• 16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform

      Fouhy, Fiona; Clooney, Adam G; STANTON, CATHERINE; Claesson, Marcus J.; Cotter, Paul D.; European Union; Science Foundation Ireland; 603038; SFI/12/RC/2273; SFI/11/PI/1137 (Biomed Central, 24/06/2016)
      Background Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA. Results The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar. Conclusions Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.
    • Characterization of Pro-Inflammatory Flagellin Proteins Produced by Lactobacillus ruminis and Related Motile Lactobacilli

      Neville, B. Anne; Forde, Brian M; Claesson, Marcus J.; Darby, Trevor; Coghlan, Avril; Nally, Kenneth; Ross, R Paul; O'Toole, Paul W.; Science Foundation Ireland; Irish Research Council for Science, Engineering and Technology; et al. (PLOS, 10/07/2012)
      Lactobacillus ruminis is one of at least twelve motile but poorly characterized species found in the genus Lactobacillus. Of these, only L. ruminis has been isolated from mammals, and this species may be considered as an autochthonous member of the gastrointestinal microbiota of humans, pigs and cows. Nine L. ruminis strains were investigated here to elucidate the biochemistry and genetics of Lactobacillus motility. Six strains isolated from humans were non-motile while three bovine isolates were motile. A complete set of flagellum biogenesis genes was annotated in the sequenced genomes of two strains, ATCC25644 (human isolate) and ATCC27782 (bovine isolate), but only the latter strain produced flagella. Comparison of the L. ruminis and L. mali DSM20444T motility loci showed that their genetic content and gene-order were broadly similar, although the L. mali motility locus was interrupted by an 11.8 Kb region encoding rhamnose utilization genes that is absent from the L. ruminis motility locus. Phylogenetic analysis of 39 motile bacteria indicated that Lactobacillus motility genes were most closely related to those of motile carnobacteria and enterococci. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644. Flagellin proteins were isolated from L. ruminis ATCC27782 and from three other Lactobacillus species, while recombinant flagellin of aflagellate L. ruminis ATCC25644 was expressed and purified from E. coli. These native and recombinant Lactobacillus flagellins, and also flagellate L. ruminis cells, triggered interleukin-8 production in cultured human intestinal epithelial cells in a manner suppressed by short interfering RNA directed against Toll-Like Receptor 5. This study provides genetic, transcriptomic, phylogenetic and immunological insights into the trait of flagellum-mediated motility in the lactobacilli.
    • Comparative Analysis of Pyrosequencing and a Phylogenetic Microarray for Exploring Microbial Community Structures in the Human Distal Intestine

      Claesson, Marcus J.; O'Sullivan, Orla; Wang, Qiong; Nikkila, Janne; Marchesi, Julian R.; Smidt, Hauka; de Vos, Willem M.; Ross, R Paul; O'Toole, Paul W.; Department of Agriculture, Food and the Marine, Ireland; et al. (PLOS, 20/08/2009)
      Background: Variations in the composition of the human intestinal microbiota are linked to diverse health conditions. Highthroughput molecular technologies have recently elucidated microbial community structure at much higher resolution than was previously possible. Here we compare two such methods, pyrosequencing and a phylogenetic array, and evaluate classifications based on two variable 16S rRNA gene regions. Methods and Findings: Over 1.75 million amplicon sequences were generated from the V4 and V6 regions of 16S rRNA genes in bacterial DNA extracted from four fecal samples of elderly individuals. The phylotype richness, for individual samples, was 1,400–1,800 for V4 reads and 12,500 for V6 reads, and 5,200 unique phylotypes when combining V4 reads from all samples. The RDP-classifier was more efficient for the V4 than for the far less conserved and shorter V6 region, but differences in community structure also affected efficiency. Even when analyzing only 20% of the reads, the majority of the microbial diversity was captured in two samples tested. DNA from the four samples was hybridized against the Human Intestinal Tract (HIT) Chip, a phylogenetic microarray for community profiling. Comparison of clustering of genus counts from pyrosequencing and HITChip data revealed highly similar profiles. Furthermore, correlations of sequence abundance and hybridization signal intensities were very high for lower-order ranks, but lower at family-level, which was probably due to ambiguous taxonomic groupings. Conclusions: The RDP-classifier consistently assigned most V4 sequences from human intestinal samples down to genuslevel with good accuracy and speed. This is the deepest sequencing of single gastrointestinal samples reported to date, but microbial richness levels have still not leveled out. A majority of these diversities can also be captured with five times lower sampling-depth. HITChip hybridizations and resulting community profiles correlate well with pyrosequencing-based compositions, especially for lower-order ranks, indicating high robustness of both approaches. However, incompatible grouping schemes make exact comparison difficult.
    • Comparing Apples and Oranges?: Next Generation Sequencing and Its Impact on Microbiome Analysis

      Clooney, Adam G; Fouhy, Fiona; Sleator, Roy D.; O'Driscoll, Aisling; STANTON, CATHERINE; Cotter, Paul D.; Claesson, Marcus J.; Science Foundation Ireland; European Union; SFI/12/RC/2273; et al. (PLOS, 05/02/2016)
      Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number ofmethodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance inmicrobiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced withMiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.
    • Effect of Lactobacillus salivarius Bacteriocin Abp118 on the Mouse and Pig Intestinal Microbiota

      Riboulet-Bisson, Eliette; Sturme, Mark H. J.; Jeffery, Ian B; O'Donnell, Michelle M.; Neville, B. Anne; Forde, Brian M; Claesson, Marcus J.; Harris, Hugh; Gardiner, Gillian E.; Casey, Patrick G.; et al. (PLOS, 17/02/2012)
      Lactobacilli are Gram-positive bacteria that are a subdominant element in the human gastrointestinal microbiota, and which are commonly used in the food industry. Some lactobacilli are considered probiotic, and have been associated with health benefits. However, there is very little culture-independent information on how consumed probiotic microorganisms might affect the entire intestinal microbiota. We therefore studied the impact of the administration of Lactobacillus salivarius UCC118, a microorganism well characterized for its probiotic properties, on the composition of the intestinal microbiota in two model animals. UCC118 has anti-infective activity due to production of the bacteriocin Abp118, a broad-spectrum class IIb bacteriocin, which we hypothesized could impact the microbiota. Mice and pigs were administered wild-type (WT) L. salivarius UCC118 cells, or a mutant lacking bacteriocin production. The microbiota composition was determined by pyrosequencing of 16S rRNA gene amplicons from faeces. The data show that L. salivarius UCC118 administration had no significant effect on proportions of major phyla comprising the mouse microbiota, whether the strain was producing bacteriocin or not. However, L. salivarius UCC118 WT administration led to a significant decrease in Spirochaetes levels, the third major phylum in the untreated pig microbiota. In both pigs and mice, L. salivarius UCC118 administration had an effect on Firmicutes genus members. This effect was not observed when the mutant strain was administered, and was thus associated with bacteriocin production. Surprisingly, in both models, L. salivarius UCC118 administration and production of Abp118 had an effect on Gram-negative microorganisms, even though Abp118 is normally not active in vitro against this group of microorganisms. Thus L. salivarius UCC118 administration has a significant but subtle impact on mouse and pig microbiota, by a mechanism that seems at least partially bacteriocin-dependent
    • Genome sequences and comparative genomics of two Lactobacillus ruminis strains from the bovine and human intestinal tracts

      Forde, Brian M; Neville, B. Anne; O'Donnell, Michelle M.; Riboulet-Bisson, Eliette; Claesson, Marcus J.; Coghlan, Avril; Ross, R Paul; O'Toole, Paul W. (Biomed Central, 30/08/2011)
      Background: The genus Lactobacillus is characterized by an extraordinary degree of phenotypic and genotypic diversity, which recent genomic analyses have further highlighted. However, the choice of species for sequencing has been non-random and unequal in distribution, with only a single representative genome from the L. salivarius clade available to date. Furthermore, there is no data to facilitate a functional genomic analysis of motility in the lactobacilli, a trait that is restricted to the L. salivarius clade. Results: The 2.06 Mb genome of the bovine isolate Lactobacillus ruminis ATCC 27782 comprises a single circular chromosome, and has a G+C content of 44.4%. In silico analysis identified 1901 coding sequences, including genes for a pediocin-like bacteriocin, a single large exopolysaccharide-related cluster, two sortase enzymes, two CRISPR loci and numerous IS elements and pseudogenes. A cluster of genes related to a putative pilin was identified, and shown to be transcribed in vitro. A high quality draft assembly of the genome of a second L. ruminis strain, ATCC 25644 isolated from humans, suggested a slightly larger genome of 2.138 Mb, that exhibited a high degree of synteny with the ATCC 27782 genome. In contrast, comparative analysis of L. ruminis and L. salivarius identified a lack of long-range synteny between these closely related species. Comparison of the L. salivarius clade core proteins with those of nine other Lactobacillus species distributed across 4 major phylogenetic groups identified the set of shared proteins, and proteins unique to each group. Conclusions: The genome of L. ruminis provides a comparative tool for directing functional analyses of other members of the L. salivarius clade, and it increases understanding of the divergence of this distinct Lactobacillus lineage from other commensal lactobacilli. The genome sequence provides a definitive resource to facilitate investigation of the genetics, biochemistry and host interactions of these motile intestinal lactobacilli.
    • Microbial Succession and Flavor Production in the Fermented Dairy Beverage Kefir

      Walsh, Aaron M.; Crispie, Fiona; Kilcawley, Kieran; O'Sullivan, Orla; O'Sullivan, Maurice G.; Claesson, Marcus J.; Cotter, Paul D.; Science Foundation Ireland; SFI/12/RC/2273; SFI/11/PI/1137; et al. (2018-11-05)
      Kefir is a putatively health-promoting dairy beverage that is produced when a kefir grain, consisting of a consortium of microorganisms, is added to milk to initiate a natural fermentation. Here, a detailed analysis was carried out to determine how the microbial population, gene content, and flavor of three kefirs from distinct geographic locations change over the course of 24-h fermentations. Metagenomic sequencing revealed that Lactobacillus kefiranofaciens was the dominant bacterial species in kefir during early stages of fermentations but that Leuconostoc mesenteroides became more prevalent in later stages. This pattern is consistent with an observation that genes involved in aromatic amino acid biosynthesis were absent from L. kefiranofaciens but were present in L. mesenteroides. Additionally, these shifts in the microbial community structure, and associated pathways, corresponded to changes in the levels of volatile compounds. Specifically, Acetobacter spp. correlated with acetic acid; Lactobacillus spp. correlated with carboxylic acids, esters and ketones; Leuconostoc spp. correlated with acetic acid and 2,3-butanedione; and Saccharomyces spp. correlated with esters. The correlation data suggest a causal relationship between microbial taxa and flavor that is supported by observations that addition of L. kefiranofaciens NCFB 2797 increased the levels of esters and ketones whereas addition of L. mesenteroides 213M0 increased the levels of acetic acid and 2,3-butanedione. Finally, we detected genes associated with probiotic functionalities in the kefir microbiome. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein and can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods. IMPORTANCE Traditional fermented foods represent relatively low-complexity microbial environments that can be used as model microbial communities to understand how microbes interact in natural environments. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein. In the process, the link between individual species, and associated pathways, with flavor compounds is revealed and several genes that could be responsible for the purported gut health-associated benefits of consuming kefir are identified. Ultimately, in addition to providing an important fundamental insight into microbial interactions, this information can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods.
    • Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

      Neville, B. Anne; Sheridan, Paul O.; Harris, Hugh; Coughlan, Simone; Flint, Harry J.; Duncan, Sylvia H.; Jeffery, Ian B; Claesson, Marcus J.; Ross, R Paul; Scott, Karen P.; et al. (PLoS, 23/07/2013)
      Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ28. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.
    • Species classifier choice is a key consideration when analysing low-complexity food microbiome data

      Walsh, Aaron M.; Crispie, Fiona; O'Sullivan, Orla; Finnegan, Laura; Claesson, Marcus J.; Cotter, Paul D.; Science Foundation Ireland; SFI/12/RC/2273; 11/PI/1137; 13/SIRG/2160 (Biomed Central, 2018-03-20)
      Background The use of shotgun metagenomics to analyse low-complexity microbial communities in foods has the potential to be of considerable fundamental and applied value. However, there is currently no consensus with respect to choice of species classification tool, platform, or sequencing depth. Here, we benchmarked the performances of three high-throughput short-read sequencing platforms, the Illumina MiSeq, NextSeq 500, and Ion Proton, for shotgun metagenomics of food microbiota. Briefly, we sequenced six kefir DNA samples and a mock community DNA sample, the latter constructed by evenly mixing genomic DNA from 13 food-related bacterial species. A variety of bioinformatic tools were used to analyse the data generated, and the effects of sequencing depth on these analyses were tested by randomly subsampling reads. Results Compositional analysis results were consistent between the platforms at divergent sequencing depths. However, we observed pronounced differences in the predictions from species classification tools. Indeed, PERMANOVA indicated that there was no significant differences between the compositional results generated by the different sequencers (p = 0.693, R2 = 0.011), but there was a significant difference between the results predicted by the species classifiers (p = 0.01, R2 = 0.127). The relative abundances predicted by the classifiers, apart from MetaPhlAn2, were apparently biased by reference genome sizes. Additionally, we observed varying false-positive rates among the classifiers. MetaPhlAn2 had the lowest false-positive rate, whereas SLIMM had the greatest false-positive rate. Strain-level analysis results were also similar across platforms. Each platform correctly identified the strains present in the mock community, but accuracy was improved slightly with greater sequencing depth. Notably, PanPhlAn detected the dominant strains in each kefir sample above 500,000 reads per sample. Again, the outputs from functional profiling analysis using SUPER-FOCUS were generally accordant between the platforms at different sequencing depths. Finally, and expectedly, metagenome assembly completeness was significantly lower on the MiSeq than either on the NextSeq (p = 0.03) or the Proton (p = 0.011), and it improved with increased sequencing depth. Conclusions Our results demonstrate a remarkable similarity in the results generated by the three sequencing platforms at different sequencing depths, and, in fact, the choice of bioinformatics methodology had a more evident impact on results than the choice of sequencer did.