Extending colonic mucosal microbiome analysis - Assessment of colonic lavage as a proxy for endoscopic colonic biopsies

This study was supported through GI Research funds and MRC Grant Ref: MR/M00533X/1 to GH.Peer reviewedPublisher PD

Development of the preterm gut microbiome in twins at risk of necrotising enterocolitis and sepsis

The preterm gut microbiome is a complex dynamic community influenced by genetic and environmental factors and is implicated in the pathogenesis of necrotising enterocolitis (NEC) and sepsis. We aimed to explore the longitudinal development of the gut microbiome in preterm twins to determine how shared environmental and genetic factors may influence temporal changes and compared this to the expressed breast milk (EBM) microbiome. Stool samples (n = 173) from 27 infants (12 twin pairs and 1 triplet set) and EBM (n = 18) from 4 mothers were collected longitudinally. All samples underwent PCR-DGGE (denaturing gradient gel electrophoresis) analysis and a selected subset underwent 454 pyrosequencing. Stool and EBM shared a core microbiome dominated by Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae. The gut microbiome showed greater similarity between siblings compared to unrelated individuals. Pyrosequencing revealed a reduction in diversity and increasing dominance of Escherichia sp. preceding NEC that was not observed in the healthy twin. Antibiotic treatment had a substantial effect on the gut microbiome, reducing Escherichia sp. and increasing other Enterobacteriaceae. This study demonstrates related preterm twins share similar gut microbiome development, even within the complex environment of neonatal intensive care. This is likely a result of shared genetic and immunomodulatory factors as well as exposure to the same maternal microbiome during birth, skin contact and exposure to EBM. Environmental factors including antibiotic exposure and feeding are additional significant determinants of community structure, regardless of host genetics

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

Peer reviewedPublisher PD

Phylotyping and Functional Analysis of Two Ancient Human Microbiomes

Background: The Human Microbiome Project (HMP) is one of the U.S. National Institutes of Health Roadmap for Medical Research. Primary interests of the HMP include the distinctiveness of different gut microbiomes, the factors influencing microbiome diversity, and the functional redundancies of the members of human microbiotas. In this present work, we contribute to these interests by characterizing two extinct human microbiotas. Methodology/Principal Findings: We examine two paleofecal samples originating from cave deposits in Durango Mexico and dating to approximately 1300 years ago. Contamination control is a serious issue in ancient DNA research; we use a novel approach to control contamination. After we determined that each sample originated from a different human, we generated 45 thousand shotgun DNA sequencing reads. The phylotyping and functional analysis of these reads reveals a signature consistent with the modern gut ecology. Interestingly, inter-individual variability for phenotypes but not functional pathways was observed. The two ancient samples have more similar functional profiles to each other than to a recently published profile for modern humans. This similarity could not be explained by a chance sampling of the databases. Conclusions/Significance: We conduct a phylotyping and functional analysis of ancient human microbiomes, while providing novel methods to control for DNA contamination and novel hypotheses about past microbiome biogeography. We postulate that natural selection has more of an influence on microbiome functional profiles than it does on the species represented in the microbial ecology. We propose that human microbiomes were more geographically structured during pre-Columbian times than today

Monitoring Bacterial Community of Human Gut Microbiota Reveals an Increase in Lactobacillus in Obese Patients and Methanogens in Anorexic Patients

Background: Studies of the bacterial communities of the gut microbiota have revealed a shift in the ratio of Firmicutes and Bacteroidetes in obese patients. Determining the variations of microbial communities in feces may be beneficial for the identification of specific profiles in patients with abnormal weights. The roles of the archaeon Methanobrevibacter smithii and Lactobacillus species have not been described in these studies. Methods and Findings: We developed an efficient and robust real-time PCR tool that includes a plasmid-based internal control and allows for quantification of the bacterial divisions Bacteroidetes, Firmicutes, and Lactobacillus as well as the methanogen M. smithii. We applied this technique to the feces of 20 obese subjects, 9 patients with anorexia nervosa, and 20 normal-weight healthy controls. Our results confirmed a reduction in the Bacteroidetes community in obese patients (p<0.01). We found a significantly higher Lactobacillus species concentration in obese patients than in lean controls (p = 0.0197) or anorexic patients (p = 0.0332). The M. smithii concentration was much higher in anorexic patients than in the lean population (p = 0.0171). Conclusions: Lactobacillus species are widely used as growth promoters in the farm industry and are now linked to obesity in humans. The study of the bacterial flora in anorexic patients revealed an increase in M. smithii. This increase might represent an adaptive use of nutrients in this population

Characterization of Bacteria in Biopsies of Colon and Stools by High Throughput Sequencing of the V2 Region of Bacterial 16S rRNA Gene in Human

BACKGROUND: The characterization of the human intestinal microflora and their interactions with the host have been identified as key components in the study of intestinal disorders such as inflammatory bowel diseases. High-throughput sequencing has enabled culture-independent studies to deeply analyze bacteria in the gut. It is possible with this technology to systematically analyze links between microbes and the genetic constitution of the host, such as DNA polymorphisms and methylation, and gene expression. METHODS AND FINDINGS: In this study the V2 region of the bacterial 16S ribosomal RNA (rRNA) gene using 454 pyrosequencing from seven anatomic regions of human colon and two types of stool specimens were analyzed. The study examined the number of reads needed to ascertain differences between samples, the effect of DNA extraction procedures and PCR reproducibility, and differences between biopsies and stools in order to design a large scale systematic analysis of gut microbes. It was shown (1) that sequence coverage lower than 1,000 reads influenced quantitative and qualitative differences between samples measured by UniFrac distances. Distances between samples became stable after 1,000 reads. (2) Difference of extracted bacteria was observed between the two DNA extraction methods. In particular, Firmicutes Bacilli were not extracted well by one method. (3) Quantitative and qualitative difference in bacteria from ileum to rectum colon were not observed, but there was a significant positive trend between distances within colon and quantitative differences. Between sample type, biopsies or stools, quantitative and qualitative differences were observed. CONCLUSIONS: Results of human colonic bacteria analyzed using high-throughput sequencing were highly dependent on the experimental design, especially the number of sequence reads, DNA extraction method, and sample type

Pyrosequencing-Based Analysis of the Mucosal Microbiota in Healthy Individuals Reveals Ubiquitous Bacterial Groups and Micro-Heterogeneity

This study used 16S rRNA-based pyrosequencing to examine the microbial community that is closely associated with the colonic mucosa of five healthy individuals. Spatial heterogeneity in microbiota was measured at right colon, left colon and rectum, and between biopsy duplicates spaced 1 cm apart. The data demonstrate that mucosal-associated microbiota is comprised of Firmicutes (50.9%±21.3%), Bacteroidetes (40.2%±23.8%) and Proteobacteria (8.6%±4.7%), and that interindividual differences were apparent. Among the genera, Bacteroides, Leuconostoc and Weissella were present at high abundance (4.6% to 41.2%) in more than 90% of the studied biopsy samples. Lactococcus, Streptococcus, Acidovorax, Acinetobacter, Blautia, Faecalibacterium, Veillonella, and several unclassified bacterial groups were also ubiquitously present at an abundance <7.0% of total microbial community. With the exception of one individual, the mucosal-associated microbiota was relatively homogeneous along the colon (average 61% Bray-Curtis similarity). However, micro-heterogeneity was observed in biopsy duplicates within defined colonic sites for three of the individuals. A weak but significant Mantel correlation of 0.13 was observed between the abundance of acidomucins and mucosal-associated microbiota (P-value  =  0.04), indicating that the localized biochemical differences may contribute in part to the micro-heterogeneity. This study provided a detailed insight to the baseline mucosal microbiota along the colon, and revealed the existence of micro-heterogeneity within defined colonic sites for certain individuals

Standard Colonic Lavage Alters the Natural State of Mucosal-Associated Microbiota in the Human Colon

Past studies of the human intestinal microbiota are potentially confounded by the common practice of using bowel-cleansing preparations. We examined if colonic lavage changes the natural state of enteric mucosal-adherent microbes in healthy human subjects.Twelve healthy individuals were divided into three groups; experimental group, control group one, and control group two. Subjects in the experimental group underwent an un-prepped flexible sigmoidoscopy with biopsies. Within two weeks, subjects were given a standard polyethylene glycol-based bowel cleansing preparation followed by a second flexible sigmoidoscopy. Subjects in control group one underwent two un-prepped flexible sigmoidoscopies within one week. Subjects in the second control group underwent an un-prepped flexible sigmoidoscopy followed by a second flexible sigmoidoscopy after a 24-hour clear liquid diet within one week. The mucosa-associated microbial communities from the two procedures in each subject were compared using 16S rRNA gene based terminal restriction fragment length polymorphism (T-RFLP), and library cloning and sequencing.Clone library sequencing analysis showed that there were changes in the composition of the mucosa-associated microbiota in subjects after colonic lavage. These changes were not observed in our control groups. Standard bowel preparation altered the diversity of mucosa-associated microbiota. Taxonomic classification did not reveal significant changes at the phylum level, but there were differences observed at the genus level.Standard bowel cleansing preparation altered the mucosal-adherent microbiota in all of our subjects, although the degree of change was variable. These findings underscore the importance of considering the confounding effects of bowel preparation when designing experiments exploring the gut microbiota

Meta-analysis of thyroidectomy with ultrasonic dissector versus conventional clamp and tie

<p>Abstract</p> <p>Background</p> <p>We conducted a systematic review to evaluate the role of Ultrasonic dissector (UAS) versus conventional clamp and tie in thyroidectomy.</p> <p>Materials and methods</p> <p>We searched for all published RCT in into electronic databases. To be included in the analysis, the studies had to compare thyroidectomy with UAS versus conventional vessel ligation and tight (conventional technique = CT). The following outcomes were used to compare the total thyroidectomy group with UAS versus CT group: operative duration, operative blood loss, overall drainage volume during the first 24 hours, transiet laryngeal nerve palsy, permanent laryngeal nerve palsy, transiet hypocalcaemia and permanent hypocalcaemia.</p> <p>Results</p> <p>There are currently 7 RCT on this issue to compare thyroidectomy with UAS versus CT. From the analysis of these studies it was possible to confront 608 cases: 303 undergoing to thyroidectomy with UAS versus 305 that were treated with CT. Actually, it was shown a relevant advantage of cost-effectiveness in patients treated with UAS; there is a statistically significant reduction of the operative duration (weighted mean difference [WMD], -18.74 minutes; 95% confidence interval [CI], (-26.97 to -10.52 minutes) (P = 0.00001), intraoperative blood loss (WMD, -60.10 mL; 95% CI, -117.04 to 3.16 mL) (P = 0.04) and overall drainage volume (WMD, -35.30 mL; 95% CI, -49.24 to 21.36 mL) (P = 0.00001) in the patients underwent thyroidectomy with UAS. Although the analysis showed that the patients who were treated with USA presented more favourable results in incidence of post-operative complications (transient laryngeal nerve palsy: P = 0.11; permanent laryngeal nerve palsy: not estimable; transient hypocalcaemia: P = 0.24; permanent hypocalcaemia: P = 0.45), these data didn't present statistical relevance.</p> <p>Conclusion</p> <p>This meta-analysis shown a relevant advantage only in terms of cost-effectiveness in patients treated with UAS; it is subsequent to statistically significant reduction of operation duration, intraoperative blood loss and of overall drainage volume during the first 24 hours. Although the analysis showed that the patients who were treated with UAS presented more favourable results in incidence of post-operative complications (transiet laryngeal nerve palsy; transiet hypocalcaemia and permanent hypocalcaemia), these data didn't present statistical relevance.</p