Hein Min Tun

Gene-centric metagenomics analysis of feline intestinal microbiome using 454 junior pyrosequencing

Abstract The feline gastrointestinal microbiota have direct influence on feline health and also human health as a reservoir for potential zoonotic pathogens and antibiotic resistant bacterial strains. In order to describe the feline gastrointestinal microbial diversity, fecal samples from cats have been characterized using both culture-dependent and culture-independent methods. However, data correlating total microbial composition and their functions are lacking. Present descriptive study evaluated both phylogenetic and metabolic diversity of the feline intestinal microbiota using GS Junior titanium shotgun pyrosequencing. A total of 152,494 pyrosequencing reads (5405 assembled contigs) were generated and classified into both phylogenetic and metabolic profiles of the feline intestinal microbiota. The Bacteroides/Chlorobi group was the most predominant bacterial phylum comprising ~68% of total classified diversity, followed by Firmicutes (~13%) and Proteobacteria (~6%) respectively. Archaea, fungi and viruses made up the minor communities in the overall microbial diversity. Interestingly, this study also identified a range of potential enteric zoonotic pathogens (0.02–1.25%) and genes involved in antimicrobial resistance (0.02–0.7%) in feline fecal materials. Based on clustering among nine gastrointestinal metagenomes from five different monogastric hosts (dog, human, mice, cat and chicken), the cat metagenome clustered closely together with chicken in both phylogenetic and metabolic level (>80%). Future studies are required to provide deeper understandings on both intrinsic and extrinsic effects such as impact of age, genetics and dietary interventions on the composition of the feline gastrointestinal microbiome.

Genomic insights into high exopolysaccharide-producing dairy starter bacterium Streptococcus thermophilus ASCC 1275

Streptococcus thermophilus ASCC 1275 (ST 1275), a typical dairy starter bacterium, yields the highest known amount (~1,000 mg/L) of exopolysaccharide (EPS) in milk among the species of S. thermophilus. The addition of this starter in milk fermentation exhibited texture modifying properties for fermented dairy foods such as yogurt and cheese in the presence of EPS as its important metabolite. In this genomic study, a novel eps gene cluster for EPS assembly of repeating unit has been reported. It contains two-pair epsC-epsD genes which are assigned to determine the chain length of EPS. This also suggests this organism produces two types of EPSs – capsular and ropy EPS, as observed in our previous studies. Additionally, ST 1275 appears to exhibit effective proteolysis system and sophisticated stress response systems to stressful conditions, and has the highest number of four separate CRISPR/Cas loci. These features may be conducive to milk adaptation of this starter and against undesirable bacteriophage infections which leads to failure of milk fermentation. Insights into the genome of ST 1275 suggest that this strain may be a model high EPS-producing dairy starter.

An extended single‐index multiplexed 16S rRNA sequencing for microbial community analysis on MiSeq illumina platforms

The primary 16S rRNA sequencing protocol for microbial community analysis using Illumina platforms includes a single‐indexing approach that allows pooling of hundreds of samples in each sequencing run. The protocol targets the V4 hypervariable region (HVR) of 16S rRNA using 150 bp paired‐end (PE) sequencing. However, the latest improvement in Illumina chemistry has increased the read length up to 600 bp using 300 bp PE sequencing. To take advantage of the longer read length, a dual‐indexing approach was previously developed for targeting different HVRs. However, due to simple working protocols, the single‐index 150 bp PE approach still continues to be attractive to many researchers. Here, we described an extended single‐indexing protocol for 300 bp PE illumina sequencing that targets the V3‐V4 HVRs of 16S rRNA. The new primer set led to increased read length and alignment resolution, as well as increased richness and diversity of resulting microbial profile compared to that obtained from150 bp PE protocol for V4 sequencing. The β‐diversity profile also differed qualitatively and quantitatively between the two approaches. Both primer sets had high coverage rates and specificity to detect dominant phyla; however, their coverage rate with regards to the rare biosphere varied. Our data further confirms that the choice of primer is the most deterministic factor in sequencing coverage and specificity.

High Molecular Weight Barley β-Glucan Alters Gut Microbiota Toward Reduced Cardiovascular Disease Risk

The physiological cholesterol-lowering benefits of β-glucan have been well documented, however, whether modulation of gut microbiota by β-glucan is associated with these physiological effects remains unknown. The objectives of this study were therefore to determine the impact of β-glucan on the composition of gut microbiota in mildly hypercholesterolemic individuals and to identify if the altered microbiota are associated with bioactivity of β-glucan in improving risk factors of cardiovascular disease (CVD). Using a randomized, controlled crossover study design, individuals received for 5-week either a treatment breakfast containing 3 g high molecular weight (HMW), 3 g low molecular weight (LMW), 5 g LMW barley β-glucan, or wheat and rice. The American Heart Association (AHA) diet served as the background diet for all treatment groups. Phases were separated by 4-week washout periods. Fecal samples were collected at the end of each intervention phase and subjected to Illumina sequencing of 16S rRNA genes. Results revealed that at the phylum level, supplementation of 3 g/d HMW β-glucan increased Bacteroidetes and decreased Firmicutes abundances compared to control (P < 0.001). At the genus level, consumption of 3 g/d HMW β-glucan increased Bacteroides (P < 0.003), tended to increase Prevotella (P < 0.1) but decreased Dorea (P < 0.1), whereas diets containing 5 g LMW β-glucan and 3 g LMW β-glucan failed to alter the gut microbiota composition. Bacteroides, Prevotella, and Dorea composition correlated (P < 0.05) with shifts of CVD risk factors, including body mass index, waist circumference, blood pressure, as well as triglyceride levels. Our data suggest that consumption of HMW β-glucan favorably alters the composition of gut microbiota and this altered microbiota profile associates with a reduction of CVD risk markers. Together, our study suggests that β-glucan induced shifts in gut microbiota in a MW-dependent manner and that might be one of the underlying mechanisms responsible for the physiological benefits of β-glucan.

Phylodynamics of H5N1 avian influenza virus in Indonesia

Understanding how pathogens invade and become established in novel host populations is central to the ecology and evolution of infectious disease. Influenza viruses provide unique opportunities to study these processes in nature because of their rapid evolution, extensive surveillance, large data sets and propensity to jump species boundaries. H5N1 highly pathogenic avian influenza virus (HPAIV) is a major animal pathogen and public health threat. The virus is of particular importance in Indonesia, causing severe outbreaks among poultry and sporadic human infections since 2003. However, little is known about how H5N1 HPAIV emerged and established in Indonesia. To address these questions, we analysed Indonesian H5N1 HPAIV gene sequences isolated during 2003–2007. We find that the virus originated from a single introduction into East Java between November 2002 and October 2003. This invasion was characterized by an initially rapid burst of viral genetic diversity followed by a steady rate of lineage replacement and the maintenance of genetic diversity. Several antigenic sites in the haemagglutinin gene were subject to positive selection during the early phase, suggesting that host‐immune‐driven selection played a role in host adaptation and expansion. Phylogeographic analyses show that after the initial invasion of H5N1, genetic variants moved both eastwards and westwards across Java, possibly involving long‐distance transportation by humans. The phylodynamics we uncover share similarities with other recently studied viral invasions, thereby shedding light on the ecological and evolutionary processes that determine disease emergence in a new geographical region.

Re-emerging of porcine respiratory and reproductive syndrome virus (lineage 3) and increased pathogenicity after genomic recombination with vaccine variant.

Porcine reproductive and respiratory syndrome virus (PRRSV) was first reported in China since late 1995 and several variants were further reported in subsequence years, causing huge economic losses to the Chinese swine industry. To date, three major lineages (lineage 3, 5.1 and 8.7) of Type 2 PRRSV were reported in China based on our global genotyping. The present study provides the epidemiology of the PRRSV in South China based on the isolates collected during 2009-2012, indicating three lineages (lineage 3, 5.1 and 8.7) of Type 2 PRRSV were still circulating in this area. Our phylogenetic reconstruction indicated that lineage 3 re-emerged in 2010 formed a huge cluster with closely related to the 2004 isolates from Hong Kong. Furthermore, the inter-lineage genomic recombination between MLV vaccine strain (lineage 5) and a recently re-emerged lineage 3 virus (QYYZ) has also been found in a farm practicing MLV vaccination. Our in vivo experiment comparing the pathogenicity and clinical presentations among currently isolated viruses indicated that pigs infected with recombinant lineage 3 virus (GM2) showed persistent higher fever compared to pigs infected by its wild counterpart (QYYZ). This study enhanced our understanding on potential importance of the recombination of PRRSV along with their evolution.

Microbial community in microbial fuel cell (MFC) medium and effluent enriched with purple photosynthetic bacterium ( Rhodopseudomonas sp. )

High power densities have been obtained from MFC reactors having a purple color characteristic of Rhodopseudomonas. We investigated the microbial community structure and population in developed purple MFC medium (DPMM) and MFC effluent (DPME) using 16S rRNA pyrosequencing. In DPMM, dominant bacteria were Comamonas (44.6%), Rhodopseudomonas (19.5%) and Pseudomonas (17.2%). The bacterial community of DPME mainly consisted of bacteria related to Rhodopseudomonas (72.2%). Hydrogen oxidizing bacteria were identified in both purple-colored samples: Hydrogenophaga and Sphaerochaeta in the DPMM, and Arcobacter, unclassified Ignavibacteriaceae, Acinetobacter, Desulfovibrio and Wolinella in the DPME. The methanogenic community of both purple-colored samples was dominated by hydrogenotrophic methanogens including Methanobacterium, Methanobrevibacter and Methanocorpusculum with significantly lower numbers of Methanosarcina. These results suggeste that hydrogen is actively produced by Rhodopseudomonas that leads to the dominance of hydrogen consuming microorganisms in both purple-colored samples. The syntrophic relationship between Rhodopseudomonas and hydrogenotrophic microbes might be important for producing high power density in the acetate-fed MFC under light conditions.

Detection of Antibiotic Resistance Genes in Source and Drinking Water Samples from a First Nations Community in Canada

ABSTRACT Access to safe drinking water is now recognized as a human right by the United Nations. In developed countries like Canada, access to clean water is generally not a matter of concern. However, one in every five First Nations reserves is under a drinking water advisory, often due to unacceptable microbiological quality. In this study, we analyzed source and potable water from a First Nations community for the presence of coliform bacteria as well as various antibiotic resistance genes. Samples, including those from drinking water sources, were found to be positive for various antibiotic resistance genes, namely, ampC, tet(A), mecA, β-lactamase genes (SHV-type, TEM-type, CTX-M-type, OXA-1, and CMY-2-type), and carbapenemase genes (KPC, IMP, VIM, NDM, GES, and OXA-48 genes). Not surprisingly, substantial numbers of total coliforms, including Escherichia coli, were recovered from these samples, and this result was also confirmed using Illumina sequencing of the 16S rRNA gene. These findings deserve further attention, as the presence of coliforms and antibiotic resistance genes potentially puts the health of the community members at risk. IMPORTANCE In this study, we highlight the poor microbiological quality of drinking water in a First Nations community in Canada. We examined the coliform load as well as the presence of antibiotic resistance genes in these samples. This study examined the presence of antibiotic-resistant genes in drinking water samples from a First Nations Community in Canada. We believe that our findings are of considerable significance, since the issue of poor water quality in First Nations communities in Canada is often ignored, and our findings will help shed some light on this important issue.

Microbial Diversity and Evidence of Novel Homoacetogens in the Gut of Both Geriatric and Adult Giant Pandas ( Ailuropoda melanoleuca )

Recent studies have described the bacterial community residing in the guts of giant pandas, together with the presence of lignocellulolytic enzymes. However, a more comprehensive understanding of the intestinal microbial composition and its functional capacity in giant pandas remains a major goal. Here, we conducted a comparison of bacterial, fungal and homoacetogenic microbial communities from fecal samples taken from two geriatric and two adult captive giant pandas. 16S rDNA amplicon pyrosequencing revealed that Firmicutes and Proteobacteria are the most abundant microbiota in both geriatric and adult giant pandas. However, members of phylum Actinobacteria found in adult giant pandas were absent in their geriatric counterparts. Similarly, ITS1 amplicon pyrosequencing identified developmental changes in the most abundant fungal classes from Sordariomycetes in adult pandas to Saccharomycetes in geriatric pandas. Geriatric pandas exhibited significantly higher abundance of a potential probiotic fungus (Candida tropicalis) as compared to adult pandas, indicating their importance in the normal digestive physiology of aged pandas. Our study also reported the presence of a lignocellulolytic white-rot fungus, Perenniporia medulla-panis, and the evidence of novel homoacetogens residing in the guts of giant pandas.

Genetic diversity and multiple introductions of porcine reproductive and respiratory syndrome viruses in Thailand

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in Thailand, causing a huge impact on the countrys swine industry. Yet the diversity and origin of these Thai PRRSVs remained vague. In this context, we collected all the Thai PRRSV sequences described earlier and incorporated them into the global diversity. The results indicated that PRRSVs in Thailand were originated from multiple introductions involving both Type 1 and Type 2 PRRSVs. Many of the introductions were followed by extensive geographic expansion, causing regional co-circulation of diverse PRRSV variants in three major pig-producing provinces. Based on these results, we suggest (1) to avoid blind vaccination and to apply vaccines tailor-made for target diversity, (2) to monitor pig importation and transportation, and (3) to implement a better biosecurity to reduce horizontal transmissions as three potentially effective strategies of controlling PRRS in Thailand.