Evaluation of <scp>DNA</scp> extraction kits and phylogenetic diversity of the porcine gastrointestinal tract based on Illumina sequencing of two hypervariable regions

Background Bacillus spp. seem to be an alternative to antimicrobial growth promoters for improving animals’ health and performance. However, there is little information on the effect of Bacillus spp. in combination with different dietary crude protein (CP) levels on the ileal digestibility and microbiota composition. Therefore, the objective of this study was to determine the effect of Bacillus spp. supplementation to low- (LP) and high-protein diets (HP) on ileal CP and amino acid (AA) digestibility and intestinal microbiota composition.MethodsEight ileally cannulated pigs with an initial body weight of 28.5 kg were randomly allocated to a row-column design with 8 pigs and 3 periods of 16 d each. The assay diets were based on wheat-barley-soybean meal with two protein levels: LP (14% CP, as-fed) and HP diet (18% CP, as-fed). The LP and HP diets were supplemented with or without Bacillus spp. at a level of 0.04% (as-fed). The apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of CP and AA was determined. Bacterial community composition from ileal digesta was analyzed by Illumina amplicon sequencing and quantitative real-time PCR. Data were analyzed as a 2 × 2 factorial design using the GLIMMIX procedures of SAS.ResultsThe supplementation with Bacillus spp. did not affect both AID and SID of CP and AA in growing pigs. Moreover, there was no difference in AID of CP and AA between HP and LP diets, but SID of cystine, glutamic acid, glycine, and proline was lower (P < 0.05) in pigs fed the HP diets. The HP diets increased abundance of Bifidobacterium spp. and Lactobacillus spp., (P < 0.05) and by amplicon sequencing the latter was identified as predominant genus in microbiota from HP with Bacillus spp., whereas dietary supplementation of Bacillus spp. increased (P < 0.05) abundance of Roseburia spp..ConclusionsThe HP diet increased abundance of Lactobacillus spp. and Bifidobacterium spp.. The supplementation of Bacillus spp. resulted in a higher abundance of healthy gut associated bacteria without affecting ileal CP and AA digestibility, whereas LP diet may reduce the flow of undigested protein to the large intestine of pigs.

show abstract

“…Extraction procedure was performed with slight modifications to manufacturer’s instructions as described by Burbach et al [32]. …”

Section: Methodsmentioning

confidence: 99%

Effect of Bacillus subtilis and Bacillus licheniformis supplementation in diets with low- and high-protein content on ileal crude protein and amino acid digestibility and intestinal microbiota composition of growing pigs

Kaewtapee

Burbach

Tomforde

et al. 2017

J Animal Sci Biotechnol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Microbial community analysis was carried out by means of high-throughput amplicon sequencing using 250 bp paired-end sequencing chemistry (MiSeq Illumina), both on the DNA and cDNA samples. The general primers 807F and 1050R, targeting the V5–V6 region of the 16S rRNA gene of both bacteria and archaea [36], were used as previously described [37]. The preparation of libraries for barcode sequencing and data-set quality filtering were carried out as described by Camarinha-Silva, et al [38].…”

Section: Methodsmentioning

confidence: 99%

Presence does not imply activity: DNA and RNA patterns differ in response to salt perturbation in anaerobic digestion

Vrieze

Regueiro

Props

et al. 2016

Biotechnol Biofuels

Self Cite

View full text Add to dashboard Cite

BackgroundThe microbial community in anaerobic digestion is mainly monitored by means of DNA-based methods. This may lead to incorrect interpretation of the community parameters, because microbial abundance does not necessarily reflect activity. In this research, the difference between microbial community response on DNA (total community) and RNA (active community) based on the 16S rRNA (gene) with respect to salt concentration and response time was evaluated.ResultsThe application of higher NaCl concentrations resulted in a decrease in methane production. A stronger and faster response to salt concentration was observed on RNA level. This was reflected in terms of microbial community composition and organization, as richness, evenness, and overall diversity were differentially impacted. A higher divergence of community structure was observed on RNA level as well, indicating that total community composition depends on deterministic processes, while the active community is determined by stochastic processes. Methanosaeta was identified as the most abundant methanogen on DNA level, but its relative abundance decreased on RNA level, related to salt perturbation.ConclusionsThis research demonstrated the need for RNA-based community screening to obtain reliable information on actual community parameters and to identify key species that determine process stability.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0652-5) contains supplementary material, which is available to authorized users.

show abstract

“…For the construction of microbial 16S rRNA amplicon libraries, the V5-V6 hypervariable regions were PCR-amplified with universal primers 807F and 1050R (Bohorquez et al 2012). The barcoding of the DNA amplicons and the addition of Illumina adaptors were conducted by PCR as described previously (Camarinha-Silva et al 2014;Burbach et al 2016). The PCR-generated amplicon libraries were subjected to 250 nt paired-end sequencing on an Illumina MiSeq platform (Illumina, San Diego, CA, USA).…”

Section: Total Dna Isolation Construction Of 16s Rrna Gene Librariesmentioning

confidence: 99%

Pristine but metal-rich Río Sucio (Dirty River) is dominated by Gallionella and other iron-sulfur oxidizing microbes

Arce-Rodríguez

Puente-Sánchez

Avendaño³

et al. 2016

Extremophiles

Self Cite

View full text Add to dashboard Cite

Whether the extreme conditions of acidity and heavy metal pollution of streams and rivers originating in pyritic formations are caused primarily by mining activities or by natural activities of metal-oxidizing microbes living within the geological formations is a subject of considerable controversy. Most microbiological studies of such waters have so far focused on acid mine drainage sites, which are heavily human-impacted environments, so it has been problematic to eliminate the human factor in the question of the origin of the key metal compounds. We have studied the physico-chemistry and microbiology of the Río Sucio in the Braulio Carrillo National Park of Costa Rica, 22 km from its volcanic rock origin. Neither the remote origin, nor the length of the river to the sampling site, have experienced human activity and are thus pristine. The river water had a characteristic brownish-yellow color due to high iron-dominated minerals, was slightly acidic, and rich in chemolithoautotrophic iron- and sulfur-oxidizing bacteria, dominated by Gallionella spp. Río Sucio is thus a natural acid-rock drainage system whose metal-containing components are derived primarily from microbial activities.

show abstract

Evaluation of DNA extraction kits and phylogenetic diversity of the porcine gastrointestinal tract based on Illumina sequencing of two hypervariable regions

Cited by 58 publications

References 51 publications

Effect of Bacillus subtilis and Bacillus licheniformis supplementation in diets with low- and high-protein content on ileal crude protein and amino acid digestibility and intestinal microbiota composition of growing pigs

Effect of Bacillus subtilis and Bacillus licheniformis supplementation in diets with low- and high-protein content on ileal crude protein and amino acid digestibility and intestinal microbiota composition of growing pigs

Presence does not imply activity: DNA and RNA patterns differ in response to salt perturbation in anaerobic digestion

Pristine but metal-rich Río Sucio (Dirty River) is dominated by Gallionella and other iron-sulfur oxidizing microbes

Contact Info

Product

Resources

About