercaptopurine, 5-amino-salicylic acid, and sul- United Kingdom71 and in the USA (40% of the CD patients fapyridine have been recently shown to inhibit MAP growth with granulomas, none of 22 non-IBD controls).72 Ryan et in vitro,81,82 it is difficult to resolve the beneficial effects of al73 detected MAP by nested PCR in 40% of the CD granu- TNF␣ blockade, which is known to cause disseminated tu- lomas (and in none of the 12 granulomatous disease controls), berculosis, but which has not been associated with MAP and Hulten et al72 detected MAP in macrophages and myo- infection in CD patients. Finally, MAP was not detected in fibroblasts. Naser et al74 detected viable MAP in peripheral CD patients in a large-scale 16S rRNA gene library study blood cells in a higher proportion of individuals with CD than (more than 15,000 small subunit ribosomal RNA genes ana- in controls. Several trials have tested the efficacy of antimy- cobacterial therapy in CD, but most of them are open to A summary of investigations relating to CD is provided criticism because of inappropriate use of antibiotics (use of ethambutol or isoniazid,75,76 which are not effective againstMAP complex infection; use of single instead of combined BOWEL COMMENSALS AND ULCERATIVE COLITIS:
antibiotic treatment77), open label design,77–79 or concomitant FECAL ANALYSES
steroid therapy.75,77,78 A recently published Australian trial There are relatively few studies of the fecal microbiota avoided these biases.80 This large placebo-controlled, double- of UC patients and it is difficult to make sense of the outcome blinded, randomized trial evaluated the efficacy of 2-year of disparate studies. Clustering of UC patients’ profiles has combination therapy with clarithromycin, rifabutin, and clo- been reported and, as in CD investigations, the composition fazimine in maintaining clinical remission following cortico- of the fecal microbiota of UC patients may be less biodiverse steroid withdrawal. The proportion of patients who relapsed (reduction in representation of Firmicutes phylum) than in the at 1, 2, and 3 years was not significantly different between the case of controls,83,84 perhaps containing an increased propor- group under an antibiotics regimen and the placebo group.
tion of bacteria not normally present in feces.43 Total Lacto- The trend observed at 1 year (39% of relapse in the antibiotic bacillus numbers have been reported to be reduced in fecal group versus 56% in the placebo group, P ϭ 0.054) was samples from patients with active UC when compared to attributed by the authors to the broad spectrum activity of those in remission (6 patients in each group).85 antibiotics against luminal organisms as induction therapy.
In a study utilizing culture methods, enteroadherent E. The authors concluded that these results did not support a coli were associated frequently with samples from UC pa- pathogenic role for MAP in CD. Moreover, although meth- tients as well as CD patients.42 Moreover, E. coli has been Inflamm Bowel Dis ● Volume 14, Number 6, June 2008 TABLE 2. Summary of Bacteriological Observations in UC
Increased concentration of total bacteria Increased concentration of total anaerobes Bacterial invasion of the mucosa and presence of bacteria No difference between injured and healthy colonic Difference between injured and healthy colonic Firmicutes (particularly C. coccoides group) Increased concentration of the pathogenic E. coli B2ϩD Enteroadherent E. coli associated with UC Increased concentration of “active” E. coli aReferences related to fecal microbiota.
bReferences related to mucosa-associated microbiota.
MAM, mucosa-associated microbes; SRB, sulfate-reducing bacteria.
shown to be more prevalent and metabolically active in the bacilli and members of clostridial cluster IV were reduced in microbiota of UC patients compared to controls.84 prevalence when ulcerated and nonulcerated tissues were Sulfate-reducing bacteria are bowel bacteria that pro- compared (average similarity: 59.9 Ϯ 26.1% and 79.2 duce hydrogen sulfide,86 which could inhibit butyrate oxida- tion and result in colonic lesions. It has been proposed, The clostridial cluster XIVa (Clostridium coccoides therefore, that in UC butyrate oxidation could be dis- group) population associated with the mucosa may have turbed.87–89 Short chain fatty acid enemas have been used reduced prevalence in UC patients.51 Rectal biopsies studied with limited success to treat refractory distal colitis.90–91 by Mylonaki et al93 showed that the epithelium-associated Pitcher et al92 showed that the microbiota of active UC counts of bifidobacteria in active and quiescent UC patients patients contained a higher concentration of sulfate-reducing were lower than in controls. Variations in the Bacteroidetes bacteria than UC patients in remission phase. Moreover, in phylum have been reported. Both increased detection50,51 and the same study 5-ASA inhibited sulfide production in a dose- reduced detection relative to controls have been reported.63 dependent manner in vitro, and stool of UC patients not Conte et al47 reported a lower occurrence of B. vulgatus in administered these drugs had a higher fecal sulfide concen- UC patients compared to controls. Finally, according to a number of authors, enterobacteria are more commonly asso-ciated with UC mucosa,50,64,93 particularly E. coli from BOWEL COMMENSALS AND ULCERATIVE COLITIS:
“B2ϩD” groups, than in healthy subjects.64 BIOPSY ANALYSES
Studies relating to UC patients are summarized in Table As in the case of CD, UC patients were reported to have 2 and a comparison with the outcomes of CD investigations higher concentrations of bacteria,48–51 particularly anaer- obes,50 associated with mucosal samples compared to con-trols. In some studies, bacterial cells were observed micro- MICROBIOLOGY OF POUCHITIS: CULTURE-
scopically in the mucosa or within crypts.48,51,93 Ott et al60 DEPENDENT APPROACH
reported a decrease in biodiversity that was comparable tothat of CD patients. In general, the mucosa-associated bacte- Luminal Microbiota
rial collection did not seem to differ between inflamed and Ruseler-van Embden et al98 analyzed the bacterial com- noninflamed tissues in UC patients,1,94,95 although Zhang et position of the ileal reservoir from patients that had under- al95 revealed differences in subdominant populations. Lacto- gone a restorative proctocolectomy either for ulcerative co- Inflamm Bowel Dis ● Volume 14, Number 6, June 2008 Bacterial Bowel Communities of IBD Patients TABLE 3. Comparisons Between UC and CD Gut Microbiota
Increased concentration of total bacteria C. leptum group and particularly F. Bifidobacteria and lactobacilli decreased Enteroadherent E. coli associated with Increased concentration of “active” E. Increased concentration of the pathogenic aReferences related to CD microbiota.
bReferences related to UC microbiota.
MAM, mucosa-associated microbes; AIEC, adherent, invasive E. coli; MAP, Mycobacterium avium subspecies pseudotuberculosis; SRB, sulfate-reducingbacteria.
litis (n ϭ 12) or familial adenomatous polyposis (n ϭ 2). The pouches of UC patients. Ohge et al100 have also shown an study was carried out at least 1 year after the surgery and 5 association between sulfate-reducing bacteria and pouches.
patients were diagnosed with pouchitis. Two fecal samples Sulfate-reducing bacteria were detected in higher numbers in were collected from each subject of the pouch control group active pouchitis patients (n ϭ 8) in comparison to patients (n ϭ 9) with an interval of at least 2 months. Plate counts without a history of pouchitis (n ϭ 8), patients with past showed large differences in the anaerobic bacterial composi- episode(s) of pouchitis (n ϭ 18), patients having an ongoing tion between 2 samples taken at different times, suggesting antibiotic treatment for pouch inflammation (n ϭ 11), and that the noninflamed pouch has a bacterial community of familial adenomatous polyposis patients (n ϭ 5). The authors unstable composition. Compared to the control group, pou- observed that this particular group of bacteria was sensitive to chitis patients showed an increased number of aerobes, de- antibiotic treatment (metronidazole or ciprofloxacin).
creased ratio of anaerobes to aerobes, less bifidobacteria and Gosselink et al101 monitored the fecal microbiota of lactobacilli, and a large number of Clostridium perfringens.
patients diagnosed with UC and having undergone a pouch Duffy et al99 compared the pouch bacterial content construction (n ϭ 13). The aim of this study was to compare from UC patients (n ϭ 10) and familial adenomatous polyp- the effect of 2 antibiotics, metronidazole and ciprofloxacin, osis (n ϭ 7). None of the patients had had a previous episode on the fecal microbiota at different times. The bacteriological of pouch inflammation. Plate enumerations did not indicate content of the pouch was analyzed at the beginning of an significant differences between the 2 groups of patients re- inflammatory episode before antibiotic treatment, during garding lactobacilli, Clostridium perfringens, Bacteroides, treatment with ciprofloxacin or metronidazole, and during and Bifidobacterium groups. Enterococci and coliforms were pouchitis-free periods. In the absence of inflammation the also similar in numbers between both groups. However, vi- pouch microbiota was characterized by the presence of lac- able sulfate-reducing bacteria were exclusively detected in tobacilli and large numbers of anaerobes. During pouchitis Inflamm Bowel Dis ● Volume 14, Number 6, June 2008 episodes there was a decrease of anaerobes, increase of aer- clone libraries. Using ␥-Proteobacteria/Enterobacteriaceae obic bacteria, lower numbers of lactobacilli, higher numbers group-specific primers, slight differences in terms of phylo- of Clostridium perfringens, and hemolytic strains of E. coli typic composition were observed between the placebo and (in half of the patients) were observed. Administration of VSL#3 groups. Enterobacter species and E. coli were mainly metronidazole eradicated the anaerobic microbiota including identified. Lactobacillus and Bifidobacterium clone libraries C. perfringens. Treatment with ciprofloxacin inhibited not generated from the VSL#3 group displayed a diverse spec- only the growth of C. perfringens but also that of coliforms, trum of species in comparison with the 2 other experimental including hemolytic strains of E. coli. However, ciprofloxacin groups (pretreatment remission [n ϭ 15] and placebo group did not significantly affect the anaerobic microbiota.
[n ϭ 5]). Some of these species were those included in theprobiotic preparation. Analysis of the mucosa-associated mi- Mucosa-Associated Bacteria
crobiota using an electrophoretic fingerprinting technique Using an ex vivo lymphocyte stimulation assay, Bell et showed that VSL#3 therapy increased the bacterial diversity al102 demonstrated the presence of proinflammatory sub- in comparison with pretreatment remission and placebo ad- stances in pouchitis-derived bacterial sonicates. Biopsy sam- ples taken from healthy pouch and pouchitis patients (UC Bacterial colonization of the pouch mucosa was mon- patients) were smeared on agar plates. Bacterial sonicates itored during the first year after pouch construction in 2 UC were prepared from a pool of colonies grown under aerobic patients.106 Neither of the patients had pouchitis during the and anaerobic conditions. Sonicates were also prepared from study. Biopsy samples from the pouches were taken at the strictly anaerobic bacteria grown on agar plates containing time of pouch construction, at closure of the ileostomy, and at metronidazole. Using lymphocyte proliferation as an indica- routine clinical examinations at 1, 3, and 12 months after tor of inflammation, the authors showed that sonicates of ileostomy closure. A variety of molecular analyses were bacteria from pouchitis samples produced an intense stimu- applied to monitor, identify, and characterize the develop- lation of the mononuclear cells. On the contrary, in sonicates ment of the bacterial microbiota in the newly formed pouch.
extracted from bacteria cultured from noninflamed pouches, Instability of the pouch bacterial community was indicated, cell proliferation was minimal. Moreover, when bacterial but most of the bacteria were affiliated with clostridial cluster sonicates were prepared from pouchitis colonies grown on XIVa, clostridial cluster IV, Bacteroides, and Enterobacteri- metronidazole-supplemented medium the proliferation effect aceae groups. Clones similar to clostridial cluster I (C. per- was abolished, suggesting inhibition of the growth of proin- fringens group) were also present in all samples from both flammatory bacteria. Although a bacterial etiology for pou- chitis was supported by this work, it was not possible to In the study of Komanduri et al,107 mucosa-associated pinpoint a particular bacterial species or group of bacteria bacteria in pouchitis was investigated. Twenty UC patients responsible for the proinflammatory effect.
having undergone proctocolectomy and ileal pouch-analanastomosis were enrolled in the study. Biopsy specimens MICROBIOLOGY OF POUCHITIS: CULTURE-
were taken from 5 patients with active pouchitis and 15 INDEPENDENT APPROACH
patients presenting without signs of pouch inflammation. A A beneficial effect of administration of the probiotic fingerprinting technique showed mucosa-associated micro- VSL#3 following antibiotic treatment has been demonstrat- biota patterns unique to each individual. Moreover, specific bacterial amplicons were unique to active pouchitis mucosa: 103,104 VSL#3 contains 300 billion viable lyophilized bac- teria per gram, comprising lactobacilli, bifidobacteria, and clostridial cluster XIVa, Enterobacteriacae, and streptococci were associated with control pouches. Streptococci were ab- randomized, placebo-controlled trial to study the impact of VSL#3 on the dominant mucosa-associated bacteria fromchronic pouchitis patients in remission induced by antibiotics.
CONCLUSIONS
Biopsy specimens collected from 15 patients before and after Microbiological analysis of bowel samples from IBD a period of 2 months therapy (10 received VSL#3 and 5 patients is plagued with sampling and technical problems. To placebo) were examined using microscopy and PCR-based the forefront are the remoteness of the fecal community from approaches. The authors observed that the mucosal micro- the sites of inflammation in the bowel, variables associated biota was mainly detected within the epithelium and nearly with the collection of biopsies from bowels that have already all bacteria were affiliated with the Enterobacteriaceae been altered by preparative measures necessary for colonos- group. Compared to the placebo group, an increase in Enter- copy, recruiting matched patients and controls in sufficient obacteriaceae within the mucosa during VSL#3 therapy was numbers to guarantee appropriate statistical power for stud- observed. Investigation at the molecular species level was ies, reliance on single samplings of patients and controls, carried out with the construction of taxonomic group-specific previous or concurrent therapeutic drugs administered to pa- Inflamm Bowel Dis ● Volume 14, Number 6, June 2008 Bacterial Bowel Communities of IBD Patients tients, and a relatively broad spectrum of “normal micro- colonoscopy on post-procedure intestinal microbiota composition. Gut. biota” compositions even in healthy humans. Technical prob- 11. Bibiloni R, Tandon P, Vargas-Voracka F, et al. Differential clustering lems include the noncultivability of a large proportion of the of bowel biopsy-associated bacterial profiles of specimens collected in bowel bacterial community, analytical reliance on a polluted Mexico and Canada: what do these profiles represent? J Med Micro- databank of 16S rRNA gene sequences, relatively shallow 12. Suau A, Bonnet R, Sutren M, et al. Direct analysis of genes encoding phylogenetic analyses due to the nature of the available 16S rRNA from complex communities reveals many novel molecular analytical tools, and PCR bias, which results in preferential species within the human gut. Appl Environ Microbiol. 1999;65:4799 – amplification of 16S rRNA gene sequences from some bac- 13. Franks AH, Harmsen HJM, Raangs GC, et al. Variations of bacterial populations in human feces measured by fluorescent in situ hybridiza- Particular strains of E. coli may be associated with tion with group-specific 16S rRNA-targeted oligonucleotide probes.
disease in a subset of CD patients and biodiversity of the Appl Environ Microbiol. 1998;64:3336 –3345.
14. Lay C, Rigottier-Gois L, Holmstrom K, et al. Colonic microbiota bowel microbiota may be reduced in IBD, especially in CD signatures across five northern European countries. Appl Environ Mi- patients. Little information that can aid clinicians in the crobiol. 2005;71:4153– 4155.
treatment of IBD has been produced, however, from phylo- 15. Miller TL, Wolin MJ. Stability of Methanobacter smithii populations in the microbial flora excreted from the human large bowel. Appl Environ genetic analyses. Thus, microbiologists must feel disappoint- Microbiol. 1983;45:317–318.
ment at their lack of achievement so far. There is much 16. Simon GL, Gorbach SL. Intestinal flora in health and disease. Gastro- disagreement between research groups as to the results of enterology. 1984;86:174 –193.
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