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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.

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.

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

Summary 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.

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.

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.

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.

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.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 of1 week and1 week post-diagnosis.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 delivery1 week (n = 6) vs.1 week (n = 4). Women who delivered1 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 of1 week latency and distinguished cases from controls (97.8%, cross-validation accuracy 92.7%, P = 0.0009).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 latency1 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.

BACKGROUND: There has been a growing interest in developing an appropriate laboratory diagnostic algorithm forClostridium difficile, mainly as a result of increases in both the number and severity of cases ofC difficileinfection in the past decade. AC difficilediagnostic 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 ofC difficileinfection. In addition, conventional reference methods forC difficiledetection (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 toxigenicC difficile.RESULT: One year of retrospectiveC difficiledata using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenicC difficile. Sixty per cent (310 of 517) of toxigenicC difficilestools 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 toxigenicC difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenicC difficilesamples.DISCUSSION: Using GDH antigen as the screening and toxin A and B as confirmatory test forC 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 toxigenicC difficilestools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted forC difficiletesting.CONCLUSION: The overview of the data illustrated the significance of each stage of this four-stepC difficilealgorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenicC difficile.

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 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.

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. (Inflamm Bowel Dis 2010;)

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

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. (Inflamm Bowel Dis 2007)