Shadi Sepehri

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.

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 Crohns 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. (Inflamm Bowel Dis 2007)

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 (Crohns 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. (Inflamm Bowel Dis 2010;)

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. Inflamm Bowel Dis 2009

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.

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 Crohns 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.

Evaluation of BacT/Alert 3D Automated Unit for Detection of Nontuberculous Mycobacteria Requiring Incubation at 30°C for Optimal Growth

ABSTRACT 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.

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.

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.