Barcoded pyrosequencing-based metagenomic analysis of the faecal microbiome of three purebred pig lines after cohabitation

Pajarillo, Edward Alain B.,Chae, Jong Pyo,Kim, Hyeun Bum,Kim, In Ho,Kang, Dae-Kyung Springer-Verlag 2015 Applied microbiology and biotechnology Vol.99 No.13

<P>The microbial communities in the pig gut perform a variety of beneficial functions. Along with host genetics and diet, farm management practices are an important aspect of agricultural animal production that could influence gut microbial diversity. In this study, we used barcoded pyrosequencing of the V1-V3 regions of the 16S ribosomal RNA (rRNA) genes to characterise the faecal microbiome of three common commercial purebred pig lines (Duroc, Landrace and Yorkshire) before and after cohabitation. The diversity of faecal microbiota was characterised by employing phylogenetic, distance-based and multivariate-clustering approaches. Bacterial diversity tended to become more uniform after mixing of the litters. Age-related shifts were also observed at various taxonomic levels, with an increase in the proportion of the phylum Firmicutes and a decrease in Bacteroidetes over time, regardless of the purebred group. Cohabitation had a detectable effect on the microbial shift among purebred pigs. We identified the bacterial genus Parasutterella as having utility in discriminating pigs according to time. Similarly, Dialister and Bacteroides can be used to differentiate the purebred lines used. The microbial communities of the three purebred pigs became more similar after cohabitation, but retained a certain degree of breed specificity, with the microbiota of Landrace and Yorkshire remaining distinct from that of their distant relative, Duroc.</P>

Quantitative Proteogenomics and the Reconstruction of the Metabolic Pathway in Lactobacillus mucosae LM1

Pajarillo, Edward Alain B.,Kim, Sang Hoon,Lee, Ji-Yoon,Valeriano, Valerie Diane V.,Kang, Dae-Kyung Korean Society for Food Science of Animal Resource 2015 한국축산식품학회지 Vol.35 No.5

Lactobacillus mucosae is a natural resident of the gastrointestinal tract of humans and animals and a potential probiotic bacterium. To understand the global protein expression profile and metabolic features of L. mucosae LM1 in the early stationary phase, the QExactive^TM Hybrid Quadrupole-Orbitrap Mass Spectrometer was used. Characterization of the intracellular proteome identified 842 proteins, accounting for approximately 35% of the 2,404 protein-coding sequences in the complete genome of L. mucosae LM1. Proteome quantification using QExactive^TM Orbitrap MS detected 19 highly abundant proteins (> 1.0% of the intracellular proteome), including CysK (cysteine synthase, 5.41%) and EF-Tu (elongation factor Tu, 4.91%), which are involved in cell survival against environmental stresses. Metabolic pathway annotation of LM1 proteome using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that half of the proteins expressed are important for basic metabolic and biosynthetic processes, and the other half might be structurally important or involved in basic cellular processes. In addition, glycogen biosynthesis was activated in the early stationary phase, which is important for energy storage and maintenance. The proteogenomic data presented in this study provide a suitable reference to understand the protein expression pattern of lactobacilli in standard conditions

Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing

Pajarillo, Edward Alain B.,Chae, Jong Pyo,Balolong, Marilen P.,Kim, Hyeun Bum,Seo, Kang-Seok,Kang, Dae-Kyung Asian Australasian Association of Animal Productio 2015 Animal Bioscience Vol.28 No.4

This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1-V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs.

Assessment of fecal bacterial diversity among healthy piglets during the weaning transition.

Alain B Pajarillo, Edward,Chae, Jong-Pyo,Balolong, Marilen P,Bum Kim, Hyeun,Kang, Dae-Kyung Institute of Applied Microbiology, University of T 2014 The Journal of general and applied microbiology Vol.60 No.4

<P>The high level of genetic diversity in the microflora of the gastrointestinal tract has the potential to provide numerous beneficial functions to the host. Thus it is now acknowledged that the complexity in animal functioning is linked to the interacting microbiome in the gut. Despite the importance of gut microbiome, there is a lack of information concerning the microbial communities in the pig gut during the weaning transition. This study describes the fecal microbial shifts of healthy piglets during the weaning transition using barcoded pyrosequencing of the prokaryotic 16S rRNA gene. Fecal samples were obtained from 15 piglets during the pre-weaning period (fourth week after birth) and post-weaning (sixth week after birth) and were subjected to community genomic DNA extraction for pyrosequencing analysis. As the piglets underwent the weaning transition a trend toward increased bacterial diversity was observed, based on species abundance as measured by the Shannon-Weaver index. Firmicutes (54.0%) and Bacteroidetes (59.6%) were the most dominant phyla during pre-weaning and post-weaning, respectively. During the weaning transition a distinct shift from Bacteroides to Prevotella as the most abundant genus was observed. Additionally, we detected a number of abundant gut bacterial species that have not been reported previously. Clostridium rectum, C. clostridioforme, C. lactatifermentans and Butyricimonas virosa were uniquely detected prior to weaning while Roseburia cecicola and Blautia wexlerae were detected during the post-weaning period only.</P>

Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing

Edward Alain B. Pajarillo,채종표,Marilen P. Balolong,김현범,서강석,강대경 아세아·태평양축산학회 2015 Animal Bioscience Vol.28 No.4

This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1–V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs.

Pyrosequencing-based Analysis of Fecal Microbial Communities in Three Purebred Pig Lines

Edward Alain B. Pajarillo,채종표,Marilen P. Balolong,김현범,서강석,강대경 한국미생물학회 2014 The journal of microbiology Vol.52 No.8

This study examined the fecal bacterial diversity of 15-weekoldpigs from three purebred lines: Duroc, Landrace, andYorkshire. Taxon-dependent and -independent analyses wereperformed to evaluate differences in the fecal bacterial communitiesand to identify bacterial genera that can be used todiscriminate breeds, following high-throughput pyrosequencingof 16S rRNA genes. Among the breeds evaluated, Landracehad the most diverse bacterial community composition. Prevotella, Blautia, Oscillibacter, and Clostridium were detectedin all samples regardless of breed. On the other hand,Catenibacterium, Blautia, Dialister, and Sphaerochaeta weredifferentially detected among breeds, as demonstrated bythe canonical loading plot. The discriminant analysis of principalcomponents plot also showed clear separation of thethree purebred pig lines, with a certain degree of similaritybetween Landrace and Yorkshire pigs and a distinct separationbetween Duroc pigs and the other two breeds. Otherfactors not related to breed, such as season or time of samplingand pen effects, may contribute to shaping the gutmicrobiota of pigs.

Quantitative Proteogenomics and the Reconstruction of the Metabolic Pathway in Lactobacillus mucosae LM1

Edward Alain B. Pajarillo,Sang Hoon Kim,Ji-yoon Lee,Valerie Diane V. Valeriano,Dae-kyung Kang 한국축산식품학회 2015 한국축산식품학회지 Vol.35 No.5

Lactobacillus mucosae is a natural resident of the gastrointestinal tract of humans and animals and a potential probiotic bacterium. To understand the global protein expression profile and metabolic features of L. mucosae LM1 in the early stationary phase, the QExactive ™ Hybrid Quadrupole-Orbitrap Mass Spectrometer was used. Characterization of the intracellular proteome identified 842 proteins, accounting for approximately 35% of the 2,404 protein-coding sequences in the complete genome of L. mucosae LM1. Proteome quantification using QExactive™ Orbitrap MS detected 19 highly abundant proteins (> 1.0% of the intracellular proteome), including CysK (cysteine synthase, 5.41%) and EF-Tu (elongation factor Tu, 4.91%), which are involved in cell survival against environmental stresses. Metabolic pathway annotation of LM1 proteome using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that half of the proteins expressed are important for basic metabolic and biosynthetic processes, and the other half might be structurally important or involved in basic cellular processes. In addition, glycogen biosynthesis was activated in the early stationary phase, which is important for energy storage and maintenance. The proteogenomic data presented in this study provide a suitable reference to understand the protein expression pattern of lactobacilli in standard conditions.

Revealing the combined effects of lactulose and probiotic enterococci on the swine faecal microbiota using 454 pyrosequencing

Chae, Jong Pyo,Pajarillo, Edward Alain B.,Oh, Ju Kyoung,Kim, Heebal,Kang, Dae‐,Kyung John Wiley and Sons Inc. 2016 MICROBIAL BIOTECHNOLOGY -BLACKWELL- Vol.9 No.4

<P><B>Summary</B></P><P>Demand for the development of non‐antibiotic growth promoters in animal production has increased in recent years. This report compared the faecal microbiota of weaned piglets under the administration of a basal diet (CON) or that containing prebiotic lactulose (LAC), probiotic <I>Enterococcus faecium </I>NCIMB 11181 (PRO) or their synbiotic combination (SYN). At the phylum level, the <I>Firmicutes</I> to <I>Bacteroidetes</I> ratio increased in the treatment groups compared with the CON group, and the lowest proportion of <I>Proteobacteria</I> was observed in the LAC group. At the family level, <I>Enterobacteriaceae</I> decreased in all treatments; more than a 10‐fold reduction was observed in the LAC (0.99%) group compared with the CON group. At the genus level, the highest <I>Oscillibacter</I> proportion was detected in PRO, the highest <I>Clostridium</I> in LAC and the highest <I>Lactobacillus</I> in SYN; the abundance of <I>Escherichia</I> was lowest in the LAC group. Clustering in the discriminant analysis of principal components revealed distinct separation of the feeding groups (CON, LAC, PRO and SYN), showing different microbial compositions according to different feed additives or their combination. These results suggest that individual materials and their combination have unique actions and independent mechanisms for changes in the distal gut microbiota.</P>

Protective effects of Bacillus subtilis against Salmonella infection in the microbiome of Hy-Line Brown layers

Oh, Ju Kyoung,Pajarillo, Edward Alain B.,Chae, Jong Pyo,Kim, In Ho,Kang, Dae-Kyung Asian Australasian Association of Animal Productio 2017 Animal Bioscience Vol.30 No.9

Objective: This study investigated the effects of Bacillus subtilis CSL2 (B. subtilis CSL2) administration before Salmonella challenge on the fecal microbiota and microbial functionality of Hy-line Brown (HLB) laying hens. Methods: Fecal samples were collected from control (CON), Salmonella-infected (SAL) and Salmonella-infected, probiotic-treated (PRO) groups before and after Salmonella challenge for microbiome analysis using 16S rRNA gene pyrosequencing. Results: Infection with Salmonella led to decreased microbial diversity in hen feces; diversity was recovered with Bacillus administration. In addition, Salmonella infection triggered significant alterations in the composition of the fecal microbiota. The abundance of the phylum Firmicutes decreased while that of Proteobacteria, which includes a wide variety of pathogens, increased significantly. Bacillus administration resulted in normal levels of abundance of Firmicutes and Proteobacteria. Analysis of bacterial genera showed that Salmonella challenge decreased the population of Lactobacillus, the most abundant genus, and increased populations of Pseudomonas and Flavobacterium genera by a factor of 3 to 5. On the other hand, Bacillus administration caused the abundance of the Lactobacillus genus to recover to control levels and decreased the population of Pseudomonas significantly. Further analysis of operational taxonomic units revealed a high abundance of genes associated with two-component systems and secretion systems in the SAL group, whereas the PRO group had more genes associated with ribosomes. Conclusion: The results of this study indicate that B. subtilis CSL2 administration can modulate the microbiota in HLB laying hens, potentially acting as a probiotic to protect against Salmonella Gallinarum infection.

Proteomic and Transcriptional Analysis of <i>Lactobacillus johnsonii</i> PF01 during Bile Salt Exposure by iTRAQ Shotgun Proteomics and Quantitative RT-PCR

Lee, Ji Yoon,Pajarillo, Edward Alain B.,Kim, Min Jeong,Chae, Jong Pyo,Kang, Dae-Kyung American Chemical Society 2013 Journal of proteome research Vol.12 No.1

<P><I>Lactobacillus johnsonii</I> PF01 has been reported to be highly resistant to bile, a key property of probiotic microorganisms. Here, we examine the nature of the bile-salt tolerance of <I>L. johnsonii</I> PF01. Growth inhibition and surface morphology and physiology aberrations were observed after overnight exposure to bile stress. Quantitative proteomic profiles using iTRAQ–LC–MS/MS technology identified 8307 peptides from both untreated PF01 cells and those exposed to 0.1%, 0.2%, and 0.3% bile salts. Some 215 proteins exhibited changed levels in response to bile stress; of these, levels of 94 peptides increased while those of 121 decreased. These were classified into the following categories: stress responses, cell division, transcription, translation, nucleotide metabolism, carbohydrate transport and metabolism, cell wall biosynthesis, and amino acid biosynthesis, and 16 of unidentified function. Analysis of the mRNA expression of selected genes by quantitative reverse transcriptase-PCR verified the proteomic data. Both proteomic and mRNA data provided evidence for increased phosphotransferase activity and cell wall biosynthesis. In addition, three bile salt hydrolases were significantly upregulated by bile exposure. These findings provide a basis for future evaluations of the tolerance of potential probiotic strains toward the various gastrointestinal challenges, including bile stress.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2013/jprobs.2013.12.issue-1/pr300794y/production/images/medium/pr-2012-00794y_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr300794y'>ACS Electronic Supporting Info</A></P>