Integrated Analysis of Human Milk Microbiota With Oligosaccharides and Fatty Acids in the CHILD Cohort.

Background: Human milk contains many bioactive components that are typically studied in isolation, including bacteria. We performed an integrated analysis of human milk oligosaccharides and fatty acids to explore their associations with milk microbiota. Methods: We studied a sub-sample of 393 mothers in the CHILD birth cohort. Milk was collected at 3-4 months postpartum. Microbiota was analyzed by 16S rRNA gene V4 sequencing. Oligosaccharides and fatty acids were analyzed by rapid high-throughput high performance and gas liquid chromatography, respectively. Dimension reduction was performed with principal component analysis for oligosaccharides and fatty acids. Center log-ratio transformation was applied to all three components. Associations between components were assessed using Spearman rank correlation, network visualization, multivariable linear regression, redundancy analysis, and structural equation modeling. P-values were adjusted for multiple comparisons. Key covariates were considered, including fucosyltransferase-2 (FUT2) secretor status of mother and infant, method of feeding (direct breastfeeding or pumped breast milk), and maternal fish oil supplement use. Results: Overall, correlations were strongest between milk components of the same type. For example, FUT2-dependent HMOs were positively correlated with each other, and Staphylococcus was negatively correlated with other core taxa. Some associations were also observed between components of different types. Using redundancy analysis and structural equation modeling, the overall milk fatty acid profile was significantly associated with milk microbiota composition. In addition, some individual fatty acids [22:6n3 (docosahexaenoic acid), 22:5n3, 20:5n3, 17:0, 18:0] and oligosaccharides (fucosyl-lacto-N-hexaose, lacto-N-hexaose, lacto-N-fucopentaose I) were associated with microbiota α diversity, while others (C18:0, 3'-sialyllactose, disialyl-lacto-N-tetraose) were associated with overall microbiota composition. Only a few significant associations between individual HMOs and microbiota were observed; notably, among mothers using breast pumps, Bifidobacterium prevalence was associated with lower abundances of disialyl-lacto-N-hexaose. Additionally, among non-secretor mothers, Staphylococcus was positively correlated with sialylated HMOs. Conclusion: Using multiple approaches to integrate and analyse milk microbiota, oligosaccharides, and fatty acids, we observed several associations between different milk components and microbiota, some of which were modified by secretor status and/or breastfeeding practices. Additional research is needed to further validate and mechanistically characterize these associations and determine their relevance to infant gut and respiratory microbiota development and health.

Impact of Saskatoon berry powder on insulin resistance and relationship with intestinal microbiota in high fat-high sucrose diet-induced obese mice.

The present study examined the impact of Saskatoon berry powder (SBp) on insulin resistance, inflammation and intestinal microbiota in diet-induced obese mice. Male C57 BL/6 J mice were fed control diet, high fat-high sucrose (HFHS) diet or HFHS+5% SBp (HFHS+B) diet for 15 weeks. The composition of fecal bacterial community was characterized using the Illumina sequencing of V4 region of 16S rRNA gene. HFHS diet increased body weight, fasting plasma glucose, cholesterol, triglycerides, insulin, homeostatic model assessment-insulin resistance, monocyte adhesion, tumor necrosis factor-α, plasminogen activator inhibitor-1, monocyte chemotactic protein-1, intracellular cell adhesion molecule-1, urokinase plasminogen activator and its receptor in plasma or aortae compared to the control diet. HFHS+B diet postponed the increase in body weight, suppressed HFHS diet-induced disorders in the metabolic and inflammatory variables. The ratio of Firmicutes/Bacteroidetes in the HFHS group was higher than that in the control group (P<.01), and that in the HFHS+B group was lower than that in the HFHS group (P<.05). The abundances of S24-7 family negatively correlated with body weight and tested metabolic or inflammatory variables. The results suggest that SBp attenuated HFHS diet-induced metabolic disorders and vascular inflammation in gut microbiota in mice.

Detection of fecal bacteria and antibiotic resistance genes in drinking water collected from three First Nations communities in Manitoba, Canada.

This study analyzed the microbiological quality of drinking and source water from three First Nations communities in Manitoba, Canada that vary with respect to the source, storage and distribution of drinking water. Community A relies on an aquifer and Community B on a lake as source water to their water treatment plants. Community C does not have a water treatment plant and uses well water. Quantification of free residual chlorine and fecal bacterial (E. coli and coliforms), as well as detection of antibiotic resistance genes (sul, ampC, tet(A), mecA, vanA, blaSHV, blaTEM, blaCTX-M, blaOXA-1, blaCYM-2, blaKPC, blaOXA-48, blaNDM, blaVIM, blaGES and blaIMP) was carried out. While water treatment plants were found to be working properly, as post-treatment water did not contain E. coli or coliforms, once water entered the distribution system, a decline in the chlorine concentration with a concomitant increase in bacterial counts was observed. In particular, water samples from cisterns not only contained high number of E. coli and coliforms, but were also found to contain antibiotic resistance genes. This work shows that proper maintenance of the distribution and storage systems in First Nations communities is essential in order to provide access to clean and safe drinking water.

Composition and Variation of the Human Milk Microbiota Are Influenced by Maternal and Early-Life Factors.

Breastmilk contains a complex community of bacteria that may help seed the infant gut microbiota. The composition and determinants of milk microbiota are poorly understood. Among 393 mother-infant dyads from the CHILD cohort, we found that milk microbiota at 3-4 months postpartum was dominated by inversely correlated Proteobacteria and Firmicutes, and exhibited discrete compositional patterns. Milk microbiota composition and diversity were associated with maternal factors (BMI, parity, and mode of delivery), breastfeeding practices, and other milk components in a sex-specific manner. Causal modeling identified mode of breastfeeding as a key determinant of milk microbiota composition. Specifically, providing pumped breastmilk was consistently associated with multiple microbiota parameters including enrichment of potential pathogens and depletion of bifidobacteria. Further, these data support the retrograde inoculation hypothesis, whereby the infant oral cavity impacts the milk microbiota. Collectively, these results identify features and determinants of human milk microbiota composition, with potential implications for infant health and development.

Invited review: Microbiota of the bovine udder: Contributing factors and potential implications for udder health and mastitis susceptibility.

Various body sites of vertebrates provide stable and nutrient-rich ecosystems for a diverse range of commensal, opportunistic, and pathogenic microorganisms to thrive. The collective genomes of these microbial symbionts (the microbiome) provide host animals with several advantages, including metabolism of indigestible carbohydrates, biosynthesis of vitamins, and modulation of innate and adaptive immune systems. In the context of the bovine udder, however, the relationship between cow and microbes has been traditionally viewed strictly from the perspective of host-pathogen interactions, with intramammary infections by mastitis pathogens triggering inflammatory responses (i.e., mastitis) that are often detrimental to mammary tissues and cow physiology. This traditional view has been challenged by recent metagenomic studies indicating that mammary secretions of clinically healthy quarters can harbor genomic markers of diverse bacterial groups, the vast majority of which have not been associated with mastitis. These observations have given rise to the concept of "commensal mammary microbiota," the ecological properties of which can have important implications for understanding the pathogenesis of mastitis and offer opportunities for development of novel prophylactic or therapeutic products (or both) as alternatives to antimicrobials. Studies conducted to date have suggested that an optimum diversity of mammary microbiota is associated with immune homeostasis, whereas the microbiota of mastitic quarters, or those with a history of mastitis, are considerably less diverse. Whether disruption of the diversity of udder microbiota (dysbiosis) has a role in determining mastitis susceptibility remains unknown. Moreover, little is known about contributions of various biotic and abiotic factors in shaping overall diversity of udder microbiota. This review summarizes current understanding of the microbiota within various niches of the udder and highlights the need to view the microbiota of the teat apex, teat canal, and mammary secretions as interconnected niches of a highly dynamic microbial ecosystem. In addition, host-associated factors, including physiological and anatomical parameters, as well as genetic traits that may affect the udder microbiota are briefly discussed. Finally, current understanding of the effect of antimicrobials on the composition of intramammary microbiota is discussed, highlighting the resilience of udder microbiota to exogenous perturbants.

Amniotic fluid proteomic signatures of cervical insufficiency and their association with length of latency.

PROBLEM: Cervical insufficiency is a precursor of preterm birth. Treatment with emergency cervical cerclage is contraindicated in the presence of intra-amniotic infection. Detecting infection with Gram stain and culture of amniotic fluid lacks sensitivity. Proteomic profiling of amniotic fluid in cervical insufficiency may help identify pregnancies best suited for emergency cerclage. METHOD OF STUDY: Thirty-two pregnant women underwent amniocentesis for routine genetic testing (n = 22) or after diagnosis of cervical insufficiency (n = 10). The proteomic profiles of the amniotic fluid samples were compared in a cross-sectional fashion, including sub-analyses of women with cervical insufficiency and latency periods of <1 week and >1 week post-diagnosis. RESULTS: Mean gestational age at diagnosis of cervical insufficiency was 21.4 weeks (95% CI 20.6-22.1). Proteomic analysis yielded 40 (7.2%, P < 0.05) differentially expressed proteins between women with delivery <1 week (n = 6) vs. >1 week (n = 4). Women who delivered <1 week had activated inflammatory response (z = 2.3, P = 6.71E-09), chemotaxis of immune cells (z = 2.9, P = 2.01E-08), and inhibited bacterial growth (z = -2.2, P = 5.82E-05). A multivariate model of eight biomarkers positively associated with cases of <1 week latency and distinguished cases from controls (97.8%, cross-validation accuracy 92.7%, P = 0.0009). CONCLUSION: In this pilot study, significant differences in the amniotic fluid proteomic profiles in cases of cervical insufficiency compared to genetic amniocentesis were observed. Proteomic signatures were predictive of achieving latency > 1 week after diagnosis of cervical insufficiency. These preliminary findings suggest that proteomic analysis may be of value in predicting outcome following cervical insufficiency and warrants further validation in larger studies.

The Prebiotic and Probiotic Properties of Human Milk: Implications for Infant Immune Development and Pediatric Asthma.

The incidence of pediatric asthma has increased substantially in recent decades, reaching a worldwide prevalence of 14%. This rapid increase may be attributed to the loss of "Old Friend" microbes from the human microbiota resulting in a less diverse and "dysbiotic" gut microbiota, which fails to optimally stimulate immune development during infancy. This hypothesis is supported by observations that the gut microbiota is different in infants who develop asthma later in life compared to those who remain healthy. Thus, early life exposures that influence gut microbiota play a crucial role in asthma development. Breastfeeding is one such exposure; it is generally considered protective against pediatric asthma, although conflicting results have been reported, potentially due to variations in milk composition between individuals and across populations. Human milk oligosaccharides (HMOs) and milk microbiota are two major milk components that influence the infant gut microbiota and hence, development of the immune system. Among their many immunomodulatory functions, HMOs exert a selective pressure within the infant gut microbial niche, preferentially promoting the proliferation of specific bacteria including Bifidobacteria. Milk is also a source of viable bacteria originating from the maternal gut and infant oral cavity. As such, breastmilk has prebiotic and probiotic properties that can modulate two of the main forces controlling the gut microbial community assembly, i.e., dispersal and selection. Here, we review the latest evidence, mechanisms and hypotheses for the synergistic and/or additive effects of milk microbiota and HMOs in protecting against pediatric asthma.

Early-Life Antibiotic Exposure, Gut Microbiota Development, and Predisposition to Obesity.

Antibiotics are often prescribed inappropriately to infants and young children, with potentially adverse effects on the developing gut microbiota and related metabolic processes. We review evidence from 17 epidemiologic studies suggesting that antibiotic exposure during critical periods of early development may influence weight gain and the development of obesity. Complementary research in both humans and rodents indicates that gut microbiota play a key role in this process, although further research is needed to confirm and characterize the causal mechanisms involved. Obesity is a complex and multifactorial condition; thus, a multipronged prevention strategy will be required to curb the current obesity epidemic. Evidence to date suggests this strategy should include the judicious use of antibiotics, especially in early life when the developing gut microbiota is particularly susceptible to perturbations with long-lasting implications for metabolic programming and obesity risk.

Carrageenan Gum and Adherent Invasive Escherichia coli in a Piglet Model of Inflammatory Bowel Disease: Impact on Intestinal Mucosa-associated Microbiota.

Inflammatory bowel diseases (IBD) including Crohn's disease (CD), and ulcerative colitis (UC), are chronic conditions characterized by chronic intestinal inflammation. Adherent invasive Escherichia coli (AIEC) pathotype has been increasingly implicated in the etiopathogenesis of IBD. In a 21-day study, we investigated the effects of AIEC strain UM146 inoculation on microbiota profile of the ileal, cecal, ascending and descending colon in a pig model of experimental colitis. Carrageenan gum (CG) was used to induce colitis in weaner piglets whereas AIEC strain UM146 previously isolated from a CD patient was included to investigate a cause or consequence effect in IBD. Treatments were: (1) control; (2) CG; (3) AIEC strain UM146; and (4) CG+UM146. Pigs in groups 2 and 4 received 1% CG in drinking water from day 1 of the study while pigs in groups 3 and 4 were inoculated with UM146 on day 8. Following euthanization on day 21, tissue mucosal scrapings were collected and used for DNA extraction. The V4 region of bacterial 16S rRNA gene was then subjected to Illumina sequencing. Microbial diversity, composition, and the predicted functional metagenome were determined in addition to short chain fatty acids profiles in the digesta and inflammatory cytokines in the intestinal tissue. CG-induced colitis decreased bacterial species richness and shifted community composition. At the phylum level, an increase in Proteobacteria and Deferribacteres and a decrease in Firmicutes, Actinobacteria, and Bacteroidetes were observed in CG and CGUM146 compared to control and UM146. The metabolic capacity of the microbiome was also altered in CG and CGUM146 compared to UM146 and control in the colon. We demonstrated that CG resulted in bacterial dysbiosis and shifted community composition similar to what has been previously observed in IBD patients. However, AIEC strain UM146 alone did not cause any clear changes compared to CG or control in our experimental IBD pig model.

Osteomyelitis due to multiple carbapenemase-producing Gram-negative bacteria: The first case report of a GES-13-producing Pseudomonas aeruginosa isolate in Canada.

A case of osteomyelitis in an infant following a burn injury sustained in Pakistan caused by a GES-13-producing Pseudomonas aeruginosa (the first reported in Canada) and an OXA-48 producing Klebsiella pneumoniae is described. The present case serves to highlight the importance of international travel as a risk factor for infection with carbapenemase-producing bacteria and the challenges in the laboratory detection of these organisms.

Les auteurs décrivent un cas d'ostéomyélite chez un nourrisson après une brûlure subie au Pakistan. Cette ostéomyélite était causée par un Pseudomonas aeruginosa producteur d'enzyme de type GES-13 (le premier déclaré au Canada) et un Klebsiella pneumoniae producteur d'enzyme de type OXA-48. Ce cas fait ressortir l'importance des voyages internationaux comme facteur de risque d'infection par des bactéries productrices de carbapénémases ainsi que la difficulté de déceler ces organismes en laboratoire.

Combination of culture, antigen and toxin detection, and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing.

BACKGROUND: There has been a growing interest in developing an appropriate laboratory diagnostic algorithm for Clostridium difficile, mainly as a result of increases in both the number and severity of cases of C difficile infection in the past decade. A C difficile diagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis of C difficile infection. In addition, conventional reference methods for C difficile detection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings. OBJECTIVE: To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenic C difficile. RESULT: One year of retrospective C difficile data using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenic C difficile. Sixty per cent (310 of 517) of toxigenic C difficile stools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenic C difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenic C difficile samples. DISCUSSION: Using GDH antigen as the screening and toxin A and B as confirmatory test for C difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenic C difficile stools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted for C difficile testing. CONCLUSION: The overview of the data illustrated the significance of each stage of this four-step C difficile algorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenic C difficile.

HISTORIQUE: L'intérêt augmente envers l'élaboration d'un algorithme de diagnostic pertinent en laboratoire pour le Clostridium difficile, surtout en raison de l'augmentation du nombre et de la gravité des cas d'infection par le C difficile depuis dix ans. Un tel algorithme diagnostique s'impose parce que les trousses diagnostiques, surtout pour déceler les toxines A et B ou l'antigène du glutamate déshydrogénase (GDH), ne suffisent pas comme test unique pour assurer le diagnostic optimal de l'infection par le C difficile. De plus, les méthodes de référence habituelles pour déceler le C difficile (p. ex., culture toxicogène et dosage de neutralisation des cytotoxines [NCT]) ne sont pas monnaie courante en laboratoire diagnostique. OBJECTIF: Analyser l'algorithme en quatre étapes utilisé aux emplacements de services diagnostiques du Manitoba pour poser un diagnostic en laboratoire de C difficile toxicogène. RÉSULTAT: Les chercheurs ont présenté les données rétrospectives sur le C difficile obtenues à l'aide de l'algorithme proposé sur une période d'un an. Sur les 5 695 coprocultures testées, 9,1 % (n=517) présentaient un C difficile toxicogène. Soixante pour cent (310 sur 517) des coprocultures de C difficile toxicogène ont été décelés après les deux premières étapes de l'algorithme. La confirmation par NCT de dosages GDH-positif, négatifs aux toxines A et B, a permis de déceler 37,7 % (198 sur 517) d'autres cas de C difficile toxicogène. La culture du troisième échantillon, prélevé chez les patients dont les deux échantillons précédents étaient négatifs, a permis de déceler 2,32 % (12 sur 517) d'échantillons de C difficile toxicogène. EXPOSÉ: En utilisant l'antigène GDH comme outil de dépistage et les toxines A et B comme test de confirmation de l'infection à C difficile, 85 % des échantillons étaient négatifs ou positifs dans un délai de quatre heures. Sans confirmation par NCT des résultats discordants de l'antigène GDH et des toxines A et B, 37 % (195 sur 517) des coprocultures de C difficile toxicogène n'auraient pas été décelées. Une fois l'algorithme effectué, il n'a fallu effectuer une culture que pour 2,72 % de tous les échantillons soumis en vue d'un test de dépistage du C difficile. CONCLUSION: L'aperçu des données fait ressortir l'importance de chaque étape de l'algorithme de C difficile en quatre étapes et la valeur du dosage de NCT et de la culture dans le cadre de l'algorithme pour assurer un diagnostic précis de C difficile toxicogène.

Establishing a clinically relevant bioburden benchmark: a quality indicator for adequate reprocessing and storage of flexible gastrointestinal endoscopes.

BACKGROUND: Microbiological surveillance of patient-ready flexible endoscopes has been suggested as a tool for endoscope reprocessing quality assurance. However, a proper guideline defining the performance and the frequency of monitoring procedures and specifying how to interpret the results is lacking. MATERIALS AND METHODS: All channels from the 20 flexible gastrointestinal endoscopes (5 gastroscopes, 9 colonoscopes, and 6 duodenoscopes) used at an endoscopy clinic were tested for the presence of bacteria and fungi early every Monday morning over a 7-month period. RESULTS: Bacteria and fungi were detected in 5.7% of the 383 channels tested. Of the 141 scopes tested, 14.1% had detectable growth in at least 1 channel. No significant relationship was detected between the scope or channel type and detection of microorganisms. Over the 7 months of testing, 99.5% of scope channels consistently demonstrated <100 cfu/mL of microbial growth. CONCLUSION: Based on our clinical findings, we recommend 100 cfu/mL as a reliable and routinely achievable cutoff for bioburden residuals in reprocessed endoscope channels. This cutoff is the same as the Canadian cutoff for dialysis water.

Characterization of Escherichia coli isolated from gut biopsies of newly diagnosed patients with inflammatory bowel disease.

BACKGROUND: Mucosal-associated Escherichia coli may play a role in the pathogenesis of inflammatory bowel diseases (IBDs). In this study we assessed mucosal-associated E. coli in adults at the time of first diagnosis. MATERIALS AND METHODS: E. coli were isolated from 59 right colon biopsies of 34 newly diagnosed adult IBD patients (Crohn's disease [CD] = 23, ulcerative colitis [UC] = 11) and 25 healthy controls (HC). Strains were serotyped, phylotyped into A, B1, B2, or D, and tested for their ability to survive in macrophages. The presence of various virulence factors was also assessed. The fimH subunit of type 1 fimbriae was sequenced and phylogenetically analyzed. RESULTS: A total of 65 E. coli were isolated from CD (29 isolates from 23 patients), UC (11 isolates from 11 patients), and HC (25 isolates from 25 subjects). All E. coli were positive for fimH, crl, and cgsA and negative for vt1, vt2, hlyA, cnf, and eae. Significant positive associations were between CD and in between CD and afae (P = 0.002), and between UC and ompA (P = 0.02), afae (P = 0.03), and USP (P = 0.04). The B2+D phylotype was significantly associated with inflammation (P = 0.04) as it was with serine protease autotransporters (SPATE), malX, ompA, and kpsMTII (P < 0.05). Macrophage survival was the highest in UC-isolated E. coli (P = 0.04). FimH amino acid substitutions N91S, S99N, and A223V were associated with IBD (P < 0.05). CONCLUSIONS: Adherent invasive E. coli are present at first diagnosis, suggesting that they may have a role in the early stages of disease onset.

Evaluation of BacT/Alert 3D automated unit for detection of nontuberculous mycobacteria requiring incubation at 30 degrees C for optimal growth.

The reliability of the BacT/Alert 3D unit for automated detection of nontuberculous mycobacteria (NTM) that grow optimally at 30 °C was assessed. This system reliably maintained a temperature of 30 °C and detected 50% of the clinical NTM strains (5 Mycobacterium marinum and 3 Mycobacterium gordonae strains) faster than 37 °C culture.

Real-time PCR assay provides reliable assessment of intrapartum carriage of group B Streptococcus.

The objective of this study was to determine the reliability of the real-time PCR assay for determining the group B Streptococcus (GBS) status of women in labor. In this prospective study we compared the results of culture and PCR testing of vaginal and rectal samples collected by nursing staff when women were in labor. Patients' charts were also reviewed to obtain relevant information about pregnancy risk factors. Our results demonstrated a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 90.5%, 96.1%, 86.4%, and 97.4%, respectively, for rapid PCR. Of the 196 women evaluated, 29 (14.8%) presented with unknown GBS status, 11 (37.9%) of whom received unnecessary intrapartum antibiotics. The rapid real-time PCR test was robust and was able to reliably detect the presence of GBS in women in labor within 1 h of specimen submission to the laboratory. We recommend that the rapid PCR test be targeted to women who present in labor with unknown GBS status. In cases where the laboratory does not offer 24-h availability of testing, sample collection followed by PCR testing the next morning is still valuable and provides reliable results 24 to 48 h faster than culture and will aid appropriate decision-making regarding continuing or stopping antibiotics for neonates of women with unknown GBS status.

Phylogenetic analysis of inflammatory bowel disease associated Escherichia coli and the fimH virulence determinant.

BACKGROUND: Evidence supports the role of adherent invasive Escherichia coli (AIEC) in the pathogenesis of inflammatory bowel disease (IBD). However, little is known about the phylogenetic structure and origin of this group of bacteria. Multi-locus sequence typing (MLST), and fimH sequence analysis were performed to elucidate the phylogenetic relationships between E. coli strains isolated from IBD tissue. METHODS: Thirty-six E. coli isolated from IBD patients and healthy individuals were used. MLST analysis of the adk, fumC, gyrB, icd, mdh, purA, and recA housekeeping genes was performed. The fimH gene was also sequenced and phylogenetically analyzed. Biochemical profiling of strains were performed using the API 20 E system. RESULTS: MLST analysis distinguished 9 new alleles and 11 new sequence types, nearly all of which belonged to IBD isolates. E. coli isolated from IBD patients were more likely to be grouped into separate clonal clusters by eBURST analysis of allelic profiles (P = 0.02). Sequencing of fimH placed putative AIEC strains into the same cluster with the uro-pathogenic E. coli CFT073 and the avian-pathogenic E. coli O1:K1:H7. CONCLUSIONS: MLST analysis suggested that E. coli isolated from IBD patients did not evolve from a unique ancestral background. Together with the fimH sequence we conclude that AIEC represent a group of bacteria that have been able to take advantage of an "IBD microenvironment" and likely shares common genes with extraintestinal pathogens like uro-pathogenic CFT073 and avian-pathogenic O1:K1:H7 E. coli. Future research should focus on genes that are unique to AIEC.

Microbial diversity of inflamed and noninflamed gut biopsy tissues in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic gastrointestinal condition without any known cause or cure. An imbalance in normal gut biota has been identified as an important factor in the inflammatory process. METHODS: Fifty-eight biopsies from Crohn's disease (CD, n = 10), ulcerative colitis (UC, n = 15), and healthy controls (n = 16) were taken from a population-based case-control study. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphisms (T-RFLP) were used as molecular tools to investigate the intestinal microbiota in these biopsies. RESULTS: ARISA and T-RFLP data did not allow a high level of clustering based on disease designation. However, if clustering was done based on the inflammation criteria, the majority of biopsies grouped either into inflamed or noninflamed groups. We conducted statistical analyses using incidence-based species richness and diversity as well as the similarity measures. These indices suggested that the noninflamed tissues form an intermediate population between controls and inflamed tissue for both CD and UC. Of particular interest was that species richness increased from control to noninflamed tissue, and then declined in fully inflamed tissue. CONCLUSIONS: We hypothesize that there is a recruitment phase in which potentially pathogenic bacteria colonize tissue, and once the inflammation sets in, a decline in diversity occurs that may be a byproduct of the inflammatory process. Furthermore, we suspect that a better knowledge of the microbial species in the noninflamed tissue, thus before inflammation sets in, holds the clues to the microbial pathogenesis of IBD.

High prevalence of Escherichia coli belonging to the B2+D phylogenetic group in inflammatory bowel disease.

BACKGROUND: It is not clear which species of bacteria may be involved in inflammatory bowel disease (IBD). One way of determining which bacteria might be likely candidates is to use culture-independent methods to identify microorganisms that are present in diseased tissues but not in controls. AIMS: (1) To assess the diversity of microbial communities of biopsy tissue using culture-independent methods; (2) to culture the bacteria found in the tissues of patients with IBD but not in the controls; (3) to identify potential virulence factors associated with cultured bacteria. METHODS: 84 biopsy specimens were collected from 15 controls, 13 patients with Crohn's disease (CD) and 19 patients with ulcerative colitis (UC) from a population-based case-control study. Ribosomal intergenic spacer analysis (RISA) was conducted to identify unique DNA bands in tissues from patients with CD and UC that did not appear in controls. RESULTS: RISA followed by DNA sequencing identified unique bands in biopsy specimens from patients with IBD that were classified as Escherichia coli. Targeted culture showed a significantly (p<0.05) higher number of Enterobacteriaceae in specimens from patients with IBD. The B2+D phylogenetic group, serine protease autotransporters (SPATE) and adherence factors were more likely to be associated with tissues from patients with UC and CD than with controls. CONCLUSIONS: The abundance of Enterobacteriaceae is 3-4 logs higher in tissues of patients with IBD and the B2+D phylogenetic groups are more prevalent in patients with UC and CD. The B2+D phylogenetic groups are associated with SPATE and adherence factors and may have a significant role in disease aetiology.