High-Throughput Quantitative Analysis of the Human Intestinal Microbiota with a Phylogenetic Microarray

Abstract

Gut microbiota carry out key functions in health and participate in the pathogenesis of a growing number of diseases. The aim of this study was to develop a custom microarray that is able to identify hundreds of intestinal bacterial species. We used the Entrez nucleotide database to compile a data set of bacterial 16S rRNA gene sequences isolated from human intestinal and fecal samples. Identified sequences were clustered into separate phylospecies groups. Representative sequences from each phylospecies were used to develop a microbiota microarray based on the Affymetrix GeneChip platform. The designed microbiota array contains probes to 775 different bacterial phylospecies. In our validation experiments, the array correctly identified genomic DNA from all 15 bacterial species used. Microbiota array has a detection sensitivity of at least 1 pg of genomic DNA and can detect bacteria present at a 0.00025% level of overall sample. Using the developed microarray, fecal samples from two healthy children and two healthy adults were analyzed for bacterial presence. Between 227 and 232 species were detected in fecal samples from children, whereas 191 to 208 species were found in adult stools. The majority of identified phylospecies belonged to the classes Clostridia and Bacteroidetes. The microarray revealed putative differences between the gut microbiota of healthy children and adults: fecal samples from adults had more Clostridia and less Bacteroidetes and Proteobacteria than those from children. A number of other putative differences were found at the genus level.

High-Throughput Sequence-Based Analysis of the Intestinal Microbiota of Weanling Pigs Fed Genetically Modified MON810 Maize Expressing Bacillus thuringiensis Cry1Ab (Bt Maize) for 31 Days

“…This deep 16S rRNA gene-sequencing approach detected 15 different bacterial phyla in 65-day-old pigs, with Firmicutes dominating, followed by Bacteroidetes and Proteobacteria. The relative distribution is in agreement with that previously observed in the large intestine of humans and pigs using 16S rRNA gene sequencing (28,29,31,38). Similarly, Poroyko et al detected 11 phyla in the ceca of 21-day-old pigs but found that Bacteroidetes dominated, followed by Firmicutes and Proteobacteria (40).…”

Numerical ecology validates a biogeographical distribution and gender-based effect on mucosa-associated bacteria along the human colon

“…However, they were unable to conclusively reveal evidence of biogeographical trends along the colonic mucosa, and suggested that higher resolution studies using microarray and/or next-generation sequencing technologies, as well as alternative methods of data analysis, were needed. Both phylogenetic microarrays (Paliy et al, 2009;Rajilic-Stojanovic et al, 2009;Kang et al, 2010) and high-throughput sequencing methods are now used more frequently to examine gut microbiome diversity, but the development of robust quantitative methods to reveal the biogeographical features of the gut microbiota is considered a 'great challenge' and 'the diversity and biogeography of the gut microbiota needs to be defined at varying scales of resolution' (Peterson et al, 2008). Although some clustering and ordination methods of numerical ecology are commonly used to compare microbial community profiles through open source software (for example, QIIME (Caporaso et al, 2010)), and increasingly used in gut microbiomics research (Fuentes et al, 2008;Biagi et al, 2010;Janczyk et al, 2010), we contend that, in addition to those methods, there are additional useful numerical ecology methods that can be used.…”

Changes in Korean Adult Females’ Intestinal Microbiota Resulting from Kimchi Intake

“…It can be seen that the kimchi intake level affects the formation of the intestinal microbiota. The intestinal microorganism microarray analysis method used in the present study identified at least three times more species of microorganisms as compared to the human intestinal microbiota microarray developed by Paliy et al [30]. Therefore, it can be seen that the customized microarray chips used in the present study are more accurate and sensitive.…”

“…The LABs to be the dominant microorganisms in the process of kimchi fermentation secrete bacteriocins which are natural antibiotics to suppress the growth of the harmful bacteria and other harmful microorganisms. In kimchi, among the LAB that comprise the dominant microorganisms in the process of fermentation, Lactobacillus and Leuconostoc produce many kinds of bacteriocins to suppress the growth of other microorganisms [11,30]. The bacteriocins produced by Lactobacillus and Leuconostoc increased in the high kimchi intake group in the present study are considered to suppress pathogenic intestinal bacteria.…”